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CONTENTS
Immunohematology
Volume 12, Number 4, 1996

ABSTRACTS

ABO genotyping by polymerase chain reaction­restriction fragment length polymorphism
N.A. Mifsud, A.P. Haddad, J.A. Condon, AND R.L. Sparrow

ABO genotyping--identification of O1, O1*, and O2 alleles using the polymerase chain reaction­sequence specific oligonucleotide (PCR-SSO) technique
N.A. Mifsud, A.P. Haddad, J.A. Condon, AND R.L. Sparrow

Rhmod phenotype: a parentage problem solved by denaturing gradient gel electorphoresis of genomic DNA
F.Jsteers, M. Wallace, M. Mora, B. Carritt, P. Tippett, and G.L. Daniels

A method to detect McLeod phenotype red blood cells
R. Øyen, M.E. Reid, P. Rubinstein, and H. Ralph

The GLAM test: a flow cytometric assay for the detection of leukocyte antibodies in autoimmune neutropenia
A. Lubenko and S. Wilson

Empirical evaluation of the transfusion medicine tutor
J.H. Obradovich, P.J. Smith, S. Guerlain, S. Rudmann, P. Strohm, J. Smith, J. Svirbely, and L. Sachs

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ABO genotyping by polymerase chain reaction-restriction fragment length polymorphism

N.A. MIFSUD, A.P. HADDAD, J.A. CONDON, AND R.L. SPARROW

Genotyping enables the identification of both maternally and paternally derived alleles. A number of protocols have been described for the genotyping of the ABO blood group system. Generally, these methods have a number of disadvantages including the use of hazardous reagents, being technically demanding, and the excessive use of materials. In this study, a relatively simple polymerase chain reaction­restriction fragment length polymorphism (PCR-RFLP) method is described. Four different amplifications were used that were specific for nucleotides sites 261, 526, 703, and 796 to distinguish the A, B, O1 and O2 alleles. The ABO genotypes of 294 random individuals were determined and were found to completely correlate with the serologic phenotypes. The protocol is applicable for investigations of weak or nonexpression of ABO alleles, paternity determinations, and population analysis. Immunohematology 1996;12:143­148.

Nicole A. Mifsud, BSc, Scientist, Red Cross Blood Bank Victoria, Development Unit, Corner of Kavanagh and Balston Streets, Southbank, Victoria, 3006, Australia; Albert P. Haddad, BSc (Hons), Senior Scientist, Red Cross Blood Bank Victoria, Development Unit; Jennifer A. Condon, BAppSc, Senior Scientist, Red Cross Blood Bank Victoria, Reference Serology Laboratory; Rosemary L. Sparrow, PhD, Development Manager, Red Cross Blood Bank Victoria, Development Unit, Southbank, Victoria, Australia.



ABO genotyping--identification of O1, O1*, and O2 alleles using the polymerase chain reaction­sequence specific oligonucleotide (PCR-SSO) technique

N.A. MIFSUD, A.P. HADDAD, J.A. CONDON, AND R.L. SPARROW

ABO polymorphism at the gene level has been investigated by molecular methods, predominantly sequencing and restriction fragment length polymorphism (RFLP). We describe the application of the polymerase chain reaction­sequence specific oligonucleotide (PCR-SSO) method, which is considered to be more versatile for large sample numbers, compared with conventional ABO genotyping by PCR-RFLP. PCR-SSO, while maintaining accurate and reliable results, reduces costs and labor. A population of 155 random individuals was investigated for the three O alleles, O1, O1*, and O2. The allelic frequencies were 35 percent, 26 percent, and 5 percent, respectively. PCR-SSO results correlated completely with both serologic and PCR-RFLP results. Immunohematology 1996;12:149­153.

Fiona J. Steers, BSc, MRC Human Biochemical Genetics Unit, Wolfson House, 4 Stephenson Way, London, NW1 2HE, UK; Maura Wallace, BSc, MRC Blood Group Unit, UK; Marialuisa Mora, PhD, Ospedali Riuniti di Bergamo, Bergamo, Italy; Ben Carritt, PhD, MRC Human Biochemical Genetics Unit, London, UK; Patricia Tippett, PhD, MRC Blood Group Unit, London, UK; Geoff Daniels, PhD, Bristol Institute for Transfusion Sciences, Southmead Road, Bristol, BS10 5ND, UK (correspondence).

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Rhmod phenotype: a parentage problem solved by denaturing gradient gel electrophoresis of genomic DNA

F.J. STEERS, M. WALLACE, M. MORA, B. CARRITT, P. TIPPETT, AND G.L. DANIELS

Initial Rh phenotyping of a man with hemolytic anemia, his wife, and son appeared to exclude paternity. No exclusion was found in other blood groups or in the human leukocyte antigen (HLA) system; excluding Rh, the paternity index was 98.58 percent. Samples from these three family members, and two other family members, were tested with additional Rh antisera. The results indicated that the propositus has an Rhmod phenotype with expression of c, weak e, and very weak D, E, and G antigens. To support this hypothesis, DNA analysis of the RHD and RHCE genes was performed on the five family members. Polymerase chain reaction (PCR) products from exons 2 and 5 were analyzed by denaturing gradient gel electrophoresis (DGGE). The DNA results corroborated the serologic findings and refuted the exclusion of paternity. Immunohematology 1996;12:154­158.

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A method to detect McLeod phenotype red blood cells

R. ØYEN, M.E. REID, P. RUBENSTEIN, AND H. RALPH

It is important to identify the McLeod phenotype in order to differentiate the McLeod syndrome from other causes of acanthocytosis, e.g., chorea acanthocytosis. A proportion of males with the McLeod phenotype have X-linked chronic granulomatous disease. Because anti-Kx + -Km, which is needed for identification, is not readily available, detection of the McLeod phenotype relies on observed weakening of Kell antigens on the individual's red blood cells (RBCs). Identification of McLeod carrier females (obligate heterozygotes) is even more difficult because only a minor subpopulation of RBCs may express the weakened Kell phenotype. RBCs from 12 sets of mother/son or father/daughter pairs were tested by standard hemagglutination tube tests and by flow cytometry using both monoclonal and polyclonal Kell system antibodies. Monoclonal anti-K14 (G10) in tests with RBCs from McLeod males reacted ± by hemagglutination (control cells 2+) and had a median fluorecence of 6­11 by flow cytometry (control cells 441). Monoclonal anti-k (F7) and human polyclonal anti-k (C30A-1) gave stronger reactions by hemagglutination with RBCs from McLeod males and were not appropriate to differentiate RBCs with the McLeod phenotype from RBCs with normal Kell antigen expression. Crisp mixed-field agglutination was obtained in tests with monoclonal anti-K14 versus RBCs from the 12 obligate carrier females. Flow cytometry using anti-K14 also clearly identified two RBC populations (median fluorescence of 6­11 and 229­382, respectively). Kell system antibodies may vary in their ability to detect a weakened expression of the corresponding antigen on RBCs with the McLeod phenotype by hemagglutination. Antibodies suitable for differentiating McLeod syndrome from other forms of acanthocytosis should be reagents that are preselected after extensive testing. In this study, flow cytometry clearly identified the two RBC populations in McLeod carrier females. Immunohematology 1996;12:160­163.

Ragnhild Øyen, Technical Director, Immunohematology Laboratory, New York Blood Center, 310 East 67th Street, New York, NY 10021; Marion E. Reid, PhD, Director, Immunohematology Laboratory; Pablo Rubinstein, MD, Member, Lindsley F. Kimball Research Institute, and Director, Immunogenetics Laboratory; and Harold Ralph, Supervisor, Special Diagnostics Laboratory, New York Blood Center, New York, NY.

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The GLAM test: a flow cytometric assay for the detection of leukocyte antibodies in autoimmune neutropenia

A. LUBENKO AND S. WILSON

The GLAM assay, a combined flow cytometric immunofluorescence test that simultaneously detects antibodies to granulocytes, lymphocytes, and monocytes, was used in the investigation of autoimmune neutropenia. This method avoids the need for a succession of immunofluorescence tests, first against granulocytes and then against lymphocytes, in order to distinguish granulocyte-specific from granulocyte/lymphocyte-reactive antibodies, such as noncomplement-fixing anti-HLA sera. Samples from 18 patients with a suspected diagnosis of autoimmune neutropenia were referred for investigation. Leukocytes were harvested in sufficient quantities from 10 of the patients such that neutrophil and lymphocyte direct antiglobulin tests (DATs) and antibody screening and identification could be undertaken (in one case the results were inconclusive). Only three of these 10 patients had DAT-positive granulocytes, and one of these three also had DAT-positive lymphocytes. One further patient demonstrated DAT-positive lymphocytes in the absence of granulocyte-bound IgG, despite a presumed diagnosis of autoimmune neutropenia, rather than pancytopenia. This was the only patient in this cohort who had demonstrable leukocyte antibodies reacting with lymphocytes but not granulocytes. Of the remaining eight patients (not evaluable for granulocyte or lymphocyte DATs), five had free leukocyte antibodies in the serum; three of these five had both granulocyte- and lymphocyte-reactive antibodies free in the serum and two only had granulocyte-specific antibodies. Immunohematology 1996;12:164­168.

Anatole Lubenko, PhD, Head, Clinical Diagnostics Department, National Blood Service--Leeds, Leeds Blood Centre, Bridle Path, Leeds LS15 7TW, West Yorkshire, UK; Sally Rosemary Wilson, BA, Medical Laboratory Scientist, Clinical Diagnostics Department, National Blood Service--Leeds, West Yorkshire, UK.

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Empirical evaluation of the transfusion medicine tutor

J.H. OBRADOVICH, P.J. SMITH, S. GUERLAIN, S. RUDMANN, P. STROHM, J. SMITH, J. SVIRBELY, AND L. SACHS

Previous research during the development of Antibody IDentification Assistant (AIDA) revealed that many medical technology students and other laboratory personnel have serious difficulties in determining the specificity of blood group alloantibodies, especially weak or multiple antibodies. Based on these previous results, AIDA was modified to provide a teaching environment for medical technology students. We report the results of a rigorous, objective evaluation of the resultant system, the Transfusion Medicine Tutor (TMT). The results show that the students who were taught by an instructor using TMT to provide the instructional environment went from 0 percent correct on a pretest case to 87 percent correct on posttests (n = 15). This increase compares with an improvement rate of 20 percent by a control group (n = 15) who used a passive version of the system with the tutoring functions turned off. Immunohematology 1996;12:169­174.

Jodi Heintz Obradovich, Cognitive Systems Engineering Laboratory, The Ohio State University, 210 Baker Systems, 1971 Neil Avenue, Columbus, OH 43210-1271; Philip J. Smith, Stephanie Guerlain, Sally Rudmann, Patricia Strohm, Jack Smith, John Svirbely, and Larry Sacks, Cognitive Systems Engineering Laboratory, The Ohio State University, Columbus, OH.

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