Real-time PCR genotyping of human platelet alloantigens HPA-1, HPA-2, HPA-3 and HPA-5 is superior to the standard PCR-SSP method.

Genotyping of the human platelet alloantigens (HPA) is useful for the diagnosis and therapy of the patients with alloimmune thrombocytopenic syndromes, such as post-transfusion refractoriness to platelets, post-transfusion thrombocytopenic purpura and foetomaternal alloimmune thrombocytopenia. We have developed, optimized and validated a new method for simultaneous genotyping of HPAs - HPA-1, HPA-2, HPA-3 and HPA-5 - by using the real-time polymerase chain reaction (PCR) based on TaqMan technology. Its performances were compared to those of the standard PCR-sequence-specific primers (SSP) method by testing 120 DNA samples. Several discrepancies between the two methods have been observed, especially in the HPA-3 genotyping. Evidently, the PCR-SSP method produced several false positive results due to its technical drawbacks. Based on our comparison, we believe that the new real-time TaqMan PCR assay for the HPA-1, HPA-2, HPA-3 and HPA-5 genotyping is faster, more reliable and reproducible, compared to the standard PCR-SSP.

Differentiation of autologous ABO, RHD, RHCE, KEL, JK, and FY blood group genotypes by analysis of peripheral blood samples of patients who have recently received multiple transfusions.

BACKGROUND: After multiple transfusions, the serologic typing of autologous blood group phenotypes is difficult, because of mixed RBC populations. The genotyping of ABO, Rh, Kell, Kidd, and Duffy systems could be used to determine autologous blood group antigen status. STUDY DESIGN AND METHODS: Blood samples from patients and donors were analyzed before and after 26 multiple-transfusion events. An average of 6.9 non-WBC-reduced RBC units with an average age of 5.9 days were administered per transfusion event. The average period of blood sampling after transfusions was 5.3 days. All samples were serologically phenotyped for ABO, Rh, Kell, Kidd, and Duffy. Pretransfusion, posttransfusion, and buccal samples from patients were genotyped for the corresponding alleles by a uniform PCR sequence-specific primer protocol that allowed their simultaneous determination within 3 hours. RESULTS: All posttransfusion samples exhibited mixed-cell populations of various blood group systems on serologic testing. Genotyping from peripheral blood produced results identical to the autologous blood group phenotypes, regardless of the amount of blood transfused or of the length of the sampling period after transfusion. CONCLUSION: A fast and reliable PCR-sequence-specific primer DNA genotyping assay for simultaneous determination of autologous ABO, Rh, Kell, Kidd, and Duffy blood groups can be performed on peripheral blood samples, even though the patients have recently received multiple transfusions.

A case of pseudothrombocytopenia after infusion of abciximab in vivo and anticoagulant-independent platelet clumping after rechallenge with abciximab in vitro.

A 45-year old man was treated for unstable angina pectoris with percutaneous transluminal angioplasty and stenting of his left anterior descending coronary artery. The procedure was followed by infusion of abciximab. The patient's automated platelet count in an EDTA-anticoagulated blood sample at admission to the hospital was normal, but dropped to 5 x 10(9)/l three hours after the procedure. The infusion of abciximab was stopped and the patient received platelet transfusions although there were no signs of bleeding. Two days later his platelet count was still low (37 x 10(9)/l) in an EDTA-anticoagulated blood sample, but normal (193 x 10(9)/l) in a heparin-anticoagulated sample. Platelet clumps were present only in the sample anticoagulated with EDTA, and pseudothrombocytopenia was diagnosed. The patient's recovery was uneventful. At follow-up visits four months and one year after discharge from hospital, the patient's blood samples were anticoagulated with three different anticoagulants: EDTA, citrate and heparin. The platelet count was normal in all three samples but after mixing with abciximab in vitro it dropped profoundly due to platelet clumping, regardless of the choice of the anticoagulant. Our report raises two points: (a) one needs to consider the possibility of pseudothrombocytopenia in a patient with a low automated platelet count after infusion of abciximab but without signs of bleeding, and (b) the in vitro results suggest that our patient who had initially responded to abciximab with pseudothrombocytopenia could develop true thrombocytopenia after repeated exposure.

Fyx is associated with two missense point mutations in its gene and can be detected by PCR-SSP.

The Duffy blood group antigens are encoded by the Duffy gene with its three major alleles: Fy*A (Fya+), Fy*B (Fyb+), and a nonexpressed Fy*Fy (Fya-b-), which is most commonly found among black people. Additionally, a fourth allele, Fyx, is found among white people and defined as weak Fyb not detectable by all anti-Fyb. Three polymerase chain reactions (PCRs) using sequence-specific priming (SSP) for detection of the major FY alleles were developed. Eighteen Fy(a-b-) samples of Tanzanian origin were correctly typed and of 300 random donors of Caucasian origin with known Fy phenotype, only four out of 59 Fy(a+b-) donors showed the discrepant DNA-type Fy(a+b+). Serologic reinvestigation by adsorption and elution techniques confirmed weakly expressed Fyb antigen in these cases and DNA sequencing of the entire Duffy gene revealed identical point mutations in all of them. Specific PCR reactions were used to reinvestigate the C265T (Arg89Cys) and G298A (Ala100Thr) substitution in the 300 samples. A298 was found to be present in both FY*X and FY*B alleles, pointing to an allelic variation among FY*B alleles. T265 was encountered exclusively in FY*X and is thought to be FY*X specific. Combining the T265 specific reaction with the three PCR-SSPs described above, we were able to correctly DNA-type all phenotypes investigated in our study.