{149} #{149} #{149} - Clinical Chemistry

ies with those by Western blot. We report the efficacy of a simple partial correction to EIA results for reducing falsepositive results caused by cross-reactivity. Serum was collected from 60 patients and divided into aliquots. One aliquot of each specimen was sent to a university laboratory for analysis of HSV1- and 2-specific antibodies by Western blot (1); the duplicate aliquots were frozen for later analysis by EIA [Sigma Immunoassay (SIA) kits for herpes 1 and herpes 2 (2)]. If EIA results were positive for either or both herpes types, the greater of the

two numerical results was multiplied by 50%. This estimated cross-reactivity was subtracted from the smaller of the two original results. SIA correctly identified (Figure 1) all patients who were HSV2-positive by Western blot (100% sensitive) but, owing to cross-reactivity, had several false-positive results (specificity = 59%). Using the 50% correctionfor cross-reactivity improved the specificity to 100%. In one case, however, these adjustment calculations changed a positive result to a false-negative outcome and another value was changed to a “borderline” negative, reducing assay sensitivity from 100% to 90%. The raw uncorrected SIA results for HSV1 IgG antibody, comparedwith Western blot (Figure 1), illustrate not only

the high proportion of false positives observed (48% specificity), but also an additional problem not observedin the HSV2 results. That is, six Western blot-positive results were negative by SIA (sensitivity = 8 1%). Correcting for cross-reactivity increased the specificity to 96%, but the sensitivity remained essentially unchanged at 79%. To adjust the uncorrected data in Figure 1, we used the simple 50% estimate of cross-reaction. At the completion of the study, we tried recalculating the actual cross-reactivi-

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ties in both assays. We found that the average crossreactivity of the HSV2 antibody in the HSV1 assay was actually greater (67%) than the average cross-reactivity of HSV1 antibody in the HSV2 assay (38%). Having achieved specificities of 96% and 100% with the simple 50% correction, we concluded that further refinement of the crossreactivity correction would have added little. The six false-negative SIA results in the HSV1 assay remain a concern.Sigma described a correlation study with HSV assays from Cordia Laboratories (2), also by ELA; of the 106 sera they analyzed, only one specimen was falsely negative by Sigma, and it was negative for both HSV1 and 2. Ashley et al. (1) also compared Western blot with an immunoassay system (3, 4). They evaluated sera from patients with culture-proven genital herpes infections for specific antibodies to HSV1 and HSV2. When serum samples were drawn 21-40 days after onset, Western blot correctly identified 99% of the HSV2-positive patients and immunoassay identified 96%. Western blot identified 94% of the HSV1-positive patients, but immunoassay failed to detect 47% (eight of 17). Because this lack of sensitivity to HSV1 antibodies has been observed in two different immunoassays, other EIA kits may also have the same problem. EIA kits for herpes antibody detection have been marketed for several years. Given the considerable technical expertise required for the implementation and interpretation of the Western blot herpes assays, we do not predict any large-scale switch away from EIA methodology.Our primary concern has been the lack of specificity due to cross-reactivity in the EJA kits already in widespread use. Kit manufacturers recommend that, when results for a patient are positive in both the type 1 and 2 assays, the results should be reported as positive for the “predominant antibody type,” the predominant antibody being the one with the greater magnitude results. We consider it more clinically relevant to use a correction factor that provides high specificity and indicates which patients are positive for either HSV1 IgG, HSV2 IgG, or both. References 1. Ashley RL, Militoni J, Lee F, Nahmias A, Corey L. Comparison of Western blot (immunoblot) and glycoprotein G-specilIc immunodot enzyme assay for detecting antibodies to Herpes Simplex Virus types 1 and 2 in human sera. J Clin Microbiol 1988;26:662-7. 2. SIA herpes 1 and herpes 2 quantitative determination of IgG antibodies to Herpes Simplex Virus in human serum. Package insert, no. 111/112. St. Louis, MO: Sigma Chemical Co. 1988. 3. Lee FK, Coleman RM, Pereira L, Bailey PD, Tatsuno M, Nahmias A. Detection of Herpes Simplex Virus type 2-specific antibody with glycoprotein G. J Clin Microbiol 1985;22:641-.4. 4. Lee FK, Pereira L, Griffin C, Reid E, Nahmias A. A novel glycoprotein for detectionof Herpes Simplex Virus type 1 specific antibodies. J Virol Methods 1986;14:111-8.

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Storage Conditions for Serum for Estimating Prostate-Specific Antigen, Brett Simm and Maree Gleeson’ (Hunter Immunol. Unit, Newcastle Mater Misericordiae Hosp., Edith St., Waratah, NSW, 2298, Australia, and’ Royal Newcastle Hosp., Newcastle, NSW, Australia) The results of quantifying serum prostate-specific antigen (PSA) are not normally required urgently, and hence, samples are commonly stored and assayed in batches to CLINICAL CHEMISTRY, Vol. 37, No. 1, 1991 113

improve cost effectiveness. Although it is recommended that samples be kept frozen (-20 #{176}C) if storage time is anticipated to be longer than 24 h (1), this may be difficult, particularly during transport, if the specimen is referred from a distant location. We investigated the effect of different temperatures and repeated freeze-thaw cycles on the stability of serum PSA, and whether refrigeration (4#{176}C) might be an adequate method of storage for periods longer than 24 h. PSA was assayed in duplicate with the Tandem-R immunoradiometric assay kit (Hybritech, San Diego, CA); in our hands, the total CV is 3.2% at 40 tg/L and 6.7% at 3.2 g/L. In a preliminary study, we aliquoted a single serum sample having an above-normal PSA concentration (222 g/L) and stored the aliquots at 37, 21, 4, and -20 #{176}C, for 1, 2, 3, 5, 7, 14, and 21 days before reassaying for PSA. As Figure 1 indicates, PSA appears more stable at lower temperatures,

(r = 0.793, slope = 0.896, intercept = 2.44, S. = 33.79). In view of these data, we suggest that samples may be stored at 4#{176}C for up to 14 days, after which time they should be frozen. Repeated freeze-thaw cycles(more than one) should be avoided because these can lead to increases or decreases in the concentration of serum PSA, which may be of clinical significance. groups

We acknowledge the excellent technical assistance provided by Michelle Terry and the Specimen Preparation Laboratory. Reference 1. Tandem-R PSA kit for the quantitative measurement of prostate-specific antigen (PSA) in serum. Method instructions sheet. San Diego, CA: Hybritech Inc., 1989.

such that samples might be stored for up to 14 days if refrigerated at 4#{176}C, and for >21 days if frozen at -20 #{176}C. To further investigate the suitability of refrigeration at An Ultraflitrate of Saliva Coilected in Situ as a 4#{176}C as a routine storage temperature, we collected 47 Biological Sample for Diagnostic Evaluation, W. serum samples over eight weeks, and stored one aliquot of Schramm and R. H. Smith (BioQuant, Inc., 1919 Green each at 4#{176}C and a second aliquot at our customary -20 #{176}C,Rd., Ann Arbor, MI 48105) for between seven and 14 days (median 11 days) before analysis. The PSA concentrations in these samples ranged For the quantitative determination of many endogenous from 0.7 to 975 pgfL (mean 275 gfL). The difference in substances and therapeutic drugs, saliva is equally suitPSA concentration between pairs of samples was always able or sometimes even superior to plasma or serum as a either