Alloimmunisation in thalassaemics: a comparison between recipients of usual matched and partial better matched blood. An evaluation at a tertiary care centre in India.

BACKGROUND: There is an ongoing controversy regarding provision of usually matched blood (i.e. matched for ABO-D antigens) or phenotypically matched blood (also matched for Rh and Kell antigens) for multiply transfused thalassaemics, especially in developing countries. A pilot study conducted at our centre revealed an alloimmunisation rate of 3.79% with Rh and Kell alloantibodies accounting for 90% of all antibodies. The present cross-sectional study was conducted to assess the impact of a policy of partial better matching (for Rh cDE and Kell antigens) of blood on alloimmunisation in thalassaemics. MATERIAL AND METHODS: In this cross-sectional study three groups of patients were considered. Group 1 comprised 211 thalassaemics who received usually matched (UM) blood until April 2009. Their rates of alloimmunisation have already been published in a prior study. Group 2 consisted of 46 thalassaemics who were enrolled after April 2009 and have received partially better matched (PBM) blood (matched for ABO, Rh cDE and Kell antigens) since the initiation of transfusion therapy. Group 3 (UM→PBM) comprised the patients from group 1 who, from April 2009, were given partial better matched blood. Antibody screening (using a 3-cell panel) and antibody identification (11-cell panel) were carried out to detect the presence of alloantibodies. RESULTS: None of the thalassaemic patients in group 2 (PBM) developed alloantibodies. Eight thalassaemics in group 3 (UM→PBM) developed new alloantibodies (after April 2009). DISCUSSION: According to the results of the present study, providing at least partially better matched blood appears to improve the efficacy of transfusion for chronically transfused thalassaemics. Large-scale, comprehensive, multicentre studies need to be carried out to formulate realistic, evidence-based, economically feasible transfusion policies for thalassaemic children based on the red blood cell antigen profile of the population.

Composition of erythrocytic (ABO, Rh-Hr, Kell, MN) group antigens characteristic of the Ozurgeti district's population.

Erythrocytic group antigens represent a genetically stably determined trait. Investigation of antigens of the said system in different regions is of the greatest importance in terms of both the creation of demographic data of the region as well as practical medicine, especially for transplantology and transfusiology. The peripheral or venous blood of 232 local natives (healthy donors) of Ozurgeti district of Guria region has been taken as the test subject. The test subject was taken by random methods in different vilifies (Bakhvi, Mshvidobauri, Ozurgeti, Likhauri, Gurianta, Bokhvauri, Dvadzu, Pampaleti) To identify the ABO, Rh-Hr, Kell, MN system antigens, an express-method using monoclonal antibodies has been applied. In studying the ABO system, it was fixed that the highest distribution frequency was characteristic of the 0(I) group (52.3±3.2%), then follows the group A(II) (38.5±3.2%). The distribution frequency of the B(III) group is (8.2±1.8%) and that of AB(IV)--(0.8±0.5). The population's 85.2±2.32% is the carrier of the Rh+ phenotypic group, while 14.7±2.3% belongs to the Rh-phenotypic group. In studying the concentration of alleles, the low concentration of p(K) allele was detected that equaled 0.2; the concentration of q(K) allele made 0.8, that of p(M)--0.65, and that of q (N) - 035.

Absence of hemolytic disease of fetus and newborn despite maternal high-titer IgG anti-Ku.

Anti-Ku seen in K(o) (Kell-null) individuals has previously been shown to cause severe hemolytic transfusion reactions. Maternal anti-Ku can cause none or moderate to severe hemolytic disease of the fetus and newborn (HDFN). In two of four previously described HDFN cases, intrauterine transfusions were required because of severe anemia. We report a case in which maternal anti-Ku did not cause HDFN. Standard serologic methods were used for RBC antibody screening and identification, adsorption and elution of RBC antibodies, and antigen typing. A gravida 3, para 3 (G3P3) woman was first evaluated in 2006 and was found to have an IgG RBC antibody that reacted against all panel RBCs in the anti-human globulin phase. A panel of RBCs treated with DTT did not react with the antibody. The antibody failed to react with one example of K(o) RBCs. The patient's RBCs typed negative for the following Kell blood group antigens: KEL1, KEL2, KEL3, KEL4, KEL6, KEL7, KEL11, KEL13, and KEL18. These results established the presence of anti-Ku in maternal serum. The newborn was group A, D+ and required phototherapy for hyperbilirubinemia, but did not require transfusion. The woman was seen again in January 2010 during the third trimester (G4P3). At this time, anti-Ku titer was 256. She delivered a healthy group O, D+ baby boy at 37 weeks' gestation. Cord RBCs were 4+ for IgG by DAT. An eluate reacted with all RBCs tested, but did not react when tested against a panel of DTT-treated RBCs. K(o) phenotype is rare to begin with, and the maternal anti-Ku formation may require more than one pregnancy. Therefore, cases that can be evaluated for anti-Ku­related HDFN are rare. Our case contributes to serologic and clinical aspects of such rare cases.

Optimal blood grouping and antibody screening for safe transfusion.

(Full text is available at http://www.manu.edu.mk/prilozi). Introduction. Blood group antigens as integrated parts of the red cell membrane have many essential functions for the cell as well as for the organism, but they are recognized as unique antigens for the purpose of safe blood transfusion. Especially in the case of those with great clinical importance because of their involvement in haemolytic transfusion reactions and hemolytic disease of the newborn, it is very important that they be correctly, and some of them routinely, typed in blood donors as well as in patients. Aim. Evaluation of Rh and Kell blood group antigen frequencies in blood donors as well as the incidence of alloimmunization in transfused patients in the Macedonian population. The need for routine typing of certain blood group antigens in addition to ABO and RhD was also evaluated. Material and method. We evaluated data from 1600 ABO/Rh and Kell typed blood donors (from January 2003 to May 2008), as well as the data from pretransfusion testing (ABO/RhD blood typing, irregular red blood cell antibody screening and compatibility testing) and antibody identification in the period from January 2005 to November 2008. All tests were performed by the DiaMed micro tube gel system. Results. The frequencies of ABO antigens were as follows: A (39.7%), O (38%), B (14.1%), AB (7.4%). The frequencies of Rh antigens were as follows: D pos. (84.2%), D neg. (15.8%), C (58.3%), c (82.4%), E (21.3%), e (97.1%). We found the following frequencies of Kell phenotypes: K+ k- (0.25%), K+ k+ (6.18%), and K- k+ (93.6%) with the total frequency of K antigen of 6.4%. Antibody screening and/or cross-match were positive in the sera from 150 transfused patients. In 75 (50%) sera the following 81 antibodies were identified: anti-K (26), -E (25), -e (1), -C (4), -c (6), -C(w) (2), -k (1), -Fy (a) (3), -Fy(b) (1), -Jk(a) (3), -Lu(b) (1), -Le(b) (2), -Le(a) (1), -M (4), -P1 (1). The most frequent alloantibody was anti-K with 32%, and anti-E with 30.8% of all identified antibodies. Conclusion. Alloimmunization to red cell antigens is still a current problem in our transfusion practice. It is obvious that the additional testing of blood donors for Rh and Kell antigens should be implemented as a routine to prevent as far as possible the incidence of alloimmunization. It would also be cost-effective, bearing in mind the additional laboratory testing necessary to provide compatible blood for alloimmunized patients. Extended blood typing should be implemented for some categories of polytransfused patients as well. This strategy is another step forward to improve the safety of blood transfusion with optimal blood grouping. Key words: Kell phenotypes frequency, alloimmunization, blood grouping, safe transfusion.

Molecular genetic blood group typing by the use of PCR-SSP technique.

BACKGROUND: DNA-based methods are useful for enhancing immunohematology typings. Ready-to-use Conformité Européenne (CE)-marked test kits based on polymerase chain reaction with sequence-specific priming (PCR-SSP) have been developed, which enable the examination of weak, unexpected, or unclear serologic findings. DEVELOPMENT AND VALIDATION: Primers were designed according to established mutation databases. Proficiency testing for CE marking was performed in accordance with Directive 98/79EC of the European Parliament and of the Council of October 27, 1998 on in vitro diagnostic medical devices using pretyped in-house and external samples. INTENDED USE: BAGene PCR-SSP kits are in vitro diagnostic devices. Genotyping of ABO and RHD/RHCE as well as HPA and KEL, JK, and FY specificities has to be performed after the conclusion of the serologic determination. APPLICATION: Ready-to-use PCR-SSP typing kits allow the determination of common, rare, or weak alleles of the ABO blood group, Rhesus, and Kell/Kidd/Duffy systems as well as alleles of the human platelet antigens. RESULTS: The investigations showed clear-cut results in accordance with serology or molecular genetic pretyping. CONCLUSION: PCR-SSP is a helpful supplementary technique for resolving most of the common problems caused by discrepant or doubtful serologic results, and it is an easy-to-handle robust method. Questionable cases in donor, recipient, and patient typing can be examined with acceptable cost.

Management of Kell isoimmunization--evaluation of a Doppler-guided approach.

OBJECTIVE: To assess the role of peak systolic velocity in the middle cerebral artery (MCA-PSV) in the management of pregnancies complicated by Kell isoimmunization. METHODS: Sixteen fetuses were monitored by conventional protocol (Group 1) and eight fetuses by an MCA-PSV-guided protocol (Group 2). The conventional protocol included a weekly ultrasound evaluation and measurement of maternal anti-Kell titers every 4-6 weeks. In Group 2 Doppler assessment of the MCA-PSV was performed at intervals of 4 to 7 days and MCA-PSV>1.5 multiples of the median (MoM) was considered as an indication for fetal blood sampling (FBS). RESULTS: No parameter emerged as a reliable predictor of isoimmunization severity in Group 1. In Group 2, no FBS was necessary in one case since the MCA-PSV values obtained during the follow-up were <1.29 MoM. In two cases the first FBS was already indicated after 1 week of follow-up, but five other fetuses were followed for 3-9 weeks before FBS was indicated. All fetuses with MCA-PSV>1.5 MoM prior to intrauterine transfusion (IUT) had severe fetal anemia on FBS. In fetuses with severe anemia on the first FBS, the MCA-PSV values 7 days before the first FBS were <1.29 MoM (four cases), between 1.29 and 1.5 MoM (two cases) and >1.55 MoM (one case). CONCLUSIONS: In the management of Kell isoimmunization invasive procedures may be avoided by implementing MCA-PSV measurements. Delineation of appropriate intervals between reassessments, the reliability of MCA-PSV following repeated IUTs, and cut-off values for FBS await further study.

Grupos sanguíneos y enfermedad / Blood groups and disease

La membrana eritrocitaria sirvió como modelo general para el conocimiento de la membrana plasmática. Algunas de sus estructuras son antígenos pertenecientes a los sistemas de grupos sanguíneos y están siendo caracterizadas molecular y funcionalmente como receptores, transportadores o enzimas, incluso como puertas de entrada para patógenos. Así, el Plasmodium vivax (causante de la malaria) requiere la glucoproteína Duffy para penetrar en el interior de los hematíes humanos, y el antígeno principal del sistema P (P1) es también el receptor para el acceso del parvovirus B19. Estos antígenos no siempre se limitan a los glóbulos rojos, sino que pueden influir en diversos tejidos, el plasma o las secreciones con importantes relaciones patogénicas. Ciertas cepas agresivas de Eschirichia coli precisan antígeno P1 para anclarse al epitelio urinario, el antígeno Lewis(b) es el receptor de Helicobacter pylori en la mucosa gástrica, el anti-B de los sujetos con los grupos sanguíneos O y A podría ayudarles a combatir las bacteriemias por E. coli, el grupo Lewis condiciona las concentraciones séricas de CA-19.9 y el efecto protector de la leche materna. Sin embargo, la principal influencia sería la hipocoagulabilidad observada en la población de grupo O (valores inferiores de factor VIII) asociada con una prevalencia menor de enfermedades tromboembólicas

The erythrocyte membrane was used as general model for the plasma membrane knowledge. Some of their structures are antigens from blood group systems being characterized at molecular and functional level as specific receptors, transporters or enzymes, even receptors for infectious agents. Plasmodium vivax malarial parasites require the Duffy blood group glycoprotein to penetrate into human red blood cells and the main antigen of P system (P1) is also the Parvovirus B19 receptor. Furthermore, these substances have an effect on several tissues, plasma and secretions involving pathogenic relationships. Certain aggressive Escherichia coli strains require the P1 antigen to attach to the urothelial cells, the Lewis(b) antigen is the gastric receptor for H. pylori, the anti-B from O or A individuals might protect them against the sepsis produced by E. coli, the Lewis group determines the CA-19.9 serum levels or the protective effect of breast milk. However, the most important effect could be the plasma hypocoagulability observed among the O blood group population (with lower factor VIII levels) in association with a reduced prevalence of thromboembolic diseases

K, Fy(a), and Jk(a) phenotyping of donor RBCs on microplates.

BACKGROUND: In many cases, the search for compatible blood for patients with clinically significant RBC alloantibodies is difficult and time-consuming. To date, it has been considered necessary only to phenotype the blood donors for ABO group and D. There has been long experience with automated routine analysis (ABO, C, c, D, E, and e typing and RBC antibody screening), using robotic dispensers and computerized interpretation of microplate results. The purpose of this study was to explore the possibilities of also phenotyping for K, Fy(a), and Jk(a), as antibodies directed against these antigens (together with Rh antigens) are the most common clinically significant alloantibodies in the Swedish population. STUDY DESIGN AND METHODS: One thousand thirty-one EDTA samples from blood donors were phenotyped for K, Fy(a), and Jk(a) by use of an IAT with PEG on microplates. The findings were compared to those using conventional IAT in tube's and the microcolumn gel test (DiaMed-ID, DiaMed). RESULTS: All typing results with the microplate method were correct. All reactions for K and Fy(a) typing could be interpreted by the computer. The results for Jk(a) were indeterminate in 1.4 percent (14/1031) of the samples. CONCLUSION: The PEG-IAT microplate method gave reliable results that were suitable for routine phenotyping, thus making available a stock of phenotyped blood at reasonable cost, ready for delivery when required.

Distribución de los fenotipos y genotipos de Sistema Kell en la población de Costa Rica / Distribution of the phenotype and genotype of Kell system in the Costa Rican population

Se analizó una muestra de sangre de 1257 personas procedentes de las siete provincias de Costa Rica, sin relación de parentesco entre ellas, para investigar los fenotipos del sistema Kell. Se encontró una frecuencia del 0,16 por ciento para Kell homocigoto, de 96,74 por ciento para el Cellano homocigoto y de 3,16 por ciento para el fenotipo heterocigoto, esto corresponde a una frecuencia génica de 0,0171 para K y de 0,9829 para k. El tamaño de la muestra se determinó de acuerdo con la fórmula n=Z2pq/d2 y la frecuenca alélica por el método de conteo de genes. A un nivel de significancia de 0,005 y 2 grados de libertad el valor de x2=10,5970 (p<0,005), indica que no hay diferencias estadísticamente significativas entre la distribución observada y la esperada. Las frecuencias encontradas en Costa Rica nos indican que este sistema tiene poca importancia clínica y en los estudios de paternidad discutida en este país. (Rev Cost Cienc Méd 1999; 20(1-2): 77-81) PALABRAS CLAVE: Sistema Kell, Fenotipos Kell, Genotipos Kell, Genes Kell Costa Rica

Antenatal genotyping of the blood groups of the fetus.

Antenatal genotyping of the fetus is now in widespread use as an aid to the clinical management in cases where there is the potential of haemolytic disease of the newborn occurring. The rapid diagnosis of an antigen-negative fetus will preclude the requirement for further, potentially risky invasive procedures being performed, whilst the determination of an antigen-positive fetus allows the potential of intensifying obstetric care for this pregnancy. Molecular genotyping is a major clinical application which has led from the determination of the molecular bases of blood group antigens expressed, most of which have been defined at the level of the gene. All assays used are dependent on the Polymerase Chain Reaction amplification of fetal DNA derived from either amniotic fluid or chorionic villi. Recent work has explored the potential of utilising fetal cells found to be present in maternal peripheral blood as a source of nucleic acid for prenatal diagnosis. Using non-invasive methods will preclude exposing mother and fetus to the potential hazards of invasive methods (amniocentesis, chorionic villus sampling and cordocentesis) which include miscarriage, fetal malformations and further maternal alloimmunisation.

Detection of Kell blood groups: molecular methods in the diagnostic laboratory.

The introduction of molecular biology techniques to the field of transfusion medicine has allowed more detailed analysis of the basis for the expression of several blood-group antigens. The application of the polymerase chain reaction for the determination of red-cell blood-group genotype is a highly sensitive technique that can be used to determine the likelihood of a fetus being affected by haemolytic disease of the fetuses or newborn. This alone makes polymerase chain reaction based techniques highly desirable for such applications. The determination of the genetic basis for the Kell/Cellano polymorphism has enabled the development of polymerase chain reaction-based techniques for genotyping. Several other red-cell blood-group antigen polymorphisms can also be analysed in this way.

Rapid detection of Rh(D)- or K-positive fetal red cells in chorion villus samples by a flow cytometric technique.

Rh(D)- and K-negative women who have become severely isoimmunized by pregnancy are at risk of fetal loss or damage in subsequent pregnancies. A flow cytometric method is described whereby the presence of Rh(D) or K antigen on fetal erythrocytes may be determined using chorion villus samples taken during the first trimester. This method has the advantage of speed and sensitivity with results being available within 2 h. Decisions as to management of the pregnancy or termination may thus be made with minimal delay.

Application of the MAIEA assay to the Kell blood group system.

The monoclonal antibody-specific immobilisation of erythrocyte antigens (MAIEA) test has been employed to investigate the Kell system using five monoclonal antibodies which recognise high frequency epitopes on the 93,000-molecular weight Kell glycoprotein: BRIC 18, BRIC 68, BRIC 107, BRIC 203 and 6-22. BRIC 107, which has anti-k-like (KEL2) specificity, identifies a distinct epitope, whilst competitive binding assays suggested that BRIC 203 (anti-Kpbc), BRIC 18, BRIC 68 and 6-22 (anti K14) comprise an overlapping set of epitopes. The MAIEA assay has been very successful in confirming the assignment of most of the Kell and para-Kell antigens to the Kell protein. Due to the competitive nature of the assay and the fact that the monoclonal antibodies bind to different regions, the results also suggest the relative positions of some of the Kell antigens on the Kell protein; these appear to be located in at least five spatially distinct regions.

[Electronic data processing-assisted serial automation of current methods in blood group serology]. / EDV-unterstützte Serienautomatisierung herkömmlicher Methoden in der Blutgruppenserologie.

The introduction of special centrifugable racks with a transparent bottom into the conventional typing of blood groups in glass tubes facilitates the simultaneous work on and reading of a maximum of 32 complete ABO, Rhesus and Kell typings in one series. As a result of the facts that it is unnecessary to label the individual tubes and that the pipetting of serum and erythrocyte suspensions is done automatically and through the unmistakable classification of the samples by means of bar-coding, the manual work is reduced to about 50%. This modified typing method combines the advantages of the conventional typing method in glass tubes with the advantages of an efficient microplate technique. There is no qualitative loss in the implementation and reading of the analysis.

Characterization of the blood group Kell (K1) antigen with a human monoclonal antibody.

A human monoclonal anti-Kell (K1) antibody secreted by an Epstein-Barr virus (EBV)-transformed B-cell line was used for binding studies and immunopurification of the K1 blood group antigen. The 125I-labeled antibody bound to 4 to 5 x 10(3) and 2.5 to 3 x 10(3) antigenic sites on K1K1 and K1K2 erythrocytes, respectively, with an affinity constant of 5 x 10(8) mol/L-1. Immunoprecipitation analysis showed that the K1 antigen is carried by a 93 Kd glycoprotein containing several cysteine residues, and approximately six N-glycosidically linked sugar chains but no detectable O-linked sugar. A minor labeled component of 32 Kd was also immunoprecipitated from K1K1 RBCs but the 93- and 32-Kd components were absent from K2K2 and Kell null erythrocytes. Under nonreducing conditions, three bands were detected at 200 (weak), 120, and 93 Kd. We suggest that the 120-Kd component represents a heterodimer of the 93- and 32-Kd proteins covalently linked by disulfide bridge(s). The 93-Kd glycoprotein is a transmembrane component which interacts with the membrane skeleton but is distinct from band 3 as shown by one-dimensional peptide mapping. The site density of K1 antigen blood group on Gerbich-negative RBCs (Ge:-2,-3) was threefold lower than on K1K1 erythrocytes, but the qualitative properties of the 93-Kd component were not modified.