International Blood Group Reference Laboratory Research Articles

Defining and finding the rare donor

Rare blood is generally defined as one that occurs at a frequency of 1:100~1000 individuals or less, and it is sometimes difficult to provide such blood types to patients because of their rarities. The Japanese Red Cross (JRC) Society lists 46 rare blood phenotypes that are divided into two categories. The rare blood types listed in Category I occur much less frequently than those listed in Category II. We screen for rare blood cells using monoclonal antibodies (MoAbs). Since 1987, our blood centre has established 93 MoAbs (32 human and 61 murine) and has provided them to the other blood centres. Many of IgG MoAbs are available on the machine by saline or bromelin method by cross‐linking with anti‐human or anti‐mouse IgG. Thus, more than 10 000 donors with rare blood phenotypes (Category I, 748; Category II, 9314) are registered in Japan. We freeze rare blood, particularly Category I types. Since 1977, a total of 576 units of rare blood with phenotypes Di(b−), D−−, Jr(a−), Ko and Lan‐, etc. have been supplied to 23 international countries. Thus, the JRC contributes to the International Panel of Donors of Rare Blood Type (IDP) which is maintained by the International Blood Group Reference Laboratory (IBGRL) in Bristol, UK. The IDP provides information on the location of rare blood donors when they cannot be found in their respective countries. We also joined the ISBT Working Party on Rare Donors which handles all matters related to rare blood. Our rare blood donor programme is successful because of international co‐operation.

Rare blood donors with irregular antibodies

Blood groups are inherited biological characteristics that do not change throughout life in healthy people. Blood groups represent antigens found on the surface of red blood cells. Kell blood group system consists of 31 antigens. Kell antigen (K) is present in 0.2% of the population (the rare blood group). Cellano antigen is present in more than 99% (the high-frequency antigen). These antigens have a distinct ability to cause an immune response in the people after blood transfusion or pregnancy who, otherwise, did not have them before. This paper presents a blood donor with a rare blood group, who was found to have an irregular antibody against red blood cells by indirect antiglobulin test. Further testing determined the specificity of antibody to be anti-Cellano. The detected antibody was found in high titers (1024) with erythrocyte phenotype Kell-Cellano+. The blood donor was found to have a rare blood group KellKell. This donor was excluded from further blood donation. It is difficult to find compatible blood for a person who has developed an antibody to the high-frequency antigen. The donor's family members were tested and Cellano antigen was detected in her husband and child. A potential blood donor was not found among the family members. There was only one blood donor in the Register of blood donors who was compatible in the ABO and Kell blood group system. For the successful management of blood transfusion it is necessary to establish a unified national register of donors of rare blood groups and cooperate with the International Blood Group Reference Laboratory in Bristol with the database that registers donors of rare blood groups from around the world.

Effect of High Throughput RHD Typing of Fetal DNA in Maternal Plasma on Use of Anti-RhD Immunoglobulin in RhD Negative Pregnant Women: Prospective Feasibility Study

Objectives To assess the feasibility of applying a high throughput method, with an automated robotic technique, for predicting fetal RhD phenotype from fetal DNA in the plasma of RhD negative pregnant women to avoid unnecessary treatment with anti-RhD immunoglobulin. Design Prospective comparison of fetal RHD genotype determined from fetal DNA in maternal plasma with the serologically determined fetal RhD phenotype from cord blood. Setting Antenatal clinics and antenatal testing laboratories in the Midlands and north of England and an international blood group reference laboratory. Participants Pregnant women of known gestation identified as RhD negative by an antenatal testing laboratory. Samples from 1997 women were taken at or before the 28 week antenatal visit. Main outcome measures Detection rate of fetal RhD from maternal plasma, error rate, false positive rate, and the odds of being affected given a positive result. Results Serologically determined RhD phenotypes were obtained from 1869 cord blood samples. In 95.7% (n=1788) the correct fetal RhD phenotype was predicted by the genotyping tests. In 3.4% (n=64) results were either unobtainable or inconclusive. A false positive result was obtained in 0.8% (14 samples), probably because of unexpressed or weakly expressed fetal RHD genes. In only three samples (0.2%) were false negative results obtained. If these results had been applied as a guide to treatment, only 2% of the women would have received anti-RhD unnecessarily, compared with 38% without the genotyping. Conclusions High throughput RHD genotyping of fetuses in all RhD negative women is feasible and would substantially reduce unnecessary administration of anti-RhD immunoglobulin to RhD negative pregnant women with an RhD negative fetus.

The utilization of rare blood donors

When transfusion is needed for a patient with a rare blood type and the corresponding antibody, it can be challenging and lead to delays in transfusion. Sometimes, the blood cannot be found. Globally, the community of rare donor facilities is extremely collaborative and is quickly engaged in locating and delivering blood to the patients in need of rare blood types. Identifying the rare donor is a resource‐intensive activity in every country especially in times of shrinking human and reagent resources. This paper will discuss the process flow for obtaining blood for patient's requiring rare types and the critical steps for each facility in the supply chain. The local facility plays a vital role in the knowledge of patient need and interaction with the patient's physician. The national facility that coordinates obtaining rare blood internationally also plays a vital role. The International Blood Group Reference Laboratory in Bristol as one of its roles, maintains the International Rare Donor Panel and also plays a vital role. These three roles hold the responsibility for collaboration to identify and obtain blood for patients in need. While there are differences in each country's definition of rare blood types, all are in agreement to assist when a country needs blood products. The International Society Blood Transfusion working party for rare donors plays a critical, collaborative role in developing processes between countries to further rare blood transfers between countries and providing expertise when needed. Outcome data are difficult to obtain, therefore success and ultimate improvement is a challenge. This challenge is one that the needs to be met to improve patient outcomes in cases where rare blood is needed throughout the world.

International Society of Blood Transfusion Committee on Terminology for Red Cell Surface Antigens: Cape Town report

International Society of Blood Transfusion Committee on Terminology for Red Cell Surface Antigens: Cape Town report

Fetal Blood Group Genotyping

Prediction of fetal blood group from DNA is usually performed when the mother has antibodies to RhD, to assess whether the fetus is at risk from hemolytic disease of the fetus and newborn (HDFN). Over the last five years RhD testing on fetal DNA in maternal plasma has been introduced. At the International Blood Group Reference Laboratory (IBGRL) we employ real-time quantitative polymerase chain reaction (RQ-PCR) to detect RHD exons 4, 5, and 10, which also reveals RHDpsi. SRY and, in RhD-negative (RhD-) females, eight biallelic polymorphisms are incorporated in an attempt to provide an internal positive control. Since 2000 we have tested 533 pregnancies for RhD. In 327 pregnancies where the RhD of the infant is known, we had one false-positive and one false-negative result. In 2004 we introduced fetal typing from DNA in maternal plasma for K, Rhc, and RhE, which represent single nucleotide polymorphisms (SNPs) on the KEL and RHCE genes. We have begun trials on an automated method for fetal RhD typing from DNA in maternal plasma. This is designed to test fetal RhD in all pregnant RhD- women, to identify the 40% with an RhD- fetus so that antenatal RhD immunoglobulin (Ig) prophylaxis can be avoided. Similar trials have already been reported by Sanquin Research Laboratories in Amsterdam.

Tumor Cells Enhance Their Own CD44 Cleavage and Motility by Generating Hyaluronan Fragments

Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that interacts with cell-surface receptors, including CD44. Although HA usually exists as a high molecular mass polymer, HA of a much lower molecular mass that shows a variety of biological activities can be detected under certain pathological conditions, particularly in tumors. We previously reported that low molecular weight HAs (LMW-HAs) of a certain size range induce the proteolytic cleavage of CD44 from the surface of tumor cells and promote tumor cell migration in a CD44-dependent manner. Here, we show that MIA PaCa-2, a human pancreatic carcinoma cell line, secreted hyaluronidases abundantly and generated readily detectable levels of LMW-HAs ranging from approximately 10- to 40-mers. This occurred in the absence of any exogenous stimulation. The tumor-derived HA oligosaccharides were able to enhance CD44 cleavage and tumor cell motility. Inhibition of the CD44-HA interaction resulted in the complete abrogation of these cellular events. These results are consistent with the concept that tumor cells generate HA oligosaccha-rides that bind to tumor cell CD44 through the expression of their own constitutive hyaluronidases. This enhances their own CD44 cleavage and cell motility, which would subsequently promote tumor progression. Such an autocrine/paracrine-like process may represent a novel activation mechanism that would facilitate and promote the malignant potential of tumor cells.

Mechanisms Involved in Enhancement of Osteoclast Formation and Function by Low Molecular Weight Hyaluronic Acid

Hyaluronic acid (HA) is a component of the extracellular matrix that has been shown to play an important role in bone formation, resorption, and mineralization both in vivo and in vitro. We examined the effects of HA at several molecular weights on osteoclast formation and function induced by RANKL (receptor activator of NF-kappa B ligand) in a mouse monocyte cell line (RAW 264.7). HA at M(r) < 8,000 (low molecular weight HA (LMW-HA)) enhanced tartrate-resistant acid phosphatase-positive multinucleated cell formation and tartrate-resistant acid phosphatase activity induced by RANKL in a dose-dependent manner, whereas HA at M(r) > 900,000 (high molecular weight HA (HMW-HA)) showed no effect on osteoclast differentiation. LMW-HA enhanced pit formation induced by RAW 264.7 cells, whereas HMW-HA did not, and LMW-HA stimulated the expression of RANK (receptor activator of NF-kappa B) protein in RAW 264.7 cells. In addition, we found that LMW-HA enhanced the levels of c-Src protein and phosphorylation of ERKs and p38 MAPK in RAW 264.7 cells stimulated with RANKL, whereas the p38 MAPK inhibitor SB203580 inhibited RANKL-induced osteoclast differentiation. This enhancement of c-Src and RANK proteins induced by LMW-HA was inhibited by CD44 function-blocking monoclonal antibody. These results indicate that LMW-HA plays an important role in osteoclast differentiation and function through the interaction of RANKL and RANK.

Genetic basis of the K0 phenotype in the Swedish population

The absence of all Kell blood group antigens (K(0) phenotype) is very rare. K(0) persons, however, can produce clinically significant anti-Ku (K5) after transfusion and/or pregnancy and require K(0) blood for transfusion. Ten alleles giving rise to the K(0) phenotype have been reported: different populations were studied although none from Scandinavia. Three K(0) samples were identified by blood banks in Sweden (Uppsala, Umeå, and Linköping) during a 20-year period. Kell antigen typing was performed with standard serologic techniques by the respective blood banks and K(0) status was confirmed by the International Blood Group Reference Laboratory in Bristol, England. Polymerase chain reaction and DNA sequencing of the KEL coding region (exons 1-19) was performed on genomic DNA. The Uppsala K(0) was homozygous for a 1540C>T substitution in exon 13, leading to an immediate stop codon. The Umeå K(0) was homozygous for 1023delG in exon 8 that results in a frameshift and a premature stop codon in exon 9. In the Linköping K(0), a previously reported mutation g>a at +1 of intron 3 was found. Two novel and one previously reported null alleles at the KEL locus are described. The identified nonsense mutations abolish expression of the Kell glycoprotein and are thus responsible for the K(0) phenotype in these Swedish families.

Reliable test for prenatal prediction of fetal RhD type using maternal plasma from RhD negative women

The objective of this study was to establish a reliable test for prenatal prediction of fetal RhD type using maternal plasma from RhD negative women. This test is needed for future prenatal Rh prophylaxis. A novel real-time PCR-based assay targeting RHD exon 7 combined with a published assay for RHD exon 10 were used to determine the fetal RHD status in DNA extracted from plasma, sampled from 56 pregnant RhD negative women in 15th-36th week of gestation. Thirty-eight samples were from ongoing pregnancies of Danish women and 21 samples from 18 pregnant women were stored anonymized samples from the International Blood Group Reference Laboratory, Bristol, United Kingdom. Prediction of fetal RhD type was compared with the serological result obtained after birth. The prediction of the fetal RhD type was in 100% concordance with the serological RhD type from the 16th week of gestation. One sample from the 15th week of gestation was inconclusive. The number of copies of fetal RHD DNA was found to increase with gestational age. Low levels of DNA were found to follow the Poisson distribution (p = 1.0000). Our set-up was very reliable for determination of fetal RhD genotype, and thus will be of value in prenatal Rh prophylaxis and in the management of immunized women.

Blood group terminology 2004: from the International Society of Blood Transfusion committee on terminology for red cell surface antigens

1 Bristol Institute for Transfusion Sciences, Bristol, UK 2 Growing your Knowledge, Spit Junction, NSW, Australia 3 American Red Cross Blood Services, Los Angeles-Orange Counties Region, Los Angeles, CA, USA 4 Biotechnology Research Centre, Auckland University of Technology, Auckland, New Zealand 5 Regional Blood Transfusion Center, Department of Clinical Immunology, University Hospital, Arhus N, Denmark 6 Department of Pathology, University Hospitals UH-2G332, Ann Arbor, Michigan, USA 7 Reference Laboratory for Immunohematology and Blood Groups, National Blood Services Centre, Tel Hashomer, Israel 8 New York Blood Center, New York, NY, USA 9 Ortho-Clinical Diagnostics, Raritan, NJ, USA 10 Drexel University College of Medicine, Philadelphia, PA, USA 11 Gamma Biologicals Inc (subsidiary of Immunocor Inc), Houston, TX, USA 12 Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, the Netherlands 13 Centre national de Reference pour les Groupes sanguines (CNTS), Paris, France 14 International Blood Group Reference Laboratory, Bristol, UK 15 Finnish Red Cross Blood Transfusion Service, Helsinki, Finland 16 South African National Blood Service, East Coast Region, Pinetown, South Africa 17 Osaka Red Cross Blood Center, Osaka, Japan 18 Blood Bank, Hospital Sirio-Libanes, Sao Paulo, Brazil 19 Rh Laboratory, University of Manitoba, Winnipeg, Manitoba, Canada

A clinical service in the UK to predict fetal Rh (Rhesus) D blood group using free fetal DNA in maternal plasma.

Antenatal determination of fetal blood group is important in pregnancies with a significant risk of hemolytic anemia due to maternal alloimmunization. The International Blood Group Reference Laboratory (part of the National Blood Service) in Bristol, UK, provides a fetal blood group genotyping service to obstetricians caring for immunized pregnant women with heterozygous partners. Since 2001, fetal D typing has been offered using free fetal DNA in maternal plasma. Real-time polymerase chain reaction (PCR) assays are performed to detect the RHD gene. To confirm the presence of fetal DNA when RHD is not detected, Y-chromosome sequences are targeted. When a D-negative female fetus is predicted, maternal buffy coat DNA is tested for eight insertion/deletion polymorphisms. Sequences that are absent from the maternal genome are then targeted in maternal plasma and are used to confirm the presence of free fetal DNA in the blood sample. Currently, 283 pregnancies have been tested, of which 50 are awaiting confirmatory results. Fetal D status was correctly predicted in 223 cases, and no result was obtainable in 7 cases. In three cases, serology on cord blood was discrepant with reported results, but all fetuses had received multiple intrauterine transfusions. The new test has significantly reduced the number of invasive procedures carried out in the UK for fetal D grouping. Antenatal anti-D prophylaxis is currently being introduced in the UK to all D-negative women; in the future, detection of fetal RHD sequences in maternal plasma may allow anti-D to be restricted to pregnancies involving a D-positive fetus.

Defective Urinary Concentrating Ability Due to a Complete Deficiency of Aquaporin-1

Aquaporin-1, the archetypal water-channel protein,1 was initially identified in red cells and renal proximal tubular epithelium.2 The gene for aquaporin-1 (AQP1) on chromosome 7 colocalizes with the Colton blood-group antigen,3,4 and the Colton blood-group antigen polymorphism was identified as a substitution of a single amino acid in an extracellular domain of aquaporin-1.5 The International Blood Group Reference Laboratory has confirmed the existence of only six kindreds who lack the Colton blood group. Members of three of these kindreds were found to be homozygous for different mutations in the AQP1 gene, and their red-cell membranes had a complete . . .

Historical Note: Past, Present and Future of the Antiglobulin Test

This is a review of two invited talks on the antiglobulin reaction on the occasion of the 50th Anniversaries of the International Blood Group Reference Laboratory, now at Bristol, and the National Blood Service, London, and coincidentally the 51st anniversary of the antiglobulin test! The first talk (as specially requested) is a very personal reminiscence of the discovery, with Rob Race and Arthur Mourant, of the antiglobulin test in blood group serology. The second talk traces developments in antiglobulin testing in general immunology using, of course, isotype-specific antiglobulin reagents as and when they became available. Special emphasis was given to: (1) testing for poorly agglutinating bacterial antibodies; (2) special procedures for measuring reaginic (IgE) antibodies to various allergens; (3) the incorporation of an antiglobulin step or stage in many of the routine automated immunoassay procedures; (4) the special experimental procedures in the form of mixed antiglobulin reactions to reveal antibodies to the surface antigens on tissue cells, and, finally, (5) by chemically coupling antiglobulin to red blood cells, a distinctive integrated immunoassay system was developed, enabling reverse passive haemagglutination as an assay for immunoglobulins, rosetting reactions to reveal native IgG receptors on lymphocytes or antibody immunoglobulin reacting with CD marker antigens; this same reagent, namely red-cell-labelled antiglobulin, can be used to detect antibodies reacting with either bacteria or antigens adsorbed on the walls of wells of microtitre plates. The need for improved stabilization and preservation of the antiglobulin-linked red cells to prolong their 'shelf-life' is stressed.

Pitching it low

<b>Objectives</b> To assess the feasibility of applying a high throughput method, with an automated robotic technique, for predicting fetal RhD phenotype from fetal DNA in the plasma of RhD negative pregnant women to avoid unnecessary treatment with anti-RhD immunoglobulin. <b>Design</b> Prospective comparison of fetal <i>RHD</i> genotype determined from fetal DNA in maternal plasma with the serologically determined fetal RhD phenotype from cord blood. <b>Setting</b> Antenatal clinics and antenatal testing laboratories in the Midlands and north of England and an international blood group reference laboratory. <b>Participants</b> Pregnant women of known gestation identified as RhD negative by an antenatal testing laboratory. Samples from 1997 women were taken at or before the 28 week antenatal visit. <b>Main outcome measures</b> Detection rate of fetal RhD from maternal plasma, error rate, false positive rate, and the odds of being affected given a positive result. <b>Results</b> Serologically determined RhD phenotypes were obtained from 1869 cord blood samples. In 95.7% (n=1788) the correct fetal RhD phenotype was predicted by the genotyping tests. In 3.4% (n=64) results were either unobtainable or inconclusive. A false positive result was obtained in 0.8% (14 samples), probably because of unexpressed or weakly expressed fetal <i>RHD</i> genes. In only three samples (0.2%) were false negative results obtained. If these results had been applied as a guide to treatment, only 2% of the women would have received anti-RhD unnecessarily, compared with 38% without the genotyping. <b>Conclusions</b> High throughput <i>RHD</i> genotyping of fetuses in all RhD negative women is feasible and would substantially reduce unnecessary administration of anti-RhD immunoglobulin to RhD negative pregnant women with an RhD negative fetus.

Use of a modified acid/EDTA elution technique

Abstract A modification of the acid/EDTA elution technique was recently developed at the International Blood Group Reference Laboratory, Bristol, UK. By altering the volumes of reagents used, maximization of elution of antibody could be achieved without loss of red cel] integrity. Immunobematology 1991;2:46–47.

Standardisation and control of blood-typing sera International and national organizations

The history of biological standardization, with the application of the principles to blood-typing serum, and the uses to which a standard preparation of blood-typing serum may be put have been considered. Reasons have been given for preferring « Unitto « Titreas a measure of agglutinating activity. International Standards for blood-typing sera that already exist have been mentioned and the procedures to be followed in producing new standards have been described. Some of the problems that may be encountered when blood grouping reagents are controlled have also been discussed. The author would like to thank Dr A. E. Mourant for all the help and advice that he has given with regard to the principles involved in biological standardization and he is most grateful to Dr E. W. Ikin who has personally undertaken all the practical work involved with regard to this matter at the International Blood Group Reference Laboratory. He is also very indebted for all the advice and co-operation that he has received from Dr D. R. Bangham, Director of the International Laboratory for Biological Standards, National Institute for Medical Research. L'auteur rappelle l'historique de la standardisation biologique et envisage l'application de ses principes à la détermination des groupes sanguins érythrocytaires en précisant les caractéristiques et l'usage d'un sérum-test standard. Il donne les raisons qui lui font préférer la notion d' « unitéà celle de « titrepour définir l'activité agglutinante d'un sérum. Les standards internationaux existant pour la détermination des groupes sanguins érythrocytaires sont mentionnés et l'auteur explique comment procéder pour produire de nouveaux standards. Il discute les principaux problèmes qui peuvent se poser lors du contrôle des sérums-tests.