Hypereosinophilia secondary to Sézary syndrome

The Sézary syndrome is a leukemic form of cutaneous T-cell lymphoma characterized by the presence of erythroderma covering at least 80% of the body-surface area, lymphadenopathy, and the presence of clonally related neoplastic T cells with cerebriform nuclei (Sézary cells) in the blood, skin, and lymph nodes. Hypereosinophilia can be caused by hematologic malignancy with clonal abnormality, which is often associated with Sézary syndrome. Sézary syndrome has rarely been reported in Korea. However, hypereosinophilia in the Sézary syndrome has not been reported in Korea. Here, we report a case of 75-year-old man with hypereosinophila, erythroderma, and cutaneous T-cell lymphoma which was finally diagnosed as Sézary syndrome.

Relationship Between Rotavirus P6 Infection in Korean Neonates and Histo-Blood Group Antigen: a Single-Center Study

Background@#Rotaviruses are a major cause of pediatric gastroenteritis. The rotavirus P[6] genotype is the most prevalent genotype isolated from Korean neonates but has rarely been reported in other countries. Histo-blood group antigen (HBGA) is known to play an important role in rotavirus infection. We investigated the relationship between rotavirus genotype and HBGA-Lewis blood type in Korean children and explored the reasons for the predominance of rotavirus P[6] strain in Korean neonates. @*Methods@#Blood and stool samples were collected from 16 rotavirus-infected patients. Rotavirus G (VP7) and P (VP4) genotyping was performed using reverse transcription-PCR and sequencing. Lewis antigen phenotypes (Lea /Leb ) were tested, and HBGA-Lewis genotype was determined by sequencing the secretor (FUT2) and Lewis (FUT3) genes. Deduced amino acid sequences and three-dimensional structures of the VP8* portion of the rotavirus VP4 protein were analyzed. @*Results@#All P[6] rotaviruses were isolated from neonates under one month of age, who were negative or weakly positive for the Leb antigen. However, 10 of the 11 non-P[6] rotaviruses were isolated from older children who were Leb antigen-positive. The VP8* amino acid sequences differed among P[6], P[4], and P[8] genotypes. Korean P[6] strains showed a unique VP8* sequence with amino acid substitutions, including Y169 > L169, which differed from the sequences of P[6] strains from other countries. @*Conclusions@#The predominance of the rotavirus P[6] genotype in Korean neonates may be related to the interaction between HBGA-Lewis antigen and the VP8* portion of the VP4 protein, and this information will be helpful in future neonatal vaccine development.

Performance Evaluation of the Automated Fluorescent Immunoassay System Rotavirus Assay in Clinical Samples

BACKGROUND: The Automated Fluorescent Immunoassay System (AFIAS) rotavirus assay (Boditech Med Inc., Chuncheon, Korea) is a new rapid antigen test for rotavirus detection. We evaluated the performance of this assay for detecting rotaviruses and their specific genotypes in clinical stool samples. METHODS: AFIAS rotavirus assay was performed in 103 rotavirus-positive and 103 rotavirus-negative stool samples (confirmed by both PCR and ELISA), and its results were compared with those of PCR, ELISA, and immunochromatographic assay (ICA). We evaluated diagnostic sensitivity/specificity, the detectability of rotavirus subtypes, lower limit of detection (LLOD), reproducibility, cross-reactivity, and interference of AFIAS rotavirus assay. RESULTS: Based on PCR and ELISA results, diagnostic sensitivity and specificity of the AFIAS rotavirus assay were both 99.0%. LLOD results showed that the AFIAS assay had sensitivity similar to or greater than ICA and ELISA. High reproducibility was confirmed, and no cross-reactivity or interference was detected. This assay could detect genotypes G1P[8], G2P[4], G3P[8], G4P[6], G4P[8], G8P[4], G8P[8], G9P[4], and G9P[8]. CONCLUSIONS: The AFIAS rotavirus assay showed high reproducibility, sensitivity, and specificity as well as excellent agreement with ELISA, PCR, and ICA. It detected the most common as well as unusual genotypes of rotavirus prevalent in Korea. It could be a useful on-site assay for rapid, convenient, and cost-effective detection of rotavirus infection.

Proposed Imprecision Quality Goals for Urinary Albumin/Creatinine Ratio

BACKGROUND: The urinary albumin/creatinine ratio (ACR) is an important indicator of albuminuria. We aimed to estimate ACR uncertainty and its impact on test results and proposed imprecision quality goals based on the estimated uncertainty. METHODS: The combined ACR uncertainty was calculated using the individual uncertainties of urinary albumin and creatinine. ACR confidence intervals (CIs) were estimated based on the expanded uncertainty. When the CI contained the ACR category boundary (30 or 300 mg/g), the cases were considered ambiguous. Quality goals for ACR were suggested using the number of ambiguous cases among actual patient results. RESULTS: The number of ambiguous cases resulting from the combined ACR uncertainty was higher than expected based on biological variation (BV) quality goals. When the ACR met BV quality specifications, we estimated that 4.8–15.5% of the results may have been misclassified. To minimize the number of ambiguous results, the minimum, desirable, and optimum quality goals were set at 34.0%, 18.0%, and 4.5%, respectively. CONCLUSIONS: We expressed ACR uncertainty using the uncertainties of urinary albumin and creatinine and assessed the impact of this combined uncertainty on the test results. Subsequently, we proposed imprecision quality goals for ACR based on ambiguous results.

Evaluation of a New Multiplex Real-Time PCR Assay for Detecting Gastroenteritis-Causing Viruses in Stool Samples

BACKGROUND: Diarrhea has been the second leading cause of death among children under the age of five, and the rapid and accurate pathogen diagnosis in patients with diarrhea is crucial for reducing morbidity and mortality. A newly developed one-step multiplex real-time PCR assay, the Allplex GI-Virus Assay, was evaluated for its ability to detect six diarrhea-causing viruses (rotavirus, norovirus genogroup I (GI) and genogroup II (GII), enteric adenovirus, astrovirus, and sapovirus) in stool samples. METHODS: The performance of the Allplex assay was compared with those of another multiplex PCR assay (Seeplex Diarrhea-V Ace Detection) and genotyping by sequencing, using 446 stool samples from patients with acute gastroenteritis. RESULTS: The overall agreement rates between the results of the Allplex and Seeplex assays were 98.7% for rotavirus, 99.1% for norovirus GI, 93.3% for norovirus GII, 98.0% for adenovirus, and 99.6% for astrovirus. The overall agreement rates between the Allplex assay and genotyping were 99.1% for rotavirus, 99.1% for norovirus GI, 98.7% for norovirus GII, 89.7% for adenovirus, 98.2% for astrovirus, and 99.8% for sapovirus. In addition, eight rotavirus genotypes, three norovirus GI genotypes, four norovirus GII genotypes, eight adenovirus genotypes, two astrovirus genotypes, and two sapovirus genotypes were detected. CONCLUSIONS: The Allplex assay showed high agreement with Seeplex and genotyping results, and was able to additionally detect sapoviruses. The Allplex assay could be useful in identifying viral gastrointestinal infections in patients with acute gastroenteritis symptoms.

Comparison of the Luminex xTAG Respiratory Viral Panel Fast v2 Assay With Anyplex II RV16 Detection Kit and AdvanSure RV Real-Time RT-PCR Assay for the Detection of Respiratory Viruses

BACKGROUND: The accurate and rapid identification of the causative viruses is important for the timely diagnosis and management of respiratory infections. Multiplex molecular diagnostic techniques have been widely adopted to detect respiratory viruses. We compared the results of a newly upgraded, multiplex, molecular bead-based respiratory viral panel (RVP) assay with the results of Anyplex II RV16 detection kit and AdvanSure RV real-time RT-PCR assay. METHODS: We tested 254 respiratory specimens and cultured viral strains using the Luminex xTAG RVP Fast v2 assay (Luminex Molecular Diagnostics, Canada) and Anyplex II RV16 detection kit and compared the results. Specimens showing discordant results between the two assays were tested with a AdvanSure RV real-time RT-PCR assay. RESULTS: Of the 254 respiratory specimens, there was total agreement in the results between the xTAG RVP Fast v2 assay and the other real-time PCR assay in 94.1–100% of the specimens. The agreement levels were relatively low (94.1–97.6%) for specimens of adenovirus, coronavirus NL63, and parainfluenza type 3. In comparison to the other assay, the xTAG RVP Fast v2 assay detected a higher number of parainfluenza type 3 (4 cases) and metapneumovirus (9 cases). CONCLUSIONS: The xTAG RVP Fast v2 assay showed comparable capabilities compared with the other assays; it will be useful for identifying respiratory viral infections in patients with respiratory symptoms. Clinicians should be aware of the characteristics of the assays they use, since different assays show different detectability for each virus.

Evaluation of the VIDAS Anti-HCV Assay for Detection of Hepatitis C Virus Infection

BACKGROUND: Anti-hepatitis C virus antibody (anti-HCV) assays are recommended for screening HCV-infected persons. The VIDAS Anti-HCV Assay (bioMérieux, France), based on the enzyme-linked fluorescence test principle, was recently introduced in Korea. We evaluated the clinical performance of the VIDAS assay. METHODS: One hundred HCV-positive and 1,002 HCV-negative blood samples confirmed by Architect anti-HCV (Abbott Laboratories, USA) and COBAS TaqMan HCV real-time PCR (Roche Diagnostics, USA) or the Procleix Ultrio Plus Assay (Gen-Probe Incorporated, USA) were obtained from the Human Serum Bank (HSB) and tested by VIDAS. In case of discrepant results, we conducted a recombinant immunoblot assay (RIBA). RESULTS: The agreement rates for known HCV-positive and HCV-negative samples between the VIDAS assay and the HSB testing were 100% (95% confidence interval [CI]: 96.4-100%) and 99.5% (95% CI: 98.8-99.8%), respectively. One of the five discrepant samples was positive for Core 2+ and NS3-2 2+ reactivity, two samples were negative, and the other two were indeterminate regarding NS4 2+ reactivity in RIBA. We observed a significant but weak positive correlation between the titers of VIDAS and Architect assays (r=0.315, P<0.001). CONCLUSIONS: The VIDAS anti-HCV assay, developed on the VIDAS automated immunoassay platform based on the ready-to-use, single-sample test concept may be useful in small-to-medium-sized laboratories. It showed good agreement with Architect anti-HCV and COBAS PCR assays and is therefore useful for detection of HCV infection. Weakly test-positive (ambiguous) samples require additional testing by another anti-HCV, RIBA, or HCV RNA assay.

Establishment of Platelet Antigen and Antibody Tests by Mixed Passive Hemagglutination with Frozen-stored Platelets / 대한수혈학회지

BACKGROUND: Platelet antigen and antibody tests have been used in platelet immunological disorders, such as neonatal alloimmune thrombocytopenia (NAIT) and post-transfusion purpura (PTP). Mixed passive hemagglutination (MPHA) method has several advantages, including frozen preservation of platelets, ability to differentiate between anti-HLA and platelet-specific antibodies, and quick and easy interpretation without expensive equipment. In this study, we intended to develop the MPHA method using indicator cells of anti-Rh(D) sensitized group O, Rh+ RBCs. METHODS: We made indicator cells sensitized with anti-Rh(D) with various strengths (1:32 to 1:256) and determined the optimal strength. We determined the sensitivity of the MPHA and compared the results using flow cytometry. We observed the changes of the reaction according to the storage time of indicator cells. RESULTS: The optimal sensitization strengths of the indicator cells were 1:192 and 1:256. MPHA showed strong positive results with 1:8,192 diluted positive control, while the detection limit of flow cytometry was 1:128. Until the second week (mean 16 days), the indicator cells showed good results comparable to those of fresh ones. CONCLUSION: We developed the MPHA method using indicator cells of anti-Rh(D) sensitized group O, Rh+ RBCs. We produced the indicator cells in our own laboratory and obtained platelet panels with rare antigen typing using frozen-stored platelets. This technology will be used effectively for detection of platelet antigens and identification of platelet antibodies and also for platelet crossmatching.

Human Platelet Antigen Genotyping Using a Multiplex Single-Base Primer Extension Reaction in Koreans / 대한수혈학회지

BACKGROUND: Alloimmunization of human platelet antigens (HPA) is associated with clinically significant disease, such as platelet refractoriness, neonatal alloimmune thrombocytopenia, or posttransfusion purpura. It is determined by single nucleotide polymorphism of genes for platelet membrane glycoprotein. To date, approximately 27 HPAs have been discovered, and their frequencies differ depending on ethnicity and country. METHODS: We conducted an investigation of prevalence of HPA in the Korean population using a multiplex single-base primer extension reaction (SNaPshot). With 84 specimens from healthy donors, HPA genotyping was performed on 11 different HPAs, including HPA-1, -2, -3, -4, -5, -6, -7, -8, -9, -13, and -15. RESULTS: A total of 90 blood samples were genotyped. The genotype frequencies of HPA were as follows: HPA-1a/1a: 100.0%, -2a/2a: 83.3%, -2a/2b: 14.3%, -2b/2b: 2.4%, -3a/3a: 39.3%, -3a/3b: 52.4%, -3b/3b: 8.3%, -4a/4a: 100.0%, -5a/5a: 95.2%, -5a/5b: 4.8%, -6a/6a: 94.0%, -6a/6b: 6.0%, -7a/7a: 100.0%, -8a/8a: 100.0%, -9a/9a: 97.6%, -9a/9b: 2.4%, -13a/13a: 100.0%, -15a/15a: 23.8%, -15a/15b: 51.2%, and -15b/15b: 25.0%. CONCLUSION: The SNaPshot assay was employed for detection of SNPs in various clinically significant HPA genes. In addition to well-known frequencies of previously reported HPA-1 to -8, this study showed frequencies of HPA-9, -13, and -15 in Koreans for the first time. The SNaPshot technique might be suitable for use in actual clinical testing in patients with platelet alloimmunization.

Prevention of ABO Incompatible Blood Transfusion: '2-2-2 Safe Blood Transfusion Campaign' / 대한수혈학회지

No abstract available.

Evaluation of Flow Cytometric Crossmatch Results in Comparison with Donor-specific Antibodies Detected by Luminex-PRA Tests in Organ Transplantation Patients / 대한이식학회지

BACKGROUND: Two of the most sensitive methods for detecting donor-specific HLA antibodies (DSAs) are solid phase panel reactive antibody (PRA) assay using Luminex platform (Luminex-PRA), and a cell-based flow cytometric crossmatch (FCXM) test. We evaluated FCXM results in relation to DSAs detected by the Luminex-PRA method in solid organ transplantation candidates or post-transplant follow-up patients. METHODS: A total of 171 donor-recipient pairs were evaluated by Luminex-PRA (LIFECODES Class I and Class II ID kits; Gen-Probe, USA) and FCXM (T- and B-cells) tests. DSA levels were analyzed using a sum of median fluorescence intensity (MFI) values, and FCXM results were analyzed using MFI ratios. RESULTS: Class I and II DSAs were detected in 11.7% (20/171) and 11.1% (19/171) of tested sera, respectively. T-FCXM was negative in 97.4% (147/151) of Class I DSA negative sera, and B-FCXM was negative in 99.3% (137/138) of Class I and II DSA negative sera. T-FCXM was positive in 91.7% (11/12) of sera with moderate to strong Class I DSAs and B-FCXM was positive in 88.9% (16/18) of sera with moderate to strong Class II and/or Class I DSAs in the evaluation of sensitivities of FCXM in relation to DSA. There were significant correlations between FCXM ratios and DSA levels for both T-FCXM (P=0.008) and B-FCXM (P97%) and the sensitivities of T- and B-FCXM were satisfactory (>88%) in detecting moderate to strong DSAs.

Clinical Relevance of Pretransplant HLA Class II Donor-specific Antibodies in Renal Transplantation Patients with Negative T-cell Cytotoxicity Crossmatches

BACKGROUND: We evaluated the clinical relevance of pretransplant donor-specific HLA antibodies (DSA) in renal transplantation patients who had negative T-cell cytotoxicity crossmatches. METHODS: From 328 consecutive renal transplant recipients, we selected 28 patients who had positive pretransplant (historical or at the time of transplantation) flow cytometry crossmatches, but negative T-cell cytotoxicity crossmatches at the time of transplantation. The presence of DSA and its level at the time of transplantation were retrospectively tested using Luminex single antigen assays. RESULTS: DSA was present in 16 (57.1%) of 28 patients. Biopsy-proven acute rejection (9 patients) occurred more frequently in patients with DSA than in those without DSA (56.3% vs. 0.0%; P=0.003). The positivity rate of class II DSA was significantly higher in patients with antibody-mediated rejection (AMR) than in those without AMR (100% vs. 21.7%; P=0.003). However, the positivity rate of class I DSA was not different between the two groups (40% vs. 40.9%). Among patients with class II DSA, those with AMR tended to have higher antibody levels (median fluorescence intensity, MFI) than those without AMR (16,359 vs. 5,910; P=0.056). A cut-off MFI value of 4,487 for class II DSA predicted the occurrence of AMR with good sensitivity and specificity (100% and 87.0%). CONCLUSIONS: In patients with negative T-cell cytotoxicity crossmatches, the presence of class II DSA and its level at the time of transplantation were associated with the occurrence of AMR. Pretransplant DSA measurement with Luminex single antigen assay would be useful in renal transplantation.

False Homozygosity Results in HLA Genotyping due to Loss of Chromosome 6 in a Patient with Acute Lymphoblastic Leukemia / 대한진단검사의학회지

Loss of heterozygosity (LOH) in chromosome 6p has been reported in a number of tumors and some hematologic malignancies, including ALL. LOH in chromosome 6p, on which the HLA genes are located, can give rise to false homozygosity results in HLA genotyping of patients with hematologic malignancies. Here we report false homozygosity results in HLA genotyping due to the loss of whole chromosome 6 in the neoplastic cells of a patient with ALL. A 33-yr-old Korean female patient was admitted for the evaluation of leukocytosis detected during a workup for headache. Her initial white blood cell count was 336.9x109/L with 84% of blasts in the differential count. Precursor-B lymphoblastic leukemia was diagnosed from a subsequent bone marrow study. HLA high-resolution genotyping of the patient was requested at the time of diagnosis for possible hematopoietic stem cell transplantation. Homozygosity results (A*02:01, B*54:01, C*08:01, DQB1*04:01) were obtained, except for the DRB1 locus (DRB1*04:05, DRB1*11:01), in sequence-based typing. Conventional karyotyping of bone marrow metaphase cells revealed chromosomal abnormalities, with loss of multiple chromosomes including chromosome 6, and reduplication of the remaining chromosomes: 29,X,+X,+8,inv(9)(p11q13),+10,+14,+18,+21[15]/58,idemX2[3]/46,XX,inv(9)[2]. LOH at the HLA region was suspected and HLA genotyping was repeated with the peripheral blood in remission state after induction chemotherapy. All 5 HLA loci were typed as heterozygous (A*02:01, A*02:06, B*40:01, B*54:01, C*03:04, C*08:01, DRB1*04:05, DRB1*11:01, DQB1*03:01, DQB1*04:01). To avoid false HLA typing results in patients with hematologic malignancies, clinicians, as well as laboratory personnel, need to be aware of such problems and take appropriate precautions.

A Case of Cimetidine-induced Immune Hemolytic Anemia / 대한수혈학회지

There have been 5 case reports about immune hemolytic anemia related with cimetidine, but there has been no such report in Korea. A sixty-four-year-old woman was admitted to the emergency department in a coma. She was diagnosed with diffuse large B-cell lymphoma 5 years ago and she achieved a complete remission after treatment. She was in a state of shock, and her blood pressure was 70/40 mmHg. The laboratory test results were consistent with hemolytic anemia (HA) as follows: hemoglobin: 1.9 g/dL, corrected reticulocyte count: 2%, total/conjugated bilirubin: 6.4/2.1 mg/dL, lactate dehydrogenase: 1,083 U/L and haptoglobin <10 mg/dL. She had a history of taking the drugs including cimetidine, acetaminophen and etizolam for two days. She urgently received transfusion of 3 units of packed red blood cells and then she regained consciousness. To investigate the relation between cimetidine and HA, we subsequently performed a drug-induced immune complex test and an antibody test by using cimetidine, AB serum and phosphate-buffered saline. The results proved that cimetidine was the cause of the HA. Patient was treated with steroid, and the following laboratory tests showed rapid improvement. This is the first case report from Korea that shows the causal relationship between cimetidine and immune HA.

Hemolytic Disease of the Newborn Associated with Anti-Jr(a) Alloimmunization in a Twin Pregnancy: The First Case Report in Korea / 대한진단검사의학회지

Jr(a) is a high-frequency antigen found in all ethnic groups. However, the clinical significance of the anti-Jr(a) antibody has remained controversial. Most studies have reported mild hemolytic disease of the newborn and fetus (HDNF) in Jr(a)-positive patients. Recently, fatal cases of HDNF have also been reported. We report the first case of HDNF caused by anti-Jr(a) alloimmunization in twins in Korea. A 33-yr-old nulliparous woman with no history of transfusion or amniocentesis was admitted at the 32nd week of gestation because of vaginal bleeding caused by placenta previa. Anti-Jr(a) antibodies were detected in a routine laboratory examination. An emergency cesarean section was performed at the 34th week of gestation, and 2 premature infant twins were delivered. Laboratory examination showed positive direct antiglobulin test and Jr(a+) phenotype in the red blood cells and the presence of anti-Jr(a) antibodies in the serum in both neonates. The infants underwent phototherapy for neonatal jaundice; this was followed by conservative management. They showed no further complications and were discharged on the 19th postpartum day. Preparative management to ensure the availability of Jr(a-) blood, via autologous donation, and close fetal monitoring must be performed even in cases of first pregnancy in Jr(a-) women.

An Evaluation of Platelet Transfusion Response Using HLA Crossmatch-compatible Donors in Patients with Platelet Refractoriness / 대한진단검사의학회지

BACKGROUND: Majority of immune-mediated platelet refractoriness is caused by HLA alloimmunization and can be effectively managed by HLA-matched platelet transfusions. However, HLA class I-typed large-sized donor registry has not been well established in Korea. We evaluated the effectiveness of platelet transfusion using HLA crossmatch-compatible donors without HLA typing. METHODS: Sixteen patients showing platelet refractoriness to random donor platelets (1 hr corrected count increment [CCI] 60%) were crossmatched with 78 platelet apheresis-eligible donors using National Institute of Health (NIH) and anti-human globulin (AHG) lymphocytotoxicity methods. NIH negative/AHG negative and NIH negative/AHG positive donors were selected as best and second choice donors, respectively. RESULTS: Eleven patients (11/16, 69%) could find NIH-crossmatch negative donors and 27 donors (27/78, 35%) belonged to the best donors. To 8 patients, 32 apheresis platelet products from 19 donors were transfused. The mean 1 hr and 24 hr CCI values from the best donors were significantly higher than those from random donors (17,893 vs 2,358, P=0.003; 8,292 vs -614, P<0.001), whereas such differences were not observed for those from the second choice donors. Platelet storage time was inversely correlated with CCI values and platelets stored < or =10 hr after collection gave significantly higher CCI values. Neither ABO match nor donor status (related vs unrelated) affected the transfusion effectiveness. CONCLUSIONS: Effective post-transfusion platelet increment using HLA crossmatch-compatible donors was attained in patients with platelet refractoriness due to HLA antibodies, and this method can be used effectively where HLA-typed platelet donor registry is not available.

ABO Genotyping using a Multiplex Single-base Primer Extension Reaction / 대한수혈학회지

BACKGROUND: ABO genotyping is being widely used in the case of ABO discrepancies and in forensic medicine. We have designed a method using a multiplex single-base primer extension reaction that has allowed us to detect six single nucleotide polymorphism (SNP) sites in the ABO gene and to determine ABO genotypes. METHODS: Genomic DNA was isolated from the peripheral blood of 75 unrelated Korean subjects. Exon 6 containing nucleotides 261 and 297 and exon 7 containing nucleotides 703, 802, 803 and 1059 were amplified using two pairs of primers. Using the products as templates, a multiplex single-base primer extension reaction was performed with six typing primers of different lengths for the six SNP sites. These reactions were performed on a PTC-200 thermal cycler (MJ Research, Waltham, MA, USA) using the SNaPshot multiplex kit (Applied Biosystems, Foster City, CA, USA), and the products were analyzed using an ABI 3130xl Genetic Analyzer (Applied Biosystems). RESULTS: The ABO genotypes determined by this method (75/75) all matched the genotypes that were determined by the use of the polymerase chain reaction using sequence-specific priming (PCR-SSP). We analyzed the peak pattern detected at each of the six SNP sites for each sample. For the smaller-sized primers, peaks were shifted to the right-side compared with the expected site and for the larger-sized primers peaks was close to the expected site. In addition, the coefficients of variation (CVs) of the smaller-sized primers were higher than the CVs of the larger-sized primers. CONCLUSIONS: We are able to detect six SNP sites in the ABO gene and to determine ABO genotypes using a multiplex single-base primer extension reaction.