UK NEQAS for H&I: SCHEME 2B - CROSSMATCHING BY FLOW CYTOMETRY
METHODOLOGY QUESTIONNAIRE
Date:
Lab Number:
Lab Name:
Please complete the following information for the methods used by your laboratory for Scheme 2B. If your
laboratory makes any changes to these methods during the assessment year, please resubmit this form with the
changes. The results of this questionnaire will be collated and made available on the UK NEQAS for H&I website.
1.
Cell preparation and controls
Cell preparation method:
Cells:
Volume/tube (l)
Cell concentration
Diluent
Serum: Volume/tube (l)
Used as supplied Yes/No:
and/or dilutions of
Source of negative control:
(e.g. NIBSC)
Number of negative control replicates:
Source of positive control:
(e.g. NIBSC)
Number of positive control replicates:
Number of crossmatch test replicates:
2.
Sensitisation stage
Temperature (0C):
Incubation time (mins):
First wash step:
Manual
Volume/tube (ml)
3.
Automated cell washer
Wash medium:
Temperature (0C)
Number of washes:
Detection Stage
Anti-human IgG reagent:
Raised in
Manufacturer:
Labelled with
Goat
FITC
Whole IgG
Sheep
Phycoerythrin
Fab2 fragment
Rabbit
Other
Dilution:
Diluted in:
Volume/tube (l)
(from working stock)
Incubation time
(mins)
Incubation temp
(ºC)
Please email the completed form to: ukneqashandi@wales.nhs.uk or return by post/fax to UK NEQAS for H&I, Welsh
Blood Service, Ely Valley Road, Talbot Green, Pontyclun CF72 9WB; Fax +44 (0) 1443 622001.
Page 1 of 4
NEQ 96, Issue 1
Effective Date 21/12/15
Ref. SOP: 133/NEQ
Lab Number:
4.
Lab Name:
Anti-T cell reagent
T-cell reagent manufacturer:
Is the anti-human IgG reagent added with the anti-T cell reagent?
Yes/No:
If No – is there a wash step before adding the anti-T cell reagent?
Yes/No:
Wash step details:
Manual
Automated cell washer
Volume/tube (ml)
Number of washes:
Wash Medium
Temperature (ºC)
Is the anti-T cell reagent added with the anti-B cell reagent:
Yes/No:
Anti-T cell reagent
Labelled with:
FITC
CD3
Phycoerythrin-cyanine 5
Other:
Yes/No:
Diluted?
If Yes - diluted in:
Other
Dilution (e.g. 1 in 10)
Volume/tube (l)
5.
Yes/No:
Incubation time
(mins)
Incubation temp
(°C)
Anti-B cell reagent
B-cell reagent manufacturer:
Is the anti-B cell reagent added with the anti-human IgG reagent? Yes/No:
If No – is there a wash step before adding the anti-B cell reagent? Yes/No:
Wash step details:
Manual
Automated cell washer
Volume/tube (ml)
Wash medium
Number of washes:
Temperature (°C)
Anti-B cell reagent
Labelled with:
Specificity:
Phycoerythrin
CD19
Diluted?
Phycoerythrin-cyanine 5
CD20
If Yes - diluted in:
Other:
Other:
Volume/tube (l)
Incubation time
(mins)
Yes/No:
Dilution (e.g. 1 in 10)
Incubation temp
(°C)
Please email the completed form to: ukneqashandi@wales.nhs.uk or return by post/fax to UK NEQAS for H&I, Welsh
Blood Service, Ely Valley Road, Talbot Green, Pontyclun CF72 9WB; Fax +44 (0) 1443 622001.
Page 2 of 4
NEQ 96, Issue 1
Effective Date 21/12/15
Ref. SOP: 133/NEQ
Lab Number:
6.
Lab Name:
Final Wash
Wash step details:
Manual
Automated cell washer
Wash medium:
Volume/tube (ml)
Number of washes
Temperature (°C)
Resuspension of cells in:
Fiaxative
Sheath fluid
Phosphate Buffered Saline
Other
7.
Volume/tube(l)
Data Acquisition
Make of flow Cytometer:
Live gated: Yes/No:
Calibration beads used: Yes/No:
Used for:
Alignment check
Type of beads:
Laser check
Type of beads:
Fluorescence check
Type of beads:
Fluorescence quantitation
Type of beads:
Events collected per sample tube
Minimum
Maximum
Total Events:
T Cells
B Cells
Parameter used to assess peak performance:
Scale used:
Median
Linear channel
Mean
Log channel
Mode
Other:
Other:
Please email the completed form to: ukneqashandi@wales.nhs.uk or return by post/fax to UK NEQAS for H&I, Welsh
Blood Service, Ely Valley Road, Talbot Green, Pontyclun CF72 9WB; Fax +44 (0) 1443 622001.
Page 3 of 4
NEQ 96, Issue 1
Effective Date 21/12/15
Ref. SOP: 133/NEQ
Lab Number:
8.
Lab Name:
Data Analysis
Parameter used to assess difference in fluorescence of anti-human IgG reagent:
Peak Shift
Median difference
Mean difference
Mode difference
% shift
Other
Minimum value for valid positive control sample e.g. 40 channel shift T Cell:
B Cell
Discriminator value taken to distinguish negative from positive:
Discriminator value to distinguish clinically significant binding of human IgG:
(if assay used for clinical purpose)
9.
Use of crossmatching by flow cytometry (mark all that apply)
Clinical / Diagnostic
Antibody screening
Research / Development
Pre-transplant crossmatching
Particular areas of use
Post-transplant monitoring
Other:
10.
When reporting the final result, is the description “positive” or “negative” adequate?
Yes/No:
If No - please say how you would ask centres to report the result so that the result can be assessed across centres:
General Comments:
Please email the completed form to: ukneqashandi@wales.nhs.uk or return by post/fax to UK NEQAS for H&I, Welsh
Blood Service, Ely Valley Road, Talbot Green, Pontyclun CF72 9WB; Fax +44 (0) 1443 622001.
Page 4 of 4
NEQ 96, Issue 1
Effective Date 21/12/15
Ref. SOP: 133/NEQ