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Protocols

Detection of γc in T-B+NK- SCIDs

Goal

This protocol describes detection of γc by whole blood flowcytometry (minimum 3 colour facs, but additional information may be gained with additional flurochromes).

Required patient material

0.5 ml EDTA-blood from patient and healthy control (does not need to be age or sex matched)

Required reagents

CellFix (BD Biosciences)

Facs Lyse (BD Biosciences)

Cell Wash (BD Biosciences)

Equipment

centrifuge, microfuge or immunofuge

*Minimum 3 colour flow cytometer (FACSScan), but more information can be gained from a 4 or 6 colour machine as for example FACSCalibur with 488nm-laser and 635nm-diode-laser or CANTO II (BD Biosciences)

* equipment of other manufacturer’s may be used

Antibodies

Antibody Clone Fluorchrome Order No. Provider
CD45 2D1 PerCP 345809 BD
G1 PE 430013 BD
Gamma chain CD132 PE 351953 BD

(The only required antibody is gc antibody as the lymphocyte population may be gated upon. Additional antibodies provide information about individual cell populations. This is useful in patients with maternal-foetal engraftment, but examining lymphocytes is sufficient to detect most γc/JAK3 SCIDs.)

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

30 minutes

Procedure

Preparation of cells

Whole blood is used.

Staining

Step 1: Label FACs Tubes (may use microcentrifuge tubes and transfer to FAC tubes later): control iso, control γc, patient (initials or other identifier like lab number) iso, patient γc.

Step 2: Add 5 ul of CD45PerCP to all tubes. Add 5 µl GI to the isotype tubes and 5 ul γc to the γc chain tubes.

Step 3: Add 100 µl of whole blood to the appropriate tubes and incubate 10 minutes at room temperature.

Step 4: After 10 minutes, add 1 ml FACs Lyse to each tube and incubate for 10 minutes, room temperature.

Step 5: After the lyse incubation, samples are peletted (centrifuged for 5 min at 300xg, 5 minutes 2000 rpm in a microfuge or 45 seconds on high in an immunofuge) and the supernatant removed.

Step 6: Wash 1 time with Cell Wash (add 1ml of wash to cell pellet, resuspend, spin and decant supernatant).

Step 7: Add 250 µl Cell Fix and acquire cells within 24 hours of staining. If not acquired immediately, cover and refrigerate.

 

Commentary: To diagnose defects in the γc the minimum that is required is to compare γc staining in lymphocytes (defined by FSC vs SSC gating or CD45 vs SSC). The addition of cell surface markers enables examination of γc in individual cell populations

 

Data acquisition

Cells in gate R1 define the lymphocyte population.

Suggested Acquisition setting: Events saved: 10000 within the lymphocyte gate with a time limit of 5 min. It is recommended to record all cells, in order to re-gate lymphoctes if required.

Lymphocyte Gate Healthy Control Lymphocyte Gate He[...]
Gamma Chain Expression Healthy Control Gamma Chain Expres[...]
Lymphocyte Gate X-linked SCID Lymphocyte Gate X-[...]
Gamma Chain Expression X-linked SCID Gamma Chain Expres[...]
Click on image to enlarge

Data analysis

Start by gating cells in the lymphocyte population based on CD45 vs SSC in the control G1 tube (or FSC vs SSC if CD45 was not included). Create a histogram and with G1/γc (FL2) on the x-axis. Only show lymphocytes (R1). Depending on software overlay the histogram with the lymphocytes from the γc tube. Draw a marker from the bottom of the G1 histogram peak (<1%) to the right side of the histogram. Obtain the statistics (%) for the G1 and γc sample. If overlaying histogram is not possible (e.g. with Diva software), create a 2nd histogram. Repeat with the patient files.

 

If cell surface markers were included (e.g. CD4, CD8, CD20), start with the isotype control sample, gate around lymphocytes (R1), then using R1, create a dot plot: surface marker vs SSC (e.g. CD4 vs SSC) and gate on the positive population (e.g CD4+) (R2). Create a histogram and with G1 (FL2) on the x-axis. Only show positive population (R2). Depending on software, overlay the histogram with the positive population from the γc tube. If overlaying histogram is not possible (e.g. with Diva software), create a 2nd histogram. Repeat with the patient files. Repeat as required for each surface marker used.

Data interpretation

The interpretation of the results requires the awareness of the medical history of the patient. The reported normal ranges are only reference values for paediatric and adults. Patients with maternal-foetal engraftment often have a small cell population showing γc staining while the majority of cells show absent gc. If surface markers are used, this positive population is usually found in the CD8+ T cells. Unexpected results need to be confirmed.

Report

Report percentage γc+ cells

 


Table gc in peripheral blood

Cell population % gc Reference range
lymphocytes >50%*
CD4+ T cells >50%
CD8+ T cells >50%
B cells >50%

(1) Gilmour et al British Journ of Haematol. 2001, 112: 671-676

Quality control

Beside standard methods of quality control, a healthy control should be run with γc staining of peripheral blood to detect mishandling in transport and staining errors. It is essential that the control sample is collected at the same time as the patient sample and they are transported together to control for delays and temperature changes in transport. Over time graphing of percent γc from all control samples enables rapid detection of technical problems such as deteriorating antibodies and drift in the flow cytometer settings among others.

Trouble shooting

Lack of γc in the control sample is usually caused by degraded antibodies, failure to add antibodies or samples that froze in transport.

Need of repetitive evaluation

no

Author(s)

Kimberly Gilmour, Centre for Immunodeficiency, Great Ormond Street Hospital, London, UK
Last update: 2010-03-31 13:00:54

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