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Suppression of Leukocytic Mitosis by Sera of Hepatitis-implicated Donors BARBARA A. MEIXA, M.D., AND HOWARD F. TASWELL, M.D. The American National Red Cross, 17th and D Streets NW, Washington, D. C. 20006, and Section of Clinical Pathology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55901 Abstract. Mella, Barbara A., and Taswell, Howard F.: Suppression of leukocytic mitosis by sera of hepatitis-implicated donors. Am. J. Clin. Path. 53: 141-144, 1970. In a blind study, 21 specimens of serum from 19 donors thought to be hepatitis carriers were incubated in cultures of normal leukocytes. Sera from 15 (79%) of the 19 donors suppressed mitosis. Immunoglobulin levels of 16 donors were determined; they were increased in 11 (69%). In 17 (89%) of the 19 suspect donors, either one or both tests were positive. Twelve specimens of serum from patients during various periods of infectious hepatitis were also tested. Mitotic suppression was found only when the sera were from patients in the preicteric or early icteric phase of the disease. Of the 23 specimens of serum used as controls, four caused mitotic suppression. from patients acutely ill with infectious hepatitis suppresses leukocytic mitosis when incubated with normal peripheral blood in vitro.a Immunoglobulin levels in blood from donors implicated as carriers of hepatitis have been reported to be above the 97.5 percentile of those in a control group. 1 Because these findings might provide a means of detecting the agent of either serum or infectious hepatitis in blood to be used for transfusion, a study was designed to determine if mitotic inhibition occurred in leukocytes of normal peripheral blood after incubation with sera from donors suspected of being carriers of hepatitis virus. If a correlation between increased immunoglobulin level and mitotic inhibition existed, a two-step test might be developed to detect carriers of the disease. The first step, immunoglobulin assay, would provide a simple, inexpensive, and relatively rapid screening test which would detect the approximately 5% of donors who have increased IgA, IgG, or IgM. The second, and perhaps more specific, test would be analysis of the effect of these sera on normal leukocytic mitosis. SERA Method and Materials A modification of the method of Moorhead and co-workers8 for culturing leukocytes and preparing chromosomes was used. The cultures were prepared with blood obtained from Red Cross staff members who had not had clinically-detectable hepatitis. The test serum (0.1 ml.) was added to the medium, the system was incubated for three days, cultures were harvested, and slides were made. Every metaphase figure on the slide was counted and karyotyped visually. A serum specimen was considered to have suppressed mitosis when there were five or fewer metaphase figures on the slide, compared with 30 to 100 for the controls. Each Received May 26, 1969; accepted for publication August 22, 1969. Supported in part by Research Grant HE-10225 from the National Institutes of Health, Public Health Service. 141 142 Vol. 53 MELLA AND TASWELL TABLE 1. Results with Donors Implicated after Single-unit Transfusions Donor Interval after Implicated Donation Immunoglobulin Level Mitotic Suppression (Mo.) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 48 48 72 3 24 48 12 1 1 4 4 20 3 4 Normal Normal Increased Increased Increased Increased Increased Increased Normal Increased Increased + +,+ +,+ + + + + + * From time of recipient's transfusion to date a new blood sample was taken from the donor. specimen of serum was tested in this manner on three separate occasions. Controls of whole blood without test serum added were prepared with every experiment. The leukocytes of the suspected carriers were not cultured. Immunoglobulin assay was performed by the method of Fahey and McKelvey.2 A blind study of 42 coded specimens of sera from the Mayo Clinic was done. Fifteen of these sera were obtained from blood donors who had been implicated in the transmission of hepatitis either on more than one occasion or after a single-unit transfusion. The other 27 sera were from a variety of control donors and patients. Twenty specimens of serum collected for the Red Cross Blood Program by cooperating blood banks were examined in the same way. Immunoglobulin assay of eight of these 20 samples had been done; 3 (nos. 3, 5, and 6 in Table 1) were duplicates of those sent from the Mayo Clinic. This was not recognized until the latter samples were decoded. Twenty-four specimens of serum were either duplicates or serial samples from 12 patients or donors. A blood donor was considered to be a hepatitis carrier when the transfusion recipient was thought to have clinical evidence of hepatitis on the basis of increased serum transaminase and bilirubin levels 15 to 180 days after receiving a unit of blood. Results Table 1 summarizes the results of the tests on sera from 14 donors implicated as hepatitis carriers after single-unit transfusions. Of the 18 specimens tested, 12 suppressed mitosis. Results with duplicate samples (donors 3, 5, and 13) agreed, except for an unexplained discrepancy with donor 6. Sera from ten of the 14 implicated TABLE 2. Results with Donors Implicated More Than Once Inter- Numval after ber Impliof cated Times Dona- ImpliDonor tion cated 15 16 17 18 (Mo.) 30 1 1 10 ? 1 Immunoglobulin Level Mitotic Suppression + + Normal Increased Normal Increased Increased Increased Increased + + + T A B L E 3 . Results with Patients Who Infectious Hepatitis Patient A B C D E F G H Interval* Immunoglobulin Level (Days) 5 2 + 1 + 1 + 4 - 15 -f- 48 +270 + 3 - 70 + 1 + 6 Normal Increased Increased Increased Increased Normal Increased Increased Increased Normal Increased Increased Had Mitotic Suppression + + + — +,+ + — — — + + + * Interval between the appearance of icterus and the withdrawal of the specimen; minus indicates that sample was taken before icterus developed. February 1970 HEPATITIS-INDUCED MITOTIC INHIBITION donors inhibited mitosis. Of the 11 implicated donors whose serum immunoglobulin levels were known, eight had increased levels. Sera from six of the 11 donors had both increased immunoglobulin levels and the ability to suppress mitosis. Table 2 summarizes the results of tests on eight serum samples taken from five donors implicated as hepatitis virus carriers after multiple-transfusion episodes. Suppression of mitosis was found with all but one of these specimens, and, in this one case (donor 18), two other specimens of serum drawn at different times did cause mitotic suppression. All three specimens of serum from this donor had greatly increased levels of immunoglobulin, as did the sera of two others of these five donors. Thus, sera from 15 (79%) of the 19 donors implicated in the transmission of hepatitis suppressed mitosis. Table 3 depicts the results of tests of sera taken from eight patients at various stages of infectious hepatitis. Suppression of mitosis was found in the first few weeks of the disease only. Four specimens (from patients A, D, and G) which suppressed mitosis were drawn between two and 70 days before icterus developed and, with one exception, these had normal immunoglobulin levels. Two of the ten specimens (from patients B and F) which were drawn prior to or during the first week of the acute disease did not suppress mitosis. The other two specimens (from patients D and E) which did not suppress mitosis were drawn seven weeks to nine months after the onset of the symptoms. All specimens had increased immunoglobulin levels except those drawn five days or more prior to the onset of symptoms. Table 4 lists the results of the studies of 23 serum specimens used as controls. Four specimens caused suppression of mitosis. They had been drawn from donors who had given numerous blood donations, none of which has been reported to have 143 TABLE 4. Results with Control Donors Immunoglobulin Level Source Normal Normal Normal Normal Cirrhosis Infectious mononucleosis Pulmonary nodule Infectious mononucleosis Hydronephrosis Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Increased Increased Increased Increased Increased — Normal Normal Increased Normal Normal Normal Normal — — — — — — — — Mitotic Suppression — + + — — — — — + + — — — — — — — — — — produced hepatitis in the recipients. Three of these were from donors deliberately selected because they had increased immunoglobulin levels. Discussion Although mitotic suppression has in the past been related to viral infections other than hepatitis, the exact cause of the suppression and why it is not always found have never been adequately explained. 7 Other observers have thought that the suppression is due to a viral effect on the DNA of the cell or incorporation of the virus into the cell, leading to abnormal replication.9 The suppression of mitosis found in the acute stages of infectious hepatitis is consistent with the results of previous serial studies.5 After the first weeks of the disease, the patients' sera have no effect on normal leukocytes; however, their own leukocytes have been shown to have aberrant chromosomal patterns for the following seven 144 Vol. 53 MELLA AND TASWELL months of convalescence.4 Studies of human volunteers have demonstrated a viremia during the late preicteric and early icteric stages of infectious hepatitis. 3 In contrast to the serum effect on mitosis, increased serum immunoglobulin levels were noted in late as well as acute stages of the disease, but not prior to the onset of symptoms. In a wide variety of diseases, immunoglobulin levels may remain increased for a prolonged period after acute infection and after the patient no longer harbors the infective agent. In contrast, mitotic inhibition has been found only when an acute viremia was thought to exist. Three specimens of serum were taken at different times from the donor who had been implicated as a hepatitis carrier on five separate occasions. Because one specimen did not suppress mitosis and two did, the factor causing this phenomenon may be transient. Most of the specimens of serum from the suspect donors were drawn three months to six years after their blood had been implicated in cases of post-transfusion hepatitis. A transient viremia could have been missed, accounting for lack of suppression of mitosis by six of the 21 specimens tested. Mitotic suppression could be caused by viremia while the increase in immunoglobulin could be a later immune reaction to the viremia, which would explain the results of this study. A toxic factor transiently present in the sera of chronic carriers could be responsible for mitotic suppression. Although many of the samples in this study had increased immunoglobulin levels, it is not likely that this alone is the toxic factor which causes suppression of mitosis. Determination of the serum immunoglobulin level is a simple, inexpensive, rapid test, readily adaptable to mass screening. T h e occurrence of mitotic suppression by the sera of nine of 11 hepatitis-impli- cated donors with increased immunoglobulin levels provides further evidence of the value of determining these levels to screen blood donors. The test for mitotic suppression is tedious. T o use it for mass screening would be impossible at this time. Attempts are being made to determine the exact cause of the phenomenon and to simplify the technics of determining its presence. If this is accomplished, a prospective study using both methods will be made. Acknowledgment. Some of the blood samples used in this study were supplied by the Central Blood Bank of Pittsburgh and the Atlanta Regional Red Cross Blood Center. References 1. Bevan, G., Taswell, H. F., and Gleich, G. J.: Serum immunoglobulin levels in blood donors implicated in transmission of hepatitis. JAMA. 203: 38-iO, 1968. 2. Fahey, J. L., and McKelvey, E. M.: Quantitative determination of serum immunoglobulins in antibody-agar plates. / . Immunol. 94: 84-90, 1965. 3. Krugman, S., and Ward, R.: Viral hepatitis, infectious hepatitis, serum hepatitis. In Infectious Diseases of Children. Ed. 3. St. Louis, C. V. Mosby Company, 1964, pp. 102-118. 4. Mella, B.: Chromosomal damage associated with infectious hepatitis and its possible teratogenic significance. Neurology (Minneap.) 18: 741-744, 1968. 5. Mella, B„ and Lang, D. J.: Chromosomal aberrations and suppression of leukocyte mitosis induced in vitro by a circulating factor associated with acute infectious hepatitis. Presented at the New York Academy of Science, November, 1966. Ann. N. Y. Acad. Sci. 155, article 3. 6. Mella, B„ and Lang, D. J.: Leukocyte mitosis: Suppression in vitro associated with acute infectious hepatitis. Science 155: 80-81, 1967. 7. Montgomery, J. R., South, M. A., Rawls, W. E., Melnick, J. L., Olson, G. B., Dent, P. B., and Good, R. A.: Viral inhibition of lymphocyte response to phytohemagglutinin. Science 157: 1068-1070, 1967. 8. Moorhead, P. S., Nowell, P. C , Mellman, W. J., Ballips, D. M., and Hungerford, D. A.: Chromosome preparations of leukocytes cultured from human peripheral blood. Exp. Cell Res. 20: 613-616, 1960. 9. Nichols, W. W.: Studies on the role of viruses in somatic mutation. Hereditas (Lund) 55: 1-27, 1966.