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Protocols

B cells 4 colour method

Goal

This protocol describes phenotyping of B cells in isolated peripheral blood mononuclear cells by 4 colour flowcytometry.

Required patient material

8ml EDTA-anti-coagulated or heparinized blood (not older than 24h)

Required reagents

Note: reagents of other manufacturer’s may be used after testing

Ficoll separation solution (1,077 g/ml; Biochrom)
RPMI 1640 (Biochrom)
Fetal calf serum
FACSFlow (BD Biosciences)

**Optilyse B (Beckman-Coulter)
**IntraPrep (Beckman-Coulter)
**Deionised water
** if cells are fixed

Equipment

Note: equipment of other manufacturer’s may be used

4 colour flow cytometer as for example FACSCalibur with 488nm-laser and 635nm-diode-laser (BD Biosciences)

Antibodies

Antibody Clone Fluorochrome Order No. Provider
CD19 JA. 119 PC7 IM3628 BC
CD21 B-Iy4 PE 555422 BD
CD27 M-T271 FITC F7178 Dako
CD38 HIT2 FITC 555459 BD
Anti-IgM Goat F(ab’)2 Cy5 109-176-129 JIR
Anti-IgD Goat F(ab’)2 PE 2032-09 SB
Anti-IgA Goat F(ab’)2 PE 2052-09 SB
Anti-IgG Rabbit F(ab’)2 FITC F0056 Dako
Isotype control Mouse-IgG1 FITC IM0639 BC
Isotype control Goat F(ab’)2 PE 0110-09 SB
Isotype control Goat F(ab’)2 Cy5 005-170-006 JIR
Provider abbreviations:
BD: BD-Biosciences; BC: Beckman-Coulter; Dako: DakoCytomation; SB: SouthernBiotech; JIR: Jackson ImmunoResearch Laboratories; * reagents of other manufacturer’s may be used after testing

Required Time

2-3 hours

Procedure

Preparation of cells
Peripheral blood mononuclear cells are separated by Ficoll density centrifugation. No further preparation is necessary. Alternative methods use whole blood for B cell staining: See “Whole blood method”  (-> Alternative protocols).

Staining

Adapted whole blood staining according to the OptiLyse B-method  (Beckman-Coulter).
Step 1: Prepare specified volumes of antibody mixture (10-20µl) as listed in table below
Step 2: 50µl cell suspension each (250.000 to 500.000 MNC) are added,, vortexed and incubated for 15-30 min on ice.
*Step 3: After thorough mixing, 25µl of Optilyse B are added to each sample, mixed again thoroughly and incubated for 15 min at room temperature.
*Step 4: Samples are mixed, 500µl deionised water are added, followed by further incubation for 15 min.
Step 5: After addition of 1ml FACSFlow each, samples are centrifuged for 5 min at 300xg.
Step 6: Cell-free supernatant is then carefully decanted or suctioned off  and cells are resuspended in 500µl FACSFlow.
*Alternatively, step 3 and 4 can be omitted.

Table Antibody mixtures for the B-cell panel

FITC 100µl PE 100µl PC7 25µl FL4 25µl
B1 Mouse-IgG1 (1:2) Goat F(ab’)2 CD19 Goat F(ab’)2 Cy5 (1:40)
B2 CD27 (1:5) Anti-IgD (1:40) CD19 Anti-IgM Cy5 (1:40)
B3 CD38 CD21 CD19 Anti-IgM Cy5 (1:40)
B4 Anti-IgG (1:20) Anti-IgA (1:40) CD19 Anti-IgM Cy5 (1:40)
Notes:
  • Stock solution, can be kept at 4°C
  • 10µl/50µl cell suspension

Commentary:

The suggested lysis method (OptilyseB) prevents contamination of the lymphocyte gate by erythrocytes and antibody-mediated cell aggregates (escapees). In addition, the cells are fixed and do not have to be processed immediately, but can be stored in the refrigerator for several hours if necessary.
Using a PC7-conjugated CD19 antibody has the advantage over PerCP- or PerCP-Cy5.5-conjugates of a brighter fluorescence and no compensation problems with respect to the IgM staining in FL4 (Cy5).

Data acquisition

Cells in gate R1 defining the lymphocyte population are displayed in a window FL3 vs. SSC containing a region R2 that includes all CD19+ B-cells. Combined regions R1 and R2 define the “B-cell” gate. A third window depicts FL1 versus FL2 and is confined to presentation of B cells.
Suggested Acquisition setting: Events saved: 5000 within the „B-cell“ gate; time limit of 5 min.. It is recommended to record all cells, or at least all lymphocytes, in order to re-examine the specificity of unexpected results with non-B cells.
Total B cells Total B cells
Naive B cells Naive B cells
Marginal zone like Marginal zone like
Switched memory Switched memory
Transitional B cells Transitional B cel[...]
Plasmablasts Plasmablasts
CD21low B cells CD21low B cells
IgA+ B cells IgA+ B cells
IgG+ B cells IgG+ B cells
Click on image to enlarge

Data analysis

Cells in the “B cell gate” using lymphocyte gate (R1) and CD19 as the line-specific marker (R2) are analyzed according to examples displayed in the windows above.
Using the “B-cell” gate, B1, B2 and B4 are analysed with quadrant statistics, while B3 is analyzed with region-statistics. B1 contains isotype controls that are used to control for compensation and non specific staining. Panel B2 separates CD27negIgM+IgD+ naïve, CD27+IgM+IgD+ Marginal zone like B cells and CD27+IgMnegIgDneg class-switched memory B cells.
Panel B3 allows the identification of CD21-CD38low CD21low B cells, IgM++CD38++ transitional B cells and IgMneg / (+) CD38+++ plasmablasts, while panel B4 determines IgMneg class-switched B- cells for IgA or IgG surface expression.

Data interpretation

The interpretation of the results requires the awareness of the medical history and age of the patient. The reported normal ranges are only reference values for adult patients. In children age related reference values need to be considered. Unexpected results need to be confirmed.

Report

Reported B-cell subpopulations are listed in the table below. Total B-cells as given as percent of lymphocytes and subpopulations as percent of B cells. For the calcuIation of absolute counts, CD19 positive B cells need to be determined by whole blood method (see for example “basic lymphocyte panel”).

Table B-cell subpopulations in peripheral blood

B-cell population Reference range
CD19+ in lymphocytes B cells 4,9-18,4 %
IgD+ CD27- in CD19+ B cells Naive B cells 42,6-82,3 %
IgD+ CD27+ in CD19+ B cells IgM-memory B cells 7,4-32,5 %
IgD- CD27+ in CD19+ B cells Class switched memory B cells and Plasmablasts 6,5-29,1 %
IgA+ in CD19+ B cells IgA+ B cells 2,7-13,8 %
IgG+ in CD19+ B cells IgG+ B cells 3,6-13,4 %
CD21low CD38- in CD19+ B cells CD21low subpopulation 0,9-7,6 %
IgM++ CD38++ in CD19+ B cells Transitional B cells 0,6-3,4 %
IgM-/(+)CD38+++ in CD19+ B cells Plasmablasts 0,4-3,6 %
Note:
Reference range: 5. - 95. percentile, based on 54 healthy donors (age range 19-61 years)

Quality control

Beside standard methods of quality control, a healthy control should be run with B-cell stainings of peripheral blood to eliminate staining errors in strongly deviating subpopulations. This is particularly important after production of fresh antibody mixtures. Samples with increased isotype staining should be repeated to control for specific staining.

Trouble shooting

Increased fluorescence of negative subpopulation probably stems from a too high concentration of antibodies. This may especially be true for anti-Ig-antisera. Poor separation of negative and positive populations on the other hand can be caused by overly diluted antibodies. These probable causes should be remembered particularly when using antibodies other than described in this protocol. Monoclonal antibodies or Antisera from different manufacturers need to be tested previously.
The gate for naïve B cells in B2 contains all transitional and most CD21low  B cells while the switched memory B cell gate contains plasmablasts which can easily separated due to their increased CD27 expression. A clear separation of all populations requires 5-6 colour staining. CD20 is not expressed on plasmablasts.

Need of repetitive evaluation

no

Author(s)

K. Warnatz M. Schlesier, CCI Freiburg
Last update: 2010-03-10 15:30:20

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