Rare Red Blood Cell Research Articles

Clinico-mutational profile and the impact of splenectomy in children with glucose-6-phosphate isomerase deficiency

BackgroundThere is a lack of literature on the role of splenectomy in hemolytic anemias due to glucose-6-phosphate isomerase (GPI) deficiency. GPI deficiency is a rare red blood cell (RBC) enzymopathy of the glycolytic pathway. Most present with severe disease requiring frequent transfusions. In this study, we report the effect of splenectomy in the world's largest cohort of GPI deficiency patients. The study aimed to describe the clinical, mutational, and laboratory parameters of patients with GPI deficiency. A comparison of the transfusion requirement pre- and post-splenectomy is also included. Material and methodsThe ambispective study was performed from 2017 to 2023. Patients with congenital non-spherocytic hemolytic anemia (CNSHA) were screened for GPI deficiency. Detailed history, including demographic, clinical data, and transfusion details, were noted. Hematological parameters and RBC enzyme activity were estimated using spectrophotometry. The genetic study was done using restriction fragment length polymorphism, and confirmation was obtained through Sanger's sequencing. Patients were followed up after splenectomy. ResultsEighteen patients were diagnosed with GPI deficiency. About 3/4th (14/18; 77.7 %) had significant hepatosplenomegaly. Median serum ferritin levels were 890 ng/ml. Seven patients were on oral iron chelation. The nutritional status assessed as per the Indian Academy of Pediatrics growth charts revealed significant growth retardation. All the patients had severe anemia (mean Hb: 6.4 g/dl) and macrocytosis (mean MCV: 129.2 fl). Laboratory features of hemolysis were evident with reticulocytosis, raised serum lactate dehydrogenase, and indirect bilirubin. The mean GPI enzyme activity was 28.75 IU/g Hb.Ten (55.5 %) patients underwent splenectomy at a median age of 7 years. Five remain transfusion-free post-splenectomy at a median follow-up of 54 months. Other 5 had a significant reduction in transfusion requirement post-splenectomy (p < 0.05). Post-splenectomy, the mean hemoglobin levels increased from 6.2 to 8.1 gm%, and the mean reticulocyte counts reduced from 7.4 % to 4.8 %. Fifteen of 18 had the pathogenic c.1040G > A(p.Arg347His) homozygous mutation. ConclusionThe study demonstrates the benefit of splenectomy in patients with GPI deficiency.

Analysis of the molecular mechanism and pedigree investigation of para-Bombay phenotype caused by combined mutations at position h649 and h768 of FUT1 gene.

The para-Bombay phenotype is a rare red blood cell phenotype characterised by the lack of ABH antigens on red blood cells, but ABH substances can be found in saliva. The aim of this research was to study the mechanism of mutation of FUT1 and FUT2 genes and the pedigree of a family with the para-Bombay phenotype. The blood group was detected by a conventional serological method, H antigen adsorption-elution test, and testing saliva for A, B, and H antigens. We amplified and sequenced the ABO, FUT1, and FUT2 genes of the proband and her family using a polymerase chain reaction method, and performed TA cloning and sequencing on the amplified products of the FUT1 gene to determine its genotype. With the conventional serological method, it was found that the red blood cell phenotype of the proband and her sister lacked H antigen, while the adsorption-elution test of H antigen could detect weak H antigen. Through FUT1 cloning and sequencing, it was found that the proband had a compound heterozygous mutation of c.649G>T and c.768delC, and the genotype was FUT1*01W.24/FUT1*01N.20; the proband's father and mother had heterozygous mutations of c.768delC and c.649G>T, and their genotypes were FUT1*01N.20/FUT1*01 and FUT1*01W.24/FUT1*01. The sister's FUT1 mutation site and genotype were the same as the those of the proband. FUT2 gene sequencing revealed that the proband and sister had a synonymous mutation of c.357C>T, while their parents both had a synonymous mutation of c.357C>T and a missense mutation of c.385A>T. The Lewis blood types of the four samples all showed Le (a-b+), all of which were secretory. Blood group serology and molecular diagnostic techniques showed that the compound heterozygous mutations of the proband and her sister were inherited from their father and mother.

Immunohematology and Transfusion Support of a Patient with Acute Myeloid Leukemia and an Anti-Vel

BackgroundTransfusion support of cancer patients presenting with red blood cell (RBC) antibodies against high-incidence antigens is a challenge for hospital-based Transfusion Services. For patients with antibodies against the high-frequency Vel antigen, only 0.0004% of blood donors will be Vel antigen-negative. Anti-Vel is notorious for looking like a benign, cold-reactive IgM antibody or a “high-titer, low-avidity” antibody; however, Vel-antibodies can fix complement at body temperatures and can cause severe hemolytic transfusion reactions. The immunohematology workup and RBC transfusion support are reported for a patient who developed an anti-Vel and other RBC antibodies during chronic transfusion therapy for myelodysplastic syndrome, with progression to acute myeloid leukemia. She was also evaluated for hematopoietic stem cell transplantation (HSCT).MethodsImmunohematologic workups were performed at a regional immunohematology reference laboratory. Since the patient had a positive autocontrol and a positive direct antiglobulin test (DAT), allogeneic adsorptions were performed at 37°C using R1R1, R2R2 and rr RBC reagents. Adsorbed plasma and eluates from adsorbed RBCs were then tested for alloantibody detection using manual tube method at both LISS and PEG reactivity. In preparation for possible HSCT, RBC alloantibody titers and monocyte monolayer assay (MMA) were performed. All samples were collected with pediatric phlebotomy tubes whenever possible.ResultsThe patient was a 66 year old woman with a myelodysplastic syndrome that transformed to acute myeloid leukemia (AML) with complex karyotype a year later. The RBC reference workup identified alloantibodies reacting to the high-incidence Vel antigen as well as low-incidence Yt(b) and Cw antigens. The patient's plasma was non-reactive with three of four Vel-negative (-) cells, whereas the reactive cell was Cw positive (+) and Yt(b+). Two allo-adsorptions were performed to remove anti-Vel. Upon repeat testing, anti-Cw was identified by LISS indirect antiglobulin test. Eluate from adsorbing RBCs was reactive with Vel- and Yt(b+) cells, suggesting presence of anti-Ytb. In preparation for HSCT, anti-Vel and anti-Cw were titered at 0 and 64, respectively. MMA for anti-Vel ranged at 0.2-2% (normal <3%). Nevertheless, the hospital-based Transfusion Service provided rare ABO/Rh Vel- CW- Yt(b-) crossmatch-compatible RBC to minimize the potential for stimulating anti-Vel, which might compromise a Vel+ HSCT. The patient required about 1 RBC unit per week to maintain hemoglobin above 4.5 g/dL. The Transfusion Service coordinated a search for antigen-negative RBCs with a national rare donor program, allowing at least 4 rare RBC units to be available at all times. Over an 18-month time period, the patient received 62 RBC Vel- units: 45 out of 62 (73%) were Cw- and 17 were Cw-unscreened but C-. Later in the patient's course, her transfusion rate increased to 2 RBC units per week. Because insufficient Vel- units were available within the USA, an international search by a rare donor program identified Vel- donors in Europe. FDA approval was obtained to import the rare units. She received 2 units from Spain and 2 units from Switzerland. Her leukemia progressed, and transfusion support was discontinued.ConclusionCoordination between hospital-based Transfusion Service, hematologists, and rare donor programs is required for rare RBC transfusion support in patients with antibodies against high-incidence antigens. A transfusion threshold of 4.5 g/dL may be tolerated when availability of rare RBC units is limited. DisclosuresNo relevant conflicts of interest to declare.

Effect of cryopreservation on a rare McLeod donor red blood cell concentrate.

Abstract Units of red blood cell (RBC) concentrates with rare phenotypes are typically not included in method validation studies for cryopreservation processes; rather, they are reserved for patients with rare blood needs. Some rare RBC phenotypes may demonstrate membrane abnormalities, like acanthocytosis as observed for RBCs with the McLeod phenotype, and are specifically banked for these rare attributes; however, the impact that rare RBC phenotypes have on post-thaw quality has not been well studied. To evaluate how a rare RBC phenotype is affected by the cryopreservation process, 4 RBC units, cryopreserved in 1993 using manual methods, were selected for evaluation. These RBCs included one with the McLeod phenotype and three with phenotypes not known to cause significant membrane changes. Post-thaw, an altered deglycerolization protocol, implemented to reduce supernatant glycerol after cryopreservation, was used before processing RBCs on an automated closed system (ACP 215; Haemonetics, Boston, MA) to accommodate the use of a closed system cell processor not available when the RBC units were previously cryopreserved. RBC quality was tested at 24 hours, 7 days, and 14 days post-deglycerolization. Before deglycerolization, an extracted sample from the thawed glycerolized RBC unit was used to obtain genetic material for phenotype confirmation. Genotyping confirmed the McLeod phenotype. When comparing McLeod with non-McLeod units, RBCs from the McLeod donor exhibited acanthocytosis, higher rigidity, and lower morphology scores than RBCs from the non-McLeod units post-deglycerolization. Hemolysis, however, was comparable across all 4 units, meeting regulatory standards. Therefore, McLeod RBCs can withstand cryopreservation, suggesting that units from these donors, glycerolized using older methods, can be deglycerolized using the ACP 215 and stored hypothermically for 14 days. It was also determined that genotyping can be performed on non-leukocyte–reduced cryopreserved RBCs, allowing for confirmation of genetic profiles of donor units banked before the implementation of molecular methods.

Recent advances in the pathophysiology of PIEZO1-related hereditary xerocytosis.

Hereditary xerocytosis is a rare red blood cell disease related to gain-of-function mutations in the FAM38A gene, encoding PIEZO1, in 90% of cases; PIEZO1 is a broadly expressed mechano-transducer that plays a major role in many cell systems and tissues that respond to mechanical stress. In erythrocytes, PIEZO1 adapts the intracellular ionic content and cell hydration status to the mechanical constraints induced by the environment. Until recently, the pathophysiology of hereditary xerocytosis was mainly believed to be based on the "PIEZO1-Gardos channel axis" in erythrocytes, according to which PIEZO1-activating mutations induce a calcium influx that secondarily activates the Gardos channel, leading to potassium and water efflux and subsequently to red blood cell dehydration. However, recent studies have demonstrated additional roles for PIEZO1 during early erythropoiesis and reticulocyte maturation, as well as roles in other tissues and cells such as lymphatic vessels, hepatocytes, macrophages and platelets that may affect the pathophysiology of the disease. These findings, presented and discussed in this review, broaden our understanding of hereditary xerocytosis beyond that of primarily being a red blood cell disease and identify potential therapeutic targets.

USING DROPLET DIGITAL PCR TO SCREEN FOR RARE BLOOD DONORS: PROOF OF PRINCIPLE

Abstract Background Digital droplet PCR (ddPCR) is a very sensitive high throughput genotyping methodology. To date, the use of ddPCR in immunohematology is restricted to fetal genotyping of red blood cell antigens. Our hypothesis is that this technology could be applied to screen for rare red blood cell genotypes, such as Di(b-). Methods Nucleic acid of 3168 donors was extracted for viral screening routine in pools of 6, which were converted into three types of 48-donor pools: control pools (only DI*B/*B samples), pools with varying amount of DI*A/*B samples (n = 1–5) and a pool with one rare DI*A/*A sample. Pools were genotyped using ddPCR to detect and quantify DI*A and DI*B alleles. Results DI*A allele was accurately detected in all pools containing Di(a + b+) samples and in the pool containing one Di(a + b-) sample. No copies were detected in the control pools (n = 60). The ratio between the number of DI*A and DI*B copies varied significantly between the pools and the triplicates. Conclusion The proposed ddPCR assay was accurate in identifying the rare DI*A allele in large pools of donors and can be applied to screen for Di(b-) phenotype. The strategy can potentially be extended to search for other rare RBC phenotypes.

Using droplet digital PCR to screen for rare blood donors: Proof of principle

BackgroundDigital droplet PCR (ddPCR) is a very sensitive high throughput genotyping methodology. To date, the use of ddPCR in immunohematology is restricted to fetal genotyping of red blood cell antigens. Our hypothesis is that this technology could be applied to screen for rare red blood cell genotypes, such as Di(b-). MethodsNucleic acid of 3168 donors was extracted for viral screening routine in pools of 6, which were converted into three types of 48-donor pools: control pools (only DI*B/*B samples), pools with varying amount of DI*A/*B samples (n = 1–5) and a pool with one rare DI*A/*A sample. Pools were genotyped using ddPCR to detect and quantify DI*A and DI*B alleles. ResultsDI*A allele was accurately detected in all pools containing Di(a + b+) samples and in the pool containing one Di(a + b-) sample. No copies were detected in the control pools (n = 60). The ratio between the number of DI*A and DI*B copies varied significantly between the pools and the triplicates. ConclusionThe proposed ddPCR assay was accurate in identifying the rare DI*A allele in large pools of donors and can be applied to screen for Di(b-) phenotype. The strategy can potentially be extended to search for other rare RBC phenotypes.

Intrapartum Management of Sickle Cell Anemia With Rare Antibody and Minimal Blood Availability

Sickle cell disease (SCD) can pose serious maternal and fetal risk in pregnancy. Transfusion, both during and outside of pregnancy, can improve patient morbidity and mortality but carries risk of alloimmunization, complicating future management. This case describes a 29-year-old gravida 1, para 0 woman with sickle cell anemia and rare red blood cell alloantibody (anti-Rh46) who presented with severe vaso-occlusive crisis at 29 weeks with hemoglobin of 7.6 g/dL. Only one unit of compatible blood existed in the country. Planning for transfusion with least-incompatible blood was made. She ultimately underwent cesarean section at 31 weeks and 2 days for abnormal fetal testing. This case highlights that blood products should be utilized judiciously because their adverse effects, like alloimmunization, can increase patient morbidity and mortality. J Med Cases. 2020;11(6):157-159 doi: https://doi.org/10.14740/jmc3479

Intrapartum Management of Sickle Cell Anemia With Rare Antibody and Minimal Blood Availability

Sickle cell disease (SCD) can pose serious maternal and fetal risk in pregnancy. Transfusion, both during and outside of pregnancy, can improve patient morbidity and mortality but carries risk of alloimmunization, complicating future management. This case describes a 29-year-old gravida 1, para 0 woman with sickle cell anemia and rare red blood cell alloantibody (anti-Rh46) who presented with severe vaso-occlusive crisis at 29 weeks with hemoglobin of 7.6 g/dL. Only one unit of compatible blood existed in the country. Planning for transfusion with least-incompatible blood was made. She ultimately underwent cesarean section at 31 weeks and 2 days for abnormal fetal testing. This case highlights that blood products should be utilized judiciously because their adverse effects, like alloimmunization, can increase patient morbidity and mortality.

The screening of rare blood donors in a highly admixed population: A new approach for Holley and Diego genotyping and impact of genomic and self-reported ancestry.

The present study aimed to develop strategies for genotyping DO*HY (Dombrock system) and DI*A/DI*B (Diego system) alleles and to evaluate the impact of genomic and self-declared ancestry on rare donor screening in admixed populations. The antigens Hy and Dib demonstrate clinical importance. The lack of antisera for the serological evaluation of these antigens makes it necessary to develop molecular methods. In addition, considering that some rare red blood cell phenotypes present differences in frequency between ethnic groups, it is important to assess the applicability of self-declared ancestry in the search for rare donors in admixed populations. DO*HY and DI*A/DI*B genotyping based on real-time polymerase chain reaction (PCR) was standardised. A total of 457 blood donors clustered by self-defined skin colour/race categories were genotyped. Furthermore, individual genomic ancestry was used in the analyses. The assays developed are reproducible and provide satisfactory results even at low concentrations of DNA, which make them useful in situations where the DNA is scarce, such as dried blood spots on filter paper, or when screening for pooled samples. No significant difference was observed in the frequencies of the DI*A, DI*B and DO*HY, comparing the self-declared White (branco) donors with those who are Black (preto) and Brown (pardo). Real-time PCR, especially using pooled samples, is a promising strategy to screen rare blood donors. Although both self-reported race/colour and some blood group phenotypes are associated with ancestry, the results point to a greater complexity in the application of self-declared race/colour in the screening of rare donors in admixed populations.

Clinical Application of Massively Parallel Sequencing in the Diagnosis of Hereditary Hemolytic and Dyserythropoietic Anemias

The Hereditary Hemolytic Anemias (HHAs) are a genetically heterogeneous group of anemias characterized by decreased red blood cell (RBC) survival because of defects in hemoglobin, RBC membrane proteins or enzymes. The diagnosis of this group of disorders is complex and challenging requiring analysis of the morphology of RBCs, hemoglobin electrophoresis, and a battery of phenotypic assays. The phenotypic analysis is often problematic in transfusion dependent patients or at times of presentation with a hemolytic crisis as transfused blood or reticulocytosis confounds diagnostic testing. Molecular genetic testing has grown in popularity in the diagnosis of hereditary hemolytic anemias as it is not affected by transfusions or other clinical variables and provides additional insight into the mechanism of the disease. We have developed a Next Generation Sequencing (NGS) panel for HHA due to RBC membrane disorders and enzymopathies and congenital dyserythropoietic anemias (CDA). CDAs, although collectively rare, are included in the panel as they are occasionally misdiagnosed as hereditary spherocytosis (HS) due to their clinical characteristics of hemolysis, increased osmotic fragility, and splenomegaly albeit with inadequate reticulocytosisWe reviewed the results of 282 sequential HHA/CDA panels testing for patients with suspected HHA or CDA diagnosis, performed and interpreted at Cincinnati Children's Hospital Medical Center between 1/2013-5/2016. Forty-three samples were omitted from the final analysis due to diagnosis of other disorders, indicating that negative results were true-negatives. For the analysis of the remaining 239 panels, all results were reviewed and categorized based on the type of testing ordered: comprehensive HHA/CDA (32 genes), RBC membrane disorders (13 genes), RBC enzyme disorders (14 genes), or CDA (6 genes). The protein-coding exons plus 25 bases of exon-intron junction as well as promoter sequences were included in the design. Genomic DNA was isolated from blood and target regions were enriched using the Haloplex technology. Enriched samples were then sequenced on an Illumina MiSeq benchtop sequencer with 150 base pair, paired-end reads. Sequencing reads were aligned to the human genome reference sequence and analysis of coverage and variants was completed using NextGENe software. All positive findings were confirmed by Sanger sequencing.These 239 panels included 159 (66.5%) comprehensive HHA/CDA panels, 41 (17.2%) RBC membrane disorder panels, 10 (4.2%) RBC enzyme disorder panels, and 29 (12.1%) CDA panels. Overall, a diagnosis was confirmed or identified in 135 (56.5%) patients with specific genotype of hereditary spherocytosis in 52 patients; hereditary elliptocytosis in 15 patients; hereditary pyropoikilocytosis in 7 patients; hereditary stomatocytosis/xerocytosis in 12 patients; South East Asian Ovalocytosis in 1 patient; G6PD deficiency in 15 patients; pyruvate kinase deficiency in 17 patients; other rare RBC enzymopathies in 6 patients; and CDA in 10 patients. The clinical performance of RBC membrane disorder and RBC enzyme disorder panels were comparable between 68-70% in reaching a final diagnosis, while CDA panel confirmed final diagnosis in only 20% of suspected cases. The overall low prevalence, complexity of diagnosis with findings of dyserythropoiesis in bone marrow studies in patients with severe HHA, and evidence of locus heterogeneity in CDA might explain this result. Among patients with suspected RBC membrane disorders, approximately 14% were eventually diagnosed with hereditary xerocytosis (HX). HX diagnosis is critical to make in such patients since splenectomy is contraindicated due to the high risk of life-threatening thrombophilia complications.In more than half (56.5%) of all cases with suspected hereditary hemolytic anemia, genetic testing provided or confirmed the diagnosis and optimized patients' clinical management. Further genetic counseling and testing for other at-risk family members was made possible by achieving molecular diagnosis. Genetic testing substantially altered management in approximately 14% of cases with suspected RBC membrane disorders due to the diagnosis of HX. In conclusion, genetic testing has a significant clinical utility and may facilitate and improve diagnosis, prognosis and management considerations in patients with hereditary hemolytic or dyserythropoietic anemia. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Recombinant blood group proteins in clinical practice – from puzzling to binary antibody testing

The main drawback of red blood cell (RBC)‐based antibody detection assays is that a positive reaction between serum and test cells does not identify the specificity of a given red blood cell antibody. When antibody mixtures or rare RBC antibodies are present, this indirect method reaches its limits. Recently, CE‐marked recombinant blood group proteins (rBGPs) have become available in sufficient quantity and quality. The advantage of rBGPs over RBCs is that proteins carrying a single antigen can be used in the antibody identification assay, so a positive test directly indicates the presence and specificity of the target antibody. The use of soluble rBGPs in addition to RBCs has been shown to increase the sensitivity and specificity of antibody detection and identification in cases with difficult‐to‐identify antibodies, resulting in increased efficiency in providing blood to immunized patients. The rBGPs have been successfully used in a wide range of solid‐phase assays (ELISA, microspheres, microarrays). Although all relevant antigen specificities have not yet been established, rBGPs have a diagnostic potential that could already be useful in antibody detection assays combining RBC‐derived antigens and recombinantly expressed antigens. Such combined assays would benefit from the potential of rBGPs for single‐step, direct antibody detection and identification, which could greatly facilitate and accelerate the identification of common and rare RBC antibodies. The clinical use of rBGPs in pretransfusion antibody screening would introduce a paradigm shift that could help to reduce the risk of haemolytic transfusion reaction, one of the leading causes of transfusion‐related death.

Changing patterns in rare red blood cell (RBC) phenotypes, and antibodies – results from melbourne RBC reference laboratory

Aim Red cell antigen phenotypes vary between different ethnic groups. The demographics of the Australian population have changed with waves of immigration. We examined whether this impacts observed patterns of red cell phenotypes and allo-antibodies. Methods We reviewed results on patient samples referred to our laboratory in two cohort periods: 1997 and 2012. Results For the 2 cohort years 1997 and 2012 we observed: (242, 537) antibodies identified in (148, 351) patient samples. Antibody frequencies: Rh (28%, 30%), K (7%, 4%), Fy (7%, 5%), Jk (6%, 4%), ≥2 alloantibodies (41%, 38%). Three patients were identified with the Jk(a-b-) phenotype in 1997 (2%), and six in 2012 (1.7%). For the Fy(a-b-) phenotype, in 1997 no patients were reported. In 2012 there were seven patients with this phenotype. Conclusion Comparing our 1997 and 2012 cohorts, we observed no major change in the pattern of the clinically significant red cell allo-antibodies. There was an increased number of patients with Fy(a-b-) phenotype. This likely reflects immigration from Africa over the last decade. Providing compatible blood for these patients can be challenging. We encourage blood donation from diverse ethnic groups, to enrich the range of red cell phenotypes available to patients.

Recombinant blood group proteins facilitate the detection of alloantibodies to high‐prevalence antigens and reveal underlying antibodies: results of an international study

Alloantibodies to high-prevalence red blood cell (RBC) antigens are not easily identified by routine serologic techniques. This multicenter study was conducted to test the effectiveness of recombinant blood group proteins (rBGPs) at regional and international RBC reference laboratories. Single or mixed soluble rBGPs (Lu, Yt, Kn, JMH, Sc, Rg, Ch, Do, and Cr) were assessed for their ability to inhibit the reactivity of antibodies to specific antigens. Initially, the effect of rBGPs was validated by testing panels of well-characterized patient serum samples containing antibodies to high-prevalence antigens in the hemagglutination inhibition assay. Subsequently, the rBGPs were prospectively used for routine antibody identification and the results were compared to those obtained with RBC-based diagnostics. Panels of predefined antibodies to high-prevalence antigens were completely and specifically neutralized by the corresponding rBGP specificities. For prospective identification, antibodies to high-prevalence antigens (n = 62) were specifically inhibited by the corresponding rBGP specificities except for some Complement Receptor 1-related antibodies, which may be directed to epitopes not expressed on the truncated recombinant Kn. In 14 cases, additional clinically relevant alloantibodies were identified. In cross-matching, the rBGPs were successfully used to inhibit the reactivity of clinically irrelevant antibodies to high-prevalence antigens to determine compatibility between donor and recipient. rBGPs enable the identification of antibodies to high-prevalence antigens without the need for rare RBC reagents, which are often unavailable. Underlying antibodies can be reliably detected and cross-matching results validated, resulting in a more efficient blood supply for immunized patients.

Transfusion Of Thawed Rare Red Blood Cell Units In Finland

The Finnish Red Cross Blood Service performs blood grouping of all blood donations in Finland and serves as a national reference laboratory in pre-transfusion testing of patients. In addition to ABO and RhD blood groups, other Rh antigens and K antigen are also determined from all red cell units. The antigens of JK, FY, and MNS blood group systems are analyzed from selected donors. The extensive donor typing procedure includes antigens from KEL, LW, LU, CO, DO, DI, YT, GE and CROM blood group systems, performed either serologically or by genotyping. Patients negative for a high frequency blood group antigen present a challenge for transfusion laboratories. A blood group is considered rare if the prevalence is 1:1000 or less, with the most common rare phenotypes in Finland being LWa neg, Jk:-3 and Pk. To ensure the availability of matching blood for patients with a rare blood group, we started a freezing program of rare blood in Finland with Haemonetics ACP 215 process in April 2010. The system is closed, therefore after thawing the units are safe for use for 7 days. 18 blood groups which were included in the program were determined based on the known rare blood groups in the Finnish population. Blood groups which are globally rare but more common in Finland, such as Jk:-3 and LWa neg, are represented in our storage and also available internationally. The aim of the present study was to analyze the status of the freezing program of rare red blood cells in Finland. For the analysis we asked the transfusion data of rare red blood cell units delivered to 10 national and 2 international hospitals. Results: In 2010-2012, altogether 204 units of rare blood were stored, including units of all the predefined rare phenotypes except Vel neg. With the exception of Vel neg, Hrs neg and Oh blood groups, we have been able to meet the need for rare blood in Finland using Finnish donors. Altogether 55 units have been thawed and distributed to 27 adult patients. The indication was delivery in 3 cases, surgery in 6 cases, and chronic anemia in 4 cases. For the present analyses the transfusion data of 49 units was available. The data of 6 Coa neg units is missing. Altogether 22 / 49 (45%) of the units were transfused. In 14 cases the blood was transfused to the patient it was intended for, and in 8 cases to another patient, 27 units were discarded. There were no transfusion associated complications. The hemoglobin response was evaluable in 7 patients. In 4 patients with a chronic anemia the median blood hemoglobin response per a unit was +8 g/l, range +2 - +16 g/l. In 3 patients with blood loss during an operation the median increase in blood hemoglobin value was +7 g/l per unit, range +4 – +12 g/l. Conclusions: Compared to the common 24 hour eligibility of thawed red cell units, our units valid for 7 days are preferable in countries like Finland with long distances and in international rare blood deliveries. We have been able to offer rare blood to Finnish patients. The hemoglobin response was reasonable and there were no transfusion associated complications. Our next challenge will be to recruit Finnish blood donors with different ethnic backgrounds (eg. immigrants) and identify their rare blood groups. We have already expanded our program to include rare combinations of common blood groups. Disclosures: No relevant conflicts of interest to declare.

Defining the clinical need for rare red blood cells

Transfusion support of patients with antibodies against high frequency (HF) antigens or a combination of antibodies that is rare is challenging and requires multidisciplinary cooperation.The treating physician should decide if there is a definitive clinical need for red blood cell (RBC) transfusion for the specific patient. According to recent clinical trials RBC transfusion is usually not indicated if the patient is haemodynamically stable and the haemoglobin (Hb) is higher than 7 g/dl. Alternative strategies for patient support might be required if rare blood is unavailable or while awaiting rare RBC unit arrival from national or international sources.

First description of phosphofructokinase deficiency in spain: identification of a novel homozygous missense mutation in the PFKM gene

Phosphofructokinase deficiency is a very rare autosomal recessive disorder, which belongs to group of rare inborn errors of metabolism called glycogen storage disease. Here we report on a new mutation in the phosphofructokinase (PFK) gene PFKM identified in a 65-years-old woman who suffered from lifelong intermittent muscle weakness and painful spasms of random occurrence, episodic dark urines, and slight haemolytic anemia. After ruling out the most common causes of chronic haemolytic anemia, the study of a panel of 24 enzyme activities showed a markedly decreased PFK activity in red blood cells (RBCs) from the patient. DNA sequence analysis of the PFKM gene subsequently revealed a novel homozygous mutation: c.926A>G; p.Asp309Gly. This mutation is predicted to severely affect enzyme catalysis thereby accounting for the observed enzyme deficiency. This case represents a prime example of classical PFK deficiency and is the first reported case of this very rare red blood cell disorder in Spain.

Recombinant blood group proteins for use in antibody screening and identification tests

The present review elucidates the potentials of recombinant blood group proteins (BGPs) for red blood cell (RBC) antibody detection and identification in pretransfusion testing and the achievements in this field so far. Many BGPs have been eukaryotically and prokaryotically expressed in sufficient quantity and quality for RBC antibody testing. Recombinant BGPs can be incorporated in soluble protein reagents or solid-phase assays such as ELISA, color-coded microsphere and protein microarray chip-based techniques. Because novel recombinant protein-based assays use single antigens, a positive reaction of a serum with the recombinant protein directly indicates the presence and specificity of the target antibody. Inversely, conventional RBC-based assays use panels of human RBCs carrying a huge number of blood group antigens at the same time and require negative reactions of samples with antigen-negative cells for indirect determination of antibody specificity. Because of their capacity for single-step, direct RBC antibody determination, recombinant protein-based assays may greatly facilitate and accelerate the identification of common and rare RBC antibodies.

Rapid detection of JMH antibodies with recombinant Sema7A (CD108) protein and the particle gel immunoassay

At present, identification of antibodies against the high-prevalence JMH antigen is difficult and limited to reference laboratories having panels of rare red blood cell (RBC) specimens in stock. Here, a novel method is described for detection of anti-JMH with particles coated with recombinant semaphorin 7A (Sema7A, CD108), the protein that carries the JMH blood group antigens. Recombinant Sema7A protein was generated and coupled onto superparamagnetic particles coated with streptavidin. The coated particles were tested in the presence of different serum and plasma samples (11 anti-JMH, 20 other antibodies, and 50 samples from nonimmunized blood donors) with the particle gel immunoassay and flow cytometry. Sema7A-coated particles reacted with all 11 samples containing anti-JMH, but not with samples lacking anti-JMH. In addition, the anti-JMH agglutination scores were higher with Sema7A-coated particles than with JMH-positive RBCs in all cases. Recombinant blood group proteins have the potential to replace RBCs as antigen carriers for identification of certain RBC alloantibodies.

Prokaryotic versus eukaryotic recombinant Lutheran blood group protein for antibody identification

At present, identification of antibodies against high-frequency antigens is limited to reference laboratories having panels of rare red blood cell (RBC) specimens in stock. Antibodies against Lu(b) are among the most frequent clinically relevant antibody specificities directed against high-frequency antigens. Soluble recombinant Lu(b) fusion proteins consisting of the first three N-terminal immunoglobulin superfamily domains and a V5-His tag were generated. Eukaryotic recombinant Lu(b) proteins were isolated from cell culture supernatant of stably transfected HEK293 cells with anti-V5 Sepharose. Prokaryotic Lu(b) fusion proteins were expressed in Escherichia coli, purified by Ni-NTA, and refolded by chromatographic procedures. Ten anti-Lu(b) serum samples, 6 anti-Lu(a) serum samples, 30 serum samples directed against other blood group antigens, 10 serum samples from patients with RBC autoantibodies, and 100 serum samples from randomly selected donors were used for antibody screening. Eukaryotic and prokaryotic recombinant Lu(b) proteins proved to be equally suited for identification of anti-Lu(b). Recombinant Lu(b) protein-based enzyme-linked immunosorbent assay correctly identified samples containing anti-Lu(b) sera, and the titers were at least two times higher than those measured by the gel agglutination-based indirect antiglobulin test. In hemagglutination inhibition assays, recombinant Lu(b) protein neutralized all anti-Lu(b), but none of the other alloantibodies decreased in reactivity. Antibody detection systems based on soluble eukaryotic or prokaryotic recombinant blood group proteins have the potential to replace current systems with rare RBCs for identification of alloantibodies against high- or low-frequency antigens. This innovation could bring routine laboratories one step closer to specialized antibody diagnostics.