Measurands

Alkaline Phosphatase (ALP)

An IFCC reference measurement procedure (RMP) is available.  IVD manufacturers should formulate reagents to conform to the RMP.

Schumann G, Klauke R, Canalias F, Bossert-Reuther S, Franck PF, Gella FJ, Jørgensen PJ, Kang D, Lessinger JM, Panteghini M, Ceriotti F. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 °C. Part 9: Reference procedure for the measurement of catalytic concentration of alkaline phosphatase International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Scientific Division, Committee on Reference Systems of Enzymes (C-RSE). Clin Chem Lab Med 2011;49:1439-46.

Alanine Aminotransferase (ALT)

The IFCC has developed reference measurement procedures for AST and ALT enzymes. The IFCC reagent formulation is generally used by IVD manufacturers with some adaptation for the technology of a given instrument system.  Standardization is thus easily achievable.  The harmonization issue is whether or not pyridoxyl-5-phosphate (P5P) is included in reagents from IVD manufacturers.  P5P is needed to fully activate the enzymes in situations when a patient has a deficiency in this vitamin as may occur in kidney failure and other conditions. A technical issue is that adding P5P to reagents reduces the reagent stability. Consequently P5P is supplied in a separate container to be mixed at the time a reagent is put into use. Furthermore, laboratories may prefer not to add P5P because there may be reagent waste in lower testing volume situations. Some countries do not typically include P5P and in other countries there is a mix of inclusion and exclusion in reagents.  Differences in vitamin deficiency between countries may contribute to different practices.  The ICHCLR recommends that manufacturers make available reagents that include P5P so that laboratories can determine if their population would benefit from its use in the reagents.  A medium priority was assigned because these two analytes are well standardized except for the P5P inclusion and the need for P5P may vary among different regions of the world.

Schumann G, Bonora R, Ceriotti F, Ferard G, Ferrero CA, Franck PF, et al. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C. International Federation of Clinical Chemistry and Laboratory Medicine. Part 4. Reference procedure for the measurement of catalytic concentration of alanine aminotransferase. Clin Chem Lab Med. 2002;40:718-24.

Albumin

Differences among results are primarily related to use of bromcresol green (BCG) or bromcresol purple (BCP) as the dye binding reagent.  There are differences in selectivity for albumin between the two reagent types (1).  A reference material for serum proteins is available from IRMM that has been shown to be commutable with clinical samples  for many common measurement procedures.  The key issue affecting harmonization is choice of dye binding reaction.  A medium priority was assigned because albumin acts as a negative acute phase reactant to inflammation and there can be substantial within person biologic variability.  The biases that are observed among measurement procedures only moderately affect clinical decisions.

Reference:

1. Bachmann LM. Yu M, Boyd JC, Bruns DE, Miller WG. State of Harmonization of 24 Serum Albumin Measurement Procedures and Implications for Medical Decisions. Clin Chem 2017;63:770-9.

Albumin

A reference system for standardization of urine albumin measurements is being developed by the Laboratory Working Group of the NKDEP in collaboration with the IFCC Working Group for Standardization of Albumin in Urine.

Alpha Fetoprotein

Alpha Fetoprotein is a tumor marker used for follow-up management of patients undergoing cancer therapy, especially for hepatocellular carcinoma, testicular and ovarian tumors (1). Most of studies report elevated AFP concentrations in approximately 70% of patients with hepatocellular carcinoma (2,3). Concentrations of AFP above the reference interval have been found in serum of patients with benign liver disease (eg. viral hepatitis, cirrhosis), gastrointestinal tract tumors and along with carcinoembryonic antigen in ataxia telangiectasia (4).

Alpha fetoprotein is measured using 2-site immunoenzymatic sandwich assays with calibration traceable to the 1st IRP WHO Reference Standard 72/225, also used by commercial manufacturers for calibration of total IgE assays.

External Quality Assessment Data from a CAP 2017 Survey of 1013 laboratories had CV <10% among participants suggesting acceptable agreement among results.

References:

1. Chen DS, Sung JL, and Sheu JC, Serum α-fetoprotein in the early stage of human hepatocellular carcinoma. Gastroenterology 1984;86:1404–9.
2. Ramsey WH, Wu GY. Hepatocellular carcinoma: update on diagnosis and treatment. Dig Dis 1995;13:81-91.
3. Stuart KE, Anand AJ, Jenkins RL. Hepatocellular Carcinoma in the United States. Prognostic features, treatment outcome, and survival. Cancer 1996;11:217-22.
4. Behne T and Copur MS. Biomarkers for Hepatocellular Carcinoma. International Journal of Hepatology 2012; 2012:1-7.

Amylase

The IFCC has developed a reference measurement procedure for amylase.  Commercial measurement procedures use different substrates that contribute to biases among procedures.  An IFCC working group is addressing harmonization of amylase measurement procedures.

Schumann G, Aoki R, Ferrero CA, Ehlers G, Ferard G, Gella FJ, Jorgensen PJ, Kanno T, Kessner A, Klauke R, Kytzia HJ, Lessinger JM, Miller WG, Nagel R, Pauwels J, Schimmel H, Siekmann L, Weidemann G, Yoshida K, Ceriotti F. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C: International Federation of Clinical Chemistry and Laboratory Medicine (IFCC): Scientific Division, Committee on Reference Systems for Enzymes (C-RSE): Part 8. Reference procedure for the measurement of catalytic concentration of alpha-amylase: [alpha-Amylase: 1,4-alpha-D-glucan 4-glucanohydrolase (AMY), EC 3.2.1.1]. Clin Chem Lab Med 2006;44:1146-55.

Anti-DNA antibody (qualitative)

Qualitative Test

Antibodies of the IgG class to double-stranded (ds, native) DNA (dsDNA) are an accepted criterion by the American College of Rheumatology for the diagnosis of systemic lupus erythematosus (SLE).

dsDNA antibodies are detectable in approximately 85% of patients with untreated SLE, and are rarely detectable in other connective tissue diseases.

In a representative 2016 College of American Pathologists Survey, 605 labs of 1020 participating labs used qualitative methods. The average consensus rate for all types of methods was 98.5 %.  This excellent consensus rate supports a low priority for harmonization of qualitative methods.

Anti-DNA antibody (quantitative)

Quantitative Test

Antibodies of the IgG class to double-stranded (ds, native) DNA (dsDNA) are an accepted criterion by the American College of Rheumatology for the diagnosis of systemic lupus erythematosus (SLE).

dsDNA antibodies are detectable in approximately 85% of patients with untreated SLE, and are rarely detectable in other connective tissue diseases. The concentrations of IgG antibodies to double-stranded (ds, native) DNA (dsDNA) in serum are known to fluctuate with disease activity in lupus erythematosus, often increasing prior to an increase in inflammation and decreasing in response to therapy.

In a representative 2016 College of American Pathologists Survey, 415 labs of 1020 participating labs used quantitative methods.  There are two types of methods used, enzyme immunoassay and multiple immunobead methods.  The CV range for enzyme immunoassay was 7.4%-82.7% and the median value was 18.5%.  The average CV of Bio-Rad multiple bead immunoassay was 8.0%, and that of Whampole multiple bead immunoassay was 28.5%.  Most participants (139 of 174) using multiple bead immunoassay used the Bio-Rad product.  The large differences among results from different quantitative methods suggests a medium priority for harmonization.

Anti-HBs Qualitative

Hepatitis B virus (HBV) infection is endemic throughout the world. Anti-HBs test has been using to identify previous exposure to hepatitis B virus and to determine adequate immunity from hepatitis B vaccination. An adequate vaccine response is defined as an anti-HBs level of 100 IU/L 4 to 8 weeks after the last of three or four vaccine injections.

Based on the World Health Organization recommendation, an Anti-HBs concentration ≥ 10 IU/L is regarded as being protective against Hepatitis B viral infection (1). Vaccinees without sufficient anti-HBs responses, so-called non-responders or low responders, undergo a special regimen of additional vaccine doses. For liver transplant recipients, quantitative measurement of anti-HBs levels is used in the management of hepatitis B immune globulin prophylaxis, which is initiated to maintain anti-HBs levels of at least 100 or 200 IU/L, according to different guidelines (2).

Harmonization for qualitative assay of anti-HBs is adequate based on proficiency testing data from the College of American Pathologists Viral Marker Survey in 2020 that showed over 99% consensus rate in about 1,500 participants using 15 assay products from 6 IVD manufacturers.

However, harmonization for quantitative assay of anti-HBs is inadequate based on Huzly et al’s findings that analysis of routine clinical samples by different systems revealed significant discrepancies for a number of sera (3). Huzly et al. reported that results for 200 serum samples showed a mean coefficient of variation(CV) between nine different measurement procedures was 47.1% (range, 15.0% to 201.0%), and when serum samples were classified according to the anti-HBs levels determined by the majority of assays (10 to 100, 101 to 1,000, and _1,000 IU/liter), the mean CV was significantly lower for the group of sera with anti-HBs levels between 101 and 1,000 IU/liter than for sera with levels between 10 and 100 or above 1,000 IU/liter. (36.2% versus 57.4% and 60.3%, respectively; P 0.003).

References:

1. World Health Organization. Hepatitis B vaccines. Weekly epidemiological record No. 40, 2009;84:405–20.
2. Jack AD, Hall AJ, Maine N, Mendy M and Whittle HC. What Level of Hepatitis B Antibody Is Protective? Journal of Infectious Diseases 1999;179:489–92.
3. Huzly D, Schenk T, Jilg,W and Neumann-Haefelin D. Comparison of Nine Commercially Available Assays for Quantification of Antibody Response to Hepatitis B Virus Surface Antigen Journal of clinical microbiology 2008;46:1298–1306.

Anti-HBs Quantitative

Hepatitis B virus (HBV) infection is endemic throughout the world. Anti-HBs test has been using to identify previous exposure to hepatitis B virus and to determine adequate immunity from hepatitis B vaccination. An adequate vaccine response is defined as an anti-HBs level of 100 IU/L 4 to 8 weeks after the last of three or four vaccine injections.

Based on the World Health Organization recommendation, an Anti-HBs concentration ≥ 10 IU/L is regarded as being protective against Hepatitis B viral infection (1). Vaccinees without sufficient anti-HBs responses, so-called non-responders or low responders, undergo a special regimen of additional vaccine doses. For liver transplant recipients, quantitative measurement of anti-HBs levels is used in the management of hepatitis B immune globulin prophylaxis, which is initiated to maintain anti-HBs levels of at least 100 or 200 IU/L, according to different guidelines (2).

Harmonization for qualitative assay of anti-HBs is adequate based on proficiency testing data from the College of American Pathologists Viral Marker Survey in 2020 that showed over 99% consensus rate in about 1,500 participants using 15 assay products from 6 IVD manufacturers.

However, harmonization for quantitative assay of anti-HBs is inadequate based on Huzly et al’s findings that analysis of routine clinical samples by different systems revealed significant discrepancies for a number of sera (3). Huzly et al. reported that results for 200 serum samples showed a mean coefficient of variation(CV) between nine different measurement procedures was 47.1% (range, 15.0% to 201.0%), and when serum samples were classified according to the anti-HBs levels determined by the majority of assays (10 to 100, 101 to 1,000, and _1,000 IU/liter), the mean CV was significantly lower for the group of sera with anti-HBs levels between 101 and 1,000 IU/liter than for sera with levels between 10 and 100 or above 1,000 IU/liter. (36.2% versus 57.4% and 60.3%, respectively; P 0.003).

Anti-Hepatitis B core (Anti-HBc total IgM and IgG)

Anti-HBc is found in serum shortly after the appearance of hepatitis B surface antigen (HBsAg) in acute HBV infections. Antibodies to HBc will persist after the disappearance of HBsAg and before the appearance of detectable antibody to HBsAg (anti-HBs).^1-7 In the absence of information about any other HBV markers, an individual with detectable levels of anti-HBc is considered to be actively infected with HBV or the infection may have resolved, leaving the person immune.⁸ The presence of anti-HBc does not differentiate between acute or chronic hepatitis B infection. HBc total Ab (IgG and IgM) is one of the important serologic markers in the diagnosis of hepatitis B viral infection status. Therefore, the medical impact is high.

Proficiency testing data from the College of American Pathologists qualitative Anti-HBc (total IgG and IgM) Survey in 2020 showed that for 18 peer groups of test kits 99.7% of approximately 1,300 results agreed for both negative and positive samples. Therefore, the status of harmonization is adequate.

JCTLM does not list any reference systems for anti-HBc.

References

1. Hoofnagle JH, Gerety RJ, Barker LF. Antibody to hepatitis B virus core in man. Lancet 1973;II:869–73.
2. Szmuness W, Hoofnagle JH, Stevens CE, et al. Antibody against the hepatitis type B core antigen. A new tool for epidemiologic studies. Am J Epidemiol 1976;104(3):256 62.
3. Hoofnagle JH, Seeff LB, Buskell-Bales Z, et al. Serologic responses in HB. In: Vyas GN, Cohen SN, Schmid R, editors. Viral Hepatitis. Philadelphia, PA: Franklin Institute Press; 1978:219–42.
4. Krugman S, Overby LR, Mushahwar IK, et al. Viral hepatitis, type B: Studies on natural history and prevention re-examined. N Engl J Med 1979;300(3):101–6.
5. Zito DR, Gurdak RG, Tucker FL, et al. Hepatitis B virus serology: Loss of antibody to surface antigen. Am J Clin Pathol 1987;88(2):229–31.
6. Gitlin N. Hepatitis B; Diagnosis, Prevention, and Treatment. Clin Chem 1997;43(8B);1500-6.
7. Koff RS. Viral Hepatitis. In: Schiff L, Schiff ER, editors. Diseases of the Liver. 7th ed. Philadelphia: JB Lippincott, 1993:492-577.
8. Dodd RY, Popovsky MA, Members of the Scientific Section Coordinating Committee. Antibodies to hepatitis B core antigen and the infectivity of the blood supply. Transfusion 1991;31(5):443–9.

Anti-Hepatitis B Surface Antigen (Anti-HBsAg)

1. Medical Impact:

Hepatitis B surface antigen (HBsAg) is the first serologic marker appearing in the serum at 6 to 16 weeks following exposure to HBV. In acute infection, HBsAg usually disappears in 1 to 2 months after the onset of symptoms with the appearance of hepatitis B surface antibody (anti-HBs Ab). Anti-HBs Ab also appears as the immune response following hepatitis B vaccination. Persistence of HBsAg for more than 6 months in duration indicates development of either a chronic carrier state or chronic HBV infection.

There has been recent renewed interest in measuring serum levels of HBsAg as a surrogate marker to predict HBsAg loss and monitor anti-HBV therapy. During the natural history of HBV infection, the loss of serum HBsAg is generally associated with their seroconversion to anti-HBs, the hallmark of a successful immunological response to HBV infection. The speed and amplitude of the decline in HBsAg levels are suspected to be a good predictors of sustained virological response and HBsAg loss.

Therefore, the concordance among different assays for HBsAg and anti-HBs Ab is very important.

2. Harmonization Status:

According to the EQA data from the College of American Pathologists in 2015 (non-commutable samples), the agreement of the test results from 11 manufacturers’ test kits was very good. The overall concordance of HBsAg from 2,252 laboratories using kits manufactured by 11 companies was 99.7% for positive samples and 99.8% for negative samples. The overall concordance of anti-HBs Ab from 1,820 laboratories using kits manufactured by 11 companies was 99.3% for the positive samples and 99.7% for the negative samples.

A comparison report in 2012 [1], the quantitation of HBsAg levels in routine clinical samples by four different test systems appeared overall to be accurate, showing low variability and little discrepancy. HBsAg levels were compared among 80 patients using Abbott Architect, Diasorin, Bio-Rad and Roche.

Another head-to-head study evaluating the performance of 3 assays (Abbott Architect, DiaSorin LiaisonXL and Roche Elecsys HBsAgII).measuring the HBsAg level [2] also showed good correlation between all current systems [2].

In 2008, a systematic and comprehensive comparison study of nine anti-HBs Ab assay kits was reported [3]. Two hundred serum samples taken from patients and health care workers were analyzed on six different automated immunoassays (Abbott Axsym, Siemens Centaur, Ortho Vitros ECI, Roche Elecsys, DiaSorin Liaison and Abbott Architect) and three enzyme immunoassays (EIAs) (DiaSorin ETI-AB-AUK-3, Siemens Enzygnost and Bio-Rad Monolisa). Although the assay specificity ranged between 96.8-100% and sensitivity ranged between 93.5-100%, a high number of sera showed discrepancies between measurements by the different systems. The classical EIAs tend to find lower anti-HBs levels than the automated systems. The serial dilution of the WHO International Standard (IS) preparation was measured accurately by most of the assays. However, the quantitative measurement of anti-HBs levels was not reliable, even though an IS standard was used for the calibration of the systems.

Another comparison study also reported that the quantification of anti-HBs can show various results according to the kind of assay kit used [4]. When serum samples from five hundred visitors to the health care centers were assayed by Roche Cobas, Siemens Enzygost, Abbott AxSYM and Roche Elecsys, the concordance rates was 92.8% and 36 samples (7.2%) showed discrepant results. The authors concluded that the quantification of anti-HBs can show various results according to the kind of assay kit used.

Reference materials are available as the 3^rd WHO IS for HBsAg, human, NIBSC code 12/226 and 2^nd WHO IS for anti-HBs Ab, human, NIBSC code 07/164 [5]. According to the WHO Expert Committee’s 54^th report on biological standardization, the WHO IS for HBsAg is intended for the calibration of secondary HBsAg reference materials by manufacturers. The WHO IS for HBsAg and anti-HBs Ab.is widely used in standardization and calibration of HBsAg and anti-HBs Ab assay kits [6].

Taken these data together, measurement procedure of HBsAg is consistent with adequate harmonization for the screening tests for HBV infection using serum or plasma samples. However, it seems that measurement procedure of anti-HBs Ab needs harmonization.

<References>

1. Tuaillon E et al. Comparison of serum HBsAg quantitation by four Immunoassays, and relationships of HBsAg level with HBV replication and HBV genotypes. PLOS ONE 2012;7:e32143.
2. Thibault V et al. Performance of HBsAg quantification assays for detection of Hepatitis B virus genotypes and diagnostic escape-variants in clinical samples. J Clin Virol 2017;89:14-21.
3. Huzly D et al. Comparison of nine commercially available assays for quantification of antibody response to hepatitis B virus surface antigen. J Clin Microbiol 2008;46:1298-1306.
4. Whang DH and Shin BM. Comparison of Four-Assay Systems for the Quantification of Hepatitis B Surface Antibody. Korean J Lab Med 2002;22:424-30 (article in Korean).
5. Wilkinson DE et al. International collaborative study on the 3rd WHO International Standard for hepatitis B surface antigen. J Clin Virol. 2016;82:173-80.
6. Ferguson M, Yu MW and Heath A. Calibration of the second International Standard for hepatitis B immunoglobulin in an international collaborative study. Vox Sanquinis 2010;99:77-84.

Anti-Hepatitis C Virus antibody (Anti-HCV Ab)

Anti-Hepatitis C Virus Antibody (Anti-HCV Ab) is the first test used for early detection of HCV infection (1). Assay performance is increased when several antigens are used to capture the antibodies.  The CDC has reported that most Anti-HCV Ab assays have greater than 95% true positive results (2). A review of College of American Pathologists proficiency testing data from 2015 showed 0.2-0.8% false negative and 0-0.3% false positive results. The current performance of Anti-HCV Ab is good and further harmonization is not needed at this time.

References

1. Testing for HCV infection: an update of guidance for clinicians and laboratories. CDC. Morbidity and Mortality Weekly Report. May 7, 2013.
2. https://www.cdc.gov/hepatitis/hcv/labtesting.htm.

Anti-myeloperoxidase (MPO) antibody, IgG

Anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitides (AAVs) are characterized by inflammation primarily of small blood vessels, including arterioles, capillaries, and venules.  The ANCA-associated vasculitides include 3 distinct diseases - microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA), and eosinophilc granulomatosis with polyangiitis (EGPA).  AAVs are so-named because they are associated with the presence of autoantibodies specific for neutrophil proteins.  ANCA can be visualized by indirect immunofluorescence (IIF) using ethanol-fixed neutrophils.  Using this approach, 2 patterns, pANCA and cANCA, can be identified.  The pANCA pattern is a perinuclear staining of the neutrophil and is associated with the presence of anti-myeloperoxidase (MPO) antibodies.  The cANCA pattern is a granular cytoplasmic stain and results from antibodies targeting proteinase 3 (PR3).

Detection of ANCA by IIF and anti-MPO/anti-PR3 antibodies by antigen-specific immunoassays are important diagnostic tools used to evaluate patients with suspected AAVs.  Recent guidelines identify the antigen-specific anti-MPO and anti-PR3 immunoassays as the optimal test for this application (1).  This recommendation is based on a study in which the diagnostic sensitivity and specificity of 8 anti-MPO antibody immunoassays was assessed (2).  The area-under-the-curves (AUCs) for the immunoassays ranged from 0.919-0.959, all of which were improved over the IIF assays.  However, the qualitative concordance of the anti-MPO antibody immunoassays in controls and in patients with MPA was 0.94 and 0.77, respectively.  This demonstrates a degree of qualitative discordance between methods, which could affect their diagnostic applications.

Anti-MPO antibody immunoassays also show poor quantitative agreement.  In another study in which 10 immunoassays were compared, quantitative differences of up to 5-fold between average concentrations were observed (3).  These quantitative differences are also evidence on the College of American Pathologists (CAP) proficiency tests.  For example, survey S2-B from 2018 included a sample positive for anti-MPO antibodies; the mean quantitative values across 7 methods ranged from 5.4-81.3, with most methods reporting in different units.  Although commutability for CAP samples has not formally been addressed, the material is sourced from patients.  The matrix of these samples should closely resemble patient samples routinely tested in the clinical laboratory.

In an attempt to address these issues, the Joint Research Center of the European Commission has developed a certified reference material (CRM) for anti-MPO antibodies (ERM-DA476/IFCC) (3, 4).  This CRM was developed from a plasmapheresis from a patient with a high-titer anti-MPO antibody.  This CRM is commutable and is designed to serve as a traceable calibrator for anti-MPO antibody immunoassays.  However, this material has not been adopted by in vitro diagnostic (IVD) manufacturers as a calibrator material, as most methods had been FDA-approved prior to its availability.  Implementation of traceability to the reference material is needed.

References:

1. Bossuyt X, Tervaert J-W, Arimura Y, et al. Revised 2017 international consensus on testing of ANCAs in granulomatosis with polyangiitis and microscopic polyangiitis. Nat Rev Rheum 13 (2017) 683-692.
2. Damoiseaux J, Csernok E, Rasmussen N, et al. Detection of antineutrophil cytoplasmic antibodies (ANCAs): a multicentre European Vasculitis Study Group (EUVAS) evaluation of the value of indirect immunofluorescence (IIF) versus antigen-specific immunoassays. Ann Rheum Dis 76 (2017) 647-653.
3. Hutu DP, Tuddenham E, Monogioudi E, et at. First steps in the standardization of immunoglobulin IgG myeloperoxidase-anti-neutrophil cytoplasmic antibody measurements. Clin Exp Immunol 183 (2015) 193-205.
4. Monogioudi E, Hutu DP, Martos G, et al. Development of a certified reference material for myeloperoxidase-anti-neutrophil cytoplasmic antibodies (MPO-ANCA). Clin Chem Acta 467 (2017) 48-50.

Antinuclear antibody (ANA)

There are two methodologies for testing for antinuclear antibodies:  a) conventional immunofluorescence (IF) of prepared, fixed cells (commonly Hep2 cells) and b) identification of specific antibodies by ELISA using beads or wells coated with known antigens.

Discrepancies within this area of testing are common. The widely accepted view is that the cell-based ANA’s provide a much wider spectrum of (auto)antigens for use in clinical labs. ELISA assays are more convenient to perform, but suffer from lack of inclusion of important antigens and therefore are more likely to give false negative results in patients with autoimmune diseases. Even among proponents and users of IF, there has been lack of agreement on some of the interpretive patterns.

Although this area is certainly in need of harmonization, there are already significant efforts underway led by experts in autoimmune testing, including International Workshops and Consensus Conferences, such as the International Consensus on ANA Patterns.

References

1. Chan EK, Damoiseaux J, Carballo OG, Conrad K, de Melo Cruvinel W, Francescantonio PL, Fritzler MJ6, Garcia-De La Torre I, Herold M, Mimori T, Satoh M, von Mühlen CA, Andrade LE: Report of the First International Consensus on Standardized Nomenclature of Antinuclear Antibody HEp-2 Cell Patterns 2014-2015. Front Immunol. 2015 Aug 20;6:412. doi: 10.3389/fimmu.2015.00412. 

2. Damoiseaux J, von Mühlen CA, De La Torre IG, et al. International consensus on ANA patterns (ICAP): the bumpy road towards a consensus on reporting ANA results. Auto Immun Highlights. 2016 Dec; 7(1): 1. Published online 2016 Jan 30. doi:  10.1007/s13317-016-0075-0.

Anti-SSA antibody IgG

SSA autoantibodies are a criteria marker for Sjögren’s Syndrome.  These antibodies are also found in other connective tissue diseases such as Systemic Lupus Erythematosus.¹  Most critical; it is known that anti-Ro/SSA positivity leads to higher risk of miscarriage and fetal cardiac malformations. Particularly, anti-p200 antibodies against a finer specificity of the Ro/SSA antigen, have been associated with congenital heart block.²

SSA autoantibodies are used as one component of a diagnostic scoring system shown in the 2016 ACR-EULAR Classification Criteria for primary Sjögren’s Syndrome: A Consensus and Data-Driven Methodology Involving Three International Patient Cohorts.  The final classification criteria are based on the weighted sum of 5 items: anti-SSA(Ro) antibody positivity and focal lymphocytic sialadenitis with a focus score ≥ 1 foci/mm2, each scoring 3; an abnormal ocular staining score ≥ 5 (or van Bijsterveld score ≥ 4), a Schirmer test ≤ 5 mm/5 min, and an unstimulated salivary flow rate ≤ 0.1 mL/min, each scoring 1. Individuals (with signs/symptoms suggestive of SS) who have a total score ≥ 4 for the items above, meet the criteria for pSS. Sensitivity and specificity against clinician-expert derived case/non-case status in the final validation cohort were high: 96% (95% CI: 92%, 98%), and 95% (95% CI: 92%, 97%), respectively.³

There is very little in the literature regarding direct comparison of assays for anti-SSA antibodies.  However, some assessment can be obtained from the College of American Pathologists (CAP) proficiency testing surveys.  Anti-SSA antibodies are included in the S2 Special Immunology survey.  This survey is conducted 3 times each year, with each challenge including 1 sample for anti-SSA antibodies.  In 2018 and 2019, the number of participants reporting results for anti-SSA antibodies ranged from 502 to 515, with the primary methods being enzyme immunoassay, fluoroenzyme immunoassay, immunoblot, and multiplex bead immunoassay.  During this time period, 4 samples (2 positive and 2 negative) demonstrated qualitative agreement ≥98.0%.  However, the remaining 2 samples were non-consensus, with only 52.6% and 77.5% of participants identifying the sample as “positive”; results from the remainder of the laboratories classified the samples as “negative” or “borderline/equivocal”.  Although not assessed as part of the CAP survey, numeric results are included, if reported by the laboratory.  For the 2 positive samples which demonstrated acceptable qualitative agreement, medians ranged from 92.9-1270 and 89.9-1126.0 with assay-specific arbitrary units (U) or units/volume (U/mL). In addition to different units, the differences in methodological approaches and in the antigens used by different methods contribute to lack of harmonization.  Although limited, this data suggests that opportunities exist to improve qualitative and quantitative harmonization across methods for detection of anti-SSA antibodies.

References:

1. Cin Exp Rheumatol. Nov-Dec 2017;35(6):929-935. Epub 2017 Jul6.
2. Arch Dermatol Res. 2016 Mar;308(2):139-43.doi: 10.1007/s00403-016-1622-2. Epub 2016 Feb 1.
3. Arthritis Rheumatol 2017 Jan;69(1): 35-45,.doi: 10.1002/art.39859. Epub 2016 Oct26.

Antistreptolysin O

Antistreptolysin O is used for diagnosis of post-streptococcal acute rheumatic fever in children, where elevated levels are used as a component of the Jones criteria. Acute rheumatic fever is uncommon in the Western world.

Literature commentary states that “the traditional ASLO assay is well standardized, but the newer latex agglutination, automated nephelometric or turbidometric assays are not.¹ These authors also point out problems with reference intervals and recommend that patients be followed in the same lab for serial levels.

The recommendation for harmonization priority is low based on few clinical indications.

Reference

1. Geerts I, De Vos N, Frans J, Mewis A. The clinical-diagnostic role of antistreptolysin O antibodies. Acta Clin Belg 2011;66:410-5.

Aspartate Aminotransferase (AST)

The IFCC has developed reference measurement procedures for AST and ALT enzymes. The IFCC reagent formulation is generally used by IVD manufacturers with some adaptation for the technology of a given instrument system.  Standardization is thus easily achievable.  The harmonization issue is whether or not pyridoxyl-5-phosphate (P5P) is included in reagents from IVD manufacturers.  P5P is needed to fully activate the enzymes in situations when a patient has a deficiency in this vitamin as may occur in kidney failure and other conditions. A technical issue is that adding P5P to reagents reduces the reagent stability. Consequently P5P is supplied in a separate container to be mixed at the time a reagent is put into use. Furthermore, laboratories may prefer not to add P5P because there may be reagent waste in lower testing volume situations. Some countries do not typically include P5P and in other countries there is a mix of inclusion and exclusion in reagents.  Differences in vitamin deficiency between countries may contribute to different practices.  The ICHCLR recommends that manufacturers make available reagents that include P5P so that laboratories can determine if their population would benefit from its use in the reagents.  A medium priority was assigned because these two analytes are well standardized except for the P5P inclusion and the need for P5P may vary among different regions of the world.

Schumann G, Bonora R, Ceriotti F, Ferard G, Ferrero CA, Franck PF, et al. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C. International Federation of Clinical Chemistry and Laboratory Medicine. Part 5. Reference procedure for the measurement of catalytic concentration of aspartate aminotransferase. Clin Chem Lab Med. 2002;40:725-33.

BCR-ABL1

BCR-ABL1 refers to a fusion gene of t(9;22), which confirms the diagnosis of chronic myelogenous leukemia (CML) and a form of acute lymphoblastic lymphoma (ALL), specifically a type of B-lymphoblastic leukemia/lymphoma. The abnormal BCR-ABL1 fusion gene codes a protein called tyrosine kinase and is effectively treated by tyrosine kinase inhibitor (TKI). Polymerase chain reaction (PCR)-based molecular testing is used to detect and measure the level of BCR-ABL1 gene transcripts, or gene product units, in a patient's blood and/or bone marrow samples.

The JRC has released the certified reference material (CRM) ERM-AD623 for the calibration of quantitative real time polymerase chain reaction (qPCR) assays measuring the BCR-ABL1 transcript level in cDNA samples from leukaemia patients. ERM-AD623 is composed of a set of 6 vials containing solutions of a common plasmid at different copy number concentrations. Each vial contains approximately 600 µL of plasmid solution.

Proficiency testing data from the College of American Pathologists Minimal Residual Disease (BCR/ABL1 p210) Survey in 2019 indicated approximately 85% or better reached consensus. For 1:10 dilution, the overall consensus for IS aligned quantitative testing for MRD-06 was 87.3% (158/181), For 1:100 dilutions, the overall consensus for IS aligned quantitative testing for MRD-04 was 85.6% (154/180). For 1:100,000 dilutions, the overall consensus for IS aligned quantitative testing for MRD-05 was 86.2% (156/181).

References:

1. Lab Tests Online – AACC https://labtestsonline.org/tests/bcr-abl1
2. White H, Deprez L, Corbisier P, et al. 2015, A certified plasmid reference material for the standardisation of BCR-ABL1 mRNA quantification by real-time quantitative PCR. Leukemia 29: 369-376

Bilirubin, conjugated

Differences among results are related to calibration traceability and the reagents used in a given measurement procedure to isolate the conjugated from unconjugated forms of bilirubin.  A medium priority was assigned because concentrations are typically interpreted relative to total bilirubin that is acceptably standardized. Small changes in conjugated bilirubin are not usually clinically meaningful.

Bilirubin, total

EQA data from two Japanese programs in 2015 that used commutable samples had 3.9% and 8.4% CV among different laboratories and methods.  EQA data from the College of American Pathologists in 2015 (non-commutable samples) had 27% difference between maximum and minimum values reported by individual laboratories and mean 7.3% CV% among mean values for different peer groups.  Taken together these data support adequate harmonization for serum or plasma total bilirubin measurement procedures.  A reference measurement procedure is listed by JCTLM.

Blood gasses (pH, pO2, pCO2, oximitry)

EQA data from the College of American Pathologists in 2018 (AQ-B) had CV 0.2-0.3% for pH, 4.2-6.6% for pCO₂, and 4.1-8.4% for pO₂ for all results among approximately 3700 laboratories using 19 different measurement procedures. Although the commutability of the aqueous samples used in the survey is unknown, the results are expected to give a good representation of agreement among different measurement procedures since blood gas instruments are typically calibrated with aqueous solutions. These data are consistent with adequate harmonization for blood gas measurements.  JCTLM does not list reference materials or measurement procedures for pH and blood gasses.  NIST has certified reference materials for pH, carbon dioxide in nitrogen and oxygen in nitrogen.

B-type Natriuretic Peptide (BNP)

B-Type natriuretic peptide (BNP) is a marker of cardiac function and is used for diagnosis, risk stratification and follow-up of patients with chronic or acute heart failure.  Laboratory assessments have determined that the agreement among results for different measurement procedures is not suitable to support uniform clinical decision values for interpretation of results (1,2).  Both a candidate reference material (2) and a candidate reference measurement procedure (3) have been recently reported. 

References

1. Clerico A, Zaninotto M, Prontera C, et al. State of the art of BNP and NT-proBNP immunoassays: The CardioOrmoCheck study. Clin Chim Acgta 2012;414:112-9.
2. Semenov AG, Tamm NN, Apple FS, et al. Searching for a BNP standard: Glycosylated proBNP as a common calibrator enables improved comparability of commercial BNP immunoassays. Clin Biochem 2017;50:181-5.
3. Torma AF, Groves K, Biesenbruch S, et al. A candidate liquid chromatography mass spectrometry reference method for the quantification of the cardiac marker 1-32 B-type natriuretic peptide. Clin Chem Lab Med 2017;55:1397-1406.

CA 125

CA 125 results are frequently different among measurement procedures.  Different antibodies are typically used that identify different epitopes in the molecule. Consequently, harmonization of results is challenging because the measurand is different. It is common laboratory practice to re-baseline results when measurement procedures change for patients who are being followed for recurrence of a tumor following treatment.  However, the comparability of results over time is compromised when a patient’s specimen is assayed by different laboratories using different measurement procedures.  In addition, IVD manufacturers are challenged to maintain long term consistency of results due to the complexity of the measurand and the regents used for the measurement procedure.  A high priority was assigned because, despite the technical challenges, harmonization would substantially improve medial decisions considering that patients may have results from different measurement procedures due to mobility or measurement procedure changes in a laboratory.

CA 15-3

CA 15-3 results are frequently different among measurement procedures.  Different antibodies are typically used that identify different epitopes in the molecule. Consequently, harmonization of results is challenging because the measurand is different. It is common laboratory practice to re-baseline results when measurement procedures change for patients who are being followed for recurrence of a tumor following treatment.  However, the comparability of results over time is compromised when a patient’s specimen is assayed by different laboratories using different measurement procedures.  In addition, IVD manufacturers are challenged to maintain long term consistency of results due to the complexity of the measurand and the regents used for the measurement procedure.  A high priority was assigned because, despite the technical challenges, harmonization would substantially improve medial decisions considering that patients may have results from different measurement procedures due to mobility or measurement procedure changes in a laboratory.

CA 19-9

CA 19-9 results are frequently different among measurement procedures.  Different antibodies are typically used that identify different epitopes in the molecule. Consequently, harmonization of results is challenging because the measurand is different. It is common laboratory practice to re-baseline results when measurement procedures change for patients who are being followed for recurrence of a tumor following treatment.  However, the comparability of results over time is compromised when a patient’s specimen is assayed by different laboratories using different measurement procedures.  In addition, IVD manufacturers are challenged to maintain long term consistency of results due to the complexity of the measurand and the regents used for the measurement procedure.  A high priority was assigned because, despite the technical challenges, harmonization would substantially improve medial decisions considering that patients may have results from different measurement procedures due to mobility or measurement procedure changes in a laboratory.

Calcium

EQA data from two Japanese programs in 2015 that used commutable samples had 1.7% and 2.3% CV among different laboratories and methods.  EQA data from the College of American Pathologists in 2015 (non-commutable samples) had 8.2% difference between maximum and minimum values reported by individual laboratories and 1.9% CV% among mean values for different peer groups.  Taken together these data support adequate harmonization for serum or plasma calcium measurement procedures.  A reference material, serum-based reference material and a reference measurement procedure are listed by JCTLM.

Calcium, ionized

Ionized calcium is used to assess calcium in situations when total calcium can be misleading such as during surgery, intensive care situations with altered blood albumin concentrations or severe hyper- or hypo-calcemia.  Ionized calcium is measured in whole blood using ion-selective electrode technology usually as part of a blood gas measurement.

Data from a 2017 College of American Pathologists survey (aqueous samples) of over 2300 labs using 18 commercially available systems had all results CVs from 3.5-3.9% for ionized calcium values of 0.99 mmol/L and 1.34 mmol/L.  However, the difference between the highest and lowest mean values for instrument groups was 8.7% for the 0.99 mmol/L sample, and 14.8% for the 1.34 mmol/L sample. Furthermore, the difference between the highest and lowest individual values was 25.6% and 20.5% for the same two samples respectively.

The biological variation model suggests desirable total error of 2.0% with 0.6% bias and 0.9% CV.  These specifications are probably not realistically achievable but suggest that the disagreement among different methods in the survey results is not adequate for clinical use.  

A candidate reference measurement procedure was developed by the IFCC but is not listed by JCTLM (1).

Reference

1. Burnett RW, et al. IFCC recommended reference method for the determination of the substance concentration of ionized calcium in undiluted serum, plasma or whole blood. Clin Chem Lab Med 2000;38:1301-14.

Carbamazepine

Carbamazepine is a commonly used anticonvulsant for the management of epilepsy and trigeminal neuralgia. Monitoring of blood levels increases the efficacy and safety of anticonvulsants. The measurement of drug blood levels may be particularly useful in cases of marked increase of seizure frequency and for verification of compliance. The usual adult therapeutic levels of carbamazepine in blood are between 4 and 12 µg/mL. Carbamazepine monitoring may be helpful to determine the cause of toxicity in cases of more than one medication. CYP3A4 inhibitors such as cimetidine, ciprofloxacin, diltiazem, omeprazole, isoniazid, ibuprofen, azoles, verapamil, and grapefruit juice can decrease carbamazepine metabolism in liver and increase carbamazepine levels in blood. In contrast, CYP3A4 inducers such as cisplatin, rifampin, phenobarbital, phenytoin, and theophylline can increase the carbamazepine metabolism and decrease carbamazepine levels (1).

In a comparability study measuring carbamazepine concentration in 74 to 77 leftover routine samples the between 6 different analyzers [Architect and AxSym (Abbott Laboratories), cobas c 501 module and COBAS INTEGRA 800 analyzer (Roche Diagnostics GmbH), Dimension Vista and Dimension Xpand (Simens Healthcare Diagnostics Inc)], systemic deviations in carbamazepine concentrations (relative mean differences) of -8.5%, -2.0% and -3.4% were found within manufacturers (cobas c 501 vs. COBAS INTEGRA 800, AxSym vs. Architect and Dimension Xpand vs. Dimension Vista, respectively). In a comparison study between 6 different analyzers, the relative mean difference for carbamazepine concentration of Dimension Xpand compared to cobas c 501 was highest at 11.4% with the 95% CI of -8.2% to +31.0% for 75 patient samples (2).

There is no suitable proficiency testing information from commutable samples.

One commutable reference material and a reference measurement procedure for carbamazepine are listed in the JCTLM database (2, 3). The reference measurement procedure (JCTLM DB identification number C14RMP5) for the quantification of carbamazepine in human serum was developed and validated by Roche Diagnostics GmbH in collaboration with the Labormedizinisches Zentrum Dr Risch Anstalt. Since the JCTLM listed certified reference material NIST SRM 1599 is no longer available, an ISO certified reference material was used for the primary calibration in the reference measurement procedure (3).

References

1. Tegretol [package insert]. East Hanover, New Jersey: Novartis Pharmaceuticals Corporation; March 2018. pharma.us.novartis.com. Accessed 16 September 2018.
2. Shipkova M, Petrova DT, Rosler AE, Orth M, Engelmayer J, Wieland E, Brandhorst G, Oellerich M. Comparability and imprecision of 8 frequently used commercially available immunoassays for therapeutic drug monitoring. Ther Drug Monit. 2014 Aug;36(4):433-41.
3. An LC-MS/MS based candidate reference method for the quantification of carbamazepine in human serum, Taibon J. et al, Clinica Chimica Acta, 2017, 475, 35-40.

Carbohydrate-deficient transferin (CDT)

A reference material for carbohydrate-deficient transferin is being developed by an IFCC working group.

Carboxyhemoglobin

Carboxyhemoglobin is measured in whole blood using multi-wavelength spectrophotometry referred to as a co-oximeter than can be stand alone or integral with blood gas instruments. Carboxyhemoglobin is used to determine exposure to carbon monoxide that can impair oxygen delivery to tissues and be fatal at elevated values.  Carbon monoxide is present from normal metabolic activity with values <1% of hemoglobin. 

Data from a 2017 College of American Pathologists survey of over 3000 labs using 12 commercially available systems had all results CVs from 3.6-7.2% for four samples with carboxyhemoglobin values of 16-51% and a CV of 11.0% at a carboxyhemoglobin value of 9.4%.  Differences between the highest and lowest mean values for instrument groups was 10-15% for four samples with carboxyhemoglobin values of 16-51%, and was 24% for the carboxyhemoglobin value of 9.4%.

These results suggest that the agreement among different methods is adequate for clinical use.

Carcinoembrionic antigen (CEA)

CEA results are frequently different among measurement procedures.  Different antibodies are typically used that identify different epitopes in the molecule. Consequently, harmonization of results is challenging because the measurand is different. It is common laboratory practice to re-baseline results when measurement procedures change for patients who are being followed for recurrence of a tumor following treatment.  However, the comparability of results over time is compromised when a patient’s specimen is assayed by different laboratories using different measurement procedures.  In addition, IVD manufacturers are challenged to maintain long term consistency of results due to the complexity of the measurand and the regents used for the measurement procedure.  A high priority was assigned because, despite the technical challenges, harmonization would substantially improve medial decisions considering that patients may have results from different measurement procedures due to mobility or measurement procedure changes in a laboratory.

Chlamydia Trachomatis PCR

Chlamydia trachomatis is the most common curable sexual transmitted bacterial infection in the world, with an estimated 4–5 million new cases each year [1]. Once detected, the infection is easily treated by a short course of antibiotic therapy. Chlamydial infection in women is commonly asymptomatic. Undetected and untreated Chlamydia infection can ascend upper genitalia that may cause pelvic inflammatory disease (PID), infertility, ectopic pregnancy and chronic pelvic pain [2,3]. Approximately 25% to 30% of women who develop acute salpingitis become infertile [4].

Multiple laboratory test options can be used to detect chlamydia. Direct detection of the pathogen using culture or nonculture methods is possible. Culture was previously considered to be the gold standard test for diagnosis of C trachomatis infection [4]. However, organisms are labile in vitro, and precise specimen collection, transportation, and processing conditions are required to maintain organism viability, which is necessary for successful culturing. In comparison, nucleic acid amplification testing (NAAT) provides superior sensitivity and specificity and is now the recommended method for diagnosis in most cases [5-7]. Immunoassays and nonamplification DNA tests are also available for C trachomatis detection, but these methods are significantly less sensitive and less specific than NAATs [4]. Serologic tests that detect a systemic immune response to infection are not recommended because of the lack of precision for the detection of an active infection [4].

The FDA of USA approved NAAT including Abbott Real Time CT/NG (Abbott Molecular Inc., Des Plaines, IL), Aptima COMBO 2 assay (Hologic/Gen-Probe Inc., San Diego, CA), BD Probe Tec ET CT/GC Amplified DNA assay (Becton Dickinson and Company, Sparks, MD), Xpert CT/NG assay (Cepheid, Sunnyvale, CA), Roche Cobas CT/NG test (Roche Diagnostics, Indianapolis, IN) [4].

The test performance of each NAAT was reported through a multisite (9 sites in USA) prospective study with 3,859 female urine specimens; 1,936 vaginal swabs; and 1,193 specimens [8]. The sensitivity and specificity of each kit were as follows: 95.6% and 99.7% for female urine, 98.6% and 99.1% for vaginal swab, respectively with Roche Cobas kit; 93.4% and 99.7% for female urine, 98.5% and 99.0% for vaginal swab, respectively with Aptima AC2 kit; 85.3% and 99.8% for female urine, 92.6% and 99.8% for vaginal swab, respectively with BD CTQ/GCQ kit; 100% and 99.7% for male urine, respectively with Roche Cobas kit; and 98.3% and 99.9 % for male urine, respectively with Aptima AC2 kit; 99.1% and 99.9% for male urine, respectively with CTQ/GCQ kit; and 99.2% and 99.9% for male urine, respectively with Abbott CT/NG kit.

CAP EQA Results on NAAT of Chlamydia trachomatis

1. Swab Specimens: The results of Chlamydia trachomatis NAAT on three swab samples using

six commercial Chlamydia Trachomatis NAAT kits and LDT assay (used by 14 institutes) in 940 CAP PT Survey participating institutes showed a 99.8% concordance rate of seven test methods, including LDT.

• 6 NAAT kits for Chlamydia Trachomatis
• Abbott m2000: 43 of 940 institutes were using this kit
• BD ProbeTec/Viper XTR (non-extracted): 71 of 940 institutes were using this kit
• BD Viper LT: 7 of 940 institutes were using this kit.
• BD Viper XTR (extracted): 46 of 940 institutes were using this kit
• Hologic Aptima Combo 2: 561 of 940 institutes were using this kit.
• Roche Cobas 4,800: 198 of 940 institutes were using this kit.

2. Urine Specimens: The results of tests on two urine samples with six commercial Chlamydia trachomatis NAAT kits* and LDT assay (used by 14 agencies) at 900 CAP PT Survey participating institutes also showed a 99.8% concordance rate of seven test methods, including LDT.

The harmonization of results for NAAT of chlamydia trachomatis at present is judged as adequate status based on CAP EQA results and the results of reference 8.

References

1. Newman L, Rowley J, Vander Hoorn S, Wijesooriya NS, Unemo M, Low N, et al. Global estimates of the prevalence and incidence of four curable sexually transmitted infections in 2012 based on systematic review and global reporting. PLoS One. 2015;10(12)
2. Land J, Van Bergen J, Morre S, Postma M. Epidemiology of chlamydia trachomatis infection in women and the cost-effectiveness of screening. Hum Reprod Update. 2009;16(2):189–204
3. World Health Organization. Global incidence and prevalence of selected curable. Sex Transm Infect. 2008:19–20.
4. Centers for Disease Control and Prevention: Sexually Transmitted Diseases Treatment Guidelines, 2010. MMWR Morb Mortal Wkly Rep 2010;59:RR12
5. Crotchfelt KA, Pare B, Gaydos C, Quinn TC: Detection of Chlamydia trachomatisby the GEN-PROBE AMPLIFIED Chlamydia trachomatisAssay (AMP CT) in urine specimens from men and women and endocervical specimens from women. J Clin Microbiol 1998 Feb;36(2):391-394
6. Gaydos CA, Quinn TC, Willis D, et al: Performance of the APTIMA Combo 2 assay for detection of Chlamydia trachomatisand Neisseria gonorrhoeaein female urine and endocervical swab specimens. J Clin Microbiol 2003 Jan;41(1):304-309
7. Chernesky MA, Jang DE: APTIMA transcription-mediated amplification assays for Chlamydia trachomatisand Neisseria gonorrhoeae. Expert Rev Mol Diagn 2006 Jul;6(4):519-525
8. Barbara VP, Kenneth F, Stephanie NT, et al: Evaluation of the Performance of the Cobas CT/NG Test for Use on the Cobas 6800/8800 Systems for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Male and Female Urogenital Samples. J Clin Microbiol 2019 Apr;57(4):1-11.

Chloride

EQA data from one Japanese program in 2015 that used commutable samples had 1.1% CV among different laboratories and methods.  EQA data from the College of American Pathologists in 2015 (non-commutable samples) had 7.7% difference between maximum and minimum values reported by individual laboratories and 1.9% CV% among mean values for different peer groups.  Taken together these data support adequate harmonization for serum or plasma chloride measurement procedures.  A reference material, serum-based reference material and a reference measurement procedure are listed.

Cholesterol, HDL

Despite the existence of a standardization program from the CDC, investigation of variability in results has shown that specimen specific variability especially when abnormal lipoproteins are present remains a measurement limitation for direct HDL cholesterol measurement procedures.  Harmonization for HDL cholesterol is dependent on developing improved reagent technology by the IVD industry to appropriately measure HDL cholesterol in the presence of other lipoproteins.  A high priority was assigned because HDL cholesterol is used in all guidelines for assessment of risk for cardiovascular disease.

Cholesterol, LDL

Despite the existence of a standardization program from the CDC, investigation of variability in results has shown that specimen specific variability especially when abnormal lipoproteins are present remains a measurement limitation for direct LDL cholesterol measurement procedures.  Harmonization for LDL cholesterol is dependent on developing improved reagent technology by the IVD industry to appropriately measure LDL cholesterol in the presence of other lipoproteins.  A medium priority was assigned because although LDL cholesterol is used in some guidelines for assessment of risk for cardiovascular disease, calculated LDL is recommended by some guidelines when triglycerides are <400 mg/dL (4.5 mmol/L) and has been shown to agree well with LDL cholesterol from the reference measurement procedure.

Despite the existence of a standardization program from the CDC, investigation of variability in results has shown that specimen specific variability especially when abnormal lipoproteins are present remains a measurement limitation for direct HDL cholesterol measurement procedures.  Harmonization for HDL cholesterol is dependent on developing improved reagent technology by the IVD industry to appropriately measure HDL cholesterol in the presence of other lipoproteins.  A high priority was assigned because HDL cholesterol is used in all guidelines for assessment of risk for cardiovascular disease.

Cholesterol, total

EQA data from two Japanese programs in 2015 that used commutable samples had 2.6% and 1.4% CV among different laboratories and methods.  EQA data from a College of American Pathologists 2018 survey with commutable samples had approximately 13% difference between maximum and minimum values reported by individual laboratories at three concentrations, and 2.0% CV% among median values for different measurement procedure groups.  Taken together these data support adequate harmonization for serum or plasma cholesterol measurement procedures.  A reference material, serum-based reference material and a reference measurement procedure are listed by JCTLM.  The CDC maintains a global network of reference laboratories and a certification program for IVD manufacturers to maintain standardized results. See the website link for additional information.

CO2, Total

It is difficult to obtain information for CO2 from EQA data because of non-commutable samples and the relative instability of CO2 in the samples.  Harmonization Oversight Group members provided data for patient sample results comparison obtained from verification of performance between pairs of measurement procedures being installed in their laboratories.  Data showed results from Abbot Architect c8000 to be a constant 4 mmol/L lower than from Siemens Advia 1800.  Data showed results from Beckman DxC to be a constant 2 mmol/L lower than from Siemens Advia XPT.  Data showed results from Beckman Olympus 2700 to be a constant 2 mmol/L lower than from Roche 702.  Taken together, these data support that the biases among several commonly used measurement procedures is large enough to affect medical decisions.  JCTLM does not list reference materials or reference measurement procedures for CO2.

Complement Total (CH50)

The complement system is part of the innate immune response, and consists of the classic, alternate, and mannose-binding lectin pathways (1).  The classic pathway is initiated by antigen/antibody cross-linking and functions through a cascade of enzymatic reactions.  The classic pathway terminates with formation of the membrane attack complex (MAC) which leads to cellular lysis.  Low total complement (CH50) can result from a genetic or acquired deficiency in one of the complement components or from consumption due to infection or chronic inflammatory condition.

Historic assays for CH50 used sheep red blood cells (RBCs) coated with antibody, with cellular lysis mediated through the MAC leading to release of hemoglobin.  Most current assays use antibody-coated liposomes, which contain glucose-6-phosphate dehydrogenase (G6PDH).  Activation of complement by the antibody on the liposomes leads to formation of the MAC and lysis of the liposomes, which releases the G6PDH.  The G6PDH reacts with G6P in the reaction, resulting in reduction of NAD to NADH, which is measured at 340 nm.

In the early 1980s, the International Union of Immunological Societies (IUIS) Complement Standardization Subcommittee initiated preparation of a reference material for CH50 and several individual complement proteins.  This material was prepared from freeze-dried human serum; it is currently included on the World Health Organization (WHO) Biological Reference Preparations list (2).  The assigned values for CH50 were based on RBC hemolytic assays.  At this time, it is unclear whether this material is still available, or whether it has been used as a traceable standard for current liposome-based assays.

The most important application of CH50 is to identify individuals with low or absent complement activity.  The College of American Pathologists (CAP) Total Hemolytic Complement (CH50) proficiency testing survey evaluates performance qualitatively, with results interpreted by the participating laboratory as “low” or “normal/high”.  Between 2018-2019, 4 out of 8 samples showed >98.0% qualitative agreement among participating laboratories (3 “normal/high” and 1 “low”).  The remaining 4 samples were split approximately 75%/25% between laboratories reporting “low” and “normal/high”, respectively.  This could reflect differences in assay standardization.  However, given that most assay-specific CV’s were <20% on the surveys, the discordant interpretations could also be caused by differences in reference ranges established by each laboratory.

References:

1. Frazer-Abel A, Sepiashvili L, Mbughuni MM, et al. Overview of laboratory testing and clinical presentations of complement deficiencies and dysregulation. Adv Clin Chem 77 (2016) 1-75.
2. https://www.who.int/bloodproducts/ref_materials/catalogue_bloodproducts.pdf

Compliment C3

Compliment C3 and C4 are used for monitoring immune and auto-immune disorders, and deficiency conditions. These lab tests are commonly performed in immunology labs.

Both measurands have reference materials listed in the JCTLM database.

Proficiency Testing Data from the College of American Pathologists Survey LN-7 (non-commutable samples) from 2016 and 2017 show good agreement among measurement procedures from different manufacturers across the range of concentrations tested.

Harmonization/standardization effort is underway as reported by:  Prohaszka Z, Nilsson B, Frazer-Abel A, Kirschfink M. Complement analysis 2016: Clinical indications, laboratory diagnostics and quality control. Immunobiology 2016;221:1247-58.

Compliment C4

Compliment C3 and C4 are used for monitoring immune and auto-immune disorders, and deficiency conditions. These lab tests are commonly performed in immunology labs.

Both measurands have reference materials listed in the JCTLM database.

Proficiency Testing Data from the College of American Pathologists Survey LN-7 (non-commutable samples) from 2016 and 2017 show good agreement among measurement procedures from different manufacturers across the range of concentrations tested.

Harmonization/standardization effort is underway as reported by:  Prohaszka Z, Nilsson B, Frazer-Abel A, Kirschfink M. Complement analysis 2016: Clinical indications, laboratory diagnostics and quality control. Immunobiology 2016;221:1247-58.

Cortisol

Cortisol is produced in the adrenal glands from pregnenolone under adreno-corticotrophic hormone stimulation and circulates in the blood. Functionally, it belongs to the glucocorticoids. About 90 % of the secreted cortisol in circulation is bound to protein, mainly to cortisol-binding globulin. The main influence of this group of hormones is on the carbohydrate metabolism: promotion of gluconeogenesis, deposition of liver glycogen, and elevation of blood glucose concentration due to decreased carbohydrate utilisation. Glucocorticoids, in addition, inhibit amino acid uptake, protein synthesis in peripheral tissues and affect fat metabolism. They also have an anti-inflammatory effect. Because of these important biological actions, measurement of serum cortisol is important to the assessment of the hypothalamic-pituitary-adrenal (HPA) axis (1).

The method of choice for measurement of serum cortisol in the majority of routine clinical laboratories is based on the immunochemical principle. IFCC decided to dedicate the 1992 Bergmeyer Conference to the standardisation of immunoprocedures and took the measurement of cortisol as one of the examples. The main objective of the IFCC Working Group for Standardisation of Cortisol Measurements (WG-SCM) being established consisted therefore in the development of a set of native, single-donation human samples (ERM-DA451/IFCC, JRC), certified for their cortisol content by a validated reference measurement procedure. In first instance, this panel serves the purpose of evaluation/verification of the metrological correctness of values obtained by routine test systems. The panel with certified reference method values could also be used in the process of assigning traceable values to manufacturers working or product calibrators.

Furthermore, the reference system is added to EQA system. For example, the “Guideline of the German Medical Association on Quality Assurance in Medical Laboratory Examinations – RiliBAEK” (2) requests that the target values for evaluation of routine test procedures for cortisol are assigned by RMP using RM as calibrators. The permissible relative deviation of the results to the target is set to +/-30% with regard to medical needs and present state of art. Data from surveys of Reference Institute for Bioanalytics in 2017 covers approximately 530 labs using 19 commercially available systems. All results CVs are from 9.5 to 12 % for four samples with cortisol concentrations from 280 to 980 nmol/L. About 96% of the participants fulfil the requirements of RiliBAEK (3).

Cortisol is one measurand of the IFCC RELA surveys. Between 3 to 7 RMP labs/candidate RMP labs are participating regularly at this annual comparison (4).

Harmonization is currently incomplete by all IVD manufacturers but the reference system tools are available for establishing metrological traceability to the SI unit for cortisol.

References:

1. Joint Research Center, ERM-DA451_IFCC CORTISOL REFERENCE SERUM PANEL, Report EUR 19007EN, 1999.
2. German Medical Associastion, Revision of the “Guideline of the German medical 600 association on quality assurance in medical laboratory examinations – Rili-BAEK” (unauthorized translation), J. Lab. Med. 39 (1) (2015) 26–69, doi:10.1515/ labmed-2014-0046.
3. Reference Institute for Bioanalytics, Surveys Hormones Group 1 – 2017, https://www.rfb.bio/cgi/surveys?searchType=rv_type&rv_type=HM&analyte=all&year=2017 (accessed 18-06).
4. IFCC, RELA – IFCC external quality assessment scheme for reference laboratories in laboratory medicine. <http://www.dgkl-rfb.de:81/index.shtml> (accessed 18-06).

C-Peptide

Insulin and C-peptide are cleaved from proinsulin and secreted in a 1:1 molar ratio, and are both important biomarkers in the pathophysiology of diabetes mellitus. Measurement of circulating insulin concentrations may improve the classification and management of diabetes and aid in defining and treating individuals with varying levels of insulin resistance. Assessment of C-peptide has clinical utility in monitoring endogenous insulin production in diabetics receiving exogenous insulin therapy, and the Diabetes Control and Complications Trial (DCCT) established the importance of C-peptide as a key biomarker of beta cell function and depletion, primarily in Type 1 diabetics. In addition, C-peptide measurements are required by CMS to approve patients for insulin pump therapy.

Insulin and C-peptide are currently being utilized in clinical trials and epidemiological studies to evaluate therapeutic and potential prevention strategies in diabetes. Standardization of insulin and C-peptide assays will allow data and information from numerous epidemiological studies and clinical trials to be aggregated and ultimately incorporated into clinical practice guidelines. In this context, assay harmonization and/or standardization of insulin and C-peptide remain critical.

There are active efforts working simultaneously to achieve standardization of both analytes. A 2016 CAP survey with presumably commutable serum pools highlighted the continuing disparity between immunoassay platforms. The insulin low and high serum pool mean concentrations across peer groups (n = 467) was 49.9 pmol/L (8.31 uIU/mL; range: 36.4 – 64.6 pmol/L) and 340.5 pmol/L (56.8 uIU/mL; range: 279.8 – 410.1 pmol/L), respectively. The C-peptide low and high serum pool mean concentrations across all peer groups (n = 285) was 0.49 pmol/L (range: 0.41 – 0.61 pmol/L) and 1.99 pmol/L (range: 1.5 – 2.37 pmol/L), respectively. Poor immunoassay specificity is likely responsible for observed analytical differences.

References:

1. Miller WG, Thienpont LM, Van Uytfanghe K, Clark PM, Lindstedt P, Nilsson G, Steffes MW; Insulin Standardization Work Group. Toward standardization of insulin immunoassays. Clin Chem. 2009 May;55(5):1011-8.
2. Marcovina S, Bowsher RR, Miller WG, Staten M, Myers G, Caudill SP, Campbell SE, Steffes MW; Insulin Standardization Workgroup. Standardization of insulin immunoassays: report of the American Diabetes Association Workgroup. Clin Chem. 2007 Apr;53(4):711-6.
3. Jones AG, Hattersley AT. The clinical utility of C-peptide measurement in the care of patients with diabetes. Diabet Med. 2013 Jul; 30(7): 803–817.
4. Little RR, Wielgosz RI, Josephs R, Kinumi T, Takatsu A, Li H, Stein D, Burns C. Implementing a Reference Measurement System for C-peptide: Successes and Lessons Learned. Clin Chem Jun 2017, clinchem.2016.269274; DOI: 10.1373/clinchem.2016.269274.

C-Reactive protein, high sensitivity

High sensitivity C-reactive protein HS-CRP) is used in some countries but not in others.  There is not global agreement on its value in assessing risk for myocardial infarction. A literature review for HS-CRP method comparisons found 7 reports from the mid-2000 years and no recent reports. In general the agreement among methods was fairly good in the range of 0-10% bias. The CDC assessed commutability for diluted CRM 470 (now replaced with ERM-DA474/IFCC) from the EU Joint Research Centre and found the diluted RM was commutable for 14 of 15 HS-CRP methods that had acceptable precision (9 were excluded).  A recent paper (1) suggests that the role of HS-CRP has narrowed to assessment of primary prevention risk when clinical decisions to initiate statin therapy are uncertain (e.g. relatively low LDLC with few other risk factors).  The available information suggests that HS-CRP is not a problem diagnostically.

(1) Ridker PM, A Test in Context: High-Sensitivity C-Reactive Protein, J Am Coll Cardiol. 2016 Feb 16;67(6):712-23. 

C-Reactive protein, inflammatory

Although there are biases in CRP results among different measurement procedures, EQA using commutable materials suggest the differences are moderate.  Results are typically interpreted semi-quantitatively to determine the degree of underlying inflammation from a disease process.  A reference material for CRP in serum is available from the EU Joint Research Centre that has been shown to be commutable for many common measurement procedures.

Creatine Kinase (CK)

An IFCC reference measurement procedure (RMP) is available.  IVD manufacturers should formulate reagents to conform to the RMP.

Schumann G, Bonora R, Ceriotti F, Clerc-Renaud P, Ferrero CA, Ferard G, et al. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C. Part 2. Reference procedure for the measurement of catalytic concentration of creatine kinase. Clin Chem Lab Med. 2002;40:635-42.

Creatinine

EQA data from two Japanese programs in 2015 that used commutable samples had 4.5% and 3.9% CV among different laboratories and methods.  EQA data from a College of American Pathologists 2018 survey (LN24-A) with commutable samples had 40%, 22%, 17%, 15%, 15% and 16% difference between maximum and minimum values reported by individual laboratories at six concentrations from 0.7, 1.4, 2.1, 2.8, 3.5 and 4.2 mg/dL respectively; and 2.2-4.4% CV among mean values for different measurement procedure groups.  Mean biases vs the reference measurement procedure were 2.8%, 1.8%, 1.0%, -0.3% -0.0% and 0.1% for the same concentrations. Taken together these data support adequate harmonization for serum or plasma creatinine measurement procedures.  The agreement at values 0.7 mg/dL suggest some improvement at lower concentrations may be indicated. A summary of creatinine standardization and eGFR reporting concluded that harmonization was adequate (1). An aggregation of EQA data between 2006 and 2011 in Italy showed with commutable materials that enzymatic methods had better accuracy than did alkaline picrate based methods presumably due to fewer specimen specific effects (2). A reference material, serum-based reference material and a reference measurement procedure are listed by JCTLM.

The acceptable harmonization of creatinine results referred to above applies when using measuring systems (i.e. reagents, calibrators, instruments from the same IVD manufacturer) supplied by IVD manufacturers from economically well developed countries with typically global distribution of measuring systems.  Harmonization has not been achieved on a global basis when reagents and calibrators are supplied by other manufacturers (3).

References:

1. Miller WG, Jones GRD, Estimated Glomerular Filtration Rate; Laboratory Implementation and Current Global Status. Adv Chronic Kidney Dis 2018;25:7-13.
2. Evaluation of the impact of standardization process on the quality of serum creatinine determination in Italian laboratories. Carobene A, Ceriotti F, Infusino I, Frusciante E, Panteghini M. Clin Chim Acta 2014;427:100-106.
3. Biljak VR, Honovic L, Matica J, Kresic B, Vojak SS. The role of laboratory testing in detection and classification of chronic kidney disease: national recommendations. Biochemia Medica 2017;27:153-176 (see Appendix 3).

CSF Proteins

IFCC has a working group addressing harmonization of CSF proteins.

Cyclosporin

Cyclosporine was the first immunosuppressant drug introduced used to prevent the body from rejecting a transplanted organ.  Therapeutic Drug Monitoring is performed for the purposes of drug dose adjustment and to ensure patients are in the correct therapeutic range.  Nonetheless, improvement in the measurement of cyclosporine is still needed in spite of the drug being used for decades.  Standardization / Harmonization of cyclosporine measurement is important for several reasons including:

• Narrow therapeutic range
• Drug toxicity (high) or inadequate immunosuppression (low)
• dose/exposure relationship is interindividually and intraindividually highly variable
• Active and inactive metabolites with variable crossreactivity depending on the method chosen
• Variability associated with pre-analysis including the whole blood extraction methods used
• Inadequate precision of many widely used methods

Various proficiency-testing programs with unclaimed commutability have demonstrated between-laboratory variability as much as 20% and CVs at lower drug concentrations frequently exceeding 20%.  This is important, as low therapeutic ranges for cyclosporine have been introduced in an attempt to reduce long-term toxicity. A recent survey (76 laboratories in 14 countries) identified three main reasons for interlaboratory variability: (1) lack of standardization of laboratory procedures and workflows starting with sample collection and handling, (2) lack of use of appropriate reference materials and (3) poor compliance with internationally accepted good laboratory practice guidelines. Although intralaboratory precision improvement is the responsibility of the individual laboratory, interlaboratory bias can only be addressed by the establishment of higher-order reference materials and/or higher-order reference procedures to ensure worldwide measurement traceability and consistency.

The IFCC Working Group on Immunosuppressive Drugs (WG-ID) is devoted to the establishment of candidate reference procedures and reference materials for immunosuppressive drugs (ISDs) as cyclosporine, sirolimus, tacrolimus, everolimus, and mycophenolic acid (MPA). Demonstration of the current state of the art in ISD – TDM by measurement comparison will define the need for harmonization or – if feasible – standardization of measurement services.

References:

1. Christoph Seger, Maria Shipkova, Uwe Christians, et al. Assuring the Proper Analytical Performance of Measurement Procedures for Immunosuppressive Drug Concentrations in Clinical Practice: Recommendations of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology Immunosuppressive Drug Scientific Committee: Ther Drug Monit 2016;38:170–189
2. Christians U, Vinks AA, Langman L, et al. The impact of laboratory practices on inter-laboratory variability in therapeutic drug monitoring of immunosuppressive drugs. Ther Drug Monit. 2015; 37:718–724.

Cytomegalovirus (CMV)

The 1^st WHO International Standard for human cytomegalovirus (CMV) was introduced in 2013.  This standard is intended for use to prepare commercially available secondary standards.  A recent report has shown that there are unresolved differences among commercially available secondary standards, all traceable to the WHO standard, when measured using real-time and digital PCR measurement procedures with the degree of bias ranging from +0.6 to -1.0 log₁₀ IU/mL. (1).   A report comparing patient sample results using two digital PCR systems and three different reagent sets found generally good agreement at higher viral loads and poorer agreement with biases on the order of 1.5 log₁₀ IU/mL among 5 of 9 combinations of instruments and reagents when viral loads were less than approximately 4 log₁₀ IU/mL (2). Comparison of whole blood and plasma CMV results between a laboratory developed test and a commercial real-time PCR method found whole-blood viral loads were on average 0.5 to 1.22 log₁₀ IU/mL higher than those in plasma (3).

1. Hayden RT, Gu Z, Sam Y, Tang L, Pounds S, Caliendo AM. Comparative Evaluation of Three Commercial Quantitative Cytomegalovirus Standards by Use of Digital and Real-Time PCR. J Clin Microbiol 2015;53:1500-5.
2. Hayden RT, Gu Z, Sam SS, Sun Y, Tang L, Pounds S, Caliendo AM. Comparative performance of reagents and platforms for quantitation of cytomegalovirus DNA by digital PCR. J Clin Microbiol 2016;54:2602–8.
3. Babady NE, Cheng C, Cumberbatch E, Stiles J, Papanicolaou G, Tang YW. Monitoring of cytomegalovirus viral loads by two molecular assays in whole-blood and plasma samples from hematopoietic stem cell transplant recipients. J Clin Microbiol. 2015;53:1252-7.

Cytomegalovirus IgG (CMV IgG)

Cytomegalovirus IgG (CMV IgG) is useful for the diagnosis of primary, acute phase infection with cytomegalovirus in patients with infectious mononucleosis, pregnant women, and transplant recipients. CMV is a significant cause of morbidity and mortality among bone marrow or solid organ transplant recipients, individuals with AIDS and other immunosuppressed patients. CMV is also responsible for congenital disease among newborns and is one of the TORCH infections (toxoplasmosis, other infections including syphilis, rubella, CMV and herpes simplex virus).  Agreement of results between methods based on CAP survey data for 2016 was 99.7%.

D-dimer

When fibrin is hydrolyzed in vivo, a spectrum of degradation products is released. The best characterized of these is D-dimer, for which clinical testing using commercial kits is commonly performed as a marker for venous thromboembolic disease and disseminated intravascular coagulation. Numerous calls for standardization of this assay have been made, which include calls for common units of measurement, age adjustments, and for common cutoffs for diagnostic use. These needs place D-dimer in a high-priority category for international harmonization/standardization.

Prior work has been performed by the International Society for Thrombosis and Hemostasis in conjunction with the WHO to develop an international standard, but that work may be inactive. Current methods use different measurement units (mg/dL or fibrinogen-equivalent units (FEU), and there is no reference measurement procedure.

Reference:

1. Lippi G, Tripodi A, Simundic AM, Favaloro EJ. International survey on D-dimer test reporting: a call for standardization. Semin Thromb Hemost. 2015;41:287–93. https://doi.org/10.1055/s-0035-154909210.1055/s-0035-1549092.

Digoxin

Digoxin is commonly used to treat atrial fibrillation, atrial flutter, and heart failure. Conventionally, based on the data from 39 patients by Smith et al., a therapeutic range of 0.8 to 2.0 mg/L was established (1). However, in post hoc analysis of the Digitalis Investigation Group (DIG) trial in 1997, which was the first large-scale prospective randomized trial for long-term treatment of digoxin on chronic heart failure, serum digoxin concentrations of 0.5 to 0.8 or 0.9 mg/L associated with the benefits of treatment with a lower risk of adverse effects (decrease in all-cause mortality and hospitalization) compared with >1.0 or 1.2 mg/L (2, 3). Thus, current data strongly support decreasing the recommended therapeutic trough serum digoxin concentration range from 0.8 to 2.0 mg/L to lower values (e.g., 0.5 to 0.8 mg/L) in the treatment of chronic heart failure (4).

In the comparability study for digoxin concentrations between 6 different analyzers [Architect and AxSym (Abbott Laboratories), cobas c 501 module and COBAS INTEGRA 800 analyzer (Roche Diagnostics GmbH), Dimension Vista and Dimension Xpand (Simens Healthcare Diagnostics Inc)], systemic deviations (relative mean differences) of -2.8%, -7.6% and 18.7% were found among manufacturers (cobas c 501 vs. COBAS INTEGRA 800, AxSym vs. Architect and Dimension Xpand vs. Dimension Vista, respectively) in 67-70 leftover routine samples for the digoxin concentration monitoring (5). The largest relative mean difference for digoxin concentration was +36.5% with the 95% CI of -17.1% to +90.1% for 67 patient samples for Dimension Vista compared to cobas c 501 (method comparisons against cobas c 501 module as arbitrary standard) (5).

There is no suitable proficiency testing information from commutable samples.

Three reference materials are listed in the JCTLM database but the commutability information is not available. Two reference measurement procedures for digoxin are listed by JCTLM.

References

1. Smith TW, Butler VP Jr, Haber E. Determination of therapeutic and toxic serum digoxin concentrations by radioimmunoassay. N Engl J Med. 1969 Nov 27;281(22):1212-6.
2. Ahmed A, Gambassi G, Weaver MT, Young JB, Wehrmacher WH, Rich MW. Effects of discontinuation of digoxin versus continuation at low serum digoxin concentrations in chronic heart failure. Am J Cardiol. 2007 Jul 15;100(2):280-4
3. Rathore SS, Curtis JP, Wang Y, Bristow MR, Krumholz HM. Association of serum digoxin concentration and outcomes in patients with heart failure. JAMA. 2003 Feb 19;289(7):871-8.
4. Goldberger ZD, Goldberger AL. Therapeutic ranges of serum digoxin concentrations in patients with heart failure. Am J Cardiol. 2012 Jun 15;109(12):1818-21.
5. Shipkova M, Petrova DT, Rosler AE, Orth M, Engelmayer J, Wieland E, Brandhorst G, Oellerich M. Comparability and imprecision of 8 frequently used commercially available immunoassays for therapeutic drug monitoring. Ther Drug Monit. 2014 Aug;36(4):433-41.

Elastase I

Stool Elastase (EI-1):  Human pancreatic elastase-1 (E1) is quite stable and remains undegraded during intestinal transit. Therefore, its concentration in feces reflects exocrine pancreatic function. During an inflammation of the pancreas, E1 is released into the bloodstream. Thus, the quantification of pancreatic elastase-1 in serum and feces allows diagnosis or exclusion of acute pancreatitis¹ 

The E1 test is used for the diagnosis or exclusion of exocrine pancreatic insufficiency in case of unexplained diarrhea, constipation, steatorrhea, flatulence, weight loss, upper abdominal pain, and food intolerances. Monitoring of exocrine pancreatic function in cystic fibrosis, diabetes mellitus or chronic pancreatitis. However, it is commonly noted that is it only helpful in detecting severe EPI, not the mild to moderate form. ^2,3

Serum Elastase, also called Pancreatopeptidase, is a protease present in pancreatic secretion with the unique ability to rapidly hydrolyze elastin. Elastase has been implicated in the pathogenesis of pulmonary emphysema, atherosclerosis and in the vascular injury of acute pancreatic necrosis.  Serum pancreatic levels quantify EL 1 for the diagnosis or exclusion of an acute pancreatitis or an inflammatory episode of chronic pancreatitis or gallstone induced pancreatitis.

Direct testing is considered the “gold standard” however it is invasive and not suited for routine use.  The indirect method using an immunoassay is commonly used.  This test can be used for patients on pancreatic enzyme replacement therapy (PERT) since it tests for human elastase-1and not porcine enzymes used in PERT.

No information on harmonization status was identified.

References:

1. Biomarkers in Toxicology, Bhupendra S. Kaphalia, 2014. (Dominguez-Munoz et al., 2006; Naruse et al., 2006)
2. Naruse, Satoru, et al, Fecal pancreatic elastase: a reproducible marer for severe exocrine pancreatic insufficiency, Journal of Gatroenterology vol 41,pp 901-908(2006)
3. Lankischa PG, et al, Faecal elastase 1: not helpul in diagnosing chronic pancreatitis associated with mild to moderate exocrine pancreatic insufficiency, Gut

Epstein-Barr Virus

Given the emergence of post-transplant lymphoproliferative disorder (PTLD) in the United States and Europe, the endemic nature of nasopharyngeal carcinoma (NPC) in Southeast Asia and other parts of the world, and the ubiquitous nature of Epstein-Barr virus (EBV) and its association with many rare and a few common pathological syndromes, there is sufficient clinical relevance as well as urgency behind the need for a reliable quantitative assay for EBV DNA. For nasopharyngeal cancer, in particular, EBV is an important prognostic marker whose clinical utility is being studied in a number of ongoing clinical trials, but it will not be possible to make optimal use and/or to properly interpret the data collected with the existing assays because of the unaccounted effects of many variables that influence assay outcomes (1). At present, there is no gold standard assay for quantification of EBV DNA for clinical or analytical purposes, and prior to 2011 there was no internationally validated reference material to calibrate EBV DNA assays or to account for the many variables that can influence EBV DNA quantification in different laboratories. Currently, there is no FDA approved assay for the detection and quantification of EBV DNA. Clinical and analytical laboratories have laboratory developed tests/assays (LDTs) for quantifying EBV DNA or have relied on a combination of commercially available research use only (RUO) and/or analyte-specific reagents (ASRs), with DNA purified from EBV-infected cells as a source of quantitative standards (ie, calibrators). Several studies have reported detailed side-by-side comparisons of the performance of some of these assays. For example, in 2009, Preiksaitis et al.(2) reported that the variation in reported results on individual samples ranged from 2.28 log10 to 4.14 log10, that more than 52.0% of all data points fell outside acceptable standards for variation (ie, variation was more than 0.5 log10 outside of the expected value), and that interlaboratory variability for replicate samples was markedly greater than intra-laboratory variability. This study demonstrated that the existing procedures could not be reliably used to provide an accurate estimate of EBV viral load.

References:

1. Kim KY, Le Q, Yom SS, Pinsky BA, et al. Current State of PCR-Based Epstein-Barr Virus DNA Testing for Nasopharyngeal Cancer. J Natl Cancer Inst.2017 Apr 1;109(4). doi: 10.1093/jnci/djx007.
2. Preiksaitis JK, Pang XL, Fox JD, et al. Interlaboratory comparison of epstein-barr virus viral load assays. Am J Transplant. 2009;9(2):269–279.

Estradiol (17-beta)

Estradiol (oestradiol) is a steroid hormone involved in female sexual development and reproductive function. Estradiol is produced by the ovaries, the adrenal gland and the placenta during pregnancy. Estradiol is responsible for initiating puberty and for triggering the menstrual cycle. Estradiol declines in post-menopausal women. Low levels cause vaginal discomfort, hot flashes and can lead to osteopenia and osteoporosis in older age.  Estrogens are used therapeutically to treat post-menopausal symptoms. Estradiol can play a role in promoting some types of breast cancer.  Estradiol is also important in male erectile function, spermatogenesis, and libido.  Estrogen likely has a causative role in male breast, prostate and possibly testicular cancers.  Aromatase is an enzyme in the synthetic pathway of estradiol. Aromatase inhibitors are used therapeutically to reduce estrogen levels in cancer treatment and for gynecomastia in children and adolescents.

Laboratory measurement of estradiol falls into two categories:  assays for high concentrations (eg 20-700 pg/mL) in pre-menopausal menstruating women; or low concentrations (eg <30 pg/mL) in post-menopausal women, men, or following aromatase inhibitor therapy.  Conventional immunoassays are generally satisfactory for higher concentrations associated with fertility assessment.  However, at low concentrations interference from estrogen metabolites and related structural steroids causes falsely elevated values with most immunoassays (1).  Clinical laboratory testing at low estradiol levels should be done by IDMS methods because of the non-selectivity of immunoassays.

EQA data with commutable samples supports reasonably good agreement among immunoassays at higher concentrations but not at lower concentrations (2).  IDMS procedures for estradiol have been developed as reference measurement procedures by several metrology institutes and are offered by some laboratories for routine testing.  JCTLM lists primary (pure substance) CRMs for preparing calibrators for the IDMS procedures, serum-based CRMs for use as trueness controls for IDMS reference measurement procedures, and several laboratories that provide reference measurement procedure testing services.  The lyophilized serum-based CRMs from the EU Joint Research Center have not been evaluated for commutability and are not intended for use as calibrators.  The CDC has a certification program for standardization of estradiol measurement procedures (3). 

Despite these resources, harmonization of results from estradiol measurement procedures, especially at lower concentrations, remains an issue for most commonly used immunoassays.  The lack of harmonization is influenced both by inadequate metrological traceability of clinical laboratory methods, and by inadequate selectivity of immunoassays for estradiol. 

References

1. Handelsman, et al. Performance of direct estradiol immunoassays with human male serum samples. Clin Chem 2014;60:510-17.
2. Accuracy based testosterone and estradiol educational discussion (ABS 2019-A). https://documents.cap.org/documents/2019-A-Accuracy-Based-Testosterone-and-Estradiol.pdf. Accessed October 29, 2019.
3. https://www.cdc.gov/labstandards/hs_standardization.html. Accessed October 29, 2019.

Ethyl Alcohol

NOTE:  the medical impact is medium for ethyl alcohol when used for medical care. However, the impact is high when used in forensic situations.

EQA data from the College of American Pathologists in 2018 (AL2-A) that used serum based samples (commutability unknown) had approximately 40% difference between maximum and minimum values at concentrations from 31-290 mg/dL reported by 3400 individual laboratories and 5.7% to 7.3% CV among all individual results. The range of values for 5 different method types was 13-19%.  Data from the 2018 (AL1-A) survey that used whole blood samples (commutability unknown) had 18-28% difference between maximum and minimum values at concentrations from 39-264 mg/dL reported by 190 individual laboratories and 3.7% to 5.0% CV among all individual results using gas chromatography measurement procedures. Although the range of individual results was larger than ideal for serum samples, the CVs for all results, the mean values for serum results among 5 different method types and the whole blood results among laboratories were consistent with adequate harmonization for serum and whole blood ethanol.  JCTLM lists a certified reference material for ethanol in water. NIST SRM 2893a Ethanol-water solution is available but not listed by JCTLM.

Ferritin

Medical Impact :

Ferritin measurements are used to diagnose iron deficiency, in the differential diagnosis of anemia including iron deficiency anemia, to monitor the response to iron therapy, and to aid in the diagnosis of iron overload including hereditary haemochromatosis. The ferritin concentration is included in the algorithm for the diagnosis of hereditary haemochromatosis  and the differential diagnosis guideline of anemia.

Harmonization Status

A comprehensive review in 2018 included 148 studies in a meta-analysis of ferritin assay performance (1). The most used methods included radiometric, non-radiometric and agglutination assays. The overall within-run imprecision (CV%) for the most frequently reported ferritin methods was 6.2±3.4% (CI 5.69±6.70%; n = 171), between-run imprecision 8.9±8.7% (CI 7.44±10.35%; n = 136), and recovery rate 95.6% (CI 91.5±99.7%; n = 94). The pooled regression coefficient was 0.985 among all methods analyzed, and 0.984 when comparing non-radiometric and radiometric methods, without statistical differences in ferritin concentration ranging from 2.3 to 1454 ng/mL (µg/L). The report concluded that the laboratory methods most frequently used to determine ferritin concentrations have comparable accuracy and performance.

Another report in 2008 (2) included 5 individual serum samples compared among 5 commonly used automated analyzers:  Abbott Architect, Beckman Access, Roche Elecsys, Siemens Centaur, Siemens Immulite 2000.  Among assay CVs were between 10-14% at concentrations from 20-293 ng/mL (µg/L) supporting good agreement among results from these measurement procedures.

Data from a 2017 College of American Pathologists survey of over 1800 labs using 14 commercially available instruments showed average 5.9% CV within instrument groups. The CAP PT materials are non-commutable so comparison among the different measurement procedure was not possible.

A reference material is available as the 3^rd WHO International Standard Ferritin, human, recombinant NIBSC code: 94/572.

References

1. Maria N. Garcia-Casal, Juan P. Peña-Rosas, Eloisa Urrechaga, Jesus F. Escanero, Junsheng Huo, Ricardo X. Martinez, Lucero Lopez-Perez. Performance and comparability of laboratory methods for measuring ferritin concentrations in human serum or plasma: A systematic review and meta-analysis. PLoS One. 2018 May 3;13(5):e0196576. doi: 10.1371/journal.pone.0196576. eCollection 2018.

2. Sheena Blackmore, Malcolm Hamilton, Anne Lee, Mark Worwood, Matthew Brierley, Alan Heath and Susan J. Thorpe. Automated immunoassay methods for ferritin: recovery studies to assess traceability to an international standard. Clin Chem Lab Med 2008;46(10):1450–1457.

Fetal fibronectin

Fibronectin (FN) is a ubiquitous extracellular matrix (ECM) glycoprotein that plays vital roles during tissue repair. Plasma fibronectin increases when there is trauma or injury.

Fetal fibronectin (fFN) has long been used as a biomarker to predict preterm labor. fFN sits at the interface between the amniotic sac and the uterine lining; thus its presence in vaginal secretions toward the end of gestation may indicate the onset of labor. Preterm labor, defined as labor that occurs before 37 weeks of gestation, is the leading cause of neonatal morbidity and mortality in the United States.¹

Anything that physically disturbs the cervix or uterus has the potential to elevate fFN levels. Semen may contain enough fFN to create a positive test result, so sexual intercourse should be avoided within 24 hours of sample collection. The patient must avoid lubricants, lotions, soaps, and douches at least a day before the test.²

Other biomarkers are increasingly used as an alternative to fFN, including placental alpha macroglobulin-1 (PAMG-1), insulin-like growth factor-binding protein-1 (IGFBP-1), and alpha-fetoprotein (AFP)¹ At Penn State University Hershey Medical Center. Annual test volume of fFN decreased from 74 in 2008 to 2 thus far in 2020, due to a rigorous specimen collection process, cost of the test, and most importantly, a lack of association between positive fFN tests and improved perinatal outcomes.³

The fetal fibronectin test should not be used as a screening test for asymptomatic women. For high-risk asymptomatic women, and especially for women with multiple pregnancies, the performance of the fetal fibronectin test was also too low to be clinically relevant. For consensual use as a diagnostic tool for women with suspected preterm labor, the best use policy probably still depends on local contingencies, future cost-effectiveness analysis, and comparison with other more recent available biochemical markers.² 

References

1. Rejwi Dahal, PhD, DABCC, Is It Time To Stop Using Fetal Fibronectin In Diagnosis of Preterm Labor? Clinical Laboratory News//AACC.org. Dec. 17, 2020. https://www.aacc.org/cln/cln-daily/2020/is-it-time-to-stop-using-fetal-fibronectin-in-diagnosis-of-preterm-labor
2. Faron G, Balepa L, et al. The fetal fibronectin test: 25 years after its development, what is the evidence regarding its clinical utility? A systematic review and meta-analysis, PMID: 29914277, DOI: 10.1080/14767058.2018.1491031
3. Yusheng Zhu PhD, speaker, shares his experience that led to discontinuation of fFN at Penn State University Hershey Medical Center. Is It Time To Stop Using Fetal Fibronectin In Diagnosis of Preterm Labor? Clinical Laboratory News//AACC.org. Dec. 17, 2020. https://www.aacc.org/cln/cln-daily/2020/is-it-time-to-stop-using-fetal-fibronectin-in-diagnosis-of-preterm-labor

04 May 2021

Fibrin Degradation Products (D-dimer)

When fibrin is hydrolyzed in vivo, a spectrum of degradation products is released. The best characterized of these is D-dimer, for which clinical testing using commercial kits is commonly performed as a marker for venous thromboembolic disease and disseminated intravascular coagulation. Numerous calls for standardization of this assay have been made, which include calls for common units of measurement, age adjustments, and for common cutoffs for diagnostic use. These needs place D-dimer in a high-priority category for international harmonization/standardization.

Prior work has been performed by the International Society for Thrombosis and Hemostasis in conjunction with the WHO to develop an international standard, but that work may be inactive. Current methods use different measurement units (mg/dL or fibrinogen-equivalent units (FEU), and there is no reference measurement procedure.

Reference:

1. Lippi G, Tripodi A, Simundic AM, Favaloro EJ. International survey on D-dimer test reporting: a call for standardization. Semin Thromb Hemost. 2015;41:287–93. https://doi.org/10.1055/s-0035-154909210.1055/s-0035-1549092.

Folate

Folic acid (folate) is important for synthesis of nucleic acids, in amino acid metabolism and for production of red blood cells. Folate is not synthesised in the body and is thus an essential nutrient.  Folate deficiency is the main clinical condition that contributes to megaloblastic anemia and neural tube defects in pregnancy.  Some countries (e.g. USA, Australia) require folate fortification of wheat flour and deficiency is less common. Other countries do not require fortification in food and deficiency is more prevalent.  Circulating folate exists in three forms. The most common (80-90%) is 5-methyltetrahydrofolate. Additional forms include unmetabolized folic acid, 5-formyltetrahydrofolate, and pteroylglutamic acid.  Folate assays use a folate-binding protein, typically beta-lactoglobulin from cow’s milk, similarly to an immunoassay format.  The measurand detected by most assays in serum is a total folate measurement that includes all three forms.

A recent report evaluated folate results standardization among 6 instrument systems from 4 major manufacturers (1).  Results for 24 human serum pools from residual samples in a clinical laboratory were used for comparison.  The WHO IS 03/178 and two levels of NIST SRM 3949 were shown to be commutable with the serum pools.  The report concluded that the mean inter-assay CV on all pools and on those with folate values >3 μg/L (n = 15) was 12.5% and 7.1%, respectively. In neither case was the performance goal of 3.0% fulfilled.  The performance goal was based on total allowable uncertainty derived from biological variation data.  The bias vs. all methods medians for samples with folate <4 μg/L was up to 1.0 μg/L for I measurement procedure and up to -0.6 μg/L for another measurement procedure.  The residual biases produced an excessive uncertainty of folate measurement on clinical samples near the important threshold for identifying deficiency.

A report from the US CDC identified inadequate agreement of folate results from different measurement procedures (2).

References:

1. Braga F, Frusciante E, Ferraro S, Panteghini M. Trueness evaluation and verification of inter-assay agreement of serum folate measuring systems. Clin Chem Lab Med 2020; ahead of print https://doi.org/10.1515/cclm-2019-0928.
2. Pfeiffer CM, Sternberg MR, Hamner HC, et al. Applying inappropriate cutoffs leads to misinterpretation of folate status in the US population. Am J Clin Nutr 2016;104:1607-15.

Fructosamine

Glycated Albumin / Fructosamine

Serum glycated albumin and fructosamine (serum glycated proteins) can be used as markers of short-term glycemia (2-3 weeks) and/or as alternate markers of glycemia when an accurate HbA1c cannot be obtained. Glycated albumin values are expressed by a ratio (%) of glycated to total albumin, and differences among individuals and albumin concentrations have negligible influences on the results. Fructosamine assays measure all serum glycated proteins and results can be affected by disorders in protein turnover. Fructosamine values can be corrected for albumin or total protein concentrations if needed. 

Review of CAP survey data (GSAB 2016) utilizes a true serum matrix for glycated albumin and fructosamine EQA. There are only 14-15 laboratories participating in this survey and only one method listed (enzymatic, presumably Asahi Kasei validated on instruments with open channels). The between laboratory CV is good, 2.7% CV (mean: 23.85) and 6.3% using the (mean: 12.10).  Many more laboratories are reporting fructosamine (n=110) proficiency testing (CAP survey FT), with a majority using the Roche fructosamine assay (n=104) compared to Diazyme (n=6). Among the Roche peer group the CVs were low for the “elevated” sample (CV: 3.4%, mean: 337.1 umol/L) and comparable for the “normal” specimen (CV: 3.3%, mean: 226.5 umol/L). In Japan, and possibly other countries, glycated albumin is used more frequently than is fructosamine.

Currently, neither glycated albumin or fructosamine are widely utilized nor are target values defined in clinical guidelines. Assays are not standardized but there are a limited number of manufacturers and for now standardization and harmonization do not present a major clinical issue.

References:

1. Juraschek SP, Steffes MW, Selvin E. Associations of alternative markers of glycemia with hemoglobin A(1c) and fasting glucose. Clin Chem. 2012;58:1648–1655.

2.0Selvin E, Francis LM, Ballantyne CM, Hoogeveen RC, Coresh J, Brancati FL, Steffes MW. Nontraditional markers of glycemia: associations with microvascular conditions. Diabetes Care. 2011 Apr;34(4):960-7.

3.0Kouzuma T, Usami T, Yamakoshi M, Takahashi M, Imamura S. An enzymatic method for the measurement of glycated albumin in biological samples. Clin Chim Acta. 2002 Oct;324(1-2):61-71.

4.0Goldstein DE, Little RR, Lorenz RA, Malone JI, Nathan D, Peterson CM, Sacks DB. Tests of glycemia in diabetes. Diabetes Care. 2004 Jul;27(7):1761-73.

Gamma glutamyl transferase (GGT)

An IFCC reference measurement procedure (RMP) is available.  IVD manufacturers should formulate reagents to conform to the RMP.

Schumann G, Bonora R, Ceriotti F, Ferard G, Ferrero CA, Franck PF, et al. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C. International Federation of Clinical Chemistry and Laboratory Medicine. Part 6. Reference procedure for the measurement of catalytic concentration of gamma-glutamyltransferase. Clin Chem Lab Med. 2002;40:734-8.

Gentamicin

Aminoglycosides are a group of antibiotics that are used to treat serious bacterial infections. The level of the prescribed aminoglycoside in the blood is measured in order to adjust doses as necessary and ensure effective treatment while avoiding toxic side effects. Gentamicin is one of the most commonly prescribed aminoglycosides, and is used to treat infections caused by certain types of Gram-negative bacteria [1].

It is important to monitor the concentration of aminoglycosides because their effectiveness depends on having an adequate level in the blood. Aminoglycosides are associated with serious toxic side effects, including damage to hearing and/or balance (ototoxicity) and acute kidney damage (nephrotoxicity). Though kidney damage caused by aminoglycosides is usually reversible, hearing and/or balance loss is frequently permanent. These side effects can occur at any time, but the risk is greater with elevated blood levels and when the drugs are given for an extended period of time. The risk of side effects is lower with some of the aminoglycosides that have been developed recently [1].

The accuracy and precision of commercial assay techniques in the measurement of gentamicin, tobramycin, amikacin, netilmicin and vancomycin in serum were investigated.  Data from the measurement of 40 external quality assessment samples from 358 laboratories providing a therapeutic drug monitoring service were analysed.  In this study, high quality of the matrix (i.e. pooled human serum with no added preservative) provides a close commutability with patient samples.  Differences between techniques in accuracy and precision were observed for all drugs.  Coefficients of variation ranged from 4.1% to 9.8%. The percentage difference in measurements from the weighed-in drug concentration ranged from –10.1% to +4.0%.  The authors concluded that all assays performed to a satisfactory standard for measurement in non-renal patients, but none met the more stringent standards desirable for monitoring patients with renal impairment, including gentamicin [2].

In addition, gentamicin levels generally agree well in the proficiency testing scheme of the German Association of Clinical Chemistry and Laboratory Medicine [DGKL; Referenzinstitut für Bioanalytik (RfB), Bonn, Germany] and in a comparable UK scheme, apart from one method.  This discrepancy appears to be mainly a consequence of the use of  gentamicin sulphate to spike samples, and is less when gentamicin base is used or in clinical samples.  There remains a need to develop a reference measurement procedures (RMP), which might help to harmonize and standardize routine serum gentamicin measurements by commercial immunoassay systems [3].

References:

1. Lab Tests Online-US. https://labtestsonline.org/tests/aminoglycoside-antibiotics
2. John F. Wilson1, Alan C. Davis and Caroline M. Tobin: Evaluation of commercial assays for vancomycin and aminoglycosides in serum: a comparison of accuracy and precision based on external quality assessment Journal of Antimicrobial Chemotherapy (2003) 52, 78–82.
3. LC-MS/MS based candidate reference method for the quantification of total gentamicin in human serum and plasma using NMR characterized calibrator material, Lucha S et al., Clinica Chimica Acta, 2017, 464, 211-217.

28 April 2021

Glucose

EQA data from two Japanese programs in 2015 that used commutable samples had 3.8% and 1.9% CV among different laboratories and methods.  EQA data from the College of American Pathologists in 2015 (non-commutable samples) had 14.1% difference between maximum and minimum values reported by individual laboratories and 3.5% CV% among mean values for different measurement procedure groups.  Taken together these data support adequate harmonization for serum or plasma glucose measurement procedures.  A reference material, serum-based reference material and a reference measurement procedure are listed by JCTLM.

Glucose

Glucose in urine is primarily an indicator of diabetes.  Glucose is one component of the common urinalysis dipstick test and gives a qualitative assessment if glucose is elevated.  Elevated urine glucose is followed up by blood tests for diabetes.  Quantitative urine glucose measurements are infrequently used to confirm renal tubular dysfunction when a patient with positive urine glucose is non-diabetic. Other biomarkers of kidney function are used to follow up an elevated urine glucose value.

Proficiency testing data from the College of American Pathologists quantitative Urine Survey in 2020 indicated the CVs for combined results from all 15 peer groups were 3.9% at 40 mg/dL, 3.1% at 73 mg/dL, and 2.4% at 269 mg/dL suggesting very good agreement in results among different measurement procedures.  For a CAP 2020 qualitative urinalysis dipstick survey, 99.8% of 2297 results agreed for a negative sample but results were more dispersed for a positive sample. For 18 peer groups, results were: 17 negative, 28 trace, 845 +1, 801 +2, 307 +3 and 295 +4.  Positive results (+1 to +4) were reported by 98% and negative or trace results by 2% of participants.  Qualitative results were more variable than quantitative results suggesting that ambiguities in interpreting a dipstick glucose should be followed up with a quantitative measurement.

JCTLM lists a certified reference material, a reference measurement procedure and a reference lab service for glucose in urine as well as other matrices.

Glucose (POCT)

Adequate

Glycated Albumin

Glycated Albumin / Fructosamine

Serum glycated albumin and fructosamine (serum glycated proteins) can be used as markers of short-term glycemia (2-3 weeks) and/or as alternate markers of glycemia when an accurate HbA1c cannot be obtained. Glycated albumin values are expressed by a ratio (%) of glycated to total albumin, and differences among individuals and albumin concentrations have negligible influences on the results. Fructosamine assays measure all serum glycated proteins and results can be affected by disorders in protein turnover. Fructosamine values can be corrected for albumin or total protein concentrations if needed. 

Review of CAP survey data (GSAB 2016) utilizes a true serum matrix for glycated albumin and fructosamine EQA. There are only 14-15 laboratories participating in this survey and only one method listed (enzymatic, presumably Asahi Kasei validated on instruments with open channels). The between laboratory CV is good, 2.7% CV (mean: 23.85) and 6.3% using the (mean: 12.10).  Many more laboratories are reporting fructosamine (n=110) proficiency testing (CAP survey FT), with a majority using the Roche fructosamine assay (n=104) compared to Diazyme (n=6). Among the Roche peer group the CVs were low for the “elevated” sample (CV: 3.4%, mean: 337.1 umol/L) and comparable for the “normal” specimen (CV: 3.3%, mean: 226.5 umol/L). In Japan, and possibly other countries, glycated albumin is used more frequently than is fructosamine.

Currently, neither glycated albumin or fructosamine are widely utilized nor are target values defined in clinical guidelines. Assays are not standardized but there are a limited number of manufacturers and for now standardization and harmonization do not present a major clinical issue.

References:

1. Juraschek SP, Steffes MW, Selvin E. Associations of alternative markers of glycemia with hemoglobin A(1c) and fasting glucose. Clin Chem. 2012;58:1648–1655.

2.0Selvin E, Francis LM, Ballantyne CM, Hoogeveen RC, Coresh J, Brancati FL, Steffes MW. Nontraditional markers of glycemia: associations with microvascular conditions. Diabetes Care. 2011 Apr;34(4):960-7.

3.0Kouzuma T, Usami T, Yamakoshi M, Takahashi M, Imamura S. An enzymatic method for the measurement of glycated albumin in biological samples. Clin Chim Acta. 2002 Oct;324(1-2):61-71.

4.0Goldstein DE, Little RR, Lorenz RA, Malone JI, Nathan D, Peterson CM, Sacks DB. Tests of glycemia in diabetes. Diabetes Care. 2004 Jul;27(7):1761-73.

Growth Hormone

The IFCC has a working group addressing harmonization of growth hormone.

Hematocrit

The first proposed reference method for hematocrit was adopted in 2001 by the ICSH (1). The method, primarily aimed at instrument manufacturers, utilized carefully defined capillary tubes and instrumentation to calculate the hematocrit as the ratio between the hemoglobin concentration of whole blood and the hemoglobin concentration of packed red blood cells with all plasma removed. By promulgating this standard, the ICSH not only endorsed a standard method but also ensured its traceability to the reference method for hemoglobinometry.

Due to its technical complexity, an additional “surrogate” reference method was developed that is more easily implemented in a standard clinical laboratory (2). This method, published in 2003, relies upon the standard spun hematocrit method with the addition of a correction factor that relates these measurements back to the reference hematocrit method. A variant of this method was endorsed in Clinical Laboratory Standards Institute document H07-A3 (3).

A recent comprehensive comparability study amongst several major hematology analyzer platforms (Abbott Cell-Dyn Sapphire, Beckman Coulter DxH 800, Siemens Advia 2120i, Sysmex XE-5000, and Sysmex XN-2000) demonstrated a high degree of agreement among hematocrit results for values between approximately 0.2 and 0.6 L/L. Average bias by bland-altman analysis ranged from 0 to 0.02 L/L with a maximum 95% limit of agreement of 0.03 L/L amongst all comparisons. Furthermore Passing-Bablok regression identified slopes between 1 and 1.04 and Kendall’s concordance coefficients were greater than 0.93 for all comparisons (4). These results are consistent with smaller studies which likewise demonstrate good comparability between analyzer pairs (5 and 6).

References

1. Van Assendelft, OW, Bull, BS, Fujimoto, K, Groner, W, Houwen, B, Van Hove, L, Klee, GG, Koepke, JA, Lewis, SM, D'Onofrio, G, Rowan, RM, Tatsumi, N & McLaren, CE 2001, 'Recommendations for reference method for the packed cell volume (ICSH Standard 2001)', Laboratory Hematology, vol. 7, no. 3, pp. 148-170.
2. Bull, BS, Fujimoto, K, Houwen, B, Klee, G, van Hove, L, van Assendelft, OW, Bunyaratvej, A, Buttarello, M, Davis, B, Koepke, JA, Lewis, SM, Machin, SJ, d'Onofrio, G, Rowan, RM & Tatsumi, N 2003, 'International Council for Standardization in Haematology (ICSH) recommendations for "Surrogate Reference" method for the packed cell volume', Laboratory Hematology, vol. 9, no. 1, pp. 1-9.
3. Bull, Brian S., and National Committee for Clinical Laboratory Standards. 2000. Procedure for Determining Packed Cell Volume by the Microhematocrit Method: Approved Standard.
4. Bruegel M, Nagel D, Funk M, Fuhrmann P, Zander J, Teupser D. Comparison of five automated hematology analyzers in a university hospital setting: Abbott Cell-Dyn Sapphire, Beckman Coulter DxH 800, Siemens Advia 2120i, Sysmex XE-5000, and Sysmex XN-2000. Clin Chem Lab Med. 2015 Jun;53(7):1057-71.
5. Woo KS, Jeong IH, An GD, Lim HH, Han JY. Performance evaluation of new Abbott Alinity hq hematology analyzer. Int J Lab Hematol. 2019 Oct;41(5):657-663.
6. Wang J, Zhao S, Su Z, Liu X. Analytical comparison between two hematological analyzer systems: Mindray BC-5180 vs Sysmex XN-1000. J Clin Lab Anal. 2019 Oct;33(8).

Hemoglobin

The peripheral blood hemoglobin level is amongst the most commonly requested laboratory tests in clinical practice. It is used in the screening, diagnosis, and management of patients with suspected or confirmed anemia.

The accepted reference procedure for hemoglobin determinations is based on a calculation from the spectrophotometric measurement of hemiglobincyanide at a wavelength of 540 nm (1,2). The analytical specifications are based on recommendations by the International Committee (now Council) for Standardization in Haemolotology (ICSH) which also put forth a preparation of hemiglobincyanide as the International Standard. These recommendations were subsequently accepted by the Expert Committee on Biological Standards of the World Health Organization (3). The Dutch National Institute for Public Health agreed to manufacture a preparation in accordance with these specifications and was accepted by WHO as the First International Reference Standard. The latest lot release has been described (4) while the JCTLM lists the previous lot, labeled CRM 522 (5).

A recent comprehensive comparability study amongst several major hematology analyzer platforms (Abbott Cell-Dyn Sapphire, Beckman Coulter DxH 800, Siemens Advia 2120i, Sysmex XE-5000, and Sysmex XN-2000) demonstrated a high degree of agreement among results. Passing-Bablok regression identified a slope of 1 and a Kendall’s concordance coefficients were greater than 0.95 for all comparisons. In an inter-instrument comparison of blood counts among routine clinical samples (n=349) maximum mean difference from the median was seen in the Sysmex XE-5000 with a hemoglobin difference of -0.07 mmol/L (range: -0.25-0.06) (6).

References

1. International Committee for Standardization in Haematology. Recommendations for reference method for haemoglobinometry in human blood (ICSH standard EP 6/2: 1977) and specifications for international haemiglobincyanide reference preparation (ICSH standard EP 6/3: 1977). J Clin Pathol. 1978 Feb;31(2):139-43.
2. Clinical and Laboratory Standards Institute. Reference and Standard Procedures for the Quantitative Determination of Hemoglobin in Blood. H15-A3. Villanova, PA, USA. December 1, 2000.
3. WHO Expert committee on biological standards: Twentieth report. WHO Technical Report Series, 384, 12-13.
4. Davis BH, Jungerius B, On Behalf of International Council for the Standardization of Haemotology (ICSH). International Council for Standardization in Haematology technical report 1-2009: new reference material for haemiglobincyanide for use in standardization of blood haemoglobin measurements. Int. Jnl. Lab. Hem. 2010, 32, 139–141.
5. Preparation and certification of a reference material of haemoglobin cyanide for standardization of blood hemoglobin measurement CRM 522; Report EUR 16101 EN European Commission bcr information reference material 1995.
6. Bruegel M, Nagel D, Funk M, Fuhrmann P, Zander J, Teupser D. Comparison of five automated hematology analyzers in a university hospital setting: Abbott Cell-Dyn Sapphire, Beckman Coulter DxH 800, Siemens Advia 2120i, Sysmex XE-5000, and Sysmex XN-2000. Clin Chem Lab Med. 2015 Jun;53(7):1057-71.

Hemoglobin A1c (HbA1c)

EQA data from a 2018 College of American Pathologists survey (GH5-B) with commutable samples had 21% difference between maximum and minimum values reported by individual laboratories and 1.7% CV among mean values for different measurement procedure groups for a sample with HbA1c 6.3% reporting units.  The mean bias vs. the reference measurement procedure was -0.2% to 0.2% reporting units for 23 measurement procedure groups. A reference material and a reference measurement procedure are listed by JCTLM.  These data support adequate standardization for HbA1c. The National Glycohemoglobin Standardization Program (USA) and the IFCC Hemoglobin Standardization Program both have global networks of reference laboratories and certification programs for IVD manufacturers to maintain standardized results. See the website links for additional information.

Hemoglobin A2 (HbA2)

The IFCC has a working group addressing harmonization of hemoglobin A2.

Hemoglobin, fetal (HbF)

Fetal hemoglobin (HbF) is a tetramer that consists of 2 alpha-globin chains and 2 gamma-globin chains. It is produced physiologically in utero and is the major hemoglobin carrier of O₂ during fetal development. Towards the end of gestation and continuing in early infancy, there is a switch of production from fetal hemoglobin to adult hemoglobin, which consists of 2 alpha-globin chains and 2 beta-globin chains¹. At birth, approximately 80% of hemoglobin is HbF.

HbF remains elevated in patients with certain thalassemias and in the related disease known as hereditary persistence of fetal hemoglobin (HPFH) ². Quantification of HbF is the standard method for diagnosis of HPFH. HbF is also measured to monitor treatment of patients with sickle cell disease (homozygous HbS) with hydroxyurea³. This drug increases production of HbF, which decreases the percentage of HbS, inhibits sickle polymerization of HbS, thereby reducing the incidence of complications arising from sickle cell anemia. An HbF target of ~20% has been proposed as clinically useful in sickle cell anemia patients³.

The most common principle for measurement of fetal hemoglobin is physical separation and quantification of the different hemoglobin fractions following red cell lysis. Results for each fraction are reported as a percentage of total hemoglobin. Absolute concentrations can be obtained by multiplying the fraction of a hemoglobin of interest by the total hemoglobin concentration (measured separately). Mass-spectrometry-based methods are also in use, but appear to be much less commonly used in clinical labs.

Separation of hemoglobin fractions may be performed by cation-exchange HPLC, capillary electrophoresis (CE), isoelectric focusing, and by acid and alkaline gel electrophoresis. All of these methods are suitable and used for identification of any hemoglobin variant, including HbF².

Proficiency Testing data from the College of American Pathologists Survey HG-B in 2017 showed that HPLC and CE have CVs ranging from 3-15% for HbA₂ in the concentration range of approximately 2% HbA₂. There were 9 commercially available IVD measurement procedures used by most of the participating labs (~400 labs). Similar performance would be expected for HbF. At higher concentrations of HbF, improved CVs would be expected.

The Kleihauer-Betke test is a morphologic assessment of the percentage of fetal red cells in the maternal circulation on a stained slide of maternal blood. Fetal hemoglobin is more resistant to acid elution than is adult hemoglobin. This differential elution forms the basis for this test. A flow cytometric approach to detecting fetal red cells in maternal circulation using an anti-HbF antibody is available⁴. Note that the Kleihauer-Betke test and its flow cytometric counterpart are used to determine the relative percent of fetal red cells, not HbF

There appears to be no systematic program for standardization for HbF. However, the degree of agreement among the current measurement principles (HPLC and CE) are probably adequate for the current clinical needs. 

References

1. Cui S, Engel JD. Reactivation of Fetal Hemoglobin for Treating beta-Thalassemia and Sickle Cell Disease. Adv Exp Med Biol 2017;1013:177-202.
2. Mosca A, Paleari R, Leone D, Ivaldi G. The relevance of hemoglobin F measurement in the diagnosis of thalassemias and related hemoglobinopathies. Clin Biochem 2009;42:1797-801.
3. Estepp JH, Smeltzer MP, Kang G, et al. A clinically meaningful fetal hemoglobin threshold for children with sickle cell anemia during hydroxyurea therapy. American journal of hematology 2017;92:1333-9.
4. Wong L, Hunsberger BC, Bruce Bagwell C, Davis BH. Automated quantitation of fetomaternal hemorrhage by flow cytometry for HbF-containing fetal red blood cells using probability state modeling. International journal of laboratory hematology 2013;35:548-54.

Hepatitis B Surface Antigen (HBsAg)

1. Medical Impact:

Hepatitis B surface antigen (HBsAg) is the first serologic marker appearing in the serum at 6 to 16 weeks following exposure to HBV. In acute infection, HBsAg usually disappears in 1 to 2 months after the onset of symptoms with the appearance of hepatitis B surface antibody (anti-HBs Ab). Anti-HBs Ab also appears as the immune response following hepatitis B vaccination. Persistence of HBsAg for more than 6 months in duration indicates development of either a chronic carrier state or chronic HBV infection.

There has been recent renewed interest in measuring serum levels of HBsAg as a surrogate marker to predict HBsAg loss and monitor anti-HBV therapy. During the natural history of HBV infection, the loss of serum HBsAg is generally associated with their seroconversion to anti-HBs, the hallmark of a successful immunological response to HBV infection. The speed and amplitude of the decline in HBsAg levels are suspected to be a good predictors of sustained virological response and HBsAg loss.

Therefore, the concordance among different assays for HBsAg and anti-HBs Ab is very important.

2. Harmonization Status:

According to the EQA data from the College of American Pathologists in 2015 (non-commutable samples), the agreement of the test results from 11 manufacturers’ test kits was very good. The overall concordance of HBsAg from 2,252 laboratories using kits manufactured by 11 companies was 99.7% for positive samples and 99.8% for negative samples. The overall concordance of anti-HBs Ab from 1,820 laboratories using kits manufactured by 11 companies was 99.3% for the positive samples and 99.7% for the negative samples.

A comparison report in 2012 [1], the quantitation of HBsAg levels in routine clinical samples by four different test systems appeared overall to be accurate, showing low variability and little discrepancy. HBsAg levels were compared among 80 patients using Abbott Architect, Diasorin, Bio-Rad and Roche.

Another head-to-head study evaluating the performance of 3 assays (Abbott Architect, DiaSorin LiaisonXL and Roche Elecsys HBsAgII).measuring the HBsAg level [2] also showed good correlation between all current systems [2].

In 2008, a systematic and comprehensive comparison study of nine anti-HBs Ab assay kits was reported [3]. Two hundred serum samples taken from patients and health care workers were analyzed on six different automated immunoassays (Abbott Axsym, Siemens Centaur, Ortho Vitros ECI, Roche Elecsys, DiaSorin Liaison and Abbott Architect) and three enzyme immunoassays (EIAs) (DiaSorin ETI-AB-AUK-3, Siemens Enzygnost and Bio-Rad Monolisa). Although the assay specificity ranged between 96.8-100% and sensitivity ranged between 93.5-100%, a high number of sera showed discrepancies between measurements by the different systems. The classical EIAs tend to find lower anti-HBs levels than the automated systems. The serial dilution of the WHO International Standard (IS) preparation was measured accurately by most of the assays. However, the quantitative measurement of anti-HBs levels was not reliable, even though an IS standard was used for the calibration of the systems.

Another comparison study also reported that the quantification of anti-HBs can show various results according to the kind of assay kit used [4]. When serum samples from five hundred visitors to the health care centers were assayed by Roche Cobas, Siemens Enzygost, Abbott AxSYM and Roche Elecsys, the concordance rates was 92.8% and 36 samples (7.2%) showed discrepant results. The authors concluded that the quantification of anti-HBs can show various results according to the kind of assay kit used.

Reference materials are available as the 3^rd WHO IS for HBsAg, human, NIBSC code 12/226 and 2^nd WHO IS for anti-HBs Ab, human, NIBSC code 07/164 [5]. According to the WHO Expert Committee’s 54^th report on biological standardization, the WHO IS for HBsAg is intended for the calibration of secondary HBsAg reference materials by manufacturers. The WHO IS for HBsAg and anti-HBs Ab.is widely used in standardization and calibration of HBsAg and anti-HBs Ab assay kits [6].

Taken these data together, measurement procedure of HBsAg is consistent with adequate harmonization for the screening tests for HBV infection using serum or plasma samples. However, it seems that measurement procedure of anti-HBs Ab needs harmonization.

<References>

1. Tuaillon E et al. Comparison of serum HBsAg quantitation by four Immunoassays, and relationships of HBsAg level with HBV replication and HBV genotypes. PLOS ONE 2012;7:e32143.
2. Thibault V et al. Performance of HBsAg quantification assays for detection of Hepatitis B virus genotypes and diagnostic escape-variants in clinical samples. J Clin Virol 2017;89:14-21.
3. Huzly D et al. Comparison of nine commercially available assays for quantification of antibody response to hepatitis B virus surface antigen. J Clin Microbiol 2008;46:1298-1306.
4. Whang DH and Shin BM. Comparison of Four-Assay Systems for the Quantification of Hepatitis B Surface Antibody. Korean J Lab Med 2002;22:424-30 (article in Korean).
5. Wilkinson DE et al. International collaborative study on the 3rd WHO International Standard for hepatitis B surface antigen. J Clin Virol. 2016;82:173-80.
6. Ferguson M, Yu MW and Heath A. Calibration of the second International Standard for hepatitis B immunoglobulin in an international collaborative study. Vox Sanquinis 2010;99:77-84.

Hepatitis C Ab qualitative

Hepatitis C (HCV) is a virus that causes an infection of the liver that is marked by liver inflammation and damage. As chronic infection with hepatitis C virus (HCV) is often asymptomatic, screening is necessary to identify most patients with infection. United States Centers for Disease Control and Prevention (CDC) recommends Hepatitis C screening at least once in a lifetime for all adults aged 18 years and older, or for all pregnant women during each pregnancy, except in settings where HCV prevalence is <0.1 %. Hepatitis C testing should be initiated with anti‑HCV test. A reactive or indeterminate/equivocal antibody test should be followed by HCV RNA testing

The British Working Standard for antibodies to Hepatitis C virus (anti-HCV) manufactured by NIBSC is available on the market (NIBSC code: 14/242-xxx). It is intended for use in the field of in vitro diagnostics, in conjunction with diagnostic immunoassay test kits/systems for the detection of anti-HCV, to monitor the performance of these systems.

Proficiency testing data from the College of American Pathologists Viral Marker Survey in 2020 indicated 99-100% reached consensus in about 2,185 participants from all method groups. The present harmonization status of Hepatitis C Ab assay is adequate based on CAP EQA results.

References:

1. Lab Tests Online – AACC https://labtestsonline.org/tests/hepatitis-c-testing
2. Centers for Disease Control and Prevention https://www.cdc.gov/hepatitis/hcv/guidelinesc.htm

Hepatitis C Virus RNA

Clinical Significance

Accurate quantitative detection of hepatitis C virus (HCV) RNA is critical for diagnosis of acute or chronic HCV infection, and for follow-up of virologic response during HCV targeted therapy. Measurement of HCV viral load for the different HCV genotypes is crucial to clinical management of HCV-infected patients, both treated and not, for disease staging, decisions regarding treatment initiation, and individualization of treatment strategy (i.e., dosage and duration based on response kinetics).

Test Methods at present

There are currently several commercially available HCV RNA assays; real-time PCR assays are generally preferred because of their wide dynamic ranges and good sensitivities. Two commercial real-time PCR platforms are mainly used in clinical laboratories, the Cobas AmpliPrep/Cobas TaqMan HCV assay (Roche Molecular Systems, Pleasanton, CA) and the Abbott RealTime HCV assay (Abbott Molecular, Des Plaines, IL).

CV data from 2016 CAP PT

1. The number of participant laboratories was 467.

2. The CV of Abbott real time PCR assay was 5%.

3. The CV of Roche AmpliPrep/Cobas TaqMan HCV assay was 4%.

4. The mean CV of Abbott and Roche was 4.5%

Conclusion

The present harmonization status of HCV RNA quantitation assay is adequate because the inter-laboratory CV of two main assay products is within 5%.

HIV-1 Genotype

Human immunodeficiency virus (HIV) genotyping is used to determine drug resistance in individuals infected with HIV.  It is tested prior to treatment initiation to determine whether the strain is resistant to antiretroviral therapy (ART) drugs. The result is used to identify and select the antiretroviral drugs that will likely be effective. [1,2]

More than 150 mutations are known to be associated with HIV drug resistance (HIVDR) and various interactions between mutations have been identified, which makes the interpretation of a genotypic resistance complicated. The minimal regions for which collection of sequence information is essential are protease (PR) coding region (codons 10 to 93), reverse transcriptase (RT) coding region (codons 41 to 238) and integrase (IN) coding region (codons 51 to 263). [2]

Currently, three commercial genotyping kits are available: ViroSeq HIV-1 (Abbott Molecular), GeneThink HIV-1 (Research Think Tank) and ATCC HIV-1 drug resistance test kit (Thermo Fisher). Some laboratories use a laboratory developed test. [3,4]

For decades, Sanger sequencing has been used as a gold standard for HIVDR genotyping. However, due to its intrinsic limitations, such as inconsistent detection of minority resistance variants (MRVs), NGS HIVDR assays are being adopted by more laboratories worldwide and may soon become the new standard for HIVDR genotyping. [5]

The ATCC produces a standard reference material: Quantitative Synthetic Human Immunodeficiency Virus 1 (HIV-1) RNA (ATCC® VR-3245SD™). This reference material is mainly constructed to quantify HIV-1 but also includes clinically significant mutation profiles to allow compatibility with the CDC genotyping assay patent, including 41L, 67N, 70R, 103N, 181C, 215Y, and 90M. [6]

External quality assurance testing has been provided through the Virology Quality Assurance (VQA) programme. The acceptance criteria require >99% nucleotide concordance scores over the entire sequence and at the drug-resistance mutation (DRM) sites. According to HIVResNet, 103 of 131 submissions (79%) had >99% nucleotide sequence identity and resistance mutation concordance, compared with consensus during 2007-2010. [7]

Data from 2020 CAP PT

1. The number of participant laboratories was 61.
2. The concordance of results from a major IVD manufacturer’s method was 100%. (n=38)
3. The concordance of LDT, Sanger was 93.8% for D67G and 100% for other mutations. (n=16)
4. The concordance of LDT, NGS was 100%. (n=3)

The present harmonization status of HIV genotyping assay is adequate based on VQA and CAP EQA results.

References:

1. Lab Tests Online – AACC https://labtestsonline.org/tests/hiv-antiretroviral-drug-resistance-testing-genotypic
2. WHO/HIVResNet HIV drug resistance laboratory operational framework. Geneva: World Health Organization; 2017. Licence: CC BY-NC-SA 3.0 IGO.
3. 2018 Handbook on HIV Drug Resistance Testing Availability, Accessibility and Capacity for Caribbean States
4. Ji, H.; Parkin, N.; Gao, F.; Denny, T.; Jennings, C.; Bremer, J.; Kantor. External Quality Assessment Program for Next-Generation Sequencing-Based HIV Drug Resistance Testing: Logistical Considerations. Viruses. 2020 May; 12(5): 556.
5. Capina, K. Li, L. Kearney, A. Vandamme, P. Harrigan, K. Laethem. Quality Control of Next-Generation Sequencing-Based HIV-1 Drug Resistance Data in Clinical Laboratory Information Systems Framework. Viruses. 2020 Jun 14;12(6):645.
6. ATCC– https://www.atcc.org/products/all/VR-3245SD.aspx#characteristics
7. Parkin, J. Bremer, S. Bertagnolio. Genotyping External Quality Assurance in the World Health Organization HIV Drug Resistance Laboratory Network During 2007–2010. Clin Infect Dis. 2012 May;54 Suppl 4(Suppl 4):S266-72.

HIV-RNA

Serum/plasma HIV is the most commonly followed biomarker of HIV infection, with defined treatment targets established by international consensus conferences. Extensive work has already been accomplished on standardization of assays for HIV. The WHO prequalifies HIV in vitro diagnostics as part of its high burden disease program. The WHO produces a standard reference material (WHO 3^rd HIV NAT). The National Institute for Biological Standards and Controls (UK) produces a reference material traceable to the WHO.

Sollis KA et al.: Systematic review of the performance of HIV viral load technologies on plasma samples. PLoS One. 2014 Feb 18;9(2):e85869. doi: 10.1371/journal.pone.0085869. eCollection 2014.

Human Chorionic Gonadotropin (hCG)

Human Chorionic Gonadotropin (hCG) is a heterogeneous mix of intact hCG (consisting of α and β subunits) as well as chemically modified and partially degraded variants.  Intact hCG, hyperglycosylated hCG (hCG-H), nicked hCG (hCGn), free β-subunit (hCGβ), nicked free beta (hCGbn), and sulfated hCG (sulfated hCG) can be detected in both serum and urine (and in CSF in certain pathological conditions); while the core fragment of hCGβ (hCGβcf) is found exclusively in urine (1-4). The proportion of each of these hCG variants depends on pregnancy gestational age, menopausal status and pathological condition.

Currently, hCG assays are not harmonized and hence yield different concentrations depending on the assay used. This poses a clinical problem for several reasons. First, serial hCG measurements are frequently utilized to diagnose ectopic pregnancy, and to follow treatment for ectopic pregnancy, molar pregnancy and various malignancies. Therefore, clinicians must be certain to follow patients using a single manufacturer's assay. Second, because different forms of hCG are elevated in different in pathological conditions, it is important that physicians are monitoring the correct hCG form. For instance, 20-40% of germ cell tumors produce exclusively hCGb (5). Therefore, it is recommended that laboratories utilize an hCG assay that recognizes both intact hCG and free beta hCG (total beta hCG) so that it can be used as both a marker of pregnancy and cancer.. Finally, the lack of standardization has an impact on manufacturers and their ability to characterize the antibodies they utilize. Several reports have demonstrated that qualitative devices do not detect the hCG isoforms that they claim to recognize (6-7). Taken together, there is a need for standardization of hCG assays (8-10).

References

1. Cole LA, Kardana A, Park SY, Braunstein GD. The deactivation of hCG by nicking and dissociation. J Clin Endocrinol Metab 1993;76:704-10.
2. Alfthan H, Haglund C, Dabek J, Stenman UH. Concentrations of human choriogonadotropin, its beta-subunit, and the core fragment of the beta-subunit in serum and urine of men and nonpregnant women. Clin Chem 1992;38:1981-7.
3. Kovalevskaya G, Birken S, Kakuma T, O'Connor JF. Early pregnancy human chorionic gonadotropin (hCG) isoforms measured by an immunometric assay for choriocarcinoma-like hCG. J Endocrinol 1999;161:99-106.
4. Sutton-Riley JM, Khanlian SA, Byrn FW, Cole LA. A single serum test for measuring early pregnancy outcome with high predictive value. Clin Biochem 2006; 39:682-687.
5. Stenman UH, Alfthan H, Hotakainen K. Human chorionic gonadotropin in cancer. Clin Biochem 2004;37:549-61.
6. Sigel CS, Grenache DG. Detection of unexpected isoforms of human chorionic gonadotropin by qualitative tests. 2007;53:989-90.
7. Cervinski MA, Lockwood CM, Ferguson AM, Odem RR, Stenman U-H, Alfthan H, Grenache DG, Gronowski AM. Qualitative point-of–care and over-the-counter urine hCG devices differentially detect the hCG variants of early pregnancy.   Clin Chim Acta 2009;406:81-5
8. Sturgeon CM, Ellis AR. Standardization of FSH, LH and hCG-Current position and future prospects. Mol Cell Endocrinol 2007;260-262:301-9.
9. Sturgeon CM, Berger P, Bidart J-M, Birken S, Burns C, Norman R, Stenman U-H, on behalf of the IFCC Working group on hCG. Clin Chem 2009;55:1484-91.
10. Gronowski AM, Grenache DG. Characterization of the hCG variants recognized by different hCG immunoassays: An important step towards standardization of hCG measurements. Clin Chem 2009;55:1447-49.

IgE, total

IgE (total) is used for diagnosis of atopic allergic diseases and hyper-IgE immunodeficiency syndromes.

A 3^rd WHO IgE international standard (11/234) is available and is used by commercial manufacturers for calibration of total IgE assays ¹.

Proficiency testing data from the from the College of American Pathologists K Survey (non-commutable samples) from 2016 and 2017 showed that there were approximately 450 labs participating and 8 peer groups for IVD measurement procedures. Most measurement procedures reported results within approximately 10-15% of each other, depending on the target concentration.

Reference

1. Thorpe SJ, Heath A, Fox B, Patel D, Egner W. The 3rd International Standard for serum IgE: international collaborative study to evaluate a candidate preparation. Clin Chem Lab Med 2014;52:1283-9.

IgG, Total

Total immunoglobulin G (IgG) measurements are used in a variety of clinical settings.  Elevated IgG (hypergammaglobulinemia) can occur in patients with infections, malignancies, autoimmune diseases, or other chronic inflammatory conditions.  In contrast, decreased concentrations of IgG (hypogammaglobulinemia) can be an indication of a primary or secondary immunodeficiency.  In the clinical laboratory, testing for total IgG is most often performed by immunoturbidimetry or immunonephelometry.

In 1993, the reference material CRM 470 was released.  CRM 470, which later became designated as ERM-DA470, was prepared from serum of healthy donors and was certified for 15 proteins, including total IgG (1).  The concentration of IgG in ERM-DA470 was determined by calibration against USNRP 12-0575C, a reference material prepared by the Centers for Disease Control.  Most clinical assays for total IgG currently on the market include calibrators and controls traceable to ERM-DA470.  Publications have demonstrated that, since ERM-DA470 became available, variation in total IgG measurements between laboratories has significantly decreased (2).  This trend is also observed on CAP proficiency testing results.  In 2018, out of 15 proficiency testing samples, 14 demonstrated all-method CVs between 4.5% and 6.1%.  One sample showed an all-method CV of 10.8%; however, quantitative results for this sample were only reported by 18.1% of participant labs, with all other reporting results as “<” values.

As the supply of ERM-DA470 has declined, ERM-DA470k/IFCC has been developed.  ERM-DA470K/IFCC was also prepared from serum of healthy donors, and has been certified for 12 proteins, with certifications for additional proteins still in progress (3).  For total IgG, the assigned concentration is based on measurements in 14 laboratories on 7 different platforms.  In ERM-DA470, the certified concentration of total IgG was 9.68 g/L, compared to 9.17 g/L in ERM-DA470k/IFCC; the mean transfer factor between the 2 CRMS was 0.948 for total IgG.  There was excellent agreement in the assigned values across laboratories and platforms, with a variation for total IgG of 1.37%. 

References:

1. Baudner S, Bienvenu J, Blirup-Jensen S, et al. The certification of a matrix reference material for immunochemical measurement of 14 human serum proteins: CRM470. Eur 15243 EN. Luxembourg: Commission of the European Communities; 1993.
2. Johnson AM, Whicher JT. Effect of certified reference material 470 (CRM 470) on national quality assurance programs for serum proteins in Europe. Clin Chem Lab Med 39 (2001) 1123-1128.
3. Zegers I, Keller T, Schreiber W, et al. Characterization of the new serum protein reference material ERM-DA470k/IFCC: Value assignment by immunoassay. Clin Chem 56 (2010) 1880-1888.

IgM, total

Total immunoglobulin M (IgM) measurements are used in a variety of clinical settings.  Elevated IgM can occur in patients with infections, malignancies, autoimmune diseases, or other chronic inflammatory conditions.  In contrast, decreased concentrations of IgM can be an indication of a primary or secondary immunodeficiency.  In the clinical laboratory, testing for total IgM is most often performed by immunoturbidimetry or immunonephelometry.

In 1993, the reference material CRM 470 was released.  CRM 470, which later became designated as ERM-DA470, was prepared from serum of healthy donors and was certified for 15 proteins, including total IgM (1).  The concentration of IgM in ERM-DA470 was determined by calibration against USNRP 12-0575C, a reference material prepared by the Centers for Disease Control.  Most clinical assays for total IgM currently on the market include calibrators and controls traceable to ERM-DA470.  Publications have demonstrated that, since ERM-DA470 became available, variation in total IgM measurements between laboratories has significantly decreased (2).  This trend is also observed on CAP proficiency testing results.  In 2018, out of 15 proficiency testing samples, 14 demonstrated all-method CVs between 5.4% and 10.2%.  For one sample, an all-method CV was not calculated, with 90.8% of participating laboratories reporting results as “<” values.

As the supply of ERM-DA470 has declined, ERM-DA470k/IFCC has been developed.  ERM-DA470K/IFCC was also prepared from serum of healthy donors, and has been certified for 12 proteins, with certifications for additional proteins still in progress (3).  For total IgM, the assigned concentration is based on measurements in 11 laboratories on 6 different platforms.  In ERM-DA470, the certified concentration of total IgM was 0.797 g/L, compared to 0.723 g/L in ERM-DA470k/IFCC; the mean transfer factor between the 2 CRMS was 0.908 for total IgM.  There was excellent agreement in the assigned values across laboratories and platforms, with a variation for total IgM of 2.11%. 

References:

1. Baudner S, Bienvenu J, Blirup-Jensen S, et al. The certification of a matrix reference material for immunochemical measurement of 14 human serum proteins: CRM470. Eur 15243 EN. Luxembourg: Commission of the European Communities; 1993.
2. Johnson AM, Whicher JT. Effect of certified reference material 470 (CRM 470) on national quality assurance programs for serum proteins in Europe. Clin Chem Lab Med 39 (2001) 1123-1128.
3. Zegers I, Keller T, Schreiber W, et al. Characterization of the new serum protein reference material ERM-DA470k/IFCC: Value assignment by immunoassay. Clin Chem 56 (2010) 1880-1888.

IgA, total

Total immunoglobulin A (IgA) measurements are used in a variety of clinical settings.  Elevated IgA can occur in patients with infections, malignancies, autoimmune diseases, or other chronic inflammatory conditions.  In contrast, decreased concentrations of IgA can be an indication of a primary or secondary immunodeficiency.  In the clinical laboratory, testing for total IgA is most often performed by immunoturbidimetry or immunonephelometry.

In 1993, the reference material CRM 470 was released.  CRM 470, which later became designated as ERM-DA470, was prepared from serum of healthy donors and was certified for 15 proteins, including total IgA (1).  The concentration of IgA in ERM-DA470 was determined by calibration against USNRP 12-0575C, a reference material prepared by the Centers for Disease Control.  Most clinical assays for total IgA currently on the market include calibrators and controls traceable to ERM-DA470.  Publications have demonstrated that, since ERM-DA470 became available, variation in total IgA measurements between laboratories has significantly decreased (2).  This trend is also observed on CAP proficiency testing results.  In 2018, out of 15 proficiency testing samples, 11 demonstrated all-method CVs between 5.1% and 10.2%.  Three samples showed all-method CVs of 56.1%, 58.1%, and 52.4%, with an additional sample not having an all-method CV calculated.  In each of these situations, a significant proportion of testing laboratories reported results as “<”, indicating a concentration at or below the lower reportable limit for most assays.

As the supply of ERM-DA470 has declined, ERM-DA470k/IFCC has been developed.  ERM-DA470K/IFCC was also prepared from serum of healthy donors, and has been certified for 12 proteins, with certifications for additional proteins still in progress (3).  For total IgA, the assigned concentration is based on measurements in 14 laboratories on 7 different platforms.  In ERM-DA470, the certified concentration of total IgA was 1.96 g/L, compared to 1.80 g/L in ERM-DA470k/IFCC; the mean transfer factor between the 2 CRMS was 0.918 for total IgA.  There was excellent agreement in the assigned values across laboratories and platforms, with a variation for total IgA of 2.36%. 

References:

1. Baudner S, Bienvenu J, Blirup-Jensen S, et al. The certification of a matrix reference material for immunochemical measurement of 14 human serum proteins: CRM470. Eur 15243 EN. Luxembourg: Commission of the European Communities; 1993.
2. Johnson AM, Whicher JT. Effect of certified reference material 470 (CRM 470) on national quality assurance programs for serum proteins in Europe. Clin Chem Lab Med 39 (2001) 1123-1128.
3. Zegers I, Keller T, Schreiber W, et al. Characterization of the new serum protein reference material ERM-DA470k/IFCC: Value assignment by immunoassay. Clin Chem 56 (2010) 1880-1888.

Insulin

Insulin and C-peptide are cleaved from proinsulin and secreted in a 1:1 molar ratio, and are both important biomarkers in the pathophysiology of diabetes mellitus. Measurement of circulating insulin concentrations may improve the classification and management of diabetes and aid in defining and treating individuals with varying levels of insulin resistance. Assessment of C-peptide has clinical utility in monitoring endogenous insulin production in diabetics receiving exogenous insulin therapy, and the Diabetes Control and Complications Trial (DCCT) established the importance of C-peptide as a key biomarker of beta cell function and depletion, primarily in Type 1 diabetics. In addition, C-peptide measurements are required by CMS to approve patients for insulin pump therapy.

Insulin and C-peptide are currently being utilized in clinical trials and epidemiological studies to evaluate therapeutic and potential prevention strategies in diabetes. Standardization of insulin and C-peptide assays will allow data and information from numerous epidemiological studies and clinical trials to be aggregated and ultimately incorporated into clinical practice guidelines. In this context, assay harmonization and/or standardization of insulin and C-peptide remain critical.

There are active efforts working simultaneously to achieve standardization of both analytes. A 2016 CAP survey with presumably commutable serum pools highlighted the continuing disparity between immunoassay platforms. The insulin low and high serum pool mean concentrations across peer groups (n = 467) was 49.9 pmol/L (8.31 uIU/mL; range: 36.4 – 64.6 pmol/L) and 340.5 pmol/L (56.8 uIU/mL; range: 279.8 – 410.1 pmol/L), respectively. The C-peptide low and high serum pool mean concentrations across all peer groups (n = 285) was 0.49 pmol/L (range: 0.41 – 0.61 pmol/L) and 1.99 pmol/L (range: 1.5 – 2.37 pmol/L), respectively. Poor immunoassay specificity is likely responsible for observed analytical differences.

References:

1. Miller WG, Thienpont LM, Van Uytfanghe K, Clark PM, Lindstedt P, Nilsson G, Steffes MW; Insulin Standardization Work Group. Toward standardization of insulin immunoassays. Clin Chem. 2009 May;55(5):1011-8.
2. Marcovina S, Bowsher RR, Miller WG, Staten M, Myers G, Caudill SP, Campbell SE, Steffes MW; Insulin Standardization Workgroup. Standardization of insulin immunoassays: report of the American Diabetes Association Workgroup. Clin Chem. 2007 Apr;53(4):711-6.
3. Jones AG, Hattersley AT. The clinical utility of C-peptide measurement in the care of patients with diabetes. Diabet Med. 2013 Jul; 30(7): 803–817.
4. Little RR, Wielgosz RI, Josephs R, Kinumi T, Takatsu A, Li H, Stein D, Burns C. Implementing a Reference Measurement System for C-peptide: Successes and Lessons Learned. Clin Chem Jun 2017, clinchem.2016.269274; DOI: 10.1373/clinchem.2016.269274.

Iron

Iron is an essential nutrient necessary for syntheses of hemoglobin that is responsible for transport of oxygen from lungs to tissues.  Iron deficiency causes anemia. Iron overload can be caused by excess ingestion or blood transfusions, and can be toxic and require procedures such as chelation to remove iron from the blood. Companion tests include ferritin that indicates iron stores in the body, transferrin that transports iron in the blood other than the iron in hemoglobin, total iron binding capacity and unsaturated iron binding capacity (see these measurands for further information).

Laboratory methods are based on chelation of iron by a number of different molecules each of which produces a chromophore that is measured spectrophotometrically.  The College of American Pathologists 2020 A Survey included results from 3464 laboratories using iron methods from 11 instrument systems that used reagent formulations with 5 different chelating molecules.  The table shows mean results for all method principles and all instruments combined.  Although the samples were of unknown commutability with clinical samples, the small CVs suggest good agreement among results from different measurement procedures using different method principles.

Iron Binding Capacity (Total): TIBC

Total iron binding capacity (TIBC) is currently used to assess iron status. Due to binding of iron to other plasma proteins (mainly albumin), TIBC methods generally overestimate the iron binding capacity of transferrin. Moreover, no generic reference values are available for TIBC (1).

The method for TIBC measurement was first described by Ramsay (2) and later refined by the International Committee for Standardisation in Haematology and described as a recommended procedure (3), revised in 1990 (4). The chemical measurement of TIBC consists of three steps : the first step involves addition of supraphysiological amounts of FeCl3 to saturate the free binding sites on transferrine (TRF) ; the second is the removal of unbound excess iron by adsorption onto solid magnesium carbonate, charcoal or an ion-exchange resin; the third is the determination of iron that is dissociated from TRF at acidic pH. Recently, direct TIBC assays have been described, most of them measure unsaturated iron binding capacity (UIBC) and calculate TIBC, rather than measure it directly (5).

One of the major drawbacks of the TIBC test is the large variation: reference ranges differ by as much as 35% between commercial methods (6), therefore a generic reference interval can not be stated. Furthermore, the chemical methods for TIBC require a relatively large sample volume and are sensitive to contamination of the laboratory ware with iron (7). This method is thus virtually obsolete.

Formulas for calculating TIBC from transferrin determination have been proposed - TIBC (µmol/L)= 25 x TRF (g/L) - , with the advantage of internationally available and accepted interim reference ranges for serum transferrin, and the introduction of the international ERM-DA470k/IFCC protein standard material (8). That is why, (excepted in certain cases with genetically controlled polymorphisms of transferrin or situations low levels transferrin), most of the laboratories use transferrin to access TIBC estimation rather than direct TIBC determination, with a good efficiency in clinical practice.

Some EQA schemes are available, but with a low number of participating laboratories.

References:

1. I Kasvosve and Joris Delanghe. Total iron binding capacity and transferrin concentration in the assessment of iron status. Clin Chem Lab Med 2002;40:1014-8.
2. Ramsay WNM. The determination of the total iron-binding capacity of serum. Clin Chim Acta 1957; 259:25–30.
3. International Committee for Standardisation in Haematology (Iron Panel). The measurement of total and unsaturated iron binding capacity in serum. Br J Haematol 1978;38:281–90.

4. Iron Panel of the International Committee for Standardisation in Haematology. Revised recommendations for the measurements of the serum iron in human blood. Br J Haematol 1990; 75:615–6
5. Bothwell TH, Jacobs RW, Kamener R. The determination of the unsaturated iron-binding capacity of serum using radioactive iron. S Afr J Med Sci 1959; 24:93–8.
6. Biochemical aspects of haematology. Fairbanks VF, Klee GG. In: Burtis CA, Ashwood ER, editors. Tietz textbook of clinical chemistry, 3rd ed. Philadelphia: W.B. Saunders Company, 1999:1703.
7. Tsung SH, Rosenthal WA, Milewski KA. Immunological measurement of transferrin compared with chemical measurement of total iron-binding capacity. Clin Chem 1975;21:1063–6.
8. Ingrid Zegers, Thomas Keller, Wiebke Schreiber et al. Characterization of the New Serum Protein Reference Material ERM-DA470k/IFCC:Value Assignment by Immunoassay. Clin Chem 2010;56:1880–8.

Iron binding capacity (unsaturated )

EQA data from the College of American Pathologists in 2015 (non-commutable samples) had 17.6% difference between maximum and minimum values reported by individual laboratories and 5.6% CV% among mean values for different peer groups.  These data are consistent with adequate harmonization for measurement procedures for serum or plasma unsaturated iron binding capacity.  No reference materials or reference measurement procedures are listed by JCTLM.

Iron saturation

Transferrin iron saturation can be calculated by using serum iron concentration and one of the following: total iron binding capacity (TIBC), unsaturated iron binding capacity (UIBC), or transferrin (TRF) concentration.

This ratio is recommended in situations involving inflammation, iron-overload situations, cases of chronic renal impairment, or when the serum ferritin result is not conclusive (normal or high concentration even though iron deficiency is strongly suspected).

TRF saturation (%) = 100 x (serum iron / TIBC)

• Iron determination : see separate page
• TIBC determination: due to binding of iron to other plasma proteins (mainly albumin), TIBC methods generally overestimate the iron binding capacity of transferrin. Moreover, no generic reference values are available for TIBC (1). One of the major drawbacks of the TIBC test is the large variation: reference ranges differed by as much as 35% between commercial methods (2). This method is now virtually obsolete (see separate page).
• Formulas for calculating TIBC from transferrin determination have been proposed with a good efficiency in clinical practice, with the advantage of internationally available and accepted interim reference ranges for serum transferrin, and the introduction of the international ERM-DA470k/IFCC protein standard material (3).

Transferrin saturations of less than 20% indicate iron deficiency, while transferrin saturations of more than 50% suggest iron overload.

The quality of the results is dependent on the quality of the two assays needed for its calculation: iron and transferrin (see separate pages).

Some EQA data show acceptable performance (e.g.: CVs around 4.5% in the French ProBioQual EQA scheme). This is consistent with adequate harmonization for measurement procedures.

References:

1. I Kasvosve and Joris Delanghe. Total iron binding capacity and transferrin concentration in the assessment of iron status. Clin Chem Lab Med 2002;40:1014-8.
2. Biochemical aspects of haematology. Fairbanks VF, Klee GG. In: Burtis CA, Ashwood ER, editors. Tietz textbook of clinical chemistry, 3rd ed. Philadelphia: W.B. Saunders Company, 1999:1703
3. Characterization of the New Serum Protein Reference Material ERM-DA470k/IFCC:Value Assignment by Immunoassay. Ingrid Zegers, Thomas Keller, Wiebke Schreiber et al. Clin Chem 2010;56:1880–8.

Lactate

EQA data from the College of American Pathologists in 2015 (non-commutable samples) had 19.8% difference between maximum and minimum values reported by individual laboratories and 5.6% CV% among mean values for different peer groups.  These data are consistent with adequate harmonization for serum or plasma lactate measurement procedures.  No reference materials or reference measurement procedures  are listed by JCTLM.

Lactate Dehydrogenase (LDH)

An IFCC reference measurement procedure (RMP) is available.  IVD manufacturers should formulate reagents to conform to the RMP.

Schumann G, Bonora R, Ceriotti F, Clerc-Renaud P, Ferrero CA, Ferard G, et al. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C. Part 3. Reference procedure for the measurement of catalytic concentration of lactate dehydrogenase. Clin Chem Lab Med. 2002;40:643-8.

Lipase

An IFCC working group is addressing harmonization of lipase. Commercial measurement procedures use different substrates that contribute to biases among procedures.

Lithium

Lithium is widely used for monitoring therapy of patients with bipolar disorders, including episodes of mania and depression, and evaluating lithium toxicity.  The most common methods are based on colorimetric dye binding with smaller numbers of labs using atomic absorption or ion exchange methods. The CV between different methods based on CAP survey data for 2016 was less than 5 % with exclusion of Vitros. Vitros had peer group CV of approximately 10% and approximately 25% lower values than other methods that could be due to non-commutability of the PT materials used.

Lp (a)

Although it was recognized nearly 20 years ago that lipoprotein(a) (Lp(a)) concentrations were elevated in patients with cardiovascular disease (CVD), the importance of Lp(a) was not strongly established due to a lack of both Lp(a)-lowering therapy and evidence that reducing Lp(a) levels improves CVD risk. More recent clinical and genetic research studies have shown that Lp(a) concentrations are causal for different CVDs, including coronary artery disease (1,2), calcified aortic valve disease (3), stroke (4), and heart failure (4.5), despite optimal LDL-C management. These insights have triggered renewed interest in Lp(a) testing and in the recent development of Lp(a) lowering medication (6,7).

Lp(a) consists of apolipoprotein (apo) B100 covalently bound to apo(a). Apo(a) contains a variable number of kringle IV repeats, a kringle V and a protease domain. Most clinical laboratories measure Lp(a) mass using automated immunoassays with polyclonal antibodies that are directed against apo (a). Despite the renewed clinical interest in Lp(a), its measurement is not well-standardized. Survey results from 2018 from SKML, the Dutch External Quality Assessment organization, using fresh frozen human samples for serum lipids, including Lp(a), for 17 laboratories found overall interlab variation ranging from 16.4% to 32.1% at Lp(a) levels of ~ 150 to 450 mg/L, whereas the interlab variations were typically smaller within manufacturers (8). These data demonstrate that in identical EQA-specimens Lp(a) mass results scatter from 150 to 300 mg/L and from 250 to 450 mg/L across labs. In another study six commercial Lp(a) immunoassays were compared and found substantial differences in mass results between assays (9).

In the 1990s standardization efforts for serum/plasma Lp(a) were initiated by the IFCC-WHO demonstrating traceability to WHO-IFCC secondary reference material “SRM2B” (10). An important limitation of the WHO-IFCC material is its unknown commutability. More recently two groups have initiated research efforts to develop a Reference Measurement System for Lp(a) based on isotope dilution mass spectrometry that should allow standardization of existing and emerging Lp(a) assays (11,12).

References:

1. Kronenberg F, Utermann G. Lipoprotein(a): resurrected by genetics. J Intern Med 2013;273:6–30.
2. Lipoprotein(a) and Cardiovascular Diseases – Revisited. Jang AY, Han SH, Sohn IS, Oh PC, Koh KK. Circ J 2020;84:867-874.
3. Cho KI, Sakuma I, Sohn IS, Jo SH, Koh KK. Inflammatory and metabolic mechanisms underlying the calcific aortic valve disease. Atherosclerosis 2018; 277: 60–65.
4. Langsted A, Nordestgaard BG, Karnstrup PR. Elevated lipoprotein(a) and risk of ischemic stroke. J Am Coll Cardiol 2019;74:54-66.
5. Ballantyne CM. Lipoprotein(a) and risk for stroke and myocardial infarction. Why aren’t we screening? J Am Coll Cardiol 2019;74:67-69.
6. Viney NJ, Yeang C, Yang X, Xia S, Witztum JL, Tsimikas S. Relationship between “LDL-C”, estimated true LDL-C, apolipoprotein B-100, and PCSK9 levels following lipoprotein(a) lowering with an antisense oligonucleotide, J Clin Lipidol 2018;3:702–710.
7. O'Donoghue ML, Fazio S, Giugliano RP, Stroes ESG, Kanevsky E, Gouni-Berthold I, et al. Lipoprotein(a), PCSK9 inhibition, and cardiovascular risk, Circulation 2019;139:1483–1492.
8. Ruhaak LR, Cobbaert CM. Quantifying apolipoprotein(a) in the era of proteoforms and precision medicine. Clin Chim Acta 2020;511:260-268.
9. Scharnagl H, Stojakovic T, Dieplinger B, Dieplinger H, Erhart G, Kostner GM, Herrmann M, Marz W, Grammar TB. Comparison of lipoprotein (a) serum concentrations measured by six commercially available immunoassays, Atherosclerosis 2019;289:206–213.
10. Tate JR, Berg K, Couderc R, Dati F, Kostner GM, Marcovina SM, et al. International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) standardization project for the measurement of lipoprotein(a). Phase 2: selection and properties of a proposed secondary reference material for lipoprotein(a). Clin Chem Lab Med 1999;37:949–58.
11. Cobbaert CM, Althaus H, Brkovic IB, Ceglarek U, Coassin S,Delatour V, et al. for the IFCC Working Group for Standardization of Apolipoproteins by Mass Spectrometry. Towards an SI-Traceable Reference Measurement System for Seven Serum Apolipoproteins Using Bottom-Up Quantitative Proteomics: Conceptual Approach Enabled by Cross-Disciplinary/Cross-Sector Collaboration. Clin Chem 2021;67:478-489.
12. Marcovina SM, Clouet-Foraison N, Koschinsky ML, Lowenthal MS, Orquillas A, Boffa MB, et al. Development of an LC-MS/MS Proposed Candidate Reference Method for the Standardization of Analytical Methods to Measure Lipoprotein(a). Clin Chem 2021;67:490-499.

03 June 2021

Magnesium

EQA data from two Japanese programs in 2015 that used commutable samples had 4.8% and 4.1% CV among different laboratories and methods.  EQA data from the College of American Pathologists in 2015 (non-commutable samples) had 12.6% difference between maximum and minimum values reported by individual laboratories and 3.4% CV% among mean values for different peer groups.  Taken together these data support adequate harmonization for serum or plasma magnesium measurement procedures.  A reference material, serum-based reference material and a reference measurement procedure are listed by JCTLM.

Methemoglobin

Methemoglobin is characterized by the presence of heme iron in the ferric state (Fe³⁺) instead of the usual ferrous (Fe²⁺) state. Under physiological conditions, methemoglobin is unable to transport O₂. Methemoglobin is measured by co-oximetry and results are usually expressed as a percentage of total hemoglobin.

Low levels of methemoglobin are formed in health by auto-oxidation of Fe²⁺ heme iron to the Fe³⁺ state. Reduction to the Fe²⁺ state is principally accomplished by an NADH-dependent cytochrome b5, methemoglobin reductase. A second reductase, NADPH-dependent methemoglobin reductase, is of minor importance in healthy subjects, but is important in patients with deficiency of the first enzyme.

Pathological elevations in methemoglobin can result rarely from congenital deficiency in NADH-dependent cytochrome b5 methemoglobin reductase or from hemoglobin variants known collectively as hemoglobin M. More commonly, methemoglobin is increased by exposure to oxidizing agents. Among these are some commonly used drugs, including nitroglycerine, nitroprusside, nitric oxide, lidocaine, prilocaine, benzocaine, phenytoin, sulfonamides. Ingestion of nitrites (e.g., in well water) can increase methemoglobin.

The reference interval for methemoglobin is <1-2% (of total hemoglobin). Elevated levels usually cause cyanosis. Clinical symptoms (dyspnea, fatigue, exercise intolerance, and syncope are associated with levels of 20-50%. Higher levels are associated with seizures, coma, metabolic acidosis and death.

Methemoglobinemia should be suspected in patients who appear hypoxic but have adequate PaO₂. Blood is characteristically muddy brown in significant methemoglobinemia.

Analysis
Methemoglobin, like oxyhemoglobin, deoxyhemoglobin, and carboxyhemoglobin, has a characteristic absorbance spectrum in the visible spectrum. Multiwavelength spectrophotometry is the analytical principle used in co-oximeters, which are widely available, and are often a component of blood gas instruments. These instruments provide rapid estimation of these hemoglobins. It should be noted that non-invasive, point-of-care pulse oximeters may not be suitable for detection of methemoglobin or carboxyhemoglobin, depending on the device design. Samples should be stored at 4 C if rapid measurement is not possible because methemoglobin is unstable at room temperature.

Harmonization
The CAP Blood Gas (“SO”) Proficiency Testing Survey results from 2020 were reviewed for participant methemoglobin performance. A major limitation of the Survey is that methemoglobin was not challenged at levels above about ~1-2%, which reflect levels seen only in health. At the physiological levels of the Survey material, the participant performance appeared to be adequate. The Survey offers no insight into assay performance at clinically significant levels (>20%). There are 6 manufacturers that produce most of the participant equipment (Avox, IL, Nova, Radiometer, Roche, and Siemens). These produce a combined 13 different instruments listed in the Survey.

More useful data are available from the Weqas program in the U.K.  A review of recent methemoglobin surveys from that program shows that selected EQA samples had values in the clinically important range of up to 29% MetHb. The types of equipment used were similar to that seen in the CAP Survey. Systematic inter-method biases were noted the clinical significance of which are uncertain, and may be due to matrix effects.  Of note is that one method consistently gave higher results that the others by 30-100% depending on the sample. However, the method had few participants (under 10), so the significance of this observation is not clear.

In summary, only one EQA/PT program was identified to this point with samples tested in the clinically important range. That one program showed consistent systematic biases between methods but these differences may be related to matrix effects in the survey materials.

References:

1. Porter WH: Methemoglobin-forming agents. In Tietz’s Clinical Chemsitry, 4th ed. (Eds. Burtis CA, Ashwood ER, Bruns DE), Elesevier Saunders, St. Louis, 2006.
2. College of American Pathologists. SO Surveys 2019-2021, Personal communication.
3. WEQAS methemoglobin EQA data, 2020-2021. Personal communication.

05 July 2021

Methotrexate

Methotrexate is a drug that is used to treat childhood acute lymphocytic leukemia, lymphoma, and cancers of the lung, head, neck, and breast.  For these applications, high doses of drug are administered.  Methotrexate is also used to treat rheumatoid arthritis and psoriasis and administered at much lower doses (1).

Methotrexate prevents cells from using folate to make DNA and RNA, slowing growth of new cancer cells. Because it can also deter growth of new skin cells, methotrexate is also used to treat psoriasis. The drug blocks several enzymes involved in the immune system and can minimize joint damage associated with rheumatoid arthritis (1).

Methotrexate levels in the blood typically rise after a dose and then fall gradually. Methotrexate is eliminated from the body by the kidneys, so any condition that decreases kidney function or interferes with drug clearance has the potential to increase blood concentrations (1).

Methotrexate should be carefully monitored when administered at high doses. Even when used correctly, it can cause significant side effects. Increased concentrations can be toxic, potentially damaging the liver, kidneys, and lungs and suppressing cell production in the bone marrow.  A drug called leucovorin (folinic acid) can be given as a "rescue" treatment to protect a person from the toxic effects of methotrexate (1).

Low-dose methotrexate therapy is rarely monitored by a methotrexate test. It is usually monitored by assessing the function of the kidney, the liver, and bone marrow cell production. Testing may include a complete blood count (CBC) to measure red blood cell counts, white blood cell counts, and platelets; a blood urea nitrogen (BUN) and creatinine to evaluate kidney function; and a liver panel (or one or more of its component tests) to evaluate liver function (1).

Current methods for the measurement of methotrexate include enzymatic and immunological assays as well as chromatographic techniques.  A study evaluated the analytical performance of the Abbott Architect and the ARK™ methotrexate immunoassay (running on the Roche Cobas© c502 analyzer) in comparison with liquid chromatography-mass spectrometry (LC-MS) and the TDx/TDxFLx Methotrexate II assay. Both assays showed strong correlations with the TDX assay and LC-MS but a positive bias of 12.2% and 20.5% in comparison to LC-MS for the Architect and ARK assay, respectively (2).

The methotrexate reagent technology from ARK Diagnostics has been adopted as an OEM product by a number of manufacturers (e.g. Siemens, Roche, Sekisui, others) (3).   Therefore, it is likely that results are comparable.

1. Lab Tests Online – AACC https://labtestsonline.org/tests/methotrexate
2. Head to head evaluation of the analytical performance of two commercial methotrexate immunoassays and comparison with liquid chromatography-mass spectrometry and the former fluorescence polarization immunoassay Günther, Viola ; Mueller, Daniel ; von Eckardstein, Arnold ; Saleh, Lanja; Zurich Open Repository and Archive University of Zurich 2015; https://doi.org/10.1515/cclm-2015-0578
3. Manufacturers’ package inserts.

Myoglobin

Myoglobin is a small cytoplasmic hemoprotein present predominantly in cardiac and skeletal muscle and rapidly released upon tissue damage. Historically myoglobin was utilized as a biomarker for early diagnosis of myocardial infarction, however the assays have low specificity due to identical cardiac and skeletal isoforms. With the advent of contemporary and high-sensitivity cardiac troponin assays there is no longer benefit in measuring myoglobin, particularly in conjunction with troponin and/or CK-MB, unless there are constraints within the laboratory to provide timely troponin testing [1-3].  Myoglobin is no longer recommended in current clinical ACC/AHA guidelines (IIIA class/level recommendation) [4]. NACB guidelines mention use of myoglobin if troponin is not available, however these guidelines are outdated and in revision [5].  Currently myoglobin is used as a biomarker of skeletal injury and is elevated in the setting of muscle trauma, resuscitation, myopathies, shock, muscular dystrophy and renal insufficiency. Extreme concentrations of serum myoglobin are notable in rhabdomyolysis, along with myoglobinuria.

There are presently ~750 labs enrolled in proficiency testing surveys through the CAP. All method CVs are <10%, and a majority are <5% within peer groups. For the qualitative optional interpretation of myoglobin, 98.8% of labs interpreted the “high” sample as abnormal and 91% classified the “low” specimen as normal, suggesting even though there are differences between quantitative myoglobin results, for the most part laboratories are interpreting or reporting myoglobin results correctly and there is no major clinical discordance (even though it should only be ordered infrequently!).

There is a urine myoglobin proficiency testing survey which has ~40 laboratories. None of the peer groups has greater than 10 participants so assay specific means cannot be determined and likely irrelevant.

References:

1. McCord J, Nowak RM, McCullough PA, et al. Ninety-minute exclusion of acute myocardial infarction by use of quantitative point-of-care testing of myoglobin and troponin I. Circulation 2001; 104:1483.
2. Eggers KM, Oldgren J, Nordenskjöld A, Lindahl B. Diagnostic value of serial measurement of cardiac markers in patients with chest pain: limited value of adding myoglobin to troponin I for exclusion of myocardial infarction. Am Heart J 2004; 148:574.
3. Kavsak PA, MacRae AR, Newman AM, et al. Effects of contemporary troponin assay sensitivity on the utility of the early markers myoglobin and CKMB isoforms in evaluating patients with possible acute myocardial infarction. Clin Chim Acta 2007; 380:213.
4. Amsterdam EA, Wenger NK, Brindis RG, Casey DE Jr, Ganiats TG, et al. 2014 ACC/AHA guideline for the management of patients with non– ST-elevation acute coronary syndromes: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines. Circulation. 2014; https://doi.org/10.1161/CIR.0000000000000134.
5. Morrow DA, Cannon CP, Jesse RL, Newby LK, Ravkilde J, Storrow AB, et al. National Academy of Clinical Biochemistry Laboratory Medicine Practice Guidelines: clinical characteristics and utilization of biochemical markers in acute coronary syndromes. Circulation, 115 (2007), pp. e356-e375.

Neisseria gonorrhea DNA by PCR

Gonorrhea is a common bacterial sexually transmitted disease (STD) that can cause serious complications if not diagnosed and treated. Gonococcal infections are typically asymptomatic in women until complications such as pelvic inflammatory disease (PID) develop.  In men, Gonnococal urethral infections cause urethritis with painful urination and, less commonly, epididymitis or disseminated gonococcal infection.  Morea rarely infections may be asymptomatic.  Gonorrhea can usually be treated with a course of antibiotics.

A vaginal swab is collected for women and first-catch urine samples can be used to test both men and women. The recommended method for detection of Gonorrheal DNA is the nucleic acid amplification test (NAAT). It is generally more sensitive and specific than other Gonorrhea tests

The FDA (USA) approved NAAT include Abbott Real Time CT/NG (Abbott Molecular Inc., Des Plaines, IL), Roche Cobas CT/NG test (Roche Diagnostics, Indianapolis, IN) Aptima COMBO 2 assay (Hologic/Gen-Probe Inc., San Diego, CA), BD MAX CT/GC, BD Probe Tec ET CT/GC Amplified DNA assay (Becton Dickinson and Company, Sparks, MD), Xpert CT/NG assay (Cepheid, Sunnyvale, CA), and binx io CT/NG Assay (binx health formerly Atlas Genetics). Sensitivity and specificity of Ng NAATs is generally >95% and >99% in swabs and male first-catch urine.   Test approval differs in other countries. 

2020 College of American Pathologists EQA Results on NAAT of Neisseria gonorrhea:

1. Swab Specimens: The results of Neisseria gonorrhea NAAT on three swab samples using six commercial Neisseria gonorrhea NAAT kits and Laboratory Developed Tests (LDT, used by 23 institutes) in 950 CAP PT Survey participating institutes showed a 99% concordance rate of seven test methods, including LDT.
2. Urine Specimens: The results of Neisseria gonorrhea tests on two urine samples with six commercial Neisseria gonorrhea NAAT kits and LDT assay (used by 39 agencies) at 950 CAP PT Survey participating institutes also showed a 99-100% concordance rate of seven test methods, including LDT.

The harmonization of results for NAAT of Neisseria gonorrhea at present is judged as adequate based on CAP EQA results and other available data.

References:

1. Lab Tests Online - https://labtestsonline.org/tests/gonorrhea-testing
2. Recommendations for the Laboratory-Based Detection of C. trachomatis and N. gonorrhoeae — 2014. MMWR Recomm Rep. 2014 Mar 14;63(RR-02):1-19.
3. Meyer, S. Buder. The Laboratory Diagnosis of Neisseria gonorrhoeae: Current Testing and Future Demands. Pathogens. 2020 Jan 31;9(2):91.

Osmolality

Osmolality is a colligative property of an aqueous solution that reflects the number of dissolved molecules in the solution.  Serum osmolality is mainly controlled by sodium, potassium, chloride, glucose and urea in serum.  Serum osmolality reflects water balance and conditions causing water overload or dehydration are reflected in decreased or increased osmolality, respectively.  The most common use of serum osmolality is to identify when a toxic substance such as ethanol, methanol isopropanol, ethylene glycol, or salicylates has been ingested.  The osmolar gap between measured osmolality and estimated osmolality calculated using serum sodium (or sodium + potassium), glucose and urea indicates the likely presence of a toxic substance, and supports follow up testing for commonly ingested substances.

Osmolality is most frequently measured using a freezing point depression approach with vapor pressure depression used by some methods.

A College of American Pathologists proficiency testing survey in 2021 with 1,830 participants reported 4 method groups with 93% of participants using the same freezing point detection instrument.  The survey included 5 samples with osmolality values from 387-599 mOsm/kg which is the same as mmol/kg water. The values in the survey samples were all elevated relative to normal values of 280-295 mmol/L likely due to preservatives used in the survey samples and to elevated concentrations for some analytes in the multi-analyte samples. Differences in mean values among the 4 measuring systems were 1.6-2.4%. The CVs within measuring system groups were 0.70-2.7% overall and 1.0-1.4% for the most commonly used method. Although the proficiency testing samples were not commutable with patients’ samples, the results are consistent with good agreement among results from different laboratories and measuring systems.

03 June 2021

Osmolality

Osmolality is a colligative property of an aqueous solution that reflects the number of dissolved molecules in the solution.  Urine osmolality is mainly controlled by creatinine and urea excreted into urine as well as sodium and chloride.  Urine osmolality is usually compared to serum osmolality to assess defects in sodium homeostasis and water reabsorption and excretion in the kidneys.

Osmolality is most frequently measured using a freezing point depression approach with vapor pressure depression used by some methods.

A College of American Pathologists proficiency testing survey in 2021 with 1,645 participants reported 4 method groups with 93% of participants using the same freezing point detection instrument.  The survey included 3 samples with osmolality values from 186-370 mOsm/kg which is the same as mmol/kg water. Differences in mean values among the 4 measuring systems were 0.8-1.2%. The CVs within measuring system groups were 0.4-1.7% overall and 0.4-1.2% for the most commonly used method. The proficiency testing samples were simulated urine but expected to reflect results for patient samples for aqueous solutions generally similar to urine. The results are consistent with good agreement among results from different laboratories and measuring systems.

03 June 2021

Oxyhemoglobin

Oxyhemoglobin is measured in whole blood using multi-wavelength spectrophotometry referred to as a co-oximeter than can be stand alone or integral with blood gas instruments. Oxyhemoglobin is the normal form of oxygen bound to hemoglobin and supplies the body with oxygen to support metabolism.  Oxyhemoglobin is normally 96-98% of the hemoglobin in red blood cells.  Reduction in oxyhemoglobin can be caused by many factors, leads to reduced oxygenation of tissues, morbidity and mortality.

Data from a 2017 College of American Pathologists survey of over 3000 labs using 13 commercially available systems had all results CVs from 1.2-2.8% for four samples with oxyhemoglobin values of 47-89%.  Differences between the highest and lowest mean values for instrument groups was 3.5-8.1% for five samples with oxyhemoglobin values of 89-48%, with CVs increasing at lower oxyhemoglobin values.

These results suggest that the agreement among different methods is adequate for clinical use.

Pancreatic lipase

An IFCC working group is addressing harmonization of pancreatic lipase.

Parathyroid hormone (PTH)

Parathyroid hormone (PTH) plays a critical role in bone and mineral homeostasis and is most frequently ordered to assess complications of chronic kidney disease and for primary hyperparathyroidism. Despite the existence of a WHO reference standard, current immunoassays for PTH produce a 4 fold difference in results for the same clinical samples. Consequently, clinical guidelines for diagnosis and treatment are compromised by the lack of harmonization among results.  Measurement issues include different molecular forms of PTH in blood and differences among the epitopes recognized by antibodies used in different measurement procedures. An IFCC Working Group on Bone Markers is addressing approaches to standardizing PTH measurement procedure results. The CDC is coordinating with the IFCC C-BM by developing reference measurement procedures.

References

1. Almond A, Ellis AR, Walker SW, Current parathyroid hormone immunoassays do not adequately meet the needs of patients with chronic kidney disease, Ann Clin Biochem 2012;49:63–67.
2. Sturgeon CM, Sprague S, Almond A, et al. Perspective and priorities for improvement of parathyroid hormone (PTH) measurement – A view from the IFCCWorking Group for PTH. Clin Chim Acta 2017;467:42-47.

05 July 2021

Phenobarbital

Phenobarbital is a long-acting barbiturate and has an anti-epileptic and sedating effects to depress the nervous system. Phenobarbital can be used to prevent seizures or to relieve anxiety. It can be prescribed to treat epilepsy and other seizure disorders by the drug effect of stabilizing electrical activity in the brain. A phenobarbital test is requested after the start of phenobarbital treatment and any time the dose is adjusted. It can also be ordered when drug side effects (such as drowsiness, headache, dizziness, depression, excitement, upset stomach, vomiting, nightmares, constipation and joint or muscle pain) are suspected. The usual therapeutic range of phenobarbital for adults taking is 15-40 mcg/ml for seizure treatment, and 5-15 mcg/ml for sedative-hypnotic use. The therapeutic range for children is 15-30 mcg/ml for seizure treatment (1).

In a comparability study for phenobarbital concentrations between 5 different analyzers  [Architect and AxSym (Abbott Laboratories), Dimension Vista and Dimension Xpand (Simens Healthcare Diagnostics Inc), cobas c 501 module (Roche Diagnostics GmbH)], the relative mean difference (method comparisons against cobas c 501 module as arbitrary standard) were less than ± 9.3% (+9.3% for Dimension Vista vs. cobas c 501, +6.8% for Dimension Xpand vs. cobas c 501, -6.1% for AxSym vs. cobas c 501, and +6.2% for  Architect vs. cobas c 501) in 58 leftover human samples (2).

There is no suitable proficiency testing information from commutable samples.

One reference measurement procedure for phenobarbital in blood serum is listed by JCTLM (3). There is no reference material or reference measurement procedure laboratory information listed at JCTLM database.

References

1. Phenobarbital, Lab Tests Online-US. https://labtestsonline.org/tests/phenobarbital Accessed 26 October 2020.
2. Shipkova M, Petrova DT, Rosler AE, Orth M, Engelmayer J, Wieland E, Brandhorst G, Oellerich M. Comparability and imprecision of 8 frequently used commercially available immunoassays for therapeutic drug monitoring. Ther Drug Monit. 2014 Aug;36(4):433-41. (Supplemental Digital Content 3, http://links.lww.com/TDM/A72; Supplemental Digital Content 4, http://links.lww.com/TDM/A73)
3. Tai SS, Yeh CY, Phinney KW. Development and validation of a reference measurement procedure for certification of phenytoin, phenobarbital, lamotrigine, and topiramate in human serum using isotope-dilution liquid chromatography/tandem mass spectrometry. Anal Bioanal Chem. 2011 Oct;401(6):1915-22.

Phenytoin

Phenytoin is commonly used anticonvulsant for the management of generalized tonic-clonic (grand-mal) seizures and complex partial (psychomotor, temporal lobe) seizures (1). Phenytoin has a narrow therapeutic window hence a fine balance must be found between efficacy and dose-related side effects (2). Phenytoin exhibits zero-order pharmacokinetics; the rate of clearance of the drug is dependent upon the concentration of drug present. Therefore, phenytoin does not have a classical half-life like other drugs, since it varies with blood concentration. As the blood concentration drops, the rate at which phenytoin is excreted increases (3). Thus, patients taking the same dosage can have up to a 50-fold difference in plasma phenytoin concentration (4). Furthermore, phenytoin is highly protein-bound and free (unbound) phenytoin is the component producing the pharmacological effect, as a result, any factor which changes the protein binding of phenytoin (e.g., drug-drug interaction, renal impairment, uremia, and critical illness, etc.) would be expected to alter the free drug levels (5).

In the comparability study for phenytoin concentrations between 6 different analyzers [Architect and AxSym (Abbott Laboratories), cobas c 501 module and COBAS INTEGRA 800 analyzer (Roche Diagnostics GmbH), Dimension Vista and Dimension Xpand (Simens Healthcare Diagnostics Inc)], systemic deviations (relative mean differences) of -3.1%, -6.1% and 5.1% were found among manufacturers (cobas c 501 vs. COBAS INTEGRA 800, AxSym vs. Architect and Dimension Xpand vs. Dimension Vista, respectively) in 50 - 63 leftover routine samples (6). The largest relative mean difference for phenytoin concentration was 12.3% with 95% CI of -9.3% to +33.9% for 52 patient samples for the Dimension Vista compared to cobas c 501 (method comparisons against cobas c 501 module as arbitrary standard) (6).

There is no suitable proficiency testing information from commutable samples.

One commutable reference material and a reference measurement procedure for phenytoin are listed in the JCTLM database.

References

1. Dilantin [package insert]. New York, New York: Pfizer Inc; Aug 2012. www.hsa.gov.sg. Accessed 3 June 2013.Accessed 3 June 2013.2.
2. von Winckelmann SL, Spret I, Willems L. Therapeutic drug monitoring of phenytoin in critically ill patients. Pharmacotherapy 2008;28(11):1391–400.
3. Richens A: Clinical pharmacokinetics of phenytoin. Clin Pharmacokinet 1979;4:153-169
4. Aronson JK, Hardman M, Reynolds DJM. ABC of monitoring drug therapy: phenytoin. BMJ 1992;305(6863):1215–8.
5. Theodore WH, Yu L, Price B, Yonekawa W, Porter RJ, Kapetanovic I, et al. The clinical value of free phenytoin levels. Acta Neurol 1985;18(1):90–3.6.
6. Shipkova M, Petrova DT, Rosler AE, Orth M, Engelmayer J, Wieland E, Brandhorst G, Oellerich M. Comparability and imprecision of 8 frequently used commercially available immunoassays for therapeutic drug monitoring. Ther Drug Monit. 2014 Aug;36(4):433-41.

Phosphate

EQA data from two Japanese programs in 2015 that used commutable samples had 3.1% and 2.0% CV among different laboratories and methods.  EQA data from the College of American Pathologists in 2015 (non-commutable samples) had 17.6% difference between maximum and minimum values reported by individual laboratories and 3.5% CV% among mean values for different peer groups.  Taken together these data support adequate harmonization for serum or plasma phosphate measurement procedures.  A reference measurement procedure is listed by JCTLM.

Platelet Count

The peripheral blood platelet count is a critical clinical parameter used in routine practice, trauma and by specialists caring for patients with complex hematological disorders. There are several commonly used technologies for assessing whole blood platelet count including microscopy, digital microscopy, immunoplatelet counting, impedance platelet counting, optical platelet counting and fluorescence platelet counting (1). Platelet counting has long been considered amongst the least harmonized particle counting procedures, particularly at the lower end of the analytical measurement range (2).

A reference measurement procedure has been developed by the German Institute for Standardization and listed on the JCTLM website with an expected uncertainty of 10% for platelet counts >30,000/uL and 3,000/uL for counts <30,000/uL (3). In addition a second reference measurement procedure has been proposed by the International Council for Standardization in Haematology (4) which is currently under review with JCTLM.

A recent comprehensive comparability study amongst several major hematology analyzer platforms (Abbott Cell-Dyn Sapphire, Beckman Coulter DxH 800, Siemens Advia 2120i, Sysmex XE-5000, and Sysmex XN-2000) using patient samples spanning the analytical measurement range demonstrated a high degree of variation among results. Passing-Bablok regression of individual platforms to the median value showed that four of the five instruments demonstrated slopes that are significantly different from 1, ranging from -0.94 to 1.1 and intercepts ranging from -2.96 to 1.31. Analyzer specific differences from the median varied across manufacturers and the analytical measurement range but ranged from 25 to -25% at normal and high platelet counts. Inter-instrument comparisons were most notably discordant at low platelet counts (<10,000/uL) where individual instrument differences from the median ranged from 50% to -50% (6). The data support the conclusion that further work is needed to optimally harmonize platelet count results for the purposes of clinical testing.

References

1. Baccini V, Geneviève F, Jacqmin H, et al. Platelet Counting: Ugly Traps and Good Advice. Proposals from the French-Speaking Cellular Hematology Group (GFHC). J Clin Med. 2020;9(3):808. Published 2020 Mar 16. doi:10.3390/jcm9030808.
2. Segal HC, Briggs C, Kunka S, et al. Accuracy of platelet counting haematology analysers in severe thrombocytopenia and potential impact on platelet transfusion. Br J Haematol. 2005;128(4):520‐ doi:10.1111/j.1365-2141.2004.05352.x.
3. Deutsches Institut für Normung e. V. (DIN). DIN 58932-5:2007-10. Haematology - Determination of the concentration of blood corpuscles in blood – Part 5: Reference method for the determination of the concentration of thrombocytes. DIN; 2007.
4. International Council for Standardization in Haematology Expert Panel on Cytometry and International Society of Laboratory Hematology Task Force on Platelet Counting. Platelet counting by the RBC/platelet ratio method. A reference method. Am J Clin Pathol. 2001;115:460–464.
5. Bruegel M, Nagel D, Funk M, Fuhrmann P, Zander J, Teupser D. Comparison of five automated hematology analyzers in a university hospital setting: Abbott Cell-Dyn Sapphire, Beckman Coulter DxH 800, Siemens Advia 2120i, Sysmex XE-5000, and Sysmex XN-2000. Clin Chem Lab Med. 2015;53(7):1057‐ doi:10.1515/cclm-2014-0945.

Potassium

EQA data from one Japanese program in 2015 that used commutable samples had 1.4% CV among different laboratories and methods.  EQA data from the College of American Pathologists in 2015 (non-commutable samples) had 7.4% difference between maximum and minimum values reported by individual laboratories and 1.6% CV% among mean values for different peer groups.  Taken together these data support adequate harmonization for serum or plasma potassium measurement procedures.  A reference material, serum-based reference material and a reference measurement procedure are listed by JCTLM.

Pregnancy-Associated Plasma Protein A

An IFCC working group is addressing harmonization of pregnancy-associated plasma protein A.

Procalcitonin

Procalcitonin (PCT) is a soluble protein whose synthesis is amplified in the presence of microbial infections and systemic inflammation. Approximately 70% of patients admitted to critical care units are started on some type of antimicrobial therapy. The most frequent indications are empiric therapy for suspected community-acquired pneumonia (CAP) or sepsis/septic shock and aggressive early use of antibacterials can lower mortality. PCT concentrations may potentially aid in determining the trajectory of disease.

Reference intervals for serum concentrations of PCT are reportedly between <0.05 ng/mL and <0.1 ng/mL and values are reflective of disease severity. It is detectable within 2-4 hours and peaks within 6-24 hours. PCT cutoffs vary with different clinical guidelines and protocols and are dependent upon the clinical indication, a few of which are listed below:

Suspected Lower Respiratory Tract Infection:

0.1 – 0.25 ng/mL     Low likelihood for bacterial infection; Antibiotics discouraged.

>0.25 ng/mL           Increased likelihood bacterial infection; Antibiotics encouraged.

Suspected Sepsis: Strongly consider initiating antibiotics in all unstable patients.

0.1 – 0.5 ng/mL       Low likelihood for sepsis; Antibiotics discouraged.

>0.5 ng/mL             Increased likelihood sepsis; Antibiotics encouraged.

>2.0 ng/mL             High risk of sepsis/septic shock; Antibiotics strongly encouraged.

Sepsis: PCT concentrations of >2.0 ng/mL predicts sepsis and values  >10 ng/mL indicate likely septic shock. Sensitivity and specificity of PCT for the diagnosis of sepsis has varied based upon population and underlying diseases. Higher PCT may be associated with a worse prognosis, but have not generally added to well validated clinical scoring systems such as APACHE or SAPS.

Bacterial Meningitis: Concentrations of <0.5 ng/mL make bacterial meningitis very unlikely. Most patients with bacterial meningitis have much higher PCT values (>5 ng/mL).

There are a variety of PCT immunoassays available, most frequently using BRAHMS PCT antibodies originally designed for use with the BRAHMS Kryptor system. Analytical comparison studies have reported classification concordance between 83 – 98% at 0.50 ng/mL, between 90 – 97% at 2.0 ng/mL and 98% at 10.0 ng/mL. The analytical results and performance varies between immunoassays and platforms.^4-5 Due to the growing utilization of PCT and implementation and integration in clinical guidelines and antimicrobial stewardship programs, the standardization/harmonization recommendation is high.

References:

1. Barlam TF, Cosgrove SE, Abbo LM, et al. Implementing an Antibiotic Stewardship Program: Guidelines by the Infectious Diseases Society of America and the Society for Healthcare Epidemiology of America. Clinical Infectious Diseases, Volume 62, Issue 10, 15 May 2016, Pages e51–e77.
2. Jensen JU, et al. Procalcitonin-guided interventions against infections to increase early appropriate antibiotics and improve survival in the intensive care unit. Crit Care Med. 2011;39:2048-58.
3. Dellinger RP, Levy MM, Rhodes A, Annane D, Gerlach H, Opal SM, et al. Surviving sepsis campaign: international guideline for management of severe sepsis and septic shock: 2012. Crit Care Med. 2013;41:580–637.
4. Dipalo M, Guido L, Micca et al. Multicenter comparison of automated procalcitonin immunoassays. Pract Lab Med. 2015;22-28.
5. Ceriotti F, Marino I, Motta A, Carobene A. Analytical evaluation of the performances of Diazyme and BRAHMS procalcitonin applied to Roche Cobas in comparison with BRAHMS PCT-sensitive Kryptor. Clin Chem Lab Med. 2018;56(1):162-169.

Prostate Specific Antigen (PSA)

PSA results are frequently different among measurement procedures.  Different antibodies are typically used that identify different epitopes in the molecule. Consequently, harmonization of results is challenging because the measurand is different. It is common laboratory practice to re-baseline results when measurement procedures change for patients who are being followed for recurrence of a tumor following treatment. However, the comparability of results over time is compromised when a patient’s specimen is assayed by different laboratories using different measurement procedures.  In addition, IVD manufacturers are challenged to maintain long term consistency of results due to the complexity of the measurand and the regents used for the measurement procedure.  A high priority was assigned because, despite the technical challenges, harmonization would substantially improve medial decisions considering that patients may have results from different measurement procedures due to mobility or measurement procedure changes in a laboratory.  Differences between measurement procedures may become increasingly important as early changes in residual PSA measurements after prostatectomy are increasingly used to make treatment decisions. 

Protein in CSF

Protein in CSF is a measure of "all" proteins present and thus not specific for any particular type of protein. Many neurologists order the test probably because it has been used for many years as a non-specific marker of increased protein in CSF.¹

Measuring CSF albumin, or even better, CSF albumin/serum albumin ratio is a better biomarker, since it specifically measures the blood-brain/CSF barrier function.  Measuring IgG and oligoclonal bands in CSF and serum are also more useful to identify inflammatory processes.

IFCC has a working group on CSF proteins for follow up information.

Reference:

1. Andersson M, Alvarez-Cermeño J, Bernardi G, Cogato I, Fredman P, Frederiksen J, Fredrikson S, Gallo P, Grimaldi LM, Grønning M, et al. J Neurol Neurosurg Psychiatry. 1994;57:897-902.

Protein S

Decreased activity of protein S is the major risk factor for VTE (venous thromboembolism) in Japan, Korea and China (1-8). The type II deficiency of protein S Tokushima (K155E/K196E) is about 2% in Japanese (2). The prevalence of protein S deficiency in Japan is approximately 10 times higher than that of Caucasians, due to not only protein S Tokushima but also other abnormalities of the protein S molecule (2, 3). About 50% of Japanese and Chinese people who developed VTE had reduced activity of protein S (3, 4). Thus, it is important to determine whether an individual is a carrier of such a thrombophilia as part of preventive medicine and for treatment of thromboses.

Protein S is recognized as an important biomarker of risk for VTE in non-Asian populations. The scientific and standardization committee of the International Society on Thrombosis and Haemostasis (ISTH) reported in 2016 that an inter-method assessment would be performed in 2017.

References

1. Shen MC, Lin JS, Tsay W: High prevalence of antithrombin III, protein C and protein S deficiency, but no factor V Leiden mutation in venous thrombophilic Chinese patients in Taiwan. Thromb. Res. 87: 377-385 (1997)
2. Kinoshita, S., Iida, H., Inoue S., et. al.: Protein S and protein C gene mutations in Japanese deep vein thrombosis patients. Clin. Biochem. 38: 908-915 (2005)
3. Hamasaki, N., Kanaji, T.: Clinical Role of Protein S Deficiency in Asian Population. pp. 597-613. in Thrombosis and Hemostasis (eds. by Tanaka K., Davie, E.W.) Springer (2008)
4. Hamasaki, N.: Unmasking Asian thrombophilia: Is APC dysfunction the real culprit? Thromb. Haemost. 10: 2016-2018 (2012)
5. Tang, L., Jian X-R, Hamasaki, N., Guo T., Wang, H-F., Lu, X., Wang Q-Y., Hu, Y.: Molecular basis of protein S deficiency in China. J. Hematol. 88: 899-905 (2013)
6. Yin,T., Miyata, T.: Dysfunction of protein C anticoagulant system, main genetic risk factor for venous thromboembolism in Northeast Asians. J. Thromb Thrombolysis 37: 56-65 (2014)
7. Kim, H-J., Seo, J-Y., Lee, Ki-O., Bang, S-H., Lee, S-T., Ki, C-S., Kim, J-W., Jung, C.W., Kim, D-K., Kim, S-H.: Distinct frequencies and mutation spectrums of genetic thrombophilia in Korea in comparison with other Asian countries both in patients with thromboembolism and in the general population. Haematologica 99: 561-569 (2014)
8. Hamasaki, N., Kuma, H., Tsuda, H.: Activated Protein C Anticoagulant System Dysfunction and Thrombophilia in Asia.  Ann. Lab. Med. 33: 8-13 (2013)

Protein, total

Urine total protein is used to assess the severity of kidney disease.  The early stages of chronic kidney disease are assessed using urine albumin.  In principle, urine total protein can assess early kidney disease, but the measurement procedures are typically not as analytically sensitive as are those for urine albumin making urine albumin the preferred test.  The common dipstick urinalysis tests for protein are more analytically sensitive to albumin than to other proteins but will detect other proteins such as Tamm Horsfall, IgG and others when present at higher concentrations associated with more advanced kidney disease.  Urine total protein is useful to assess severity of nephrotic syndrome when large amounts of protein, 1 or more grams per 24 hours, are excreted.  The ratio of albumin to total protein in urine is helpful to distinguish between predominantly glomerular disease or if another etiology is contributory to the disease (1).

Urine total protein is also elevated in patients with multiple myeloma which causes increased immunoglobulins and in particular light chains in the urine.  Higher amounts of urine protein are associated with more advanced myeloma. Tests for serum immunoglobulins, light chains and other biomarkers are used for diagnosis and staging of multiple myeloma (2).

One of the challenges to harmonization of urine total protein methods is that the measurand can be a number of different proteins not a single molecule. Another challenge is the relatively non-selective methods used (dye binding or protein aggregation) give different measurement responses for different proteins.  These two challenges mean there is no suitable reference material, although albumin is commonly used, and using a reference material would give different calibration relationships for different proteins seen in different pathologic conditions.

A College of American Pathologists EQA survey in 2018 included a presumably commutable freshly collected unmodified urine sample (3). Results from 2917 participants using 20 different instrument/method combinations at an approximate urine protein concentration of 350 mg/L had a 48% difference between the highest and lowest median values and an 84% difference between the highest and lowest values reported.  Although this disagreement among methods is large, the recommendation for low priority medical impact and inactive harmonization activity reflects the challenges mentioned to develop a practical reference system, and that current medical use considers relatively large amounts or large changes in urine protein when assessing kidney disease (1).

References:

1. Miller WG, Inker LA. Laboratory Evaluation of Kidney Function. In Contemporary Practice in Clinical Chemistry, 3rd Edition, ed. by Clarke W, AACC Press 2016.
2. Durie BGM, et al. International uniform response criteria for multiple myeloma. Leukemia 2006;20:1467-73.
3. College of American Pathologists Urine Survey, A mailing 2018. https://documents.cap.org/documents/2018-b-urine-chemistry.pdf

Protein, total

EQA data from two Japanese programs in 2015 that used commutable samples had 2.3% and 1.3% CV among different laboratories and methods.  EQA data from the College of American Pathologists in 2015 (non-commutable samples) had 7.7% difference between maximum and minimum values reported by individual laboratories and 2.0% CV% among mean values for different peer groups.  Taken together these data support adequate harmonization for serum or plasma total protein measurement procedures.  JCTLM does not list a reference material or reference measurement procedures for total serum proteins.  A bovine serum albumin solution certified reference material is listed by JCTLM and a biuret reagent measurement procedure is widely used as a comparative method for total protein in calibration hierarchies.

Pseudocholinesterase

Pseudocholinesterase (PCHE) testing is used to monitor for exposure to organophosphorus insecticides and preoperative screening for patients with a history or a family history of prolonged paralysis and apnea after the use of the muscle relaxant, succinylcholine, for anesthesia.

Although the pseudocholinesterase test has been used for the diagnosis of acute exposure of organophosphorus insecticides its medical impact is low because cases of acute organophosphorus exposure are not frequent, and acute organophosphorus exposure can be diagnosed easily by the patient’s symptoms.

Information on agreement of results among different measurement procedures is not readily available. The number of laboratories participating in a CAP proficiency testing survey in 2020 was only 29 representing 5 measurement procedures from 4 major IVD manufacturers. The results varied from 6.0 U/mL to 7.7 U/mL for 4 of 5 measurement procedures, and from 10.4 U/mL to 11.1 U/mL for the fifth measurement procedure. Normal pseudocholinesterase is method dependent but typically 5-12 U/mL and deficiency causes a 50% or greater reduction in activity. Since pseudocholinesterase is interpreted as a substantial decrease in activity from that of normal individuals, the harmonization of results appears adequate for clinical use of the test procedure.

Quinidine

Quinidine, a hydantoin anticonvulsant, is used alone or with phenobarbital or other anticonvulsants to manage tonic-clonic seizures, psychomotor seizures, neuropathic pain syndromes including diabetic neuropathy, digitalis-induced cardiac arrhythmias, and cardiac arrhythmias associated with QT-interval prolongation (1). 

Quinidine acts on sodium channels on the neuronal cell membrane, limiting the spread of seizure activity and reducing seizure propagation. The antiarrhythmic actions are mediated through effects on sodium channels in Purkinje fibers. Quinidine may also act on the slow inward calcium current (ICa), the rapid (IKr) and slow (IKs) components of the delayed potassium rectifier current, the inward potassium rectifier current (IKI), the ATP-sensitive potassium channel (IKATP) and Ito (1).

Plasma concentrations of quinidine are now mostly determined by automated enzyme immunoassays (EIA) and HPLC with greater specificity.  Furthermore, several of the metabolites of quinidine are active, and accumulate to clinically significant concentrations during chronic therapy. (2)

Plasma quinidine results determined by a commercially available enzyme immunoassay were compared with results from a quinidine-specific high performance liquid chromatographic (HPLC) assay. During a clinical study, 16 patients with stable ventricular arrhythmias were treated with three oral quinidine preparations given during three consecutive 3-day periods. The overall mean (+/- SD) quinidine concentrations by EIA and HPLC were 2.16 +/- 0.58 and 1.81 +/- 0.60, respectively, n = 816, with a mean overall EIA/HPLC ratio of 1.23 +/- 0.18.  Mean ratios in individual patients ranged from 1.01 to 1.56; the average of mean individual ratios was 1.23 +/- 0.13. The EIA assay, which also reports dihydroquinidine and small amounts of quinidine metabolites as quinidine, reported quinidine values that averaged 1.2-fold greater than results from a quinidine-specific HPLC method (3). Quinidine is implicated in a number of clinically significant drug interactions, which may increase requirements for monitoring (2).

There is no reference material, reference measurement procedure and reference measurement procedure laboratory information listed at JCTLM database.

References

1. Quinidine: https://go.drugbank.com/drugs/DB00908
2. J. Campbell & K. M. Williams: Therapeutic drug monitoring: antiarrhythmic drugs; British Journal of Clinical Pharmacology 2001; 52:21S–34S.
3. C R Lehmann, K J Boran, W P Pierson, A P Melikian, G J Wright: Quinidine assays: enzyme immunoassay versus high performance liquid chromatography; Therapeutic Drug Monitoring 1986; 8(3):336-9.

03 June 2021

Rheumatoid factor (RF)

RF is an immunoglobulin, usually IgG, directed against IgM. It is detectable in about 71% of patients with active rheumatoid arthritis, but can also be present in healthy individuals and in other rheumatological diseases, leading a diagnostic specificity of 83%. Diagnostic use is usually in conjunction with citrullinated cyclic peptide (CCP). Current assay methodologies use EIA, ELISA, nephelometry, and latex agglutination. There is no standard reference material available, and there is little expressed concern about the need for harmonization in the literature or by the professional rheumatology organizations.

Rotavirus Ag

Rotaviruses are the most important pathogens in non-bacterial gastroenteritis in children from 6 months to 3 years old. Early and reliable detection of rotaviruses is important for avoiding further infection. In addition, accurate and rapid diagnosis of Rotavirus diarrhea can reduce unnecessary use of antibiotics and ultimately reduce drug resistance. Detecting rotavirus antigen by enzyme immunoassay has nearly 100% sensitivity and 92% specificity when compared to transmission electron microscopy, the method initially used to detect virus in fecal and intestinal biopsy specimens and the standard to which rotavirus diagnostic tests are compared (1).

Seven commercial Rotavirus antigen assays were compared with in-house PCR methods. The assay sensitivities were 80% to 100%, while the specificities were 99.4% to 100% for 6 of 7 assays (2). These 6 EIA assays were reliable for Rotavirus antigen detection. Harmonization of Rotavirus antigen assay results is adequate for their medical use.

1. Mitchell DK, Jiang X, Matson DO: Gastrointestinal infections. In Essentials of Diagnostic Virology. Edited by A Storch, Churchill Livingstone. 2000 pp 82-84.

2. Ye S, Lambert SB, Grimwood K, Roczo-Farkas S, Nimmo GR, Sloots TP, Kirkwood CD, Whilley DM. Comparison of Test Specificities of Commercial Antigen-Based Assays and In-House PCR Methods for Detection of Rotavirus in Stool Specimens. Journal of Clinical Microbiology 2015;53(1):295-297.

Sirolimus

Sirolimus (Rapamune®, rapamycin) is a potent immunosuppressive drug for immunosuppressive therapy following kidney transplantation. Monitoring of whole blood concentrations of sirolimus during immunosuppressive therapy has become standard practice.  Therapeutic Drug Monitoring is performed for the purposes of drug dose adjustment and to ensure patients are in the correct therapeutic range.  Nonetheless, improvement in the measurement of sirolimus is still needed in spite of the drug being used for many years.  Standardization / Harmonization of sirolimus measurement is important for several reasons including:

• Narrow therapeutic range
• Drug toxicity (high) or inadequate immunosuppression (low)
• dose/exposure relationship is interindividually and intraindividually highly variable
• Active and inactive metabolites with variable crossreactivity depending on the method chosen
• Variability associated with pre-analysis including the whole blood extraction methods used
• Inadequate precision of many widely used methods [1]

Sirolimus quantification can be accomplished by high performance liquid chromatography (HPLC), tandem mass spectrometric detection (LC/MS/MS) and by commercially available immunoassays.  In a study comparing a selected commercially available immunoassay to HPLC and LC/MS/MS methods at several sites, the general trend was for a positive bias by the immunoassay with patient samples.  The correlation slopes showed significant differences between the clinical sites, ranging from 1.00 to 1.36.  Correlation coefficients were higher between the immunoassay and the LC/MS/MS laboratories (range 0.91–0.96) relative to HPLC/UV laboratories (0.79–0.84). There were also some noteworthy differences in correlation slopes between the laboratories for the comparisons with LC/MS/MS and LC/UV. Differences in calibration of the LC based assays are a likely reason for the differences.  Across all laboratories, there was an approximately 25% positive bias for the slope between the immunoassay and the LC methods.  Because the immunoassay antibody crossreacts with metabolites of the parent drug, the IA was shown to over-estimate clinical and proficiency samples prepared using pooled patient samples. This 25% bias is consistent with the estimated distribution pattern of sirolimus metabolites in the blood of kidney transplant patients and the characterized cross-reactivity of the IA with some of the metabolites. [2]

A recent survey (76 laboratories in 14 countries) identified three main reasons for interlaboratory variability: (1) lack of standardization of laboratory procedures and workflows starting with sample collection and handling, (2) lack of use of appropriate reference materials and (3) poor compliance with internationally accepted good laboratory practice guidelines. Although intralaboratory precision improvement is the responsibility of the individual laboratory, interlaboratory bias can only be addressed by the establishment of higher-order reference materials and/or higher-order reference procedures to ensure worldwide measurement traceability and consistency. [3]

The IFCC Working Group on Immunosuppressive Drugs (WG-ID) is devoted to the establishment of candidate reference procedures and reference materials for immunosuppressive drugs (ISDs) as cyclosporine, sirolimus, tacrolimus, everolimus, and mycophenolic acid (MPA). Demonstration of the current state of the art in ISD – TDM by measurement comparison will define the need for harmonization or – if feasible – standardization of measurement services.

References:

1. Christoph Seger, Maria Shipkova, Uwe Christians, et al. Assuring the Proper Analytical Performance of Measurement Procedures for Immunosuppressive Drug Concentrations in Clinical Practice: Recommendations of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology Immunosuppressive Drug Scientific Committee: Ther Drug Monit 2016;38:170–189
2. Wilson, F. Johnston, D. Holt, M. Moreton, J. Engelmayer, J.-M. Gaulier, H. Luthe, P. Marquet, D. Moscato, M. Oellerich, R. Mosso, F. Streit, M. Brunet, C. Fillee, R. Schmid, P. Wallemacq, G. Barnes: Multi-center evaluation of analytical performance of the microparticle enzyme immunoassay for sirolimus.  Clinical Biochemistry 39 (2006) 378–386.
3. Christians U, Vinks AA, Langman L, et al. The impact of laboratory practices on inter-laboratory variability in therapeutic drug monitoring of immunosuppressive drugs. Ther Drug Monit. 2015; 37:718–724.

Sodium

EQA data from one Japanese program in 2015 that used commutable samples had 1.8% CV among different laboratories and methods.  EQA data from the College of American Pathologists in 2015 (non-commutable samples) had 6.7% difference between maximum and minimum values reported by individual laboratories and 1.6% CV% among mean values for different peer groups.  Taken together these data support adequate harmonization for serum or plasma sodium measurement procedures.  A reference material, serum-based reference material and a reference measurement procedure are listed by JCTLM.

Syphilis

See Treponema pallidum Antibodies

Tacrolimus

Tacrolimus is the calcineurin inhibitor used for patients receiving solid organ transplantation. Since it is recommended that minimizing tacrolimus dosage regimen be used to prevent rejection of the transplanted organ while reducing the nephrotoxic risk of drug, accurate and precise measurement of the tacrolimus concentration in whole blood is essential in optimal immunosuppressive therapy. However, the current tacrolimus quantification methods based on LC-MS and immunoassay are not traceable to an accepted reference LC-MS method or reference material, so tacrolimus concentration values may not be comparable between methods and laboratories.

In the comparability study (to 22 clinical laboratories in 14 countries) for tacrolimus concentrations between 3 different assays (Abbott ARCHITECT, LC-MS, and Siemens Dade Dimension) using 4 pooled whole blood samples from patients undergoing routine tacrolimus TDM (therapeutic drug monitoring), the relative mean bias to EM-IDMS (exact-matching isotope dilution mass spectrometry) at the LGC as a reference value ranged from 2.5% to 9.2% for ARHCITECT  (n=17), -4.3% to -2.8% for LC-MS (n=9), and -0.3% to 6.4% for Dade Dimension (n=5), respectively. Total error across the 4 pooled samples ranged from 17.6% to 21.4% for ARCHITECT, 28.0% to 33.4% for LC-MS, and 17.6% to 54.0% for Dade Dimension (1).

The IFCC Working Group on Immunosuppressive Drugs (WG-ID) is devoted to the establishment of candidate reference procedures and reference materials for immunosuppressive drugs (ISDs) as cyclosporine, sirolimus, tacrolimus, everolimus, and mycophenolic acid (MPA) (2). Demonstration of the current state of the art in ISD – TDM by measurement comparison will define the need for harmonization or – if feasible – standardization of measurement services.

There is no suitable proficiency testing information from commutable samples.

One reference material listed at JCTLM database is ERM-DA 110a (LGC Limited, UK), which was found to be commutable for using isotope dilution mass spectrometry reference method and routine laboratory LC-MS/MS applied to both the reference material and patient samples (3). There is no reference measurement procedure and reference measurement procedure laboratory information listed at JCTLM database.

References:

1. Levine DM, Maine GT, Armbruster DA et al. The need for standardization of tacrolimus assays. Clin Chem. 2011 Dec;57(12):1739-47.
2. Christoph Seger, Maria Shipkova, Uwe Christians, et al. Assuring the Proper Analytical Performance of Measurement Procedures for Immunosuppressive Drug Concentrations in Clinical Practice: Recommendations of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology Immunosuppressive Drug Scientific Committee: Ther Drug Monit 2016;38:170–189.
3. ERM-DA110, Tacrolimus in whole human blood (LGC Limited, UK) https://www.lgcstandards.com/GB/en/Tacrolimus-in-whole-human-blood/p/ERM-DA110)

Theophylline

Theophylline is used to prevent and treat wheezing, shortness of breath, and chest tightness caused by asthma, COPD (chronic bronchitis, emphysema), and other lung diseases. It works in the airway by relaxing muscles and opening air passages and to make it easy to breathe. Theophylline testing is used for monitoring the amount of theophylline in the blood. The test may be requested when a patient has symptoms of suspected theophylline toxicity or a patient is not responding as expected to therapy. The usual adult therapeutic levels of theopylline in blood are between 10 and 20 mg/L to be used as a bronchodilator. Levels higher than 20 mg/L may show symptoms associated with acute theophylline toxicity (low blood pressure, rapid heart rate, restlessness, tremors, nausea, abdominal pain, confusion, etc.) (1).

In a comparability study for theophylline concentrations between 6 different analyzers  [Architect and AxSym (Abbott Laboratories), cobas c 501 module and COBAS INTEGRA 800 analyzer (Roche Diagnostics GmbH), Dimension Vista and Dimension Xpand (Simens Healthcare Diagnostics Inc)], the relative mean difference (method comparisons against cobas c 501 module as arbitrary standard) were less than ± 8.2% (-8.2% for AxSym vs. cobas c 501, -7.4% for Architect, -3.5% for Dimension Vista and -0.5% for Dimension) in 68 to 79 leftover human samples (2).

In German EQA schemes, the results of laboratories are generally evaluated using materials (pooled serum, lyophilized, spiked with theophylline, no additives) with GC-IDMS reference measurement procedure values assigned. In 2019, the concentrations of the EQA materials ranged from 8.4 mg/L to 27.4 mg/L. The deviation of the different test systems varied from -7% to +11% (except one test system using mass spectrometry (Chromsystems) which deviated up to +21%) (3).

One reference material listed in the JCTLM database is a high-purity material used as a primary calibrator for IDMS reference measurement procedures. Two and three reference measurement procedures for theophylline in blood plasma and blood serum, respectively, are listed by JCTLM. One reference measurement procedure laboratory is listed in the JCTLM database.

References

1. Theophylline and Caffeine - Lab Tests Online-UK. https://labtestsonline.org.uk/tests/theophylline-and-caffeine. Accessed 09 June 2019
2. Shipkova M, Petrova DT, Rosler AE, Orth M, Engelmayer J, Wieland E, Brandhorst G, Oellerich M. Comparability and imprecision of 8 frequently used commercially available immunoassays for therapeutic drug monitoring. Ther Drug Monit. 2014 Aug;36(4):433-41.
3. Surveys Therapeutic drug monitoring – Reference Institute for Bioanalytics. https://www.rfb.bio/cgi/surveys?searchType=rv_type&rv_type=AK&analyte=all&year=2019. Accessed 11 December 2019.

Theophylline

Theophylline is used to prevent and treat wheezing, shortness of breath, and chest tightness caused by asthma, COPD (chronic bronchitis, emphysema), and other lung diseases. It works in the airway by relaxing muscles and opening air passages and to make it easy to breathe. Theophylline testing is used for monitoring the amount of theophylline in the blood. The test may be requested when a patient has symptoms of suspected theophylline toxicity or a patient is not responding as expected to therapy. The usual adult therapeutic levels of theopylline in blood are between 10 and 20 mg/L to be used as a bronchodilator. Levels higher than 20 mg/L may show symptoms associated with acute theophylline toxicity (low blood pressure, rapid heart rate, restlessness, tremors, nausea, abdominal pain, confusion, etc.) (1).

In a comparability study for theophylline concentrations between 6 different analyzers  [Architect and AxSym (Abbott Laboratories), cobas c 501 module and COBAS INTEGRA 800 analyzer (Roche Diagnostics GmbH), Dimension Vista and Dimension Xpand (Simens Healthcare Diagnostics Inc)], the relative mean difference (method comparisons against cobas c 501 module as arbitrary standard) were less than ± 8.2% (-8.2% for AxSym vs. cobas c 501, -7.4% for Architect, -3.5% for Dimension Vista and -0.5% for Dimension) in 68 to 79 leftover human samples (2).

In German EQA schemes, the results of laboratories are generally evaluated using materials (pooled serum, lyophilized, spiked with theophylline, no additives) with GC-IDMS reference measurement procedure values assigned. In 2019, the concentrations of the EQA materials ranged from 8.4 mg/L to 27.4 mg/L. The deviation of the different test systems varied from -7% to +11% (except one test system using mass spectrometry (Chromsystems) which deviated up to +21%) (3).

One reference material listed in the JCTLM database is a high-purity material used as a primary calibrator for IDMS reference measurement procedures. Two and three reference measurement procedures for theophylline in blood plasma and blood serum, respectively, are listed by JCTLM. One reference measurement procedure laboratory is listed in the JCTLM database.

References

1. Theophylline and Caffeine - Lab Tests Online-UK. https://labtestsonline.org.uk/tests/theophylline-and-caffeine. Accessed 09 June 2019
2. Shipkova M, Petrova DT, Rosler AE, Orth M, Engelmayer J, Wieland E, Brandhorst G, Oellerich M. Comparability and imprecision of 8 frequently used commercially available immunoassays for therapeutic drug monitoring. Ther Drug Monit. 2014 Aug;36(4):433-41.
3. Surveys Therapeutic drug monitoring – Reference Institute for Bioanalytics. https://www.rfb.bio/cgi/surveys?searchType=rv_type&rv_type=AK&analyte=all&year=2019. Accessed 11 December 2019.

Thyroid stimulating hormone (TSH)

The literature reports moderate biases among different measurement procedures for TSH.  An IFCC committee is working to harmonize results from measurement procedures for TSH.

Thyroxine (T4)

The literature reports substantial biases among different measurement procedures for T4.  An IFCC committee is working to harmonize results from measurement procedures for T4.

Thyroxine, free (FT4)

The literature reports substantial biases among different measurement procedures for FT4.  An IFCC Committee for Standardization of Thyroid Function Tests is working to harmonize results from measurement procedures for FT4. The CDC is collaborating with the IFCC C-STFT by providing reference measurements to PT/EQA providers and launching a CDC FT4 standardization program in 2021.

05 July 2021

Tobramycin

Tobramycin is an aminoglycoside antibiotic to be used for treatment of infections by susceptible strains of aerobic gram-negative bacteria such as Acinetobacter, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Providencia stuartii, and Serratia (1-3). Tobramycin can cause reversible nephrotoxicity similar to other aminoglycosides as well as irreversible vestibulocochlear toxicity, and neuromuscular blockade (2,3).

Tobramycin tests are requested after the start of tobramycin treatment and any time the dose is adjusted. Tests may also be requested in clinical situations such as suspected infection in premature infants and neonates or patients with renal impairment, signs or symptoms of toxicity, coexistence of diseases which may increase risk of toxicity or coadministration of drugs with the potential to potentiate toxicity (1-3). The use of peak and trough levels is recommended to guide tobramycin dosing. The recommended therapeutic range is 5-10 mcg/ml for peak levels and trough level < 1 mcg/mL; the toxic level is >12 mcg/ml (4).

In a comparability study for tobramycin concentrations between 5 different analyzers  [Architect and AxSym (Abbott Laboratories), Dimension Vista and Dimension Xpand (Simens Healthcare Diagnostics Inc), Cobas c 501 module (Roche Diagnostics GmbH)], the relative mean difference (method comparisons against Cobas c 501 module as arbitrary standard) were less than ± 9.7% (-2.0% for Dimension Vista vs. Cobas c 501, +3.1% for Dimension Xpand vs. Cobas c 501, +9.7% for AxSym vs. Cobas c 501, and -0.7% for  Architect vs. Cobas c 501) in 47 residual human samples (5).

There is no suitable proficiency testing information from commutable samples. In a multilaboratory commutability evaluation study in the Netherland within the framework of the Dutch Calibration 2000 project, commutable proficiency testing materials for tobramycin have been developed and future studies will indicate whether such materials could be used for harmonization between laboratories and methods (6).

There is no reference material, reference measurement procedure or reference measurement procedure laboratory information listed in the JCTLM database.

References

1. Tobramycin Level, Medscape. https://emedicine.medscape.com/article/2090440-overview Updated: Sep 29, 2020
2. Hammet-Stabler C, Johns T. Laboratory Guidelines for Monitoring Antimicrobial Drugs. Clin Chem. 1980. 44:5:1129-1140.
3. Isaacs D, Elliott E, Gilbert R, Moyer V, Pichichero M. Aminoglycosides: Dosing and Monitoring Blood Levels. Evidence-based Pediatric Infectious Diseases, Appendix 2. 2008. 301-305.
4. Pagana KD, Pagana TJ, Pagana TN. Mosby's Diagnostic & Laboratory Test Reference. 14th ed. St. Louis, MO: Elsevier; 2019. 333.
5. Shipkova M, Petrova DT, Rosler AE, Orth M, Engelmayer J, Wieland E, Brandhorst G, Oellerich M. Comparability and imprecision of 8 frequently used commercially available immunoassays for therapeutic drug monitoring. Ther Drug Monit. 2014 Aug;36(4):433-41. (Supplemental Digital Content 3, http://links.lww.com/TDM/A72; Supplemental Digital Content 4, http://links.lww.com/TDM/A73)
6. Robijns K, Boone NW, Jansen RT, Kuypers AW, Neef C, Touw DJ. Commutability of proficiency testing material containing tobramycin: a study within the framework of the Dutch Calibration 2.000 project. Clin Chem Lab Med 2017;55:212–7.

28 April 2021

Toxoplasma gondii IgG

Toxoplasma gondii is an intracellular protozoan parasite capable of infecting warm-blooded animals including humans. Primary Toxoplasma infections in humans are usually asymptomatic and once the host develops antibodies, the infection enters a dormant phase [1].

Primary maternal infection can lead to a risk of congenital infection, fetal death or serious congenital abnormalities, particularly brain and Central Nervous System (CNS) damage [2,3]

In contrast to the benign infection seen in the general population, immunocompromised patients may experience severe Toxoplasmosis with CNS involvement. Toxoplasmosis is of great importance in the management of AIDS patients. Various studies indicate 3–38% of AIDS patients may develop cerebral toxoplasmosis from the reactivation of a quiescent viable parasitic focus [4,5].

The 2020 CAP EQA results for commutable serum samples participated 573 laboratories using more than 15 commercial kits showed 99.8% concordance rate. Therefore, the harmonization status for the Toxoplasma gondii IgG test is judged as adequate.

References

1. Dubey JP, Beattie CP. Toxoplasmosis of animals and man. CRC Press, Boca Raton, 1988. 1–220.
2. Remington JS, Desmonts G. Toxoplasmosis in: Remington JS, Klein JP. Infectious diseases of the fetus and newborn infant. Ed. WB Saunders, Philadelphia, 1990. 89–195.
3. Dunn D, Wallon M, Peyron F, Petersen E, Peckham C, Gilbert R. Mother-to-child transmission of toxoplasmosis: risk estimates for clinical counselling. Lancet,1999; 353, 1829–1833.
4. Luft BJ, Remington JS. Toxoplasmic encephalitis in AIDS. Clin Infect Dis 1992; 15: 211-222.
5. Israelski DM, Chmiel JS, Poggensee L, Phair JP, Remington JS. Prevelence of toxoplasma infection in a cohort of homosexual men at risk of AIDS and toxoplasmic encephalitis. J Acquir Immune Defici Syndr 1993; 6: 414-418.

04 May 2021

Transferrin

Transferrin is the main transport protein for iron in blood.  Transferrin is used to understand iron deficiency anaemia.  In healthy individuals, transferrin is approximately one-third saturated with iron.  In iron deficiency, all of the stored iron is used and the body tries to compensate by producing more transferrin to increase iron transport. While the serum iron level continues to decrease, the transferrin level increases. Thus, the amount of transferrin available to bind iron increases and the amount of transferrin saturated with iron (i.e., percent transferrin saturation) decreases.  Transferrin is usually ordered with iron to estimate the percent saturation, and with ferritin to assess the reserve of iron in bone marrow.

Transferrin is also used to assess iron overload in cases of suspected toxicity due to excess iron ingestion.  In this case, the transferrin saturated with iron will approach or exceed 100% and chelation treatment is needed to reduce the toxicity from iron overload.

JCTLM lists ERM-DA470k/IFCC, human serum, as a commutable CRM for use in calibration hierarchies. Its predecessor, ERM-DA470, also included transferrin so a reference system has been in place for many years.

Results for approximately 1700 participants using 12 measurement procedures in one CAP Survey in 2020 had CVs from 3.9-6.9% for results for 5 samples with concentrations 66-254 mg/dL from all method groups; and results for 295 participants in another CAP survey had CV 6.5% at a concentration 69 mg/dL for results from all method groups.   The commutability status of these samples is not known but the small CVs are consistent with good agreement among 10 measurement procedures.

Limited data was available comparing methods using patient samples. A comparison between Abbott Architect and Dade-Behring BN II had a total error of 16% (1).

References

1. Mali B, Armbruster D, Serediak E, Ottenbreit T. Comparison of immunoturbidimetric and immunonephelometric assays for specific proteins. Clin Biochem 2009;42:1568–71.

Treponema pallidum Antibodies

Syphilis is a sexually transmitted infection caused by the bacterium Treponema pallidum. Patients may seek evaluation for symptoms or signs of primary infection (e.g., chancre), secondary infection (e.g., diffuse rash), or tertiary infection (e.g., symptoms of aortic insufficiency). The diagnosis of syphilis is most commonly made by serologic testing. There are two types of serologic tests for syphilis: nontreponemal tests and treponemal tests.

Nontreponemal tests (also known as tests for reagin antibodies) are based upon the reactivity of serum from infected patients to a cardiolipin-cholesterol-lecithin antigen. All nontreponemal tests measure both immunoglobulin (Ig) G and IgM antiphospholipid antibodies formed by the host in response to lipoidal material released by damaged host cells early in infection and lipid from the cell surfaces of the treponeme itself.  Although these screening tests are nonspecific, and therefore not definitive, they have traditionally been used for initial syphilis screening due to their relatively low cost, ease of performance, and ability to be quantified for the purpose of following response to therapy.

Nontreponemal tests include:

• Rapid plasma reagin (RPR)
• Venereal Disease Research Laboratory (VDRL)
• Toluidine Red Unheated Serum Test (TRUST)

All treponemal tests use T pallidum or its components as the antigen. Treponemal tests have historically been more complex and expensive to perform than nontreponemal tests. Thus, they have traditionally been used as confirmatory tests for syphilis when the nontreponemal tests are reactive.

Treponemal tests include:

• Fluorescent treponemal antibody absorption (FTA-ABS)
• Microhemagglutination test for antibodies to T. pallidum (MHA-TP)
• pallidum particle agglutination assay (TPPA)
• pallidum enzyme immunoassay (TP-EIA)
• Chemiluminescence immunoassay (CIA)

The NIBSC had released WHO International Standard 1st IS for human syphilitic plasma IgG and IgM (code: 05/132). This standard can be used to calibrate the venereal disease research laboratory test (VDRL) and the rapid plasma reagin test (RPR) and the Treponema pallidum passive particle agglutination assay (TPPA). In addition, this standard can be used as a positive control in the Fluorescent Treponema Antibody (FTA) assay, the 19SIgM FTA and IgM and IgG enzyme immunoassays.

Proficiency testing data from the College of American Pathologists Syphilis Serology Survey in 2020 indicated approximately 95% or better reached consensus. The item includes VDRL (Serum, CSF), MHA-TP/TP-PA/PK-TP/TPHA, RPR, EIA (IgG, Total Antibody, Reagin), USR, FTA-ABS, CMIA, Multiplex Flow Immunoassay, and TP-LIA IgG. The present harmonization status of Treponema pallidum is adequate based on CAP EQA results.

References:

1. UpToDate- https://www.uptodate.com/contents/syphilis-screening-and-diagnostic-testing?search=syphillis&source=search_result&selectedTitle=1~150&usage_type=default&display_rank=1
2. Sensitivity and Specificity of Treponemal-specific Tests for the Diagnosis of Syphilis. Clin Infect Dis. 2020 Jun 24;71(Suppl 1):S13-S20.

05 July 2021

Triglycerides

EQA data from two Japanese programs in 2015 that used commutable samples had 2.7% and 2.0% CV among different laboratories and methods.  EQA data from a College of American Pathologists 2018 survey with commutable samples had approximately 17% difference between maximum and minimum values reported by individual laboratories at three concentrations, and 2.3% CV% among median values for different measurement procedure groups.  Taken together these data support adequate harmonization for serum or plasma triglyceride measurement procedures.  A reference material, serum-based reference material and a reference measurement procedure are listed by JCTLM.  The CDC maintains a global network of reference laboratories and a certification program for IVD manufacturers to maintain standardized results. See the website link for additional information.

Triiodothyronine, free (FT3)

A report that used a fresh-frozen presumably commutable sample in a College of American Pathologists Survey in 2003 found an approximate 2-fold difference in free-T3 results among 11 clinical laboratory immunoassay measurement procedures (1).  A recent review reported that free-T3 results continue to have discrepancies among clinical laboratory immunoassay measurement procedures (2).  To some extent, method specific reference intervals compensate for biases among different measurement procedures. The review reported that multiple studies have shown that T3, free-T3 and free-T4 for approximately 20% of patients on therapy for hypothyroidism are within the reference intervals by immunoassays when the values are below the reference interval by an ultrafiltration LC-MS/MS measurement procedure.  Free thyroid hormone measurement by immunoassay is a challenging technology because of the influence of various binding protein abnormalities that alter the recovery and thus compromise the suitability of the reference interval determined for these methods. 

References

1. Steele BW, Wang E, Klee GG, et al. Analytic Bias of Thyroid Function Tests. Analysis of a College of American Pathologists Fresh Frozen Serum Pool by 3900 Clinical Laboratories. Arch Pathol Lab Med 2005;129:310-7.

2. Welsh KJ, Soldin SJ. Diagnosis of endocrine disease: how reliable are free thyroid and total T3 hormone assays? Eur J Endocrinol 2016;175:R255-63.

Troponin I, cardiac (cTnI)

Troponin I has historically had large biases among different measurement procedures. Efforts to harmonize troponin I have gone on for many years and among method variability has improved.  Use of the 99th percentile for identifying a positive result has helped to mitigate differences in values among different measurement procedures. The current development of more sensitive assays will add to the importance to harmonize results for use in clinical practice guidelines.  At this time an IFCC working group is addressing harmonization of Troponin I.  A high priority was assigned because of the very common use of Troponin I for diagnosing myocardial infarction.

Troponin T

Troponin T is available from a single IVD manufacturer, consequently harmonization is adequate.

Urea N (BUN)

EQA data from two Japanese programs in 2015 that used commutable samples had 3.0% and 2.4% CV among different laboratories and methods.  EQA data from the College of American Pathologists in 2015 (non-commutable samples) had 22.8% difference between maximum and minimum values reported by individual laboratories and 7.0% CV% among mean values for different peer groups.  Taken together these data support adequate harmonization for serum or plasma urea (or urea nitrogen) measurement procedures.  A reference material, serum-based reference material and a reference measurement procedure are listed by JCTLM.

Uric acid

EQA data from two Japanese programs in 2015 that used commutable samples had 2.6% and 2.0% CV among different laboratories and methods.  EQA data from the College of American Pathologists in 2015 (non-commutable samples) had 9.2% difference between maximum and minimum values reported by individual laboratories and 2.3% CV% among mean values for different peer groups.  Taken together these data support adequate harmonization for serum or plasma uric acid measurement procedures.  A reference material, serum-based reference material and a reference measurement procedure are listed by JCTLM.

Valproic Acid

Valproic acid is used primarily to control seizures by decreasing the severity and frequency.  It can also be prescribed for bipolar disorder, especially mania, and can be used for recurrent migraine headaches. A valproic acid test is requested when the valproic acid treatment for patient is started or the medication is changed. It can also be ordered when drug side effects (such as gastrointestinal symptoms, dizziness, unusual weight gain or loss, tremors, blurred or double vision, mood swings, unusual bruising and bleeding, etc.) are suspected. The usual therapeutic levels of total valproic acid (bound and unbound) in blood are between 50 and 100 mg/L for the treatment of epilepsy. A free valproic acid test may be ordered in conditions to affect the protein-bound drug balance, such as low protein level of hypoalbuminemia, which can increase the risk of valproic acid overdose in blood (1).

In the comparability study for valproic acid concentrations between 5 different analyzers  [Architect and AxSym (Abbott Laboratories), Dimension Vista and Dimension Xpand (Simens Healthcare Diagnostics Inc), cobas c 501 module (Roche Diagnostics GmbH)], the relative mean difference (method comparisons against cobas c 501 module as arbitrary standard) were less than ± 12.7% (+12.7% for Dimension Vista vs. cobas c 501, +6.8% for Dimension Xpand vs. cobas c 501, -7.6% for AxSym vs. cobas c 501, and -4.7% for  Architect vs. cobas c 501) in 79 to 80 leftover human samples (2).

There is no suitable proficiency testing information from commutable samples. In a multilaboratory commutability evaluation study in the Netherland within the framework of the Dutch Calibration 2000 project, the commutable proficiency testing materials for carbamazepine, valproic acid and tobramycin have been developed and future studies will indicate whether the commutable materials could be used for harmonization between laboratories and methods (3, 4).

The reference material SRM 1599 (anticonvulsant drug level assay standard) produced by NIST was discontinued and it was delisted from JCTLM database on 21 April, 2020 (5). NIST does not intend to replace this SRM. In practice, many IVD manufacturers use a reference material from the US Pharmacopeia (USP39-NF) to prepare calibrators in human serum.

There is no reference material, reference measurement procedure or reference measurement procedure laboratory information listed at JCTLM database.

References

1. Valproic acid, Lab Tests Online-US. https://labtestsonline.org/tests/valproic-acid, Accessed 21 June 2020.
2. Shipkova M, Petrova DT, Rosler AE, Orth M, Engelmayer J, Wieland E, Brandhorst G, Oellerich M. Comparability and imprecision of 8 frequently used commercially available immunoassays for therapeutic drug monitoring. Ther Drug Monit. 2014 Aug;36(4):433-41. (Supplemental Digital Content 3, http://links.lww.com/TDM/A72; Supplemental Digital Content 4, http://links.lww.com/TDM/A73)
3. Robijns K, Boone NW, Kuypers AW, Jansen RT, Neef C, Touw DJ. A multilaboratory commutability evaluation of proficiency testing material for carbamazepine and valproic acid: A study within the framework of the Dutch Calibration 2000 Project. Ther Drug Monit 2015;37:445–50.

Vancomycin

Vancomycin is one of the most widely used antimicrobial agents for treatment of gram-positive infections including methicillin-resistant Staphylococcus aureus (MRSA), and highly utilized for therapeutic drug monitoring (TDM) purposes for dose-adjustment and to ensure patients are in an appropriate therapeutic range. Vancomycin TDM primarily aims at balancing therapeutic efficacy against the risk of nephrotoxicity. Several studies have associated high vancomycin trough concentrations with a greater incidence of nephrotoxicity, and low trough concentrations with an increased occurrence of resistant strains of S. aureus and complications from reoccurring infections. However, a plethora of analytical methodologies are available and utilized in clinical practice and clinical trials.

Based on these issues the American Society of Health-System Pharmacists, the Infectious Diseases Society of America, and the Society of Infectious Diseases Pharmacists have published expert panel recommendations for vancomycin TDM, recommending trough serum concentrations of vancomycin of 15–20 mg/L in complicated infections (1,2).

Substantial between-method differences in vancomycin immunoassays exist, as well as bias between immunoassays and LC-MS/MS methods, likely due to differences in specificity to quantitate the free and bound forms of vancomycin. The analytical differences have been shown to cause significant clinical discordance, particularly in neonates and individuals with renal disease (3,4).

In the comparison study for vancomycin concentrations in 4 different immunoassays [Architect i2000SR (Abbott Laboratories), Cobas 8000 c702 (Roche Diagnostics GmbH), Ortho Vitros 5000 (Ortho Clinical Diagnostics) and Dimension Vista 15000 assay (Simens Healthcare Diagnostics Inc)] to LC-MS/MS assay, a mean proportional difference was within 10% for 3 out of 4 immunoassays except the Cobas 8000 assay that showed a substantial proportional difference of +22% as compared to LC-MS/MS for 99 leftover lithium-heparin blood samples from 68 different patients (4).

In the comparability study for vancomycin concentrations between 6 different analyzers [Architect and AxSym (Abbott Laboratories), cobas c 501 module and COBAS INTEGRA 800 analyzer (Roche Diagnostics GmbH), Dimension Vista and Dimension Xpand (Simens Healthcare Diagnostics Inc)], systemic deviations (relative mean differences) of -7.4%, -7.0% and 12.2% were found among manufacturers (cobas c 501 vs. COBAS INTEGRA 800, AxSym vs. Architect and Dimension Xpand vs. Dimension Vista, respectively) in 80-82 leftover human samples (5).  The largest relative mean difference for vancomycin concentration was -19.9% with the 95% CI of -44.3% to +4.5% for 81 patient samples for Architect compared to cobas c 501 (method comparisons against cobas c 501 module as arbitrary standard) (5).

There is no suitable proficiency testing information from commutable samples.

A LC-MS/MS reference measurement procedure for vancomycin in fresh, or frozen, human blood serum is listed by JCTLM (6).

References

1. Rybak M, Lomaestro B, Rotschafer JC, et al: Therapeutic drug monitoring of vancomycin in adult patients: A consensus review of the American Society of Health-System Pharmacists, the Infectious Diseases Society of America, and the Society of Infectious Diseases Pharmacists. Am J Health Syst Pharm 2009;66:82-98.
2. Ye Z-K, Li C, Zhai S-D (2014) Guidelines for Therapeutic Drug Monitoring of Vancomycin: A Systematic Review. PLoS ONE 9(6): e99044. doi:10.1371/journal.pone.0099044.
3. Crandon JL, MacVane SH, Nicolau DP. Clinical laboratory-based assay methodologies may underestimate and increase variability of vancomycin protein binding in hospitalized patients. Pharmacotherapy. 2014 Feb; 34(2):203-9.
4. Oyaert M, Peersman N, Kieffer D, et al. Novel LC-MS/MS method for plasma vancomycin: Comparison with immunoassays and clinical impact. Clinica Chimica Acta. 2015;441:63-70.
5. Shipkova M, Petrova DT, Rosler AE, Orth M, Engelmayer J, Wieland E, Brandhorst G, Oellerich M. Comparability and imprecision of 8 frequently used commercially available immunoassays for therapeutic drug monitoring. Ther Drug Monit. 2014 Aug;36(4):433-41.
6. König K, Kobold U, Fink G, Leinenbach A, Dülffer T, Thiele R, Zander J, Vogeser M. Quantification of vancomycin in human serum by LC-MS/MS. Clin Chem Lab Med. 2013 Sep;51(9):1761-9.

Vitamin B12

Vitamin B12 (cobalamin) is a water-soluble vitamin that is plays a critical role in hematopoietic and neural function due to its role as a cofactor for methionine synthase and methylmalonyl-CoA mutase (1).  There are a number of notable causes of vitamin B12 malabsorption or deficiency, including pernicious anemia.  Thus, accurate measurement would theoretically provide a method to screen for low serum levels in the setting of other symptoms such as megaloblastic anemia.  However, the limitations of current assays have negatively affected their clinical utility.  Notably, given the functional relationship between B12 deficiency and elevated methylmalonic acid (MMA), MMA measurement has been proposed as a surrogate marker in order to overcome known deficiencies in B12 measurement by immunoassay.

Two independent studies (2,3) using 69 and 24 serum samples, respectively, have evaluated B12 immunoassay measurements using platforms from 4 major manufacturers: Abbott, Beckman Coulter, Roche, and Siemens.   In both cases, three platforms (Abbott, Roche, Siemens) showed acceptable agreement (in the case of (3), +/- 17.7%), while one (Beckman Coulter Unicel) exhibited significant negative bias (-108 pmol/L at 329 pmol/L) relative to the others.  Although some have argued that this performance represents an adequate level of harmonization (4), the current state of the field prevents the effective establishment of a global cutoff.  Notably, even when acceptable agreement was seen, within-method variability was >=9% for three of the evaluated methods.  For three pooled serum samples, commutability was assessed: two liquid-pooled serum samples were found to be commutable, while a lyophilized pool exceeded the standards for commutability.  

A reference material is available from WHO as IS 03/178, Vitamin B12, Serum Folate and holoTC (International standard). Its commutability with clinical samples has not been evaluated.

References:

1. Stapler SP. Clinical practice.  Vitamin B12 deficiency.  N Engl J Med.  2013; 368(2): 149-60.
2. Ispir E, Serdar MA, Ozgurtas T, Gulbahar O, Akin KO, Yesildal F, Kurt I. Comparison of four automated serum vitamin B12 assays.  Clin Chem Lab Med.  2015; 53:1205-13.
3. Kristensen GB, Rustad P, Berg JP, Aakre KM. Analytical Bias exceeding desirable quality goal in 4 out of 5 common immunoassays: results of a native single serum external quality assessment for cobalamin, folate, ferritin, thyroid-stimulating hormone, and free T4 analysis.  Clin Chem.  2016; 62:1255-63.
4. Ferraro S, Panteghini M. Folate and vitamin B12 assays after recalibration to the WHO International Standard 03/178: making the interpretation as simple as possible, but not simpler. Clin Chem Lab Med. 2019; 57(8): 1112-1114.

Vitamin D

Serum total 25-hydroxyvitamin D [25(OH)D] concentration is used to assess an individual’s vitamin D status. Vitamin D deficiency has long been associated with osteoporosis, bone fractures, and osteomalacia.⁽¹⁾  Vitamin D deficiency has been associated with obesity, diabetes, hypertension, depression, fibromyalgia, chronic fatigue syndrome, osteoporosis and neuro-degenerative diseases including Alzheimer’s disease. Vitamin D deficiency has also been associated with increased risk of the development of cancers, especially breast, prostate, and colon cancers.⁽²⁾  Vitamin D may play a role in controlling the immune system (possibly reducing one’s risk of cancers and autoimmune diseases), increasing neuromuscular function and improving mood, protecting the brain against toxic chemicals, and potentially reducing pain.⁽³⁾   However, it should be noted that while these associations have been noted in epidemiological studies there is limited evidence for beneficial effects of vitamin D supplementation other than on bone and muscle health.

 Research has consistently shown that there is a great deal of variation in 25(OH)D assays. This assay variation impedes pooling of 25(OH)D results from different studies in systematic reviews for the specific purpose of determining dose-response and/or clinical cut-points. The VDSP was established to correct this problem.⁽⁴⁾

There are currently three organizations that are working on various approaches to harmonize the measurement of vitamin D assays. Their activities are both complimentary and different in focus.

1) NIH Vitamin D Standardization Programme (VDSP)

The objective of the programme is to improve the detection, evaluation, and treatment of vitamin D deficiency and insufficiency by promoting the standardized laboratory measurement of serum total 25-hydroxyvitamin D [25(OH)D]. VDSP promotes the standardized laboratory measurement of total 25(OH)D (a measure of vitamin D status) to improve clinical and public health practice worldwide.

• VDSP Objectives
1. Standardize vitamin D measurement in national health surveys worldwide.
2. Promote standardized 25(OH)D measurement by assay manufacturers and clinical and research laboratories.
3. Conduct an international research program devoted to 25(OH)D and its laboratory measurement.

All VDSP programs are open to assay manufacturers, clinical and commercial laboratories, national survey laboratories, and research laboratories.

2) Centre for Disease Control (CDC) (https://www.cdc.gov/labstandards/vdscp.html)

CDC’s Vitamin D Reference Method Laboratory and the Vitamin D Standardization–Certification Program (VDSCP) work to

• provide reference measurements for total 25-hydroxyvitamin D (sum of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3),
• assess the accuracy and precision of vitamin D tests, and monitor their performance over time, and
• provide technical support to external quality assurance programs, proficiency testing programs, and research studies that ensure vitamin D tests are accurate and reliable.

3) The IFCC Committee on Bone Metabolism

Objectives related to Vitamin D include the following: (https://www.ifcc.org/ifcc-scientific-division/sd-committees/c-bm/)

• Re-evaluate current VDSP performance guidelines for 25(OH)D
• Establish VDSP performance guidelines for 24,25(OH)2D, C3-epimer and vitamin D binding protein
• Post-survey of the standardization effects
• Propose services to reassess the true value of 25OHD obtained in former epidemiological or interventional studies that had used non-standardized methods

A potential exists for duplication of effort and services among the three programs. ICHCLR encourages the programs to combine efforts where possible to avoid conflicting recommendations.

 References:

1. Cleveland Clinic.org
2. Naeem, A, /Vitamin D Deficiency – An Ignored Epidemic; Int J Health Sci (Qassim). 2010 Jan; 4(1): V-VI
3. Iqbal R, Khan A. Possible causes of vitamin D deficiency. J Pak Med Asso. 2010;60(1):1–2. 
4. Phinney K, et al, Value of Assignment of Vitamin D Metabolites in Vitamin D Standariztion Program (VDSP) Serum Samples, J AOAC Int. 2017 September 01; 100(5): 1253-1259. Dpo“10.5740/jaoacint.17-0204.

28 April 2021