H. W. Reesink

Confirmation of hepatitis C virus infection by new four-antigen recombinant immunoblot assay

A new four-antigen recombinant immunoblot assay (4-RIBA) for confirmation of hepatitis C virus (HCV) C-100 enzyme-linked immunosorbent assay (ELISA) reactivity was tested in stored serum samples (1984-86) of blood donors and recipients and compared with results from polymerase chain reaction (PCR) analysis of fresh (1990) plasma samples in donors and recipients from the original study. Of 37 HCV C-100 ELISA-positive blood products, 8 were 4-RIBA positive, of which 7 were implicated in post-transfusion non-A, non-B hepatitis (PT-NANBH) and/or PCR confirmed recipient HCV infection. Of 9 recipients with PT-NANBH, 8 were reactive in 4-RIBA (6 positive and 2 indeterminate). With fresh plasma samples, 3 donors and 6 recipients who were 4-RIBA positive were also PCR positive. 4 4-RIBA indeterminate and 78 4-RIBA negative samples of donors and recipients were PCR negative. Of 6 4-RIBA positive recipients, 5 were PCR positive four to six years later. 1.6% of the 383 recipients became chronically infected with HCV. The new 4-RIBA represents a candidate confirmation test to discriminate between infective and non-infective HCV C-100 ELISA-positive blood donors.

Reliability of polymerase chain reaction for detection of hepatitis C virus

The polymerase chain reaction (PCR) is used to detect hepatitis C virus (HCV) RNA, and the results of this assay may have a bearing on management of patients. We tested 31 laboratories for performance of HCV PCR with a coded panel that comprised 4 HCV-positive plasma samples, 6 HCV-negative samples, and two dilution series of HCV-positive plasma. 15 (48%) laboratories had faultless results with both dilution series, and 16 (52%) laboratories reported erroneous results with one or both series. 10 (32%) laboratories had faultless results when testing undiluted plasma samples, 11 (35%) produced a false-negative result with a weak-positive sample, and 10 (32%) produced false negative and/or false positive results. Only 5 (16%) laboratories performed faultlessly with the entire panel of samples. Reports of presence of HCV should be interpreted with care until reliable HCV-RNA detection becomes widely available.

PREVENTION OF THE HBsAg CARRIER STATE IN NEWBORN INFANTS OF MOTHERS WHO ARE CHRONIC CARRIERS OF HBsAg AND HBeAg BY ADMINISTRATION OF HEPATITIS-B VACCINE AND HEPATITIS-B IMMUNOGLOBULIN: Double-blind Randomised Placebo-controlled study

Newborn infants of Chinese HBeAg-carrier mothers in Hong Kong were randomly assigned to one of four study groups. Group I was treated with 3 micrograms heat-inactivated hepatitis B (HB) vaccine at birth and at 1, 2, and 6 months thereafter, in conjunction with seven monthly HBIg injections; group II was treated according to the same vaccine schedule but received only one HBIg injection at birth; group III received only the vaccine, at months 0, 1, 2, and 6; and group IV received placebos for both vaccine and HBIg. The first set of injections was given within 1 h after birth. Comparisons were made in the 140 children who were at least six months old at the close of the trial (495 days). In all three treatment groups development of the persistent carrier state was significantly (p less than or equal to 0.0001) less frequent than in controls (2.9%, 6.8%, and 21.0% versus 73.2%). Although vaccination alone was significantly less protective than vaccination plus multiple HBIg injections (p = 0.03), the degree of protection was still remarkable. 12 months after the first set of injections 96-100% of the infants in the three treatment groups were anti-HBs positive; the geometric mean titres of anti-HBs in the three groups did not differ significantly. This indicates that even high doses of HBIg do not interfere with the anti-HBs response to the vaccine. Probable intra-uterine HB infections were observed in 3 infants. No serious side-effects were observed from the interventions, even in the babies with intra-uterine infections who had received HBIg and HB-vaccine at birth. To prevent development of the persistent HBsAg carrier state, and thereby the consequent chronic liver disease and/or primary carcinoma of the liver, HB vaccine and HBIg should be administered as soon as possible after birth to all newborn infants at risk of perinatal hepatitis B infection.

Infectivity of blood seropositive for hepatitis C virus antibodies

Stored serum samples from 5150 blood product transfusions and 383 recipients were tested for antibodies to hepatitis C virus (anti-HCV) by a recombinant enzyme-linked immunosorbent assay (ELISA) as part of a prospective study on post-transfusion non-A, non-B hepatitis (NANBH). Donor cofactors associated with HCV infectivity of anti-HCV-positive blood products were raised alanine aminotransferase concentrations (6 of 9 infective vs 1 of 26 not infective); a mean ELISA optical density/cut-off ratio greater than or equal to 2 (7 of 9 vs 9 of 26); both preceding factors (together in 6 blood products, all of which transmitted infection); and persistent donor anti-HCV seropositivity. Use of anti-HCV screening to prevent post-transfusion NANBH was compared with measurement of alanine aminotransferase concentrations: a corrected efficacy of 63% and 65%, a specificity of 93% and 64%, and a positive predictive value of 16.2% and 3.6% were found, respectively; 0.7% or 3.8% of blood donations, respectively, would be discarded. Blood donor screening for anti-HCV is recommended to reduce the incidence of post-transfusion NANBH.

ANTI-HEPATITIS C ANTIBODIES AND NON-A, NON-B POST-TRANSFUSION HEPATITIS IN THE NETHERLANDS

In a prospective study carried out in the Netherlands (1984-86) to establish the incidence of post-transfusion hepatitis non-A, non-B (PTH-NANB) in patients undergoing open heart surgery, 393 patients received 5315 blood product transfusions. PTH-NANB developed in 9 patients (index cases); stored serum samples from these patients and from 9 control patients, matched for age, sex, and number of blood product transfusions, as well as serum samples of all implicated blood products, were selected retrospectively. Sera were tested under code with a radioimmunoassay for the detection of antibodies to hepatitis C virus (anti-HCV). PTH-NANB patients received 151 blood product transfusions and control patients 140. 4 of 9 PTH-NANB patients (3/5 chronic, 1/4 acute resolved hepatitis) and 0/9 controls seroconverted. 7 of the transfusions given to PTH-NANB patients but none of those given to control patients were anti-HCV positive. In 7 of 9 serum sets from PTH-NANB index cases plus implicated donors, either a donor or the recipient was anti-HCV positive. Among the donors implicated in transmission of PTH-NANB there was a strong correlation between raised alanine aminotransferase levels and the presence of anti-HCV antibodies.

Sexual transmission of hepatitis C virus

We tested 50 heterosexual partners of hepatitis C viraemic (HCV) individuals, using second generation HCV antibody assays and a validated polymerase chain reaction assay. In none of them were HCV antibodies or HCV-RNA detected. The median duration of the sexual relationship was 13 years. This study, with the most sensitive techniques for detection of HCV, indicates that the risk of sexual transmission of HCV is absent or very low.

Platelet activation during preparation of platelet concentrates: a comparison of the platelet-rich plasma and the buffy coat methods

The activation of platelets during the preparation of platelet concentrates (PCs) by two methods was compared. To eliminate interdonor differences, 2 units of whole blood were pooled and subsequently divided into two batches. From one batch, the platelets were harvested as pelleted platelets from platelet‐rich plasma (PRP) and from the other as nonpelleted platelets from the buffy coat (BC). The activation of platelets in these PCs was studied immediately after preparation and during storage for up to 9 days at 22°C with gentle agitation. The binding of monoclonal antibodies (MoAbs) against the GP IIb/IIIa complex and against activation‐dependent antigens (GMP 140 from the alpha granules and a 53‐kDa glycoprotein from the lysosomal granules) was measured. β‐thromboglobulin (β‐TG) release was also determined. Disc‐to‐sphere transformation was quantitated by measuring on an aggregometer the difference in light transmission during stirring at different rates and also by light microscopy. Immediately after preparation, platelets derived from PRP had a more spheric morphology (p < 0.01), had a higher β‐TG release (p < 0.01), bound more MoAbs against GP IIb/IIIa (p < 0.01), and expressed more GMP 140 and 53‐kDa glycoprotein (p < 0.01) than did BC‐ derived platelets. However, these differences had disappeared after 2 days of storage. It was concluded that, immediately after preparation, PRP‐derived platelets are more activated than BC‐derived platelets. This is most likely a result of the pelleting that follows the second high‐speed centrifugation of the PRP.

International collaborative study on the second EUROHEP HCV-RNA reference panel

Eighty-six laboratories participated in a collaborative study and tested the second EUROHEP HCV-RNA reference panel. The coded panel comprised 4 HCV-RNA positive plasma samples (one weak positive), 6 HCV-RNA negative plasma samples and two dilution series of HCV-RNA genotype 1 and 3 plasma standards. The 86 laboratories submitted 136 coded data forms for evaluation. Of these data sets 99 were tested using a PCR assay developed in-house, 28 using a commercially available HCV-PCR test (AMPLICOR, Roche Diagnostic Systems) and 9 using other amplification methods. Twenty-two data forms (16%) had faultless results, 39 (29%) missed the weak positive sample only and 75 data sets (55%) had false positive and/or false negative results. Participants using the commercial HCV-PCR test tended to reach a sufficient quality score more often than investigators using assays developed in-house (64% versus 45%, P = 0.11). The UNG system in the commercial HCV-PCR test did not prevent five laboratories generating false-positive results in the 6 HCV-RNA negative samples. Among the laboratories with satisfactory results, up to 10000-fold differences in sensitivity were observed in the dilution series. The 50% and 90% laboratories detection endpoints in the dilution series of the HCV genotype 1 plasma standard were approximately 600 genome equivalents per ml (geq/ml) and 7750 geq/ml according to a standard applied in a signal amplification assay (bDNA, Chiron). Our results suggest that the detection efficiency for genotype 3 by commercial HCV-RNA assays is lower than by the in-house assays. Internationally characterized HCV-RNA plasma standards should be made available for validation and standardization of HCV-RNA assays for HCV diagnosis and virological safety testing of blood products.

Prevention of hepatitis B virus carrier state in infants according to maternal serum levels of HBV DNA

235 infants of HBeAg-carrier mothers in Hong Kong were assigned to four study groups. Groups I, II, and III received hepatitis-B (HB) vaccine at birth and at 1, 2, and 6 months. Group I also received seven monthly injections of HB immunoglobulin (HBIg), and group II received one HBIg injection at birth. Group III received vaccine only and group IV received placebo for both vaccine and HBIg. At the age of 3 years, all infants of the three treatment groups were significantly protected against the HB virus (HBV) carrier state compared with the placebo group (p less than 0.0001); the protective efficacy rates in groups I, II, and III were 87%, 80%, and 65%, respectively. At all times, group I was significantly better protected than group III. In groups III and IV, infants of mothers with serum HBV DNA levels of 5 pg/ml or above were at a significantly higher risk of acquiring the HBV carrier state than those whose mothers had HBV DNA levels below 5 pg/ml. This difference was not significant in groups given HBIg. Of the 183 infants who initially escaped HBV infection, 73 (40%) had transient and 8 (4%) chronic HBV infection between 6 and 36 months. Vaccinated infants who had actively formed anti-HBs remained well protected against the HBV carrier state. However, infants in groups I and II with no active anti-HBs response to vaccine became at risk for the HBV carrier state when the passively acquired anti-HBs antibodies had disappeared. HBIg should be included in HB vaccination schedules for all infants of HBeAg-positive mothers.

Prevention of Yersinia enterocolitica growth in red-blood-cell concentrates

In response to concern about Yersinia enterocolitica contamination of blood products, we have studied the effects on Y enterocolitica growth of holding whole blood at 22 degrees C for 20 h and then removing leucocytes. Thirty pools of three bags of blood were inoculated with Y enterocolitica (2 x 10(1)-3 x 10(4) colony-forming units/ml). One bag in each pool was processed to red-blood-cell concentrate after 6 h at 4 degrees C (RBC); the other two were held at 22 degrees C for 20 h before processing to buffy-coat-depleted RBC (BCd-RBC). One of these bags was then depleted of leucocytes by filtration (Ld-RBC). All bags were stored at 4 degrees C for 5 weeks. RBC bags showed Y enterocolitica growth after the shortest storage times, followed by BCd-RBC then Ld-RBC (p less than 0.03-0.001). We recommend that whole blood should be held at 22 degrees C to make use of inherent bactericidal activity; leucocytes should then be removed.