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.