Healthy Blood Donor Samples Research Articles

Development of an immunoassay for quantification of soluble human CD40L (CD154) in plasma and serum samples

When the membrane protein CD40 ligand (CD40L) on activated T cells binds the receptor CD40 on B-cells, it provides a co-stimulatory signal for B cell activation. Dysregulation of the CD40L:CD40 axis is associated with inflammatory and autoimmune diseases. The presence of soluble CD40L (sCD40L) in plasma is implicated in several diseases, from cardiovascular and autoimmune diseases to different types of cancer, and sCD40L has been suggested as a valuable marker of disease. If sCD40L is to be used as a biomarker, being able to precisely measure and quantify the levels of sCD40L in human blood samples is of utmost importance. We demonstrate the development of a sandwich-type time-resolved immunofluorometric assay for quantification of sCD40L in plasma or serum samples. For this, we generate 29 monoclonal anti-CD40L antibodies, and from these, we select the optimal combination of capture antibody and detection antibody. A number of variables were tested: the influence of the type of sample (comparing 3 different blood collection tubes for serum sampling and 4 different types of tubes for plasma sampling), the influence of freeze-thaw cycles, the influence of sampling time during night and day, and the influence of centrifugation of the samples. We found a very similar level of sCD40L in paired EDTA plasma and serum samples. Out of 100 healthy blood donor samples 61 had a level of sCD40L below the detection level of the assay, whereas the remaining 39 samples had ranging levels of sCD40L from 1.14 to 33.14 ng/mL. In summary, we present a time-resolved immunofluorometric assay based on paired monoclonal antibodies, ensuring high specificity, sensitivity, and homogeneity. The Eu3+-based assay additionally provides consistent assay readouts due to the extended decay time not seen in standard enzyme-linked immunosorbent assays. The assay paves the way for specific and consistent quantification of sCD40L in human plasma and serum samples.

Characterization of the Human Blood Virome in Iranian Multiple Transfused Patients.

Blood transfusion safety is an essential element of public health. Current blood screening strategies rely on targeted techniques that could miss unknown or unexpected pathogens. Recent studies have demonstrated the presence of a viral community (virobiota/virome) in the blood of healthy individuals. Here, we characterized the blood virome in patients frequently exposed to blood transfusion by using Illumina metagenomic sequencing. The virome of these patients was compared to viruses present in healthy blood donors. A total number of 155 beta-thalassemia, 149 hemodialysis, and 100 healthy blood donors were pooled with five samples per pool. Members of the Anelloviridae and Flaviviridae family were most frequently observed. Interestingly, samples of healthy blood donors harbored traces of potentially pathogenic viruses, including adeno-, rota-, and Merkel cell polyomavirus. Viruses of the Anelloviridae family were most abundant in the blood of hemodialysis patients and displayed a higher anellovirus richness. Pegiviruses (Flaviviridae) were only observed in patient populations. An overall trend of higher eukaryotic read abundance in both patient groups was observed. This might be associated with increased exposure through blood transfusion. Overall, the findings in this study demonstrated the presence of various viruses in the blood of Iranian multiple-transfused patients and healthy blood donors.

Serological Evidence of Zika Virus Infection in Febrile Patients and Healthy Blood Donors in Sabah, Malaysian Borneo, 2017-2018.

Several Zika virus (ZIKV) seroprevalence studies have been conducted in Africa, Asia, Oceania, the Americas, and the Caribbean. However, studies on ZIKV seroprevalence are limited in Malaysia though several studies have shown that the disease is endemic in the Malaysian state of Sabah. To evaluate the seroprevalence of ZIKV infection, 818 serum samples were collected from febrile patients and healthy blood donors from the Kudat and Kota Kinabalu districts in Sabah from 2017 to 2018. They were screened for ZIKV infection by IgM and IgG ELISA, and positive ZIKV IgM samples were subjected to a 90% neutralization test for confirmation. Twenty-four (6% [95% CI 4 to 8]) confirmed and two (0.5% [95% CI 0.13 to 1.8]) probable ZIKV infections were detected among 400 febrile illness patients. Of 418 healthy blood donor samples, six (1.4% [95% CI 0.65 to 3]) were determined as confirmed ZIKV infections and six (1.4% [95% CI 0.65 to 3]) indicated probable ZIKV infection. This is the first study on the seroprevalence of ZIKV infections among patients and healthy blood donors in Sabah. Compared with previous studies in Malaysia, this study shows that the incidence of ZIKV infection has increased. It also suggests that a sero-surveillance system is essential to determine the circulation of ZIKV in Sabah, Malaysia.

Somatic STAT3 Mutations in CD8+ T Cells of HTLV-2 Positive Blood Donors

Introduction: T-cell large granular lymphocyte (T-LGL) leukemia is a rare lymphoproliferative disorder with recurrent somatic STAT3 mutations. It has been suggested that viral antigens act as the initial stimuli resulting in clonal expansion of CD8+ cells in the disease. However, less is known whether chronic exposure to viral antigens is associated with acquisition of somatic mutations in CD8+ T cells among individuals without clinically detectable lymphoproliferations.Human T-cell leukemia virus type 2 (HTLV-2) antibody positivity has been detected in patients with T-LGL leukemia. Here, we examined whether CD8+ T cells from HTLV-2 positive healthy blood donors harbor somatic mutations in STAT3 or other immune-associated genes, potentially identifying individuals at risk of subsequent lymphoproliferative diseases.Methods: We analyzed HTLV-2 infected (n=30) and uninfected (n=35) healthy blood donor samples obtained from University of California San Francisco and Vitalant Research Institute, which were enrolled in the United States-based HTLV Outcomes Study (HOST) cohort. All cases had serologic evaluation for HTLV-2 infection at the time of sampling. We examined somatic mutations of STAT3 in CD4+ and CD8+ T-cell populations using ultra-deep targeted amplicon sequencing. In addition, we applied a custom sequencing panel covering the coding regions of 2,533 immune-related genes to characterize a larger spectrum of somatic mutations in CD8+ T cells.Results: Somatic STAT3 mutations were detected in CD8+ but not in CD4+ T cells of four (13.3%, 4/30) HTLV-2 positive healthy blood donors (p.Y640F, p.N647I, p.D661Y, and p.Y657_K658insY with variant allele frequencies of 11.9%, 0.5%, 4.9%, and 1.2%, respectively) using amplicon sequencing. The detected STAT3 mutations have been previously described and reported in T-LGL leukemia. Total white blood cell and lymphocyte counts were similar between STAT3 mutated and non-mutated cases. No STAT3 mutations were discovered in HTLV-2 negative donors with amplicon sequencing.Of the 28 HTLV-2 positive cases, 19 had at least one somatic variant in CD8+ T cells based on the immunogene panel sequencing (n=28). 8 cases had variants in genes previously identified in T-LGLL (STAT3, KMT2D, TYRO3, DIDO1, BCL11B, CACNB2, KRAS, LRBA and FANCA), and 5 cases had variants in genes involved in JAK-STAT signaling (NFKBIA, PIK3R5, MAPK14, EP300, MPL, IFNAR1, IL6ST and IL20RA). Three recurrently mutated genes were detected: VWF, SMAD7 and MXRA5. The four HTLV-2 positive blood donors harboring STAT3 mutations had more somatic mutations (median=6) than HTLV-2 positive donors without STAT3 mutations (median=1, p=0.06).Conclusion: We report the presence of somatic gain-of-function STAT3 mutations in CD8+ T cells of 13% of HTLV-2 positive healthy blood donors. We identified additional somatic mutations in genes associated with JAK-STAT signaling, immune regulation and lymphoproliferation in CD8+ T cells of HTLV-2 positive cases. While STAT3 activation, with or without mutations, is considered as a hallmark of T-LGLL, our results reveal the presence of STAT3 mutations in CD8+ T cells of healthy blood donors harboring HTLV-2 without clinical history of lymphoproliferative disease. Additional research is warranted to elucidate whether HTLV-2 carriers harboring STAT3 and other mutations are at increased risk of subsequent T-LGL leukemia or other lymphoproliferative diseases. DisclosuresMustjoki: Pfizer: Research Funding; BMS: Research Funding; Novartis: Research Funding; Janpix: Research Funding.

Past and Future Perspectives for Hepatitis B and C in Pakistan.

BackgroundHepatitis B and C are viral infections of the liver transmitted by blood contamination. These infections are endemic in Pakistan and put a tremendous burden on its healthcare system. We conducted this study to assess the prevalence of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections in Gujranwala, Pakistan, from 2010 to 2015 and determine the trend of future infections for a prediction of the disease burden by 2030 so policymakers can make informed decisions.MethodsWe conducted a retrospective cross-sectional study of 66,308 healthy blood donor samples at District Headquarters Teaching Hospital in Gujranwala from January 2010 to December 2015. Samples were screened for HBV and HCV using the kit method, and data were analyzed using IBM SPSS Statistics for Windows, version 20.0 (IBM Corp., Armonk, NY). We applied a least squares regression to our results to predict HBV and HCV incidence in 2030.ResultsA total of 715 samples (1.08%) were positive for HBV and 1,846 samples (2.78%) were positive for HCV. Our projections indicate that 3.25% of patients in Pakistan will be positive for HBV, and 6.36% will be positive for HBC by 2030.ConclusionWe found an unexpectedly greater burden of HBV and HCV in the recent past than at current levels. The predicted percentages of future burden over the next decade were alarmingly high. These data necessitate implementing preventive and therapeutic measures by policymakers to reduce the disease burden and mortality in Pakistan.

Molecular Detection and Genotyping of Human Herpes Virus 8 in a sample of Iraqi Blood Donors

Human herpes virus-8 (HHV-8) infection has increased recently in Arabic countries. HHV-8 in healthy persons does not necessarily cause life-threatening infection, and however, it causes a more severe infection among immunocompromised patients. The distribution of HHV-8 genotypes varies according to ethnicity and depends on the geographic region prior rapid development of global travel. A cross sectional prospective study included a hundred healthy blood donor samples with a mean age of (36.60±10.381), 81% were positive for molecular detection of HHV-8 DNA. PCR results for HHV-8 were strongly related with risk factors such as the number of sexual relations, previous surgeries, blood transfusion, dental operation, and the number of blood donations. In this study, genotypes (A, B, C and D) were detected, largely associated with blood donors residences and distributed to areas of Iraq through a map. Genotype A comprised 28 (34.6%) of blood donors and for genotype C it was 16 (19.8%) and both genotypes were found to be the predominant genotypes, followed by genotype B of 7 (8.7%) and D of 2 (2.5%), the latter is included into Mixed genotypes of 8 (9.9%), whereas, 22 (27.2%) were undetermined genotypes. Efforts should focus on these findings, which may indicate that Iraq is an endemic region of HHV-8 infection.

Detection of pre-existing SARS-CoV-2-reactive T cells in unexposed renal transplant patients

BackgroundRecent data demonstrate potentially protective pre-existing T cells reactive against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in samples of healthy blood donors, collected before the SARS-CoV-2 pandemic. Whether pre-existing immunity is also detectable in immunosuppressed patients is currently not known.MethodsFifty-seven patients were included in this case–control study. We compared the frequency of SARS-CoV-2-reactive T cells in the samples of 20 renal transplant (RTx) patients to 20 age/gender matched non-immunosuppressed/immune competent healthy individuals collected before the onset of the SARS-CoV-2 pandemic. Seventeen coronavirus disease 2019 (COVID-19) patients were used as positive controls. T cell reactivity against Spike-, Nucleocapsid-, and Membrane- SARS-CoV-2 proteins were analyzed by multi-parameter flow cytometry. Antibodies were analyzed by neutralization assay.ResultsPre-existing SARS-CoV-2-reactive T cells were detected in the majority of unexposed patients and healthy individuals. In RTx patients, 13/20 showed CD4+ T cells reactive against at least one SARS-CoV-2 protein. CD8+ T cells reactive against at least one SARS-CoV-2 protein were demonstrated in 12/20 of RTx patients. The frequency and Th1 cytokine expression pattern of pre-formed SARS-CoV-2 reactive T cells did not differ between RTx and non-immunosuppressed healthy individuals.ConclusionsThis study shows that the magnitude and functionality of pre-existing SARS-CoV-2 reactive T cell in transplant patients is non-inferior compared to the immune competent cohort. Although several pro-inflammatory cytokines were produced by the detected T cells, further studies are required to prove their antiviral protection.Graphic abstract

Efficient Identification of High-Titer Anti-Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Antibody Plasma Samples by Pooling Method.

The ongoing COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has elicited a surge in demand for serologic testing to identify previously infected individuals. In particular, antibody testing is crucial in identifying COVID-19 convalescent plasma, which has been approved by the Food and Drug Administration under the Emergency Use Authorization for use as passive immunotherapy for hospitalized patients infected with COVID-19. Currently, high-titer COVID-19 convalescent plasma can be qualified by Ortho's Vitros COVID-19 IgG antibody test. To explore the use of an efficient testing method to identify high-titer COVID-19 convalescent plasma for use in treating COVID-19-infected patients and track COVID-19 positivity over time. We evaluated an enzyme-linked immunosorbent assay (ELISA)-based method that detects antibodies specific to the SARS-CoV-2 receptor binding domain (RBD) with individual and pooled plasma samples and compared its performance against the Vitros COVID-19 IgG antibody test. Using the pooled RBD-ELISA (P-RE) method, we also screened more than 10 000 longitudinal healthy blood donor samples to assess seroprevalence. P-RE demonstrates 100% sensitivity in detecting Food and Drug Administration-defined high-titer samples when compared with the Vitros COVID-19 IgG antibody test. Overall sensitivity of P-RE when compared with the Vitros COVID-19 IgG antibody test and our individual sample RBD-ELISA (I-RE) were 83% and 56%, respectively. When screening 10 218 healthy blood donor samples by P-RE, we found the seroprevalence correlated with the local infection rates with a correlation coefficient of 0.21 (P < .001). Pooling plasma samples can be used to efficiently screen large populations for individuals with high-titer anti-RBD antibodies, important for COVID-19 convalescent plasma identification.

Frequency of hepatitis B virus-DNA among hepatitis B surface-Ag negative, anti-hepatitis B core antibody-positive blood donors in Yazd, Iran.

BACKGROUND:The diagnosis of hepatitis B infection in most blood transfusion centers is based on hepatitis B surface (HBs) antigen detection by an enzyme immunoassay method. This study aimed to determine the frequency of hepatitis B core (HBc) antibody, HBs antibody, and hepatitis B virus (HBV) DNA among HBs antigen-negative healthy blood donors of Yazd province, Iran.MATERIALS AND METHODS:This cross-sectional study was conducted on 1500 healthy blood donor samples negative for HBs antigen, hepatitis C virus antibody, human immunodeficiency virus antigen/antibody, and rapid plasma regain tests. All samples were screened for HBc antibody test. HBs antibody and real-time polymerase chain reaction were performed for HBc antibody-positive samples.RESULTS:HBc antibody was positive in 74 (4.9%) samples and 11 (14.9%) of 74 positive samples for HBc antibody were negative for HBs antibody. Sixty-three (85.1%) positive samples for HBc antibody had HBs antibody titer over 10 IU/L, and 43 (58.1%) had HBs antibody titer over 100 IU/L. There was no hepatitis B DNA-positive sample in the present study.CONCLUSIONS:The study results suggest that there is a very low risk for transmission of HBV through blood donors of Yazd, Iran.

Consecutive reactive results in screening of transfusion transmitted infections: Family history of blood donors is also important

BackgroundEnzyme linked immunosorbent assay (ELISA) test is used for screening of transfusion transmitted infections (TTI) in blood donors. Consecutive reactive results in ELISA is due to sample/reagent carryover or donor related. In this study we tried to find out the possibilities of family history/close contacts with patients of hepatitis among these consecutive reactive donors. AimTo analyze the consecutive reactive results in ELISA tests for TTI testing on samples of healthy blood donors. Material and methodsA retrospective observational study was conducted from January 2016 to July 2018 in a tertiary care hospital, North India. Consecutive reactive results by fourth generation ELISA for TTIs screening were evaluated for possible reasons. Confirmation tests were not done. Reactive donors were contacted telephonically for relevant history of close contact with infected personnel. ResultsOut of 53,740 donations 1,061 were reactive for TTIs during our study period. Prevalence of Hepatitis B (HBV), Human Immunodeficiency (HIV) and Hepatitis C (HCV) virus infection in blood donors were 1.27%, 0.20% and 0.50% respectively. Consecutive reactive results for HBV were 9.20% (63/685), for HCV 6.0% (16/266) and nil for HIV. There was no sample carryover in this. Out of 79 consecutive reactive donors 69 donated for same patients and 32 were related with infected patient which are statistically significant (p < 0.0001). DiscussionThis study recommends that in analysis of consecutive positive results in ELISA along with looking for procedure/sample error, there is also a need to take retrospective history of donors for close contact with infected patients.

Development and evaluation of latex agglutination test coating with recombinant antigen, LipL32 for serodiagnosis of human leptospirosis.

Leptospirosis is a widespread zoonotic disease caused by Leptospira interrogans. Symptoms of disease range from mild symptoms to serious complications including, jaundice, pulmonary hemorrhage, renal and hepatic failure, which may prove fatal. Clinical presentations of this disease are similar with other febrile illness. Therefore, rapid and appropriated laboratory diagnostic tests are needed to aid clinical case identification. As these reasons, objective of this study is to develop and evaluate a simple latex agglutination test coating with recombinant leptospiral antigens, LipL32 for serodiagnosis of human leptospirosis. Firstly, lipl32 gene was amplified from genomic DNA of Leptospira interogans serovar Pyrogenes. Then PCR product of lipl32 gene was ligated with pGEX-2T plasmid, generating pGRK32 recombinant plasmid. Recombinant GST-LipL32 protein was overexpressed and subsequently purified by using Glutathione-Agarose Resin. Recombinant GST-Lipl32 protein was coated on latex beads for development latex agglutination test (LAT). The relative sensitivity, specificity and accuracy of the developed LAT were compared with indirect immunofluorescences assay (IFA) for detection of anti-leptospiral antibodies in 30 human leptospirosis samples, 30 healthy blood donor samples, 10 dengue fever positive samples, 10 scrub typhus positive samples, and 10 melioidosis samples. Results showed that the developed LAT showed sensitivity, specificity and accuracy: 66.66%, 86.66%, and 80.00%, respectively, comparing with IFA method. Moreover, Kappa analysis showed agreement rate of the two methods were 0.421. It concluded that our developed gave compatible result with IFA. Additionally, Our LAT are simple, rapid and suitable for detection in the field. However, for better sensitivity, diagnostic specificity, positive predictive value, negative predictive value, accuracy and Cohen’s kappa comparison should be done in larger amounts of sera samples.

Detection of Merkel Cell Polyomavirus DNA in Serum Samples of Healthy Blood Donors.

Merkel cell polyomavirus (MCPyV) has been detected in 80% of Merkel cell carcinomas (MCC). In the host, the MCPyV reservoir remains elusive. MCPyV DNA sequences were revealed in blood donor buffy coats. In this study, MCPyV DNA sequences were investigated in the sera (n = 190) of healthy blood donors. Two MCPyV DNA sequences, coding for the viral oncoprotein large T antigen (LT), were investigated using polymerase chain reaction (PCR) methods and DNA sequencing. Circulating MCPyV sequences were detected in sera with a prevalence of 2.6% (5/190), at low-DNA viral load, which is in the range of 1–4 and 1–5 copies/μl by real-time PCR and droplet digital PCR, respectively. DNA sequencing carried out in the five MCPyV-positive samples indicated that the two MCPyV LT sequences which were analyzed belong to the MKL-1 strain. Circulating MCPyV LT sequences are present in blood donor sera. MCPyV-positive samples from blood donors could represent a potential vehicle for MCPyV infection in receivers, whereas an increase in viral load may occur with multiple blood transfusions. In certain patient conditions, such as immune-depression/suppression, additional disease or old age, transfusion of MCPyV-positive samples could be an additional risk factor for MCC onset.

Abstract 224: Enrichment and isolation of uncontaminated breast cancer cells from human blood samples

Abstract Cancer cells in peripheral blood, known as circulating tumor cells (CTCs), play an important role in tumor dissemination. CTCs are shed from either the primary tumor or its metastases and circulate in the peripheral blood of patients; thus they can be regarded as “liquid biopsies” of metastasizing cells. Although the exact origin and physiology of CTCs is unknown, a fraction of these cells are thought to be viable metastatic precursors capable of initiating a clonal metastatic lesion. The molecular characterization of CTCs is important because it may provide insights into the molecular biology of metastasis, the association of their molecular profiles with treatment outcomes, and reveal the presence of potential therapeutic targets. The process of CTC enrichment represents a significant step toward the isolation of CTCs from whole blood. Given that there are approximately 5×109 erythrocytes and 107 leukocytes per ml of whole blood, purification must represent a reduction of many orders of magnitude. In this study, we show a method to isolate CTCs which combines a microfluidic cell separation device (Parsortix system) as an enrichment method followed by the DEPArray selection system. The Parsortix system is versatile in its ability to separate, capture and harvest CTCs in an enriched form compatible with the needs of downstream processes such as DEPArray. Here we demonstrate enrichment of CTCs via the Parsortix system using both breast cancer cell spiked healthy blood donor samples and blood from metastatic breast cancer patients. For these experiments, two milliliters of blood were processed. The cells were harvested and stained to identify CTCs in the enriched harvest product. Cells were stained with fluorescently labeled monoclonal antibodies specific for pan cytokeratin (CK-8/18/19-PE), leukocyte common antigen (CD45-APC), and nuclear stained with [4′,6-diamidino-2-phenylindole (DAPI)]. With spiked samples, tumor cells were isolated by DEPArray selection, which yielded a pure population of tumor cells for molecular characterization. Tumor cells were defined by presence of a clear DAPI-stained nucleus, CK-PE-positive cytoplasm and CD-45-APC negative. We demonstrated that by using a combination of enrichment and isolation/selection methods, we are able to isolate single, uncontaminated tumor cells to achieve single cell molecular analysis. The use of single cells is emerging as a powerful approach to molecular analysis in oncology, and this study demonstrates its potential application with circulating tumor cells. Citation Format: Sandra V. Fernandez, Christopher Wagner, Zahida Parveen, Lucy Aburto, Carmela Paolillo, George Hvichia, Zhaomei Mu, Laura Austin, Massimo Cristofanilli. Enrichment and isolation of uncontaminated breast cancer cells from human blood samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 224. doi:10.1158/1538-7445.AM2015-224

Anticoagulant Proteins in a Population of Mexican Mestizo Donors

To determine the activity of antithrombin (AT), protein C (PC), and protein S (PS), as well as the frequency of deficiencies of these proteins in a population of healthy Mexican mestizo blood donors. AT, PC, and PS were determined from 1,502 plasma samples of healthy blood donors by using commercial kits in a coagulometer 4 STA (Diagnostica Stago, Asnières, France). A total of 741 women and 761 men were under study. They were divided into age range groups (18-24, 25-34, 35-44, 45-54, and 55-64 years). Activity of AT, PC, and PS was determined. For AT, activity values were specific for each age group according to gender when it had to do with PS, as well as when PC was determined. Frequencies of AT, PC, PS, and activated PC resistance activity deficiencies were obtained from reference levels (RLs) and average levels of this study. Differences were found between both frequencies for AT, PC, and PS, and the average levels obtained were used in this study. The frequencies of the activity deficiencies obtained through the values gotten in this population were: AT, 0.6%; PC, 1.06% (which is higher than the one obtained using the RLs described by commercial kits 0.33% and 0.66%, respectively); and PS, 1% (which is less than 4.5%). It is necessary to know the characteristics and biological behavior of the coagulation proteins in the Mexican population because the RLs used have been established for populations that are genetically different.

Utility of quantitative flow cytometry immunophenotypic analysis of CD5 expression in small B-cell neoplasms.

The value of assessing CD5 expression in the differential diagnosis of small B-cell neoplasms is well established. Assessment is usually done qualitatively. To assess CD5 expression levels by quantitative flow cytometry immunophenotyping and to determine possible differences among various small B-cell neoplasms. We performed 4-color flow cytometry analysis on specimens of peripheral blood and bone marrow aspirate and quantified CD5 expression in various small B-cell lymphomas and leukemias. We also assessed CD5 levels in peripheral blood samples of healthy blood donors. Cases of chronic lymphocytic leukemia and mantle cell lymphoma had higher levels of CD5 compared with control B cells (P < .001). Cases of marginal zone lymphoma and hairy cell leukemia had CD5 levels similar to control B cells (P = .35 and P = .14, respectively), whereas cases of follicular lymphoma and lymphoplasmacytic lymphoma had significantly lower CD5 levels than control B cells (P < .001 and P = .04, respectively). In B-cell neoplasms, a high level of CD5 expression was correlated with a homogeneous pattern of positive events, whereas lower CD5 levels were correlated with heterogeneous patterns of positive events. Using flow cytometric immunophenotypic analysis to quantify CD5 levels can aid in diagnosis. CD5 expression levels are higher in patients with chronic lymphocytic leukemia and mantle cell lymphoma, and expression is observed in a homogeneous pattern, as compared with other B-cell neoplasms that are either negative for CD5 or express CD5 at lower levels with a heterogeneous pattern. However, there is some overlap in CD5 expression levels between a subset of atypical chronic lymphocytic leukemia and marginal zone lymphoma cases.

Characterization of Tat Antibody Responses in Chinese Individuals Infected with HIV-1

HIV-1 Tat is an important regulatory protein involved in AIDS pathogenesis. However, the immunoprofiles of anti-Tat responses remain unclear. We analysed the immunoprofiles of the anti-Tat antibody responses and the neutralizing activities. Out of 326 HIV-1-seropositive individuals, 12.9% were positive for anti-Tat antibodies. We found six different immunological profiles of anti-Tat antibody responses: full-potential response, combined response, N-specific response, C-specific response, full-length Tat-specific response and Tat-related response. These responses represent two types of anti-Tat responses: the major complete response and the alternative C-prone response. A Tat-neutralizing activity is significantly higher in anti-Tat-seropositive samples than anti-Tat-negative or healthy blood-donor samples, and significantly correlates with the anti-Tat reactivities. The data here could contribute to a better understanding of the significance of anti-Tat responses in preventing HIV pathogenesis and could be useful for designing more effective vaccines in the future.

The Monitoring Interest of BK Virus Load in Renal Allograft Tunisian Recipients: Prospective Study

BK virus (BKV) may cause nephropathy in renal transplant recipients receiving immunosuppressive therapy, resulting in renal dysfunction and, possibly graft loss. However, the positive and negative predictive values of BK viral load are still controversial. In this prospective, single-center study, BKV DNA was measured 1, 3 and 6 months after transplantation. The viral load in urine and plasma was quantified with the real-time Q-PCR (Argen kit) in 73 renal allograft recipients Three of them showed acute rejection. To determine the cutoff value of viral load, 60 sera samples of healthy blood donors, matched for age and sex, were tested. The mean plasmatic viral load one month posttransplantation was statistically higher in renal transplant recipients (17.23 copies/ml) compared to that in controls (2 copies/ml) (p: 0.06). This difference of the distribution of viremia values is more evident in the third and sixth month (p: 0.002 and 0.010 respectively). Furthermore, analysis of the kinetic of viral load revealed an average rise of viremia at 3 months (1589.14 copies/ml) followed by its decrease at 6 months (249.75 copies/ml). However, the difference was not statistically significant. The same is true for the distribution of values of viruria and in all cases the average viral load was statistically higher in urine than in plasma. In addition, this study did not shown significant relationsheep between viremia/viruria and the occurrence of acute rejection, the renal function deterioration, the source of allograft or immunosuppressive therapy protocol. If the results of this study demonstrate the importance of the replication of BKV in renal transplant patients from the first month compared to that in immunocompetent subjects, the screening of the DNA of this virus does not appear to have a prognostic value in the occurrence of acute rejection. However, the plasma and urine monitoring of BKV load beyond 6 months, not appear to exclude the relationsheep between these two biomarkers and the occurrence of chronic graft dysfunction.

False-negative serology in patients with acute parvovirus B19 infection

Background Acute parvovirus B19 (B19V) infection is characterized by high-level viremia. Antibodies against the capsid proteins VP1 and VP2 may complex with B19V-particles thereby becoming undetectable in diagnostic tests. Objectives We intended to obtain data on the frequency of false-negative serology in acute B19V-infection. Study design 129 plasma or serum samples of healthy blood donors and of patients with suspected B19V-infection were analyzed for B19V-DNA by qPCR and VP1/VP2-specific IgG and IgM by ELISA. Eleven of these samples were derived from four pregnant women with previous contact to B19V-infected individuals. Using acidic conditions virus/antibody-complexes were disrupted and detected by WesternLine and ELISA. Results 83/118 samples were derived from acutely infected individuals displaying viremia (10 3–10 12 geq/mL). In 24/83 viremic samples (28.9%) VP1/VP2-specific IgM and IgG were undetectable in ELISA, but could be demonstrated to be complexed with B19V-particles. Each 7/83 (8.4%) was IgM-positive/IgG-negative and IgM-negative/IgG-positive, in 45/83 samples (54.2%) IgG and IgM could be detected. 35 samples did not contain B19V-DNA; five of these were from seronegative persons. Analyzing consecutive sera derived from four pregnant women, B19V-DNA was demonstrated in 10/11 samples, B19V-specific IgG- and IgM-antibodies were detectable in 10/11 and 4/11 samples, respectively. In 2/4 women seroconversion was observed, but IgM was not detected in 50% of the samples. B19V-specific IgG but not IgM was detectable in 2/4 women. Conclusion Acute B19V-infection cannot be diagnosed by exclusive analysis of B19V-specific antibodies. Only the combination of assays for detection of B19V-DNA and antibodies enables correct serodiagnosis.

Abstract 1284: Analysis of circulating CD70+ lymphocytes and CD70 transcript expression in a phase 1 clinical trial of the CD70-targeting antibody-drug conjugate SGN-75

Abstract Aberrant expression of CD70 has been reported in various cancers, e.g., non-Hodgkin lymphoma (NHL), renal cell carcinoma (RCC), and pancreatic carcinoma. An auristatin-based anti-CD70 drug conjugate, SGN-75 (h1F6-mcMMAF), has demonstrated antitumor activity in preclinical experiments modeling these malignancies and is currently being evaluated in a phase 1 clinical trial in relapsed/refractory NHL or metastatic RCC. Tumor expression aside, CD70 is expressed on a low and consistently detectable percentage of circulating normal T and B cells. Hence, we explored the utility of monitoring CD70+ lymphocytes in patients treated with SGN-75 as a potential pharmacodynamic marker for SGN-75. A flow cytometry assay utilizing an anti-CD70 monoclonal antibody whose binding to CD70+ cells is minimally affected in the presence of SGN-75 was developed. Applying this method, 8 of 8 patients (4 NHL and 4 RCC) with pre- and post-treatment blood samples demonstrated decreases in the percentage of CD70+ T and B cells post treatment with SGN-75. Conversely, there were no changes in the total lymphocytes or the activated CD25+ T lymphocytes, demonstrating target specificity of SGN-75. To complement the flow cytometry assay, a real-time quantitative PCR (qPCR) assay to measure CD70 mRNA in peripheral blood was developed. Amplification of CD70 mRNA was performed by a single-step reverse transcriptase PCR reaction with 97% amplification efficiency. A correlation between CD70 mRNA and protein expression was then established. CD70 mRNA expression in 13 tumor cell lines with CD70 receptor copy numbers ranging from 2,000 to 189,000 per cell showed significant correlation (ρ=0.8517, p=0.0002) between CD70 mRNA and cell surface protein expression. Similarly, correlation (ρ=0.8125, p=0.0004) was seen between CD70 mRNA expression levels and the percentage of CD70+ lymphocytes in healthy blood donor samples ranging from 1 to 10% CD70+ cells. Analysis of peripheral blood spiked with increasing numbers of the CD70+ 786-O or Caki-1 RCC cells showed corresponding increases in qPCR signals for CD70 mRNA. A reduction of CD70 mRNA was detected in 5 (2 NHL and 3 RCC) of 7 (2 NHL and 5 RCC) patients post SGN-75 treatment. Of the 5 patients showing reduction of CD70 mRNA 3 (2 NHL, 1 RCC) also had available blood samples for flow cytometric analysis; all 3 demonstrated a corresponding decline in the frequency of CD70+ circulating lymphocytes post SGN-75 treatment. Our data suggest that changes in the frequency of circulating CD70+ cells assayed by flow cytometry and CD70 mRNA level assay by qPCR can be potentially used as pharmacodynamic biomarkers for SGN-75 in clinical trials. Ongoing and future studies include correlating these biomarker changes with the pharamcokinetics of SGN-75 and clinical antitumor activity to assist in selecting the optimal dose and schedule for future development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1284. doi:10.1158/1538-7445.AM2011-1284

Screening for G6PD Deficiency in Blood Donor Population

Dear Editor, Glucose-6-phosphate dehydrogenase deficiency is the most common enzyme deficiency worldwide. Approximately 400 million people are said to be affected worldwide. According to world health organization 7.5% of world population are carriers of glucose-6-phosphate dehydrogenase deficiency and 2.9% were G6PD deficient [1]. It causes spectrum of diseases including neonatal hyperbilirubinemia, acute hemolytic and chronic hemolytic anemia. Usually the affected individuals are asymptomatic unless they are provoked by oxidative stress [2]. This enzyme deficiency is very prevalent in individuals of Africa, America, Mediterranean, and East Asia. In India the incidence of G6PD has been variably reported as 0–37% in different castes and communities. Higher incidence of G6PD deficiency is seen in north and west India (15%) as compared to south India (1–2%) [3]. It implies that this inherited metabolic disorder is an important health problem in India and it is necessary to carryout large scale screening in the whole population. Severe manifestations of G6PD deficiency can be prevented if necessary precautions are taken, hence this study was done to determine the incidence of G6PD deficiency in asymptomatic population represented by healthy blood donors and to characterize the demographic profile and laboratory profile of such group. The study was conducted on 2005 healthy blood donor samples. This screening program included 2000 male and 5 female blood donors who came to blood bank for donation of blood. These donors were from different part of India. The G6PD activity was assessed in their red cells using anticoagulated (EDTA) venous blood. Sixteen blood donors out of 2005 screened by methaemoglobin reduction test were found to be G6PD deficient. All of them were male. These individuals were clinically normal and had no significant past history. The incidence of G6PD deficiency was 0.8% in the sampled population. Among this 16 G6PD deficient subjects two blood donors were from West Bengal, one from Kerala and the rest of the 13 subjects were from Karnataka. The mean Haemoglobin values of the normal donor population vs. G6PD deficient donor population were compared using unpaired t test. The difference in the mean Hb value was not statistically significant (P value > 0.5). Sixteen units of blood were transfused to patients. There was two [12.5%] transfusion related reaction among the patients who received the G6PD deficient blood. One of the patients, who was transfused following upper Gastro intestinal bleeding, had itching and rashes all over the body after transfusion of 50 ml blood. The reaction workup done ruled out haemolytic reaction. A probability of allergic reaction was considered. As in this patient transfusion was stopped and he did not receive further transfusions for next 48 h, his Hb fell from 9.0 to 7.7 gm/dl. The second patient was a neonate undergoing surgery for a ruptured meningomyelocoele. The blood was transfused during surgery following which the baby developed mottling and passed red colored urine. A hemolytic transfusion was suspected. The transfusion reaction workup showed haemolysis in the post transfusion sample and hemoglobinuria. But blood bag showed no hemolysis. However as pretransfusion sample also showed haemolysis, a hemolytic reaction could not be conclusively reported. Hemoglobin dropped from 12.2 to 8.0 gm/dl. The beneficial effects of blood transfusion, for the typical 70 kg human each unit of whole blood should increase the hematocrit by 3–5% or hemoglobin by 1–1.5 gm/dl [4].Testing for G6PD deficiency is not routinely practiced for donors units [2]. Various studies have reported a mild increase in bilirubin, LDH, and decrease in hemoglobin following the transfusion of G6PD deficient blood. This is especially in neonates (preterm). Transfusion of G6PD deficient red cells to the premature infants has been associated with hemolytic and severe hyperbilirubinemia requiring exchange transfusion [5]. In our study also one neonate developed suspected hemolytic transfusion reaction following transfusion of G6PD deficient blood. Under normal circumstances the transfusion of G6PD deficient blood is harmless for most of the individuals until they are exposed to certain oxidative drugs. This study was an attempt to find out the prevalence of G6PD deficiency in our population. This will help us to quantitate the magnitude of the problem in the population and initiate measures to screen the population or a selected group in the population. The prevalence of G6PD being very low in this study (0.8%) and as many people remain asymptomatic unless challenged with oxidative drugs or infection, screening the whole population for G6PD deficiency is not warranted. As one of the neonates receiving G6PD deficient blood developed suspected hemolytic reaction, screening of blood to be transfused for select population like neonates is advisable.