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Letters to the Editor |
Clin. Chem. Lab., Satakunta Central Hosp., FIN-28500 Pori, Finland
To the Editor
We recently found unsatisfactory agreement of luteinizing
hormone (hLH) results of the hLH Spec assay (Wallac) and Opus Magnum
hLH assay (Behring Diagnostics) (Fig. 1
A). Particularly disturbing were the occasional marked
discrepancies.
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According to the manufacturers, both assays are standardized against
the WHO 2nd International Standard for pituitary hLH for immunoassay
(code 80/552). Both assays are claimed to be highly specific, with
cross-reactivities for hCG of <0.02% (AutoDELFIA) and <0.12% (Opus
Magnum); their respective detection limits are 0.05 and 0.6 IU/L. Among
35 samples (Fig. 1A
) striking discrepancies were seen in 3, for which
the Opus results were below the stated detection limit (0.6 IU/L)
whereas the DELFIA results were 5.3, 26.1, and 35.3 IU/L.
An immunologically anomalous, biologically active common genetic variant has been described in the Finnish population (1)(2)(3) and recently in other populations as well (4)(5)(6). The frequency of the variant, either heterozygous or homozygous, is 8–30% (7).
For further analyses we sent the samples to Kim Pettersson at the
University of Turku. Use of the double-monoclonal I3/A2 intact LH assay
described by that laboratory earlier (2) yielded an
excellent correlation with the results from the Opus Magnum assay (Fig. 1B
). This double-monoclonal I3/A2 intact LH assay, in which the capture
antibody detects an epitope present only in the intact dimer whereas
the tracer antibody is directed against the alpha subunit, has been
shown to be totally unreactive with the hLH variant (8).
As shown in Fig. 1C
and D, hLH in the three anomalous samples did not
react in this assay, indicating a homozygous variant hLH; 9 other
samples from the 35 women appeared to be heterozygous. The data in
Figs. 1C
and 1D
suggest that the Opus Magnum assay uses at least one
antibody similar to those in the double-monoclonal I3/A2 intact LH
assay.
References
Behring Diagnostics Inc., 151 University Ave., Westwood, MA 02090
To the Editor:
Glycoprotein hormones like LH occur as a complex mixture of circulating isoforms. Standardization and agreement among immunoassays is notoriously problematic, most probably attributable to the use of highly specific but different monoclonal antibodies in the various assays. Recent publications cited by Eskola-Williams above have demonstrated that certain LH tests underestimate or even completely fail to detect LH in some individuals who were shown by functional bioassays to have physiologically active molecules. Nucleotide mutations within codons 8 and 15 in the LH ß-subunit gene have been described and appear to account for the majority of these immunologically unrecognizable LH isoforms. The frequency of this variant form of LH has been shown to be relatively high in the Finnish population, ~28%.
The OpusTM LH immunoassay (Behring Diagnostics) utilizes a polyclonal/monoclonal antibody pair, both specific to the ß-subunit. The monoclonal antibody was selected for its high degree of specificity for LH. Method comparison studies were performed with several commercially available assays, including DELFIA, and initial correlation coefficients were quite good (all r >0.9, many >0.95). Additionally, serial blood samples were obtained from 20 different women throughout the course of their menstrual cycles and clinically evaluated for LH, follitropin (FSH), and estradiol on the Opus and other commercial systems. The Opus LH assay correctly identified the follicular, ovulatory, and luteal phase of the menstrual cycle in all cases, peaking on the same day as or one day earlier than the estradiol peak. In certain instances, however, the recovery of LH was lower that in some other commercial kits. The low bias was not attributed to calibration because recoveries were variable within the same method comparison. Discrepancies in recovery were believed to result from the differential recognition of various LH isoforms in individual patients. We did not, however, observe any samples in which the Opus assay totally failed to recognize LH.
Variable recoveries among LH assays utilizing monoclonal antibodies are common and, given that the clinical utility of the assay was demonstrated, the absolute value for LH seemed less important. As literature accounts became increasingly available describing the LH variant commonly seen in the Finnish population, however, more investigation into the specificity of the Opus reagents was undertaken. Results suggest that the antibody used in our current kit does not recognize the LH variant described in the Finnish study conducted by Eskola-Williams. Why we did not observe the degree of discordance described in the Finnish study is probably related to the relatively low frequency of the variant in the population we sampled, and similar specificity of the reagents used in several of the other assays. Indeed, good correlation was observed when the Opus assay was compared with the I3/A2 "intact" LH assay described in the Finnish study.
We agree that the value of a substantial number of LH samples will be underestimated in the Finnish population. The number of samples testing negative (homozygotes) for LH, however, will be relatively small. The references cited by Eskola-Williams describe the 28% population frequency as consisting of 24% heterozygous and 4% homozygous. According to our studies with menstrual cycle panels, accurate clinical information regarding cycle phase can still be obtained in most cases. We also agree that failure to recognize physiologically active LH, even in a small subset of patients, is undesirable, and test results not agreeing with clinical and other biochemical data should be interpreted with caution. A second-generation Opus LH assay has been developed that recognizes the LH variant described here while retaining excellent specificity. This new assay is currently available for research evaluation and will be commercially available beginning in the second quarter of 1997.
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) homozygotes, (
) heterozygotes, and (x)
normal type for the variant LH allele. Sb =
standard error of slope,
Sy/x = standard error of
estimates.