Studies of Donors Who Transmit Posttransfusion Hepatitis

Studies of Donors Who Transmit Posttransfusion Hepatitis

E. TABOR, J. H. HOOFNAGLE, L. A. SMALLWOOD, J. A. DRUCKER, G. C. PINEDA-TAMONDONG, L. Y. NI, T. J. GREENWALT,

L. F. BARKER AND R. J. GERETY

From the Hepatitis Branch, Division of Blood and Blood Products, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland, and the American National Red Cross,

Washington, D.C.

Sera and qwstbnnalres were evaluated retrospectively h m 128 volunteer blood donors whose blood had been implicated in cases of clinkally recognized post- transfusbn hepatitis in recipients of one- or two-unit bbod transfusions between 1971 and 1977. Serologk markers of hpatltis B virus (HBV) infectbn were found in 23 per cent, compared to 9.7 per cent of 3,230 prospective blood donors. The prevalence of antibody to hepatltls A virus was simllar among impllcated donors

pllcated donors Witb (41%) and Without (44%) HBV markers. Among implicated donors, none had a history at the tlme of donation of having had clinically recognizable hepatitis, 93 per cent had no history of prior blood transfusion, and 80 per cent had normal hepatk enzymes. Data from this study con6rm that non-A, wn-B hepatitis has been a common form of posttransfusion hepatltis in recent years, since 77 per cent of thes impllcated donors had w HBV srobgk markers. In addition, these donors could not be distinguished by age, race, sex, history of clinkd hepatitis or of prior blood transfusion, or in most cases by hepatk enzyme levels.

(44%), ProepsCtiVe dowrs (5896)s Md .mOng thoao h-

IN THE YEARS since the discovery of hepatitis B surface antigen ( H B s A ~ ) , ~ and hepatitis A virus (HAV),6 the use of serologic tests to diagnose hepatitis B virus (HBV) and HAV infections have revealed that many cases of hepatitis are not caused by either of these v i r u s e ~ . ~ * ~ ~ This “non-A, non-B hepatitis” is transmitted by a virus or vi- ruses2*12~27 and accounts for as many as 89 per cent of cases of posttransfusion hepa- ti ti^.'.^^ In the present study, we evaluated sera and histories from a group of volunteer blood donors whose blood had been screened and found nonreactive for HBsAg by the most sensitive available methods at the time of donation in the years 1971 to 1977, and

Received for publication October 5, 1978; accepted December 13, 1978.

whose blood was transfused alone or with one additional unit to patients who subsequently developed clinically recognizable posttransfusion hepatitis. This study was a retrospective effort to evaluate certain criteria that might identify donors whose blood transmits hepatitis, particularly those transmitting non-A, non-B hepatitis.

Methods

Sera and responses to questionnaires were obtained retrospectively from 128 former blood donors who had been listed on the American National Red Cross Donor Deferral Registry between January 1, 1971 and June 30, 1977, after patients transfused with a single unit of their blood (48 cases) or a single unit of their blood and the blood of one other donor (80 cases) developed clinically recognizable posttransfusion hepatitis. All donors gave fully informed written consent prior to entry in this study. At the time of original donation, each met all American National Red Cross criteria for healthy donors, and each unit of their blood had been tested for HBsAg prior to transfusion using the most sensitive test available at the time, counterelectrophoresis (CEP) prior to 1973, CEP or radioimmunoassay (RIA) during 1973, and RIA beginning in 1973. The interval between the donation implicated in a case of posttransfusion hepatitis, and the col- lection of serum for this study, was one year for 21 donors, two years for 47 donors, and greater than two years for 60 donors. In most cases, sera were immediately frozen

at -20 C, and subsequently stored at -70 C. Sera were tested for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) by a biochromatic assay13 (ABA- 100, Abbott Labora- tories, North Chicago, IL); for HBsAg by RIAIS (Ausria 11, Abbott Laboratories), CEP,’ and re- versed passive hemagglutination2’ (Auscell,

Transfusion November-Dcccmber 1979 725 Volume 19

Number 6

Transfusion November-December 1979

726 TABOR ET AL.

Table 1. Donors Implicated in Posttransfusion Hepatitis ~

Serologic Analyses of Recent Serum Samples from Donors

Year Total No. Anti-HBc Anti-HBs Anti-HBs Implicated Donors HBsAg Alone' and Anti-HBc Alone

- 1971 8 1 1 1972 18 1 2 1 1973 17 2 1 4 1

1 1974 23 1 3 3 1975 32 -

1976 16 2 2 1977 3

2 1

- - - - - - - - - - - - Undetermined 11 -

126 implicated donors tested for anti-HBc.

Abbott Laboratories); antibody to HBsAg (anti-HBs) by RIA8 (Ausab, Abbott Laboratories) and by passive hemagglutination28 (Virgo Re- agents, Electro-Nucleonics, Bethesda, MD); antibody to hepatitis B core antigen (anti-HBc) by complement fixationS*l0 and a solid phase RIA (reagents kindly provided by Dr. L. R. Overby, Abbott Laboratories);2o and hepatitis B e antigen (HBeAg) and its antibody (anti-HBe) by agar gel diffusion (AGD).16-*e Antibody to hepatitis A virus (anti-HAV) was detected by immune adherence hemagglutination (IAHA) using hepatitis A antigen prepared in our laboratory from the stools of experimentally infected chirnpanzee~,~~*l~ and by solid phase RIA (courtesy of Dr. Richard Decker, Abbott Laboratories).

Sera and responses to questionnaries were also obtained from 3,230 prospective American Na- tional Red Cross volunteer blood donors. These prospective donors were obtained in three cate- gories: 1) 1,151 prospective donors rejected because of having a history of clinical hepatitis; 2) 993 prospective donors rejected because of a history of having been transfused with blood or

plasma, but having no history of clinical hepatitis; and 3) 1,086 acceptable blood donors, chosen at random from donors appearing daily at the same donation centers. Sera from prospective blood donors were tested for HBsAg and anti-HBs. Additional testing was done on sera from a representative selection of these donors (298, including 101 with a history of hepatitis, % with a history

. of transfusion, and 101 with no such histories), including complement fixation tests for anti- HBc,*JO and RIA and IAHA for anti-HAV.*7Jg Statistical significance of differences in serologic test results between implicated donors and prospective donors was determined using chi-square and contingency tables.

Results Implicated Donors

Serologic markers of HBV were detected in 29 (23%) of 128 donors whose blood was implicated in the transmission of posttransfusion hepatitis after one- or two-unit transfusions (Ta- bles 1 and 2). Three implicated donors were

Table 2. HEY and HAY Markers in Sera of Donors ~ ~

Total Anti-HBc No. HBsAg Alone Anti-HBs

Donors (%I (%I (%I Donors Implicated after Transfusion of

One-unit 48 1 (2) 4 (8) 9 (19) Two-units 80 2 (2) 5 (6) 8 (10)

History of Hepatitis 1151 11 (1) History of Transfusion 993 l(O.1) No History of Hepatitis or Transfusion 1 086 2 (0.2)

Prospective Donors 179 (15.7) 60 (6.1) 63 (5.8)

Tested in representative sera, see text.

Volume 19 Number 6 DONORS IMPLICATED IN HEPATITIS 727

HBsAg-positive, seventeen had anti-HBs, and nine had anti-HBc alone. HBcAg was detected in the serum of one of the HBsAg-positive donors; anti-HBe was detected in the sera of two donors.

Anti-HAV was detected in the sera of 55 (44%) of 126 donors tested, including 12 (41%) of 29 with HBV markers and 43 (44%) of 97 without HBV markers. The percentage of implicated donors with anti-HAV increased with age (Fig. 1).

AST and ALT were determined on sera from 123 implicated donors (Fig. 2). Elevated AST or ALT, defined as greater than 40 IU/ml, was detected in five (1%) of 26 implicated donors with HBV markers, none of whom had HBsAg, and in 19 (20%) of 97 implicated donors without HBV markers. The greatest elevations (AST 227 IU and ALT 3 18 IU) were found in a donor without HBV markers.

Although none of the 128 implicated donors gave a history at the time of donation of having had clinical hepatitis, such a history was elicited later, at the time of this study, from four implicated donors, which had occurred five, eight, twenty and an undetermined number of years, respectively, prior to being implicated in posttransfusion hepatitis (Fig. 2). Two had anti-HBs, and two had no HBV markers. A history of prior transfusion was obtained from eight implicated donors, all without HBV markers; in each case the transfusion was received greater than five years prior to their blood being implicated in posttransfusion hepatitis (Fig. 2). Of the 128 implicated donors, 126 (99%) considered themselves to be in good health. The age, race and sex of implicated donors without HBV markers did not differ significantly from those with HBV markers or from the prospective donors.

AGE IN YEARS OF IMPLICATED DONORS

FIG. 1 . Striped area indicates the relationship of age to detection of anti-HAV. Total height of bar = relationship of age to probable transmission of non-A. non-B hepatitis; as indicated by the absence of HBV markers. * = number of implicated donors in each age group.

Prospective Donors Serologic markers of HBV infection were de-

tected in 316 (%) of 3,230 prospective donors. HBsAg was detected in 14 (0.4%) of the 3,230 prospective donors (compared to implicated donors, p < 0.02) (Table 2). Anti-HBs was detected in 302 prospective donors (%; not a signif- icant difference from implicated donors). Anti- HBc was not detected in the absence of other markers for HBV infection in any of 298 selected sera from prospective donors. Hepatic enzyme levels were not available on sera from prospec-

O L

FIG. 2. Characteristics 3 of implicated donors with 1 (m) and without (0) HBV markers. %

1

kabm - ~ I I z 8 0

ELEVATED HISTORY OF HISTORY OF ANTI-HAV AST OR ALT CLINICAL TRANSFUSION - - ~

HEPATITIS

728 TABOR ET AL. Transfusion November-December 1979

tive donors. Anti-HAV was detected in 158 (58%) of 273 representative prospective donors tested.

Recipients of Implicated Donor Blood HBsAg tests of sera from recipients of im-

plicated donor blood at the time of hepatitis had been done by RIA in only 25 cases. Of these, 12 recipients had HBsAg-positive hepatitis, and 13 had HBsAg-negative hepatitis. In 103 recipients, the type of hepatitis was not determined. The 13 recipients with HBsAg-negative hepatitis received only blood from implicated donors with no HBV markers.

Discussion

Implicated Donor Serology

In the present study, 77 per cent of donors whose blood was implicated in the transmission of posttransfusion hepatitis between 1971 and 1977 had no serologic markers for HBV infection, suggesting that the majority transmitted non-A, non-B hepatitis. The finding of HBsAg in two per cent of implicated blood donors is significantly higher than the 0.4 per cent found in the prospective donors and among similar reported group^.^^.^^ The detection of HBsAg, anti- HBc or anti-HBs in 23 per cent of these donors many years later does not prove that HBV was responsible for the hepatitis, and thus the number transmitting non-A, non-B hepatitis may have been even greater than the 77 per cent suggested by the serologic data.

The transmission of non-B, posttransfusion hepatitis most likely was not due to HAV.22*33.P5 This is confirmed by the similar prevalence of anti-HAV in this study among donors with HBV markers and donors with no HBV markers, which was consistent in both groups with the prevalence in the general population.22 The prevalence of anti- HAV increased with age among implicated donors (Fig. l), as reported for the general population in the U.S.,22 and strongly suggests that the acquisition of anti-HAV in these donors was unrelated to the transmis-

sion of posttransfusion hepatitis by their blood.

Some of the implicated donors were clearly asymptomatic carriers of the non-A, non-B agent or agents. This has been documented by the transmission of non-A, non-B hepatitis to chimpanzees using human s e n ~ m , ~ , * ~ including two sera chosen from implicated donors with elevated AST and ALT in this

Although elevated enzymes did not identify all of the donors who transmitted non-A, non-B hepatitis in the present study, elevated levels of AST or ALT in 20 per cent of the implicated donor sera months to years after these donors were implicated in the transmission of post-transfusion hepatitis strongly suggests that these donors were chronic carriers of the non-A, non-B hepatitis agent.

All specimens in this study were obtained from months to years after volunteer donors were implicated in the transmission of clinically recognized hepatitis. AST and ALT in the semm specimens obtained do not necessarily reflect the donor's hepatic enzyme levels at the time of transmitting hepatitis. Enzyme levels in some donors may have returned to normal, or may not have been elevated at the time of donation despite the transmission of hepatitis. Similarly, the presence of HBV markers in the sera studied does not necessarily identify donors who transmitted HBV infections, since they may have acquired these markers before or since transmitting hepatitis. The detection of anti- HBc alone in nine of 29 implicated donors with HBV markers, suggests that some HBsAg-negative donors implicated in the transmission of hepatitis B may be low-level carriers potentially detectable using tests for anti-HBc." However, the total absence of HBV markers in many implicated donors probably indicates that such donors did not transmit HBV infection. This retrospective study evaluated donors whose blood was implicated in cases of clinically recognizable posttransfusion hepatitis. The actual num-

Volume 19 Numkr 6 DONORS IMPLICATED IN HEPATITIS 729

ber of donors implicated in non-A, non-B hepatitis may be different if recipients with subclinical hepatitis and asymptomatic sero- conversions could be identified. This study ,also evaluated only volunteer donors; whether a study would yield similar results in a paid donor population cannot be determined.

The data presented do suggest that the exclusion of donors with an elevated AST or ALT would have identified approximately 20 per cent of the implicated donors transmitting either hepatitis B or non-A, non-B hepatitis, although admittedly a larger percentage may have had elevated AST or ALT at the time of donation. The data also in- dicate that elevated AST and ALT would not have discriminated between donors transmitting non-A, non-B hepatitis and those transmitting hepatitis B. Serum enzyme levels for determining donor eligibility have never received widespread acceptance, although most studies have evaluated their use only as a screening device for HBV infection. 14*18

Implicated Donor Histories The donor history of hepatitis appears not

to be a useful screening test for those transmitting non-A, non-B hepatitis, since none of the implicated donors had had a history of clinically recognizable hepatitis at the time of blood donation. This may reflect the fre- quent occurrence of asymptomatic cases of non-A, non-B hepatitis in the general population, which may act as a reservoir for the disease, as with HAV and HBV infections.

A history of prior transfusion did not ap- pear to identify those implicated donors who transmitted posttransfusion hepatitis. In 90 per cent of implicated donors with no HBV markers, and in 100 per cent of implicated donors with HBV markers, no prior transfusions had been received. Eight donors without HBV markers had had transfusions greater than five years previously. If these transfusions were responsible for their

non-A, non-B infections, then the infections lasted greater than five years. This time period, although protracted, is consistent with human chronic non-A, non-B infections demonstrated by chimpanzee inoculations .2,27

The use of nonspecific criteria such as hepatic enzyme tests, history of clinical hepatitis, history of prior transfusion, sub- jective assessment of health, or donor age did not permit the identification of donors who were more likely to transmit non-A, non-B hepatitis. Specific identification of donors whose blood is potentially infectious for non-A, non-B hepatitis may have to await development of a specific serologic screening test.

Prospective Donor Histories Before the discovery of HBsAg as a

marker for the presence of HBV in blood, it was decided that exclusion of blood donors with a history of clinically recognizable hepatitis or of a recent transfusion would decrease the risk of posttransfusion hepatitis. This became required in the federal regulations for blood and plasma collec- t i ~ n . ~ . ~ The basis for these regulations has not been evaluated since the development of sensitive serologic tests, although a small study conducted among hospital staff did not find a history of hepatitis associated with increased prevalence of HBsAg, but did find a greater prevalence of anti-HBs.I4 In the prospective donor group in the present study, a history of hepatitis was associated with a higher prevalence of HBsAg or anti- HBs than was found in prospective donors without such a history, but was similar to that found in implicated donors. Although a history of hepatitis may exclude units of blood with the potential to transmit HBV infection, the need for it as a means of preventing hepatitis B is diminished by the use of sensitive tests for HBsAg. Whether it would eliminate infectious units with HBsAg in low titers undetectable by RIA cannot be determined. Prospective

730 TABOR ET AL.

donors with a history of prior transfusion did not differ in the prevalence of HBsAg or anti-HBs from those with no such history. Exclusion of donors with a history of transfusion appears less useful as a means of screening for HBV infectious donors.

Acknowledgments The authors thank Mr. A. J. Shawver, Mr. D. Gil-

bert, and Mr. J. Thiel for their excellent technical assistance, Mr. L. Fraise for data processing, Dr. Suresh Rastogi for statistical consultation, and Dr. W. Mallin and Dr. N. Pollok for their assistance in the administration of this study, conducted in part under Food and Drug Administration contract X223-74- 1103.

References 1. Alter, H. J., P. V. Holland, and R. H. Purcell:

The emerging pattern of post-transfusion hepatitis. Am. J. Med. Sci. 270:329. 1975.

2. - , R. H. Purcell, P. V. Holland, and H. Pop- per: Transmissible agent in non-A, non-B hepatitis. Lancet 1:459, 1978.

3. Blumberg, B. S.: Polymorphisms of the serum pro- teins and the development of iso-precipitins in transfused patients. Bull. NY Acad. Med. 40: 377, 1964.

4. Code of Federal Regulations. Volume 2 1: Food and Drugs. Section 640.3, 1975, p. 80.

5. [bid., Section 640.63, p. 87. 6. Peinstone, S. M., A. Z. Kapikian, and R. H. Pur-

cell: Hepatitis A: Detection by immune electron microscopy of a virus-like antigen associated with acute illness. Science 182:1026. 1973.

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9. Hoofnagle, J. H., R. J. Gerety. and L. F. Barker: Antibody to hepatitis-B core antigen. In: Trans- missible Disease and Blood Transfusion. T. J. Greenwalt, and G. A. Jamieson, Eds. New York, Grune and Stratton, 1975, p. 43.

10. -, R. J. Gerety, and L. F. Barker: Antibody to hepatitis-B-virus core in man. Lancet 2: 869, 1973.

11. -, R. J. Gerety, L. Y. Ni, and L. F. Barker: Antibody to hepatitis B core antigen. A sensitive indicator of hepatitis B virus replication. N. Engl. J. Med. 290:1336, 1974.

Trensfusion November-December 1979

12. - , R. J. Gerety, E. Tabor.et a/.: Transmission of non-A, non-B hepatitis. Ann. Intern. Med. 87:14, 1977.

13. Humoller, F. L., J. M. Holthaus, and J. R. Walsh: Improved method for the colorimetric deter- mination of glutamic-oxalacetic transaminase activity. Clin. Chem. 3703, 1957.

14. Lewis, T. L., H. J. Alter, T. C. Chalmers, et al.: A comparison of the frequency of hepatitis-B antigen and antibody in hospital and nonhospital personnel. N. End. J. Med. 289647, 1973.

15. Ling, C. M., and L. R. Overby: Prevalence of hepatitis B virus antigen as revealed by direct radioimmune assay with lWI-antibody. J. Im- munol. 1099334, 1972.

16. Magnius, L. 0.: Characterization of anew antigen- antibody system associated with hepatitis B. Clin. Exp. Immunol. 20:209, 1975.

17. Miller, W. J., P. J. Provost, W. J. McAleer, et al.: Specific immune adherence assay for human hepatitis A antibody. Application to diagnostic and epidemiologic investigations. Proc. SOC. Exp. Biol. Med. 149254, 1975.

18. Miller, W. V., L. E. Watson. P. V. Holland, and J. H. Walsh: Evaluation of the effectiveness of hepatitis screening tests. Vox Sang 21: 1, 1971.

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DONORS IMPLICATED IN HEPATITIS 73 1

27. - , R. J. Gerety, J. A. Drucker, ef al.: Trans- mission of non-A, non-B hepatitis from man to chimpanzee. Lancet 1:463, 1978.

28. Vyas, G. N., and N. R. Shulman: Hemagglutina- tion assay for antigen and antibody associated with viral hepatitis. Science 170:332, 1970.

Edward Tabor, M.D., Research Investigator, Hepa- titis Branch, Bureau of Biologics, 8800 Rockville Pike, Bethesda, Maryland 20014 (reprint requests).

Jay H. Hoofnagle, M.D., Senior Investigator, Liver Unit, NIAMDD, National Institutes of Health, Bethesda, Maryland 20014.

Linda A. Smallwood, M.S., Microbiologist, Hepa- titis Branch, Bureau of Biologics, 8800 Rockville Pike, Bethesda, Maryland 20014.

Jacques A. Drucker, M.D., Pediatric Resident, H o p ital de Clocheville, Boulevard Btranger, 37000 Tours, France.

Geronima C. Pineda-Tamondong, M.D., Medical As- sociate, American National Red Cross, 1730 E Street, N.W., Washington, D.C. 20006.

Louisa Y. Ni, M.S., Senior Technical Associate, American National Red Cross, 1730 E Street, N.W., Washington, D.C. 20006.

Tibor J. Greenwalt, M.D., Senior Scientific Advisor, American National Red Cross, 1730 E Street, N.W., Washington, D.C. 20006.

Lewellys F. Barker, M.D., Vice Resident, Blood Services, American National Red Cross, 1730 E Street, N.W., Washington, D.C. 20006.

Robert J. Gerety, M.D., Ph.D., Director, Hepatitis Branch, Bureau of Biologics, 8800 Rockville Pike, Bethesda, Maryland 20014.

Documents

Studies of Donors Who Transmit Posttransfusion Hepatitis