Hepatitis B e Antigen in Volunteer and Paid Blood Donors

E. TABOR, M. GOLDFIELD, H. C. BLACKANDR. J. GERETY

From the Hepatitis Branch, Division of Blood and Blood Products, Bureau of Biologics. Food and Drug Administration, Bethesda. Marvland;

and the New Jersey State Department of Health, Trenton. New Jersey

Sera from 200 volunteer blood donors and 200 paid blood donors, all positive for hepatitis B surface antigen (HBsAg), were tested for the presence of hepatitis B e antigen (HBeAg). HBeAg was detected in 31 HBsAg- positive paid donors (15%). and in 11 HBsAg-positive volunteer donors (5%) by agar gel diffusion. The pres- ence of HBeAg was associated with higher titers of HBsAg. No significant difference was found in the preva- lence of antibody to HBeAg (anti-HBe) in the two donor groups. Rheumatoid factor was not associated with the presence or absence of HBeAg or anti-HBe, indicating that HBeAg is probably not an anti-1gC. Thesedatasup- port the epidemiological evidence that paid blood donors appear to be more likely than volunteer donors to transmit hepatitis B virus infection to recipients of their blood.

THE RISK of hepatitis B virus (HBV) infec- t ion af ter transfusion with blood from paid d o n o r s appears to be greater t h a n that follow- ing receipt of blood from volunteer donors.” Because of this risk, blood must be labeled according t o its source, as ei ther “volunteer” or “paid.”6 The present s tudy was undertaken t o evaluate whether t h e statistical risk of hepatitis B transmission by blood obtained from paid donors is reflected in a greater prevalence of t h e hepatitis B e ant igen (HBeAg), an antigen associated with an in- creased risk of t ransmit t ing HBVinfection.’6v2’

Materials and Methods Serum samples were obtained at the time of

blood donation from 200 volunteer blood donors and 200 paid blood donors whose blood was rejected when it was found to be HBsAg-positive by counterelectrophoresis (CEP) or radioimmu- noassay (RIA). The number of donors screened by each method was the same in both groups of donors. Each serum sample was stored at -20 C

Received for publication November 29. 1978; accepted February 9,1979.

and was subsequently tested for HBsAg by RIA,” counterelectro horesis (CEP),’ and complement fixation (CF). HBsAg subtyping was performed by immunodiffusion.14 Antibody to HBsAg (anti- HBs) was tested by RIA.” Antibody to hepatitis B core antigen (anti-HBc) was detected by CF.’* Rheumatoid factor (RF) was tested by a latex agglutination slide test (RA-Test, Hyland Labora- tories, Costa Mesa, CA).23

Serum samples were tested under code for HBeAg and its antibody (anti-HBe) using un- diluted specimens in an agar gel diffusion Agarose plates were made with 0.6% agarose (In- dubiose A 37; L‘industrie Biologique Francaise, Gennevilliers, France), with one drop of 10% sodium azide per 500 ml of agarose. Agarose(3 ml) was poured into small (35 X 10 mm) tissue culture plates (no. 0001; Falcon Plastics, Oxnard, CA). Wells were punched in a hexagonal configuration; each well was 5 mm in diameter, and the centers of the wells were 8 mm apart. Reagent anti-Hie (-0.07 ml) was placed in the center well, and - 0.07 ml reagent HBeAg and samples to be tested were placed in the peripheral wells. (HBeAg and anti-HBe reagents were standardized with reagents provided by Dr. George L. Le Bouvier and Mr. Alan Williams, Yale University School of Medi- cine, New Haven, CT). Plates were incubated in a moist chamber a t 24 C, and read at 24-hour inter- vals for seven days.

The presence of HBeAg was determined by the appearance of a precipitin line with reagent anti- HBe and confirmed by a line of identity with re- agent HBeAg. HBeAg/ 1, HBeAg/Z,and HBeAg/3 components were detected as described previ- o ~ s l y . * ~ ~ * ~ The presence of anti-HBe was deter- mined by the appearance of a precipitin line with reagent HBeAg and a line of identity with reagent anti-HBe.

Statistical significance of the serologic differ- ences between volunteer and paid donors was de- termined using chi square and contingency tables.

Results HBeAg was detected in 31 HBsAg-positive paid

donors (15%) and in 11 HBsAg-positive volunteer

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HBeAg ANTI-HBe Fici. I . HBeAg and Anti-HBe in HBsAg-positive

volunteer (0) and paid (m) blood donors.

donors (5%) (p<O.OOI) (Fig. 1). The HBeAg/I component was detected in all H BeAg-positive donors. The HBeAg/2 component was detected in two HBeAg-positive volunteer donors and the HBeAg/3 component was not detected in any (Table 1). Among both groups of donors, the

presence of HBeAg was associated with higher titers of HBsAg (Fig. 2). HBeAg wasdetected in 11 of 103 volunteer donors with an HBsAg C F titer 21:32 (11%), but in none of the 97 volunteer donors with an HBsAg C F titer < 1:32 (p<O.Ol). HBeAg was detected in 27 of 131 paid donors with HBsAg C F titer 21:32 (21%), but in only four of the 69 paid donors with HBsAg C F titer <1:32 (6%) (p<O.Ol). The increased prevalence of HBeAg among paid donors was detected a t al- most every HBsAg titer (Fig. 2). The HBeAgl1, HBeAg/2. and HBeAg/3 components and their antibodies were not consistently associated with HBsAg titer. The prevalence of HBeAg was inde- pendent of anti-HBc titer among both donor groups.

Anti-HBe was detected in 122 HBsAg-positive volunteer donors (61%) and in 96 HBsAg-positive paid donors (48%) (not a significant difference) (Fig. 1 ) . No significant difference was found be- tween the two donor groups in the prevalence of anti-HBe/ I or anti-HBe/2 (Table I ) . Anti-HBe/2 alone was detected much less frequently than anti-HBe/I alone. AntiHBel3 could not be de- tected in any donor sera. In the serum of four volunteer donors with HBeAg/1 and ten paid donors with HBeAg/l, anti-HBe/2 was also detected. In general, detection ofanti-HBe was not significantly associated with HBsAg titer, but it was less frequently detected among those with higher HBsAg titers in both groups (Fig. 3). The prevalance of anti-HBe was independent of anti- HBc titer among both donor groups.

N o association could be demonstrated between the HBsAg subtypes and the prevalence of HBeAg or anti-HBe, or any of the components of HBeAg

Table 1. HBeAg, Anti-HBe, and HBsAg Subtype in the Serum of HBsAg-positive Volunteer and Paid Blood Donors

TotalHBsAg+ HBeAg/l anti-HBe/l* anti-HBe/2* donors alone HBeAg/l + 2 alone alone anti-HBe/l + 2*

21

Subtype adw 86 4 0 41 3 11 Subtype ayw 59 3 2 24 a 3 Subtype adr 2 1 0 0 0 0 Undetermined 53 1 0 19 6 7

6

- 17 - 84 - 2 - 9 - 200 - Volunteer Donors

- - 20 - 70 - 0 - 31 - Paid Donors 200

Subtype adw 84 12 0 37 7 5 Subtype ayw 69 14 0 13 9 1 Subtype adr 1 0 0 0 0 0 Undetermined 46 5 0 20 4 0

* Without detectable HBeAg

I94 HBeAg IN BLOOD DONORS 1 ranrluwm March-April 19RO

L c

i <1:4 la 1:16 192 1S4 1:128 la >1512

HBsAg TITER

FIG. 2 . Relationship of HBsAg titer and HBeAg in volunteer ( 0 ) and paid (m) blood donors.

(1 :4 1s

1

W13

on w4

1:16 1 3 2 1s 1:128 1.m ,1512 HBsAg TITER

FIG. 3 . Relationship of HBsAg titer and anti-HBe in volunteer (0) and paid (m) blood donors.

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195

or their corresponding antibodies among either volunteer or paid donors (Table 1). The HBsAg subtypes adw and ayw were similarly distributed in both volunteer (86 adw, 59 ayw, 53 undetermined) and paid (84 adw, 69 ayw, 46 undetermined) donor groups. Three donors had HBsAg subtype adr; HBeAg was detected in one.

The presence of anti-HBs was a repeatable finding in 12 of the 400 HBsAg-positive donors (3%). Although presumed to be heterotypic anti- body,z5 the coincident anti-HBs was in most cases of such low titer that subtyping could not be per- formed. HBeAg could not be detected in any of these 12 donors. Anti-HBe was detected in five of seven volunteer donors with coincident HBsAg and anti-HBs(71%), and in one of five paid donors with simultaneous HBsAgand anti-HBs (20%). N o single anti-HBe specificity was more prevalent in this group.

Rheumatoid factor (RF) was equally prevalent among volunteer and paiddonors. Among HBeAg- positive donors it was more prevalent among paid donors (Fig. 4). No significant difference was found in the prevalence of R F among HBeAg- positive donors when compared to anti-HBe- positive volunteer or paid donors, or to those with

neither HBeAg or anti-HBe. No association was found between R F and the HBeAg/ 1 , HBeAg/2, and HBeAg/3 components or their antibodies.

Discussion

HBeAg is a soluble antigen,” distinct from the other known HBV-associated antigens, but intimately associated with HBV infection. It has been found in some patients with chronic persistent o r chronic aggressive hepa- titis B.’,” In contrast, anti-HBe is found more frequently in the sera of asymptomatic car- riers of HBsAg.”I7 The high prevalence of HBeAg among HBsAg carriers in hemodia- lysis units and the increased infectivity attrib- uted to their sera,” suggested that HBeAg might be an indicator of increased HBV infec- tivity. Recent studies have shown an associa- tion between HBeAg, the presence of intact HBV particles, DNA p~ lymerase ‘” ’~~ and

FIG. 4. Relationship of rheumatoid factor (RF) to HBeAg and anti-HBe in vol- unteer (0) and paid (m) blood donors.

HBeAg ANTI-HBe NEITHER

196 HBeAg IN BLOOD DONORS I ranstusion March -Apri l I980

increased risk of HBV transmission from infected mothers to their newborn infants.""' HBeAg has also been shown to be associated with increased transmission of HBV by acci- dental needlestick.'

More than twice as many recipients of HBsAg-negative units of blood from paid donors develop HBV infection, compared to those receiving H BsAg-negative blood from volunteer donors." It is not known whether HBsAg-positive paid donors also might be more infectious as a group than HBsAg- positive volunteer donors, and to determine this we compared HBsAg-positive volunteer and paid donors for HBeAg, a marker for increased infectivity. In the present study, HBeAg was found more often among HBsAg- positive paid donors than among HBsAg- positive volunteer donors. Another recent study also found a higher prevalence of HBeAg among paid donors than volunteer donors, but the two groups in that study were from different geographic locations."

The detection of HBeAg was found to be directly related to HBsAg titer in this study, as has been ~uggested.',~~'' The association of HBeAg with increased infectivity x * 1 6 * 2 1 and increased numbers of intact HBV particles 2",27

may reflect the association of HBeAg with the presence of higher HBsAg titers. Even when more sensitive assays for HBeAg are devel- oped, a similar association may be found between high titers of HBeAg and high titers of HBsAg, as suggested in a recent study by Mushahwar et ~ 1 . ' ~ In this study, HBeAg was more prevalent among paid than volunteer donors at almost every HBsAg titer and thus the greater prevalence of HBeAg among paid donors could not be ascribed solely to higher HBsAg titers.

The existence of a t least three components of HBeAg and their corresponding antibodies have been d e ~ c r i b e d I ~ * ~ ~ + ~ ~ but their clinical significance is not understood. In this study, no difference was found between volunteer and paid donors with regard to the compo- nents of HBeAg or specificities of anti-HBe

nor were these related to HBsAg titer or the detection of RF. Thus, the presence or one or more of these components could not explain the higher prevalence of HBeAg among paid blood donors.

A recent hypothesis suggested that HBeAg might be anti-IgG4.'' Mushahwar et al." have shown that H BsAg-negative, R F-positive sera do not react in assays for HBeAg or anti- HBe. We evaluated the donors in this study for the presence of RF, an anti-lgG. N o asso- ciation was found between R F and the pres- ence or absence of HBeAg or anti-HBe, suggesting that HBeAg is not an anti-IgG. The finding of Chadwick et al.4 who detected HBeAg in an HBsAg-positive patient with agarnmaglob~linemia~ also suggests that HBeAg is not an antibody. The high preva- lence of R F in patients with liver diseases, regardless of the HBsAg status of the patients, has been well-documented.13 The greater prevalence of R F in HBeAg-positive paid donors compared to HBeAg-positive volun- teer donors here therefore may reflect a greater severity of liver injury13 among the paid donors.

The simultaneous detection of HBeAg/ 1 and anti-HBel2 has not been previously reported. In this study, such a serologic pat- tern was detected in three per cent of HBsAg- positive donors, and was a repeatable finding. The fact that HBeAg/l, HBeAgl2, and HBeAg/3 form three distinct, parallel pre- cipitin lines in agar gel diffusion, strongly suggests that they are completely separate antigens, and may be produced or removed independently ofeach other. This finding may represent a stage in the elimination of HBeAg and conversion to anti-HBe,' and anti-HBe/Z may be present in undetectable titers in other HBeAg/ 1 -positive individuals. The finding of anti-HBe/l and 2 in 21 (10%) volunteer donors and six (3%) paid donors suggests that clearance of preexisting HBeAg/ 1 and HBeAg/2 may have occurred in more of the volunteer donors than paid donors. This appears to clarify the mechanism by which

Volume 20 Number 2 TABOR ET AL. I97

HBeAg is cleared from the circulation, a change which may signal the development of a more favorable p r o g n o s i ~ . ’ ~ ~ ~ ’ ~

The nature of HBeAg is not well-defined, and this makes it difficult t o explain why HBeAg was more prevalent among HBsAg- positive paid donors than among volunteer donors. This study shows that the greater prevalence cannot be attributed solely t o higher HBsAg titers, even though HBeAg and HBsAg titers were closely associated, since HBeAg was more prevalent among paid donors a t almost every HBsAg titer. Nor could the greater prevalence be ascribed to the presence of any of the three known HBeAg components, or to the simultaneous detec- tion of HBeAg/l and anti-HBe/2 in some donors. The role of the donors’ ages” was not evaluated.

Acknowledgment

The authors thank Mr. A. J . Shawver and Mr. D. Gil- bert for technical assistance.

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I98 HBeAg 1N BLOOD DONORS

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I ransfusion March-April 1980

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

Martin Goldfield, M.D., Professor of Epidemiology, School of Public Health, University of South Carolina, Columbia, S C 29208

Henry C. Black, M.S., D.V.M., (Formerly) Research Scientist, New Jersey State Department of Health ( Deceased )

Robert J. Gerety, M.D., Ph.D., Director, Hepatitis Branch, Bureau of Biologics.