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Protocols

Bone marrow precursor B cells 4 colour method

Background

The analysis of B cell precursors can be helpful in the context of the differential diagnosis of patients with severe combined immunodeficiency and agammaglobulinemia. 
The protocol has been published under: 

Noordzij JG, De Bruin-Versteeg S, Verkaik NS, et al. The immunophenotypic and immunogenotypic B-cell differentiation arrest in bone marrow of RAG deficient SCID patients corresponds to residual recombination activities of mutated RAG proteins. Blood. 2002;100:2145-2152

Goal

This protocol describes 4 colour flow cytometric immunophenotyping of precursor B-cells in bone marrow samples.

Required patient material

Heparinized bone marrow or frozen bone marrow mononuclear cells

Equipment

4 colour flow cytometer for example FACSCalibur

Antibodies

Table Antibodies

Antibody Clone Fluorchrome Order No. Provider
CD10 J5 FITC
CD20 Leu16 PE
CD19 SJ25C1 APC
CD34 HPCA-2 FITC
IgD FITC
IgM MB-11 PE
CD36 CLB-IVC7 FITC
CD19 Leu12 PE
IgM FITC
CD179a HSL96 PE
CD22 S-HCL-1 APC
TdT hTdT-6 FITC
CD79a HM47 PE
CD3 SK7 PerCP
CD33 PerCP
CD16 PerCP

Table 1

Required Time

3 hours

Procedure

- - -

Staining

25μl of whole lysed bone marrow (20x106 cells/ml) or thawed bone marrow mononuclear cells is incubated for 10 minutes at room temperature with combinations of optimally titrated monoclonal antibody (mAb) (25μl of each antibody is used) (See Table 2) and washed with PBS-BSA 

Labeling 1-4

Labelings for membrane-bound antigens (labelings 1-4) were directly analyzed by flow cytometry using FACSCalibur (Becton Dickinson, San Jose, CA).

Labeling 5-8

Labelings involving intracellular staining of Cy CD79a, CyIgm, and CyVpreB,  and intranuclear staining of terminal deoxynucleotidyl transferase (TdT) (labelings 4-8), first the membrane labelings need to be performed followed by permeabilization of the BM cells using IntraPrep Permeabilization Reagent (Immunotech, Marseille, France), and subsequent intracellular staining.

 

Data acquisition

If possible, acquire 10,000 B-cell specific events (CD19 or CD22 positive; negative for the mixture CD3/CD33/CD16).

Pro B cells Pro B cells
Pre B 1 and pre B 2 cells Pre B 1 and pre B [...]
Pre B 1 cells Pre B 1 cells
Pre B 2 cells Pre B 2 cells
Immature and mature B cells Immature and matur[...]
Immature B cells Immature B cells
Mature B cells Mature B cells
Click on image to enlarge

Data analysis

The composition of the BM lymphocyte gate in healthy children is highly variable, because of “contamination” with T lymphocytes, NK cells, myeloid precursors, and normoblasts. Therefore, a mixture of CD3, CD33 and CD16 was used for exclusion. B-cells are analyzed in a purified B-cell gate (Figure 1).

CD22 is also expressed on basophils, therefore CD36 was used as exclusion marker for the CD22 gate, resulting in increased purity of the B-cell gates.

The entire B-cell compartment is defined as being CD22-positive. The CyCD79a and the CD19 fractions need to be recalculated within the CD22 B-cell compartment.

The gating strategy is depicted in figure 1: 

 

Figure 1: Gating strategy

Legend: Flow cytometric analysis of bone marrow of a healthy individual. The composition of the precursor B-cel compartment is analyzed in a lymphogate and a purified CD19 gate. The order of the B-cell differentiation stages are indicated by an arrow. A. Labeling with CD10 and CD20 (labelling 1). B. Labeling with cytoplasmic Igm and surface membrane IgM (labelling 5).

Data interpretation

The gating is described in Data analysis, the differential expression of the markers for the respective B cell precursor subpopulation summarized in Figure 2.

 

Figure 2: Expression pattern of specific markers on B cell precursor cells in the bone marrow

 

The mature B-cell population in BM varies between samples because of blood contamination To correct for this variance the mature B-cell population needs to be excluded from calculations. Consequently, the percentages of stages 1 to 8 were recalculated and set at 100% (Figure 3).

Figure 3: Composition of the precursor B-cell compartment (excluding mature B-cells) in healthy children.

Need of repetitive evaluation

no

Author(s)

Dr. Mirjam van der Burg
Last update: 2012-11-04 22:41:25

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