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.

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 PCR Analysis for Bcl-2/IgH in a Phase III Study of Yttrium-90 Ibritumomab Tiuxetan As Consolidation of First Remission in Patients With Follicular Lymphoma

2009 ◽  
Vol 27 (36) ◽  
pp. 6094-6100 ◽  
Author(s):  
Lindsey Goff ◽  
Karin Summers ◽  
Sameena Iqbal ◽  
Jens Kuhlmann ◽  
Michael Kunz ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Follicular Lymphoma ◽  
Quantitative Polymerase Chain Reaction ◽  
Phase Iii ◽  
Pcr Analysis ◽  
Control Group ◽  
Ibritumomab Tiuxetan ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Yttrium 90

Purpose The randomized First-Line Indolent Trial (FIT) was conducted in patients with advanced follicular lymphoma (FL), to evaluate the safety and efficacy of yttrium-90 (90Y) ibritumomab tiuxetan given as consolidation of complete or partial remission. This study of minimal residual disease was undertaken in parallel, to determine the rate of conversion from bcl-2 polymerase chain reaction (PCR) –detectable to –undetectable status and the corresponding effect on progression-free survival (PFS). Patients and Methods Blood samples from 414 patients (90Y-ibritumomab, n = 208; control, n = 206) were evaluated using real-time quantitative polymerase chain reaction (RQ-PCR); 186 were found to have the bcl-2 rearrangement and were thus eligible for inclusion in the RQ-PCR analysis. Results Overall, 90% of treated patients converted from bcl-2 PCR–detectable to –undetectable disease status, compared with 36% in the control group. Treatment significantly prolonged median PFS in patients converting to bcl-2 PCR-undetectable status (40.8 v 24.0 months in the control group; P < .01, hazard ratio [HR], 0.399). In patients who had bcl-2 PCR-detectable disease at random assignment, treatment significantly prolonged median PFS (38.4 v 8.2 months in the control group; P < .01, HR, 0.293). Conclusion Eradication of PCR-detectable disease occurred more frequently after treatment with 90Y-ibritumomab tiuxetan and was associated with prolongation of PFS.

The role of intraocular f luid polymerase chain reaction in the diagnosis, treatment and monitoring of cytomegalovirus retinitis

2021 ◽  
pp. 380-383
Author(s):  
B.S. Pershin ◽  
◽  
A.A. Maschan ◽  
V.Y. Makhmutov ◽  
M.A. Ilushina ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Stem Cell ◽  
Retinal Detachment ◽  
Cytomegalovirus Retinitis ◽  
Chain Reaction ◽  
Hematopoietic Stem ◽  
Key Words Cytomegalovirus ◽  
Polymerase Chain ◽  
Intraocular Fluid

Purpose. To study the possibilities of a new method of CMRR treatment in the prevention of irreversible blindness. Material and methods. 74 patients with cytomegalovirus retinitis, frolicking after hematopoietic stem cell transplantation. The first group (9 people, 15 eyes) consisted of children, whose treatment was carried out under ophthalmoscopic control. The second group (65 people, 114 eyes) consisted of children in whom the control of the effectiveness of treatment was carried out using PCR of aqueous humor in real time. Results. In the first group, retinal detachment was diagnosed in three out of fifteen eyes, accounting for 20%. In the second group, the incidence of retinal detachment was 3.5% of 114 eyes. Among patients receiving treatment under ophthalmoscopic control, CMRR relapses were detected in 5 cases, which amounted to 33.3%. In children, whose treatment was controlled by intraocular fluid PCR, relapses were diagnosed in 22 cases, which amounted to 19.29%. Conclusions. Intravitreal administration of antiviral drugs under the control of polymerase chain reaction is a more effective method of treating cytomegalovirus retinitis than intravitreal administration under ophthalmoscopic control. Key words: cytomegalovirus retinitis, intraocular fluid polymerase chain reaction.

Comparison of Clostridium Difficile Associated Diarrhea Detection by Enzyme Linked Immunosorbent Assay (EIA) and Polymerase Chain Reaction (PCR) in Autologous Hematopoietic Stem Cell Transplant (AHSCT) Recipients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4739-4739
Author(s):  
Kenneth Utz ◽  
Christopher Frei ◽  
Bonita Neumon ◽  
Brenda L. Frye ◽  
Deanna Schneider ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Stem Cell ◽  
Clostridium Difficile ◽  
Enzyme Linked Immunosorbent Assay ◽  
Repeated Testing ◽  
Chain Reaction ◽  
Hematopoietic Stem ◽  
Common Cause ◽  
Polymerase Chain ◽  
Clostridium Difficile Associated Diarrhea

Abstract Abstract 4739 Background: Diarrhea is a frequent complication of autologous hematopoietic stem cell transplantation and is a common cause of morbidity. Clostridium difficile associated diarrhea (CDAD) is the most common cause of nosocomial infections. Detection of CDAD is widely performed using enzyme linked immunosorbent assay (EIA) to C. difficile toxins A and B because of its technical ease of use and rapid results. The sensitivity of EIA is 60%, but the sensitivity increases with repeated testing. Recently, detection of CDAD by Polymerase Chain Reaction (PCR) methodology has resulted in rapid detection of amplified C. difficile toxin (CDT) B chromosomal material and has been shown to be more sensitive than EIA in patients suspected of CDAD; however, no comparison of the two methodologies has been performed in recipients of an autologous hematopoietic AHSCT. Methods: This retrospective analysis of AHSCT recipients at our institution evaluates the incidence of CDAD when using either EIA or PCR. CDAD was assessed at the onset of diarrhea in both cohorts. The time period of interest was from 7 days prior to transplantation to 30 days posttransplanation. In the EIA cohort, three consecutive liquid stools were tested for the presence of CDT A and B by EIA. In the PCR cohort, only one liquid stool was sent for sampling. Our institution adopted PCR methodology on 1 February 2010. Demographic and clinical information were collected to assess CDAD risk. Successful treatment of CDAD was defined as resolution of diarrhea within seven days. Previously published data from our institution have shown the incidence of CDAD to be 15% when using EIA methodology. Nominal data were analyzed by Fisher's Exact Test and continuous data were analyzed by Student's T-Test. Results: A total of 159 patients were screened; and 16 patients were excluded because they did not have a CDAD test performed. Seventy-three were included in the EIA cohort and 69 were included in the PCR cohort. Clinical characteristics were similar between the groups. Known risk factors for CDAD including days in the hospital, days of neutropenia, and days of prophylactic and intravenous antibiotics used were similar between the cohorts. The incidence of a positive CDAD test was higher in the EIA cohort compared to the PCR cohort (18% vs. 7% [p = 0.049]). The duration of diarrhea was longer in the EIA cohort as compared to the PCR cohort (7.3 days vs. 5.5 days [0.016]). Of the three consecutive EIA tests sent, the first was positive only 30% (4/13) of the time. Metronidazole was the first-line agent used in all cases. Response to therapy was poor in both the EIA and PCR cohorts with rates of 30% and 20%, respectively. No fatalities occurred in either group as a result of CDAD. Conclusion: The incidence of diarrhea was significantly shorter in the PCR cohort as compared to the EIA cohort. The duration of diarrhea was significantly shorter in the PCR group as compared to the EIA cohort. It is possible that repeated testing for CDAD by EIA decreases the specificity of the test or that the EIA is detecting a non-toxigenic C. difficile enterotoxin. Because our patient population has additional causes of diarrhea including mucosal irritation caused by chemotherapy, additional studies are needed to discern the discrepancies between both methodologies. Disclosures: No relevant conflicts of interest to declare.

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