Phenotyping autologous red cells within 1 day after allogeneic blood transfusion by using immunomagnetic isolation of reticulocytes

BACKGROUND: When a transfused patient develops multiple or weak blood group antibodies, posttransfusion phenotyping is useful in antibody identification. To perform a correct phenotyping after transfusion, isolation of autologous red cells is necessary. However, mature autologous red cells are impossible to separate from their donor counterparts. Since the proportion of autologous reticulocytes compared to donor reticulocytes increases rapidly after transfusion, selective isolation of reticulocytes provides autologous cells for antigen typing. STUDY DESIGN AND METHODS: Extensive phenotyping was performed on red cells from 10 surgical patients before transfusion and on red cells and reticulocytes after the transfusion of 5 or more red cell units. Reticulocytes were isolated by using an antibody against the human transferrin receptor coupled to magnetic beads. RESULTS: The data showed nearly full agreement between pretransfusion phenotyping of red cells and posttransfusion typing of reticulocytes. Correct phenotyping of transferred patients could be obtained 8 to 10 hours after transfusion using isolated reticulocytes. CONCLUSION: This method is helpful in selecting compatible blood when patients have developed antibodies and have an urgent need for further transfusions.     

A flow cytometric method for phenotyping recipient red cells following transfusion

BACKGROUND: Reticulocyte phenotyping is used for transfused patients, who have red cell antibodies, to match blood for subsequent transfusion. Current methods are labor-intensive and require a significant amount of sample. STUDY DESIGN AND METHODS: A simple dual- color flow cytometry method developed for antigen typing of reticulocytes in mixed red cell populations is reported. Antigens were labeled by an indirect immunofluorescence technique using undiluted reagent sera as the primary label, biotinylated goat anti-human IgG as the secondary label, and avidin-phycoerythrin as the fluorescent stain. Reticulocytes were labeled with a thiazole orange fluorescent stain. Reticulocyte identification and antigen typing were performed on 319 samples to establish the validity of the procedure. Mixed red cells were prepared in all possible c antigen combinations to simulate transfusion concentrations of 25, 50, and 75 percent. RESULTS: The anti- c flow cytometry profiles readily distinguished between antigen- positive and antigen-negative populations and allowed the detection of reticulocytes at all simulated transfusion concentrations. Similar results were obtained in experiments using C, K, s, Fya, Fyb, Jka, or Jkb sera against equal volumes of antigen-positive and -negative cells. Anti-S gave inconsistent results. The in vitro results were confirmed in 19 transfused patients who had received red cells antigenically different from their own as well as cells from 1 chimera blood donor. CONCLUSION: This method provides a simpler, safer, less labor- intensive, and less subjective technique requiring far less sample volume than current methods for antigen typing of reticulocytes in mixed red cell samples from recently transfused patients.     

Flow cytometric analysis of reticulocytes using an RNA-binding fluorochrome.

The reticulocyte count is an important parameter in the diagnosis of anaemia. The commonly used microscopic counting technique is, however, laborious and hampered by poor precision and low sensitivity. An alternative method has recently been developed, which is based on flow cytometric identification and quantification of reticulocytes after staining with an RNA-binding fluorochrome, thiazol orange. The two methods were compared both in a sample of healthy individuals and in patient specimens. The correlation between the methods was good, but the flow cytometric analysis consistently gave values approximately 1.5 times higher (reticulocytes in per cent of erythrocytes) than the microscopic technique. The coefficient of variation was significantly lower for the flow cytometric technique. Reference values for the reticulocyte count were determined and the effects of storage of specimens were evaluated. It is concluded that flow cytometric analysis of reticulocytes is more sensitive and has a better precision that the standard microscopic procedure.     

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.     

Quantitation of passively acquired human immunodeficiency virus (HIV) antibodies in AIDS patients transfused with a plasma that is rich in HIV antibodies

BACKGROUND: Passive immunotherapy in human immunodeficiency virus (HIV) infection is based on transfusions of plasma that is rich in HIV antibodies. STUDY DESIGN AND METHODS: To verify whether the clearance of transfused antibodies will maintain an elevated titer of specific antibodies between biweekly transfusions of plasma, the titers of anti- HIV-1 in plasma and in transfusion recipients were measured. Samples from 12 recipients were analyzed by automated scanning of Western blot, before transfusion and at Days 2, 7, and 14 after transfusion. RESULTS: The p24 antibody became detectable or higher than the baseline after transfusion and remained detectable until the second transfusion. Anti- p24 titers were variable and dependent on the antibody titer of the transfused plasma and the baseline p24 antigen titer. CONCLUSION: Biweekly transfusion of plasma with a high anti-HIV titer maintains a high anti-p24 titer between transfusions in AIDS patients treated with passive immunotherapy.     

Life-threatening adverse reaction followed by thrombocytopenia after passive transfusion of fresh frozen plasma containing anti-CD36 (Nak) isoantibody

BACKGROUND: Anti-CD36 isoantibody in blood recipients is reported to cause refractoriness to platelet (PLT) transfusions and posttransfusion purpura-like syndrome. There are few reports, however, about the effects of passively transfused blood products containing this isoantibody on recipients. CASE REPORT: A 67-year-old Japanese woman underwent brain surgery. On the 6th postoperative day, the patient experienced tightness of the chest and nausea after receiving a transfusion of fresh frozen plasma (FFP). When she manifested hypotension, the transfusion was discontinued. No cutaneous manifestation was observed. The patient's condition gradually improved soon after the administration of steroids. RESULTS: Her pretransfusion PLT count was 17.1 x 10(4) per microL. It decreased to 1.9 x 10(4) per microL 12 hours after transfusion and recovered to 15.4 x 10(4) per microL 8 days after transfusion. The donor of the FFP had a Type I CD36 deficiency. Flow cytometric analysis identified anti-CD36 isoantibody in the FFP. The cross-match between the patient's PLTs and the FFP was positive. The FFP induced the aggregation of PLTs derived from healthy adults. CONCLUSION: This is the first reported case of life-threatening adverse effects and thrombocytopenia caused by passively transfused anti-CD36 isoantibody. The possibility of passive infusion of this antibody should be considered in the evaluation of life-threatening transfusion reactions followed by thrombocytopenia.     

Rapid clearance of transfused murine red blood cells is associated with recipient cytokine storm and enhanced alloimmunogenicity

BACKGROUND: Fourteen‐day stored red blood cells (RBCs) containing an RBC‐specific transgenic antigen (HOD) induce a recipient proinflammatory cytokine storm and are significantly more immunogenic compared to fresh RBCs. Given that recipient mice clear transfused stored RBCs more rapidly than fresh RBCs, we hypothesized that rapid RBC clearance was associated with adverse transfusion outcomes. STUDY DESIGN AND METHODS: HOD RBCs were treated by two distinct methods known to lead to rapid posttransfusion RBC clearance: phenylhydrazine or heat. HOD antigen expression was analyzed on the treated cells before transfusion, and RBC recovery, recipient cytokine response, and recipient anti‐HOD alloimmunization response were measured after transfusion. RESULTS: Phenylhydrazine and heat treatment each led to near complete RBC clearance in recipients by 24 hours posttransfusion, without significantly altering HOD antigen expression on the transfused RBCs. Recipients of phenylhydrazine‐ or heat‐treated RBCs had elevated circulating levels of keratinocyte‐derived chemokine/CXCL‐1, monocyte chemoattractant protein‐1, and interleukin‐6 after transfusion. Furthermore, phenylhydrazine‐ or heat‐treated RBCs were significantly more immunogenic than control RBCs, with a mean 25.1‐ and 10.3‐fold enhancement, respectively, of anti‐HOD alloimmunization magnitude by flow cytometric crossmatch. CONCLUSIONS: Three separate insults to RBCs (storage, phenylhydrazine, or heat treatment) result in rapid posttransfusion clearance, with a recipient proinflammatory cytokine storm and enhanced alloimmunogenicity. These data are consistent with the hypothesis that rapid clearance of RBCs is causally involved in these outcomes and suggest that human donor RBCs with favorable posttransfusion clearance profiles may be less immunogenic.     

Hemolysis of “stress” reticulocytes: a source of erythropoietic bilirubin formation

The formation of bilirubin-(14)C was measured in rats given transfusions of red blood cells containing (14)C-labeled hemoglobin heme. Per cent conversion of hemoglobin-(14)C to bilirubin was 4 times greater with transfusion of "stress" reticulocytes from rats responding to hemorrhage than with normal reticulocytes from unstimulated donors. When the increased number of labeled reticulocytes produced by hemorrhaged donors was also considered, the total magnitude of labeled bilirubin formation was almost 20 times higher with stress as compared to normal reticulocytes. The findings were not influenced by splenectomy of either donor or recipient rats, iron loading of donors, or bleeding of recipients. However, bilirubin-(14)C formation fell off progressively as studies were performed at longer intervals after erythroid stimulation.Total bilirubin-(14)C formation in rats transfused with stress reticulocytes was compared to the production of early-labeled bilirubin from all potential sources in intact rats bled according to the same schedule used in the transfusion experiments. It is estimated that degradation of hemoglobin from sress reticulocytes accounts for virtually the entire rise in erythropoietic bilirubin formation from 24 to 96 hr after glycine-2-(14)C administration, but that additional sources make a major contribution before that time. These findings are consistent with the concept that destruction of immature erythroid cells in the peripheral blood, and probably in the bone marrow, accompanies the physiologic response to erythroid stimulation.     

Transfusion experience with platelet concentrates stored for 24 to 72 hours at 22 degrees C. Importance of storage time

We studied the transfusion response from random donor platelet concentrates in 15 stable multitransfused, thrombocytopenic patients by comparing the platelet counts measured before and 20 hours after transfusion. The observed platelet increments were corrected (corrected increment, C.I.) for the number of units of platelet concentrate transfused and the patient's body surface area in square meters (platelets/microliter per unit/m2). Using platelet concentrates stored for less than 24 hours, the patients achieved a median C.I. of 9500 (range: 5000–18,000). When platelet concentrates stored for 24 to 48 hours or 48 to 72 hours were given, the median C.I. markedly decreased to 1000 (range: 0–4800) and 0 (range: 0–5100), respectively (p less than 0.001). These differences could not be explained by further recipient alloimmunization. Transfusion with platelet concentrates less than 24 hours old on a second occasion, bracketing the transfusions of older platelet concentrates, resulted in a median C.I. of 7200 (range: 5400–14,500). Similar results were obtained in three patients when HLA- identical sibling platelet concentrates were employed. In vitro tests, including pH, morphology, and aggregation, demonstrated no statistically significant differences among the platelet concentrates stored for less than 24 hours, 24 to 48 hours, and 48 to 72 hours. These studies suggest that, although platelet concentrates can be stored for 72 hours without loss of in vitro function, the in vivo recovery is significantly diminished after 24 hours of storage, and preferably patients should not be transfused prophylactically with platelet concentrates greater than 24 hours old.     

The Transfusion of Leukocytes from Donors with Chronic Myelocytic Leukemia to Patients with Leukopenia*

Leukocytes were collected from donors with chronic myelocytic leukemia by plasmapheresis and transfused into severely leukopenic recipients. The median transfusion of 7 × 10¹⁰ granulocytes (range.15 to 35 × 10¹⁰) resulted in a median increase in circulating granulocytes of 1,000 per cu. mm. (range 0 to 19,000), one hour after injection. The posttransfusion increment was directly related to the number of cells injected. Only 4.8 per cent of the injected cells were recovered in the circulating blood volume at one hour (range 0–37 per cent). The per cent recovery was directly related to the pretransfusion granulocyte count of the recipient; the more severe the recipient's leukopenia, the lower was the per cent recovery of transfused cells. The transfused granulocytes disappeared from the recipients' circulation with a half time of 24 hours. A number of other factors were found to influence the results of transfusion, such as: the antileukemic drugs given, the presence of fever, the transfusion vehicle (i.e., saline concentrates or whole plasma), the ABO red cell compatibility of donor and recipient, and the sequence of transfusion. Clinical responses as measured by disappearance of fever were seen in 54 per cent of the recipients. The fraction of febrile patients responding increased as the dose of leukocytes transfused increased. Severe reactions, manifested by dyspnea, cyanosis, and lung infiltrates were seen in a small percentage, but febrile reactions occurred in 67 per cent of afebrile patients.     

Donor WBCs can persist and transiently mediate immunologic function in a murine transfusion model: effects of irradiation, storage, and histocompatibility

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-<1 cell/microL) in female Balb/c recipient mice blood over 6 weeks. Donor WBC survival kinetics displayed an early MHC-dependent phase, which was followed by a more rapid phase that was not influenced by donor-recipient MHC differences. All donor WBCs were cleared within 24 to 48 hours. DNCB sensitivity was passed through transfusion, where it was transiently expressed in naive recipients. CONCLUSION: The clearance of donor WBCs in the murine transfusion model is much slower than that in humans. Allogeneic donor WBC clearance may be biphasic, involving MHC-dependent as well as MHC-independent mechanisms. DNCB sensitivity can be transferred transiently to a naive recipient.     

Blood transfusion utilization and recipient survival at Hospital das Clinicas in São Paulo, Brazil

BACKGROUND: The characteristics of blood recipients including diagnoses associated with transfusion and posttransfusion survival are unreported in Brazil. The goals of this analysis were: 1) to describe blood utilization according to clinical diagnoses and patient characteristics and 2) to determine the factors associated with survival of blood recipients. STUDY DESIGN AND METHODS: A retrospective cross‐sectional analysis was conducted on all inpatients in 2004. Data came from three sources: The first two files consist of data about patient characteristics, clinical diagnosis, and transfusion. Analyses comparing transfused and nontransfused patients were conducted. The third file was used to determine survival recipients up to 3 years after transfusion. Logistic regression was conducted among transfused patients to examine characteristics associated with survival. RESULTS: In 2004, a total of 30,779 patients were admitted, with 3835 (12.4%) transfused. These patients had 10,479 transfusions episodes, consisting of 39,561 transfused components: 16,748 (42%) red blood cells, 15,828 (40%) platelets (PLTs), and 6190 (16%) plasma. The median number of components transfused was three (range, 1‐656) per patient admission. Mortality during hospitalization was different for patients whose admissions included transfusion or not (24% vs. 4%). After 1 year, 56% of transfusion recipients were alive. The multivariable model of factors associated with mortality after transfusion showed that the most significant factors in descending order were hospital ward, increasing age, increasing number of components transfused, and type of components received. CONCLUSION: Ward and transfusion are markers of underlying medical conditions and are associated with the probability of survival. PLT transfusions are common and likely reflect the types of patients treated. This comprehensive blood utilization study, the first of its kind in Brazil, can help in developing transfusion policy analyses in South America.     

Flow cytometric determination of RBC survival in autoimmune hemolytic anemia

BACKGROUND: In cases of warm autoimmune hemolytic anemia (WAIHA), crossmatch incompatible RBCs are most often used for transfusion. The determination of the in vivo survival of transfused and autologous RBCs in WAIHA is helpful in the assessment of the efficacy of transfusion and other therapeutic interventions. CASE REPORT: A 38-year-old man presented with acute WAIHA, thrombocytopenia, and neutropenia. Steroids and IVIG therapy were ineffective, and the patient received RBCS: Because of increasing hemolysis and persisting thrombocytopenia, splenectomy was performed, resulting in partial remission. Further improvement was achieved by immunosuppressive therapy. MATERIALS AND METHODS AND RESULTS: Survival of transfused and autologous RBCs was determined, using a flow cytometric method based on the determination of different blood group antigens of patient and donor RBCS: The survival of autologous and transfused RBCs before splenectomy was determined on two consecutive days. The life span of autologous RBCs remained rather stable at 69 and 64 hours on Days 10 and 11, respectively, whereas the life span of transfused RBCs decreased from 186 hours to 25 hours. After splenectomy, the life span of transfused RBCs almost normalized: 43 days at postsplenectomy Day 3 and 87 days at postsplenectomy Day 69. CONCLUSION: Flow cytometry was successfully used to determine changing hemolytic activity during the clinical course of WAIHA. Additionally, the survival of transfused RBCs could be measured, which may be helpful to judge for the compatibility of allogeneic RBCS: Thus, we were able to show the therapeutic inefficacy of steroids and immunoglobulins, and quick improvement after splenectomy.     

Effects of transfusion on human erythrovirus B19-susceptible or -infected pediatric recipients in a genotype 3-endemic area

BACKGROUND: Human erythrovirus (parvovirus) B19 is transmitted by transfusion of blood, blood components, and plasma derivatives and is resistant to most viral inactivation methods. B19 genotype 3 is prevalent in Ghana, and no related clinical information is available. STUDY DESIGN AND METHODS: This study assessed the transmission of B19 genotype 3 by transfusion and the potential effect of transfused B19 antibodies in viremic recipients. Immunological aspects of B19 genotype 3 infection in children mainly transfused for acute malarial anemia were examined. Molecular and serologic methods adapted to genotype 3 were developed and used. RESULTS: Among 114 donor-recipient pairs from Ghana, two donations contained B19 DNA and specific antibodies, and no evidence of transmission was found. B19 immunoglobulin G (IgG)-containing whole blood was transfused to 14 B19 DNA-positive recipients. Three recipients with detectable levels of IgG to B19 failed to clear viremia 1 to 2.3 months after transfusion. Ten recipients without IgG to VP2 before transfusion cleared the virus but failed to develop an immune response to B19 within 1 to 2 months after transfusion. Only 1 patient who received little specific IgG by transfusion produced detectable antibodies. CONCLUSION: Low levels of B19 genotype 3 DNA associated with specific IgG are not infectious by transfusion. Viral clearance and apparent down regulation of immune response to B19 may be related to removal of the viral antigens by transfused antibodies and/or immunomodulatory effect of transfusion.     

Transfusion transmission of highly prevalent commensal human viruses

BACKGROUND: Anellovirus species Torque teno virus (TTV), Torque teno mini virus (TTMV), and Torque teno midi virus (TTMDV) and flavivirus GBV‐C are highly prevalent and genetically diverse chronic human viral infections that have not yet been associated with disease. STUDY DESIGN AND METHODS: To determine if these commensal viruses are transmitted by blood transfusions, we genetically analyzed viral species in cryopreserved samples from blood donors and corresponding pre‐ and posttransfusion samples from recipients enrolled in the Transfusion‐Transmitted Viruses Study cohort. RESULTS: All 24 individuals in 12 donor‐recipient pairs were infected with TTV, while 16 were infected with TTMV, 15 with TTMDV, and four with GBV‐C. None of the 12 informative cases of TTV transfusion or eight cases of TTMV transfusion, where the donor and recipient viruses could be genetically differentiated, resulted in detectable transmissions in which the donor viruses were detected in the recipient by direct sequencing of the polymerase chain reaction products. Of the five informative cases of TTMDV transfusion, including two cases of transfusion into TTMDV‐negative recipients, one case of superinfection was seen with both the recipient and the donor viral variants detected in the transfusion recipient for at least 11 days posttransfusion. Three donor‐recipient pairs were informative for GBV‐C transmission with only one transfusion into a GBV‐C–negative recipient resulting in a transiently detected infection. CONCLUSIONS: Transmission of the common commensal anelloviruses and GBV‐C during transfusion was detected in 2 of 12 already infected or uninfected recipients. Underestimation of the true rate of viral transmission may be due to limitations in detecting donor viral variants present as minority variants in the already infected transfusion recipients.     

Alloimmunization to transfused platelets requires priming of CD4+ T cells in the splenic microenvironment in a murine model

BACKGROUND: Alloantibodies are a clinically significant sequelae of platelet (PLT) transfusion, potentially rendering patients refractory to ongoing PLT transfusion support. These antibodies are often IgG class switched, suggesting the involvement of CD4+ T‐cell help; however, PLT‐specific CD4+ T cells have not been visualized in vivo, and specifics of their stimulation are not completely understood. STUDY DESIGN AND METHODS: A murine model of alloimmunization to transfused PLTs was developed to allow in vivo assessment and characterization of CD4+ T cells specific for PLT major histocompatibility complex (MHC) alloantigen. PLTs were harvested from BALB/c mice, filter leukoreduced, and transfused into C57BL/6 recipients. PLT‐specific CD4+ T‐cell responses were visualized by using a T‐cell receptor transgenic mouse that detects peptide from donor MHC I presented on recipient MHC II. Antibody responses were determined by indirect immunofluorescence using BALB/c donor targets. RESULTS: C57BL/6 recipients of BALB/c leukoreduced PLT transfusions produced BALB/c antibodies, with proliferation of antigen‐specific CD4+ T cells seen in the spleen but not lymph nodes or liver. Depletion of recipient CD4+ cells or splenectomy independently abrogated the alloantibody response. CONCLUSION: We report a novel model to study antigen‐specific CD4+ T cells during alloimmunization to PLT transfusion. The presented data support a critical role for CD4+ T‐cell help in the humoral response to PLT transfusion and establish the spleen as a required microenvironment for effective CD4+ T‐cell priming against donor PLT–derived MHC I.     

Selective loss of calcium permeability on maturation of reticulocytes.

Calcium and sodium permeability of human reticulocytes have been studied and compared to mature erythrocytes. Mature erythrocytes had extremely low Ca2+ permeability which was less than 0.1% of values published for squid axon or HeLa cells. Calcium entry was markedly increased in reticulocyte-rich suspensions and the uptake was linearly related to the percentage of reticulocytes present. The data suggest that reticulocytes are 43-fold more permeable to Ca2+ than mature cells although their Ca2+ concentration is not increased. Sodium influx into reticulocyte-rich suspensions was also increased in direct proportion to the percent of reticulocytes present. Reticulocytes are sixfold more permeable to Na+ than mature cells so the ratio of Ca2+:Na+ permeability falls by sevenfold as the reticulocyte changes to an erythrocyte. [3H]Ouabain binding was increased in reticulocyte-rich cell suspensions and the correlation suggested a value of about 4,000 sites per reticulocyte compared with 362+/-69 per mature cell. Maturation of the human reticulocyte produces disproportionate changes in cation permeability and in particular a selective loss of Ca2+ permeability.     

Survival of fresh human platelets in a rabbit model as traced by flow cytometry

BACKGROUND: In the event of hemorrhage and blood loss, platelets play a vital role in the coagulation process. However, there are currently no acceptable protocols for long-term storage of platelets. As a first step toward testing the efficacy of stored platelets or platelet substitutes in vivo, a flow cytometric technique was developed to detect human platelets in rabbit blood. STUDY DESIGN AND METHODS: Human platelets were transfused to rabbits whose reticuloendothelial system was inhibited by the administration of ethyl palmitate. Because human and rabbit platelets display surface molecules with different epitopes, human platelets were selectively labeled with antibodies specific for glycoprotein IX (CD42a). As this antibody does not label rabbit platelets, it allows discrimination of human from rabbit platelets in samples of rabbit blood containing both types of platelets. RESULTS: Survival of human platelets in rabbits was monitored by flow cytometry and fluorescence microscopy in blood drawn at various times after the platelet transfusion. Fresh human platelets transfused to untreated control rabbits (n = 3) were removed from circulation within 10 minutes of the completion of the transfusion. Fresh platelets (1 day old) transfused to rabbits treated with ethyl palmitate (n = 5) survived for 24 hours with an average half-life of 8.6 hours. In contrast, 8-day-old platelets were cleared from the circulation sooner with an average half- life of 2.9 hours (n = 4). CONCLUSION: This report describes a rapid and efficient method of assessing the survival of human platelets in a rabbit model using flow cytometry. This technique will enable the monitoring in rabbits of human platelets prepared by various preservation protocols.     

Determination of reticulocytes: three methods compared.

Determination of reticulocytes in peripheral blood is a valuable tool for getting information about erythropoiesis of an individual. For many years, reticulocyte numbers were quantified manually by means of a microscope after staining with supravital dyes. However, this method is tedious and shows low reproducibility. Therefore, several methods for the automated determination of reticulocytes have been established in laboratory routine within the last years. The aim of this study was to compare three of these automated methods for reticulocyte analysis. Reticulocytes were determined in 130 subsequent routine samples by means of an ABX Pentra 120 Retic haematological analyser, a Coulter EPICS XL MCL flow cytometer and a Coulter STKS haematology system, using the fluorescent dye thiazole orange or the supravital dye new methylene blue for reticulocyte staining, respectively. The reticulocyte concentrations were slightly lower for the Coulter STKS haematology system (mean +/- SD 1.89+/-1.32%) when compared with the Coulter EPICS XL MCL flow cytometer or the ABX Pentra 120 Retic haematological analyser (2.11+/-1.25% and 2.12+/-1.15%, respectively). The correlations between all methods were significant (r(s) > or = 0.843, p < 0.001). Small intercepts were, however, observed in the correlation plots between the values obtained by means of the Coulter STKS haematology system and those obtained by the other two methods. Within-batch coefficients of variation were 6.0%, 6.9% and 7.8% for the ABX Pentra 120 Retic haematological analyser, the Coulter STKS haematology system and the Coulter EPICS XL MCL flow cytometer, respectively. The corresponding between-batch coefficient of variation values were 6.8%, 4.9% and 5.3% as well as 14.1%, 7.6% and 6.1% for the low, medium and high control levels determined by means of the ABX Pentra 120 Retic haematological analyser and the Coulter STKS haematology system, respectively. These data suggest that all three methods allow the efficient and reliable determination of reticulocyte counts under clinical routine conditions. However, although the obtained data are very similar, differences exist which should be taken into account for the normal values of the different methods.     

Recipients potentially infected with parvovirus B19 by red blood cell products

BACKGROUND: Since 2000, blood donor screening for parvovirus B19 (B19) by nucleic acid testing (NAT) at the Ulm Institute has been conducted 6 to 8 weeks postdonation, that is, after transfusion of cellular blood products, whereas at the Frankfurt Institute all donations are screened before releasing any blood product. In this study, we evaluated the infectivity of B19‐positive blood products in relation to the virus concentration in the transfused blood component. STUDY DESIGN AND METHODS: Recipients were classified into two groups (A, transfused with blood products with B19 virus load less than 10⁵ IU/mL; and B, transfused with blood products with B19 virus load greater than 10⁵ IU/mL). Phylogenetic analyses were done for B19 DNA–positive donor and recipient pairs in the variant VP‐1u genome region. All samples were investigated for immunoglobulin (Ig)M and IgG B19 antibodies. RESULTS: B19 DNA was detected in 9 of 18 recipients of red blood cells (RBCs) from Group B, whereas none of the 16 recipients of RBCs from Group A were positive for B19 DNA (p = 0.016). Phylogenetic analysis demonstrated identical genomic sequences between the donors and recipients. Because recipient B19 DNA and antibody results were not available before transfusion, we interpret our overall data to indicate equivocal evidence of B19 transmission by RBC transfusion. CONCLUSION: B19 transmission by cellular blood products correlates with the virus concentration and the concentration of neutralizing antibodies. Thus, blood donor screening for B19 by minipool NAT should be done to supply at‐risk patients (e.g., immunosuppressed patients) with B19‐negative blood components.