Impact of donor-specific antibody with low mean fluorescence intensity on allograft outcomes in kidney transplant.

BACKGROUND: Immune-mediated rejection is the most common cause of allograft failure in kidney transplant (KT) patients. Exposure to alloantigen, including human leukocyte antigen (HLA), results in the production of donor-specific antibodies (DSA). There are limited data about low levels of mean fluorescence intensity (MFI) DSA, especially post-transplantation. This study evaluated allograft outcomes in KT patients with low MFI DSA. METHODS: From January 2007 to December 2021, KT patients who were tested for post-transplant DSA at Ramathibodi Hospital, Bangkok, Thailand, with the DSA MFI ≤ 1000 were evaluated. These KT patients were categorized into two groups: very low DSA (VLL; MFI = 1-500) and low DSA (LL; MFI = 501-1000). All KT patients were evaluated for the primary outcomes, such as the incidence of acute rejection, serum creatinine levels at one and five years after transplantation as well as allograft and patient survivals. RESULTS: Among 36 KT patients 25 were included as those with VLL and 11 as those with LL. The LL group had significantly higher T-cell mediated allograft rejection (TCMR) than the VLL group (45% vs. 12%, P = 0.04). In addition, 10 patients, 5 in the VLL group and 5 in the LL group developed antibody-mediated allograft rejection (ABMR). Both TCMR and ABMR were confirmed by biopsy results. There was a trend toward higher MFI in KT patients with ABMR than without ABMR (P = 0.22). At 5 post-transplant years, serum creatinine, allograft and patient survivals were comparable between these two groups. Furthermore, the univariate and multivariate analyzes revealed that the LL group was a high risk for rejection. CONCLUSION: Low MFI DSA values after transplantation may be associated with a higher incidence of rejection, but this finding did not show differences in allograft and patient survival in this study's analysis.

Impact of HLA Eplet Mismatch on De Novo Donor Specific Antibody Formation After Kidney Transplantation.

BACKGROUND: HLA eplet mismatching is an alternative approach to assess the risk of developing de novo donor-specific antibodies (dnDSA) in kidney transplantation. This strategy may offer more precise risk stratification than conventional approaches. This study aimed to find the association between HLA eplet mismatches and dnDSA formation in Thai kidney transplant recipients. METHODS: A retrospective cohort study of kidney transplant recipients transplanted between 2000 and 2021 at Ramathibodi Hospital was performed. Recipients with pretransplant panel reactive antibody >0% or without DSA testing post-transplant were excluded. One hundred fifty recipients were included in the final study. High-resolution HLA typing was imputed by the HaploStat application. HLA eplet mismatch analysis was conducted using HLAMatchmaker. The association between the number of eplet mismatches and the risk of dnDSA formation was assessed by Cox regression analysis. RESULTS: Of 150 recipients, 43 were dnDSA-positive, and 107 were dnDSA-negative patients. Compared with the dnDSA-negative group, patients with class II dnDSA had significantly more HLA-DR/DQ antibody (Ab)-verified eplet mismatches (6 [IQR 4-8] vs 4 [IQR 1-7], P = .045). The receiver operating characteristics analysis showed that the HLA-DQ Ab-verified eplet mismatches ≥2 were the best predictive of HLA class II dnDSA development. The number of HLA-DQ Ab-verified eplet mismatches ≥2 had the highest hazard rate of HLA class II dnDSA occurrence (adjusted HR, 3.74; 95%CI, 1.24-11.24, P = .019). CONCLUSIONS: HLA-DQ Ab-verified eplet mismatches are significantly associated with class II dnDSA development. Our data supports the utility of HLA eplet mismatching for donor-recipient risk assessment.

A 46-Year-Old Thai Woman with Secondary Acquired Pure Red Cell Aplasia Due to Treatment with Recombinant Erythropoietin While on Dialysis for End-Stage Renal Disease Who Recovered Following ABO-Incompatible Kidney Transplantation.

BACKGROUND Pure red cell aplasia (PRCA) is an uncommon cause of anemia in end-stage kidney disease (ESKD). It is attributed to recombinant human erythropoietin (rHuEPO) administration. Although immunosuppression is the mainstay therapy, its effectiveness varies from 30% to 70%. PRCA in ESKD has been reported to improve following kidney transplantation. CASE REPORT A 46-year-old woman with ESKD secondary to lupus nephritis was treated for uremia at our center. She developed severe anemia. Bone marrow aspiration and biopsy revealed a reduction of erythroid precursors, consistent with PRCA. Because she had no sibling's blood group matched with her, ABO-incompatible kidney transplantation was an option for treatment. She underwent a desensitization protocol consisting of rituximab 375 mg/m2, tacrolimus, mycophenolate mofetil, and prednisolone 4 weeks before surgery, in addition to 3 sessions of double-filtration plasmapheresis (DFPP) every other day followed by intravenous immunoglobulin (IVIG) and 1 session of specific immunoadsorption (Glycosorb® B column) at pre-transplant day -1. She also received low-dose rabbit anti-thymocyte globulin (rATG) (Thymoglobulin®) (total 2.0 mg/kg). Maintenance therapy included tacrolimus, mycophenolate mofetil, and prednisolone. Allograft function normalized a few days after transplantation and her Hb gradually increased. CONCLUSIONS We report a rare case of PRCA in a patient with ESKD undergoing ABO-incompatible kidney transplantation. The outcome was satisfactory, with complete correction of anemia and kidney function.

Misidentification of preformed anti-HLA-DP antibodies leads to antibody-mediated kidney transplant rejection: a case report.

BACKGROUND: Patients who are HLA-sensitized are at high risk for early antibody-mediated rejection (AMR) and worse outcomes. Therefore, it is crucial to detect the presence of donor-specific antibodies (DSAs) using pretransplant antibody identification and crossmatch assays. An error in antibody identification can lead to disastrous clinical outcomes. We present a case of acute AMR associated with preformed HLA-DPα and HLA-DPß DSAs that were not identified before transplantation. CASE PRESENTATION: A 27-year-old woman received a second kidney transplant from a deceased donor. Her pretransplant panel-reactive antibody level was 94%. The complement-dependent cytotoxicity crossmatch was negative for T and B cells at the time of transplantation. She experienced early acute AMR proven by a kidney biopsy. Single antigen bead testing of the patient's serum at the time of rejection as well as the pre-second transplant serum revealed strong antibodies against the DPA1*01:03 and DPB1*02:01 alleles in the second donor. These antibodies were not identified by phenotypic bead assay during the patient's time on the waiting list. The patient was treated with plasmapheresis and anti-thymocyte globulin. However, she experienced abdominal pain on day 37 post-transplantation. Surgical exploration revealed a laceration on the transplanted kidney, which was then repaired. Subsequently, infected hematoma was suspected and the transplanted kidney was removed. CONCLUSION: The present case highlights the clinical significance of preformed HLA-DPα and HLA-DPß DSAs. Accuracy in determination of HLA antibodies before transplantattion is critical for transplant outcome. HLA-DP typing and single antigen bead testing are recommended for a precise antibody interpretation, especially in highly sensitized patients. Careful interpretation of antibody testing results is essential for the success of organ transplantation.

Associations between HLA-B27 subtypes and outcomes in Thai children with enthesitis-related arthritis.

OBJECTIVE: Expression of human leukocyte antigen B27 (HLA-B27) has been identified as a predictor of severe disease in enthesitis-related arthritis (ERA) patients. However, the associations between HLA-B27 subtypes and outcomes of this disease are still unclear. Here, we examined the distributions of HLA-B27 subtypes among ERA patients and the associations with disease outcomes. METHODS: This was a historical cohort study of ERA patients. Patients were followed from diagnosis to the most recent visit. Relationships between outcomes and the HLA-B27 subtype were assessed by mixed-effect regression, Kaplan-Meier survival, and Cox proportional hazards regression analyses. RESULTS: Of the 66 ERA patients, 50 HLA-B27-positive (86% male) and 16 HLA-B27-negative (69% male) patients were included in this study. Patients with HLA-B27-positive were classified into HLA-B*27:04-positive (84%), including combined HLA-B*27:04 and HLA-B*27:07 (2%), and HLA-B*27:04-negative (16%), including HLA-B*27:05 (10%), HLA-B*27:06 (2%), HLA-B*27:07 (2%), and HLA-B*27:15 (2%). HLA-B*27:04-positive (83.3%) and HLA-B*27:04-negative patients (100%) had refractory disease more than HLA-B27-negative patients (37.5%, p = 0.001). HLA-B*27:04-negative patients (57%, 1.73 years) had relapsing disease more and earlier than HLA-B*27:04-positive (35%, 5.54 years) and HLA-B27-negative patients (40%, 6.92 years; p < 0.001). Furthermore, HLA-B*27:04-negative was predictors of refractory disease (HR 4.56, 95%CI 1.40-14.87; p = 0.012) and relapsing disease (HR 3.80, 95% CI 1.18-12.30; p = 0.026). The duration before anti-tumor necrosis factor treatment initiation > 1 year was also a predictor of refractory disease (HR 116.08, 95% CI 14.67-918.26; p < 0.001). CONCLUSION: HLA-B*27:04 was the most common HLA-B27 subtype in Thai ERA patients. HLA-B*27:04-negative was associated with more unfavorable outcomes than HLA-B*27:04-positive and HLA-B27-negative patients. Key Points • Most ERA patients in Thailand had HLA-B27-positive, and HLA-B*27:04 was the most common HLA-B27 allele in these patients. • The outcomes of ERA were associated with the presence of HLA-B27 and its subtypes. • HLA-B*27:04-negative patients had unfavorable outcomes, including refractory and relapsing disease, compared to HLA-B*27:04-positive and HLA-B27-negative patients.

Double-Filtration Plasmapheresis Plus Low-Dose Anti-thymocyte Globulin and Tacrolimus in Asian Living-Donor Kidney Transplantation With Donor-Specific Anti-HLA Antibody.

BACKGROUND: Pretransplant desensitization protocols, including plasmapheresis, intravenous immunoglobulin, induction antibody therapy, and intensive maintenance immunosuppression, are generally employed in kidney transplant recipients who have positive status for donor-specific anti-HLA antibody (DSA). To avoid serious infectious complications, the authors designed a novel low-dose protocol in Thai patients undergoing DSA+ living-related kidney transplantation (LRKT). METHODS: A retrospective cohort study of the patients who underwent DSA+ LRKT was conducted. The novel protocol consisted of 3 to 5 sessions of pretransplant double-filtration plasmapheresis (DFPP) with or without low-dose intravenous immunoglobulin together with low-dose anti-thymocyte globulin (ATG) induction (1-1.5 mg/kg/d for 3-4 days) and low-dose tacrolimus (Tac) (trough level 5-10 ng/mL), mycophenolate, and prednisolone. RESULTS: The study included 17 patients. The lymphocyte crossmatch via complement-dependent cytotoxicity was negative in 12 patients and positive for B cell immunoglobulin M in 5 patients. The novel desensitization protocol resulted in a decrease of at least 50% of DSA mean fluorescence intensity from baseline (from 4320 ± 549 before DFPP to 1601 ± 350 before transplantation, P < .005) and successful kidney transplantation with good allograft function in all cases. Early DSA rebound was observed in 3 patients after transplantation, and kidney biopsy revealed subclinical antibody-mediated rejection in 1 patient and diffuse C4d staining without cell infiltration in 2 patients. There were good long-term outcomes in patient and graft survival (100% and 94.1%, respectively). Only 1 allograft loss occurred because of nonadherence. The majority of patients have stable allograft function with serum creatinine less than 1.5 mg/dL. However, infections, including CMV and other organisms, were commonly observed. CONCLUSIONS: Desensitization protocol with DFPP, low-dose ATG, and Tac provides excellent outcomes in living donor kidney transplantation in highly sensitized Asian populations.

Comparison Between Phenotypic Bead Assay and Single Antigen Bead Assay for Determining Specificity of HLA Antibodies in Kidney Transplant Waiting List.

OBJECTIVES: The determination of unacceptable antigens in patients on kidney transplant waiting list is a critical laboratory investigation in sensitized patients. The Luminex single antigen bead (SAB) assay has high sensitivity and accuracy. However, several countries have not yet implemented SAB testing for waitlisted patients because of limited financial resources. In Thailand, specificities of HLA antibodies are identified by using a phenotypic bead assay. The aim of this study was to evaluate the performance of the phenotypic bead assay for determining HLA antibody specificities when compared with the SAB assay. METHODS: A total of 254 sera from patients awaiting kidney transplantation were included. Of 254 sera, 206 and 171 were positive for HLA class I and II antibodies, respectively. Antibody specificities of sera that were tested with both phenotypic and SAB assay were analyzed. The performances of the phenotypic bead assay were compared with those of the SAB assay as the gold standard by using estimation of pooling sensitivity, specificity, and accuracy. RESULTS: The sensitivity, specificity, and accuracy of the phenotypic bead assay for determining HLA class I antibodies was 53.9%, 93.0%, and 78.1%, respectively. The sensitivity, specificity, and accuracy of the phenotypic bead assay for determining HLA class II antibodies were 57.3%, 94.9%, and 81.4% respectively. CONCLUSION: In waitlisted kidney transplant patients, the phenotypic bead assay had high specificity and moderate accuracy when compared with the SAB assay. However, the low sensitivity of the test suggests that the use of the phenotypic assay for determining HLA specificities should be applied with caution in sensitized patients.

Antibody-Mediated Rejection Due to Donor-Specific HLA-DQB1 and DQA1 Antibodies After Kidney Transplantation: A Case Report.

BACKGROUND: Donor-specific HLA antibody (DSA) is associated with the risk of allograft loss due to antibody-mediated rejection (ABMR). The majority of de novo DSA after kidney transplantation is directed toward donor HLA-DQ antigens. A HLA-DQ antigen is a heterodimer consisting of an alpha and beta chain. Traditionally, HLA-DQA1 typing has not been part of the pretransplant evaluation. Therefore, DQ alpha proteins are not usually taken into account in the interpretation of HLA-DQ antibody reactions. METHODS: We hereby present a case of a kidney transplant recipient with 0% pretransplant panel reactive antibody. She received kidney allograft from her husband. Two years after transplantation, she experienced abdominal swelling, and enlargement of transplanted kidney was identified. A biopsy of the allograft kidney demonstrated chronic active ABMR. DSAs were investigated using immunoglobulin G (IgG) and C1q single antigen bead (SAB) assay. HLAMatchmaker analysis was performed to identify eplets that explain the antibody reactivity patterns. RESULTS: The IgG SAB analysis of a patient's serum at the time of rejection showed positive reactions with all DQ2-carrying beads with mean fluorescence intensity (MFI) > 10000. However, the C1q assay demonstrated strong reaction to only HLA-DQA1∗05:01-DQB1∗02:01-carrying bead with MFI = 22462, whereas weak or no reactions against other HLA-DQ2-carrying beads were found. High-resolution HLA typing revealed that HLA-DQA1∗05:01 and DQB1∗02:01 were mismatched donor antigens. HLAMatchmaker analysis showed that the antibodies were reactive toward 40GR3 eplet on DQA1 and 45GE3 eplet on DQB1. CONCLUSIONS: This case highlights the clinical significance of antibodies specific to both DQ alpha and DQ beta chains after kidney transplantation.

Genetic profiles of killer-cell immunoglobulin-like receptors and HLA ligands in Thai blood donors.

Killer-cell immunoglobulin-like receptors (KIRs) play an important role in natural killer (NK) cell regulation. Interaction of KIRs with human leukocyte antigen (HLA) class I molecules can transmit signals to regulate the function of NK cells. In this study, the diversities of KIR genes and their ligands in 500 Thai blood donors were investigated. The coexistence of inhibitory KIRs (iKIR), activating KIRs (aKIR) and their ligands in the same individuals were also analyzed. Overall, 36 KIR genotypes were identified. The most common genotype was genotype AA1 (40.8%). All individuals carried at least one iKIR-HLA pair whereas 18% of the individuals lacked aKIR-HLA pair. The most common compound KIR-HLA profile was the presence of 3 iKIR-HLA pairs with 1 aKIR-HLA pair (21.4%). The most common compound gene profile of KIR-HLA pairs was the combined presence of KIR2DL3-C1, 3DL1-Bw4, 3DL2-A3/A11 and the full length KIR2DS4-its ligands (8%). This study provided a comprehensive analysis of the KIR-HLA profiles in Thai blood donors in regards to KIR genotypes, HLA ligands, KIR-HLA ligand pairs and compound gene profiles of both iKIRs and aKIRs and their ligands. These findings will be useful as baseline information for further studies in the associations of KIR genes and various diseases.

Association of soluble human leukocyte antigen-G with acute tubular necrosis in kidney transplant recipients.

BACKGROUND: Human leukocyte antigen (HLA)-G is a nonclassical HLA class I molecule that displays strong immune-inhibitory properties and has been associated with allograft acceptance. However, there are conflicting data on the correlation of soluble HLA-G (sHLA-G) and acute rejection and no data on the correlation with acute tubular necrosis in kidney transplantation. OBJECTIVE: To evaluate the association of sHLA-G level in early post-transplant period and allograft rejection/ and acute tubular necrosis (ATN) in kidney transplant recipients. METHODS: The sera procured before transplantation and serially on day 3 and day 7 after transplantation from 76 kidney transplant recipients were analyzed for the level of sHLA-G by enzyme-linked immunosorbent assay. RESULTS: The levels of sHLA-G from three serial sera did not differ between patients with acute rejection and patients without rejection. However, the sHLA-G levels on day 3 post-transplant and day 7 post-transplant in patients with ATN were significantly higher than that in patients without ATN (16.3 vs 9.85 U/ml, p = 0.018, for day 3 post-transplant and 12.47 vs 5.42 U/ml, p = 0.044, for day 7 post-transplant). In addition, the ROC analysis of sHLA-G for identifying patients with ATN showed that the area under curve was 0.67 (95% confidence interval 0.54-0.80). CONCLUSIONS: There was no significant difference for sHLA-G levels between patients with acute rejection and without rejection. Interestingly, high levels of sHLA-G in day 3 and day 7 after transplantation were associated with acute tubular necrosis. Our findings raise the question whether the increased levels of sHLA-G in patients with acute tubular necrosis after transplantation might be a result of ischemia and reperfusion injury.

Pre-transplant donor specific antibody and its clinical significance in kidney transplantation.

BACKGROUND: The traditional method for assessing HLA antibodies in recipient serum samples is the complement-dependent cytotoxicity testing (CDC). Recently, the highly sensitive microbead-based Luminex assay was introduced and can detect low levels of anti-HLA Abs. OBJECTIVE: To determine the impact of pretransplant donor-specific HLA antibodies (DSA) detectable by Luminex, despite a negative CDC crossmatch, on the outcomes of kidney transplantation. The correlation and cut-off value of panel reactive antibody (PRA) and DSA was also evaluated. METHODS: Pre-transplant sera from 116 kidney transplant recipients with a negative CDC crossmatch were assessed for donor-specific HLA antibodies by using Luminex single antigen beads. The patients received kidney transplants at Ramathibodi Hospital between January 2003 and December 2007. The results were correlated with kidney graft outcomes. RESULTS: DSA were found in 24.1% (28/116) of all recipients. Of the twenty-eight DSA positive patients, four developed antibody-mediated rejection (AMR) (4/28 = 14.3%). All these 4 patients had positive C4d staining in their biopsies. Of the eighty-eight DSA negative patients, two developed AMR (2/88 = 2.3%). The AMR occurred more frequently in the DSA positive group than in the DSA negative group (14.3% versus 2.3%. The patient and graft survival were similar in both groups. The strength of pre-transplant DSA was not associated with the incidence of rejection episodes. CONCLUSION: There was a higher incidence of AMR in patients with pre-transplant DSA despite a negative CDC crossmatch. However, pre-transplant DSA detected by Luminex had no statistically significant impact on delayed graft function, patient survival and graft survival.

Distribution of killer cell immunoglobulin-like receptor genes in Thai blood donors.

BACKGROUND: Killer cell Immunoglobulin-like Receptors (KIRs) are members of a group of molecules expressed on the surfaces of natural killer (NK) cells and some T cells. KIRs recognize MHC class I molecules on target cells. The interaction of these molecules regulates NK cell reactivity. The KIR gene cluster is highly polymorphic in individuals and different populations. OBJECTIVE: Determine the frequencies and diversities of KIR genes among the Thai population. RESULTS: Seventeen KIR genes and common subtypes were identified in 500 healthy Thai blood donors by PCR-SSP. The framework genes KIR2DL4, KIR3DL2, KIR3DL3, and KIR3DP1 were present in all individuals (100%). The observed frequencies of KIR genes vary in the presented population. The most frequent non-framework KIR gene was KIR2DLI (98.4%) while the least frequent was KIR2DL5B (24.2%). CONCLUSION: It was observed that the Thai population shows polymorphism of the KIR genes and the diversities of KIR genes in Thai differed from other populations. These data might be of benefit to future studies of the KIR gene and its association with diseases.

Antigen-driven patterns of TCR bias are shared across diverse outcomes of human hepatitis C virus infection.

Hepatitis C virus (HCV) infection causes significant morbidity and mortality worldwide. T cells play a central role in HCV clearance; however, there is currently little understanding of whether the disease outcome in HCV infection is influenced by the choice of TCR repertoire. TCR repertoires used against two immunodominant HCV determinants--the highly polymorphic, HLA-B*0801 restricted (1395)HSKKKCDEL(1403) (HSK) and the comparatively conserved, HLA-A*0101-restricted, (1435)ATDALMTGY(1443) (ATD)--were analyzed in clearly defined cohorts of HLA-matched, HCV-infected individuals with persistent infection and HCV clearance. In comparison with ATD, TCR repertoire selected against HSK was more narrowly focused, supporting reports of mutational escape in this epitope, in persistent HCV infection. Notwithstanding the Ag-driven divergence, T cell repertoire selection against either Ag was comparable in subjects with diverse disease outcomes. Biased T cell repertoires were observed early in infection and were evident not only in persistently infected individuals but also in subjects with HCV clearance, suggesting that these are not exclusively characteristic of viral persistence. Comprehensive clonal analysis of Ag-specific T cells revealed widespread use of public TCRs displaying a high degree of predictability in TRBV/TRBJ gene usage, CDR3 length, and amino acid composition. These public TCRs were observed against both ATD and HSK and were shared across diverse disease outcomes. Collectively, these observations indicate that repertoire diversity rather than particular Vß segments are better associated with HCV persistence/clearance in humans. Notably, many of the anti-HCV TCRs switched TRBV and TRBJ genes around a conserved, N nucleotide-encoded CDR3 core, revealing TCR sequence mosaicism as a potential host mechanism to combat this highly variant virus.

TCD8 response in diverse outcomes of recurrent exposure to hepatitis C virus.

To analyse the immune correlates in a setting of recurrent exposure to hepatitis C virus (HCV), we studied T(CD8) responses in injecting drug users (IDUs) with different disease outcomes. Ex vivo HCV-specific T(CD8) responses assessed by interferon-gamma (IFNgamma) enzyme-linked immunospot (ELISPOT) were comparable in human lymphocyte antigen (HLA)-matched IDUs with spontaneous HCV clearance or persistent infection. A detailed characterization of these T(CD8) cells in age and HLA-matched IDUs demonstrated that HCV clearance and protection from reinfection correlated with HCV-specific T(CD8) cells that could proliferate in vitro, possessed cytotoxic potential and produced IFNgamma and tumour-necrosis factor-alpha, rather than with the circulating frequency of responding T(CD8) cells determined ex vivo. While validating the importance of multifunctional T(CD8) in mediating protection in IDUs with recurrent exposure to HCV our findings highlight that the magnitude and/or breadth of HCV-specific T(CD8) determined in ex vivo ELISPOT may not be the sole determinant of protection especially in a setting of recurrent exposure.

Fiber-modified recombinant adenoviral constructs encoding hepatitis C virus proteins induce potent HCV-specific T cell response.

Hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTLs) play an important role in HCV clearance. The frequency of HCV-specific T(CD8) in peripheral blood of HCV-infected donors is very low and HCV cannot be cultivated for reinfection of antigen presenting cells, making it difficult to detect T(CD8) of broad HCV specificities from peripheral blood mononuclear cells (PBMCs). We have developed a recombinant adenoviral system that efficiently reactivates and expands HCV-specific CTLs from PBMCs of HCV-infected donors. Replication-incompetent adenoviruses expressing individual HCV proteins (core and NS3) were produced and PBMCs from HCV-infected donors were transduced with these recombinant adeno-HCV constructs to stimulate HCV-specific CTL populations. T cells expanded from adeno-HCV stimulated cultures were potent producers of HCV-specific IFN-gamma and TNF-alpha and efficiently lysed target cells pulsed with HCV peptides. These constructs could stimulate T(CD8) directed towards multiple HCV peptides while preserving the determinant hierarchy. This approach therefore overcomes some of the shortcomings of the selective expansion of CTLs with peptide-based vaccine strategies. These findings provide an effective approach for the expansion of HCV-specific CTLs from PBMCs of HCV-infected patients and have potential for immunotherapeutic/vaccine development.

Reversed passive hemagglutination test fails to detect HBsAg in a number of HBeAg positive sera.

In endemic areas of hepatitis B virus (HBV) infection, perinatal transmission from asymptomatic HBsAg carrier mothers to infants plays a major role in the transmission of HBV. HBeAg indicates a high level of viral replication and infectivity. Most of the infants born to HBeAg positive mothers become carriers. Prenatal screening of HBsAg would identify infected mothers and thus allow preventive administration of immunoglobulin and immunization to the newborns. Reversed passive hemagglutination assay (RPHA) is commonly used in Thailand for HBsAg screening. However this method has low sensitivity and gives false negative results. Therefore, infants born to HBsAg false negative mothers would not receive proper immunization. This study reveals the rate of false negative results for HBsAg by RPHA in high infectivity sera. Of 985 sera which were HBsAg positive by ELISA, 70 (7.1%) were negative for HBsAg by RPHA. Of these 70 false negative sera, 7 (10%) were HBeAg positive. Our results indicate that RPHA is a less sensitive method for detection of HBsAg than ELISA. RPHA can give false negative results even in sera with high HBV infectivity. Therefore, RPHA should be replaced by EIA for prenatal HBsAg screening or any other screening for HBV infection whenever possible.