Q&A column

Editor: Frederick L. Kiechle, MD, PhD

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Q. Is the evaluation of gene copies by RT-PCR or multiplex ligation-dependent probe amplification a qualitative or quantitative assay? Copy number analysis of genes or chromosomes determines a numerical value, with a normal autosomal count being two. However, an FDA-approved microarray test (CytoScan Dx assay, Thermo Fisher Scientific) is labeled as a qualitative assay for the detection of copy number variations.

A. September 2020—This is an interesting question. The intended use section of the FDA’s decision summary for the CytoScan Dx assay refers to the product as a qualitative assay. The summary’s test principles section explains that CytoScan Dx “reports the copy number state (loss, gain), copy number (i.e., 0, 1, 2, 3, or 4 or greater), and position/location of chromosomal segment copy number changes across the queried genome.”¹

For a genotyping or sequencing test that determines germline gene copy number from an assay with numerical output, I have used the term semiquantitative. An article I found helpful in answering this question (Jennings, et al.) defines semiquantitative testing only in general terms.² It does not indicate whether semiquantitative is a standard designation for DNA-based tests.

Following are explanations of the three types of assays used for DNA-based tests.

Quantitative assay: an assay that gives an exact numeric quantitative measure of the amount of a substance in a sample. Examples include viral load and tumor burden determinations.

Qualitative assay: an assay that, in general, gives a pass or fail, or positive or negative, or some sort of narrow qualitative gradation rather than an exact quantity. An example is traditional genotyping.

Semiquantitative assay: an assay in which quantitative data are used to produce a qualitative result. For example, data from multiplex ligation-dependent probe amplification, qPCR, etc., are reported as zero, one, two, or three copies, as determined from a numerical output range.

Genotyping does not require precise measurement across the full range of output possibilities. The alternate allele can be present in zero, one, two, or three copies (or more if the laboratory validates greater copy numbers) but not 2.1, 2.5, or 3.15 copies. Therefore, the data, although quantitative in nature, are used to reach a qualitative determination.

1. U.S. Food and Drug Administration. Evaluation of automatic class III designation for Affymetrix CytoScan Dx Assay decision summary. www.accessdata.fda.gov/cdrh_docs/reviews/K130313.pdf.
2. Jennings L, Van Deerlin VM, Gulley ML. Recommended principles and practices for validating clinical molecular pathology tests. Arch Pathol Lab Med. 2009;133(5):743–755.

Lora J. H. Bean, PhD
Senior Director, Laboratory Quality Assurance
Clinical Laboratory Director, PerkinElmer Genomics
Duluth, Ga.
Member, CAP/ACMG Biochemical and Molecular Genetics Committee

Q. How does using sodium heparin, in an attempt to reduce EDTA-induced platelet clumps, affect the platelet count?

A. There is little data on the use of sodium heparin to mitigate EDTA-induced platelet clumps. However, studies show that sodium heparin can have a significant effect on the platelet count.

Thompson, et al., measured the effects of a variety of anticoagulants on complete blood count parameters every two hours for an eight-hour period using whole blood collected from nine healthy volunteers.¹ In that study, sodium heparin anticoagulant generated greater variation in platelet count and mean platelet volume (MPV) than did a variety of citrate- and EDTA-based anticoagulants, while other CBC parameters were relatively stable. The platelet count showed an overall 16 percent mean decrease in sodium heparin in the first two to four hours after collection, as compared with a 2.5 to 6.5 percent decrease or increase among samples collected in the other anticoagulants. A cause for the decreased platelet count in the setting of sodium heparin is not described but is presumed to be due to agglutination.

In parallel with the decrease in platelet count in sodium heparin in this study was an increase in mean platelet volume. Morphologic visualization showed a platelet shape change, from discoid to spherical, underlying this transient increase in MPV. MPV also showed an inverse correlation with plasma osmolality, but it was not significantly altered by plasma pH, which was highest with sodium heparin. Sodium heparin also reportedly affected the accuracy of the white blood cell count but not the red blood cell count.

A 2019 study reports the effects of anticoagulants on the platelet count in healthy subjects and patients with thrombocytopenia from a variety of causes.² Sodium heparin anticoagulant was not assessed in that study. Nevertheless, the platelet count is variably affected by anticoagulation in vitro. The most well-known form of laboratory-induced pseudothrombocytopenia, caused by antibody-mediated platelet agglutination upon exposure to K3EDTA anticoagulant and found in a subset of the population, is well described in the literature. See also CAP TODAY coverage of this topic.^3-9

Additional research demonstrating the feasibility of using sodium heparin to achieve an accurate platelet count in individuals whose platelets clump in EDTA could be useful to many laboratories, and the CAP Hematology/Clinical Microscopy Committee encourages its undertaking.

1. Thompson CB, Diaz DD, Quinn PG, Lapins M, Kurtz SR, Valeri CR. The role of anticoagulation in the measurement of platelet volumes. Am J Clin Pathol. 1983;80(3):327–332.
2. Podda GM, Pugliano M, Casazza G, et al. Measurement of platelet count with different anticoagulants in thrombocytopenic patients and healthy subjects: accuracy and stability over time. Haematologica. 2019;104(12):e570–e572.
3. Kroft SH. Q&A Column. CAP TODAY. 2001;15(4):94–95.
4. Ansari-Lari MA, Fuller P. Q&A Column. CAP TODAY. 2009;23(1):96.
5. Etzell JE. Q&A Column. CAP TODAY. 2014;28(1):66.
6. Etzell JE, Perkins S. Q&A Column. CAP TODAY. 2014;28(6):84–85.
7. Darden T. Q&A Column. CAP TODAY. 2016;30(8):79.
8. Mahe E. Q&A Column. CAP TODAY. 2017;31(2):64.
9. Chandler WL. Best of Q&A Series. CAP TODAY. 2019;33(6):66.

Alexandra E. Kovach, MD
Associate Professor of Clinical Pathology
Keck School of Medicine, University of Southern California
Director of the Hematology Laboratory,
Children’s Hospital Los Angeles
Member, CAP Hematology/Clinical Microscopy Committee

Q. How do you know whether thyroid-stimulating hormone isoforms have been measured in an assay when the TSH levels are very high and free T4 is considerably less than the reference interval (i.e. less than 50 percent of the reference interval)?

A. Measurement of thyroid-stimulating hormone is recommended as a first-line test to screen for thyroid dysfunction, followed by measurement of free T4 in cases with abnormal TSH levels.¹ An elevated TSH with a suppressed free T4 is most often characteristic of primary hypothyroidism. In such cases, clinical correlation should identify patient history and symptoms consistent with hypothyroidism.

However, there are several conditions in which measurement of TSH is not the only indicator of thyroid dysfunction. Clinical correlation is always indicated to determine whether preanalytical or analytical issues have resulted in incongruous results.

In people with high TSH concentrations and low free T4 concentrations, it is prudent to exclude medication noncompliance in those treated with thyroxine. If primary hypothyroidism is not suspected, it is necessary to rule out such factors as use of other medications or the presence of heterophile or human anti-mouse antibodies, which may cause falsely high or falsely low results, depending on the nature of the antibody. Anti-TSH autoantibodies and macro-TSH are also possible causes of unexpectedly elevated TSH concentrations.² However, patients with central hypothyroidism can have high circulating concentrations of TSH due to overproduction of bioinactive TSH isoforms.¹

TSH is a heterogeneous molecule, primarily owing to its high degree of glycosylation. It has three N-glycosylation sites, and the specific carbohydrate structure of the glycans at these sites appears to affect bioactivity and antibody recognition.^3,4 This may have biological relevance, as multiple studies have suggested that TSH produced in hypothyroid individuals is more sialylated and less fucosylated than TSH from euthyroid individuals.^5,6 These highly sialylated and afucosylated isoforms of TSH have been reported to be less bioactive than euthyroid pituitary TSH.

Literature suggests that variation in TSH glycosylation affects the results of TSH immunoassays. One case report highlighted a clinically euthyroid-appearing patient with profoundly elevated TSH and low free T4. The report concluded that the elevated TSH in this patient was due to variation in glycosylated isoforms of TSH, based on studies in which the patient’s TSH was shown to bind to anti-TSH antibodies with similar affinity to recombinant TSH.⁷ Recombinant TSH, which is typically produced in Chinese hamster ovary (CHO) cells and has a high sialic acid content, has higher affinity to anti-TSH antibodies than does the pituitary-derived WHO international reference preparation for TSH.^8-10 Other antibody-binding studies using monoclonal anti-TSH antibodies from a variety of manufacturers also suggest that the degree of sialylation and fucosylation of TSH affects antibody epitope recognition.⁴

Immunoassay methods for TSH historically have not been well harmonized,^11,12 but harmonization efforts have increased in recent years. While this appears to have improved the accuracy of TSH measurements among euthyroid individuals, whether assays are equally sensitive to the differentially sialylated and fucosylated isoforms reported in hypothyroid patients is still unclear.¹³ Contemporary TSH assays are standardized to the WHO IRP for 80/558; however, the specific capture and detection antibody pair used may affect the ability of an assay to detect specific isoforms.⁴ Information regarding the degree to which specific assays are sensitive to specific isoforms of TSH is, however, very limited. Most assay package inserts do not report the epitope specificity of the monoclonal antibody pairs used, and there is a dearth of literature regarding the ability of major manufacturers’ assays to detect TSH isoforms. Further, many isoforms exist due to the microheterogeneity of glycosylated isoforms of TSH, and evaluation of each is challenging.

Bioassays for TSH have been suggested as a means to identify whether circulating TSH is bioactive, as highly sialylated TSH isoforms, such as recombinant TSH or hypothyroid TSH, have been suggested to be more immunoreactive but less bioactive than TSH from euthyroid individuals.¹⁴ However, these assays are nonstandardized, complex to perform, and not widely or routinely available. Mass spectrometry has been employed in studies to identify glycoforms of TSH,¹⁵ but methods have not been developed for clinical use.

In summary, the response of TSH immunoassays to differently glycosylated isoforms of TSH is not well defined. This issue highlights the role of careful clinical correlation in interpreting laboratory results. Incongruence between TSH measurements and the clinical picture should prompt further investigation to rule out preanalytical causes of discordant results, such as medication, and lead the laboratory to investigate other types of analytical interferences, such as biotin, interfering antibodies, etc. Elevated TSH concentrations secondary to detecting TSH isoforms is, at this point, a diagnosis of exclusion.

However, as the phenomenon of variation in glycosylated TSH isoforms in hypothyroidism is relatively well documented, future harmonization efforts should address the issue. Future studies should evaluate the ability of TSH assays to detect not only euthyroid pituitary TSH but also TSH from recombinant sources or hypothyroid sources that exhibit alternate glycosylation patterns.

1. Garber JR, Cobin RH, Gharib H, et al. Clinical practice guidelines for hypothyroidism in adults: cosponsored by the American Association of Clinical Endocrinologists and the American Thyroid Association [published correction appears in Endocr Pract. 2013;19(1):175]. Endocr Pract. 2012;18(6):988–1028.
2. Spencer CA. Assay of thyroid hormones and related substances. In: Feingold KA, ed. Endotext. MD Text; 2017. www.endotext.org/chapter/assay-of-thyroid-hormones-and-related-substances3.
3. Szkudlinski MW, Fremont V, Ronin C, Weintraub BD. Thyroid-stimulating hormone and thyroid-stimulating hormone receptor structure-function relationships. Physiol Rev. 2002;82(2):473–502.
4. Donadio S, Morelle W, Pascual A, Romi-Lebrun R, Michalski JC, Ronin C. Both core and terminal glycosylation alter epitope expression in thyrotropin and introduce discordances in hormone measurements. Clin Chem Lab Med. 2005;43(5):519–530.
5. Trojan J, Theodoropoulou M, Usadel KH, Stalla GK, Schaaf L. Modulation of human thyrotropin oligosaccharide structures—enhanced proportion of sialylated and terminally galactosylated serum thyrotropin isoforms in subclinical and overt primary hypothyroidism. J Endocrinol. 1998;158(3):359–365.
6. Schaaf L, Trojan J, Helton TE, Usadel KH, Magner JA. Serum thyrotropin (TSH) heterogeneity in euthyroid subjects and patients with subclinical hypothyroidism: the core fucose content of TSH-releasing hormone-released TSH is altered, but not the net charge of TSH. J Endocrinol. 1995;144(3): 561–571.
7. Gauchez AS, Pizzo M, Alcaraz-Galvain D, et al. TSH isoforms: about a case of hypothyroidism in a Down’s syndrome young adult. J Thyroid Res. 2010;2010:703978.
8. Donadio S, Pascual A, Thijssen JH, Ronin C. Feasibility study of new calibrators for thyroid-stimulating hormone (TSH) immunoprocedures based on remodeling of recombinant TSH to mimic glycoforms circulating in patients with thyroid disorders. Clin Chem. 2006;52(2):286–297.
9. Gaines Das RE, Bristow AF. The second international reference preparation of thyroid-stimulating hormone, human, for immunoassay: calibration by bioassay and immunoassay in an international collaborative study. J Endocrinol. 1985;104(3):367–379.
10. Canonne C, Papandreou MJ, Medri G, Verrier B, Ronin C. Biological and immunochemical characterization of recombinant human thyrotrophin. Glycobiology. 1995;5(5):473–481.
11. Rawlins ML, Roberts WL. Performance characteristics of six third-generation assays for thyroid-stimulating hormone. Clin Chem. 2004;50(12):2338–2344.
12. Silvio R, Swapp KJ, La’ulu SL, Hansen-Suchy K, Roberts WL. Method specific second-trimester reference intervals for thyroid-stimulating hormone and free thyroxine. Clin Biochem. 2009;42(7–8):750–753.
13. Thienpont LM, Van Uytfanghe K, Van Houcke S; IFCC Working Group for Standardization of Thyroid Function Tests. Standardization activities in the field of thyroid function tests: a status report. Clin Chem Lab Med. 2010;48(11):1577–1583.
14. Estrada JM, Soldin D, Buckey TM, Burman KD, Soldin OP. Thyrotropin isoforms: implications for thyrotropin analysis and clinical practice. Thyroid. 2014;24(3):411–423.
15. Morelle W, Donadio S, Ronin C, Michalski JC. Characterization of N-glycans of recombinant human thyrotropin using mass spectrometry. Rapid Commun Mass Spectrom. 2006;20(3):331–345.

Sridevi Devaraj, PhD, D(ABCC)
Director, Clinical Chemistry, Texas Children’s Hospital, Professor, Pathology and Immunology
Baylor College of Medicine
Member, CAP Clinical Chemistry Committee