Hsueng-Mei Liu

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.

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.

Two novel Jknull alleles derived from 222C>A in Exon 5 and 896G>A in Exon 9 of the JK gene

BACKGROUND: Polynesian Jknull 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 Jknull mutation. Five others were noted to be unreported heterozygous Polynesian Jknull mutation. An investigation was then conducted to characterize the underlying mechanism leading to this particular Jknull genotype.

Novel polymorphisms in exons 6 and 7 of A/B alleles detected by polymerase chain reaction-single strand conformation polymorphism

Background and Objectives  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 have been defined. Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) and analysis by PCR sequence specific primers (SSP) are commonly conducted for genotyping but have the limitation of being unable to detect unknown substitution(s) in amplified DNA fragments, whereas PCR–single strand conformation polymorphism (SSCP) can be used for both.

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.

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.

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.

Association of IL-6 C-572G Gene Polymorphism with Anti-E Production

Background: Interleukin 6 (IL-6) is involved in regulation of immunoglobulin production. The aim of this study was to investigate the association between IL-6 single nucleotide polymorphisms (SNPs) in the IL-6 promoter and anti-E in red blood cell (RBC) transfusion recipients. Methods: 50 healthy subjects, 54 patients with RBC alloantibody anti-E (responders), and 45 patients without alloantibody (non-responders) were recruited. All patients were E antigen-negative. Results: All healthy subjects and patients had GG at -174 position of IL-6 gene. In our healthy subjects, the frequency of the -572 CC genotype was 58%, that of the -572 CG genotype 38%, and that of the -572 GG genotype 4%. The frequency of G allele of -572 SNP in responders was significantly higher than that in non-responders, (31.5 vs. 16.7%; p = 0.020). The frequency of -572 G-positive genotypes (CG and GG) in responders was also significantly higher than that in non-responders, (55.6 vs. 31.1%; p = 0.016). The relative risk of RBC alloimmunization for patients with the -572 G-positive genotype was significantly higher than that of patients with the -572 CC genotype, (1.771 vs. 0.640; p = 0.016). Conclusion:IL-6 C-572G gene polymorphism is significantly associated with anti-E production, with the allele G as a risk allele.

A Novel A(superscript el) 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(superscript 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(superscript 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(superscript 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(superscript el) allele that differs molecularly from seven A(superscript el) alleles reported in the literature.