scholarly journals Phenotype analysis of hematopoietic CD34+ cell populations derived from human umbilical cord blood using flow cytometry and cDNA-polymerase chain reaction

Blood ◽  
1994 ◽  
Vol 83 (8) ◽  
pp. 2103-2114 ◽  
Author(s):  
SJ Thoma ◽  
CP Lamping ◽  
BL Ziegler
Keyword(s):  
Flow Cytometry ◽  
Polymerase Chain Reaction ◽  
Cell Surface ◽  
Chain Reaction ◽  
Hematopoietic Stem ◽  
Hla Dr ◽  
Cd34 Cells ◽  
Rare Populations ◽  
Pcr Method ◽  
Polymerase Chain

Abstract A strategy to phenotype rare populations of hematopoietic cells expressing the cell-surface marker CD34 was studied. The antigenic phenotype of umbilical core blood (CB) CD34+ cells was investigated using flow cytometry and compared with the mRNA-phenotype determined by cDNA-polymerase chain reaction (cDNA-PCR) analysis. The cDNA-PCR method allowed an mRNA evaluation of small numbers of cells. Monoclonal antibodies and oligonucleotide primers that recognize myeloid, lymphoid, erythroid and platelet/megakaryocytic cell membrane antigens or corresponding mRNA transcripts were used. Evaluation by flow cytometry showed that the vast majority of CD34+ CB cells coexpressed CD38, CD18, HLA-DR, and CD33. Rare subpopulations of CD34+CD38-, CD34+CD18-, CD34+HLA-DR-, and CD34+CD33- were also identified. A large proportion of CD34+ CB cells expressed CD13, CD45R, and to a lesser extent CD71. The CD36, CD51, and CD61 antigens were identified on a small number of CD34+ cells. The three-color flow cytometry analysis showed that CD34+ cells stained with antibodies to CD61 and CD36 or CD51 can be divided into subsets that may represent progenitor cells committed to the erythroid and/or megakaryocytic lineage. A variety of other lineage-specific cell-surface antigens including pre-T-cell marker CD7 and markers of early B cells, ie, CD10 and CD19, were not coexpressed with CD34+. Using the cDNA-PCR it was seen that the mRNA phenotype of a small number of sorted CD34+ cells (purity > 98%) was negative for the markers CD2, CD14, CD16, CD20, CD21, CD22, CD41b, and glycophorin A that are expressed on differentiated cells but positive for CD34, CD7, CD19, CD36, and CD61. The results suggest that circulating CD34+CD7+ and CD34+CD19+ CB cells cannot be distinguished by flow cytometry but can be detected by cDNA-PCR. This indicates that CB either contains very low numbers of these progenitors or that the antigen density of CD7 and CD19 on CD34+ cells is below the detection limit of the flow cytometer. In contrast to flow cytometry, cDNA-PCR allows the phenotypic analysis of cells even if their number is small. Thus, the cDNA-PCR method can be useful in linking phenotype analyses, ie, markers of differentiation, to studies on gene expression within rare populations of hematopoietic stem cells.

scholarly journals Phenotype analysis of hematopoietic CD34+ cell populations derived from human umbilical cord blood using flow cytometry and cDNA-polymerase chain reaction

Blood ◽  
1994 ◽  
Vol 83 (8) ◽  
pp. 2103-2114 ◽  
Author(s):  
SJ Thoma ◽  
CP Lamping ◽  
BL Ziegler
Keyword(s):  
Flow Cytometry ◽  
Polymerase Chain Reaction ◽  
Cell Surface ◽  
Chain Reaction ◽  
Hematopoietic Stem ◽  
Hla Dr ◽  
Cd34 Cells ◽  
Rare Populations ◽  
Pcr Method ◽  
Polymerase Chain

A strategy to phenotype rare populations of hematopoietic cells expressing the cell-surface marker CD34 was studied. The antigenic phenotype of umbilical core blood (CB) CD34+ cells was investigated using flow cytometry and compared with the mRNA-phenotype determined by cDNA-polymerase chain reaction (cDNA-PCR) analysis. The cDNA-PCR method allowed an mRNA evaluation of small numbers of cells. Monoclonal antibodies and oligonucleotide primers that recognize myeloid, lymphoid, erythroid and platelet/megakaryocytic cell membrane antigens or corresponding mRNA transcripts were used. Evaluation by flow cytometry showed that the vast majority of CD34+ CB cells coexpressed CD38, CD18, HLA-DR, and CD33. Rare subpopulations of CD34+CD38-, CD34+CD18-, CD34+HLA-DR-, and CD34+CD33- were also identified. A large proportion of CD34+ CB cells expressed CD13, CD45R, and to a lesser extent CD71. The CD36, CD51, and CD61 antigens were identified on a small number of CD34+ cells. The three-color flow cytometry analysis showed that CD34+ cells stained with antibodies to CD61 and CD36 or CD51 can be divided into subsets that may represent progenitor cells committed to the erythroid and/or megakaryocytic lineage. A variety of other lineage-specific cell-surface antigens including pre-T-cell marker CD7 and markers of early B cells, ie, CD10 and CD19, were not coexpressed with CD34+. Using the cDNA-PCR it was seen that the mRNA phenotype of a small number of sorted CD34+ cells (purity > 98%) was negative for the markers CD2, CD14, CD16, CD20, CD21, CD22, CD41b, and glycophorin A that are expressed on differentiated cells but positive for CD34, CD7, CD19, CD36, and CD61. The results suggest that circulating CD34+CD7+ and CD34+CD19+ CB cells cannot be distinguished by flow cytometry but can be detected by cDNA-PCR. This indicates that CB either contains very low numbers of these progenitors or that the antigen density of CD7 and CD19 on CD34+ cells is below the detection limit of the flow cytometer. In contrast to flow cytometry, cDNA-PCR allows the phenotypic analysis of cells even if their number is small. Thus, the cDNA-PCR method can be useful in linking phenotype analyses, ie, markers of differentiation, to studies on gene expression within rare populations of hematopoietic stem cells.

Quantitative assessment of hematopoietic chimerism after bone marrow transplantation by real-time quantitative polymerase chain reaction

Blood ◽  
2002 ◽  
Vol 99 (12) ◽  
pp. 4618-4625 ◽  
Author(s):  
Mehdi Alizadeh ◽  
Marc Bernard ◽  
Bruno Danic ◽  
Charly Dauriac ◽  
Brigitte Birebent ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Stem Cell ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Assessment ◽  
Quantitative Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Hematopoietic Stem ◽  
Pcr Method ◽  
Polymerase Chain

We have developed a real-time quantitative polymerase chain reaction (PCR) assay using TaqMan technology (Applied Biosystems, Foster City, CA) for monitoring donor cell engraftment in allogenic hematopoietic stem cell transplant recipients. For this purpose, we selected 19 specific sequence polymorphisms belonging to 11 human biallelic loci located on 9 different chromosomes. Using a set of specially designed primers and fluorogenic probes, we evaluated the 19 markers' informativity on a panel of 126 DNA samples from 63 recipient/donor pairs. In more than 90% of these pairs, discrimination between recipient and donor genetic profile was possible. By using serial dilutions of mixed DNAs, we evaluated the linearity and sensitivity of the method. A linear correlation with rhigher than 0.98 and a sensitivity of 0.1% proved reproducible. Fluorescent-based PCR of short tandem repeats (STR-PCR) and real-time PCR chimerism assay were compared with a panel of artificial cell mixtures. The main advantage of the real-time PCR method over STR-PCR chimerism assays is the absence of PCR competition and plateau biases, and results evidenced greater sensitivity and linearity with the real-time PCR method. Furthermore, different samples can be tested in the same PCR run with a final result in fewer than 48 hours. Finally, we prospectively analyzed patients who received allografts and present 4 different clinical situations that illustrate the informativity level of our method. In conclusion, this new assay provides an accurate quantitative assessment of mixed chimerism that can be useful in guiding early implementation of additional treatments in hematopoietic stem cell transplantation.

scholarly journals Lack of t(14; 18) Polymerase Chain Reaction-Positive Cells in Highly Purified CD34+ Cells and Their CD19 Subsets in Patients With Follicular Lymphoma

Blood ◽  
1997 ◽  
Vol 89 (10) ◽  
pp. 3763-3768 ◽  
Author(s):  
Maria Teresa Voso ◽  
Stefan Hohaus ◽  
Marion Moos ◽  
Rainer Haas
Keyword(s):  
Polymerase Chain Reaction ◽  
Follicular Lymphoma ◽  
Progenitor Cells ◽  
Significant Proportion ◽  
Cell Subset ◽  
Cell Fraction ◽  
Chain Reaction ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Polymerase Chain

Follicular lymphoma (FL) is characterized in a significant proportion of cases by the t(14; 18) chromosomal translocation, which results in the juxtaposition of the oncogene bcl-2 to the joining region of the immunoglobulin heavy chain (IgH) gene. Molecular sequence analysis indicates that the t(14; 18) rearrangement occurs in a B-lymphoid progenitor cell at the time of IgH rearrangement. We were interested whether hematopoietic stem and progenitor cells as characterized by CD34 expression bear the translocation. Bone marrow (BM)-CD34+ cells were enriched from 14 patients with FL whose BM was known to be positive for bcl-2/IgH (major breakpoint region [MBR]). Six patients were in complete remission (CR), two patients were in partial remission (PR), and six patients had active disease. Six patients had histological BM involvement when the samples were obtained. Using an immunomagnetic selection device (MINIMACS), a mean purity of 88.7% ± 4% CD34+ cells was achieved. The CD34+ cells were further enriched by fluorescence activated cell sorting (FACS) using CD34 fluorescein isothiocyanate (FITC)- and CD19 phycoerythrin (PE)-conjugated antibodies. The IgH gene was rearranged in the CD34+/CD19+ cell subset of all patients assessed by polymerase chain reaction (PCR). This population is thought to represent the progenitor stage at which the bcl-2/IgH translocation occurs. The unseparated BM mononuclear cell fraction from all 14 patients was positive for bcl-2/IgH using a nested PCR, but the BM-CD34+ cell fraction and the respective CD34+/CD19+ subset were negative in 13 of these 14 patients. The one patient with a positive PCR signal in the CD34+ cell subset had a relapse with BM involvement. We conclude that CD34+ progenitor cells including CD34+/CD19+ B-cell progenitors are not involved in the malignant cell clone. These data are in agreement with a transgenic mouse model, which indicates that the malignant phenotype in FL is sustained by mature B cells.

scholarly journals Lack of t(14; 18) Polymerase Chain Reaction-Positive Cells in Highly Purified CD34+ Cells and Their CD19 Subsets in Patients With Follicular Lymphoma

Blood ◽  
1997 ◽  
Vol 89 (10) ◽  
pp. 3763-3768 ◽  
Author(s):  
Maria Teresa Voso ◽  
Stefan Hohaus ◽  
Marion Moos ◽  
Rainer Haas
Keyword(s):  
Polymerase Chain Reaction ◽  
Follicular Lymphoma ◽  
Progenitor Cells ◽  
Significant Proportion ◽  
Cell Subset ◽  
Cell Fraction ◽  
Chain Reaction ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Polymerase Chain

Abstract Follicular lymphoma (FL) is characterized in a significant proportion of cases by the t(14; 18) chromosomal translocation, which results in the juxtaposition of the oncogene bcl-2 to the joining region of the immunoglobulin heavy chain (IgH) gene. Molecular sequence analysis indicates that the t(14; 18) rearrangement occurs in a B-lymphoid progenitor cell at the time of IgH rearrangement. We were interested whether hematopoietic stem and progenitor cells as characterized by CD34 expression bear the translocation. Bone marrow (BM)-CD34+ cells were enriched from 14 patients with FL whose BM was known to be positive for bcl-2/IgH (major breakpoint region [MBR]). Six patients were in complete remission (CR), two patients were in partial remission (PR), and six patients had active disease. Six patients had histological BM involvement when the samples were obtained. Using an immunomagnetic selection device (MINIMACS), a mean purity of 88.7% ± 4% CD34+ cells was achieved. The CD34+ cells were further enriched by fluorescence activated cell sorting (FACS) using CD34 fluorescein isothiocyanate (FITC)- and CD19 phycoerythrin (PE)-conjugated antibodies. The IgH gene was rearranged in the CD34+/CD19+ cell subset of all patients assessed by polymerase chain reaction (PCR). This population is thought to represent the progenitor stage at which the bcl-2/IgH translocation occurs. The unseparated BM mononuclear cell fraction from all 14 patients was positive for bcl-2/IgH using a nested PCR, but the BM-CD34+ cell fraction and the respective CD34+/CD19+ subset were negative in 13 of these 14 patients. The one patient with a positive PCR signal in the CD34+ cell subset had a relapse with BM involvement. We conclude that CD34+ progenitor cells including CD34+/CD19+ B-cell progenitors are not involved in the malignant cell clone. These data are in agreement with a transgenic mouse model, which indicates that the malignant phenotype in FL is sustained by mature B cells.

Validation of allele-specific polymerase chain reaction for DNA typing of HLA-B27

1995 ◽  
Vol 41 (5) ◽  
pp. 687-692 ◽  
Author(s):  
H M Steffens-Nakken ◽  
G Zwart ◽  
F A van den Bergh
Keyword(s):  
Flow Cytometry ◽  
Polymerase Chain Reaction ◽  
Cross Reactivity ◽  
Single Step ◽  
Specific Method ◽  
Hla B27 ◽  
Chain Reaction ◽  
Specific Polymerase Chain Reaction ◽  
Pcr Method ◽  
Polymerase Chain

Abstract To find a specific method for HLA-B27 typing for the diagnosis of rheumatic disorders, we extensively tested the single-step B27-specific polymerase chain reaction (PCR) described by Dominguez et al. (Immunogenetics 1992;36:277-82). This method, which relies on specific primer recognition of a sequence in the third exon (unique to the B27-allele), was used for screening of 270 characterized blood samples, 57 of which were B27-positive. The method proved to be both sensitive and specific: It unambiguously identified all B27-positive samples and produced no false-positive results. For approximately 1% of the samples, we had to repeat DNA isolation and PCR to obtain a clear control amplification signal. In contrast to the specificity of the PCR method, parallel-performed flow cytometry gave ambiguous results in 3% of the samples because of antibody cross-reactivity. Flow cytometry and the PCR method described were similar in labor and costs. Therefore, we conclude that the proposed single-step PCR is feasible in a routine laboratory and would improve the reliability of HLA-B27 typing.

scholarly journals Three Severe Cases of Viral Infections with Post-Kidney Transplantation Successfully Confirmed by Polymerase Chain Reaction and Flow Cytometry

2018 ◽  
Vol 8 (3) ◽  
pp. 198-206
Author(s):  
Keita Nakanishi ◽  
Hiroshi Kaito ◽  
Miki Ogi ◽  
Denshi Takai ◽  
Junya Fujimura ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Polymerase Chain Reaction ◽  
Kidney Transplantation ◽  
Viral Infections ◽  
Specific Treatment ◽  
Epstein Barr Virus Infection ◽  
Chain Reaction ◽  
Barr Virus ◽  
Polymerase Chain ◽  
Epstein Barr

Viral infections in patients with post-kidney transplantation are often difficult to diagnose as well as treat. We herein report three cases with severe viral infections after kidney transplantation. All their causative pathogens could be detected promptly by polymerase chain reaction and flow cytometry during the early stages of infection. These examinations would also be of great use to monitor therapeutic responses and disease activity. It is indeed true that no specific treatment is available for most of the viral infections, but we should be aware that some infections, such as Epstein-Barr virus infection, can be treatable with prompt and specific treatment, such as rituximab.

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