Letters to the Editor

EDITOR’S NOTE: In order to permit timely publication of correspondence, the references have not been verified as they have been for articles appearing in TRANSFUSION, and, therefore, the accuracy of cited references in Letters to the Editor is the sole responsibility of the authors.

Specificity of enzyme immunoassay for hepatitis B core antibody used in screening blood donors

To the Editor: We were interested to see that Lee et al.’ attempted to

demonstrate the specificity of their sera found positive for anti-HBc by “neutralization” with absorption-column purified HBcAg. One caveat that applies to this approach is that neutralization can be considered “specific” only when it relates to antibody ablating an antigen reaction. The ac- tivity of a nonspecific inhibitor or a cross-reacting antibody will be blocked by an antigen as if it were a genuine an- tibody. Therefore, blocking the reaction of a putative “an- tibody” (even with highly purified antigen) is not directly comparable with neutralizing a putative antigen with spe- cific authenticated antibody and cannot distinguish between specific antibody activity and nonspecific inhibition.

The authors reported that 7.8 percent of their HBsAg- positive specimens were negative for anti-HBc, which lends firm support to the view of many that an anti-HBc test should not replace HBsAg screening of blood donors. This recommendation has been confirmed previou~ly.~”

JOHN BARBARA, MA, MSc, PHD, FIBIOL MARCELA CONTRERAS, MD

North London Blood Transfuswn Centre, Deansbrook Road, Edgware

Middksa, HA8 9BD UK

References I . Lee H-S, Rajagopalan MS, Chien D, Cordell R, Perkins HA,

Vyas GN. Specificity of enzyme immunoassay for hepatitis B core antibody used in screening blood donors. Transfusion

2. Barbara JAJ, Tedder RS, Briggs M. Anti-HBc testing alone not a reliable blood donor screen. Lancet 1984;1:346.

3. CouroucC A-M, Drouet J, LeMarrec N, Drouet A, Soulier JP. Blood donors positive for HBsAg and negative for anti-HEk antibody. Vox Sang 1985;49:26-33.

1987;27: 103-6.

The above ktter was sent to Dr. Lee and coikagues, who offered the following reply.

The generic issue of valid serologic methods for demon- strating the specificity of antibodies against antigens is an important consideration. However, the caveat raised by Barbara and Contraras may be mitigated by the following explanation.

According to Sternberger,’ none of the current tests for specificity of immunoassays except immunoblot (with certain limitations) can fully exclude the possibility of cross-reacting antibodies. The anti-HBc test (Corzyme, Ab- bott Laboratories, North Chicago, IL) is a competitive inhi- bition assay in which the unknown antibody in a test serum ablates the reaction of immobilized HBcAg with standard- ized human IgG anti-HBc. Additionally, the use of a mono- clonal anti-HBc as an alternative probe has not shown any differences in testing of the Corzyme reactive serums (per- sonal communication, Dr. Decker, Abbott Laboratories). Thus, the Corzyme configuration currently offers the best available immunochemical specificity. The antigen we used

for the absorption test is a homogeneous protein of approx- imately 22,000 daltons with N-terminal sequence analysis showing seven amino acid residues of beta galactosidase followed by the HBcAg gene product of 185 residues ex- pressed by the transfected E coli Therefore, the absorbtive removal of the putative anti-HBc from a reactive serum in our experiments is specific. The concurrent anti-HBc test- ing of the absorbed and unabsorbed serums provided the results that were analytically maneuvered by us to yield an amplified discrimination of the specific reaction. Semanti- cally, had we used the term Absorption Index (A11 rather than Neutralization Index (NI), the concern voiced by Bar- bara and Contreras may have been avoided.

Occurrence of HBsAg for a period preceding appearance of anti-HBc was documented by Krugman et aL2, followed by several reports including the ones cited by Barbara and Contreras. The page limitations of the Rapid Communica- tions Section in Transfusbn precluded our listing all reports. However, we entirely agree with Barbara and Contreras that anti-HBc testing should not replace HBsAg screening.

HYO-SUK LEE, MD, PHD University of California

San Francisco, CA 94143 DAVID CHIEN, PHD Chiron Corporation

Emeryvilk?, CA 94608 GIRISH N. VYAS, PHD

Irwin Memorial Blood Bank San Francisco, CA 941 18

References I . Sternberger LA. Immunocytochemistry. New York: John Wiley,

2. Krugman S , Overby LR, Mushahwar IK, et al. Viral hepatitis, 1986231.

type B. N Eng J Med 1979;300101-6.

HBV DNA in blood donors To the Editor:

Posttransfusion hepatitis is still a major problem in hemotherapy. Development of a carrier state, active hep- atitis and related cirrhosis may lead to mortality. Potential etiologic agent, that have been identified include hepatitis B virus, hepatitis A virus, cytomegalovirus and Epstein-Barr virus; others that seem to be viral in origin have been called non-A, non-B (NANB).’ Prospective studies of hepatitis in recipients of blood transfusions* showed that hepatitis B oc- curred in 15 percent of cases, NANB in 80 to 90 percent; the other etiologic agents were very rare. Several investiga- tors have identified “virus B-like particules” in patients with NANB hepatitis, but because of lack of a sensitive, reliable and reproducible assay these data are still controver~ial.~

Recent advances in recombinant DNA technology and the use of an HBV DNA probe in a DNA-DNA hybridization technique enabled us to reexamine the role of virus B as a major agent in posttransfusion hepatitis. We investigated 1000 units of blood collected from volunteer donors using a

84

TRANSFUSION 1988-Vol. 28. No. I LETTERS TO THE EDITOR 85

molecular hybridization technique and compared the results with a conventional assay.

A spot method was used for the hybridization tech- nique. DNA was directly trapped in a nitrocellulose filter (Schleicher-Shuhl). The filtration apparatus was a Manifold I1 (Schleicher-Shuhl). Fifty pl of each sample to be tested was deposited in a well on the filter and allowed to filter under vacuum. The denaturation of the samples was per- formed by soaking the filter with 0.5M Tris HCI (pH 7.6) and 3M NaCl for 5 minutes. The filter was removed un- der continuous vacuum, blotted between two sheets of ab- sorbant filter paper and dried for two hours at 70°C.

The viral probe was obtained by insertion of the com- plete viral genome in pBR 322. After cleavage with EcoR I restriction endonuclease, the insert was radiolabeled by ran- dom priming. The specific activity of the probe was greater than 2X108 cpm per pg ADN and this probe was used at a concentration of lo6 cpm per pl in hybridization buffer. The filters were sequentially washed in SXSSC, 0.1 per- cent SDS, 30 minutes at 42°C; lXSSC, 0.1 percent SDS at 42OC; and O.lXSSC, 0.1 percent SDS 30 minutes at 55°C.

One sample was positive by both the conventional assay and the hybridization spot technique. No serum positive for HBV DNA and negative for HBsAg was found. Moreover, in a group of 30 asymptomatic carriers (HBsAg positive, HBeAg negative, anti-HBe negative, anti-HBc positive with a follow-up of two years at least), all were HBV DNA negative and there were no discrepancies between the HBe marker and HBV DNA, as has been reported in patients with chronic hepatitis40r in hemodialyzed patients.’ There is no reason to suppose that hepatitis B virus could be detected by hybridization but not by presence of HBsAg. Nor does it seem that an important part of the so-called NANB hepatitis is of B virus origin. This investigation did not provide clinical information on the etiology of the acute hepatitis called “non-A, non-B.”

C. FEREC C. VERLINGUE

J.P. SALEUM Centre Departmental de Transfusion Sanguine

46 Rue Felix L.e Dantec Brest

France

References I . Goldfield M, Bill J, Black H, et al. Hepatitis associated with the

transfusion of HB Ag negative blood. In: Vyas GN, Perkins HA, Smid R, eds. Hepatitis and blood transfusion. New York: Grune and Stratton, 1972:353-61.

2. Aach RD, Lander JJ, Sherman LA, et al. Transfusion transmit- ted viruses: interim analysis of hepatitis among transfused and non transfused patients. In: Vyas GN, Cohan SN, Schmid R, eds. Viral hepatitis: a contemporary assessment of etiology, epi- demiology, pathogenesis and prevention. Philadelphia: Franklin Institute Press, 1978:383-96.

3. Dienstag JL. Non A, non B hepatitis. 11. Experimental trans- mission putative virus agents and markers and prevention. Gas- troenterology 1983;85:743-68.

4. Scotto J, Hadchonal M, Hery C, Yvart J. Tiollais P, Brechot C. Detection of hepatitis B virus DNA in serum by a simple spot hybridization technique: comparison with results for other viral markers. Hepatology 1983;3:279-84.

5. Ferec C, Cledes J. Verlingue C, Morin JF, Saleun JP. Hepatitis B DNA in patients with renal failure comparison with the usual serological markers. IRCS Med Sci 198614: 1055.

Tn-polyagglutinable donors and the antiglobulin compatibility test

To the Editor: We describe an additional problem that can arise if

the antiglobulin compatibility test is not performed. Dur- ing investigation of an unexpected positive compatibil- ity test, we discovered that the donor’s red cells were Tn-polyagglutinable. This finding was overlooked by the donor center since they used enzyme-treated red cells to determine the ABO group and Rh type of the donor.

Incompatibility between the patient’s serum and the donor’s red cells was initially noted only in the indi- rect antiglobulin test. Subsequent investigation showed very weak reactivity at room temperature with the majority of patients’ sera tested and with anti-A,B reagent. If blood had been issued following an immediate spin com- patibility test, this unit would have been transfused as com- patible. It should be noted that a number of normal healthy donors have been found with Tn-polyagglutinable red cells. It has also been shown that Tn-polyagglutinable red cells may be rapidly destroyed when injected into a normal individual.’

R. ANDORKA, MT(ASCP) A. WAHEED, MS, MT(ASCP)SBB

M. S. KENNEDY, MD Transfuswn Service

The Ohio State University Hospitals 410 West 10th Avenue Columbus, OH 43210

References 1. Myllya G, Furuhjelm U, Nordling S, et al. Persistent mixed field

polyagglutinability: electrokinetic and serological aspects. Vox Sang 1971;20:7-23.

Level of probability in antibody identification To the Editor:

Hams and Hochman’ recently reconsidered p values in testing blood group antibodies and correctly concluded that the serologist’s use of Fisher’s exact test led to “undue con- servatism in interpretation of agglutination patterns and re- sults in unnecessary additional testing to confirm the iden- tity of blood group antibodies.” May we introduce a note of caution? Although the level p < 0.05 used by Hams and Hochman is acceptable as a level of “significance” when considering the results of an experiment comparing two things, that is not the case for the initial testing of an unknown antibody. When a patient’s serum is tested against a panel of red cell samples, the comparisons are done af- ter the work and there are many different comparisons to be made. Taking a 0.05 level of significance will lead to a “false positive” in about one of every 20 cases, which will lead to misidentification of some antibodies. The level of probability considered to be “significant” depends on the number of ways in which the results of the work can be compared. Race and Sanger’ record that they do not get too excited until p < 0.01. At this level they recommend that the investigation be restarted, using fresh material, making a comparison between two things (the agglutination results and the presence or not of a selected antigen); a probability level of p < 0.05 may then be considered “significant.”

Incidentally, the exact test was also independently dis- covered by two friends of Fisher’s, F. Yates and J. 0.