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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 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.

Author(s): Sabino, EC; Ribeiro, AL; Lee, TH; Oliveira, CL; Carneiro-Proietti, AB; Antunes, AP; Menezes, MM; Ianni, BM; Salemi, VM; Nastari, L; Fernandes, F; Sachdev, V; Carrick, DM; Deng, X; Wright, D; Goncalez, TT; Murphy, EL; Custer, B; Busch, MP; Chagas Study Group of the NHLBI Retrovirus Epidemiology Donor Study-II, International Component | Abstract: BackgroundThe significance of detection of Trypanosoma cruzi DNA in blood of antibody-positive patients for risk of development of Chagas heart disease is not well established. The objective of this study was to compare detection of T. cruzi DNA with known clinical and laboratory markers of Chagas cardiomyopathy (CC) severity.MethodsThis is a case-control study nested within a retrospective cohort developed in Brazil to understand the natural history of Chagas disease. The study enrolled 499 T. cruzi seropositive blood donors (SP-BD) and 488 frequency matched seronegative control donors (SN-BD) who had donated between 1996 and 2002, and 101 patients with clinically diagnosed CC. In 2008-2010 all enrolled subjects underwent a health questionnaire, medical examination, electrocardiograms and echocardiograms and polymerase chain reaction (PCR) analyses. A blinded panel of three cardiologists adjudicated the outcome of CC. Trypanosoma cruzi kinetoplast minicircle sequences were amplified by real-time PCR using an assay with a sensitivity of one parasite per 20 mL of blood. All testing was performed on coded samples.ResultsRates of PCR detection of T. cruzi DNA were significantly (P = 0.003) higher in CC patients and SP-BD diagnosed with CC (79/105 [75.2 %]) compared with SP-BD without CC (143/279 [51.3%]). The presence of parasitaemia was significantly associated with known markers of disease progression such as QRS and QT interval duration, lower left ventricular ejection fraction, higher left ventricular index mass, and elevated troponin and NTpro-BNP levels.ConclusionTrypanosoma cruzi PCR positivity is associated with presence and severity of cardiomyopathy, suggesting a direct role of parasite persistence in disease pathogenesis.

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 and Objectives Transfusion is associated with a risk of infection andalloimmunization. Pathogen reduction using riboflavin and UV light (Mirasoltreatment) inactivates pathogens and leucocytes. With increasing adoption of thetechnology in clinical use, regulatory agencies have recommended the introduc-tion of quality control measures to monitor pathogen reduction efficacy. Wesought to develop a real-time PCR-based assay to document the impact of patho-gen reduction on the mitochondrial genome in blood components.Materials and Methods DNA was extracted from platelet and plasma componentsbefore and after treatment with riboflavin and UV light. Inhibition of PCR ampli-fication of mitochondrial DNA (mtDNA) in short- and long-amplicon targetregions, ranging from under 200 base pairs (bp) to over 1800 bp, was measuredin treated relative to untreated components.Results Pathogen reduction of platelets using riboflavin and UV light resulted ininhibition of PCR amplification of long-amplicon mtDNA targets, demonstratingapproximately 1 log reduction of amplification relative to untreated products.Amplification of short-amplicon mtDNA targets was not affected by treatment.Evaluation of 110 blinded platelet samples from the PREPAReS clinical trialresulted in prediction of treatment status with 100% accuracy. Pathogen reduc-tion of plasma components resulted in similar levels of PCR inhibition, whiletesting of 30 blinded plasma samples resulted in prediction of treatment statuswith 93% accuracy.Conclusion A differential sized amplicon real-time PCR assay of mitochondrialDNA effectively documents nucleic acid damage induced by Mirasol treatment ofplatelets. The use of the assay for plasma product pathogen reduction requiresfurther investigation.Keywords: mitochondrial DNA, pathogen reduction, plasma, platelets, quantita-tive PCR.

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.

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.

Frequent blood donors become iron deficient. HFE mutations are present in over 30% of donors. A 24-month study of 888 first time/reactivated donors and 1537 frequent donors measured haemoglobin and iron status to assess how HFE mutations impact the development of iron deficiency erythropoiesis. Donors with two HFE mutations had increased baseline haemoglobin and iron stores as did those with one mutation, albeit to a lesser extent. Over multiple donations haemoglobin and iron status of donors with HFE mutations paralleled those lacking mutations. The prevalence of HFE mutations was not increased in higher intensity donors. Thus, in general, HFE mutations do not temper donation-induced changes in haemoglobin and iron status. However, in Black donors there was an increase of H63D carriers at baseline, from 3·7% in first time/reactivated donors to 15·8% in frequent donors, suggesting that the relative effects of HFE mutations on iron absorption may vary between racial/ethnic groups. In secondary analyses, venous haemoglobin decreased more slowly in donors with ferritin ≥12μg/l; and haemoglobin recovery time was shorter in donors with reticulocyte haemoglobin (CHr) ≥32·6pg, indicating that these biochemical measures are better indicators of a donor's response to phlebotomy than their HFE mutation status.

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

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.

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.

Blood transfusion is a newly recognized cause of microchimerism, the stable persistence of a minor population of allogeneic cells. Relatively recent advances in polymerase chain reaction technology have spawned new information about the frequency and aetiology of transfusion-associated microchimerism (TA-MC). Although conceptually related to fetal-maternal microchimerism, TA-MC is a distinct and separate entity. Evidence of TA-MC has been strongest among patients with severe traumatic injuries who receive relatively fresh blood products shortly after an episode of massive haemorrhage. The presence of a focal deficit in the cellular immunologic repertoire prior to transfusion that happens to match a blood donor's human leucocyte antigen type also appears to be an important predisposing factor. TA-MC seems to be common (affecting approximately 10% of transfused injured patients), enduring (lasting years to decades) and pronounced (involving up to 5% of circulating leucocytes and multiple immunophenotypic lineages suggestive of haematopoietic engraftment). Further study of this topic may reveal important information regarding potential clinical consequences of TA-MC, as well as basic haematologic and immunologic processes.

The presence of a population of allogeneic cells underlies and partially defines both microchimerism (MC) and graft-versus-host disease (GVHD). Although the relationship between these two apparently distinct clinical entities is not well understood, it is plausible that progenitor cell dose, histocompatibility, recipient immune status and genetic profile all help determine whether allogeneic cells die, persist as a minor population or proliferate in a GVHD response. Although MC has been described, at low levels, in association with pregnancy and autoimmune disease, much higher levels of MC have recently been found in association with blood transfusion. This transfusion-associated microchimerism (TA-MC) has thus far been identified only in patients multiply transfused for severe traumatic injury, which is well documented to induce broad short-term suppression of both the innate and adaptive arms of the host immune system. In this review, we will summarize the limited available data concerning the clinical, immunologic and genetic settings in which TA-MC and GVHD develop and examine the factors that may influence their development. We will not address humoral alloimmunization as a result of transfusion here because its relationship to donor leucocyte persistence is unknown.

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.

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.

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.

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.

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.