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Isolation of Precursor B-cell Subsets from Umbilical Cord Blood
Published on: April 16, 2013
Immunophenotypic analysis of CD19+ precursors in normal human adult bone marrow: implications for minimal residual
J Ciudad1, A Orfao, B Vidriales
1Servicio General de Citometría, University of Salamanca, Spain.
Insights
This study identifies normal B-cell maturation patterns in adult bone marrow, revealing infrequent B-cell phenotypes crucial for monitoring minimal residual disease (MRD) in precursor-B-acute lymphoblastic leukemia (precursor-B-ALL). These findings aid in detecting residual leukemia cells post-treatment.
Area of Science:
- Immunology
- Hematology
- Cell Biology
Background:
- Normal B-cell differentiation antigen expression sequences are not fully understood.
- Identifying rare B-cell subsets in normal bone marrow (BM) is critical for detecting minimal residual disease (MRD) in precursor-B-acute lymphoblastic leukemia (precursor-B-ALL).
Purpose of the Study:
- To analyze antigen expression sequences in normal adult BM CD19+ B cells.
- To identify infrequent or undetectable B-cell phenotypes for MRD detection in precursor-B-ALL.
Main Methods:
- Analysis of adult BM samples from 33 healthy volunteers using triple staining and flow cytometry.
- A sensitive two-step acquisition procedure was employed to identify and characterize very low-frequency cells.
Main Results:
- Five distinct CD19+ B-cell subsets were identified based on maturation markers (CD34, CD10, CD20, CD22).
- Several B-cell phenotypes common in precursor-B-ALL are rare or absent in normal BM differentiation.
- Specific co-expression patterns (e.g., CD19/CD117, CD20/CD34, CD22/CD34) were found at very low frequencies (< 1 x 10^-3), with CD19/CD13 and CD19/CD33 also infrequent and lacking defined populations.
Conclusions:
- Normal adult BM B-cells exhibit consistent maturation patterns in phenotype and distribution.
- Deviations from these normal patterns can serve as a valuable tool for monitoring MRD in precursor-B-ALL patients achieving complete remission.
Background And Objective:
Normal B-cell differentiation has been characterized extensively, but discrepancies persist regarding the exact sequence of antigen expression. Few systematic studies focusing on identification of the minor or undetectable B-cell subsets in normal human bone marrow (BM) which are frequently found in leukemic cells have been performed. Such studies could help to monitor minimal residual disease (MRD) in precursor-B-acute lymphoblastic leukemia (precursor-B-ALL). The aim of the present study was to analyze the sequence of antigen expression among normal human CD19+ B cells from adult BM. Our major goal was to identify infrequent and undetectable B-cell phenotypes that could be used for the detection of MRD in patients with precursor-B-ALL.
Design And Methods:
Adult BM samples from a total of 33 healthy volunteers were analyzed using triple stainings, and measured by flow cytometry. A sensitive method based on the two-step acquisition procedure was used for the identification and characterization of cells present at very low frequencies.
Results:
Five different subsets of CD19+ cells were identified in normal BM samples according to their degree of maturation: 1) CD19+/CD34+/CD10-/CD20-/CD22dlm+ (0.5 +/- 0.4% B cells); 2) CD19+/CD34-/CD10++/CD20-/CD22dlm+ (3.4 +/- 2.7%); 3) CD19+/CD34-/CD10+/CD20-/CD22dlm+ (3.5 +/- 2.2%); 4) CD19+/CD34-/CD10+/CD20+,++/CD22dlm+ (21 +/- 11%), and 5) CD19+/CD34-/CD10-/CD20++/CD22+ (73 +/- 19%). We observed that several B-cell phenotypes are frequent among precursor-B-ALL, but are infrequent or undetectable in normal human B cell differentiation. Accordingly, in all normal BM samples analyzed, less than 4 x 10(-5) cells co-expressed CD19 and CD117; CD20strong+/CD34+ and CD22strong+/CD34+ events were found at frequencies less than 5 x 10(-4), while CD20+/CD34+ phenotypes were found in less than 1 x 10(-3) BM cells. Although both CD19+/CD13+ and CD19+/CD33+ events were found at frequencies of up to 3 x 10(-3), they never formed a well-defined population of cells and therefore these latter phenotypic patterns could also be of use for MRD investigation in CD13+ and/or CD33+ precursor-B-ALL cases.
Interpretation And Conclusions:
Our results show that in adult BM normal B-cells display constant patterns of maturation as regards both their phenotypic characteristics and their relative distribution. Abnormalities in these patterns provide a potentially useful tool for monitoring MRD in precursor-B-ALL patients who achieve cytomorphologic complete remission.
