Measurands

Akaline 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-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.

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.

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.

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

Adequate

Blood gasses (pH, pO2, pCO2, oximitry)

Adequate

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

Adequate

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. 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. CCLM 2000;38:1301-14.

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.

Chloride

Adequate

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.  Harmonization for HDL cholesterol is dependent on improved reagent technology 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.  Harmonization for LDL cholesterol is dependent on improved reagent technology 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.

Cholesterol, total

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.

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.

Creatine Kinase-MB (CK-MB)

Adequate

Creatinine

Adequate

CSF Proteins

IFCC has a working group addressing harmonization of CSF proteins.

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.

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.

Ethyl Alcohol

Ethyl Alcohol

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

Folate is required for the normal production of red blood cells (RBCs). Complications resulting from a deficiency in folate are associated with increased risks of adverse health outcomes from:

• megaloblastic anemia, which means the RBCs are larger than normal and not fully developed
• low levels of white blood cells and platelets
• serious birth defects in the spinal cord and brain of a developing fetus, which are called neural tube defects.

According to the CDC, the folate assays have been plagued with comparability issues over many years.  Performance has improved, but the lack of a NIST reference material with certified concentrations for total folate and the various folate forms makes harmonization difficult.  CDC is closely collaborating with NIST on such a serum material which may be available soon but may not have certified values for total folate.  Whole blood folate is another set of difficulties (different folate forms depending on MTHFR genotype, different polyglutamates that need to be deconjugated to monoglutamates, plus the usual folate instability issues that are also present to some extent with serum).

The 2015 College of American Pathologists Ligand Survey showed large within-platform (~7–30% and ~10–50% for serum and RBC folate, respectively) and even larger across-platform variability (~1.5- to 6-fold and ~8- to 40-fold differences for serum and RBC folate), respectively. Although the PT materials were likely non-commutable with patient samples, the large differences among measurement procedures  is likely consistent with a need for harmonization.

References:

1. 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.

Glucose

Adequate

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.

HBV Ag and anti-HBs Ab

Hemoglobin A1c (HbA1c)

Adequate

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 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 Ag with HIV-1 and HIV-2 Abs

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 antibodies to myeloperoxidase

In development.

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 binding capacity (unsaturated )

Lactate

Adequate

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.

Magnesium

Adequate

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.

NT-proBNP

NT-proBNP is available from a single IVD manufacturer, consequently harmonization is adequate.

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 is addressing approaches to standardizing PTH measurement procedure results.

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.

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

Adequate

Potassium

Adequate

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

Adequate

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.

Sodium

Adequate

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 is working to harmonize results from measurement procedures for FT4.

Treponema pallidum (syphilis)

Triglycerides

Adequate

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)

Adequate

Uric acid

Adequate

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.