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Background Some people rapidly develop iron deficiency anemia following blood donation, while others can repeatedly donate without becoming anemic. Methods Two cohorts of blood donors were studied. Participants (775) selected from a 2-year longitudinal study were classified into six analysis groups based on sex, donation intensity, and low hemoglobin deferral. Associations with iron supplement use, cigarette smoking, and four genetic variants of iron metabolism were examined at enrollment and with longitudinal regression models. An unbiased assessment of genetic variability and ability to repeatedly donate blood without experiencing low hemoglobin deferral was conducted on participants (13,403) in a cross-sectional study who were examined by genome wide association (GWA). Results Behaviors and genetic variants were associated with differences in hemoglobin and ferritin change following repeated donation. At least weekly iron supplement use was associated with improved status in first-time donors, while daily use was associated with improved status in high-intensity donors. Cigarette smoking was associated with 0.5 g/dL increased hemoglobin in high-intensity donors. A736V in TMPRSS6 was associated with a rapid drop in hemoglobin and ferritin in first-time females following repeated donation. Conversely, the protective TMPRSS6 genotype was not enriched among high-intensity donors. H63D in HFE was associated with increased hemoglobin in female high-intensity donors. However, no differences in genotype between first-time and high-intensity donors were found in GWA analyses. Conclusion Behavioral and genetic modifiers contributed to first-time donor hemoglobin and iron status, while iron supplement use was more important than underlying genetics in high-intensity donors.

Background Babesia microti is a parasite that infects red blood cells (RBCs) in mammals. It is transmitted to humans by tick bites, transfusion, organ transplantation, and congenital acquisition. Although the Babesia natural history and seroprevalence in donors have been well described, gaps in knowledge relevant to transfusion remain. Study design and methods Mice were infected with dilutions of parasitized blood to address the minimal infectious dose and the kinetics of parasitemia by quantitative polymerase chain reaction (qPCR) and of antibodies by enzyme immunoassay. Results In immunocompetent DBA/2 mice infected with 100 parasitized RBCs (pRBCs) and in immunodeficient NSG mice infected with 63 pRBCs, parasitemia was detectable in five of five mice each. Peak parasitemia up to 2 × 107 pRBCs/mL at 2 to 3 weeks or 5 × 108 pRBCs/mL at 6 weeks was observed for DBA/2 and NSG mice, respectively. Protracted fluctuating parasitemia was observed for 8 months in DBA/2 mice, whereas NSG mice exhibited a high-plateau parasitemia. Antibody titers continued to increase until 6 to 18 weeks in DBA/2 mice and remained high through 6 months. This study also investigated the analytical performance of Babesia assays that detect parasite DNA or RNA using a blinded panel. A Babesia assay targeting parasite RNA was approximately 10-fold more sensitive compared to qPCR targeting DNA. Conclusion The mice in this study were highly susceptible to Babesia infection using as few as 1 to 2 log pRBCs and maintained chronic parasitemia. If the infectious dose in human transfusion recipients is comparably low, a highly sensitive assay targeting parasite RNA may safeguard the blood supply, particularly before antibody detection.

BACKGROUND Alloimmunization through blood transfusion, transplantation, or circulating fetal cells during pregnancy is a significant concern. Some exposed individuals make alloantibodies while others do not, implying variation in genetic risk factors. STUDY DESIGN AND METHODS We conducted a genomewide association study (GWAS) of 9,427,497 single-nucleotide polymorphisms (SNPs) to identify genetic variants for HLA alloimmunization in previously pregnant blood donors with (n = 752) and without (n = 753) HLA Class I or II alloantibodies. RESULTS A SNP in the neurexophilin 2 (NXPH2) gene surpassed genome-wide significance (p = 2.06 × 10-8 ), with multiple adjacent markers p

Background Following transfusion, donor white blood cells (WBCs) can persist long-term in the recipient, a phenomenon termed transfusion-associated microchimerism (TA-MC). Prior studies suggest TA-MC is limited to transfusion following traumatic injury, and is not prevented by leukoreduction. Study design and methods We conducted a prospective cohort study at a major trauma center to evaluate TA-MC following injury. Index samples were collected upon arrival, prior to transfusion. Follow-up samples were collected at intervals up to one year, and beyond for those testing positive for TA-MC. TA-MC was detected by real-time quantitative allele-specific polymerase chain reaction assays at the HLA-DR locus and several polymorphic insertion deletion sites screening for non-recipient alleles. Results A total of 378 trauma patients were enrolled (324 transfused cases and 54 non-transfused controls). Mean age was 42 ± 18 years, 74% were male, and 80% were injured by blunt mechanism. Mean Injury Severity Score was 20 ± 12. Among transfused patients, the median (interquartile range) number of red cell units transfused was 6 (3,12), and median time to first transfusion was 9 (0.8,45) hours. Only one case of long-term TA-MC was confirmed in our cohort. We detected short-term TA-MC in 6.5% of transfused subjects and 5.6% on non-transfused controls. Conclusions In contrast to earlier studies, persistent TA-MC was not observed in our cohort of trauma subjects. Short-term TA-MC was detected, but at a lower frequency than previously observed, and rates were not significantly different than what was observed in non-transfused controls. The reduction in TA-MC occurrence may be attributable to changes in leukoreduction or other blood processing methods.

BACKGROUND Babesia microti is the foremost infectious risk to the US blood supply for which a Food and Drug Administration (FDA)-licensed test is unavailable for donation screening. Characterization of the antibody response to B. microti and correlation with parasitemia is necessary to guide screening and donor management policies. STUDY DESIGN AND METHODS During an FDA licensure trial, blood donors were prospectively screened (July-November 2013) using a B. microti-specific antibody enzyme immunoassay (EIA, Immunetics) in highly endemic (New York [NY]; n = 13,688), moderately endemic (Minnesota [MN]; n = 4583), and nonendemic (New Mexico [NM]; n = 8451) regions. Blood donors with repeat-reactive (RR) results participated in a 12-month prospective cohort study using B. microti EIA, immunofluorescent assay, polymerase chain reaction (PCR), blood smear, and clinical questionnaire. RESULTS Thirty-seven (61.67%; 24 NY, seven MN, six NM) of 60 eligible RR donors enrolled in the study; 20 of 37 (54%) completed the 12-month follow-up visit of which 15 (75%) were still seroreactive. Nine PCR-positive donors were identified during index screening; five participated in the follow-up study, three were PCR positive at 6 months, and two remained positive at final follow-up (378 and 404 days). Most RR donors displayed low-level seroreactivity that was either stable or waning during follow-up. The level and pattern of reactivity correlated poorly with PCR positivity. CONCLUSION The findings indicate prolonged seropositivity in blood donors. Although rare, asymptomatic, persistent PCR positivity supports the current policy of indefinite deferral for donors with a history of babesiosis or positive test results. Repeat testing by PCR and serology will be necessary if reinstatement is to be considered.

BACKGROUND Pathogen inactivation methods are increasingly used to reduce the risk of infections after transfusion of blood products. Photochemical treatment (PCT) of platelets (PLTs) and plasma with amotosalen and ultraviolet A (UVA) light inactivates pathogens and white blood cells through formation of adducts between amotosalen and nucleic acid that block replication, transcription, and translation. The same adducts block the amplification of nucleic acids using polymerase chain reaction (PCR) in a manner that correlates with the number of adducts formed, providing a direct quality control (QC). Current QC measures for PCT rely on indirect methods that measure the delivered UVA dose or percent residual amotosalen after illumination, rather than directly measuring nucleic acid modification. STUDY DESIGN AND METHODS Endogenous mitochondrial DNA (mtDNA), which is detectable in PLT and plasma units, was chosen as a target for the quantification of photochemically induced modifications. DNA was extracted from untreated or amotosalen and UVA–treated PLTs or plasma, and mtDNA fragments of variable lengths were quantified using a real-time PCR inhibition assay. RESULTS PCT induced increasing real-time PCR inhibition of mtDNA amplification for larger amplicon sizes. Amplification was unaffected by treatment with amotosalen or UVA alone, whereas up to 3 log inhibition was observed after PCT. Blinded PCR testing of a panel of 110 samples each, from PLT or plasma components prepared for routine use within a blood center, allowed 100% discrimination between untreated and treated units. CONCLUSION Our initial findings indicate that an adequately sensitive, quantitative real-time PCR inhibition assay targeting mtDNA could provide a valuable tool to confirm and monitor PCT.

Background SFTSV, an emerging tick-borne pathogen that can cause fatal severe fever with thrombocytopenia syndrome (SFTS), was first identified in China in 2009. Limited evidence suggests that SFTSV can be transmitted between humans via blood contact raising concerns over transfusion safety. A study of donor samples from three Chinese blood centers was conducted to investigate the seroprevalence and rate of SFTSV viremia among Chinese blood donors.

Background Previous reports of West Nile virus (WNV) RNA persistence in blood compartments have raised concerns around the remaining risk of WNV transfusion transmission. This study characterized the dynamics of WNV viremia in blood compartments in a longitudinal cohort of 54 WNV-infected blood donors. Study Design and Methods Blood samples were collected throughout the year after WNV RNA–positive blood donation (index) and characterized for WNV immunoglobulin (Ig)M and IgG antibodies and for WNV RNA by real-time reverse transcription–polymerase chain reaction. WNV viral loads were compared in plasma and whole blood samples and correlated with blood groups and clinical outcomes. Results WNV RNA persisted in the red blood cell (RBC) compartment up to 3 months postindex in 42% of the donors. Donors with the highest WNV RNA levels in plasma at index maintained the highest WNV RNA levels in whole blood over the 3 months postindex. Blood group A donors maintained higher postindex WNV viral load in whole blood than blood group O individuals (p = 0.027). Despite a trend for WNV RNA to persist longer in whole blood from symptomatic subjects, no significant association was found between WNV RNA levels in whole blood and disease outcome. Conclusion This study confirmed that WNV RNA persists in the RBC fraction in whole blood and further suggested that the level of persistence in whole blood may be a reflection of initial viral burden in plasma. The association with blood groups suggests that WNV adherence to RBCs may be mediated by molecules overrepresented at the surface of blood group A RBCs.

Chronic inflammation may contribute to human immunodeficiency virus (HIV) persistence through a number of potential pathways. We explored the impact of immunosuppressant therapy on peripheral blood measures of HIV persistence following kidney transplantation. Stored plasma and peripheral blood mononuclear cells prior to transplantation and at weeks 12, 26, 52 and 104 posttransplant were obtained from 91 transplant recipients. In a multivariate model, higher pretransplant plasma HIV RNA level (p < 0.0001) and a longer duration of follow-up posttransplant (p = 0.09) were associated with higher posttransplant plasma HIV RNA levels. A higher baseline HIV DNA (p < 0.0001) was significantly associated with higher HIV DNA levels posttransplant, while higher CD4+ T cell count (p = 0.001), sirolimus use (p = 0.04) and a longer duration of follow-up (p = 0.06) were associated with lower posttransplant HIV DNA levels. The association between sirolimus exposure and lower frequency of cells containing HIV DNA levels posttransplant suggest that the immune-modifying drugs may affect the level of HIV persistence during effect therapy. Future studies of sirolimus as a reservoir-modifying agent are warranted.

Background Dengue viruses (DENV-1-4) pose a transfusion-transmission risk. This study estimated the dengue RNA detection period in asymptomatic blood donors and relationships between donor viremia and dengue incidence during a large epidemic. Methods Donor samples from the 2012 dengue transmission season in Rio de Janeiro, Brazil, were tested for DENV RNA by a transcription-mediated amplification (TMA) assay, with DENV types and viral loads determined by polymerase chain reaction. Samples collected during the first and last weeks of enrollment were tested for DENV immunoglobulin (Ig) G and IgM to estimate incidence during the study period, which was analyzed relative to nucleic acid amplification technology (NAT) yield to estimate the duration of NAT-detectable viremia and compared with reported clinical dengue cases in Rio. Results Samples from 16 241 donations were tested; 87 (0.54%) were confirmed as DENV-4 RNA positive. Dengue IgM-positive/IgG-positive reactivity increased from 2.8% to 8.8%, indicating a 6.2% incidence (95% confidence interval [CI], 3.2%-9.1%) during the study period. Based on these data, we estimated a 9.1-day period (95% CI, 4.4-13.9 days) of RNA detectable with TMA. With 100 475 reported cases of clinical dengue, 1 RNA-positive donation was identified per 800 DENV cases. Conclusions These parameters allow projections of dengue incidence from donor NAT yield data and vice versa, and suggest that viremic donations will be rare relative to clinical disease cases.

BACKGROUND: At most blood centers in the United States routine testing of donations for Trypanosoma cruzi using an enzyme-linked immunosorbent assay (ELISA) is followed by supplemental testing by radioimmunoprecipitation assay (RIPA). The objective of this study was to report the results of routine testing and risk factor data from allogeneic blood donors. STUDY DESIGN AND METHODS: T. cruzi testing data from January 2007 through December 2009 were analyzed, and risk factor interviews and follow-up studies were conducted on seroreactive donors. Prevalences of confirmed infection and risk factors associated with infection were assessed using logistic and multivariable logistic regression. RESULTS: Of 2,940,491 allogeneic donations from 1,183,076 donors, 305 (0.01% per donation tested and 0.026% per blood donor) were repeat reactive (RR) and 89 of those were confirmed positive by RIPA, yielding an overall seroprevalence of 1 per 33,039 donations and 1 per 13,292 donors. Country of birth and US blood center location differences in the seroprevalence of T. cruzi were evident. The odds of confirmed infection were highest if the donor reported having been bitten by the reduviid (kissing) bug (odds ratio [OR], 76.1; 95% confidence interval [CI], 11.1-3173) followed by having lived in a rural area of Latin America (OR, 38.6; 95% CI, 15.1-102.5). In multivariable analyses, having spent 3 months or more in Mexico or Central and/or South America was associated with the highest odds of RIPA-confirmed infection (OR, 8.5; 95% CI, 2.7-26.5). Polymerase chain reaction (PCR) testing of ELISA RR donors exhibited low sensitivity (1/22 [4%] RIPA-confirmed donors was PCR positive). CONCLUSION: Risk factors for confirmed infection in US blood donors are consistent with the known epidemiology of Chagas disease. Blood donors or transfusions do not substantially contribute to the burden of T. cruzi infection in the United States.

BACKGROUND: Xenotropic murine leukemia virus (MLV)-related virus (XMRV) and other related MLVs have been described with chronic fatigue syndrome and certain types of prostate cancer. In addition, prevalence rates as high as 7% have been reported in blood donors, raising the risk of transfusion-related transmission. Several laboratories have utilized microneutralization assays as a surrogate marker for detection of anti-MLV serologic responses—with up to 25% of prostate cancer patients reported to harbor neutralizing antibody responses. STUDY DESIGN AND METHODS: We developed a high-throughput microneutralization assay for research studies on blood donors using retroviral vectors pseudotyped with XMRV-specific envelopes. Infection with these pseudotypes was neutralized by sera from both macaques and mice challenged with XMRV, but not preimmune serum. A total of 354 plasma samples from blood donors in the Reno/Tahoe area were screened for neutralization. RESULTS: A total of 6.5% of donor samples gave moderate neutralization of XMRV, but not control pseudotypes. However, further testing by Western blot revealed no evidence of antibodies against MLVs in any of these samples. Furthermore, no evidence of infectious virus or viral nucleic acid was observed. CONCLUSION: A microneutralization assay was developed for detection of XMRV and can be applied in a high-throughput format for large-scale studies. Although a proportion of blood donors demonstrated the ability to block XMRV envelope-mediated infection, we found no evidence that this inhibition was mediated by specific antibodies elicited by exposure to XMRV or MLV. It is likely that this moderate neutralization is mediated through another, nonspecific mechanism.

BACKGROUND Transfusion-associated microchimerism (TA-MC), the persistence of significant levels of donor leukocytes in blood recipients for prolonged periods, has been demonstrated following non-leukoreduced and leukoreduced transfusion to patients with severe traumatic injury. Development of TA-MC has not been rigorously studied in settings that do not involve massive trauma where the blood is leukoreduced and irradiated.

BACKGROUND: Despite implementation of targeted individual-donor nucleic acid test (NAT) screening of blood donors for West Nile virus (WNV), three “breakthrough” WNV transfusion transmission cases were reported (2004-2008), suggesting that current plasma-based assays are unable to detect all WNV-infectious donations. A 2007 report found that 19 of 20 red blood cell components from WNV-infected donors contained 1 log higher viral load than plasma components. This study's aim was to further establish the value of screening whole blood relative to plasma for WNV RNA by generating differential viral loads on paired samples derived from blood screening tubes. STUDY DESIGN AND METHODS: WNV RNA–positive donors identified by routine NAT screening were enrolled and quantitative viral data were generated using cross-sectional (index-donation) and longitudinal (follow-up) specimens. A real-time reverse transcription–polymerase chain reaction viral load assay was used on both study sample sets and replicate qualitative NAT screening assays were also used on the longitudinal study samples. RESULTS: For the cross-sectional study, seronegative index donations (n = 29) had WNV RNA concentrations fourfold higher in plasma than in whole blood, whereas for seropositive donations (n = 13), the WNV RNA concentrations were 10-fold higher in whole blood than in plasma. All 10 longitudinal study participants were seropositive throughout the follow-up study; whole blood viral load was consistently greater than plasma viral load (mean difference, 343 copies; p

Highly active antiretroviral therapy (HAART) has been effective in decreasing morbidity and mortality associated with human immunodeficiency virus (HIV) infection [1]. However, a significant proportion of individuals are unable to achieve a normal CD4+ T cell count despite prolonged viral suppression with effective HAART. In one study, ∼25% of patients who started HAART at a CD4+ T cell count of 500 cells/mm3 even after more than 7–10 years of HAART [2]. Moreover, having a suboptimal CD4+ T cell response has been associated with significant clinical consequences, including increased AIDS-related and non–AIDS-related morbidity and mortality [3–6]. The mechanisms of suboptimal immune recovery are not completely understood. Persistent T cell activation may be causally related to the inability to reconstitute normal CD4+ T cell counts, perhaps owing to its effect on lymphoid tissue architecture [7–11]. Moreover, ongoing low-level viral replication may be the proximal cause of persistent activation during HAART [12–15]. Several studies have attempted to address this issue [15–18], although none have focused on the host responses to HIV in both blood and gut-associated lymphoid tissue (GALT). We conducted a randomized, double-blinded, placebo-controlled study of raltegravir intensification to assess whether ongoing viral replication contributes to immune activation and suboptimal immune recovery during HAART. Our secondary objective was to explore the host determinants of viral persistence in both blood and GALT.

Objective: Transient HIV infections have been invoked to account for the cellular immune responses detected in highly virus-exposed individuals who have remained HIV-seronegative. We tested for very low levels of HIV RNA in 524 seronegative plasma samples from 311 highly exposed women and men from three longitudinal HIV cohorts. Design: Two thousand and seventy-three transcription-mediated amplification (TMA) HIV RNA tests were performed for an average of 3.95 TMA assays per plasma sample. Quadruplicate TMA assays, analyzing a total of 2 ml of plasma, provided an estimated sensitivity of 3.5 HIV RNA copies/ml. Results: Four samples from individuals who did not seroconvert within the following 6 months were positive for HIV RNA. For one sample, human polymorphism DNA analysis indicated a sample mix-up. Borderline HIV RNA detection signals were detected for the other three positive samples but further replicate TMA testing yielded no positive results. Nested PCR assays (n = 254) for HIV proviral DNA in peripheral blood mononuclear cells (PBMCs) from these three individuals were negative. Conclusion: Transient viremia was not reproducibly detected in highly HIV-exposed seronegative men and women. If transient infections do occur, plasma HIV RNA levels may remain below the detection limits of the sensitive assay used here, be of very short duration, or viral replication may be restricted to mucosal surfaces or their draining lymphoid tissues.

Introduction Because the receptor for Parvovirus B19 (B19V) is on erythrocytes, we investigated B19V distribution in blood by in-vitro spiking experiments and evaluated viral compartmentalization and persistence in natural infection.

HAART can effectively reduce plasma HIV RNA levels to below the level of detection in most HIV-infected patients. The degree to which residual low-level viremia persists during HAART remains unclear.We identified 180 individuals (median duration of HIV infection 12 years) who had at least two consecutive plasma HIV-1 RNA levels below the level of detection (50-75 copies/ml) while taking antiretroviral drugs; 36 of 180 had been virologically suppressed for more than 5 years. Longitudinal plasma samples that were taken from these individuals during periods of viral load suppression were selected and analyzed. The isothermal transcription-mediated amplification (TMA) (limit of detection3.5 copies RNA/ml) assay was used to measure persistent viremia. A 'detuned' EIA assay was used to obtain quantitative HIV antibody levels.A total of 1606 TMA assays were performed on 438 specimens in 180 HAART-suppressed individuals (median 3 replicates per specimen). In the first year of viral suppression, plasma RNA levels declined significantly (P = 0.001), but after month 12 there was no evidence for a continued decline (P = 0.383). In the first year of viral suppression, HIV antibody levels also declined (P = 0.054), but after month 12 there was no evidence for a continued decline (P = 0.988).Viremia continued to decline during the first 12 months after viremia became undetectable using conventional methods, and then remained stable. HIV antibody levels also decreased in the first year of viral suppression and then remained stable. Viremia and the HIV-associated host response appear to achieve a steady-state 'set-point' during long-term combination therapy.

There is increasing use of highly sensitive testing with polymerase chain reaction (PCR) to study white cell microchimerism after transfusion and transplantation. This study investigated possible artifactual sources of allogeneic sample contamination before PCR testing.Quantitative Y-chromosome PCR was used to study microchimerism among transfused patients with sickle cell disease (SCD) and thalassemia by using residual specimens from the clinical laboratory. High levels of circulating male white cells among transfused patients with SCD but not thalassemia led to concern over the artifactual origin of male cells. To investigate, paired specimens were collected from 26 female SCD patients: one specimen underwent processing only for PCR, while the other underwent testing in the clinical laboratory before PCR as a process control. All laboratory instruments were also assessed for their ability to impart male allogeneic cells to aliquots of female blood.Thirty-three (31%) of 107 SCD samples, but 0 of 20 thalassemia samples, gave a high-level PCR signal. One of 26 paired samples that was not exposed to clinical laboratory equipment had low-level PCR positivity while 10 of the 26 became strongly positive after testing on a blood cell analyzer and a reticulocyte analyzer. Sixteen of 32 female samples became positive after reticulocyte analysis, while none became positive after blood cell analysis. Samples from thalassemia patients tested PCR-negative because reticulocyte counts had not been performed.Allogeneic cell contamination is common with clinical laboratory equipment. These samples may not be suitable for microchimerism studies. In addition to method controls, process controls should be employed where appropriate.

Author(s): Kwaan, Nicholas; Lee, Tzong-Hae; Chafets, Daniel M; Nass, Catharie; Newman, Bruce; Smith, James; Garratty, George; Murphy, Edward L; HTLV Outcomes Study (HOST) Investigators | Abstract: BackgroundThe human T lymphotropic virus (HTLV)-I or -II proviral load (VL) may be linked to viral pathogenesis, but prospective data on VL and disease outcomes are lacking.MethodsUsing data from a prospective cohort study of HTLV disease outcomes, we examined baseline VLs with real-time quantitative polymerase chain reaction in 122 HTLV-I- and 319 HTLV-II-infected subjects and serial VLs over the course of 6 visits in a subset of 30 HTLV-I- and 30 HTLV-II-infected subjects. Cox and logistic-regression models were used to test baseline associations, and repeated-measures analysis was used to study variations in VL over time.ResultsOver the course of a median of 10.4 years, HTLV-I VLs decreased slightly (slope, -0.017 log(10) copies/10(6) peripheral blood mononuclear cells [PBMCs]/year; P=.042) and HTLV-II VLs did not change (slope, -0.019 log(10) copies/10(6) PBMCs/year; P=.165). Changes in VL over time were associated positively with alcohol use (P=.07) and negatively with black race (P=.03) for HTLV-I and positively with smoking (P=.08) for HTLV-II. In the larger group, there was no association between baseline VL and disease outcomes. In the smaller group with serial VL data, there was an association between increasing VL and bladder or kidney infections for both HTLV-I (P=.005) and HTLV-II (P=.022).ConclusionsHTLV VLs are stable over time, but alcohol and tobacco intake may affect the progression of VLs. The association between increasing VLs and bladder/kidney infection may be explained by early HTLV-related neuropathologic progression.

BACKGROUND: Transfusion of trauma patients can result in long-term survival of donor white blood cells (WBCs) or “transfusion-associated microchimerism” (TA-MC). The aim was to determine whether leukoreduction of blood transfusions, advocated to reduce the immunomodulatory effect of transfusion, decreases the likelihood of developing TA-MC. STUDY DESIGN AND METHODS: A subgroup of trauma patients from a randomized trial was examined, evaluating the risk of infection following leukoreduced versus nonleukoreduced blood transfusion. Patients’ blood was sampled at least 1 month after hospital discharge, and TA-MC was assessed with quantitative allele-specific polymerase chain reaction detection of differences at the HLA-DR locus or a panel of insertion-deletion polymorphism loci distributed throughout the chromosomal complement. At the time of blood sampling, a scripted interview was used to ascertain symptoms suggestive of chronic graft-versus host disease (cGVHD). RESULTS: For 67 patients evaluated, the mean age was 43 ± 17 years and mean Injury Severity Score was 24 ± 12. Median time from injury to blood sampling for TA-MC was 240 (interquartile range, 116-360) days. Nine of 32 patients (28%) in the nonleukoreduced transfusion group developed TA-MC compared to 13 of 35 patients (37%) in the leukoreduced group (p = 0.43). Subjects with TA-MC were no more likely than subjects without TA-MC to have at least one symptom suggestive of cGVHD (64% vs. 76%, respectively). CONCLUSIONS: TA-MC seems to be a prevalent condition among injured patients at the second of two regional trauma centers evaluated, suggesting that it is a common phenomenon after transfusion in the setting of injury. Although leukoreduction removes greater than 99.9 percent of donor WBCs, it fails to prevent or even substantially reduce the likelihood of developing TA-MC. TA-MC does not appear to be strongly associated with symptoms suggestive of cGVHD several months after transfusion.

Cellular immune responses to Kaposi sarcoma-associated herpesvirus (KSHV), the etiological agent of KS and several other malignancies, are incompletely characterized. We assessed KSHV-specific interferon- gamma enzyme-linked immunospot responses in a cohort of 154 individuals, using overlapping peptide sets spanning the KSHV-encoded latency-associated nuclear antigen (ORF73) and the minor capsid glycoprotein (ORF65). Among KSHV-seropositive subjects, ORF73-specific responses dominated over responses to ORF65 and were preferentially detected in human immunodeficiency virus-coinfected individuals who had elevated levels of cell-associated KSHV DNA, indicating that the viral antigen burden may have been driving these responses. Responses to both ORF73 and ORF65 were also detected in several KSHV-seronegative subjects who were at increased risk for KSHV infection, which demonstrates that cellular immunity can be found in the absence of detectable humoral responses. These data have implications for the reliable identification of KSHV infection and may help guide the design of immune-based therapeutic and prophylactic interventions.

BACKGROUND: Long-term white blood cell (WBC) microchimerism (MC), of at least 2 years, has been reported in trauma patients receiving fresh nonleukoreduced (non-LR) blood. It is unknown, however, whether this occurs with LR blood products that are nearly devoid of WBCs. Twenty-seven patients transfused with LR and non-LR blood products were studied after severe traumatic injury. A secondary aim was to explore donor-recipient mixed lymphocyte reactivity in vitro. STUDY DESIGN AND METHODS: To quantify MC, allele-specific real-time polymerase chain reaction assays were developed targeting HLA Class II sequence polymorphisms. Extensive validation showed that these assays reliably detect a single copy of target sequence in a complex allogeneic background without false positivity. RESULTS: At a median follow-up of 26 months (range, 24-39 months), long-term MC was observed in 3 of 20 patients (15%) who received non-LR blood products and 2 of 7 (29%) who received LR blood products. The maximum MC ranged from 0.40 to 4.90 percent of circulating WBCs and appeared, by Class II genotype analysis, to be attributable to a single donor. CONCLUSION: It is concluded that robust levels of long-term MC, apparently traceable to a single donor, occur at similar frequency despite leukoreduction of transfused blood products. Exploratory analysis of donor-recipient mixed lymphocyte reactivity suggests that long-term MC may require a state of bidirectional tolerance before transfusion.

Author(s): Roucoux, Diana F; Wang, Baoguang; Smith, Donna; Nass, Catharie C; Smith, James; Hutching, Sheila T; Newman, Bruce; Lee, Tzong-Hae; Chafets, Daniel M; Murphy, Edward L; HTLV Outcomes Study Investigators | Abstract: BackgroundCross-sectional studies support sexual transmission of human T lymphotropic virus (HTLV)-I/II; however, prospective incidence data, particularly for HTLV-II, are limited.MethodsA cohort of 85 HTLV-positive (30 with HTLV-I and 55 with HTLV-II) blood donors and their stable (gor=6 months) heterosexual sex partners were followed biannually over the course of a 10-year period.ResultsFour of 85 initially seronegative sex partners of HTLV-I and -II carriers seroconverted, for an incidence rate (IR) of 0.6 transmissions/100 person-years (py) (95% confidence interval [CI], 0.2-1.6). This includes 2 HTLV-I transmissions/219 py (IR, 0.9 transmissions/100 py [95% CI, 0.1-3.3]) and 2 HTLV-II transmissions/411 py (IR, 0.5 transmissions/100 py [95% CI, 0.06-1.8]), with no significant difference by HTLV type. There were 2 male-to-female (IR, 1.2 transmissions/100 py [95% CI, 0.1-4.3]) and 2 female-to-male (IR, 0.4 transmissions/100 py [95% CI, 0.05-1.6) transmissions. HTLV-I or -II proviral load was 2 log10 lower in newly infected partners than in index positive partners who transmitted HTLV (P=.007).ConclusionsThe incidence of sexual transmission of HTLV-II may be similar to that of HTLV-I, and female-to-male transmission may play a more important role than previously thought. HTLV-I and -II proviral load may be lower in sexually acquired infection, because of a small infectious dose.

High human T lymphotropic virus (HTLV)-I provirus load (VL) has been associated with an increased risk of HTLV-associated myelopathy, but little is known about variation in HTLV-I or -II VLs by demographic characteristics and risk behaviors.We measured HTLV-I and HTLV-II VLs in a large cohort of 127 HTLV-I-seropositive and 328 HTLV-II-seropositive former blood donors, by use of real-time polymerase chain reaction using tax primers. Multivariable linear regression was used to control for confounding by relevant covariates.The mean VLs were 3.28 log(10) copies/10(6) peripheral blood mononuclear cells (PBMCs) (range, 0.5-5.3 log(10) copies/10(6) PBMCs) for HTLV-I and 2.60 log(10) copies/10(6) PBMCs (range, 0.05-5.95 log(10) copies/10(6) PBMCs) for HTLV-II (P.0001). HTLV-II VLs were higher in those subjects with subtype A infection (mean, 2.82 log(10) copies/10(6) PBMCs) than in those with subtype B infection (mean, 2.29 log(10) copies/10(6) PBMCs) (P=.005). Higher HTLV-I VL was associated with previous receipt of a blood transfusion (P=.04), and lower HTLV-II VL was associated with female sex (P=.007). These associations persisted in virus-specific multivariate linear regression models controlling for potential confounding variables.VL was significantly higher in HTLV-I than in HTLV-II infection and was higher in HTLV-II subtype A than in HTLV-II subtype B infection. Chronic HTLV VLs may be related to the infectious dose acquired at the time of infection, with higher VLs following acquisition by blood transfusion and lower VLs following sexual acquisition.

BACKGROUND: Accurate typing of a patient’s RBCs in the setting of prior transfusion or a hemolytic transfusion reaction is crucial in the selection of compatible blood but is time consuming, technically difficult, and sometimes impossible. To address this problem, a simple, rapid, and inexpensive quantitative PCR method was developed to identify the single nucleotide polymorphism (SNP) of the Kidd blood group. We applied this method in a clinical investigation of 54 multiple-transfusion patients. STUDY DESIGN AND METHODS: Patients were eligible if they had received at least one RBC transfusion within 30 days and had a sample referred to our regional reference lab for assistance with compatibility testing requiring reticulocyte separation, hypotonic saline treatment, or chemical modification to remove IgG. We compared serologic result to the normalized quantitative PCR. For discrepants, or where no serologic type could be assigned, DNA sequencing characterized the patient’s Kidd SNP. RESULTS: Of the 54 patients, the reference lab could assign a serologic Kidd type for 33. Quantitative PCR assigned a Kidd type for 53 of the 54. In three cases, where serology and PCR were discrepant, and for all cases where serology could not assign a Kidd type, DNA sequencing verified the Kidd typing assigned by PCR. CONCLUSION: A simple, rapid, and accurate technique has been developed. The assay performs well in the clinical setting. With further study, and inclusion of other blood group systems, this may become an important supplemental technique for selected patients in the immunohematology reference laboratory. ntibodies to the Kidd blood group antigens are among the most problematic in clinical transfusion practice. Molecular genotyping of blood groups, including Kidd, using restriction fragment length polymorphism 1,2 and sequence-specific

BACKGROUND: As part of assessing the possibility of transfusion transmission of human herpesvirus 8 (HHV-8 or Kaposi's sarcoma-associated herpesvirus), HHV-8 seroprevalence was estimated among US blood donors, the performance of HHV-8 serologic tests was compared, and the presence of HHV-8 DNA was tested for in donated blood. STUDY DESIGN AND METHODS: Replicate panels of 1040 plasma specimens prepared from 1000 US blood donors (collected in 1994 and 1995) and 21 Kaposi's sarcoma patients were tested for antibodies to HHV-8 in six laboratories. HHV-8 PCR was performed on blood samples from 138 donors, including all 33 who tested seropositive in at least two laboratories and 22 who tested positive in at least one. RESULTS: The estimated HHV-8 seroprevalence among US blood donors was 3.5 percent (95% CI, 1.2%-9.8%) by a conditional dependence latent-class model, 3.0 percent (95% CI, 2.0%-4.6%) by a conditional independence latent-class model, and 3.3 percent (95% CI, 2.3%-4.6%) by use of a consensus-derived gold standard (specimens positive in two or more laboratories); the conditional dependence model best fit the data. In this model, laboratory specificities ranged from 96.6 to 100 percent. Sensitivities ranged widely, but with overlapping 95 percent CIs. HHV-8 DNA was detected in blood from none of 138 donors evaluated. CONCLUSIONS: Medical and behavioral screening does not eliminate HHV-8-seropositive persons from the US blood donor pool, but no viral DNA was found in donor blood. Further studies of much larger numbers of seropositive individuals will be required to more completely assess the rate of viremia and possibility of HHV-8 transfusion transmission. Current data do not indicate a need to screen US blood donors for HHV-8.

BACKGROUND : New-generation RBC filters reduce WBC concentrations by 4 to 5 logs and may prevent or decrease transfusion complications such as HLA alloimmunization, nonhemolytic febrile reaction, and transfusion-transmitted infections. The residual level of WBC subsets may influence efficacy of WBC reduction for preventing various complications. This study analyzed subsets of residual WBCs in WBC-reduced RBC components prepared for a large, multicenter prospective study. STUDY DESIGN AND METHODS : The Viral Activation Transfusion Study (VATS) assessed the impact of WBC reduction in HIV-1-infected patients undergoing RBC transfusion. WBC-reduced RBC from 11 clinical sites with variable filtration practices were sorted into “low,”“middle,” and “high” groups based on residual WBC concentration. Subsets were isolated from units by immunocapture (anti-CD4-, anti-CD8-, anti-CD15-, and anti-CD19-coated magnetic beads) and quantified by PCR amplification. RESULTS : After validation studies confirming test methodology, 105 VATS WBC-reduced RBC samples were analyzed. Concentrations of subsets in low and middle residual WBC groups were very low in contrast to relatively high concentrations in the high group. Although highly significant differences were identified between the middle and high groups for total WBCs and all subsets, no single subset predominated. CONCLUSION : These results suggest that overall efficacy of WBC filtration correlates with removal of WBC subsets.

A linked donor-recipient study was conducted during epidemics in 2 cities in Brazil to investigate transfusion-transmitted (TT) dengue virus (DENV) by DENV RNA-positive donations.During February-June 2012, samples were collected from donors and recipients and retrospectively tested for DENV RNA by transcription-mediated amplification. Recipient chart review, using a case (DENV positive)-control (DENV negative and not known to be exposed) design, was conducted to assess symptoms.Of 39 134 recruited blood donors, DENV-4 viremia was confirmed in 0.51% of donations from subjects in Rio de Janeiro and 0.80% of subjects in Recife. Overall, 42 DENV RNA-positive units were transfused into 35 recipients. Of these, 16 RNA-positive units transfused into 16 susceptible recipients were identified as informative: 5 cases were considered probable TT cases, 1 possible TT case, and 10 nontransmissions. The TT rate was 37.5% (95% confidence interval [CI], 15.2%-64.6%), significantly higher than the viremia rate of 0.93% (95% CI, .11%-3.34%) in nonexposed recipients (P.0001). Chart review did not find significant differences between cases and controls in symptoms or mortality.During a large epidemic of DENV-4 infection in Brazil,0.5% of donations were RNA positive, and approximately one third of components resulted in TT. However, no significant clinical differences were evident between RNA-positive and RNA-negative recipients.

Seaberg and Thio [1] refer to evidence that suggests that route of infection may play a role in hepatitis C virus (HCV) spontaneous clearance. We did not examine the role of the route of infection on HCV clearance in HIV controllers in our study. HCV clearance differs by HIV status, but little prospective data are available to assess clearance by transmission route. One retrospective analysis showed that presumed sexually acquired HCV had higher rates of spontaneous clearance than parenterally acquired HCV [2]. Other studies have not shown this [3,4]. In prospective studies of HCV monoinfected patients, there is no evidence that clearance differs by transmission route [5,6]. Less examined is how genetic variations and mode of transmission impact divergent HCV outcomes. Seaberg et al. [7] found a higher magnitude of association between IL28B CC and HCV clearance in men MSM with reported injected drug use (IDU) than in those without, but this analysis includes MSM with and without HIV. We examined these factors in HIV controllers, the study population examined in our study ‘Human Leukocyte Antigen (HLA) B*57 Does Not Fully Explain Hepatitis C Clearance in HIV Controllers’ [8]. People with the IL28B CC (vs. CT or TT) allele are significantly more likely to spontaneously clear HCV infection [9,10]. In our study, the presumed mode of HCV acquisition did not modify the association between IL28B genotype and HCV clearance: there was no difference in the effect of IL28B CC (vs. CT/TT) on HCV clearance between those with a history of IDU [prevalence ratio 2.98; 95% confidence interval (CI) 1.57–5.64] and those without reported IDU history (prevalence ratio 2.51; 95% CI 1.52–4.16; test for heterogeneity P=0.93). Unlike the study by Seaberg et al. [7], we found no evidence that the impact of IL28B CC (vs. CT/TT) on HCV clearance was modified by IDU history among MSM (prevalence ratio in non-IDU MSM 2.13; 95% CI 1.25–3.65; prevalence ratio in IDU and MSM 1.57; 95% CI 0.36–6.86). HLA B*57 has been shown to be highly enriched in people who innately control their HIV infection [11,12], prompting questions about whether this may hold true for other viral infections, including HCV. We found no evidence that HLA B*57 is associated with clearance in HIV-infected patients (adjusted prevalence ratio 1.36; 95% CI 0.71−2.60, P = 0.35) [8]. Although power was limited, we also found no evidence for a protective role of HLA B*57 in any subgroup of interest. For example, patients with HLA B*57 had similar prevalence of HCV clearance to those without HLA B*57 among both HIV controllers (31 vs. 34%, P = 0.83) and noncontrollers (29 vs. 27%, P = 0.79). HLA B*57 does not explain the increase in HCV clearance in controllers in our cohort. We also found no association between HLA B*57 and HCV clearance in those with and without an IDU history (prevalence ratio 1.18; 95% CI 0.56−2.51 vs. prevalence ratio 1.19; 95% CI 0.62−2.28) and in MSM vs. non-MSM exposure groups (prevalence ratio 0.98; 95% CI 0.49−1.99 vs. prevalence ratio 1.36; 95% CI 0.68−2.70). The table presenting the multivariate analysis in the original article was mis-printed and omitted the significance of IL28B CC on clearance. An erratum will correct this. Seaberg and Thio [1] state that we did not discuss the finding that HIV controllers were more likely to clear HCV when negative for HLA B*57. The original publication states that HLA B*57 cannot explain the increased prevalence of HCV clearance in HIV controllers in our cohort. We agree with our colleagues that this is perhaps the most important novel finding from our study. It suggests that other unmeasured factors associated with HIV control must also contribute to HCV clearance in this setting. Future host genetic studies of HIV/HCV-coinfected controllers and noncontrollers may help shed light on these factors, potentially identifying novel genetic factors that contribute to the clearance and/or control of multiple viral pathogens. Nevertheless, as pointed out, HLA B*57 has been associated with HCV clearance in other studies [13,14]. Our study does not discount those studies, as a larger sample size might have revealed a more significant effect of HLA B*57. However, our study does suggest that there are factors other than HLA B*57 that contribute to HCV clearance in HIV controllers. The evidence of differential outcome of HCV in association with route of exposure is inconclusive; further research is necessary to improve our understanding of this potential effect and putative mechanisms involved. We agree that further research is needed to understand why HIV controllers are more likely to clear HCV, as this cannot be entirely explained by enrichment for HLA B*57.

BACKGROUND: As universal leukocyte (WBC) reduction (ULR) is being considered as a new standard, few data are available on the performance of WBC-reduction filtration in routine practice. The performance of WBC-reduction in RBCs, using varied filtration practices, in meeting the current FDA requirement (

BACKGROUND: Allogeneic blood transfusion is common in the treatment of neonatal anemia of prematurity or anemia due to multiple phlebotomies. The immune response of neonates to passenger leukocytes from allogeneic red cells was investigated. STUDY DESIGN AND METHODS: Fourteen infants (4 male, 10 female) prospectively were randomly assigned to receive either white cell-reduced (Group 1) or non-white-cell reduced (Group 2) irradiated blood. Blood samples were taken before and at various time intervals after transfusion (Days 1, 5-7,and 10-14). Cord blood from 11 healthy term infants was used for comparison. The following surface markers were used to assess immune modulation by flow cytometry: CD45RA/CD45RO, CD4/CD8, CD25/CD28, CD3/DR, CD14/B7, and CD3/CD56+CD16. Donor cell microchimerism was studied using semi-quantitative polymerase chain reaction Y-chromosome detection in female infants who received male donor blood. Donor and recipient HLA class II typing was performed with polymerase chain reaction with sequence specific primers. RESULTS: The lymphocyte counts in both groups were significantly increased after transfusion, and there was a significant increase in lymphocytes expressing CD45RA, CD3–/CD16+CD56, CD80, and CD3–/DR on Day 14. The premature infants' pretransfusion natural killer cell population (CD3–/CD16+CD56) was significantly lower than that of term infants, but it reached a similar level by Days 10-14. CD8 subpopulations were increased but not CD4+ cells. Two female infants (of 6) had circulating Y chromosomes 1 day after transfusion, and most of the infants effectively cleared the donor cells within 24 hours of transfusion. Two Group 2 infants who by chance received presumably HLA-haploidentical donor blood developed necrotizing enterocolitis. CONCLUSION: Blood transfusion alters immune cell antigen expression in premature neonates and may initially be immunostimulatory and later immunosuppressive.

A small percentage (1-5%) of people living with HIV are able to intrinsically control their HIV infection over long periods of time without the use of antiviral therapy (ART) [1]. Two groups of “HIV controllers” have been described: (1) Elite controllers, who have HIV antibodies (anti-HIV), but plasma HIV RNA levels below the detection limits of commercially available assays, and; (2) Viremic controllers, who have measurable, but very low levels of HIV RNA (< 2000 copies/mL). HIV controllers represent “outliers” in the clinical HIV spectrum, and are considered very important for studies of host immunological and genetic factors that control viral replication, with the expectation that they will inform vaccine and drug development, or perhaps identify strategies to achieve a “functional cure [1, 2].” Interestingly, HIV controllers may also be more likely to spontaneously clear hepatitis C virus (HCV) infection. While not observed in all studies [3, 4], most studies suggest a significantly higher rate of spontaneous HCV clearance in HIV controllers (23-39%) [5, 6] than in monoinfected groups (25%) [7] and HIV non-controllers (6.6%-10%) [1, 5]. This has led several groups to examine host genetic factors that might contribute to the control of both virus infections. For example, HLA B*57, the major histocompatibility class I allele, is highly enriched in HIV controllers and is also associated with spontaneous HCV clearance [8-10]. In contrast, HLA Cw*04 has been associated with faster progression of both HIV disease and chronic HCV infection [11, 12]. The IL28B CC genotype has been shown to be significantly associated with spontaneous control of HCV [13-16], conferring as much as 3.1 higher odds of clearance compared to CT and TT genotypes [17, 18]. Whether the CC genotype of IL28B is also enriched in HIV controllers remains controversial. A relatively small Spanish study found that the CC genotype of IL28B was enriched in Caucasian HIV controllers relative to non-controllers [4], while other studies in primarily African-American cohorts have not observed this [6, 19]. Furthermore, none of the much larger genome-wide association studies have found an association between the IL28B (CC) genotype and HIV control, at least at the genome-wide level of significance [20-23]. In one study the IL28B CC genotype has been associated with higher all-cause mortality among all people living with HIV [24]. In this study, we investigated the degree to which spontaneous clearance of HCV could be explained by HLA B*57 and/or IL28B CC genotype in a well characterized cohort of HIV controllers.

(See the editorial commentary by Henrich and Gandhi on pages 1189–93 and the major article by Yukl et al 1212–20.) T-cell immune activation, defined by coexpression of CD38 and HLA-DR on CD4+ and CD8+ T cells, has been linked to morbidity and mortality in untreated human immunodeficiency virus (HIV) infection, both AIDS-related and non-AIDS-related [1, 2]. In patients receiving HIV antiretroviral therapy (ART), higher levels of T-cell activation has been linked to diminished CD4+ T-cell count recovery [3–5], surrogate markers of cardiovascular disease [6, 7], and increased mortality [8]. ART-mediated virologic suppression reduces both CD4+ and CD8+ T-cell activation levels [3, 9]. However, patients frequently continue to exhibit T-cell activation levels higher than those seen in HIV-negative controls, indicating that ART reduces but does not fully reverse HIV-related immune activation [3, 9, 10]. A key unanswered question, therefore, is whether ART initiated early (within the first months after HIV infection) can reduce on-therapy immune activation more than ART initiated during chronic HIV infection. A related question is whether early initiation of ART durably limits the size of persistent HIV reservoirs. Cellular reservoirs of latent genomically integrated HIV are established quickly after infection [11–14], but studies investigating whether ART initiated in the first few months after infection can limit the size of the established reservoir have reached inconsistent conclusions. One study found that ART initiated 2 years. We assessed T-cell activation levels and measured the HIV reservoir (based on both HIV DNA and cell-associated RNA levels) to study the impact of early ART on these outcomes.

One hundred thirty-two recipients of blood components that retrospectively tested positive for antibody to human immunodeficiency virus type 1 (anti-HIV-1) were identified. Fourteen (11%) remained seronegative throughout follow-up. Donor and recipient characteristics that could have influenced transmission were examined. Attributes did not differ for infected and uninfected recipients. Peripheral blood mononuclear cells (PBMC) from uninfected recipients were HIV-1-negative by DNA amplification and culture but were susceptible to in vitro infection. Transmitting and nontransmitting donors at donation differed only for HIV-1 RNA positivity. By immunocapture reverse transcriptase-polymerase chain reaction, 6 of 11 transmitters and 0 of 11 nontransmitters tested RNA-positive (P = .02). A more sensitive quantitative RNA assay detected RNA in all donation sera, but median levels were higher in transmitting than nontransmitting sera (P = .01). Median CD4 cell counts were lower for transmitting than nontransmitting donors at enrollment (P = .02). Level of viremia is an important determinant of HIV infection by blood transfusion.

Background Babesia microti, the most frequently implicated pathogen in transfusion-transmitted babesiosis, is widely endemic in the Northeast and upper Midwestern United States. High seroprevalence in endemic areas limits antibody-based donor screening. A high-performance molecular test is needed to identify donors in the preseroconversion window phase as well as to discriminate past serologic exposure with parasite clearance from continued parasitemia. Study Design and Methods Frozen Babesia-spiked whole blood was microcentrifuged, and the supernatant transferred and microcentrifuged again to concentrate the parasite. The DNA was extracted and amplified using real-time polymerase chain reaction (PCR) using Babesia-specific primers. The assay was employed in three series of experiments: 1) a validation and optimization spiking experiment, 2) a blinded serial dilution probit analysis to determine the limit of detection, and 3) evaluation of two blinded panels of clinical samples from possible babesiosis cases. Results At a decreasing inoculum of 445, 44.5, and 4.45 copies/mL, the assay had positive rates of 100, 97.5, and 81%, respectively. The blinded probit analysis demonstrated a detection rate of 95 and 50% at 12.92 and 1.52 parasites/2 mL of whole blood, respectively. Evaluation of clinical samples showed 13 of 21 samples to be positive, with a range of 85 to 4.8 million parasites/mL. There were no positives detected among 48 healthy donors Conclusion We have developed a highly sensitive and specific, quantitative real-time PCR-based assay for detection of B. microti that could have a useful role in blood screening. It can also be employed broadly to understand Babesia epidemiology, disease pathogenesis, and host immunology.

Storage of lymphocytes for later use in prospective epidemiologic studies of blood donors and transfusion recipients has been limited by the cost of separating peripheral blood mononuclear cells (PBMCs). When the Transfusion Safety Study began in 1985, it was decided to establish a cell repository of cryopreserved buffy coat (BC) samples, and thus far over 20,000 samples have been accumulated from enrolled subjects. To determine if these specimens could be used for polymerase chain reaction, a simple thawing and pelleting technique for recovering hemoglobin-free total white cells (WBCs) was developed. To validate the technique, parallel analysis was conducted of BCs, whole blood (WB), and PBMC samples from human immunodeficiency virus type 1 (HIV-1)-seropositive subjects. Immediate postthaw cell courts of 29 frozen-thawed (F-T) WB and BC samples averaged 90 percent of the prefreeze (input) values. Representative WBC populations were obtained by immediate pelleting. Amplification of HIV-1 gag sequences from F-T BCs and F-T WB was 94 and 75 percent, respectively, which is as sensitive as that obtained with freshly separated PBMC lysates. Quantitative HIV-1 proviral load analysis by serial dilution of 23 F-T BCs and 8 WB lysates showed results comparable to those obtained with lysates of fresh PBMCs. Values for WBC differential and immunophenotyping could be applied to express viral load relative to total WBCs, PBMCs, or CD4+ cells. These results establish the basis for simplified virologic analysis of cryopreserved BC or WB specimens.

Evidence of frequent HIV-1 infections in antibody-negative, high-risk individuals (so-called 'silent' infections) remains controversial. To evaluate whether these discrepant results may be the consequence of intermittent detection of rare infected cells (low viral load) preceding seroconversion, we developed a modification of the polymerase chain reaction (PCR) technique which enabled analysis of 10-fold greater amounts of cellular DNA per reaction than standard PCR (2 x 10(6) rather than 0.2 x 10(6) input cells). This technique allowed consistent detection of HIV-1 provirus in two seropositive individuals who had repeatedly tested negative by standard-input PCR. However, results were negative when high-input PCR was applied to 51 specimens from 39 selected high-risk seronegative individuals. These results suggest that variations in viral load preceding or in the absence of seroconversion probably do not explain discrepant evidence regarding silent HIV-1 infection.

In settings where maternal antiretroviral treatment is not used, about a quarter of infants born to HIV+ women become HIV-infected before or at birth, as demonstrated by polymerase chain reaction (PCR) tests. Specifically, in Malawi, site of the current study, approximately 6–8% of infants were infected in utero, defined as being HIV PCR-positive in umbilical cord blood. Of uninfected infants, 15 to 20% became infected perinatally, defined by being PCR-negative in cord blood but PCR-positive in the first tested blood sample taken 1–2 months after birth1, 2. Thus, the two thirds of the infants who are diagnosed as infected by 1–2 months of age acquired HIV at or very near the time of birth. The route of perinatal transmission seems likely to be across the placenta. There is little support for infection in the birth canal1, 3, and breast feeding in the first weeks of life appears to contribute little to perinatal infection risk4. One plausible mechanism for perinatal transmission is by transfer of HIV-infected maternal cells into the fetal circulation at or near the time of delivery. Several reports describe the detection of fetal DNA in the mother’s circulation during pregnancy and this approach has been applied to detect genetic abnormalities in the fetus5–6. Transplacental microtransfusions from the mother to the fetus have also been documented using hybridization7 and by PCR testing of DNA extracted from cells obtained from fresh cord blood, where frequency was estimated to be 1 maternal cell per 1000 to 10,000 child cells in mid-trimester fetuses8. However, obtaining this level of sensitivity is challenging when the target DNA is not greatly different from an overwhelming background of quite similar DNA, such as when blood from first degree relatives (i.e., mother and child pairs) is admixed. We report here a semi-quantitative approach using DNA extracted from dried blood spots (DBS), in which levels below 1 added cell per 1000 cells were reliably detected. We further used this technique to examine DBS made from umbilical cord blood to examine whether finding maternal DNA sequences was associated with perinatally-acquired HIV infection in the infant.

BACKGROUND: Dengue fever and hemorrhagic disease are caused by four dengue virus (DENV) serotypes (DENV-1 to -4), mosquito-borne flaviviruses with increasing incidence, and expanding global distributions. Documented transfusion transmission of West Nile virus raised concern regarding transfusion-transmitted DENV. METHODS: A DENV RNA assay was developed based on transcription-mediated amplification (TMA) blood screening assays routinely used by blood centers worldwide. Sensitivity was established by endpoint dilution analyses of DENV-1 RNA transcript and pedigreed tissue culture standards for all four DENV-serotypes. Frozen plasma samples were tested from 2994 donations from Honduras (September 2004-January 2005), 4858 donations from Brazil (February-April 2003), and 5879 donations from Australia (March-September 2003). Type-specific polymerase chain reaction (PCR) assays were used to quantify and genotype TMA repeat-reactive samples; viral cultures, type-specific antibody, and antigen assays were also performed. RESULTS: The TMA assay detected 14.9 copies per mL DENV-1 transcript (95% detection limit), with comparable sensitivity for all four serotypes. Honduran donors yielded 9 TMA repeat-reactive samples (0.30%); 8 were confirmed by PCR, with 3 DENV serotypes detected and viral loads from fewer than 3 × 104 to 4.2 × 104 copies per mL; and 4 samples yielded infectious virus. Three (0.06%) Brazilian samples tested repeat-reactive; 2 (0.04%) were PCR-positive (serotypes DENV-1 and -3; 12 and 294 copies/mL). No Australian donor samples tested repeat-reactive. CONCLUSION: Dengue viremia rates among asymptomatic blood donors ranged from 0.30 percent in Honduras to 0.04 percent in Brazil. Future studies are needed to establish rates of transfusion transmission by viremic donations and clinical consequences in recipients.

BACKGROUND: Blood transfusion after traumatic injury can result in microchimerism (MC) of donor white cells (WBCs) in the recipient as late as 2 to 3 years postinjury, the longest prospective follow-up to date. The purpose of this study was to determine how long transfusion-associated MC lasts after traumatic injury. STUDY DESIGN AND METHODS: A group of US combat veterans who received transfusions who responded to a recruitment notice was retrospectively evaluated. Their blood was sampled, and MC was assessed by quantitative allele-specific polymerase chain reaction detection of differences at the HLA-DR locus or a panel of insertion-deletion polymorphism loci. Results of veterans were compared to those from an age- and gender-matched blood donor control group, from whom WBCs were retrieved from leukoreduction filters. RESULTS: Among 163 combat veterans who received transfusion and 150 control subjects who did not receive transfusions, 16 (9.8%) of the veterans and 1 (0.7%) control subject had evidence of MC (relative risk, 14.7; 95% confidence interval, 2.0-110). The veterans with MC included 3 who served in WWII (7% of subjects from that conflict), 5 in Korea (18%), and 6 in Vietnam (7%). CONCLUSIONS: Transfusion for combat-related injury can result in MC that lasts for 60 years, suggesting that it may involve permanent engraftment. MC is rare among male blood donors who did not receive transfusions, who are probably representative of individuals who have not had postnatal allogeneic exposures.

BACKGROUND: Blood donor parvovirus B19 DNA prevalence with sensitive nucleic acid test assays has recently been demonstrated to be higher than that found with assays designed to detect high viral titers in the plasma manufacturing sector. STUDY DESIGN AND METHODS: Stored plasma aliquots from 5020 donations collected between 2000 and 2003 at seven US blood centers were tested. Testing was performed with a real-time B19 DNA polymerase chain reaction (PCR; TaqMan, Applied Biosystems) assay with a 50 percent limit of detection (LOD) of 1.6 IU per mL (95% confidence interval [CI], 1.2-2.1 IU/mL) and a 95 percent LOD of 16.5 IU per mL (95% CI, 10.6-33.9 IU/mL). Confirmation and quantitation of B19 DNA was accomplished by retesting of two additional subaliquots. Confirmed-positive specimens were tested for the presence of anti-B19 immunoglobulin M (IgM) and IgG with FDA-licensed assays. RESULTS: B19 DNA prevalence was 0.88 percent (95% CI, 0.64%-1.2%). Among the 23 donations with B19 DNA titers of at least 20 IU per mL, the median DNA concentration was 105 IU per mL with an interquartile range of 42 to 481 IU per mL; the highest value was 1869 IU per mL. All B19 DNA–positive donations were positive for the presence of IgG and 10 (23%) were also positive for the presence of IgM; IgM seropositivity was associated with increasing DNA levels (p = 0.0013). CONCLUSION: Low-level B19 DNA was detected in nearly 1 percent of donations. The 23 percent of DNA-positive donations with both IgM and IgG B19 antibody most likely represent acute resolving infection, whereas those with IgG but no IgM are most consistent with a more chronic and possibly persistent phase of B19 infection.

BACKGROUND: The characterization of microchimerism (MC) by gene amplification has been limited by few allogeneic markers, ascertainment bias, and assay analytic performance. To address this, a panel of 12 MC assays based on insertion-deletion (InDel) polymorphisms had been optimized. STUDY DESIGN AND METHODS: The InDel assays were validated with comprehensive in vitro spiking studies at the stochastic limit of detection. Their ability was also determined to ascertain MC of unknown source genotype with both theoretical and actual donor-recipient pairs, and the assays were applied to a clinical population of 73 trauma patients who received transfusions where MC was previously characterized by HLA-based assays alone. RESULTS: In the stochastic spiking experiments, all assays were sensitive to a single copy of target DNA, and no false-positive amplification occurred among 1128 samples studied. Among 219 theoretical donor-recipient pairs, informative alleles existed for 99.5 percent with both InDel and HLA compared to 91.3 percent with HLA alone. In the clinical population, 33 cases of MC were detected (9 more cases than by HLA-DR alone) in the nonleukoreduced (non-LR) group and 8 cases (1 more case than by HLA-DR) in the LR group for the short-term follow-up. Among 27 long-term follow-up samples, 8 cases were detected overall (3 more cases than by HLADR alone). CONCLUSION: It is concluded that an InDel-based assay panel has excellent technical performance characteristics while also allowing for ascertainment of some MC cases not detectable with HLA alone. The tandem use of both the InDel and the HLA provides a powerful tool for the enhanced ascertainment of MC. himerism occurs in the settings of maternalfetal cell exchange, 1,2 hematopoietic stem cell

BACKGROUND: In some patients multiply transfused to treat severe trauma, white blood cells (WBCs) from a single blood donor can persist for years, constituting up to 5 percent of all circulating WBCs. The immunologic mechanisms responsible for this are not known but, if understood, might allow manipulation of the human immune system to induce microchimerism for a variety of therapeutic purposes. To better characterize these mechanisms, a murine transfusion model was developed with a panel of immunologic knockouts as transfusion recipients. By conducting a systematic series of transfusion experiments, the purpose was to determine which recipient immune cell population, when abrogated, could lead to prolonged survival of donor cells (microchimerism). STUDY DESIGN AND METHODS: Blood was transfused from normal donors to knockout recipients in syngeneic, allogeneic, and xenogeneic settings. Donor WBC survival was evaluated by quantitative polymerase chain reaction, and recipient lymphocyte subsets by fluorescence-activated cell sorting. RESULTS: In the syngeneic setting, donor WBCs persisted in C2ta, RAG-1, and TCR knockout recipients. Allogeneic donor WBCs persisted in RAG-2 and RAG-2/Common gamma knockout recipients. Xenogeneic donor WBCs required RAG-2/Common gamma and RAG-2/Pfp double knockouts to persist. CONCLUSION: It is concluded that donor-recipient major histocompatibility complex (MHC) concordance alone is not sufficient to achieve microchimerism. Further, the degree of recipient immune compromise necessary to achieve persistent microchimerism is directly proportional to the degree of donor-recipient MHC disparity.

BACKGROUND: The Viral Activation Transfusion Study was a prospective, randomized, double-blind comparison of transfusion with WBC-reduced versus non-WBC-reduced RBCs to HIV+ patients. The primary study characterized the effect of transfusion on HIV and CMV activation by monitoring viral load changes. The present study analyzed HBV, HCV, HTLV-I and -II, and human herpes virus-8 (HHV-8) viral load before and after transfusion to evaluate the further hypothesis that global immune stimulation following allogeneic RBC transfusion results in activation and increased viral proliferation of chronic viral infections other than HIV and CMV. STUDY DESIGN AND METHODS: Baseline samples from 519 to 523 subjects were screened for HBV, HCV, HTLV-I and -II, and HHV-8 infection, and baseline, serial weekly, and quarterly blood samples from infected subjects in the non-WBC-reduced arm were evaluated for changes from baseline in viral nucleic acid and ALT levels. RESULTS: Seroprevalence of HBV, HCV, HTLV-I and -II, and HHV-8 was 68, 25, 5, and 30 percent, respectively. No significant induction of HBV, HCV, HHV-8, or HTLV-I and -II viral replication following allogeneic transfusion of non-WBC-reduced blood was observed. A significant, albeit small, association was observed between transfusion and ALT. CONCLUSIONS: Based on these results and our previous finding that no adverse effect on HIV and CMV viral load and disease progression results from allogeneic transfusion, no evidence is found to support the selective use of WBC-reduced blood components for HIV-infected patients.

BACKGROUND: Fetal genetic material is detectable in the maternal circulation and has been used for noninvasive prenatal diagnosis. However, few data are available concerning its quantity and natural history during gestation. STUDY DESIGN AND METHODS: This study prospectively characterized the kinetics of cellular and cell-free fetal DNA in the circulation of 25 healthy women during and after uncomplicated pregnancy. Real-time kinetic PCR was used to quantitate human Y-chromosome sequences, and liquid oligomer hybridization with 32P-labeled probes was used to verify the identity of amplified products. RESULTS: In all male pregnancies, but no female pregnancies, low-level fetal Y-chromosome DNA was detected in both cellular and cell-free compartments beginning at 7 to 16 weeks but increasing steadily after 24 weeks and reaching a peak at parturition. The fetal DNA decreased rapidly after birth. CONCLUSION: Fetal genetic material can be detected throughout pregnancy, and its quantity is a function of gestational age and of whether the plasma or cellular compartment is examined. Both the absolute quantity of fetal DNA and its ratio to total DNA (maternal + fetal) are greater in the plasma than in the cellular compartment. Fetal DNA is cleared rapidly from both compartments after parturition, which suggests that turnover is dynamic. Because they provide prospective and quantitative data concerning fetal DNA levels, these observations and kinetic PCR methods may have implications for noninvasive prenatal diagnosis. Further studies will be needed to determine the immunologic implications of fetal-maternal DNA exchange and cellular microchimerism.

BACKGROUND Donor WBCs are responsible for numerous transfusion complications, but little is known concerning the natural history of their clearance following transfusion or of their function in the recipient's circulation. A murine transfusion model was developed to investigate the effects of blood component characteristics and histocompatibility on donor WBC survival kinetics and function. STUDY DESIGN AND METHODS To investigate the effects of storage and irradiation, fresh whole blood and blood stored for 1, 2, and 6 weeks at 4 degrees C, all from male C57b (H2K(b)) mice, was transfused to female Balb/c (H2K(d)) mice. To study the effect of histocompatibility, blood was also transfused from C57b mice to Balb/c, FVB, C3H, and SW (outbred) mice. To investigate the xenogeneic setting, blood from humans, rats, and rabbits was transfused to Balb/c mice. Samples were collected weekly after transfusion, and the donor WBCs were analyzed, targeting the Y-chromosome with quantitative PCR. To investigate donor WBC function, dinitrochlorobenzene (DNCB) sensitivity was induced in donor and recipient mice, and the transfusion recipients were observed for hypersensitivity to DNCB. RESULTS Donor WBCs had reduced in vivo survival equivalent to their period of storage ex vivo at 4 degrees C. Irradiation of donor blood produced no observable difference in donor WBC survival. Allogeneic male donor WBCs persisted (100

BACKGROUND Plasma and serum samples have been used to detect cell-free genomic DNA in serum or plasma in certain pathologic conditions such as systemic lupus erythematosus, pulmonary embolism, and malignancies, as well as in fetal cell chimerisms in maternal serum and/or plasma. In this study, baseline concentrations of cell-free DNA in serum and plasma samples were evaluated for the study of posttransfusion chimerism. STUDY DESIGN AND METHODS DNA was extracted from fresh or stored (4 degrees C for 1-6 days) normal donor serum or plasma samples (ACD; EDTA) by using reagents from an HIV assay kit. After incubation and washing of samples, purified DNA was amplified with HLA DQ-alpha primers (GH26 and 27) or human Y-chromosome primers (SA and SD) to quantitate the concentration of genomic DNA. RESULTS Fresh serum samples had concentrations of cell-free DNA that were about 20-fold higher than the concentrations in fresh plasma samples. The concentration of cell-free genomic DNA in serum samples increased daily, to a level more than 100 times baseline after clotted blood tubes were stored at 4 degrees C for 4 to 5 days. There was a small increase in cell-free plasma DNA in stored ACD whole blood samples. Male WBCs, spiked into fresh nonanticoagulated female blood, were lysed during the process of clotting, with male DNA liberated into the serum samples. CONCLUSION Most cell-free DNA in serum samples is generated during the process of clotting in the original collection tube. The concentration of cell-free genomic DNA in fresh plasma is probably the same as that in circulation. Consequently, while serum samples should not be used to monitor the concentration of cell-free DNA in a patient's circulation, serum collected from sample tubes containing clots (i.e., without anticoagulant), 3 to 5 days after the date of phlebotomy, could be useful as a source of DNA with which to screen for posttransfusion microchimerism.

BACKGROUND: Suitable detection methods are needed to support larger studies of microchimerism and the allogeneic exposures that may be etiologically related to it. STUDY DESIGN AND METHODS: A twotier PCR strategy for microchimerism detection was developed on the basis of the observation that assay sensitivity for the detection of microchimerism depends on the specificity with which primer pairs recognize sequences unique to the minor population. First, specimens are tested to determine the host HLA class II genotype by using a locus-specific PCR strategy with low sensitivity for microchimerism. Then, a sequence-specific PCR analysis having high sensitivity for detection of microchimerism is applied to detect and quantitate the minor population. Locus-specific, group-specific, and sequence-specific amplification strategies for the detection of distinct minor WBC populations prepared ex vivo were compared. In addition, 39 clinical samples from patients with known transfusion-associated microchimerism and 20 umbilical cord blood (CB) specimens containing maternal WBCs were studied. RESULTS: Locus-specific amplification detected 17 (94%) of 18 cases in which microchimerism was present at 10 percent, but only 1 of 51 cases with microchimerism ≤1 percent. Group-specific amplification detected all 63 cases with minor populations present at ≥0.10 percent, but only 16 of 21 cases at the 0.01 percent level. Sequence-specific amplification detected 100 percent of cases down to the 0.01 percent level. When applied to clinical samples, locus-specific amplification reliably identified the major population but proved insensitive to low-level minor populations. CONCLUSIONS: For the detection of microchimerism, assay sensitivity is a function of amplification strategy. These results suggest a simple approach to population screening for microchimerism: the background population of WBCs is typed by a locus-specific method, while minor population(s) can then be sought by using one or several sequence-specific amplifications.

BACKGROUND: Although inactivation of enveloped viruses transmitted by plasma derivatives has been successful, no methods for virus inactivation or removal have been established for platelet concentrates or red cell (RBC) components. Relatively little is known regarding the extent or significance of virus interactions with the cellular constituents in these components. STUDY DESIGN AND METHODS: Units of whole blood were collected from six HIV type 1 (HIV-1)-positive, asymptomatic individuals and separated into peripheral blood mononuclear cells (PBMNCs), cell-free plasma, white cell-reduced platelet concentrate, and white cell-reduced RBCs. DNA and RNA polymerase chain reaction and virus culture methods were used to study the compartmentalization of HIV-1 immediately after component preparation and after storage. RESULTS: As expected, HIV DNA and infectious virus were detected in fresh blood and in PBMNCs, and virion- associated RNA was detected in fresh plasma from all six donors. The levels of viral nucleic acids in these preparations remained relatively stable with 4 degrees C storage, whereas infectivity of PBMNCs was rapidly lost. Washed RBCs tested negative for HIV in all assays at all time points. Platelets retained high levels of HIV RNA (but not infectivity) after extensive washing, as well as after storage at 4 and 22 degrees C. High-level platelet-associated HIV-1 was also demonstrated in samples collected during early seroconversion. Periseroconversion and postseroconversion levels of platelet-associated HIV-1 correlated with the level of plasma viremia and with the rate of progression to AIDS. Cell-free virus from donor plasma and tissue culture fluid rapidly and firmly attached to platelets from noninfected donors. Infectivity of tissue culture virus bound to platelets was demonstrated in vitro. CONCLUSION: Significant levels of HIV-1 are associated with platelets during all stages of infection. Platelet- associated HIV could either mediate virus clearance or facilitate virus dissemination and expanded tropism. Finally, virus inactivation research must address virus associations with platelets.