Genomic analysis of KEL*03 and KEL*04 alleles among Thai blood donors.

Background: The Kell blood group system is clinically important in transfusion medicine, particularly in patients with antibodies specific to Kell antigens. To date, genetic variations of the Kell metallo-endopeptidase (KEL) gene among Thai populations remain unknown. Objective: This study aimed to determine the frequencies of KEL*03 and KEL*04 alleles among Thai blood donors using an in-house polymerase chain reaction-sequence-specific primer (PCR-SSP) method. Methods: Blood samples obtained from 805 unrelated central Thai blood donors at a blood bank in Pathumthani, Thailand, from March 2023 to June 2023, were typed for Kpa and Kpb antigens using the column agglutination test, and the results for 400 samples were confirmed using DNA sequencing. A PCR-SSP method was developed to detect the KEL*03 and KEL*04 alleles, and genotyping results were validated using known DNA controls. DNA samples obtained from Thai donors in central (n = 2529), northern (n = 300), and southern (n = 427) Thailand were also genotyped using PCR-SSP for comparison. Results: All 805 (100%) donors had the Kp(a-b+) phenotype. The PCR-SSP genotyping results agreed with the column agglutination test and DNA sequencing. All 3256 Thai blood donors had the homozygous KEL*04/KEL*04 genotype. Frequencies of the KEL*03 and KEL*04 alleles among Thai donors differed significantly from those of Japanese, Native American, South African, Brazilian, Swiss, and German populations. Conclusion: This study found a 100% KEL*04 allele frequency in three Thai populations. These data could provide information on KEL*03 and KEL*04 allele frequencies to estimate the risk of alloimmunisation in Thai populations. What this study adds: This study demonstrates that in-house PCR-SSP can be used to determine KEL*03 and KEL*04 alleles to predict Kpa and Kpb antigens. Even though only homozygous KEL*04/KEL*04 genotypes were found among Thai donor populations, the established PCR-SSP method may be useful for estimating the risk of alloimmunisation in other populations.

[Application of Modified DTT Methods to Reduce the Interference of Daratumumab on Serological Detection].

OBJECTIVE: To modified the classic dithiothreitol (DTT) method for treating red blood cells (RBCs) in Technical Manual of American Association of Blood Banks（AABB） and evaluate its application value in pre-transfusion examination of patients treated with daratumumab. METHODS: The classic 0.2 mol/L DTT method was improved in terms of PBS, DTT concentration, donor RBCs concentration (suspended/packed) and sample processing time. The modified DTT methods and AABB classic DTT method were applied to the blood matching tests of 12 multiple myeloma patients treated with daratumumab. The effect of treating panel RBCs with modified DTT methods on the detection of other irregular antibodies was evaluated by using antiserum and antibody reagents with known antibody properties. RESULTS: Two modified DTT methods were established (method 1: changed the concentration of DTT to 0.01 mol/L; method 2: changed the concentration of DTT to 0.02 mol/L and replaced the packed RBCs with 3% RBCs suspension). The optimal treatment time was 35 min for the modified DTT methods. At this time, the pan-agglutination caused by daratumumab was eliminated, but the detection of antibodies such as anti-E, anti-JKa, anti-M were not affected, and the titer of anti-K antibodies was only slightly decreased. CONCLUSION: The modified DTT methods were effective, which can eliminate the interference of daratumumab while retaining the activity of the Kell blood group system, and can replace the current classic DTT method in AABB Technical Manual.

Frequency of Kell and Rh alloantibodies in Iranian Thalassemia Patients in Khorasan Razavi Province, Iran.

Background: Thalassemia is an inherited disease with anemia and hemolysis. Blood transfusion is a routine treatment for thalassemia patients; alloimmunization is one of the complications of blood transfusion, which is very serious for these patients, especially girls and young women. Materials and Methods: In this cross-sectional study, 446 thalassemia patients were examined. Demographic information of patients was extracted and recorded. The phenotype of ABO, Rh, and Kell antigens (tube method) with antisera from IMMUNDIANOSTICA Company (Germany) and the frequency of alloantibodies were determined. Results: 55.8% of the studied individuals were male, and 44.2% were female. Mean age of the studied patients was 19.94±10.63. The alloantibodies were detected in 7.5% of cell-pack receivers. The most prevalent phenotype of the ABO system was the O blood group (37.4%), and the most abundant antigen of the Rh group was 'e', which was found in 99.8% of the studied population. The most common alloantibody detected was Anti K (38.2%); concerning kell phenotype, (K_k+) and (K+k+) were found in 99.3% and 0.7% of patients, respectively. The frequency of Anti-D, Anti-C, Anti-c, and Anti-E was 23.5%, 14.7%, 2.9%, and 14.7%, respectively. Conclusion: According to the results of this paper, finding the compatible packed cells in terms of Kell and Rh systems antigens in addition to the ABO blood group is recommended to decrease the rate of alloantibodies in thalassemia patients.

K antigen serological screening results in 20 354 population from Qingdao area / 中国输血杂志

【Objective】 To perform serological screening of K antigen in Qingdao area and analyze the results. 【Methods】 From March to June 2023, 16 201 samples of patients treated in our hospital and applied for blood grouping, and 4 153 samples of blood donors sent to the clinic by Blood Transfusion Department during the same period were detected for Rh and K antigen by Ortho BioVue System Rh/K Cassette. 【Results】 A total of 18 out of 16 201 patient samples and 8 out of 4 153 blood donor samples were positive for K antigen, with the positive rate at 0.127 7%. 【Conclusion】 There was K antigen in Qingdao population, and the positive frequency of K antigen was different from previous reports. It is of great significance to construct rare blood group bank in this area and formulate reasonable blood donation and transfusion strategy for population with K antigen, so as to avoid unexpected antibody production of Kell blood group system, prevent hemolytic disease of newborn and ensure the safety of blood use.

Non-Invasive Fetal K Status Prediction: 7 Years of Experience.

Introduction: In the Kell blood group system, the K and k antigens are the clinically most important ones. Maternal anti-K IgG antibodies can lead to the demise of a K-positive fetus in early pregnancy. Intervention can save the fetus. Prenatal K status prediction of the fetus in early pregnancy is desirable and gives a good basis for pregnancy risk management. We present the results from 7 years of clinical experience in predicting fetal K status as well as some theoretical considerations relevant for design of the assay and evaluation of results. Methods: Blood was collected from 43 women, all immunized against K, at a mean gestational age of 18 weeks (range 10-38). A total of 56 consecutive samples were tested. The KEL *01.01 /KEL *02 single nucleotide variant that determines K status was amplified from maternal plasma DNA by PCR without allele specificity. The PCR product was sequenced by NGS technology, and the number of sequenced KEL *01.01 and KEL *02 reads were counted. Prediction of the fetal K status was based on this count and was compared with the serologically determined K status of the newborns. Results: All fetal K predictions were in accordance with postnatal serology where available (n = 34), using our current data analysis. Conclusion: We have developed an NGS-based method for the non-invasive prediction of fetal K status. This approach requires special considerations in terms of primer design, stringent preanalytical sample handling, and careful analytical procedures. We analyzed samples starting at GA 10 weeks and demonstrated the correct prediction of fetal K status. This assay enables timely clinical intervention in pregnancies at risk of hemolytic disease of the fetus and newborn caused by maternal anti-K IgG antibodies.

Novel KEL allele associated with loss of Kpb identified in a white blood donor.

The importance of identifying variant alleles among blood donors is significant to the safety of transfusion for recipients. Molecular methods have become more prominent in the routine process of antigen typing donor units. Some variant antigens cannot be detected using only serologic methods. Molecular testing allows the determination of nucleotide sequences that are used to predict a phenotype. Antigens of the Kell blood group system are known for being highly immunogenic and causing adverse reactions upon antibody formation. A female white blood donor who typed Kp(b-) using serologic methods on multiple donations since 2005 was the subject of a typing discrepancy investigation. Routine genotyping using a commercial genotyping kit (HemoID DQS Panel; Agena Bioscience, San Diego, CA) predicted the donor to type Kp(a+b+). Investigation of the discrepancy between these two results identified a rare single nucleotide variant in the KEL gene at nucleotide position c.948G>T that alters amino acid residue 316 from tryptophan (Trp) to cysteine (Cys). After discovery of the novel allele, adsorption and elution studies were performed to see if there was weakened Kpb expression. The elution studies yielded negative results, which indicated that Kpb is not expressed. The KEL transcripts expressed by the donor were determined using cDNA analysis, and the predicted amino acid sequence of the novel allele was modeled to investigate the impact of the amino acid sequence on the structure of the KEL polypeptide. Both SWISS-MODEL and Robetta software were used to evaluate the impact of the p.Trp316Cys on the three-dimensional protein structure. There was no conformational change noted with SWISS-MODEL, whereas the Robetta software showed a significant conformational change compared with the normal Kp(b+) reference sequence. Because the donor is homozygous for variants associated with k and Jsb expression, it was not possible to determine whether the novel allele is associated with loss of Kpb only or loss of all Kell antigens.

Noninvasive diagnostics of fetal KEL*01.01 allele from maternal plasma of immunized women using digital PCR protocols.

Allo-antibodies produced by K-negative pregnant women against a fetal K antigen from the Kell blood group system may cause hemolytic disease of the fetus and newborn (HDFN). Predicting the fetal K antigen using noninvasive prenatal testing (NIPT) is important for decisions concerning management of pregnancies. Digital and droplet digital PCR techniques permit the detection of fetal single nucleotide variant with a higher specificity and sensitivity than real-time polymerase chain reaction (PCR). AIM: The aim was to evaluate and compare protocols for fetal KEL*01.01 genotyping using different assays and digital PCR platforms. METHODS: DNA isolated from 59 pregnant women (9-39 weeks of gestation, 49 with anti-K) was tested using home-made and custom-ordered KEL*01.01/KEL*02 assays with Droplet Digital™ and QuantStudio™3D. The results were compared with fetal/neonatal genotypes/phenotypes. RESULTS: Fetal KEL*01.01 results using all tested protocols were concordant with fetal/neonatal KEL*01.01 genotypes/phenotypes. None of the tested combinations of assays or digital PCR platforms gave false KEL*01.01-negative results, but inconclusive KEL*01.01 reads were observed in all tested protocols. For 36 cases compared using two digital PCR platforms and assays, there were not statistically significant differences in a level of fetal KEL*01.01 fraction (p < .72). CONCLUSION: Independent of the applied dPCR and ddPCR platforms and KEL*01.01 assays, prediction of the fetal KEL*01.01 is highly reliable. Before implementation in routine practice further validation of the KEL*01.01 protocol with a larger group of pregnant women should be performed.

Análise do perfil de Rh e Kell em doadores de sangue em Porto Alegre, Rio Grande do Sul / Phenotypic profile analysis of Rh and Kell blood group systems among donors in Porto Alegre, Rio Grande do Sul

Introdução: A hemoterapia é uma prática terapêutica pelo meio de transfusão sanguínea. Devido ao baixo estoque de bolsas de sangue e o aumento de pacientes crônicos e emergenciais, se faz necessária a realização de testes imuno-hematológicos para minimizar os riscos de reações transfusionais e aloimunizações em doadores e receptores de sangue. Deste modo, no estudo foi avaliada a prevalência dos antígenos dos sistemas Rh e Kell em doadores de sangue de Porto Alegre ­ RS.Métodos: Estudo quantitativo, transversal e retrospectivo que foi realizado através da análise das informações dos doadores de sangue contidas no banco de dados do Hemocentro do Estado do Rio Grande do Sul, nos anos de 2018 e 2019.Resultados: Das 6.479 amostras fenotipadas, quanto ao sistema Rh, 44,6% são Rh positivo e 55,4% são Rh negativo. As frequências dos antígenos encontradas foram de, CC 10,1%, Cc 27%, cc 62,9%, EE 1,2%, Ee 13,9%, ee 84,9%. E, para o sistema Kell, K1 positivo 7,1% e K1 negativo 92,9%.Conclusões: Antígenos do sistema Rh e Kell exibem um grande nível de imunogenicidade e uma forte ligação com a Doença Hemolítica do Recém-nascido, podendo ocorrer a sensibilização em pacientes caso não haja a compatibilidade sanguínea. Este estudo ressalta a importância da implementação da fenotipagem eritrocitária em doadores de sangue, sugere-se mais estudos com períodos distintos para a pesquisa de resultados satisfatórios.

Introduction: Hemotherapy is a therapeutic practice consisting of blood transfusion. Low blood supply and an increase in chronic and emergency patients have made it necessary to conduct immunohematology tests to minimize the risks of adverse reactions and alloimmunization in donors and recipients. Therefore, this study aimed to assess the prevalence of Rh and Kell blood group antigens among blood donors in Porto Alegre, Rio Grande do Sul, Brazil.Methods: We conducted a quantitative, cross-sectional, retrospective study. Information from blood donors included in the Rio Grande do Sul's Blood Center database from 2018 to 2019 were analyzed.Results: A total of 6,479 samples were phenotyped, of which 44.6% were Rh-positive and 55.4% were Rh-negative. Antigen prevalence was CC (10.1%), Cc (27%), cc (62.9%), EE (1.2%), Ee (13.9%), and ee (84.9%). As for the Kell group, 7.1% were K1-positive and 92.9% were K1-negative.Conclusions: The Rh and Kell antigens are highly immunogenic and have a strong link with the hemolytic disease of the newborn. Sensitization may occur in patients if there is no blood compatibility. This study highlights the importance of implementing erythrocyte phenotyping in blood donors. Further studies should be conducted in different time frames to achieve satisfactory results.

An Easy Multiplex PCR-SSP Assay for the Genotyping of KEL1 and KEL2 in Multi-transfused Patients.

Kell blood group system consists of 34 antigens. KEL1 and KEL2 are the most clinically important antigens of this system, causing hemolytic disease of the fetus and newborn (HDFN) and transfusion reaction. A total of 200 samples from blood donors were tested serologically for the presence of KEL1 and KEL2 antigens on erythrocytes. Genomic DNA was analyzed by PCR-SSP method to determine the Kell genotype. A multiplex PCR-SSP assay was designed and tested to genotype KEL1/KEL2 alleles in a single reaction. PCR genotyping revealed samples as; KEL2/KEL2 (93.5%) and KEL1/KEL2 (6.5%), while no sample determined as KEL1/KEL1. A 100% concordance observed between PCR and serological results. Multiplex PCR accurately diagnosed Kell genotype. Kell blood group genotyping by PCR-SSP can be used as an alternative method, especially in multi-transfused patients where serological findings are ambiguous.

The prenatal intervention of pregnancy complicated with anti-Kell isoimmunization: a review.

Since the first case of the hemolytic disease of the fetus and newborn (HDFN) caused by anti-K was reported in 1946, the fetal diagnosis of K-HDFN has made rapid progress from invasive immunological and biochemical tests to noninvasive Doppler ultrasound and molecular biology techniques. However, its treatment, especially prenatal intervention, has developed slowly compared with RhD-HDFN. This review attempts to clarify the current key points and explore the direction of the next phase by systematically reviewing the development of diagnosis and treatment of this disease, which involving multidisciplinary participation.

Genomic analyses of KEL alleles in alloimmunized thalassemia patients from Iran.

OBJECTIVES: Serological methods are unreliable for red blood cells (RBCs) antigen typing in multi-transfused thalassemia patients due to the presence of donor RBCs in the recipient's circulation and interfering antibodies. Kell blood group system is important in transfusion medicine and Kell antibodies have shown as the most prevalent antibodies in thalassemia patients. We intended to determine the genotype of Kell antigens among Iranian alloimmunized thalassemia patients using molecular methods and compare the results with serological phenotyping. METHODS: Two hundred thalassemia patients participated in this study. Blood group phenotype was performed by the serological method, while the genotype was determined for KEL*01, KEL*02, KEL*03, and KEL*04 alleles using PCR-Sequence Specific Primer (PCR-SSP) method. The genotypes of patients with incompatibility between phenotype and genotype were re-evaluated by Restriction Fragment Length Polymorphism-PCR (RFLP-PCR) and confirmed by DNA sequencing in all cases. RESULTS: Ten patients were found with discrepancies between genotype and phenotype; however, there was a complete agreement between the results of SSP-PCR, RFLP-PCR, and DNA sequencing. Six discrepancies were found in the KEL*01/KEL*02 allele when serologically phenotyped as K-k+. One patient with K-k- and three patients with Kpa-Kpb + phenotype were identified as KEL*01/KEL*02 and KEL*03/KEL*04, respectively. CONCLUSION: It can be concluded that molecular genotyping is more reliable compared with the serological method, especially in the patients who have received multiple transfusions. Therefore, using a combination of these techniques can lead to a better matched transfusion in these patients.

Association between HLA-DRB1*01 and HLA-DRB1*15 with alloimmunisation in transfusion-dependent patients with thalassaemia.

BACKGROUND: Alloantibody production is one of the most challenging complications in transfusion-dependent thalassaemia patients. Haemolytic anaemia, an increase in blood consumption, difficulty in haematopoietic stem cell transplantation and reduced quality of life are consequences of alloimmunisation. The most predisposed antigens (Ags) for alloantibody development are Rh and Kell blood group Ags. OBJECTIVE: The aim of the present study is to evaluate any correlation between HLA-DRB1 alleles and Rh and Kell alloantibodies. MATERIALS AND METHODS: Fifty-two non-responders (control) and 54 responders (case) were enrolled in this study. Alloantibody detection was performed using the tube method. Genotyping of HLA-DRB1*01 and HLA-DRB1*15 was conducted by single-specific primer-polymerase chain reaction. RESULTS: In the responder group, 77.8% were hyper-responders (more than one alloantibody), and only 22.2% were mono-responders. Most detected alloantibodies were Anti-K (94.4%), followed by Anti-E (64.8%), Anti-C (29.6%) and Anti-D (25.9%). There was a significant difference in HLA-DRB1*15 between responder and non-responder groups, 73.7% vs 26.3%, respectively. (P = .029, OR = 3.290; 95%CI). Our results showed that HLA-DRB1*15 was more frequent in hyper-responders than mono-responders (92.9% vs 7.1%) (P = .007). The greatest HLA-DRB1*15 was seen in Anti-K (P = .014, odds ratio [OR = 3.784]; 95% confidence interval [CI]) and Anti-E (P = .011, OR = 3.609; 95%CI) alloantibodies. There is no association between HLA-DRB1*01 and alloimmunisation. CONCLUSION: Our findings showed that there is a significant correlation between HLA-DRB1*15 and Anti-K and Anti-E alloantibodies. These findings can be useful in detecting susceptible thalassaemic patients and improving transfusion management.

XKR6 rs7014968 SNP Increases Serum Total Cholesterol Levels and the Risk of Coronary Heart Disease and Ischemic Stroke.

The X Kell blood group complex subunit-related family member 6 (XKR6) gene single-nucleotide polymorphisms (SNPs) have been associated with serum lipid profiles and the risk of coronary heart disease (CHD) and ischemic stroke (IS) in several previous studies, but the association between the XKR6 rs7014968 SNP and serum lipid levels and the risk of CHD and IS has not been detected previously. This study aims to explore the association between the XKR6 rs7014968 SNP and serum lipid traits and the susceptibility to CHD and IS in the Guangxi Han Chinese population. Snapshot technology was used to determine the genotypes of the XKR6 rs7014968 SNP in 624 controls, 588 patients with CHD, and 544 patients with IS. The XKR6 rs7014968C allele carriers in the control group had higher serum total cholesterol (TC) levels than the C allele noncarriers (P = .025). The XKR6 rs7014968C allele carriers also had an increased risk of CHD and IS (P < .05-.01). Stratified analysis showed that the patients with the rs7014968C allele in the female, age >60 years, body mass index (BMI) >24 kg/m2, and hypertension subgroups had a higher risk of CHD than those in the subgroup counterparts. The patients with the rs7014968C allele in the male, BMI > 24 kg/m2, smoker, and hypertension subgroups also had a higher risk of IS than those in the subgroup counterparts. These results suggest that the XKR6 rs7014968 SNP is likely to increase the risk of CHD and IS by increasing serum TC levels in our study populations.

Regulatory SNP rs5743417 impairs constitutive expression of human ß-defensin 1 and has high frequency in Africans and Afro-Americans.

The prediction of regulatory single nucleotide polymorphisms (rSNPs) in proximal promoters of disease-related genes could be a useful tool for personalized medicine in both patient stratification and customized therapy. Using our previously reported method of rSNPs prediction (currently a software called SNPClinic v.1.0) as well as with PredictSNP tool, we performed in silico prediction of regulatory SNPs in the antimicrobial peptide human ß-defensin 1 gene in three human cell lines from 1,000 Genomes Project (1kGP), namely A549 (epithelial cell line), HL-60 (neutrophils) and TH 1 (lymphocytes). These predictions were run in a proximal pseudo-promoter comprising all common alleles on each polymorphic site according to the 1,000 Genomes Project data (1kGP: ALL). Plasmid vectors containing either the major or the minor allele of a putative rSNP rs5743417 (categorized as regulatory by SNPClinic and confirmed by PredictSNP) and a non-rSNP negative control were transfected to lung A549 human epithelial cell line. We assessed functionality of rSNPs by qPCR using the Pfaffl method. In A549 cells, minor allele of the SNP rs5743417 G→A showed a significant reduction in gene expression, diminishing DEFB1 transcription by 33% when compared with the G major allele (p-value = .03). SNP rs5743417 minor allele has high frequency in Gambians (8%, 1kGP population: GWD) and Afro-Americans (3.3%, 1kGP population: ASW). This SNP alters three transcription factors binding sites (TFBSs) comprising SREBP2 (sterols and haematopoietic pathways), CREB1 (cAMP, insulin and TNF pathways) and JUND (apoptosis, senescence and stress pathways) in the proximal promoter of DEFB1. Further in silico analysis reveals that this SNP also overlaps with GS1-24F4.2, a lincRNA gene complementary to the X Kell blood group related 5 (XKR5) mRNA. The potential clinical impact of the altered constitutive expression of DEFB1 caused by rSNP rs5743417 in DEFB1-associated diseases as tuberculosis, COPD, asthma, cystic fibrosis and cancer in African and Afro-American populations deserves further research.

First report of a KELnull phenotype in Peru and a lesson of invisible genetic disparity.

Blood banks in developing countries have limited capability to typify common blood groups creating disparities in the access to blood units for patients with rare blood genotypes. We report the case of a Peruvian woman with metastatic breast cancer with KELnull phenotype (K0), a rare blood group characterized by the lack of expression of all Kell antigens on the red blood cells (RBCs). The molecular studies identified that the patient's RBCs were homozygous for the nonsense c.1546C > T mutation predicted to encode p.Arg516Ter (KEL*02 N.17 allele), which confirmed the K0 phenotype. We conducted a local and international search of compatible blood units. Finally, the Japanese Red Cross donated the blood units for the patient. We present here the first report for a K0 phenotype in Peru and the challenging genetic disparities that many patients have to face to access to blood units in our country.

Frecuencia y procedencia del antígeno Kell en mujeres donantes de sangre durante los años 2016-2017 / Frequency and origin of the Kell antigen in female blood donors during 2016-2017

Resumen Introducción: El sistema Kell está formado por dos antígenos principales: el Kell (K) y el Cellano (k), estos son capaces de causar reacciones graves, tales como reacción hemolítica postransfusional y la enfermedad hemolítica del recién nacido. Los antígenos de este sistema son altamente inmunogénicos lo que les confiere el tercer lugar en importancia clínica. Objetivo: Determinar la frecuencia del antígeno Kell y procedencia de las mujeres donantes de sangre con antígeno Kell positivo en el Hemocentro del Centro Oriente Colombiano (HCOC). Metodología: Estudio descriptivo de corte transversal que incluyó 186 donantes voluntarias de sangre del Hemocentro Centro Oriente Colombiano, se realizó la fenotipificación del antígeno Kell, utilizando la técnica Aglutinación en lámina, la cual se basa en enfrentar glóbulos rojos del donante con anticuerpo monoclonal anti K. Se calculó la frecuencia fenotípica del antígeno Kell, en porcentajes y para el procesamiento de la información se utilizó el paquete estadístico SPSS versión 21.0 en español donde se realizó todo el análisis de los datos de la población. Resultados: Se procesaron 177 muestras obtenidas en 9 campañas de donación de sangre realizadas en diferentes municipios del departamento de Boyacá, obteniéndose una frecuencia fenotípica del 7,5% para el antígeno Kell, en la población de mujeres donantes de sangre del HCOC, siendo esta similar con la frecuencia encontrada en Colombia y Latinoamérica. Conclusión: Se determinó que la frecuencia del antígeno Kell en las mujeres donantes de sangre del HCOC fue del 7,5%, y se logró identificar que no existe una relación estadísticamente entre la procedencia y la presencia del antígeno Kell en las donantes, lo anterior está relacionado con el mestizaje y los procesos de migración.

Abstract Introduction: The Kell system consists of two major antigens: Kell (K) and Cellano (K), which are capable of causing serious reactions, such as posttransfusion hemolytic reaction and hemolytic disease of the newborn. The antigens of this system are highly immunogenic which gives them the third place in clinical importance. Objective: To determine the frequency of Kell antigen and origin of blood donors in the Hemocenter of the Centro Oriente Colombiano (H.C.O.C). Methods: Cross-sectional descriptive study involving 186 blood donors from the Centro Oriente Colombian Hemocenter, phenotyping of the Kell antigen was carried out, using the technique Aglutination in lamina, which is based on facing donor red blood cells with anti-K monoclonal antibody. Calculated the phenotypic frequency of the Kell antigen in percentages and for the processing of the information was used the statistical package SPSS version 21.0 in Spanish where all the analysis of the data of the population was carried out. Results: 177 samples obtained in 9 blood donation campaigns were carried out in different municipalities of the department of Boyacá, obtaining a phenotypic frequency of 7.5% for the Kell antigen in the population of female HCOC blood donors. Similar to the frequency found in Colombia and Latin America. Conclusion: It was determined that the frequency of Kell antigen in the female HCOC donors was 7.5%, and it was possible to identify that there is no statistically relation between the origin and the presence of Kell antigen in the donors, Is related to mestizaje and migration processes.

Extended Donor Typing by Pooled Capillary Electrophoresis: Impact in a Routine Setting.

BACKGROUND: PCR with sequence-specific priming using allele-specific fluorescent primers and analysis on a capillary sequencer is a standard technique for DNA typing. We aimed to adapt this method for donor typing in a medium-throughput setting. METHODS: Using the Extract-N-Amp PCR system, we devised a set of eight multiplex allele-specific PCR with fluorescent primers for Fya/Fyb, Jka/Jkb, M/N, and S/s. The alleles of a gene were discriminated by the fluorescent color; donor and polymorphism investigated were encoded by product length. Time, cost, and routine performance were collated. Discrepant samples were investigated by sequencing. The association of new alleles with the phenotype was evaluated by a step-wise statistical analysis. RESULTS: On validation using 312 samples, for 1.1% of antigens (in 5.4% of samples) no prediction was obtained. 99.96% of predictions were correct. Consumable cost per donor were below EUR 2.00. In routine use, 92.2% of samples were successfully predicted. Discrepancies were most frequently due to technical reasons. A total of 11 previously unknown alleles were detected in the Kell, Lutheran, and Colton blood group systems. In 2017, more than 20% of the RBC units prepared by our institution were from donors with predicted antigen status. A steady supply of Yt(a-), Co(a-) and Lu(b-) RBC units was ensured. CONCLUSIONS: Pooled capillary electrophoresis offers a suitable alternative to other methods for extended donor DNA typing. Establishing a large cohort of typed donors improved transfusion support for patients.

Anti Kpa alloantibody: Development of a rare alloantibody in a non-Hodgkin's lymphoma patient of Indian origin.

The Kell blood group system is complex, contains many antigens, highly immunogenic, and potent in triggering immune reactions. Antibodies to Kell blood group system are the most common immune red cell antibodies, following ABO and Rh. However, among the anti-Kell antibodies; anti-Kpa is extremely rare. We report an interesting case of Anti-Kpa in a 59-year-old female patient of Non-Hodgkin's Lymphoma, post radiotherapy, who first developed warm autoantibody and later developed anti-Kpa alloantibody on multiple transfusions.

Silent KEL alleles identified from Japanese individuals with the Ko phenotype.

BACKGROUND AND OBJECTIVE: The rare Ko phenotype lacks all 36 antigens in the Kell blood system. The molecular basis of the Ko phenotype has been investigated, and more than 40 silent KEL alleles are reported by many investigators. The majority of silent alleles are the KEL*02 background. Here, we report molecular genetic analysis of the KEL gene in Japanese individuals with the Ko phenotype. MATERIALS AND METHODS: The Ko phenotype was screened from Japanese blood donors for several years using monoclonal anti-Ku or anti-K14 by an automated blood grouping system PK7300. Kell-related antigens were typed by standard tube tests. Genomic DNA was extracted from the blood samples, and KEL gene was analysed by polymerase chain reaction (PCR) and Sanger sequencing. RESULTS: We collected 35 Ko blood samples with K-k-, Kp(a-b-), Js(a-b-) and K14-. PCR and sequence analysis revealed that 11 individuals were homozygous for a mutant KEL allele with a c.299G>C (p.Cys100Ser) mutation (rs. 200268316). Three individuals were homozygous for the KEL*02N.24 allele that is c.715G>T (p.Glu239*), and one individual was homozygous for the KEL*02N.40 allele that is c.1474C>T (p.Arg492*). Five individuals were homozygous for novel KEL alleles with single-nucleotide mutations, four individuals had a c.2175delC (p.Pro725 fs*43), and one individual had a c.328delA (p.Arg110 fs*79). The remaining 15 individuals were compound heterozygous, and eight new alleles were identified from them. CONCLUSIONS: We identified three known and ten new silent KEL alleles from Japanese individuals with the Ko phenotype. The KEL allele with the c.299G>C (p.Cys100Ser) mutation was the most frequent.