| Abstract|| |
BACKGROUND: In multi-transfused patients, alloimmunization usually interferes with accurate phenotyping of blood group antigens and proper selection of blood components. Therefore, DNA-based blood group typing including minor blood groups such as Kell, Kidd, and Duffy systems can overcome the limitations of serological methods and improves the efficiency of the transfusion process.
AIM OF THE WORK: The goal of this study was to determine the allelic frequencies of genetic polymorphisms in Kell, Kidd, and Duffy genes and to compare red blood cell (RBC) molecular genotyping to serological phenotyping in chronically transfused patients.
STUDY DESIGN AND SUBJECTS AND METHODS: A cross-sectional study was carried out on 100 repeatedly blood-transfused Egyptian patients. Patient samples were subjected to ABO, Rh, Kell, Kidd, and Duffy hemagglutination phenotyping in addition to the identification of Kell (K, k), Kidd (JKa, JKb), and Duffy (Fya, Fyb) polymorphisms by polymerase chain reaction-restriction length polymorphism. Patient samples were also subjected to screening and identification of RBC antibodies.
RESULTS: Serological identification of minor blood groups revealed that in Kidd blood group the most common antigen pattern is JK (a + b−) being 34%, in Duffy FY (a + b−) being 26% and for Kell; 96% were K antigen positive and 92% were K antigen negative. As regards genotyping, the most frequent genotypes of the Kell, Kidd, and Duffy systems were KEL02/KEL02 (98%), JK01/JK02 (42%), and FY02/FY02 (60%), respectively. The discrepancy between genotyping and phenotyping was also found in 76% of patients in the Kidd system, 92% of patients in Duffy system, and 2% in Kell blood group system.
CONCLUSION: Extended blood group genotyping including Kell, Kidd, and Duffy groups is superior to serological analysis and can be used as a valuable tool to predict definitive blood grouping, especially in alloimmunized multi-transfused patients to select blood units for perfect cross-matching and preventing alloimmunization.
Keywords: Alloimmunisation, Duffy, Kell, Kidd, multitransfused
|How to cite this URL:|
Salem DD, Habashy DM, ElSayed HTN, Mohamed DM, Youssef I, Atif HM. Identification of molecular alleles of Kell, Kidd, and Duffy in multi-transfused patients with undetermined phenotypes: An approach to reduce alloimmunization. Asian J Transfus Sci [Epub ahead of print] [cited 2023 Feb 8]. Available from: https://www.ajts.org/preprintarticle.asp?id=356889
| Introduction|| |
Blood transfusion is a life-saving therapy for hematologic diseases, hemoglobinopathies, organ transplantation, renal failure, and various types of cancers. However, patients requiring chronic blood transfusion are at high risk of alloimmunization.
The development of red blood cell (RBC) antibodies complicates the selection of compatible blood units and increases the rate of life-threatening delayed hemolytic transfusion reactions (HTRs).
The most important unexpected alloantibodies are directed toward the Rh (D, C, E, c, and e) and Kell (K) antigens, followed by antigens of the Duffy, Kidd, and MNS blood group systems. Accurate identification of these antigens by phenotypic and genotypic profiling can reduce the risk of alloimmunization and improves therapeutic processes in repeatedly transfused patients.
These antigens are encoded by highly polymorphic genes and they are population-specific so, they can be used as markers for ethnicity. The majority of blood group polymorphisms are associated with a single point mutation or single-nucleotide polymorphisms in the gene encoding the protein carrying the blood group antigen or the enzymes catalyzing the addition of monosaccharide onto a nascent blood group oligosaccharide moiety (i.e., ABO system).
Serological techniques have been considered the gold standard methods used to determine the phenotype of patients however, they have significant limitations. It was obvious that hemagglutination techniques might fail to detect the phenotype in presence of donor-derived erythrocytes from previous transfusions or in cases with hematopoietic stem cell recipients. Moreover, weakly or rarely reactive antigens and insufficient antibodies formation in infants are among the challenges that serological testing confront. Finally, misinterpretation of results could be due to inadequate mixing of RBCs and antisera., Therefore, red cell genotyping platforms have become widely available in immunohematology circumstances in which traditional serologic techniques are difficult or impossible to perform.
This study aimed to compare the genotypic polymorphisms and serologic phenotypes of the Kell, Duffy, and Kidd blood group systems in chronically transfused Egyptian patients.
| Subjects and Methods|| |
This cross-sectional study was conducted on 100 multitransfused patients (more than 1 unit of blood in 1 month, or at least 10 units within 3 months) recruited nonrandomly, attending the Haematology/Oncology Unit of Ain Shams University Paediatric Hospital. The selected patients were of different diagnoses.
Informed consent was obtained from all enrolled patients and/or their legal guardians. The study was approved by the Scientific and Ethical Committee, Ain-Shams University (FMASU 338/2017), and was in accordance with the declaration of Helsinki.
All the included clinical and transfusion data of the enrolled patients were collected from the medical records of the central blood bank and the hematology departments in ASUH, laying stress on age, sex, consanguinity, splenectomy status, age at first transfusion, the total number, and type of transfused RBC units, frequency of blood transfusions per year and transfusion-related side effects, especially history signifying antibody-dependent reactions.
Patients' blood samples were collected in ethylenediaminetetraacetic acid (EDTA) and plain tubes for ABO/RhD blood grouping and antibody screening/identification, respectively according to the standard protocols of the central blood bank of ASUH. A separate EDTA blood sample was used for genotyping by restriction length polymorphism-polymerase chain reaction (RFLP-PCR). All tests were performed on the same day of collection except PCR samples were centrifuged and the separated plasma was frozen at −80°C till use.
Two milliliters of EDTA anticoagulated venous blood were collected from each patient to perform ABO/RhD blood grouping by the gel card agglutination method using the ABO-RhD/reverse grouping system (ABO forward/Reverse Grouping and Rh phenotype ID-Cards provided by Diamed system, Switzerland). RBCs extended phenotyping Rh (C, c, E, e) and phenotyping for Kell, Kidd, and Duffy was carried out by hemagglutination technique using Diamed anti-sera (e.g., anti-K, anti-k, anti-Fya, anti-Fyb, anti-JKa, and anti-JKb) using ID-Card Antigen provided by Diamed system, Switzerland.
The antibody-screening test was done by indirect antihuman globulin gel card method (IAT) using an antibody screening panel (DiaCell I + II + III; Diamed, Switzerland). While, antibody identification was performed using the indirect antiglobulin test using an extended commercial 11-cell antibody identification panel (DiaPanel; Diamed, Switzerland). An auto control, using the patient's cells and serum, was added for testing side by side with each screen to exclude the existence of autoantibodies.
Minor blood group genotyping identification
The Kell blood group antigens K and k, Kidd blood group antigens JKa and JKb, and Duffy blood group system antigens FYa and FYb polymorphisms were identified by PCR-RFLP analysis which includes four steps: DNA extraction, amplification, restriction enzyme digestion, and finally electrophoresis.
The DNA was extracted from peripheral blood leukocytes using a DNA extraction kit (Thermo Scientific Gene JET Whole Blood Genomic DNA Purification Mini Kit). Afterward, each PCR reaction contained 10 μL of DNA template, 25 μL of master mix (Thermoscientific DreamTaq Green PCR Master Mix ×2), 1 μL of each forward and reverse primers [Table 1] and the final volume was completed to 50 μL by nuclease-free water with annealing at 62 °C.
|Table 1: Primer sequences for restriction fragment length polymorphism-polymerase chain reaction of Kell, Kidd and Duffy blood group system antigens|
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Following the manufacturer's instructions, the amplification products were then digested by restriction endonuclease (MnlI, BanI [BshNI] and Mva [BsmI]) for Kidd, Duffy, and Kell respectively (FastDigest, Thermo Scientific). The fragments were then separated electrophoretically by running the gel for 10 min at 70 V followed by 20 min at 90 V for Kell and Duffy and 40 min at 80 V for Kidd identification.
Interpretation of PCR result
The digestion product for the Kidd blood group alleles was 246 bp in size and the MnlI digestion resulted in 110, 80, and 56 bp fragments. For the Duffy blood group alleles, the PCR product was 392 bp in size, and the BanI digestion resulted in 306 bp or 210 bp and 86–96 bp fragments. Finally, for the Kell blood group alleles the PCR product was156 bp in size and the BsmI digestion resulted in 100 and 56 bp fragments [Figure 1].
|Figure 1: Gel electrophoresis showing blood group genotyping. (a) Duffy blood group genotyping. Sample 12: Fy01,02; Samples 11, 13, 14 and 17: Fy 02,02. (b) Kell blood group genotyping. All sample are Kell02,02. (c) Kidd blood group genotyping. Sample 28 and 33:JK01, 02; Sample 25,31 and 32: JK01,01; Samples 23 and 26: JK02,02|
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Data were analyzed using Statistical Package for Social Science (IBM SPSS) version 23. Qualitative data were presented as numbers and percentages, while quantitative variables were described as mean and standard deviation in parametric data or median and interquartile range (IQR) in nonparametric one (IQR; the difference between 25th and 75th centiles).
A comparison between two groups with qualitative data was done using the Chi-square test (χ2). Fisher's exact test was used instead of χ2 test when the expected count in any cell was found <5. Comparison between two independent groups regarding quantitative data with parametric distribution was done using an Independent t-test, while the comparison of quantitative data with nonparametric distribution among two groups was done using the Mann–Whitney test. A P < 0.05 was considered the cut-off value for significance in all analyses.
| Results|| |
The present study was conducted on 100 repeatedly blood transfused patients with a male-to-female ratio 1:1.5. Their median age was 10 years with the 25th and 75th percentile IQR 6 and 14 years, respectively. Patients' demographic and clinical data are listed in [Table 2].
The most frequent blood group was O (57%), while the least one was AB (4%). 92% of the patients were Rh positive, with the most frequent antigen was e (98%) followed by C (96%), c (94%), and the least one being E (42%).
The antibody screening tests revealed clinically significant both allo and autoantibodies in 14% and 16% of patients, respectively. Eighteen different alloantibodies were detected, with the most encountered one being anti-Kell1 (K) in 42.9%. Finally, 4 patients (22%) showed double antibodies (2 with anti-E, K and 2 had anti-E, C).
The median age of patients who developed alloantibodies was 18 years (IQR: 15–50) and for those who did not develop alloantibodies was 9 years (IQR: 5-13). Moreover, alloantibodies formation was significantly higher in old, female, transfusion-dependent, ß-Thalassemia major patients and those with splenectomy (P = 0.000, 0.034, 0.011, 0.001, and 0.000, respectively).
Maximum numbers of alloantibodies were detected in O-positive group patients. Furthermore, a significant difference was shown as regards Kidd blood group genotyping and formation of both auto and alloantibodies (P = 0.022 and 0.002, respectively) with the commonest genotypes 02.02 and 01.01, respectively. On the other hand, 12 patients out of 16 and 8/14 who developed either auto or alloantibodies were 02.02 Duffy genotype with no significant difference detected. Finally, all patients who developed either auto or alloantibodies (16/16 and 14/14) were 02.02 Kell2 genotype.
Serological testing for Kell, Kidd, and Duffy revealed that 90% were Kell2 (k) antigen positive, 34% were JK1 (a + b−) and finally, 26% were FY (a + b−) [Table 3].
|Table 3: Phenotype and genotype frequencies of Kell, Kidd, and Duffy blood group systems|
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DNA genotyping using PCR-RFLP showed that the most common genotype frequencies observed in Kell, Kidd, and Duffy systems were Kell02/Kell02 (92%), JK01/02 (42%), and Fy02/Fy02 (60%), respectively [Table 3].
Serological testing did not identify the patients' phenotype in 2%, 46%, and 72% of patients in the Kell, Kidd, and Duffy blood group system respectively. In contrast, the genotyping overcomes this problem and raised the percentage of patients' phenotype identification to 100%.
Moreover, discrepancies between genotyping and serological testing showed significant differences with the highest value among the Duffy blood group system being 92% (P = 0.011), followed by 76% for the Kidd blood group system (P = 0.146). On the other hand, 98% of the Kell blood group system showed similarities between both genotyping and serological testing [Table 4].
|Table 4: Comparison between blood group phenotyping and genotyping of Kell, Kidd, and Duffy blood systems|
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Genotype, demographic, and clinical data association
Kidd JK-3 (01.02 genotype) occurred mostly in males, with positive consanguinity, suffering from ß-thalassemia major and nonsplenectomized (P = 0.008, 0.000, 0.002, and 0.032).
In the Duffy blood group, most of our female patients that were presented with ß thalassemia had a 02.02 genotype (P = 0.009 and 0.010, respectively).
| Discussion|| |
The transfusion of blood and blood components has become an integral part of the management of patients with hematologic disorders and malignancies. Many of those patients require a blood transfusion during their illness or maybe lifetime.
RBCs alloimmunization resulting from the genetic disparities between donor and recipient is one of the risks of long-term transfusion therapy. Our study confirmed this finding, as 14% and 16% of our patients suffered from either auto and/or alloantibodies, respectively. This was consistent with the study done among the Egyptian population in 2014, where the researchers detected alloantibodies in transfusion-dependent patients in the frequencies of 10.5%. In disagreement with us, Sahu et al. in 2020 and Abdulquader et al. in 2020 detected lower frequencies of both auto (6.6% and 4%) and alloantibodies (7.5% and 5.8%)., On the other hand, a higher rate of alloimmunization was found in studies from Taiwan. These discrepancies could be attributed to the difference in the adopted protocols in the transfusion centers, where using filtered leukodepleted RBCs was shown to reduce alloimmunization in multi-transfused patients. Heterogeneity and racial diversity between donors and recipients were also considered contributing factors. Moreover, the age of starting blood transfusion may play a role in this issue.
To reduce RBCs alloimmunization as well as HTRs, antigen typing to provide phenotype-matched donors is mandatory. Unfortunately, in general, ABO and Rh D antigens are the only ones tested. On the other hand, many other red cell antigens which are highly immunogenic such as Kell, Duffy, Kidd, and Rh antigens besides Rh D are not matched and this mismatch predisposes to alloimmunization. It is of interest that our study revealed the predominance of anti-Kell1 (42.9%), followed by 28.6% with Anti-C, 14.3% anti-JK1, and 7.1% anti-Fy a.
Antigen distribution caused by different population-related events such as natural selection and gene flow in descendant populations contributes to high differences in RBCs antigens frequencies observed in unrelated ethnic groups. Classic serological techniques are the gold standard one used to type several red cell antigens. However, they have many limitations and drawbacks. Thus, the application of molecular techniques for blood group polymorphisms can make predicting blood group genotypes and phenotypes possible.
Serological testing for Kell, Kidd, and Duffy revealed that 96% were Kell2 (k) antigen-positive, 34% were JK1 (a + b−), and finally, 26% were FY (a + b−). On the other hand, serology failed to identify the phenotype in 2%, 46%, and 72% of Kell1 (K), Kidd, and Duffy, respectively, where the mixed field was detected.
A high prevalence of k antigen was also recorded by Costa et al., in 90% of multi-transfused patients. Our study detected Kell1 antigen in 8%, in accordance with the percentage detected in both the Sudanese (5.6%) and Caucasian (9%) populations,, but in disagreement with the Egyptian study done in 2018, where 23.6% of a studied Egyptian population were Kell1 antigen positive. This variation could be attributed to the sample size difference.
In India, Shah et al., in 2020 results were in line with the present study as regards the prevalence of JKa + b− (33.3%), but their results disagree with ours regarding the Duffy system as Fya + b + was the predominant phenotype (42%). These variations could be due to ethnic effects.
Genotyping revealed that the most common genotypes were Kell02, 02 (92%), JK01,02 (42%), and Fy02,02 (60%) for Kell, Kidd, and Duffy systems, respectively. Costa et al., 2016 agreed with our results as Kell02,02 and JK01,02 were demonstrated in 90% and 58%, respectively. Furthermore, Jalali et al. in 2020 detected JK01,02 in 47.5% of the studied subjects. On the other hand, results of the present study were different compared to previous studies where Fy01,02 and Fy02,02 were found in 50.9% and 10.5% of the patients, respectively. These differences could be attributed to the high polymorphism among RBCs antigens as well as the variation of alleles distribution between different populations and ethnic groups.
A significant discrepancy was found between serological and molecular analysis, with the highest rate of discordance found in the Duffy system (92%, P = 0.011), the Kidd system (76%, P = 0.146), and finally Kell system (2%, P < 0.001). These results coincided with Shah et al., in 2020 and Gholami et al. in 2021 who showed that Duffy had the highest mismatch between serological and molecular methods (66.7% and 34.8%, respectively) in multi-transfused thalassemia patients, while Kidd mismatch was (59.6 and 26.1%, respectively) in the studied patients., No Kell mismatch was detected in both studies. Zarghamian et al., in 2021, also found no discrepancy between the serological and molecular typing of Kell antigens indicating that genotyping can be used as an alternative method to identify the phenotype in instances such as in those patients on regular transfusion.
The disagreement between serological and molecular typing in our study was in the range of worldwide reported frequency of genotype-phenotype discrepancy being found in 15%–90% of multi-transfused patients. Compared to previous studies, our results may be different due to differences in ethnicity, variable sample size, and different age groups. This is in addition to variant alleles that may cause loss or weakening of antigen expression leading to discordant results between genotype and phenotype.
In the end, determining the accurate phenotype of blood group antigens plays a critical role in transfusion. However, in multi-transfused patients, MF agglutination due to the presence of donor RBCs interferes with the interpretation of the patients' blood phenotype and further selection of antigen-matched blood products for those patients. In such circumstances, genotyping will be helpful in the identification of their phenotype and prevention of alloimmunization. The present study succeeds to prove this theory as PCR-RFLP identifies the extended blood group antigens accurately in 100% of the patients.
| Conclusion|| |
The occurrence of mixed field reactions in serological typing hampers the exact red cell phenotype identification, especially in multi-transfused patients. These limitations highlight the importance of performing DNA-based molecular analysis in the determination of extended blood group antigens that can assist in the selection of well-matched donor-recipient RBCs units for transfusion with a subsequent decrease in alloimmunization and HTRs. Furthermore, genotyping can help to build up the blood group datasets in each country and provides an important reference standard for studying diseases associated with blood group systems.
Limitation of the study
As genotyping was not routinely introduced in our hospital during the study, it was difficult to define the percentage of alloimmunization before and after genotyping introduction.
Ethics approval and consent to participate
Informed written consents were obtained from all enrolled patients. The study was approved by the Scientific and Ethical Committee, Ain Shams University (FMASU 338/2017), and was in accordance with the declaration of Helsinki.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding on reasonable request.
All authors have read and approved the final manuscript submission. IY and DH conceptualized and designed the study. DS and HA contributed to data interpretation and manuscript writing. HS performed the technical work. DM selects cases and collects clinical data.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Dalia Diaa ElDine Salem,
Faculty of Medicine, Ain Shams University, El Abasseya Square, Cairo 11566
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3], [Table 4]