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.
Two whole blood protocols (ultracentrifugation and a rapid RBC lysis/removal protocol) were evaluated using quantitative real-time PCR. Whole blood (WB) was spiked with known concentrations of B19V and recovery in various blood fractions was determined. The rapid RBC lysis/removal protocol was then used to compare B19V concentrations in 104 paired whole blood and plasma samples collected longitudinally from 43 B19V infected donors with frozen specimens in the REDS Allogeneic Donor and Recipient Repository (RADAR).
In B19V spiking experiments, ~one-third of viral DNA was recovered in plasma and two-thirds was loosely bound to erythrocytes. In the IgM positive stage of infection in blood donors when plasma B19V DNA concentrations were > 100 IU/mL, median DNA concentrations were ~30-fold higher in WB than in plasma. In contrast, when IgM was absent and when the B19V DNA concentration was lower, the median whole blood to plasma ratio was ~1. Analysis of longitudinal samples demonstrated persistent detection of B19V in WB but declining ratios of WB/plasma B19V with declining plasma VL levels and loss of IgM-reactivity.
The WB/plasma B19V DNA ratio varies by stage of infection. Further study is required to determine if this is related to the presence of circulating DNA-positive erythrocytes derived from B19V infected erythroblasts, B19V-specific IgM mediated binding of virus to cells, or other factors.
Antibodies specific for the neutrophil antigen HNA-3a cause severe, sometimes fatal transfusion-related acute lung disease (TRALI) when transfused, but it has not been possible to screen blood donors for anti-HNA-3a because using neutrophils as targets was impractical and molecular properties of the antigen were unknown. Recently it was shown that HNA-3a is carried on choline transporter–like protein-2 (CTL2) and that the HNA-3a/b phenotype is closely correlated with an R154Q amino acid polymorphism in CTL2. However, it has not been shown by direct experiment that R154 is essential for the HNA-3a epitope.
STUDY DESIGN AND METHODS
Preliminary attempts to express recombinant full-length CTL2 (R154) recognized by anti-HNA-3a were unsuccessful. We therefore tested HNA-3a–specific antibodies from donors implicated in TRALI reactions for reactivity against chemically synthesized linear and cyclic CTL2 peptides containing R154 or Q154.
Nine of 20 HNA-3a antibodies recognized the R154, but not the Q154 version of a cyclic 36-residue CTL2 peptide (D131-K166). However, 11 others failed to distinguish between the two versions of this peptide.
The findings provide direct evidence that R154 in the context of CTL2 D131-K166 is necessary to create the HNA-3a epitope but, in the context of cyclic CTL2 peptide D131-K166, is sufficient to detect only about one-half of the HNA-3a–specific antibodies implicated in TRALI. It is likely that fragments of CTL2 longer than can be made on a large scale with an automated synthesizer will be needed to produce a target capable of detecting all examples of anti-HNA-3a in donated blood.
Protein S-nitrosylation (the binding of a nitric oxide group to a cysteine thiol) is a major mechanism through which the ubiquitous cellular influence of nitric oxide is exerted. Disruption of S-nitrosylation is associated with a wide range of pathophysiological conditions. Hemoglobin exemplifies both of these concepts. It is the prototypical S-nitrosylated protein in that it binds, activates, and deploys nitric oxide. Within red blood cells, hemoglobin is S-nitrosylated during the respiratory cycle and thereby conveys nitric oxide bioactivity that may be dispensed to regulate local blood flow in the physiological response known as hypoxic vasodilation. Hemoglobin thus both delivers oxygen directly and delivers vasoactivity to potentially optimize tissue perfusion in concert with local metabolic demand. Accordingly, decreased levels of S-nitrosylated hemoglobin and/or impaired delivery of red blood cell-derived nitric oxide bioactivity have been observed in a variety of disease states that are characterized by tissue hypoxemia. It has been shown recently that storage of blood depletes S-nitrosylated hemoglobin, accompanied by reduced ability of red blood cells to induce vasodilation. This defect appears to account in significant part for the impaired ability of banked red blood cells to deliver oxygen. Re-nitrosylation can correct this impairment and thus may offer a means to ameliorate the disruptions in tissue perfusion produced by transfusion.
Red cell transfusion is associated with lung injury in susceptible hosts, although many cases do not meet criteria for transfusion related acute lung injury. Patients with underlying pulmonary fibrosis can exhibit precipitous deteriorations in respiratory status of unknown etiology defined as acute exacerbations due to superimposed lung injury syndrome. It is unclear whether red cell transfusion is associated with acute exacerbation of underlying pulmonary fibrosis.
We describe a patient who underwent an uneventful elective left total hip replacement but developed anemia post-operatively. Twenty-four hours following transfusion of her fifth non-leukoreduced AS-5 red cell unit, she developed new bilateral airspace infiltrates associated with progressive hypoxemia. These RBC units were 35-38 days old. Despite supportive care and diuresis, patient remained profoundly hypoxemic with infiltrates that progressed to fibrosis.
The patient had mild sub-clinical lower-lobe predominant interstitial pulmonary fibrosis but developed diffuse bilateral ground glass opacities with areas of consolidation 24 h after receiving her last RBC unit. Transbronchial biopsy of the right lower lobe showed active organizing pneumonia and underlying interstitial fibrosis, supporting the clinical diagnosis of acute exacerbation of pulmonary fibrosis. The bronchoalveolar lavage showed progressive bloody effluent, consistent with diffuse alveolar hemorrhage, a marker of lung injury. There was no evidence of viral inclusions, fungal elements, pneumocystis, or bacterial organisms.
Transfusion of multiple units of aged RBCs was temporally associated with an acute exacerbation and rapid progression of underlying sub-clinical pulmonary fibrosis.
transfusion; acute exacerbation; pulmonary fibrosis; lung injury; red blood cells
Marrow damage from chemo- and radiation therapies has been suggested to affect quality and quantity of Hematopoietic stem cell (HSC) products. We tested the hypothesis that CD34+ cells (HSC) from low mobilizers are qualitatively inferior to HSC from high mobilizers.
Materials and Methods
HSC quality was defined by proportion of primitive HSC subsets (CD34+CD38−, CD34+HLA-DR− and CD34+ in G0 stage of cell cycle), the proportion of HSCs that express CXCR4 and CD26 homing proteins and days, to neutrophil and platelet engraftments post transplant. HSC content and CD34 subsets analyses were performed using flow cytometry following ISHAGE protocol. We evaluated the HSC quantity and quality of 139 autologous filgrastim mobilized HSC products. Patients were categorized into low, moderate and high mobilizers if their total HSC collection was <3 ×106/kg, ≥3 ×106/kg and <5 ×106/kg, and ≥5 × 106/Kg respectively.
The median number of primitive CD34 subsets increases with increasing HSC numbers and this association was statistically significant (p = 0.001). However, when the ratios of the primitive CD34 subsets to total HSC counts were compared among the mobilization groups, the ratios were not significantly different. Co-expression of neither CD26 nor CXCR4 with CD34 antigen correlated with HSC mobilization. Evaluation of days to neutrophil engraftment among the mobilization groups did not show a statistically significant difference (p = 0.1). However, days to platelet engraftment among the mobilization groups was statistically significantly different (p = 0.05).
The quality of HSCs from low mobilizers was comparable to HSCs from high mobilizers.
Hematopoietic stem cells; mobilization; Poor Mobilizers; CD34+ Cells
Reports of monosomy 7 in patients receiving granulocyte colony stimulating factor (G-CSF) have raised concerns that this cytokine may promote genomic instability. However, there are no studies addressing whether repeated administration of G-CSF produces monosomy 7 aneuploidy in healthy donors.
Study Design and Methods
We examined chromosomes 7 and 8 by fluorescent in situ hybridization (FISH) in CD34+ cells from 35 healthy hematopoietic stem cell transplant (HSCT) donors after G-CSF administration for 5 days, and by spectral karyotyping analysis (SKY) in four individuals to assess chromosomal integrity. We also studied 38 granulocyte donors who received up to 42 doses of G-CSF and dexamethasone (Dex) using FISH for chromosomes 7 and 8.
We found no abnormalities in chromosomes 7 and 8 in G-CSF mobilized CD34+ cells when assessed by FISH or SKY, nor did we detect aneuploidy in G-CSF/Dex treated donors.
G-CSF does not promote clinically detectable monosomy 7 or trisomy 8 aneuploidy in HSCT or granulocyte donors. These findings should be reassuring to healthy HSCT and granulocyte donors.
There are multiple benefits to transfusing only ABO identical blood components. Historically our institution routinely transfused ABO non-identical platelets (PLTs) and cryoprecipitate to surgical patients. In April 2005, we implemented a policy of transfusing only ABO identical components whenever feasible, regardless of outdating/logistic considerations.
Technical staff closely monitored product usage and adjusted blood center orders based on recent utilization and planned transfusions. When unable to provide ABO identical PLTs ABO compatible platelets were washed to remove incompatible plasma. Data on outdating were collected for eighteen months before and after implementation. We compared transfusion reaction and red cell alloimmunization incidence for four years preceding (2001–2004) and subsequent (2006–2009) to implementation.
In the year following implementation, only 11 of 410 surgical patients received ABO non-identical platelets (2.7%). There was a 5.6% increase in outdating of platelets. Transfusing ABO identical components was associated with significant reductions in febrile (−46%; 8.0 to 4.3 per 10,000 components; p<0.0001) and allergic transfusion reactions (−23%; from 7.0 to 5.4 per 10,000 components; p=0.025). A progressive reduction in de novo red cell alloimmunization incidence also occurred (−50% by 2009; p=0.03).
Providing ABO identical platelets to almost all patients was feasible in our setting by changing ordering and inventorying procedures, and making the ABO identical policy a staff priority. Unexpected and striking reductions in febrile and allergic reactions, and red cell alloimmunization were observed, of uncertain causal relationship to this ABO policy change, which will require further study.
Transfusion related acute lung injury (TRALI) has been associated with both HLA and HNA antibodies. HNA antibody frequency, specificity, and demographic associations have not been well defined in the blood donor population.
A subset of 1171 donors (388 non-transfused males, 390 HLA antibody negative females with three or more pregnancies, and 393 HLA antibody positive females with three or more pregnancies) from a larger leukocyte antibody prevalence study (LAPS) was tested for IgG and IgM HNA antibody using a granulocyte immunofluorescence flow cytometry assay. Additional testing on selected samples included monoclonal antibody immobilization of granulocyte antigen – flow cytometry and granulocyte genotyping.
Eight samples were HNA antibody positive (prevalence 0.7% [95% CI, 0.3 - 1.3%]). Three HNA antibodies (one IgG and two IgM) were found in non-transfused males (prevalence 0.8% [95% CI, 0.2 - 2.2%]); all were pan-reactive or non-specific. One HLA antibody negative previously pregnant female had an IgG HNA antibody with HNA-1a specificity (prevalence 0.3% [95% CI, 0.01-1.4%]). Four HLA antibody positive previously pregnant females demonstrated HNA antibodies, three IgG and one IgM (prevalence 1% [95% CI, 0.3 - 2.6%]). Two of these were HNA-1a specific, one HNA-4a specific, and one non-specific.
HNA antibodies occur with low frequency in the donor population and are present in both male and female donors. Despite the implementation of TRALI reduction strategies, HNA antibodies are still present in donor blood products. Though our data do not create a case for urgent implementation of donor HNA antibody testing, future new developments for high throughput HNA antibody screening, including for HNA-3a, may warrant reconsideration.
Blood group A and B antigens are expressed only weakly on platelets (PLTs) of most individuals but are very strongly expressed on PLTs from approximately 1 percent of normal subjects (Type II high expressers). The implications of this trait for transfusion medicine are undefined.
STUDY DESIGN AND METHODS
A family was studied in which two Group B infants were born with neonatal thrombocytopenia, whereas a third infant whose blood group was A2 had a normal PLT count at birth.
Serologic studies demonstrated a maternal antibody that reacted strongly with PLTs from the father and the two group B children in flow cytometry and with GPIIb/IIIa from their PLTs in solid-phase assays. No PLT-specific antibodies were detected in maternal serum sample, but it contained a high-titer immunoglobulin G antibody specific for blood group B. All PLT-reactive antibody in the mother’s serum was removed by absorption with pooled, washed group A and B red cells (RBCs). Studies with monoclonal anti-B and measurement of serum B-glycosyltransferase activity showed that the father and both group B children were Type II high expressers of blood group B.
The findings indicate that high-titer blood group antibodies acquired from the mother can cause thrombocytopenia in infants possessing the Type II high-expresser phenotype despite competition for antibody binding by blood group antigens expressed on RBCs and other tissues.
Recent reports suggest that maternal immunization against low-frequency, platelet (PLT)-specific glycoprotein (GP) polymorphisms is a more common cause of neonatal alloimmune thrombocytopenia (NATP) than previously thought.
STUDY DESIGN AND METHODS
Serologic and molecular studies were performed on PLTs and DNA from three families in which an infant was born with apparent NATP not attributable to maternal immunization against known PLT-specific alloantigens.
Antibodies reactive only with paternal PLTs were identified in each mother. In Cases 2 (Kno) and 3 (Nos), but not Case 1 (Sta), antibody recognized paternal GPIIb/IIIa in solid-phase assays. Unique mutations encoding amino acid substitutions in GPIIb (Case 2) or GPIIIa (Cases 1 and 3) were identified in paternal DNA and in DNA from two of the affected infants. Antibody from all three cases recognized recombinant GPIIIa (Case 1 [Sta] and Case 3 [Nos]) and GPIIb (Case 2, Kno) mutated to contain the polymorphisms identified in the respective fathers. None of 100 unselected normal subjects possessed the paternal mutations. Enzyme-linked immunosorbent assay and flow cytometric studies suggested that failure of maternal serum from Case 1 (Sta) to react with paternal GPIIIa in solid-phase assays resulted from use of a monoclonal antibody AP2, for antigen immobilization that competed with the maternal antibody for binding to the Sta epitope.
NATP in the three cases was caused by maternal immunization against previously unreported, low-frequency GP polymorphisms. Maternal immunization against low-frequency PLT-specific alloantigens should be considered in cases of apparent NATP not resolved by conventional serologic and molecular testing.
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 post-transfusion RBC survival were analyzed by flow cytometry. RBC alloimmunization was monitored by measuring circulating anti-HEL immunoglobulin levels.
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.
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.
Several differences exist between antigens on transfused red blood cells (RBCs) and other immunogens, including anatomical compartmentalization. Whereas antigens from microbial pathogens and solid organ transplants drain into local lymph nodes, circulating RBCs remain segregated in the peripheral circulation, where they are consumed by antigen-presenting cells (APCs) in the spleen and liver. Accordingly, it was hypothesized that the splenic APCs play a central role in primary alloimmunization to transfused RBCs.
STUDY DESIGN AND METHODS
Recipient mice were splenectomized and transfused with transgenic RBCs expressing the membrane-bound hen egg lysozyme (mHEL) model RBC antigen. In some experiments, mHEL-specific CD4+ T cells were adoptively transferred into recipient mice to allow investigation of helper T-cell responses. Unmanipulated or sham-splenectomized mice served as controls. Recombinant murine cytomegalovirus expressing mHEL (mHEL-MCMV) was used as a control non-RBC immunogen. Humoral responses were measured by mHEL-specific enzyme-linked immunosorbent assay and flow cytometric–based RBC cross-match.
Control animals synthesized detectable anti-HEL immunoglobulin (Ig)G after a single mHEL RBC transfusion. mHEL-specific CD4+ T cells underwent robust expansion, and adoptive transfer of CD4+ T cells resulted in a 1000-fold increase in anti-HEL IgG. In contrast, minimal anti-HEL IgG was detectable in splenectomized mice, mHEL-specific CD4+ T cells did not proliferate, and adoptive transfer did not increase anti-HEL IgG. However, anti-HEL IgG response after exposure to mHEL-MCMV was equivalent in control and splenectomized mice.
Together, these findings illustrate the distinct properties of transfused RBCs as immunologic stimuli, with the spleen playing a critical role in primary RBC alloimmunization at the level of CD4+ T-cell activation.
Maternal immunization against low-frequency, platelet (PLT)-specific antigens is being recognized with increasing frequency as a cause of neonatal alloimmune thrombocytopenia (NAIT).
STUDY DESIGN AND METHODS
Serologic and molecular studies were performed on PLTs and DNA from two families in which an infant was born with severe thrombocytopenia not attributable to maternal immunization against known PLT-specific alloantigens.
Antibodies reactive only with paternal PLTs were identified in each mother using flow cytometry and solid-phase assays. Unique mutations encoding amino acid substitutions K164T in glycoprotein (GP)IIb (Case 1) and R622W in GPIIIa (Case 2) were identified in paternal DNA and in DNA from the affected infants. Each maternal antibody recognized recombinant GPIIb/IIIa mutated to contain the polymorphisms identified in the corresponding father. None of 100 unselected normal subjects possessed these paternal mutations.
Severe NAIT observed in the affected infants was caused by maternal immunization against previously unrecognized, low-frequency antigens created by amino acid substitutions in GPIIb/IIIa (αIIb/β3 integrin). A search should be conducted for novel paternal antigens in cases of apparent NAIT not explained on the basis of maternal-fetal incompatibility for known human PLT antigens.
Recent reports have shown that the HNA-3a leukocyte antigen, a target for antibodies that cause severe transfusion-related acute lung injury, correlates with an arginine 154 (rather than glutamine) polymorphism in choline transporter–like protein 2 (CTL2) but did not show directly that R154 determines HNA-3a. CTL2 peptides containing R154 are recognized by only half of HNA-3a antibodies studied to date. Constructs that react with all HNA-3a antibodies are needed to fully define the HNA-3a epitope.
STUDY DESIGN AND METHODS
HEK293 cells were transfected with cDNA encoding full-length CTL2 linked to green fluorescent protein (GFP). Transfectants were selected for GFP expression and tested with antibodies specific for HNA-3a and -3b.
Each of 20 HNA-3a antibodies reacted preferentially with HEK293 cells expressing the R154 CTL2 construct. An HNA-3b antibody reacted only with CTL2 (Q154).
These findings provide direct evidence that R154 in the context of full-length CTL2 is both necessary and sufficient to create the HNA-3a epitope but suggest that posttranslational modifications of the protein, for example, S–S bonds or addition of glycans, are necessary for recognition of HNA-3a by many antibodies. This could complicate development of an assay for large-scale screening of blood donors to detect anti-HNA-3a.
Measurement of red blood cell (RBC) survival (RCS) is important for investigating pathophysiology and treatment of anemia. Our objective was to validate the multidensity biotin method for RCS determination in sheep, a commonly used model of RBC physiology. [14C]Cyanate served as the reference method for long-term RCS because the 51Cr method (the reference method for humans) is not reliable in sheep.
STUDY DESIGN AND METHODS
Aliquots of autologous RBCs from eight adult sheep were labeled with [14C]cyanate and four separate densities of biotin (BioRBCs) and reinfused. Short-term RCS was assessed by posttransfusion recovery at 24 hours (PTR24); long-term RCS was assessed by the time to 50% survival (T50) and mean potential life span (MPL).
Values for PTR24 of the four BioRBC densities were not different. Values for RCS as reflected by T50 and MPL were nearly identical for [14C]cyanate and the two intermediate-density BioRBC populations. In contrast, the lowest-density BioRBC population survived slightly longer (p < 0.01), but with a difference of no clinical significance. The highest-density BioRBC population importantly shortened RCS (p < 0.01 compared to the two intermediate densities).
This study provides evidence that BioRBCs labeled at four biotin densities can be used to independently and simultaneously measure short-term RCS and that BioRBCs labeled at the three lowest biotin densities can be used to accurately and simultaneously measure long-term RCS. Because the sheep RBC model is comparable to humans, this nonradioactive method has promise for use in RBC kinetic studies in neonates and pregnant women.
Safe, accurate methods to reliably measure circulating red blood cell (RBC) kinetics are critical tools to investigate pathophysiology and therapy of anemia, including hemolytic anemias. This study documents the ability of a method using biotin-labeled RBCs (BioRBCs) to measure RBC survival (RCS) shortened by coating with a highly purified monomeric immunoglobulin G antibody to D antigen.
STUDY DESIGN AND METHODS
Autologous RBCs from 10 healthy D+ subjects were labeled with either biotin or 51Cr (reference method), coated (opsonized) either lightly (n = 4) or heavily (n = 6) with anti-D, and transfused. RCS was determined for BioRBCs and for 51Cr independently as assessed by three variables: 1) posttransfusion recovery at 24 hours (PTR24) for short-term RCS; 2) time to 50% decrease of the label (T50), and 3) mean potential life span (MPL) for long-term RCS.
BioRBCs tracked both normal and shortened RCS accurately relative to 51Cr. For lightly coated RBCs, mean PTR24, T50, and MPL results were not different between BioRBCs and 51Cr. For heavily coated RBCs, both short-term and long-term RCS were shortened by approximately 17 and 50%, respectively. Mean PTR24 by BioRBCs (84 ± 18%) was not different from 51Cr (81 ± 10%); mean T50 by BioRBCs (23 ± 17 days) was not different from 51Cr (22 ± 18 days).
RCS shortened by coating with anti-D can be accurately measured by BioRBCs. We speculate that BioRBCs will be useful for studying RCS in conditions involving accelerated removal of RBCs including allo- and autoimmune hemolytic anemias.
Increased rates of RBC alloimmunization in patients with sickle cell disease may be due to transfusion frequency, genetic predisposition, or immune dysregulation. To test the hypothesis that sickle cell pathophysiology influences RBC alloimmunization, we utilized two transgenic mouse models of sickle cell disease.
Study Design and Methods
Transgenic sickle mice, which express human α and βS globin, were transfused with fresh or 14-day stored RBCs containing the HOD (hen egg lysozyme, ovalbumin, and human Duffyb) antigen; some recipients were inflamed with poly (I:C) prior to transfusion. Anti-HOD alloantibody responses were subsequently measured by ELISA and flow crossmatch; a cohort of recipients had post-transfusion serum cytokines measured by bead array.
Both Berkeley and Townes homozygous (SS) and heterozygous (AS) mice had similar rates and magnitude of anti-HOD RBC alloimmunization following fresh HOD RBC transfusion compared with control animals; under no tested condition did homozygous SS recipients make higher levels of alloantibodies than control animals. Unexpectedly, homozygous SS recipients had blunted cytokine responses and lower levels of anti-HOD alloantibodies following transfusion of 14-day stored RBCs, compared with control animals.
In sum, homozygous βS expression and the ensuing disease state are not alone sufficient to enhance RBC alloimmunization to transfused HOD RBCs in 2 distinct humanized murine models of sickle cell disease under the conditions examined. These data suggest other factors may contribute to the high rates of RBC alloimmunization observed in humans with sickle cell disease.
red blood cells (sickle cell disease); alloimmunization; transfusion medicine
Plasma is vital for the resuscitation of injured patients and to restore necessary pro-coagulants, especially factors II, V, VII, X and XIII; however, female plasma has been implicated in the majority of transfusion-related acute lung injury (TRALI) cases and male-only plasma transfusion regimens have significantly decreased the incidence of TRALI. Little is known about the Human plasma proteome, and no comparisons have been made between male and female plasma; therefore, we hypothesize that there are significant differences between plasma from male and female donors.
5 units of fresh frozen plasma (FFP) each were collected from nulliparous female donors and male donors and the proteome was analyzed by depleting the 14 most common proteins by immunoaffinity columns followed by protein separation by one dimension gel electrophoresis, tryptic digestion of the proteins, analysis of the peptides by liquid chromatography tandem mass spectrometry and identification employing human protein sequence databases.
female plasma, vs. males contained pregnancy zone protein (419-580-fold), factor V (2-fold), α1-antitrypsin (2-fold), β2-microglobulin (2-fold), and complement factors H and C4B (1.5-2-fold) at significantly higher concentrations than males and males contained significant increases in Fc binding protein (2-fold), protein Z-dependent protease inhibitor (2-fold), phosphatidylinositol-glycan specific phospholipase (4-fold), protein S-100 (3-fold) and transgelin-2 (14-fold) vs. females (p<.005). The increases in factor V, α1-antitrypsin, and β2-microglobulin were confirmed by an activity assay or immunoblots. We conclude that there are proteomic differences between male and female plasma which could be exploited to improve clinical outcomes in transfused patients.
Xenotropic murine leukemia virus (MLV)-related virus (XMRV) and other related MLVs have been described with chronic fatigue syndrome (CFS) 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 micro-neutralization assays as a surrogate marker for detection of anti-MLV serological responses – with up to 25% of prostate cancer patients reported to harbor neutralizing antibody responses.
STUDY DESIGN AND METHODS
We developed a high-throughput micro-neutralization 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 pre-immune serum. 354 plasma samples from blood donors in the Reno/Tahoe area were screened for neutralization.
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.
A micro-neutralization 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/MLV. It is likely that this moderate neutralization is mediated through another, non-specific mechanism.
High-throughput micro-neutralization assay; XMRV; MLV; Pseudoviruses; Donor screening
This study investigated the effect of blood donation environment, fixed or mobile with differing sponsor types, on donation return time.
STUDY DESIGN AND METHODS
Data from 2006 through 2009 at six US blood centers participating in the Retrovirus Epidemiology Donor Study-II (REDS-II) were used for analysis. Descriptive statistics stratified by whole blood (WB), plateletpheresis (PP), and double red blood cell (R2) donations were obtained for fixed and mobile locations, including median number of donations and median interdonation interval. A survival analysis estimated median return time at fixed and mobile sites, while controlling for censored return times, demographics, blood center, and mandatory recovery times.
Two-thirds (67.9%) of WB donations were made at mobile sites, 97.4% of PP donations were made at fixed sites, and R2 donations were equally distributed between fixed and mobile locations. For donations at fixed sites only or alternating between fixed and mobile sites, the highest median numbers of donations were nine and eight, respectively, and the shortest model-adjusted median return times (controlling for mandatory eligibility times of 56 and 112 days) were 36 and 30 days for WB and R2 donations, respectively. For PP donations, the shortest model-adjusted median return time was 23 days at a fixed location and the longest was 693 days at community locations.
WB, PP, and R2 donors with the shortest time between donations were associated with fixed locations and those alternating between fixed and mobile locations, even after controlling for differing mandatory recovery times for the different blood donation procedures.
The consequences of temporary pre-donation donor deferrals are unsatisfactorily understood. Previous studies have found that deferral negatively impacts future return for donation in both first time and repeat donors. However, the applicability of these findings across centers has not been established.
Using a cohort design, presenting donors with a temporary deferral in the years 2006 – 2008 in 1 of 6 categories (low hematocrit, blood pressure or pulse, feeling unwell, malaria travel, tattoos/piercing and related exposures, or couldn’t wait/second thoughts) were passively followed for up to a 3-year period for the time to first return after the expiration of the deferral at 6 US blood centers. Time-to-event methods were used to assess return following the receipt of each deferral. We also analyzed which donor characteristics are associated with return following temporary deferral using multivariable logistic regression.
Of 3.9 million donor presentations, 505,623 resulted in deferral in 1 of the 6 categories. Low hematocrit was the most common deferral, had the shortest median time to return, and largest cumulative number of donors returning. Deferrals of shorter duration had better return. Longer term deferrals (up to 1-year in length) had the lowest cumulative return which did not exceed 50% during the study period for malaria travel or tattoo/piercing and related exposures. In multivariable logistic regression modeling, return following deferral was associated with previously identified factors such as repeat donor status, older age, and higher educational attainment regardless of the type of deferral. In addition, return was associated with having been born in the USA, Asian race/ethnicity, and donation at fixed sites regardless of the type of deferral.
The category of temporary deferral influences the likelihood of future return, but the demographic and donation factors associated with return are consistent regardless of the deferral.
Due to their homology, close proximity and opposite orientation, RHD and RHCE can exchange nucleotides giving rise to variant alleles. Some of these variants encode the so-called partial phenotypes. The DIII partial D category has been subdivided into DIIIa, DIIIb, DIIIc, DIII type 4, DIII type 6, and DIII type 7. During DNA-based screening tests, we identified a second example of DIII type 7 in a Dce donor from South Africa. Our study describes hemagglutination tests on this sample and raises a question regarding the molecular basis of the originally defined DIIIb category.
Materials and methods
Hemagglutination and DNA testing were performed by standard techniques.
RBCs from this DIII type 7 donor typed D+C−E−c+e+G−, DAK+ and did not react with anti-D made by people with the DIII phenotype. The allele is RHD*DIII 150C, 178C, 201A, 203A, 307C, 410T, 455C, 602G, 667G).
Based on the serotype and ethnicity (black African), it is likely that DIII type 7 is the originally defined DIIIb category.
DIII phenotype; RHD allele; Rh blood group system; RHD variant; partial D phenotype