Red Cell Phenotype Research Articles

Red blood cell phenotyping after transfusion: an in vitro model

Recipients red blood cell (RBC) phenotyping using serologic techniques, within 3 months of a transfusion, is considered unreliable. We conducted in vitro experiments to determine how long recipients RBC phenotyping results would be compromised. In vitro models were created to mimic in vivo posttransfusion ratios of "transfused" RBCs with either a single or a double dose of an antigen at 10-day intervals from day 0 to day 90 in hypothetical recipients with varying weights and hematocrits (Hct) receiving varying numbers of RBC units. In general, a reliable recipient RBC phenotype was possible earlier after transfusion in larger recipients, those with higher Hct, and those transfused with fewer RBC unit and if the transfused units had the antigen of interest in single, rather than double, dose. We believe that a reliable RBC phenotype, using routine serologic techniques, can often be obtained well before 3 months after transfusion. Similar studies with other donors, antigens, antisera, and methods and in actual patients will be useful.

Comments on: Molecular matching of red blood cells is superior to serological matching in sickle cell disease patients

Comments on: Molecular matching of red blood cells is superior to serological matching in sickle cell disease patients

Detection of alloimmunization to ensure safer transfusion practice.

Background:Serological safety is an integral part of overall safety for blood banks. Emphasis is on the use of routinue Red Blood Cell (RBC) antibody screen test, at set time intervals, to reduce risks related to alloantibodies. Also emphasis is on importance of issuing antigen negative blood to alloantibody positive patients. Effect of using leucodepleted blood on the rate of alloimmunization is highlighted. The concept of provision of phenotypically matched blood is suggested.Materials and Methods:Antibody screen test is important to select appropriate blood for transfusion. Repeat antibody screen testing, except if time interval between the earlier and subsequent transfusion was less than 72 hours, followed by antibody identification, if required, was performed in patients being treated with repeat multiple blood transfusions. Between February 2008 and June 2009, repeat samples of 306 multi-transfused patients were analyzed. Search for irregular antibodies and reading of results was conducted using RBC panels (three-cell panel of Column Agglutination Technology (CAT) and two cell panel of the Solid Phase Red Cell Adherence Technology (SPRCAT). Specificities of antibodies were investigated using appropriate panels, 11 cell panel of CAT and 16 cell panel of SPRCA. These technologies, detecting agglutination in columns and reactions in solid phase, evaluate the attachment of irregular incomplete antibody to antigen in the first phase of immunological reaction more directly and hence improve the reading of agglutination. Three to four log leuco reduced red blood cells were transfused to patients in the study using blood collection bags with integral filters.Results:Alloimmunization rate of 4.24% was detected from 306 multiply transfused patients tested and followed up. The Transfusion therapy may become significantly complicated.Conclusion:Red cell antibody screening and identification and subsequent issue of antigen negative blood have a significant role in improving blood safety. Centers that have incorporated antibody screen test and identification have ensured safe transfusion. Identified patients should be flagged in a database and information shared. Such patients can be given carry-on cards and educated about the names of the identified antibodies. Full red cell phenotyping of individuals, patients and donors, can be feasibility.

Transfusion Practices in the Management of Sickle Cell Disease: A Survey of Florida Hematologists/Oncologists

The purpose of this study was to characterize transfusion practices in the management of sickle cell disease and to identify factors attributing to differences in prescribing practices among Florida hematologists/oncologists. A cross-sectional study was performed in 2005-2006 utilizing a mail survey. The survey instrument addressed practice characteristics, sickle cell patient populations, transfusion settings, indications and techniques, red blood cell phenotype specifications/modifications, use of practice guidelines, and educational resource utilization. One hundred fifty two physicians (75% adult-oriented, 25% pediatric) completed the survey. Non-academic practice settings (78 %) were the primary location. Pediatric practices had a larger percentage of patients with overt strokes, and receiving hydroxyurea therapy than adult-oriented practices. The majority of survey respondents did not request limited phenotypically matched red blood cells on a routine basis. The majority of pediatric practices (60%) had individually defined transfusion practice guidelines in contrast to 8% of adult-oriented practices. There were statistically significant differences for pediatric and adult-oriented practices in managing certain acute and chronic transfusion indications. Analysis of clinical vignette data revealed variation among hematologists/oncologists in the transfusion management of common clinical scenarios. The study underscores the need for the development and dissemination of comprehensive sickle cell transfusion guidelines and protocols.

Seventy-five genetic loci influencing the human red blood cell

Anaemia is a chief determinant of global ill health, contributing to cognitive impairment, growth retardation and impaired physical capacity. To understand further the genetic factors influencing red blood cells, we carried out a genome-wide association study of haemoglobin concentration and related parameters in up to 135,367 individuals. Here we identify 75 independent genetic loci associated with one or more red blood cell phenotypes at P < 10(-8), which together explain 4-9% of the phenotypic variance per trait. Using expression quantitative trait loci and bioinformatic strategies, we identify 121 candidate genes enriched in functions relevant to red blood cell biology. The candidate genes are expressed preferentially in red blood cell precursors, and 43 have haematopoietic phenotypes in Mus musculus or Drosophila melanogaster. Through open-chromatin and coding-variant analyses we identify potential causal genetic variants at 41 loci. Our findings provide extensive new insights into genetic mechanisms and biological pathways controlling red blood cell formation and function.

Red Cell Alloimmunization in Sickle Cell Disease: Benefit of Extended Crossmatching in Adults

Abstract 4761 Background:Patients with sickle cell disease (SCD) receive frequent transfusions and have been observed to have a high rate of transfusion reactions and alloantibody formation after transfusion. Alloimmunization to antigens of the Rh system, Kell, Duffy, MNSs, and Kidd are most common. It has become accepted practice at most sickle cell centers to match for C, E, and Kell antigens in order to prevent alloimmunization. Most studies of the effectiveness of red cell (RBC) phenotype matching have been in pediatric patients and have shown a decrease in alloimmunization beyond those specific antigens that are pre-emptively matched. Leukoreduction (LR) of blood products is known to decrease HLA alloimmunization and may also reduce the risk of RBC antibody formation. It is also unclear whether there is value in continuing phenotype matching beyond the pediatric age and whether new RBC antibodies may be prevented by LR, independent of phenotype matching. Patients with SCD at our institution are routinely matched for C, E, and Kell antigens and given leukoreduced blood. Here, we study our past experience in reducing the frequency of new alloantibody formation. Methods:We reviewed charts of patients with SCD, including sickle cell anemia, sickle/thalassemia, and SC disease, who were transfused at Thomas Jefferson University (TJU) and had documentation of presence or absence of alloantibodies on presentation to our center. Data extracted from chart included patient's age, disease type, known antibodies, and time of first documentation of antibody. Antibodies were considered to be definitely unrelated to transfusion at TJU if they were present before first transfusion at TJU or if a previous screen not showing the antibody in question had been performed and no transfusions given between that screening and the development of the relevant antibody. LR was used routinely at TJU starting in 2003 and was used in selected cases, including many sickle patients, earlier. In 1996, TJU began crossmatching for C, E, and Kell antigens. Results:68 charts of patients with SCD transfused at least once at TJU were reviewed, including 8 patients undergoing routine exchange transfusion for stroke prevention. Of these, 33 (48.5%) had no alloantibodies detected at any time. Of the remaining 35, 16 had 1 antibody, 8 had 2 antibodies, and 11 had 3 or more antibodies, including 3 patients with 8–10 each. A total of 92 antibodies were identified among these 35 patients, 60 of which were not related to transfusion at TJU. Of the remaining 32 antibodies, the most common antibodies identified were C (5 instances) and E (6 instances). Antibodies to Kell, Fya, and Jkb developed in 3 patients apiece. Two instances each of C, E, and Kell antibodies developed after the initiation of crossmatching for these antigens, but in all but one case, the records suggested that the antibody was most likely developed by transfusion at an outside institution not practicing phenotype matching, but this could not be confirmed (i.e. the patient was not transfused at TJU for >1 year prior to the positive screen appearing, but there were no screens in between to identify whether the antibody became evident ). One instance of C developed after emergency transfusion without extended crossmatch and thus the antibody likely developed from transfusion at TJU.Patients who developed antibodies had a higher number of transfusions than non-immunized patients (median 117 vs 74 units, respectively) and were older (38 vs 35 years, respectively), but neither was statistically significant (p>0.1 in each). Of 8 patients undergoing chronic RBC exchange transfusion, 3 have no alloantibodies, 3 had antibodies that developed from transfusion elsewhere, 1 had a new alloantibody but had prior alloimmunization and 1 developed new alloantibodies at TJU. Conclusions:Our results confirm that C, E, and Kell continue to be the most common antigens leading to alloimmunization in transfused adult SCD patients. A combination of LR and extended phenotype matching appears to be effective in reducing antibody formation compared to historical reports. Our retrospective study has limitations, notably the possibility that patients may have been transfused at other institutions, which may not use phenotype matching or leukoreduced products. Further prospective studies are indicated to aid in determining whether phenotype matching is justified in adult sickle cell patients in the face of growing global LR. Disclosures:No relevant conflicts of interest to declare.

Editorial introductions

Current Opinion in Hematology was launched in 1994. It is part of a successful series of review journals whose unique format is designed to provide a systematic and critical assessment of the literature as presented in the many primary journals. The field of hematology is divided into nine sections that are reviewed once a year. Each section is assigned a Section Editor, a leading authority in the area, who identifies the most important topics at that time. Here we are pleased to introduce the Section Editors for this issue. SECTION EDITORS Andrea BacigalupoAndrea BacigalupoDr Bacigalupo is the Head of the Division of Hematology and Stem Cell Transplants at Ospedale San Martino, Genova, Italy. He graduated in 1973 from the Genova Medical School, Italy, and started an allogeneic transplant program in 1976. He has conducted several clinical trials in patients with aplastic anemia and in patients undergoing allogeneic transplants. His main interests are immune control of hematopoiesis, bone marrow failure, GvHD and related complications, and graft versus leukemia reactions. He has also been interested in cytomegalovirus and Epstein–Barr virus infections, looking at early diagnosis and prospective studies on prophylaxis and pre-emptive therapy. Dr Bacigalupo has served as a board member of the European Group for Bone Marrow Transplantation (EBMT) for several years, as EBMT Secretary, Chairperson of the Aplastic Anemia Working Party and then as President for the 1998 to 2002 term. He served as President of the Italian Group of Bone Marrow Transplantation (GITMO) for the 2002 to 2005 term. He has been a member of the Italian National Health Council for two terms (2000–2006). He has served on the Regimen Related Toxicity Working Group of the Center for International Blood and Marrow Transplant Research (CIBMTR). He is a member of several national and international hematology societies and is Associate Editor of the journal Bone Marrow Transplantation and Biology of Blood and Marrow Transplantation. He serves on the Board of Haematologica and of the Journal of Clinical Oncology. Dr Bacigalupo has published over 400 articles in peer-reviewed journals. Martin L. OlssonMartin L. OlssonDr Olsson is a Professor of Transfusion Medicine at Lund University, Sweden, and the Medical Director of the Nordic Reference Laboratory for Genomic Blood Group Typing in Lund, Sweden. He received his M.D. and Ph.D. at Lund University and has been a guest scientist at the Bristol Institute of Transfusion Science, UK, and a visiting Associate Professor/Lecturer at Harvard Medical School, Boston, MA, USA. His main clinical and research interests are in the molecular genetics of red cell surface markers with a special emphasis on carbohydrate histo-blood group systems and blood typing. His scientific contributions have focused on clarifying the correlation between red cell phenotypes and the underlying genetic polymorphisms, as well as glycosyltransferase function in different blood groups. He is also interested in pathogen-related aspects of blood group antigens and different mechanisms of protection against hemolysis. Dr Olsson serves as Associate Editor of Transfusion Medicine and is a member of the Editorial Board of Transfusion. His awards include the Jean Julliard Prize from the International Society of Blood Transfusion, the Race and Sanger Award from the British Blood Transfusion Society, the Fernström Prize at Lund University, the Hain Foundation Prize and the Claes F. Högman's Honorary Lectureship.

Ex vivo erythrocyte generation and blood doping.

Ex vivo erythrocyte generation and blood doping.

A direct blood polymerase chain reaction approach for the determination of GP.Mur (Mi.III) and other Hil+ Miltenberger glycophorin variants

GP.Mur (Mi.III) is a glycophorin B-A-B hybrid sialoglycoprotein expressing several potent immunogens, including Mi(a), Mur, and Hil. GP.Mur is considered one of the most important red blood cell (RBC) phenotypes in blood banking in Southeast Asia. However, there are no antibodies commercially available for the screening of GP.Mur RBCs. To develop a direct blood polymerase chain reaction (PCR) approach for the screening of GP.Mur cells, we first confirmed the genomic sequence differences among four GP.Mur and three Mi(a-) samples by sequencing their GYP.Mur and GYPB genes. With these data, we designed PCR primers that best discriminate GYPB and GYP.Mur. Our primer design also allows the detection of other Hil+ glycophorin variants. We also constructed two plasmids--pGBi2i3 and pMiIIIi2i3--which serve as the negative and positive control DNA, respectively, for the PCR procedure. Additionally, we designed a control PCR to be run side by side with the typing PCR. Because of the high specificity of our primers, we found it unnecessary to extract DNA from blood samples for PCR. We have tested this PCR method on 379 fresh and frozen blood samples. The results were further validated by serology and DNA sequencing and were shown to be completely accurate in our hand. We also found that the rapid genotyping method--high-resolution melting--can be a timesaving alternative for DNA sequencing. This direct blood PCR approach for determination of GP.Mur and related Hil+ phenotypes is reliable and economical and is expected to be useful for blood banking in Southeast Asia.

The challenge and paradox in serology RhD typing for blood donors and patients.

Selecting reagents for RhD typing poses an ongoing challenge and is a paradox for transfusion medicine. Red cells from the majority of blood donors and patients are readily defined as D positive or D negative. However, a spectrum of partial D, weak D or very weak (DEL) red cell phenotypes are found in individuals who carry RHD genetic variants. The challenge in typing blood donors is to select appropriate typing reagents and testing strategies with sufficient sensitivity to detect these partial and weak D types. Ideally blood donations from such individuals should be labelled as D-positive. The paradox arises in patient management where individuals whose red cells carry partial and weak D antigen types are managed as D-negative. Hospital serology in these situations is detuned to type these patients as D negative1–3. This is potentially important for pregnant women to ensure those with partial D antigen phenotypes receive anti-D prophylaxis to guard against haemolytic disease of the foetus and newborn (HDFN). RhD immunisation has been reported in carriers of partial D phenotypes and can be associated with HDFN. One further challenge arises because the frequency of partial and weak D types varies for different populations. Indeed, the advent of molecular typing has provided a basis for classification for the range of alleles present in different populations4–6. However, molecular typing as an adjunct to serology is resource intensive and, currently restricted to specialised reference laboratories. In this edition Kulkarni et al. report a serological study to resolve 60 cases, in an Indian population, referred because of discrepant RhD serological typing7. Extended serological testing using a commercial panel, comprising 12 monoclonal antibodies, defined partial and weak D types present for 93% (56/60) of the samples. In addition to providing insight into the range of variants present, the authors observed distinct reactivity patterns for a subset of monoclonal antibodies. For all samples one monoclonal antibody consistently typed all samples as D negative and three monoclonal antibodies consistently typed all samples as D positive. The authors propose that a streamlined testing strategy using this subset of monoclonal antibodies provides a strategy for resolution of samples with D typing discrepancies. For this case series, this strategy provided an evidence base for D assignment required to ensure safe clinical management for both patients and donors. There are partial D and weak D types, not represented in this cohort, which would have been accurately signalled on the basis of reactivity patterns different from the one observed. The authors show these among the patterns in Table II. It would be appropriate to apply extended serological and molecular typing to such samples. As molecular typing becomes more readily available, a combination of enhanced serological and molecular typing strategies promises to overcome the ongoing challenge and paradox in RhD typing for donors and patients. It is therefore important to document evidence for the range of partial and weak D types in various population groups and document evidence for the performance of typing reagents. Accumulated evidence of the type presented by Kulkarni et al., will contribute to selection of optimised serological and molecular typing strategies in the future.

Red cell phenotyping of blood from donors at the National blood center of Malaysia

Background:Human blood groups are polymorphic and inherited integral structures of the red cell membrane. More than 300 red cell antigens have been identified and further categorized into 30 major discrete systems. Their distribution varies in different communities and ethnic groups.Aims:This work was set to determine the prevalence of red cell phenotypes in donors from the major ethnic groups in Malaysia, namely, Malays, Chinese, and Indians.Materials and Methods:The work utilized the dextran acrylamide gel technique in which four types of gel cards were used to identify the blood groups of 594 subjects collected at the National Blood Transfusion Centre, Malaysia.Results:Blood group O and CDe/CDe (R1R1) were the most common in all ethnic groups. The cde/cde (rr) was more prevalent amongst Indians. The rare phenotypes found were cDE/cDE(R2R2) and cDE/CDE(R2Rz). With the Lewis system, the distribution of Le(a-b+) was similar among the ethnic groups. The rarest phenotype Fy(a-b-) was discovered in two donors. Jk(a-b-) was found in seven Malays and in two Indians. In the MNSs system, MN was common in Malays and Chinese, while the MM was more common among Indians. The rare SS was found in 19 donors. Malay and Chinese subjects had high P1 Negative blood but Indians showed high P1 positive blood. Within the Kell System, the very rare KK type was found in six subjects.Conclusions:The results obtained serve as an established database for the distribution of red cell phenotypes based on the blood group systems of donors from the major ethnic groups in Malaysia.

Applying molecular immunohematology discoveries to daily transfusion practice

Applying molecular immunohematology discoveries to daily transfusion practice

ENU Mutagenesis in the Mouse for Identification of Genes Regulating Erythropoiesis: a Mouse Mutant with An Activating Mutation of the KCl Cotransporter, KCC1 Causing Dehydrated Red Cells

Abstract 684Genome-wide phenotype-driven studies have emerged as a powerful tool for the identification of novel genes in a biological process of interest, new functions for existing genes and the establishment of mouse models of human disease. Recent advances in exomic and whole genome sequencing has simplified mutation detection, making genetic screens in the mouse more facile. We have employed a large-scale mutagenesis screen in mice using the chemical mutagen N-ethyl-N-nitrosourea (ENU) to identify genes regulating erythropoiesis. In this dominant screen, G1 progeny were screened at 7 weeks of age by automated hematological analysis for isolated abnormalities of red cell indices (HCT, RCC and MCV) defined as greater than three standard deviations from the wild type population. Here, we describe the RBC10 mouse line, in which homozygotes had mild anemia with microcytosis, target cells, spherocytes, reticulocytosis and increased resistance to osmotic stress. A combination of SSLP and SNP mapping isolated the genetic mutation to a 3.65 Mb region on chromosome 8. Whole genome Next Generation Sequencing of the mapped interval identified an A to T nucleotide substitution within the coding region of the potassium chloride (KCl) co-transporter, Slc12a4 (KCC1), resulting in a non-conservative amino acid substitution from methionine to lysine at position 935 within the C-terminal cytoplasmic domain. KCC1 and KCC3 function are the major erythroid KCl co-transporters that export KCl in response to hypotonic swelling. We hypothesized that the M935K was an activating mutation of KCC1, based upon the microcytosis and resistance to osmotic stress, which contrasts with the macrocytic phenotype of red cells lacking both KCC1 and KCC3. 86Rb efflux assay confirmed increased KCC1 activity of RBC mutant red cells. We propose that the M935K mutation increases activity of KCC1 by preventing phosphorylation of the nearby threonine reside, a critical regulatory target of the serine-threonine kinase, WNK1. Disclosures:No relevant conflicts of interest to declare.

ROCK1 Functions As a Critical Regulator of Stress Erythropoiesis and Survival by Regulating p53

Abstract 916Erythropoiesis is a dynamic, multistep process in which hematopoietic stem cells differentiate toward a progressively committed erythroid lineage through intermediate progenitors. Under normal physiologic condition, erythropoiesis takes place primarily in the bone marrow of humans and mice. Efficient stress erythropoiesis is crucial to survival and recovery from various pathophysiologic conditions including blood loss, anemia, and therapeutic procedures used in the treatment of hematological malignancies such as chemotherapy and stem cell transplantation. Impaired stress erythropoiesis can be fatal under these conditions. In order to develop improved strategies to stimulate stress erythropoiesis in patients with such conditions, it is critical to identify the molecular mechanisms that regulate this process. While many of the downstream signaling molecules and pathways have been elucidated in steady state erythroid cell development, the major regulators under stress conditions remain to be defined. The small families of Rho GTPases and their downstream effectors, Rho kinases have been implicated in regulating various cellular functions including actin cytoskeleton organization, cell adhesion, and cell motility in non-hematopoietic cells and inflammatory cells. Rho kinases (ROCK1 and ROCK2) belong to a family of serine/threonine kinases, and their physiologic role in erythropoiesis is not known. Utilizing mice deficient in the expression of ROCK1, we demonstrate no significant difference with respect to erythroid parameters in peripheral blood under steady-state conditions (n=19 mice for each genotype). The frequency of Ter119+, CD71+ or double-positive erythroid cells in the bone marrow and spleen of ROCK1-deficient mice was comparable to wild type (WT) controls at basal levels (n=7). In response to myelotoxic stress, ROCK1 deficient Ter119 positive cells demonstrated enhanced survival and recovery compared to WT controls (n=3–6, *p< 0.05). Further, using a phenylhydrazine (PHZ)-induced murine model of hemolytic anemia, we demonstrate that ROCK1-deficient mice exhibit increased red blood cells and enhanced hematocrits relative to WT mice (n=6, *p< 0.05). In addition, ROCK1−/− mice support efficient erythro-splenomegaly, and exhibit a threefold increase in splenic weight after PHZ injection compared to WT controls (n=6, *p< 0.05). Flow cytometric analysis revealed increased frequency of Ter119/CD71 double positive erythroid progenitor pool in ROCK1−/− spleens after PHZ treatment compared to WT (n=6, *p< 0.05). Furthermore, histopathological analysis of WT and ROCK1−/− spleens following PHZ treatment revealed a defined population of white pulp and red pulp in control spleens but enhanced extramedullary erythropoiesis in ROCK1−/− spleens (n=3, *p< 0.05). In vitro colony forming assays showed that ROCK1-deficient splenocytes generated more erythroid colony forming units (CFU-Es) and eventually generated more erythroid-burst forming units (BFU-Es) compared to WT splenocytes in response to different concentrations of EPO and combination of EPO and SCF (n=3, *p< 0.05). Deficiency of ROCK1 also resulted in enhanced survival of mice treated with PHZ compared to controls (n=17 mice per group, *p< 0.05). The enhanced survival of ROCK1-deficient mice in response to PHZ was associated with reduced reactive oxygen species (ROS) levels compared to WT (n=6 mice per group, *p< 0.05). Bone marrow transplantation studies revealed that enhanced stress erythropoiesis in ROCK1-deficient mice is stem cell autonomous (n=6 mice per genotype, *p< 0.05). Remarkably, the red cell phenotype observed in ROCK1−/− mice is similar to that reported in mice deficient in p53. We show that ROCK1 binds p53 directly and regulates its stability and expression (n=3). In absence of ROCK1, p53 phosphorylation and expression is reduced in ROCK1−/− erythroblasts (n=3). Our findings reveal that ROCK1 functions as a physiologic regulator of p53 under conditions of erythroid stress. These findings are expected to offer new perspectives on stress erythropoiesis and may provide a potential therapeutic target in human disease characterized by anemia. Disclosures:No relevant conflicts of interest to declare.

Editorial introductions

Current Opinion in Hematology was launched in 1994. It is part of a successful series of review journals whose unique format is designed to provide a systematic and critical assessment of the literature as presented in the many primary journals. The field of hematology is divided into nine sections that are reviewed once a year. Each section is assigned a Section Editor, a leading authority in the area, who identifies the most important topics at that time. Here we are pleased to introduce the Section Editors for this issue. Section Editors Andrea BacigalupoAndrea BacigalupoDr Bacigalupo is the Head of the Division of Hematology and Stem Cell Transplants at Ospedale San Martino, Genova, Italy. He graduated in 1973 from the Genova Medical School, and started an allogeneic transplant program in 1976. He has conducted several clinical trials in patients with aplastic anemia and in patients undergoing allogeneic transplants. His main interests are immune control of hematopoiesis, bone marrow failure, GvHD and related complications, and graft versus leukemia reactions. He has also been interested in cytomegalovirus and Epstein–Barr virus infections, looking at early diagnosis and prospective studies on prophylaxis and pre-emptive therapy. Dr Bacigalupo has served as a board member of the European Group for Bone Marrow Transplantation (EBMT) for several years, as EBMT Secretary, Chairperson of the Aplastic Anemia Working Party and then as President for the 1998–2002 term. He served as President of the Italian Group of Bone Marrow Transplantation (GITMO) for the 2002–2005 term. He has been a member of the Italian National Health Council for two terms (2000–2006). He has served on the Regimen Related Toxicity Working Group of the Center for International Blood and Marrow Transplant Research (CIBMTR). He is a member of several national and international hematology societies and is Associate Editor of the journal Bone Marrow Transplantation and Biology of Blood and Marrow Transplantation. He serves on the Board of Haematologica and of the Journal of Clinical Oncology. Dr Bacigalupo has published over 400 articles in peer-reviewed journals. Martin L. OlssonMartin L. OlssonDr Olsson is a Professor of Transfusion Medicine at Lund University, Lund, Sweden and has been a guest scientist at the Bristol Institute of Transfusion Science and a visiting Associate Professor/Lecturer at Harvard Medical School, Boston, MA, USA. He received his medical degree and Ph.D. at Lund University. His main clinical and research interests are in the molecular genetics of red cell surface markers with a special emphasis on carbohydrate histo-blood group systems and blood typing. His scientific contributions have focused on clarifying the correlation between red cell phenotypes and the underlying genetic polymorphisms, as well as glycosyltransferase function in different blood groups. He is also interested in pathogen-related aspects of blood group antigens and different mechanisms of protection against hemolysis, including exoglycosidase-treated red cells for ABO-universal transfusion. Dr Olsson serves as Associate Editor of Transfusion Medicine and is a member of the Editorial Board of Transfusion. His awards include the Jean Julliard Prize from the International Society of Blood Transfusion, the Race and Sanger Award from the British Blood Transfusion Society, the Fernström Prize at Lund University and the Hain Foundation Prize.

Risk factors for alloimmunization in patients with sickle cell anemia

Summary Objective To determine erythrocyte phenotyping in blood donors and patients with sickle cell anemia (SS) treated at Hemocentro of Alagoas and describe the frequency and factors associated with erythrocyte alloimmunization. Methods Cross-sectional study with 102 SS patients and 100 blood donors. The following tests were performed: erythrocyte phenotyping, Direct and Indirect antiglobulin test, and detection of irregular antibodies by panel of phenotyped red blood cells. Data were compared by Mann-Whitney, qui-square or Fisher's exact tests. Factors associated with alloimmunization were studied by univariate and multiple logistic regression analysis. Results The most frequent antigens found in patients and blood donors were: c, e, M, s, JK(a). Significant differences were observed between the frequency of the phenotype of patients and donors in regard to antigens s, FY(a) and JK(b). Of 79 transfused patients, 10 presented positive Indirect Coombs. Thirteen alloantibodies were found, 7 of the Rh system, 2 of Kell and 4 were not identified. Factors associated with alloimmunization were the period of time between the last transfusion and the date of the test and more than 10 red blood cell transfusions. Patients who received more than 10 transfusions were 16.39 (95% CI: 2.23–120.59) times more likely to be alloimmunized than patients with fewer transfusions. Conclusion The prevalence of alloimmunization in SS patients was 12.7%, with 70% of antibodies belonging to the Rh and Kell systems. This study shows the importance of performing erythrocyte phenotyping in blood donors and receptors to decrease the risk of alloimmunization.

Molecular blood typing augments serologic testing and allows for enhanced matching of red blood cells for transfusion in patients with sickle cell disease

Sickle cell disease (SCD) patients have dissimilar red blood cell (RBC) phenotypes compared to the primarily Caucasian blood donor base due, in part, to underlying complex Rh and silenced Duffy expression. Gene array-based technology offers high-throughput antigen typing of blood donors and can identify patients with altered genotypes. The purpose of the study was to ascertain if RBC components drawn from predominantly Caucasian donors could provide highly antigen-matched products for molecularly typed SCD patients. SCD patients were genotyped by a molecular array (HEA Beadchip, BioArray Solutions). The extended antigen phenotype (C, c, E, e, K, k, Jk(a) , Jk(b) , Fy(a) , Fy(b) , S, s) was used to query the inventory using different matching algorithms; the resulting number of products was recorded. A mean of 96.2 RBC products was available for each patient at basic-level, 34 at mid-level, and 16.3 at high-level stringency. The number of negative antigens correlated negatively with the number of available products. The Duffy silencing mutation in the promoter region (67T>C) (GATA) was found in 96.5% of patients. Allowing Fy(b+) products for patients with GATA increased the number of available products by up to 180%, although it does not ensure prevention of Duffy antibodies in all patients. This feasibility study provides evidence that centers with primarily Caucasian donors may be able to provide highly antigen-matched products. Knowledge of the GATA status expands the inventory of antigen-matched products. Further work is needed to determine the most clinically appropriate match level for SCD patients.

Red blood cell phenotype matching for various ethnic groups

Abstract Patients requiring chronic transfusion support are at risk of alloimmunization after red blood cell (RBC) transfusion because of a disparity between donor and recipient antigen profiles. This research explored the probability of obtaining an exact extended phenotype match between blood donors randomly selected from our institution and patients randomly selected from particular ethnic groups. Blood samples from 1,000 blood donors tested by molecular method were evaluated for the predicted phenotype distribution of Rh, Kell, Kidd, Duffy, and MNS. A random subsample of 800 donor phenotypes was then evaluated for the probability of obtaining an exact match with respect to phenotype with a randomly selected patient from a particular ethnic group. Overall, there was a greater than 80 percent probability of finding an exact donor-recipient match for the K/k alleles in the Kell system. The probability ranged from 3 percent to 38 percent, depending on the ethnicity and disparities in phenotypic profiles, for the Rh, Kidd, Duffy, and MNS systems. A significant donor-recipient phenotype mismatch ratio exists with certain blood group antigens such that, with current routine ABO and D matching practices, recipients of certain ethnic groups are predisposed to alloimmunization. Immunohematology 2011; 27:12–19.

Manual Chronic Partial Exchange In Children and Adolescents with Sickle Cell Disease: Impact on Clinical Outcome and Iron Overload

AbstractAbstract 4821 Background:Red cell exchange transfusion is frequently used in the management of patients with sickle cell disease either electively or therapeutically to maintain an hemoglobin S (Hb S) level < 30–50%. This target is often difficult to maintain. In order to assess the effects of chronic partial exchange transfusion (CPET) a) on level of Hb and Hb S, b) on iron overload c) the need for chelation, d) on risk of long term adverse events and e) clinical outcome, we analyzed the data of sickle cell disease patients treated by long term CPET in our center. Methods/subjects:In the cohort of 163 SCD patients followed at University Children's Hospital at Brussels (Belgium), 10 benefit from CPET. Main reasons for CPET were neurologic disease (4), frequent ACS (3), previous severe hepatic cholestasis (2), leg ulcer (1) and pulmonary hypertension (1). The median age at start of treatment was 13 years (range 4 –19). These patients (6 males and 4 females) account for 248 exchanges during a median follow-up of 20 months (range 6– 36). These exchanges are until now performed manually and the volume exchanged is calculated taking into account the Hb level and the last HbS percentage. It is usually between 30 and 40 ml/kg BW. Except if severe anemia occurs, the goal of these exchanges is to keep a constant hematocrit level. All patients had a full red cell phenotype performed and received blood matched for ABO, Rhesus, Kell and Duffy antigens systems. The estimation of iron balance (iron intake- iron removed) was calculated yearly. Results:The pre-exchange Hb value was 9.5 g/dl (median; range: 7.7–10.9 g/dl) and the mean post value was 9.4/dl (range: 8.4– 11.1 g/dl). These values are not statistically different (p> 0.05). The majority of patients (9/10) are reached an HbS < 50% when measured 3–5 weeks after PET (just before the next procedure) with a median HbS value of 40% (range: 30–54). At start of CPET program, the median ferritin level was 439 ng/ml (range: 80 – 1704). Five patients had already a ferritin > 500 ng/mL due to numerous previous transfusions. At last evaluation, the median ferritin did not change significantly and was 531 ng/ml (range 84– 3840). The two patients with ferritin higher than 1000 ng/ml start chelation with good result for one. One The mean annual net RBC load were 1.72 ml RBC/kg/yr provided approximately 1.85 mg of iron/kg/yr. Individual data are given in table 1. CPET-treated patients exchanged showed clinical improvement with disappearance of SCD crisis and related complications. The procedure was well tolerated by most patients, and adverse effects were limited to mild hypotension (3/10). No autoimmune hemolysis or allo-immunisation was documented in this cohort. All children remained negative for HIV and hepatitis C virus infections. Conclusion: Manual CPET seems to be safe to prevent middle-term iron overload and the need of elation therapy in most of patients. CPET can therefore be recommended for SCD patients who required decreased in Hb S levels either prophylactically or therapeutically. Manual are safe, effective and easy to use when mechanized exchanges are not possible for technical reasons. Disclosures:No relevant conflicts of interest to declare.

The structural basis of blood group A-related glycolipids in an A3 red cell phenotype and a potential explanation to a serological phenomenon

Glycolipids from the red cells of a rare blood group A subgroup individual, expressing the blood group A(3) phenotype with the classical mixed-field agglutination phenomenon, A(2(539G>A))/O(1) genotype, and an unusual blood group A glycolipid profile, were submitted to a comprehensive biochemical and structural analysis. To determine the nature of blood group A glycolipids in this A(3) phenotype, structural determination was carried out with complementary techniques including proton nuclear magnetic resonance (1D and 2D), mass spectrometry (MS) (nano-electrospray ionization/quadrupole time-of-flight and tandem mass spectrometry) and thin layer chromatography with immunostaining detection. As expected, total blood group A structures were of low abundance, but contrary to expectations extended-A type 2 and A type 3 glycolipids were more dominant than A hexaglycosylceramides based on type 2 chain (A-6-2 glycolipids), which normally is the major A glycolipid. Several para-Forssman (GalNAcβ3GbO(4)) structures, including extended forms, were identified but surmised not to contribute to the classic mixed-field agglutination of the A(3) phenotype. It is proposed that the low level of A antigen combined with an absence of extended branched glycolipids may be the factor determining the mixed-field agglutination phenomenon in this individual.