Blood Group Variants in an Egyptian Individual: A Case Study

INTRODUCTION

An ABO discrepancy was detected in the teaching laboratory of a medical laboratory sciences (MLS) program when a student performed a type and antibody detect test (ADT) on their own blood specimen as part of an educational exercise. Following the initial results, an institutional review board (IRB) application was submitted and approved to further investigate the discrepancy (IRB-AY23-24-299). Subsequent serologic and molecular analyses revealed the presence of additional rare blood group alleles. The participant was found to have the rare A₂B blood type with anti-A1 antibodies, a partial c antigen, and the GATA-box silencing mutation, contributing to the limited literature on blood group diversity in the student’s Egyptian ancestry.

Managing blood transfusion in individuals with rare blood types presents unique challenges. Individuals exhibiting certain ABO discrepancies can complicate compatibility testing and increase the risk of hemolytic transfusion reactions unless they are accurately detected and the underlying cause identified.¹ Additionally, inheritance of genetic variants, including partial c antigen expression, can lead to the formation of alloantibodies that complicate the acquisition of compatible blood. This case study highlights the critical role of combining serologic and molecular testing in identifying blood group genetic variants, underscores the value of hands-on learning in MLS programs, and demonstrates how educational laboratory activities can lead to clinically significant discoveries.

MATERIALS AND METHODS

After collecting the subject’s blood in a tube containing ethylenediaminetetraacetic acid (EDTA), a 2% to 5% RBC suspension was made using physiologic saline and was washed once prior to testing to achieve optimal testing results. ABORh forward and reverse typing, Dolichos biflorus lectin testing, and the ADT were initially performed manually using tube technique with low ionic strength solution as the enhancement media as part of a routine educational activity.

After IRB approval was obtained, additional EDTA specimens were collected and labeled with fictitious data to protect the anonymity of the subject. All serologic testing was repeated for confirmation using well-controlled antisera in a local hospital’s immunohematology laboratory, and all results were concordant with previous testing. Separate refrigerated whole-blood specimens collected in EDTA were sent to the New York Blood Center and LifeShare Blood Services for ABO and additional blood group genetic testing, respectively.

Genetic testing was performed using Sanger sequencing (SS) to determine the subject’s ABO alleles on a refrigerated specimen of whole blood collected in EDTA. After extracting the DNA from the whole-blood specimen, the DNA was amplified using polymerase chain reaction (PCR) to target the ABO gene. The final genotype was determined by examining the ABO promoter, enhancer, exons 1 to 7, and flanking intron regions.

Additional blood-group genotyping using targeted next-generation sequencing (NGS) was performed to detect the single-nucleotide polymorphisms. The DNA was extracted from a refrigerated whole-blood specimen and then amplified using targeted multiplex PCR. The individual reactions were combined to create an NGS library. After quantification, the NGS library was loaded on a sequencer for data acquisition, and the data were analyzed using specialized software designed to assign genotypes and generate the predicted phenotypes.

Capture solid phase technology (Immucor/Werfen) was used to determine whether the anti-A1 antibody isotype was completely IgG or if it contained an IgG component. The procedure was performed according to the manufacturer’s instructions using a semi-automated Capture workstation (Immucor/ Werfen). Reagent A₁ cells were immobilized to the bottom of the microstrip wells, and a drop of the manufacturer’s positive and negative control serum were added to their respective cells. The subject’s plasma was collected from an EDTA specimen and was added to 3 microstrip wells to perform the isotype identification in triplicate to ensure reproducibility. After performing the subsequent steps according to the manufacturer’s instructions, an illuminated light box was used to read the reactions. The presence of a smooth monolayer of cells indicated a strongly positive reaction, and tight cell buttons demonstrated a negative reaction.

RESULTS

The subject’s ABORh-forward type demonstrated an AB-positive blood type, while the reverse type displayed an unexpected 2+ positive hemagglutination reaction with the A₁ cells using the manual tube technique. The RBCs were then tested with 2 different lots of D. biflorus lectin, and the cells were nonreactive, indicating the absence of A₁ antigens.

Additionally, the subject’s plasma consistently demonstrated 2+ positive reactions when tested with 2 additional lots of A₁ cells and negative reactions with 2 different lots of A₂ cells using the manual tube technique. Subsequently, the ADT demonstrated all negative reactions at room temperature, ruling out cold reactive unexpected antibodies.

The subject’s ABO genotype, ABO*A2.01/B0.1, was determined with SS. In addition, the extended blood group genotype was found to be RHCE*01.20.01/RHCE*02. C+, E-, e+, V+, VS+ and partial c+, and the FY*02N.01 GATA box mutation was detected.

On completion of the peripheral blood smear, hypochromia was noted, along with few ovalocytes and acanthocytes (Figure 1). Additionally, the automated complete blood cell (CBC) results generated from a Sysmex XS-1000i, indicating that the mean corpuscular volume (MCV) is slightly less than the lower limit of the reference interval, corresponding with the mild microcytosis observed (Table 1).

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Figure 1.

Peripheral blood smear. Left arrow indicates a hyphochronic RBC, and the right arrow indicates an ovalocyte.

Because the capture solid phase method used in this study detects IgG only, the lack of reaction of the subject’s anti-A1 antibodies indicates that the antibodies do not contain an IgG component and are, therefore, presumed to be predominantly of the IgM isotype.

DISCUSSION

This case study, examining an ABO discrepancy discovered in an educational exercise, emphasizes the importance of implementing pretransfusion testing protocols in clinical curriculum. This analysis exemplifies a practical scenario that has the potential to be encountered in a routine laboratory setting and yields blood grouping results that are underexplored in the Egyptian population. Genotype testing revealed the subject to be A₂B and Rh positive with the expression of a partial c antigen. The absence of prior pregnancies or transfusions, combined with the characterization of anti-A1 antibodies as wholly or partially IgM, suggests natural antibody stimulation and adds to the limited published data regarding the tendency of A₂B individuals forming non-RBC immune anti-A1 antibodies. Reported ABO blood group distributions across various North African countries are similar to those observed in Egypt. For example, the frequency of the AB-positive type is reported as 5.00% in Tunisia, 4.28% in Algeria, and 5.00% in Sudan.^18⇓-20 Although expression of the partial c antigen is uncommon and poorly documented in the literature, it poses a potential risk for transfusion incompatibility because partial c antigen expression is associated with a higher risk of alloimmunization.¹³ Furthermore, the frequency of the FY*02N.01 allele within the Egyptian population is also currently unknown.

CONCLUSION

This case study highlights the importance of investigating blood group genetic variations across populations. The data collected provide preliminary insight into potential causes of discrepancies encountered in the immunohematology laboratory. In addition, this case highlights the utility of combining traditional serologic testing with molecular methods to achieve comprehensive blood typing. Currently, blood group allelic frequencies within the Egyptian population are not well documented. Further research is needed to underscore the significance of our findings. Expanding the sample size would enhance the reliability and applicability of our results, particularly in addressing ABO discrepancies and partial Rh antigen expression among different ethnic groups.