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P Yam

Publications and source records attributed to P Yam.

13 recordsLinked to original sources
PubMed1991

Amplification by the polymerase chain reaction of hypervariable regions of the human genome for evaluation of chimerism after bone marrow transplantation.

We combined the polymerase chain reaction (PCR) with oligonucleotide hybridization as a novel and sensitive technique to evaluate posttransplant chimerism. Specific oligonucleotides for hybridization were synthesized homologous to tandemly repetitive core sequences of regions with a variable number of tandem repeats (VNTRs). Polymorphisms at such loci result from allelic differences in the number of repeats. Primers flanking the repeat region of each of the corresponding VNTRs were used for amplification. Recipient and donor pretransplant DNA and recipient posttransplant DNA were amplified. The resultant fragments were analyzed after gel electrophoresis either by hybridization in-gel or after Southern transfer. To confirm our findings, we also performed standard assays of restriction fragment length polymorphisms (RFLPs). Evaluation of 13 selected cases indicated mixed chimerism (4), complete chimerism (5), recurrence of leukemia (2), and endogenous repopulation of hematopoiesis (2) after marrow transplantation. Sensitivity of the method was determined by mixing various proportions of recipient and donor DNA; the limit of detection of the minor component in a mixture was 0.1%. PCR data correlated with RFLP data in all cases except two in which PCR proved more sensitive than RFLP. PCR amplification of VNTRs combined with oligonucleotide hybridization is a novel technique for documenting posttransplant chimerism and has advantages over RFLP analysis: high sensitivity, use of small amounts of DNA (250 ng), ease of preparation of DNA, elimination of need for restriction enzymes, and the ability to complete studies in 2 days.

Base Sequence↗
PubMed1990

Bone marrow transplantation for severe aplastic anemia. Effect of a preparative regimen of cyclophosphamide-low-dose total-lymphoid irradiation and posttransplant cyclosporine-methotrexate therapy.

Twenty-nine patients with severe aplastic anemia were entered into a study of pre- and posttransplant immunosuppressive therapy for bone marrow transplantation. Four of twenty-five previously transfused recipients prepared with cyclophosphamide 200 mg/kg and total-lymphoid irradiation 3 Gy experienced graft failure, indicating that this regimen was inadequate to ensure sustained engraftment. Posttransplant treatment with cyclosporine and methotrexate resulted in an actuarial incidence for grade greater than or equal to 2 graft-versus-host disease of 22 +/- 16%. Actuarial survival was 78 +/- 15%. These data indicate that more effective treatment is necessary to prevent graft failure, but since many patients can be successfully retransplanted, overall survival is comparable to other recent studies.

Adolescent↗
PubMed1989

Detection of herpes simplex virus in clinical specimens using a DNA probe after centrifugal inoculation of A549 cells.

Two methods for detection of herpes simplex virus (HSV) in 216 clinical specimens were compared: (a) 24-well plate centrifugation using A-549 cells followed by nucleic acid hybridization (Ortho Diagnostic Systems, Inc., Raritan, NJ) after incubation for 16 to 18 h, and (b) conventional tube cell culture using A-549 cells. HSV was identified by conventional tube cell culture in 44 of 216 specimens (20%) and in 36 specimens (17%) by the centrifugation-hybridization method (P less than 0.01). HSV was recovered by tissue culture from all specimens positive by centrifugation-hybridization. The sensitivity, specificity, and positive and negative predictive values of the centrifugation-hybridization technique for detection of HSV in clinical specimens were 82, 100, 100, and 96%, respectively. Centrifugal inoculation of A549 cells in 24-well plates followed by nucleic acid hybridization after overnight incubation should not replace conventional tube cell culture for detection of HSV in clinical specimens.

Cells, Cultured↗
PubMed1989

Use of deoxyribonucleic acid probes in the identification of cell origin and detection of cellular contamination in human lymphoblastoid cell lines.

Established lymphoblastoid cell lines have provided a valuable reference source for studying neoplastic lymphoproliferative disorders in humans. However, two major problems are associated with the establishment and growth of these cell lines: (a) the established cell line may not represent the original neoplastic clone, and (b) contamination of the established cell line with the other cell lines may occur. Lymphoblastoid cell lines "W" and "SP5" were established from splenectomy specimens of two patients with hairy cell leukemia. Both cell lines displayed B cell characteristics by immunophenotypic and Ig gene rearrangement studies. The banding patterns of the rearranged Ig genes (heavy and light chains) in the W cell line were different and in the SP5 cell line were identical with the corresponding untransformed splenic cell lines, indicating that cell line SP5 did and cell line W did not represent the original neoplastic clone. Continuous cultures of some of the subclones derived from cell line W and SP5 led to the growth of the cell lines W15T, W17T, and SP5T which all demonstrated T cell features based on immunophenotypic and T cell receptor rearrangement studies. However, the T cell receptor alpha and beta rearranged bands as well as bands generated by hybridization with highly polymorphic DNA probes p YNH24 and 0-3315-32 in these three lines and a human T cell leukemia line (CEM), were identical indicating that W15T, W17T and SP5T cell lines were contaminated with CEM. Studies of gene establishment patterns and DNA polymorphisms by Southern blotting are effective methods to establish clonal identity and to rule out cellular contamination in lymphoblastoid cell lines.

B-Lymphocytes↗
PubMed1987

Development of a single probe for documentation of chimerism following bone marrow transplantation.

Although numerous genetic markers are available for studying chimerism after bone marrow transplantation (BMT), there remains a need for a practical and highly informative method that is applicable in the early posttransplantation period. Using DNA restriction-fragment-length polymorphisms (RFLPs), we have evaluated the feasibility of developing a single synthetic oligonucleotide probe to study post-BMT chimerism. We have thus tested three candidate probes, termed O-3315-32, O-3315-80, and O-AY-29, that are homologous to tandemly repetitive sequences. Our results demonstrated donor-specific and recipient-specific fragments in 11 of 11 HLA-matched sibling pairs tested using probes O-3315-32 and O-3315-80. When probe O-AY-29 was used, 14 of 17 sibling pairs showed both donor and recipient markers, one had only a recipient marker, and two were identical. We showed that each of the three synthetic probes was effective in documenting donor marrow engraftment, mixed hematopoietic chimerism, the patient's pre-BMT phenotype (by using cultured skin fibroblasts obtained after BMT), and the origin of the malignant hematopoietic cells (i.e., of donor or recipient origin) in patients who developed recurrent hematologic malignancy following BMT. Compared with the use of cloned genomic probes, there are several important advantages to the use of synthetic oligonucleotide probes in studying post-BMT chimerism. Synthetic probes have absolute hybridization specificity and can be designed to suit the purposes of an individual study, since they have adjustable specificity that can be altered by changes in the length of the probe and by changes in the hybridization temperature. A single synthetic probe analogous to several highly polymorphic loci can have a polymorphism information content sufficiently high so that all but a small percentage of BMT patients could be followed easily; for example, if a probe were complementary to three highly polymorphic unlinked loci, it would discriminate approximately 98% of sibling donor/recipient pairs. This would be accomplished using only one restriction-endonuclease digestion and only one gel electrophoresis. Since other genetic markers, e.g., red blood cell antigens, immunoglobulin allotypes, and chromosome analysis, are not uniformly informative and, in some cases, cannot be used in the early posttransplantation period, the use of synthetic oligonucleotide probes for analysis of DNA RFLP is emerging as the method of choice for studies of post-BMT chimerism. This method will allow for the development of new knowledge that has not been possible with previous methods.

Bone Marrow↗
PubMed1979

[The radioactive antiglobulin (Coombs') test].

Weak red cell sensitization by hemolyzing antibodies may not be detected by the standard antiglobulin test. For the diagnosis of certain immune hemolytic anemias more sensitive methods are needed. Anti-IgG was purified as F(ab)2 by pepsin digestion and labelled with 125 I using the chloramine T method. 15 X 10(7) cells were washed and suspended in 0.1 ml of 6% albumin in saline. After incubation with 0.1 ml of 125 I-anti-IgG F(ab')2 for 30 minutes, the cells were washed and the uptake of radioactivity was counted using a gamma-counter. Standard cells were coated with a known concentration of an IgG anti-D preparation in the range of 50--1000 molecules per red cell. The sensitivity for the detection of red cell sensitization was at least 50 molecules per red cell. Because of varying binding ratios between the labelled anti-IgG reagent and different red cell antibodies the determination of the exact number of IgG molecules on the red cells is difficult. However, this very sensitive and reproducible method shows practical advantages in comparison with a complement fixation antibody consumption method with which corresponding results were obtained. Beside the evaluation of antiglobulin test negative autoimmune hemolytic anemias, this method may be applied for the determination of the specificity of weak red cell alloantibodies not detectable by standard blood bank procedures.

Anemia, Hemolytic, Autoimmune↗
PubMed1975

The relationship between hemolytic and immunodiffusion methods for measurement of C4 in patients with immunologic disorders.

A comparison of radial immunodiffusion and functional (hemolytic) assays for C4 revealed a highly significant correlation between the two methods in patients with a variety of immunologic disorders. Immunodiffusion technics are more convenient than hemolytic assays for most clinical laboratories. C4 assays are useful for the presumptive diagnosis of hereditary angioneurotic edema, whereas C3 is usually normal in such patients. C4 is usually more sensitive than C3 in a variety of immunologic disorders, although in some patients the opposite is true, especially in hypocomplementemic nephritis.

Angioedema↗