Evaluation of seven PCR-based assays for the analysis of microchimerism

Objective: The presence of small numbers of cells of donor origin in the circulation of recipients of organ transplants (microchimerism) may correlate with immunologic tolerance. As part of our ongoing studies on microchimerism, we evaluated the utility of seven PCR-based assays for the detection of the less abundant DNA in paired mixtures (100 ng total DNA).

Design and methods: DNA samples were screened to identify pairs informative for one or more PCR assays. DNA mixtures from the informative pairs were then analyzed using at least one assay. The assays were based on the X-Y homologous region; a Y chromosome microsatellite locus; three autosomal microsatellite loci; the D1S80 minisatellite locus; and sequence specific oligonucleotide probe (SSOP) analysis of the HLA DRB1 locus.

Results: About 0.1% of male DNA against a background of female DNA was detectable using primers for the X-Y homologous region, but the sensitivity was increased to 0.0001% using nested primers for the Y chromosome microsatellite marker. Analysis of the minor DNA component was difficult with the three autosomal microsatellite assays because of the presence of shadow bands. Similar problems with the D1S80 assay were resolved using more stringent PCR conditions, and the sensitivity was 0.1%. Using the DRB1 locus, we were able to detect 1% DNA in the mixed samples.

Conclusions: These studies show that: (a) nested PCR for the Y chromosome is the most sensitive assay for the detection of microchimerism; (b) D1S80 is a useful marker for microchimerism; (c) additional optimization of analytical conditions is required if autosomal microsatellite markers and the SSOP assay are to be used for microchimerism analysis.     

Direct assessment of cytomegalovirus transfusion‐transmitted risks after universal leukoreduction

BACKGROUND: Cytomegalovirus (CMV) transfusion‐transmitted disease (TTD) remains a clinical concern. Universal leukoreduction has become one of the main strategies for the prevention of CMV‐TTD. Through prospective clinical follow‐up and testing of transfusion recipients (TRs), the risk for CMV‐TTD was studied. STUDY DESIGN AND METHODS: Transfused units were all leukoreduced and not prospectively screened for CMV. For TRs with negative baseline CMV testing results (CMV total antibody and DNA), all follow‐up TR samples were tested for CMV total antibody and DNA, and retained linked donor serum samples were tested for CMV total antibody. In cases when CMV‐TTD was suspected, donor sera were also tested for CMV DNA and selected TR samples were tested for CMV immunoglobulin M antibody. Evaluable transfusion was defined as a transfusion with TR sample(s) collected 14 to 180 days posttransfusion. RESULTS: Forty‐six TRs were negative for CMV at baseline. There were 1316 evaluable cellular blood transfusions to these TRs. Of 1316 evaluable cellular products, 460 (35%) were positive for CMV total antibody tested using linked donor samples. Three cases of probable CMV‐TTD were found; however, there was no definitive proof from donor follow‐up that they were transfusion associated. CONCLUSION: Among all 46 baseline seronegative recipients and 1316 evaluable transfusions, the calculated overall CMV‐TTD risk was up to 6.5% (95% confidence interval [CI], 1.0%‐18.0%) in terms of TRs and up to 0.23% (95% CI, 0.06%‐0.62%) in terms of non–CMV‐screened leukoreduced cellular products. In summary, after universal leukoreduction, CMV‐TTD, while uncommon, may still occur.     

Presence of donor- and recipient-derived DNA in cell-free urine samples of renal transplantation recipients: urinary DNA chimerism.

BACKGROUND: Previous studies have indicated that microchimerism is present in body tissues, peripheral blood, and plasma of recipients after organ transplantation. We hypothesize that donor-derived DNA may also be present in cell-free urine of renal transplant recipients and that the concentrations of urine DNA may be correlated with graft rejection. METHODS: Thirty-one female patients who had renal transplantation were enrolled in the study. In women with male organ donors, the SRY gene on the Y chromosome was used as a marker for donor-derived DNA. Real-time quantitative PCR for the SRY and beta-globin genes was carried out on cell-free urinary DNA from these patients. Serial urine samples from a female renal transplant recipient undergoing an acute rejection episode were also collected and analyzed with the beta-globin quantitative PCR system. RESULTS: SRY sequences were detected in the urine of 14 of 17 female patients with male organ donors. None of the 14 patients with female organ donors had detectable SRY sequences in urinary DNA. The median fractional concentration of donor-derived DNA was 8.7% (interquartile range, 1.9-26.4%). During the acute rejection episode, urinary concentrations of the beta-globin gene were markedly increased, with the concentrations returning rapidly to normal following antirejection treatment. CONCLUSIONS: Our results demonstrate that urinary DNA chimerism is present following renal transplantation. The measurement of urinary DNA using quantitative PCR may be useful for the diagnosis and monitoring of graft rejection.     

Microchimerism of maternal origin persists into adult life

Recent studies indicate that fetal cells persist in maternal blood for decades after pregnancy. Maternal cells are known to engraft and persist in infants with immunodeficiency, but whether maternal cells persist long-term in immunocompetent offspring has not specifically been investigated. We developed sensitive human leukocyte antigen-specific (HLA-specific) PCR assays and targeted nonshared maternal HLA genes to test for persistent maternal microchimerism in subjects with scleroderma and in healthy normal subjects. Nonshared maternal-specific DNA was found in 6 of 9 scleroderma patients. In situ hybridization with double labeling for X and Y chromosome-specific sequences revealed female cells in peripheral blood samples from 2 male scleroderma patients. HLA-specific PCR also frequently revealed persistent maternal microchimerism in healthy control subjects. The mean age of all subjects with maternal microchimerism was 28 years (range: 9-49 years). With few exceptions, mothers of subjects with persistent maternal microchimerism were HLA incompatible with subjects for class I and class II alleles. These results clearly indicate that HLA-disparate maternal cells can persist in immunocompetent offspring well into adult life. The biological significance of maternal microchimerism and whether it might contribute to autoimmune disease requires further investigation.     

Immune modulation and microchimerism after unmodified versus leukoreduced allogeneic red blood cell transfusion in cancer patients: results of a randomized study

BACKGROUND: Transfusion of red blood cells (RBCs) has been associated with immunomodulatory effects. Persistence of donor cells in the recipient may be contributive. STUDY DESIGN AND METHODS: A randomized single-center trial was conducted to compare microchimerism and immune responses in 35 patients undergoing cancer surgery and transfused perioperatively with either unmodified RBCs (UN-RBCs, n = 18) or leukoreduced RBCs (LR-RBCs, n = 17). Biologic parameters included microchimerism assessment peripheral blood mononuclear cell (PBMNC) phenotyping, cytokine production by stimulated PBMNCs, FoxP3 gene expression, and T-cell repertoire (TCR) analysis. RESULTS: Microchimerism was documented in 8 of 18 patients after UN-RBC transfusion while absent after LR-RBC transfusion (0/17; p = 0.001). After UN-RBC transfusion, microchimerism was associated with increased interleukin (IL)-10 production (p = 0.02), reduced TCR alteration (p = 0.04), and reduced CD56+ cell counts (p = 0.02) when compared to recipients without evidence for microchimerism. FoxP3 gene expression did not differ significantly between both treatment groups nor with the presence or absence of microchimerism in the UN-RBC group. Finally, after an initial early decrease after surgery and transfusion, IL-12 production increased and more significantly so after UN-RBC transfusion versus LR-RBC transfusion (p = 0.05). CONCLUSION: UN-RBC-induced microchimerism is associated with specific immunomodulatory effects in cancer patients who received transfusions during surgery.     

High-level long-term white blood cell microchimerism after transfusion of leukoreduced blood components to patients resuscitated after severe traumatic injury

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.     

Transmission of cytomegalovirus (CMV) infection by leukoreduced blood products not tested for CMV antibodies: a single-center prospective study in high-risk patients undergoing allogeneic hematopoietic stem cell transplantation (CME)

BACKGROUND: Measures to prevent transfusion‐transmitted cytomegalovirus (TT‐CMV) infection after hematopoietic stem cell transplantation (HSCT) include transfusion of CMV antibody–negative blood units and/or transfusion of leukoreduced cellular blood products. We assessed the incidence of TT‐CMV in CMV‐seronegative patients receiving CMV‐seronegative HSC transplants, who were transfused with leukoreduced cellular blood products not tested for anti‐CMV. STUDY DESIGN AND METHODS: In a prospective observational study between 1999 and 2009, all HSCT patients received leukoreduced cellular blood products not tested for anti‐CMV. Patients were screened for CMV serostatus and CMV‐negative recipients of CMV‐negative transplants were systematically monitored for TT‐CMV clinically and by CMV nucleic acid testing. Anti‐CMV antibodies (immunoglobulin [Ig]G and IgM) were assessed after three time intervals (Interval 1, study inclusion to Day +30 after HSCT; Interval 2, Day +30‐Day +100; Interval 3, after Day +100). RESULTS: Among 142 patients treated with allogeneic HSCT, 23 CMV‐negative donor‐patient pairs were identified. These 23 patients received 1847 blood products from 3180 donors. All patients remained negative for CMV DNA and none developed CMV‐associated clinical complications. This results in a risk for TT‐CMV per donor exposure of 0% (95% confidence interval, 0.0%‐0.12%). However, 17 of 23 patients seroconverted for anti‐CMV IgG, but none for anti‐CMV IgM. CMV IgG seroconverters received significantly more transfusions per week than nonconverters. CONCLUSION: The risk of TT‐CMV is low in high‐risk CMV^neg/neg HSCT patients transfused with leukoreduced blood products not tested for anti‐CMV. The cause of anti‐CMV IgG seroconversion is most likely passive antibody transmission by blood products.     

配偶供肾移植后微嵌合体发生与急性排斥反应

背景:微嵌合作为移植物与受者之间的双相细胞移动的标志,在移植免疫耐受中的作用日益受到重视。目的:探讨夫妻生活与嵌合体的发生,与肾移植后急性排斥反应及其他相关性的研究。方法:将接受肾脏移植的女性受者(有过生育史的女性除外)分为丈夫活体供肾组、无关男性尸体供肾组,并设立接受妻子活体供肾的对照组。STR方法检测女性受者体内男性供者来源的Y染色体反映微嵌合体的存在,与急性排斥反应发生的关系,并比较配偶间供肾效果的差异。结果与结论:尽管配偶间供肾移植存在供者年龄偏大以及人类白细胞抗原错配率较高的因素,但与接受无关男性尸肾移植的女性受者相比,接受丈夫活体供肾移植的女性更易检测出微嵌合体,而且肾移植后恢复情况好,急性排斥反应发生率低。而与接受妻子供肾的丈夫相比,接受丈夫供肾的妻子肾移植效果好。说明夫妻间长期相处导致女性接受丈夫体液的机会多,由此产生免疫耐受对于肾移植后人/肾的相容性好,急性排斥反应小。     

Lung transplantation complicated by graft-versus-host disease and confounded by incidental transfusion-associated macrochimerism

BACKGROUND: Passenger lymphocyte–mediated graft‐versus‐host disease (GVHD) in solid organ transplantation (SOT‐GVHD) is considered a rare complication, particularly among recipients of lung allografts. The risk of transfusion‐associated GVHD (TA‐GVHD) in solid organ transplants is also considered rare. The suspicion of either may be heralded by signs and symptoms of GVHD in the company of a population of passenger lymphocytes in excess of 1 percent (microchimerism). This case report illustrates the challenge of a patient who presented with macrochimerism both from the lung transplant allograft and from transfusions. STUDY DESIGN AND METHODS: Chimerism assessments of the pre‐ and posttransplant donor lung, and the recipient's aplastic marrow, were made using DNA‐based polymerase chain reaction testing. RESULTS: Macrochimerism was observed in both the posttransplant aplastic host marrow and the engrafted donor lung, with the former predominantly consisting of lung donor lymphocytes and the latter a mixture of lung and presumably transfusion source donor lymphocytes. The pretransplant donor lung exhibited no GVHD‐like pathology. CONCLUSION: This case demonstrates SOT‐GVHD, with the unusual feature of concomitant macrochimerism from transfusions. SOT‐GVHD likely predisposed this patient to the observed transfusion‐associated macrochimerism. However, the dissociation between transfusion‐attributable macrochimerism and attributable pathology is intriguing. Furthermore, the risk spectrum of transfusion‐associated macrochimerism and TA‐GVHD in solid organ transplant recipients with and without the complication of SOT‐GVHD is unknown and warrants further study.     

Minimum conditions of major histocompatibility complex compatibility and recipient immune compromise required to establish donor white blood cell persistence in a murine transfusion model

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.     

Trypanosoma cruzi infection in North America and Spain: evidence in support of transfusion transmission (CME)

BACKGROUND: The United States, Canada, and Spain perform selective testing of blood donors for Trypanosoma cruzi infection (Chagas disease) to prevent transfusion transmission. The donor, product, and patient characteristics associated with transfusion‐transmitted infections are reviewed and the infectivity of components from donors with serologic evidence of infection is estimated. STUDY DESIGN AND METHODS: A systematic review of transfusion‐transmitted T. cruzi cases and recipient tracing undertaken in North America and Spain is described. Cases were assessed for the imputability of the evidence for transfusion transmission. RESULTS: T. cruzi infection in 20 transfusion recipients was linked to 18 serologically confirmed donors between 1987 and 2011, including 11 identified only by recipient tracing. Cases were geographically widely distributed and were not associated with incident or autochthonous infections. Index clinical cases were described only in immunocompromised patients. All definite transmissions (n = 11) implicated apheresis or whole blood–derived platelets (PLTs), including leukoreduced and irradiated products. There is no evidence of transmission by red blood cells (RBCs) or frozen products, while transmission by whole blood transfusion remains a possibility. Recipient tracing reveals low component infectivity from serologically confirmed, infected donors of 1.7% (95% confidence interval [CI], 0.7%‐3.5%) overall: 13.3% (95% CI, 5.6%‐25.7%) for PLTs, 0.0% (95% CI, 0.0%‐1.5%) for RBCs, and 0.0% (95% CI, 0%‐3.7%) for plasma and cryoprecipitate. CONCLUSIONS: T. cruzi is transmitted by PLT components from some donors with serologic evidence of infection. Evidence of transmission before the implementation of widespread testing in the countries studied is sparse, and selective testing of only PLT and fresh whole blood donations should be considered.     

Microchimerism decades after transfusion among combat-injured US veterans from the Vietnam, Korean, and World War II conflicts

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.     

Implementation of a pediatric trauma massive transfusion protocol: one institution's experience

BACKGROUND: Massive transfusion protocols (MTPs) with fixed ratios of blood products may improve outcomes in coagulopathic adult trauma patients. However, there is a paucity of data on transfusion support protocols for pediatric trauma patients, whose mechanisms of injury may differ from those seen in adults. We hypothesized that an MTP would improve outcomes in children, through a balanced blood product resuscitation. STUDY DESIGN AND METHODS: A pediatric trauma MTP, with a fixed ratio of red blood cells (RBCs) : fresh‐frozen plasma (FFP) : platelets : cryoprecipitate in quantities based on the patient's weight, was initiated at a pediatric hospital. Data on clinical status, resuscitation volumes, and hospital course were collected and compared to data from pre‐MTP trauma patients requiring transfusion. RESULTS: Fifty‐three patients were enrolled over a 15‐month period and compared to 49 pre‐MTP patients. Seventy‐two percent of MTP patients had at least one coagulation value outside of the normal range upon emergency department (ED) arrival, and the median time to FFP transfusion decreased fourfold after MTP implementation (p < 0.0001). A total of 49% of MTP patients received greater than 70 mL/kg blood products, and the 24‐hour median FFP : RBC transfusion ratio was twofold higher in these patients than the pre‐MTP cohort (median, 1:1.8 vs. 1:3.6; p = 0.002). No improvement in mortality was observed after MTP implementation, taking into consideration injury severity, prothrombin time, and partial thromboplastin time. CONCLUSIONS: A pediatric trauma MTP is feasible and allows for rapid provision of balanced blood products for transfusion to coagulopathic children. Larger studies are warranted to determine whether such protocols will improve outcomes for pediatric trauma patients.     

Transfusion‐transmitted anaplasmosis from leukoreduced red blood cells

BACKGROUND: Human granulocytic anaplasmosis (HGA) is a tick‐borne rickettsial infectious disease. To date four cases of transfusion‐transmitted anaplasmosis (TTA) have been described in the literature, and only one from leukoreduced red blood cells (RBCs). CASE REPORT: A 64‐year‐old patient with acute gastrointestinal blood loss was admitted to the hospital and received 5 units of prestorage leukoreduced RBCs. He was stabilized and discharged. He developed headache, fever, and chills 2 days after discharge and was readmitted. On Day 5 of his second admission polymorphonuclear leukocytes containing morulae consistent with HGA were reported in the peripheral smear. RESULTS: Samples from the recipient tested positive by polymerase chain reaction (PCR) for Anaplasma phagocytophilum, the causative agent of HGA and a segment from one of the five donors tested positive by both serology and PCR. CONCLUSION: Leukoreduction theoretically reduces the risk of TTA but does not interdict all infections. TTA requires consideration in recipients of RBC transfusion with unexplained fever.     

Institutional variation in hemotherapy for solid organ transplantation

BACKGROUND: Solid organ allograft recipients may require large amounts of blood components. The modification of components to make them safer for iatrogenically immunosuppressed transplant patients increases workload demands on blood banks and transfusion services. STUDY DESIGN AND METHODS: Institutions within the United States and Canada providing hemotherapy as support for transplant recipients were surveyed for their transfusion practices. RESULTS: Responses from 25 institutions provide the data for this report. In 1991, the mean intraoperative red cell requirements ranged from <1 unit for renal allograft recipients to 17.3 units for liver transplant recipients. The latter group also required the greatest amounts of platelets, fresh-frozen plasma, and cryoprecipitate. More than 75 percent of responding institutions provided either cytomegalovirus-seronegative or white cell-reduced cellular components to pediatric recipients of liver allografts and to both adult and pediatric recipients of heart, lung, and heart-lung allografts. The use of irradiated cellular blood components, although uncommon, was greatest in heart transplant recipients. The use of pretransplantation transfusions for immunomodulation was generally limited to patients awaiting a living-donor renal transplant. CONCLUSION: Transfusion practices varied among the institutions, but the majority provide cytomegalovirus-safe cellular blood components to heart and lung allograft recipients and to pediatric transplant patients. Gamma-radiated cellular components are not routinely provided to patients undergoing solid organ transplantation. Liver allograft recipients require the greatest amount of hemotherapeutic support.     

Results of lookback for Chagas disease since the inception of donor screening at New York Blood Center

BACKGROUND: Chagas disease is a parasitic infection by Trypanosoma cruzi, typically transmitted via infected triatomine bug fecal contamination of bite sites. Other routes of infection include congenital, oral, organ transplantation, and blood product transmission. STUDY DESIGN AND METHODS: From 2007 until 2011, New York Blood Center screened donations for the presence of T. cruzi antibodies using a Food and Drug Administration–approved test. Confirmatory testing was performed and recipients of units donated by confirmed‐positive donors were investigated via lookback. RESULTS: A total of 204 donors were T. cruzi antibody positive representing 0.019% of all donors during this time period (1,066,516 unique donors screened). Of the enzyme‐linked immunosorbent assay–reactive donors, 77 were confirmed positive by radioimmunoprecipitation assay (0.007%). At least 154 units from 29 of the confirmed‐positive donors had been transfused to 141 recipients. At the time of lookback, 48 of the 141 recipients were alive and seven underwent T. cruzi screening. Two recipients were found to be immunofluorescence assay (IFA) positive. Both IFA‐positive recipients received a leukoreduced apheresis platelet unit (two separate donations) from the same confirmed positive donor, a 72‐year‐old immigrant from Argentina. CONCLUSIONS: Lookback analysis was able to identify the first two cases of probable transfusion‐transmitted T. cruzi infection since implementation of the national screening program, which increases the total number of reported cases in the United States to 8.     

Cost-effectiveness of additional blood screening tests in the Netherlands

BACKGROUND: During the past decade, blood screening tests such as triplex nucleic acid amplification testing (NAT) and human T‐cell lymphotropic virus type I or I (HTLV‐I/II) antibody testing were added to existing serologic testing for hepatitis B virus (HBV), human immunodeficiency virus (HIV), and hepatitis C virus (HCV). In some low‐prevalence regions these additional tests yielded disputable benefits that can be valuated by cost‐effectiveness analyses (CEAs). CEAs are used to support decision making on implementation of medical technology. We present CEAs of selected additional screening tests that are not uniformly implemented in the EU. STUDY DESIGN AND METHODS: Cost‐effectiveness was analyzed of: 1) HBV, HCV, and HIV triplex NAT in addition to serologic testing; 2) HTLV‐I/II antibody test for all donors, for first‐time donors only, and for pediatric recipients only; and 3) hepatitis A virus (HAV) for all donations. Disease progression of the studied viral infections was described in five Markov models. RESULTS: In the Netherlands, the incremental cost‐effectiveness ratio (ICER) of triplex NAT is €5.20 million per quality‐adjusted life‐year (QALY) for testing minipools of six donation samples and €4.65 million/QALY for individual donation testing. The ICER for anti‐HTLV‐I/II is €45.2 million/QALY if testing all donations, €2.23 million/QALY if testing new donors only, and €27.0 million/QALY if testing blood products for pediatric patients only. The ICER of HAV NAT is €18.6 million/QALY. CONCLUSION: The resulting ICERs are very high, especially when compared to other health care interventions. Nevertheless, these screening tests are implemented in the Netherlands and elsewhere. Policy makers should reflect more explicit on the acceptability of costs and effects whenever additional blood screening tests are implemented.     

IMMUNOHEMATOLOGY: Storage of murine red blood cells enhances alloantibody responses to an erythroid‐specific model antigen

BACKGROUND: Red blood cell (RBC) alloimmunization can be a serious complication of blood transfusion, but factors influencing the development of alloantibodies are only partially understood. Within FDA‐approved time limits, RBCs are generally transfused without regard to length of storage. However, recent studies have raised concerns that RBCs stored for more than 14 days have altered biologic properties that may affect medical outcomes. To test the hypothesis that storage time alters RBC immunogenicity, we utilized a murine model of RBC storage and alloimmunization. STUDY DESIGN AND METHODS: Blood from transgenic HOD donor mice, which express a model antigen (hen egg lysozyme [HEL]) specifically on RBCs, was filter leukoreduced and stored for 14 days under conditions similar to those used for human RBCs. Fresh or 14‐day‐stored RBCs were transfused into wild‐type recipients. The stability of the HOD antigen and posttransfusion RBC survival were analyzed by flow cytometry. RBC alloimmunization was monitored by measuring circulating anti‐HEL immunoglobulin levels. RESULTS: Transfusion of 14‐day‐stored, leukoreduced HOD RBCs resulted in 10‐ to 100‐fold higher levels of anti‐HEL alloantibodies as detected by enzyme‐linked immunosorbent assay than transfusion of freshly collected, leukoreduced RBCs. RBC expression of the HOD antigen was stable during storage. CONCLUSIONS: These findings demonstrate that HOD murine RBCs become more immunogenic with storage and generate the rationale for clinical trials to test if the same phenomenon is observed in humans. Length of storage of RBCs may represent a previously unappreciated variable in whether or not a transfusion recipient becomes alloimmunized.     

Transfusion-related immunomodulation by platelets is dependent on their expression of MHC Class I molecules and is independent of white cells

BACKGROUND: Transfusion‐related immunomodulation (TRIM) has been correlated with the presence of white cells (WBCs) in blood transfusions, but the role of components such as platelets (PLTs) in mediating TRIM has not been extensively examined. We designed a murine PLT transfusion model to study whether leukoreduced PLTs mediate TRIM effects. STUDY DESIGN AND METHODS: CBA recipient mice were administered four weekly transfusions of either fresh (4 hr) or aged (24 and 72 hr) donor leukoreduced PLTs from allogeneic BALB/c mice and then transplanted with skin grafts from donor‐matched mice. TRIM was measured by comparing the times to graft rejection and these were correlated with immunoglobulin G (IgG) antibody development measured by flow cytometry. RESULTS: Compared with nontransfused control recipients, four transfusions of fresh, extremely leukoreduced (<0.05 WBCs/mL), allogeneic PLTs significantly (p < 0.002) reduced the recipient's ability to reject donor‐matched skin grafts (survival >49 days compared with <14 days in nontransfused controls) despite the presence of high‐titered serum IgG donor antibodies. In contrast, however, aged PLTs or fresh PLTs devoid of MHC Class I molecules were unable to affect skin graft survival nor stimulate antibody production. The PLT age‐related inability to induce TRIM was shown to be due to loss of PLT‐associated MHC Class I molecules; soluble supernatant MHC molecules that were transfused were unable to induce TRIM. CONCLUSION: These results suggest that fresh PLTs can induce TRIM independently of WBCs due to their MHC antigen expression whereas aging results in loss of MHC and ability to mediate TRIM. The findings support the concept that either active MHC removal from fresh PLTs or passive removal by, for example, storage, may reduce any deleterious effects of TRIM in transfusion recipients.