A 90-year-old male patient with one history of blood transfusion was admitted to the hospital for prostatitis and acute retention of urinary. After evaluation by the urologist, he underwent the proposed surgery. Routine blood analysis revealed a hemoglobin (Hb) level of 106g/L, and 2 units of red blood cells were applied for blood preparation before operation. The patient was group A, RhD positive, with positive result of unexpected antibody screening. Crossmatching tests were performed on four donors, revealing major matching incompatibilities, which needs further testing.

The patient's antibody screening showed a positive result with the microcolumn agglutination anti-human globulin(MA-AHG) test and a negative result with the saline medium, while direct antiglobulin test (DAT) was negative. Alloantibody was suspected in this patient . Antibody identification was conducted using a sixteen-panel cells, resulting in positive findings for MA-AHG with agglutination observed at 2+. Since the patient's DAT was negative, autoantibodies were ruled out. Additionally, the absence of anti-Kpb, anti-Jsb and anti-Lub antibodies were confirmed through antigen typing in this patient. Crossmatching tests were conducted with 40 donors, revealing 2+ to 3+ agglutination on the major, while all minor results were negative. Therefore, it is suspected that the patient produces combined antibodies or high-prevalence antigen antibodies.

Rh typing for patients’ red blood cells demonstrated agglutination with IgM monoclonal anti-D, while no agglutination was observed with IgM monoclonal anti-C, anti-c, anti-E, and anti-e, indicating that the Rh blood group as D-- phenotype. There was no difference in agglutination intensity IgM monoclonal anti-D by direct agglutination between D antigen in this patient and normal individual, so titer detection was performed. The titer of human anti-D (The reagent was prepared in our laboratory from an individuals who was RhD negative and produced anti-D) detected by red blood cells from DCCee blood donors with the same ABO type as the patient was 4, whereas the titer of human anti-D detected by this patient's red blood cells was 16. To evaluate the expression of D antigen, we designed absorption and elution tests, these tests were conducted using two types of red blood cells and human anti-D serum. O+ red blood cells were utilized for anti-D titers in the elution solution. The anti-D titer of elution solution from the red blood cell classified as DCCee was 2, while the titer of elution solution from this patient's red blood cell was 4. This indirectly indicates a higher expression of the D antigen in this patients' red blood cells compared to normal D+ red blood cells. The red blood cells with three different Rh phenotypes (DCCee, DccEE, dccee) were washed to absorb this patient serum, and acid release experiments were conducted. The pattern of absorbtion and elution solution of the three types was similar, albeit with varying intensities, raising suspicion of a combined composite antibody or anti-Hr0 of the Rh blood group system. Refer to Fig. 1 for the detection process.

Flow chart of antibody identification

By analyzing the exon 1–7 sequence of the RHCE gene, homozygous mutations were identified in exons 1, 2, and 5. The specific mutation sites can be seen in Fig. 2. Referring to the RH (ISBT004) Blood Group Alleles: RHCE (004RHCE Alleles v4.0-20180208) data from the ISBT website, using RHCE*01 (RHCE*ce, NM_020485 (mRNA)) as the reference sequence, and considering the sequencing results, it was observed that the 5th exon of the RHCE gene in this patient was substituted with the 5th exon of the RHD gene. The remaining sequence aligned with the characteristics of the RHCE*C allele, leading to the gene being named RHCE*CE-D(5)-CE.

Results from Rh gene sequencing (EXON1, EXON2, and EXON5). The black arrow indicates the base mutation site. No mutations were detected in EXON3-4 and EXON6-10