Clinical application of a flow cytometric direct antiglobulin test.

BACKGROUND: The clinical application of flow cytometric direct antiglobulin test (FC-DAT) has rarely been evaluated for patients with various diseases including immune and nonimmune hemolytic anemia. STUDY DESIGN AND METHODS: Blood samples from 380 patients with a variety of diseases were studied using the tube direct DAT and FC-DAT. The results of tube DAT and FC-DAT were compared. The predictive values of DAT for hemolysis were evaluated. RESULTS: Of 57 patients with autoimmune hemolytic anemia (AIHA), 6 of the 17 with a negative tube DAT (immunoglobulin G [IgG]) had a positive FC-DAT (IgG) and 23 of the 36 patients with a negative tube DAT (complement 3d [C3d]) had a positive FC-DAT (C3d). In 57 patients with AIHA, the incidence of positive results of FC-DAT (IgG) and tube DAT (IgG) were similar (42 positive vs. 40 positive); but in 323 patients without AIHA, the incidence of positive FC-DATs (IgG) was higher than that of tube DAT (IgG; 47 positive vs. 9 positive). The higher incidence of positive FC-DAT (C3d) than that of tube DAT (C3d) was seen in patients with AIHA (42 positive vs. 21 positive) as well as in patients without AIHA (61 positive vs. 5 positive). Both DAT (IgG) and DAT (C3d) positive has highest positive predictive value for hemolysis, followed by DAT (IgG) alone positive and DAT (C3d) alone positive. CONCLUSIONS: FC-DAT is a complementary test for diagnosing AIHA. There is a synergistic effect of the red blood cell-bound IgG and complement in predicting hemolysis.

Two novel Jk(null) alleles derived from 222C>A in Exon 5 and 896G>A in Exon 9 of the JK gene.

BACKGROUND: Polynesian Jk(null) is well known for its mutation as Intron 5 g>a at the 3' splice acceptor site. After sequencing analysis, however, it was noticed that only three of eight samples with the Jknull phenotype carried typical homozygous Polynesian Jk(null) mutation. Five others were noted to be unreported heterozygous Polynesian Jk(null) mutation. An investigation was then conducted to characterize the underlying mechanism leading to this particular Jk(null) genotype. STUDY DESIGN AND METHODS: Genomic DNA covering 5'-untranslated region exons and intervening introns of the JK gene was amplified by polymerase chain reaction, and the fragments were directly sequenced. The sequencing results were compared with those published in literature and related biologic Web sites. RESULTS: In all five samples with a heterozygous Polynesian Jk(null) mutation, additional mutations were identified. Two samples carried missense mutations: 222C>A (Asn74Lys) in Exon 5 and 499A>G (Met167Val) in Exon 7. Three others had missense mutation 896G>A (Gly299Glu) in Exon 9. These substituted amino acids were located either near or at transmembrane domains, respectively. In addition, two polymorphic nucleotides at positions -103 (a>g) and -119(c>a) from the 3' end of Intron 1 were also Polynesian mutation-related. CONCLUSIONS: In contrast to the typical homozygous Polynesian Jk(null) mutation, two novel heterozygous Jk(null) alleles were noted to be associated with the Jknull phenotype. One carried missense mutation 222C>A in Exon 5, and the other had 896G>A missense mutation in Exon 9. These findings may have implications in designing a molecular screening assay for people with the Jknull phenotype.

A Novel Ael allele derived from a unique 816insG in Exon 7 of the ABO gene.

The ABO blood group system is the most important blood group system in transfusion medicine. In addition to the major A, B and O alleles, many rare alleles with weak expression of the A or B antigens on RBCs have been defined. We report here the molecular analysis of a novel A(el) allele. Exons 6 and 7 of the ABO gene were PCR-amplified, cloned and sequenced for the propositus, Mr C, who is a 56-year-old Taiwanese male and was incidentally observed to have an A(el) phenotype. His direct family members including wife, son and daughter were subsequently enrolled for further study. Three hundred random blood donors of AB phenotype served as control. A novel A(el) allele was uncovered from the propositus and his daughter, of which a unique 816insG mutation occurred on the A102 background that results in a frame shift leading to a 37-amino acid longer polypeptide than the normal A1 transferase, a finding similar to that of Ael01 allele with 804insG. We found that the C family carried a novel A(el) allele that differs molecularly from seven A(el) alleles reported in the literature.

Y-chromosome short tandem repeats analysis to complement paternal lineage study: a single institutional experience in Taiwan.

BACKGROUND: Highly polymorphic autosomal short-tandem-repeat (STR) analysis can be useful in most kinship testing. Y-chromosome-specific STRs, in contrast, have been increasingly applied for the verification of equivocal paternal genetic transmissions. STUDY DESIGN AND METHODS: A total of 338 unrelated males were first typed for the 9-loci Y-STR European minimal haplotype (minHt). Samples with haplotypes that were found at least two times were subject to further study by a commercially available 17-Y-STR multiplex set (AmpFlSTR Yfiler). A separate clinical study for 113 various kinship identifications of male genetic transmission were then conducted by a panel consisting of 18 autosomal STRs and complemented by both Y-STR multiplex sets and their respective results compared. RESULTS: For the 338 individuals, a total of 270 haplotypes were identified after the minHt study, of which 234 were unique. Among the rest of the 104 samples, AmpFlSTR Yfiler identified 82 other unique haplotypes. Altogether, 324 different haplotypes were observed; 316 (97.5%) were unique whereas 8 were shared by two to seven times. The haplotype diversities for the minHt and the AmpFlSTR Yfiler were 99.75 and 99.96 percent, respectively, whereas the powers of discrimination (PDs) were 79.88 and 95.86 percent, respectively. Despite a lower PD for minHt, there was no discrepancy on the clinical setting for personal identification between the two Y-STR sets in an allegedly true male lineage transmission involving 66 cases with 24 father-son, 19 siblings, 9 uncle-nephew, 8 grandfather-grandson, 3 cousins, and 3 half-siblings. For 47 other cases with a false allegation of paternity, exclusion was made for all without ambiguity by either Y-STR panel. CONCLUSION: The 9-loci minHt Y-STR set is adequate to complement conventional autosomal STRs for kinship studies where Y-lineage transmission is concerned.

Unappreciated HLA antibodies in adult immune thrombocytopenic purpura.

BACKGROUND/PURPOSE: Immune thrombocytopenic purpura (ITP) is an autoimmune disease. Platelet refractoriness is frequently seen in patients with ITP. Platelets express platelet-specific antigens and human leukocyte antigens (HLA). Platelet antibodies to platelet-specific antigens and HLA may be present, but HLA antibodies in patients with ITP have rarely been reported. METHODS: Sera from 44 adult patients with ITP were screened for platelet antibodies by two flow cytometric assays. In method I, platelets from normal donor platelets were used as target cells to screen both platelet-specific antibodies and HLA class I antibodies. In method II, the FlowPRA Class I Screening Test kit was used to screen HLA class I antibodies. Fluorescein isothiocyanate (FITC)-conjugated sheep anti-human IgG Fc was used as the staining reagent in both methods. The negative serum control was from one of the normal males with AB blood group who had never received a transfusion. Sera from a pool of five highly sensitized patients were used as the positive control. RESULTS: Of the 44 sera from patients with ITP, 31 (70.5%) were method I positive, and 28 (63.6%) were method II positive. There was no significant difference between the results of method I and method II (p = 0.439). The distribution of the results of these two tests was: both tests positive in 22 sera, method I positive and method II negative in nine sera, method I negative and method II positive in six sera, and both tests negative in seven sera. The mean platelet counts of patients with positive (41.0 +/- 40.0 x 10(9)/L) and negative (40.4 +/- 26.8 x 10(9)/L) tests by method I did not differ significantly (p = 0.643). The mean platelet counts of patients with (36.7 +/- 31.5 x 10(9)/L) and without (48.1 +/- 43.6 x 10(9)/L) HLA class I antibodies did not differ significantly (p = 0.59). CONCLUSION: HLA class I antibodies are frequently found in ITP. The screening of platelet antibodies including platelet-specific antibodies and unappreciated HLA class I antibodies is warranted in patients with ITP.

Screening for platelet antibodies in adult idiopathic thrombocytopenic purpura: a comparative study using solid phase red cell adherence assay and flow cytometry.

BACKGROUND: Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disorder caused by antiplatelet autoantibodies. In this study, we compared 2 methods for screening serum platelet antibodies in patients with ITP. METHODS: A total of 44 adult patients were clinically classified with ITP. We used 2 indirect tests to detect human leukocyte antigen antibodies and/or platelet-specific antibodies in their sera. In method I, we used solid phase red cell adherence (SPRCA) assay. In method II, by flow cytometry, platelets from plateletpheresis components were used as target cells, and fluorescein isothiocyanate-conjugated sheep anti-human IgG Fc was used as the staining reagent. Positive results were defined as any test with the percentage of fluorescence exceeding the reference range by 3% or more in method II. Direct tests detecting platelet-associated IgG on platelets of patients with ITP were done by flow cytometry. RESULTS: Serum specimens from 44 adult patients with ITP (28 female, 16 male) were tested. SPRCA assay could only detect platelet antibodies in 22 patients (50%). By method II, 31 serum specimens (70.5%) yielded positive results. There was a difference between the results of the SPRCA test and method II, with a high degree of significance (p < 0.001) by the McNemar test. No significant difference in platelet counts was observed for patients with and without discernible platelet antibodies by SPRCA assay (p = 0.90). The direct test was positive in 12 patients (66.7%) out of 18 ITP patients tested. CONCLUSION: Flow cytometry is more sensitive than SPRCA assay for detecting platelet antibodies. Detection of platelet antibodies is useful in explaining the immune mechanism and platelet transfusion refractoriness in ITP.

Revisiting of preoperative blood ordering policy--a single institute's experience in Taiwan.

BACKGROUND: If unnecessary blood orders can be reasonably waived, it will reduce both workload and financial expenditure. A review of the surgical blood ordering practice is, therefore, mandatory. METHODS: Routine preoperative blood orders were retrospectively audited. After receiving the requests, we usually performed only type and screen tests without cross matching until an actual need for transfusion occurred. Transfusion probability (number of patients transfused / number of procedures x 100) was calculated. One unit of donation was defined as 500 mL whole blood. If surgical procedures were associated with insignificant blood loss (number of units transfused < or = 1) and transfusion probability was less than 5%, then it was considered to be safe to disregard a preoperative blood order. RESULTS: The blood ordering practices for 5,472 patients who received various surgical procedures were reviewed over a period of 48 operation days. Neither preoperative requests for preparation of red cells nor transfusion was made in 3,482 patients. Preoperative requests for preparation of red cells were made in 1,990 patients, but only 751 (37.74%) actually received blood transfusion on the day of the operation. Analysis showed that it would have been safe to disregard a preoperative blood order for ophthalmic surgery, ear surgery, nose surgery (endoscopic sinus surgery, submucosal turbinectomy), microlaryngoscopic surgery, tracheostomy, thyroidectomy, mastectomy, laparoscopic cholecystectomy, hemicolectomy, hernioplasty, arthroscopic surgery, laminectomy, laparoscopically assisted vaginal hysterectomy, vasectomy and varicose vein surgery. CONCLUSION: A review of preoperative blood orders has identified certain surgical procedures with insignificant blood loss and low transfusion probability, for which preoperative blood orders may be safely disregarded in order to reduce unnecessary laboratory workload while not jeopardizing patient safety.

A novel cis-AB allele derived from a unique 796C>A mutation in exon 7 of ABO gene.

BACKGROUND: The cis-AB phenotype is very rare, and only three genotypes that correspond to specific ABO allele changes have been reported. Cis-AB01 involves the A102 allele with a nonsynonymous substitution G803C in exon 7, whereas cis-AB02 and cis-AB03 involve different nonsynonymous substitutions A796C and C700T, respectively, on the B101 allele background. The nucleotide substitutions give rise to a change of the respective glycosyltransferase, resulting in varying bifunctional AB transferase activities. STUDY DESIGN AND METHODS: Two cis-AB phenotypes were identified in a Taiwanese C. family and two unrelated individuals, respectively. Serologic studies, molecular cloning, and sequencing of exon 6 and exon 7 were carried out to determine their respective phenotypic characteristics and cis-AB alleles. A cohort of 300 AB-phenotype, healthy random individuals served as controls. RESULTS: A novel cis-AB allele is uncovered out of the three family members, of which a 796C>A substitution occurs predicting an amino acid change at residue 266 of leucine to methionine on the background of A102 allele. It is serologically like cis-AB03, an A2B phenotype, but molecularly different. Both of the two unrelated individuals are of cis-AB01 allele, and all of the 300 AB blood group controls are excluded cis-AB phenotype. CONCLUSION: The C. family described carries a novel cis-AB allele that differs molecularly from all previously reported cis-AB alleles.

HLA-DR typing by polymerase chain reaction amplification with sequence-specific primers in paternity disputes.

BACKGROUND: HLA-DR typing was generally performed by serology before. HLA-DRB1 typing can be achieved by polymerase chain reaction amplification with sequence specific primers (PCR-SSP). METHODS: In this study, primers for "low-resolution" HLA-DR typing by PCR-SSP were synthesized upon our request by a company in Taiwan. Twenty-eight DNA samples from international standardized DNA Reference Panel and 20 DNA samples with known serological typing were used as control. We conducted HLA-DR PCR-SSP typing on 18 samples from 6 true paternity trios, 16 samples from 8 true duos, 27 from 9 false trios, and 8 from 4 false duos. These DNAs from disputed paternity families had been previously tested for the parentage using polymerase chain reaction (PCR)-amplified short tandem repeat (STR) analysis. RESULTS: No false positive nor false negative results were obtained in typing 28 positive control DNA samples from international standardized DNA Reference Panel for HLA Class II. Among the 20 DNA samples typed by microlymphocytotoxicity technique, the discordant typing results between HLA-DR PCR-SSP typing and serological typing were found in 3 (15%). In the family of true paternity, HLA-DR typing could not exclude any of alleged fathers and the pattern of inheritance was consistent with autosomal codominant. By HLA-DR typing alone, paternity in 2 alleged fathers out of 9 false trios and 2 alleged fathers out of 4 false duos could not be excluded. CONCLUSIONS: Precise HLA-DR typing can be achieved by PCR-SSP analysis. Economic considerations preclude HLA-DR typing in routine parentage tests where STR typing is performed first.

PCR with sequence-specific primer-based simultaneous genotyping of human platelet antigen-1 to -13w.

BACKGROUND: Accurate human platelet antigen (HPA) typing is important for patients with diagnosis of alloimmune thrombocytopenic syndromes and provision of HPA-matched blood components for these patients. STUDY DESIGN AND METHODS: Thirteen sequence-specific primers (SSPs) designed on the basis of known published polymorphisms for HPA-1 to HPA-13w, respectively, were employed for simultaneous HPA genotyping. All PCR amplifications were carried out with identical cycling conditions in 96-well plates containing primer mixtures. A total of 300 blood samples from unrelated volunteer donors in Taiwan were included in the study. RESULTS: All primers had specific amplification products. The typing results were available within 4 hours each time for up to four blood samples tested. Among the 13 HPAs, HPA-3 had the greatest heterozygosity with a gene frequency of 0.3267, 0.4967, and 0.1767 for HPA-3a/HPA-3a, HPA-3a/HPA-3b, and HPA-3b/HPA3-b, respectively. For the remaining 12 HPAs, the predominance of a/a homozygosity was noted for HPA-1, -2, -4, -5, and -6, with a frequency ranging from 0.9200 to 0.9967. The frequency of a/a homozygosity was 1.0000 for HPA-7w to -13w, except for HPA-10w, for which one case was observed to be HPA-10aw/HPA-10bw heterozygous. Excluding HPA-3, b/b homozygosity was noted in only one case (HPA-6b/HPA-6b). The prevalence rates of HPA-1 to -13w in this study were consistent with previous reports using different methods. CONCLUSION: An extended, streamlined PCR-SSP protocol for simultaneous genotyping of HPA-1 to HPA-13w was established. This allows fast and reliable diagnosis of alloimmune thrombocytopenia, and is readily applicable to large-scale genetic population studies.