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SCHEME 2B - CROSSMATCHING BY FLOW CYTOMETRY - METHODOLOGY 2017 1. Cell preparation Lab Code
Cell preparation method
Cell vol (μl)
Cell concentration
Diluent
Serum vol (μl)
Used as supplied
Diluted
Local negative control
Replicates
Local positive control
No of pos control replicates
No of test replicates
9 11 14 15 19 20 23 24 25 28 34 35 38 39 41 42 45 48 51 54 58 62 101 112 114 115 117 118 119 120 122 126 130
Density gradient centrifugation
200 30 30 T: 25, B:50
2-3x10^6 5x10^6 10^7 10x10^6
RPMI with 10% FCS PBS/Azide (0.1%) PBS/BSA/Azide FACS diluent
50 20 40 T: 25, B: 50
Yes Yes Yes Yes
1:2
Biosera AB Serum (unsensitised male blood donors) NIBSC In-house
5 4 2 2
Pooled serum of sensitised patients Pooled highly sensitised patient sera In-house In-house
2 1 weak, 1 strong 2 2
4 2 2 2
50 50 50 50 30 50 50 30 50 25 50 40 30 50 100 50 50 100 25 100 250,000
5x10^6 5x10^6 2.5x10^6 5M 16x10^6 6x10^6 5x10^6 5M 1x10^7 10x10^6 4x10^6 2.5x10^6 5x10^6 2x10^6 2x10^6 5.7x10^6 5M
PBS PBS/1%FBS HBSS 1% PBSA PBA TBS PBS+1% FCS PBS azide RPMI 2% FBS/PBS 1% FCS/PBS TPM Local flow diluent PBS PBS 0.1% azide 1% normal goat + 1% normal mous serum PBS 1% NBCF in PBS (PBSCS)
Yes Yes Yes
In-house NIBSC NIBSC NIBSC Biorad Local NIBSC NIBSC NIBSC Seralab
NIBSC In-house NIBSC and In-house In-House Campath Local In-house In-House In-House In-house
PBS AB sera
200,000 1x10^6 200000 5M 3x10^5
RPMI with 20% FBS PBS PBS PBS1x -BSA1% PBS+2% FCS
2 2 1 2 2 2 3 4 3 3 3 2 2 2 1 3 2 2 2 2 1 3 2 3 2 1 weak, 1 strong
4 3 3 3 3 3 3
10x10^6 2.5x10^5
50 50 50 50 50 50 50 20 50 50 50 10 20 50 50 50 50 50 30 20 25 50
133 136 138 139 142 143
Buffy coat (without density gradient) Ficoll hypaque separation
144 145 147 149 154 157 159 160 163 167 169 176 186 190 191 193 194 195 201 202 204 206 209 218 220 227 235 238 245 246 252 260 262 271 284 293 297 341 351 358
Ficoll separation Ficoll Ficoll Ficoll
Lymphoprep
Lymphoprep Lymphocyte separation medium (Lympholyte) Lymphocyte separation medium Lymphocyte density gradient method Carbonyl iron lympholyte + Dextran sulphate Ficoll density gradient Density gradient centrifugation Lympholyte Density gradient
Dendity gradient centrifugation Ficoll Hypaque Gradient centrifugation Concentartion gradient-lympholyte-H + Pronase Treatment Density gradient separation Ficoll 200 Density gradient Cell wash centrifugation suspended in Hanks
10 100-120 100 50
0.25-0.3x10^6
Yes Yes
Local and NIBSC NIBSC Local SLI NIBSC NIBSC
100 20 50
No No Yes Yes No Yes Yes Yes
Human serum AB Pooled AB sera NIBSC NIBSC Local + NIBSC AB+ male AB serum NIBSC
4 3 2 3 3 6 3 2 3 3 3 2 2 3 6 4 3 2 2 2 2 3 2 4 2 2
In-House
2
In-house
1 weak, 1 strong 2
AB serum AB serum AB serum AB serum
PRA positive (>10000MFI or 100%+) Hypersensitized sera pool Hypersensitized sera pool Hyperimmunixed patient pool
In-house
1 3 1 2 3 2
Local pooled sera
1 2 1 1 1 1
2 2 1 1 1 2
Local Local Human serum AB Biorad Commercial In-house Local Local NIBSC
1 1 2 1 2 2 3 2 2
Local Biorad Serum from polcry immunised patients Biorad Commercial Hyperimmunixed patient pool Local Biotest NIBSC
2 1 2 1 2 2 2 1 2
2 1 2 1
1 1 3 2 2 2 3 1
Local serum Local sera from immunized patients Pool hyperimmunised Pooled PRA positive sera 2 pooled sera In-house Local Local PC
1 2 2 2 2 1 1 1
1 1 3 2 2 2 2 1
Yes Yes Yes Yes Yes Yes Yes Yes Yes
2 Local and NIBSC Local pool Local In-House + Human monoclonal antibodies In-House poitive pool In-House Pool patients Pooled sensitized patients NIBSC and In-House Local pool Local Patient pool Hyperimmunised serum Local
3 3 2 2 2 3 2 2 2 2 2 2 2
50
Yes
50 100 100 50 2000 40
Visual check 2.5x10^6 10^7 6000 6-15 3x10^6
RPMI RPMI PBS RPMI-SVF (5%) PBS FBS
50 20 80 50 20 50
Yes No Yes Yes Yes Yes
25 50 100 50 20 15 100 50 0
15x10^6 1x10^7 5000 1x10^7 15x10^6 1.5x10^5 3000 10^6 0.2x10^6
PBS PBS RPMI PBS PBS BSA 1% PBS PBS BSA 1% RPMI+2%FBS
25 50 50 50 20 50 50 50 50
Yes Yes Yes Yes Yes Yes Yes Yes Yes
PBMC isolation (Lymphoprep Ficoll Ficoll Isolated cells sent were used. Washed with PBS Manual
100 100 100 100 100 100 25 50
2E6/ml 5M 200000 2-3x10^5 3000-5000 2400-3800 20000 1-1.5x10^7
TBS
100 25 50 20 20 20 50 50
Yes Yes
NACL PBS RPMI PBS RPMI 20% medium (IMDM+FCS)
Yes Yes Yes Yes
AB donor pool Pool sera for AB donors SAB male AB Negative, PRA negative sera Pooled sera In-house NIBSC Local NC
Ficoll separation PBMC (Ficoll gradient)
50 25
6000 10000
RPMI PBS+FCS
50 50
Yes Yes
Pool of AB sera from healthy blood donors Local neg sera pooled
2 1
Poll of sera from hypersensitized recipients Local positive sera pooled
1 1
1 2
Ficoll Pronase-treatment STEMCELL Ficoll hypaque Lymphocytes Separation Medium Whole blood Ficoll
10000 6x10^6 600000 10M 2500 400000
2% FCS0, 5% NaN3-PBS NaCl 0.9% RPMI+FCS RPMI PBS RPMI
1 2 2 1 2 2 1
RPMI + Human SAB 10% PBS 2% FBS RPMI
1 2 2 3
Local In-house In-house Hypersensitized patient Pool hyperimmunised patient's sera NIBSC NIBSC+BAG PRA >80% serum Pooled positive sera Immunised patient In-house Local + NIBSC
1 2 2 1 2 2 1
500000 250000 5x10^6
Local In-house In-house AB non sensitized donor Pool AB human sera NIBSC NIBSC AB + Healthy serum Ab male Human SAB AB NHS Invitrogen NIBSC
2 4 2 2 2 3 4
Ficoll Ficoll hypaque Mononuclear cells isolated by density centrifugation over Ficoll
400 15 50 50 50 100 150 25 5 50 Dried pellet 50
1 1 1 2
1 2 2 2
Density gradient centrifugation using Ficoll Ficoll hypaque Density gradient centrifugation
50 50 200 100
5500 2-2.5x10^6 5x10^6
PBS+2% FCS
Local NC HLA Class I+II Local Inno-Train
2 2 1 1
Local PC HLA Class I+II Local Inno-Train
1 1 1 1
2 2 2 1
Ficoll layering (Lymphoprep) and magnetic nanoparticle separation
Cell wash centrifugation suspension in RPMI+2%FBS Lymphocyte separation medium
PBS +1% BSA
200 15 50 50 50 75 150 25 20 50 25 50 50 50 20 50
Yes Yes Yes Yes Yes Yes
1/2
1:8
2
1/2
Yes Yes
Yes Yes Yes
1/16
2 2 2 2
SCHEME 2B - CROSSMATCHING BY FLOW CYTOMETRY - METHODOLOGY 2017 2. Sensitisation Stage Temp
3. Detection Stage:
Lab Code
Time (mins)
Manual Automated washer
Wash medium
(OC)
Vol / tube (ml)
Washes
9 11 14 15 19 20 23 24 25 28 34 35 38 39 41 42 45 48 51 54 58 62 101 112 114 115 117 118 119 120 122 126 130 133 136 138 139 142 143 144 145 147 149 154 157 159 160 163 167 169 176 186 190 191 193 194 195 201 202 204 206 209 218 220 227 235 238 245 246 252 260 262 271 284 293 297 341 351 358
30 30 30 30
37 37 RT RT
Manual Manual Manual Automated
PBS Cold PBS/Azide
15 30 30 15 30 30 30 30 30 20 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30
37 37 37 37 20 37 37 22 37 22 22 RT 21 RT 22 37 37 Ambient 4 RT 4 24 RT RT RT 22
Automated Automated Automated Automated Manual Automated Manual Manual Manual Automated Manual Manual Manual Manual Yes Manual Manual Manual Automated Manual Manual Manual Manual Manual Manual
Manufacturer
Raised in
Labelled with
Dilution
Diluted in
Vol (μl)
Inc time (mins)
Inc temp
100 4 2 3
3 3 2 3
RT 22 RT RT
Life Technologies Sigma Sigma Sigma
Goat Goat Goat Goat
FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment
1:20 1/10 1/23 1:180
PBS PBS/Azide PBS/BSA/Azide FACS diluent
2-8 4 RT RT 4 4 4 22 RT 22
Beckman Coulter Sigma Sigma Sigma DAKO Sigma Sigma Southern Biotech Sigma Sigma
RT 21 RT 4 4 22 Ambient Ambient RT 4 24 RT RT RT 22
1:20 1:40 1/15 1/50 1:20 1/32 1:300 1/50 1:20 1:2000 1/10 1/50 Neat 1:10 1:20 1:50
Goat Goat Goat Goat
FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC/Whole IgG FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment Strepavidin FITC/Fab2 fragment/ Biotin IgG FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment
CD3-PC5 and CD19-PE (detection cocktail) PBS PBS 1% PBSA PBA TBS dH2O PBS azide 1% FBS in PBS buffer 2% FBS/PBS PBS PBA
DAKO Sigma Sigma DAKO Life Technologies Invitrogen DAKO Jackson BD BD Jackson
Goat Goat Goat Goat Rabbit Goat Goat Goat Goat Goat Goat Goat Rabbit Rabbit Goat Goat Rabbit Goat Goat Rabbit Goat
Jackson DAKO Sigma Biorad Sigma Beckman Coulter DAKO Biorad Sigma Jackson
Goat Rabbit Rabbit Rabbit Goat Goat Goat Rabbit Goat Goat Goat Goat Rabbit Sheep Goat Goat
FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment
50 20 115 100 50 5 50 5 5 100 50 50 100 4 90 10 50 4 5 100 5 12 10 80 20 20 10 200 10 50 50 50 25 50 20 20 10 0.5 10 25 50 100 50 50 20 100 50 20 20
30 20 20 15 30 15 30 30 15 30 30 30 20 20 10 15 45 30 30 30 30 30 30 10 20 30 30 1.15, 2.10 30 30 30 20 20 40 20 20 30 20 15 30 20 30 20 30 20 30 30 30 30
4 4 4 4 4 2-8 4 2-8 4 4 4 RT 4 4-8 22 22 Ice 4 4 4 4 22 4 4 4 4 4 RT RT 4 4 4-8 Ice 4 4 5 4 4 2-4 4 4 4 4 20 4 4 4 4 4
2 1 BD LWA 1 2.5 5 1.5 4 4 4 2 1 2 3 4 4 4 2-3 0.8 150 0.4 2 3 2 2 1
2 3 4ml x3 3 2 2 3 2 3 3 2 2 3 2 2 3 3 2 2 3 3 3 4 3 3 3
30 30 30 30 30 30 20 30 30 30 30 30 30 30 30 30
RT 22 RT 2 22 4 24 RT RT 4 RT 20 4 22 37 Ambient
Manual Manual Manual Manual Manual Manual Manual Manual Manual Manual Manual Manual Manual Manual Manual Manual
2 2 200 4 0.2 0.2 1.5 4 3 2.5 3 1 2 1 2 2
3 2 4 1 3 3 2 3 2 3 2 2 2 3 2 3
RT 22 RT 4 22 4 2-4 RT RT 22 RT 20 4 4 RT 20
PBS PBS PBS PBS-BSA1% PBS 0.02% NaN3 2% FBS PBS-BSA1% BD cell wash PBS 1X/BSA 0.5%
30 30 30 30 30 30 30 30
20 4 20 RT 22 22 22 22
Manual Manual Manual Automated Manual Manual Manual Manual
PBS/BSA/Azide PBS PBS-BSA1% PBS-BSA PBS PBS
3 2 0.2 5 2 2 4 4
3 2 4 2 3 2 2 3
18 4 20 RT RT 22 22 22
DAKO Beckman Coulter Jackson Jackson Jackson Beckman Coulter DAKO Jackson
Rabbit Goat Goat Rabbit Goat Goat Rabbit Goat
FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment Phycoerythrin FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment
20 30 30 30 30 30 30 30
20 4 4 RT 4 5 22 22
30 30
22 20
Manual Manual
PBS PBS+FCS
0.2 0.2
4 2
22 20
Beckman Coulter DAKO
Goat Rabbit
60 30 30 30 30 30 30 30 30 30 30 30
37 4 22 4 22 22-24 20 RT 22 20-25 4 22
Manual Manual Manual Manual Manual Manual Manual Manual Manual Manual Manual Manual
PBS 2% FCS0, 5% NaN3-PBS PBS PBS PBS PBS + SVF FACS Flow PBS x1 Cell wash BD
2 2 3 2 2 2 3 3 2 2 3 2
RT 4 22 4 22 22-24 20 RT 22 20-25 20-25 22
Abcam Jackson Jackson DAKO Biorad DAKO
PBS 2% FBS Cell wash BD
5 200 0.2 1 4 3 10 1 1 3 0.5 2
30 30 30 30
22 22-25 4 RT
Manual Manual Manual Manual
PBS PBS+2%FCS Cold PBS Azide PBS+1% BSA
0.2 1 1 1
3 3 5 3
22 22-25 4 RT
PBS PBS PBS/FBS BD cell wash 1% PBSA PBA TBS PBS+1% FCS PBS azide 1% FBS in PBS buffer 1% FCS/PBS PBA Local flow diluent PBS PBS 0.1% azide PBS PBSCS PBS PBS DPBS without Ca++ PBS+2% FBS PBS PBS PBS PBS1x - BSA1% PBS+2% FCS Washing buffer PBS PBS/azide/FCS 3% PBS PBS BD pharmingen stain buffer
TBS
Temp (OC)
BioSciences
PBS PBS 0.1% azide 1% normal goat + 1% normal mous serum PBS
(OC)
Lot dependent 1/60 1/30 1:160
PBS PBS PBS
1/50, 1/100 Optimum dilution after titration 1/80 1:50
PBS H2O distiled 1ml PBS1x - BSA1% PBS+2% FCS
1/80 1/20 1/50
Washing buffer PBS Gelatine/PBS/azide
1/40
PBS
1:25 1:40 1/500 1:40 1:250 1:3 1/500 1:80 1:200
PBS PBS PBS PBS PBS-BSA1% PBS PBS-BSA1% PBS PBS-BSA1%
1/200
PBS + FBS 8%
1/40 1/60 1/100 1/50 1:100
PBS PBS-BSA1% PBS PBS PBS
5 50 40 5 50 50 100 5
FITC/Fab2 fragment FITC/Fab2 fragment
1/400 1/100
PBS PBS
10 100
30 20
22 4
Phycoerythrin/Fab2 fragment Phycoerythrin/Fab2 fragment Phycoerythrin/Fab2 fragment Phycoerythrin/Fab2 fragment FITC/Fab2 fragment FITC/Whole IgG FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment
512 1/10 1/10 1/80
PBS-CD3-CD19 PBS PBS PBS
Jackson Biorad Jackson DAKO
Goat Goat Goat Rabbit Sheep Rabbit Mouse Rabbit Goat Sheep Goat Rabbit
1x 1.5 1/100 1/100
PBS x1 dH2O PBS PBS 2% FBS
5 50 10 25 100 10 5 50 5 100 20 10
30 30 30 30 20 30 10 30 30 20 20 30
RT 4 4 4 22 22-24 20 RT 22 4 4 4
Invitrogen DAKO Jackson
Goat Goat Rabbit Goat
FITC/Fab2 fragment FITC/Fab2 fragment FITC/Fab2 fragment FITC
1/40 1000
PBS PBS+2%FCS
1:100
PBS+1%BSA
50 3 50
30 30 30 30
4 4 4 RT
Invitrogen Jackson DAKO DAKO Coulter
SCHEME 2B - CROSSMATCHING BY FLOW CYTOMETRY - METHODOLOGY 2017 4. Anti-T cell reagent Lab Code
Manufacturer
IgG added
Wash step
with anti-T
before adding
Wash step
Wash medium
cell reagent anti-T cell reag
9 11 14 15 19 20 23 24 25 28 34 35 38 39 41 42 45 48 51 54 58 62 101 112 114 115 117 118 119 120 122 126 130 133 136 138 139 142 143 144 145 147 149 154 157 159 160 163 167 169 176 186 190 191 193 194 195 201 202 204 206 209 218 220 227 235 238 245 246 252 260 262 271 284 293 297 341 351 358
Beckman Coulter BD DAKO Beckman Coulter BD BD BD BD BD DAKO Beckman Coulter DAKO BD DAKO Beckman Coulter DAKO Beckman Coulter BD Beckman Coulter Beckman Coulter Iotest Beckman Coulter BD BD BD Beckman Coulter Beckman Coulter Immunostep BD BD DAKO Coulter BD BD BD BD Beckman Coulter Beckman Coulter BD Beckman Coulter BD Beckman Coulter
BD Beckman Coulter Beckman Coulter BD BD Beckman Coulter
Yes No Yes No Yes Yes Yes No Yes Yes No No No Yes No No Yes Yes No Yes No Yes No Yes Yes No No Yes Yes Yes Yes Yes Yes No Yes Yes No Yes Yes Yes Yes Yes No Yes No No
Yes
Yes Yes Yes Yes Yes
Number of
Temp
tube
washes
(OC)
(ml)
Manual Manual Yes
Vol /
PBS Cold PBS/Azide
Automated
Automated
Manual Manual Manual Manual Manual Manual
100 4 3
1% PBSA
PBS azide 1% FBS in PBS buffer
1
1.5 4 4
3 1 1
3
3 1 1
RT 22
PBS
3
1 2
No No Yes Yes Yes Yes
Phycoerythrin R-Phycoerythrin Phycoerythrin-cyanine 5 RPE
4
4
2
22
Manual Manual
DPBS without Ca++ PBS+2% FBS
150 0.4
1 1
4 4
Manual Manual Manual
PBS PBS
3 2 2
2 3 3
RT RT RT
Manual
Washing buffer
2
2
RT
Manual
Yes
No
No Yes No
Yes
BD
No Yes Yes Yes No Yes Yes Yes
Beckman Coulter BD
Yes Yes
No
BD eBioscience Beckman Coulter BD Beckman Coulter Beckman Coulter BD BD BD BD Biosciences BD BD
No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
Yes
BD BD Miltenyi
Yes Yes Yes Yes
Yes
PBS
4
1
4
BD pharmingen stain buffer
0.2 20
2 1
4 2-4
1 3 1 2 1 3 2
RT 22 RT 20 4 4 RT
1 1
18 4
Manual Manual Manual Manual Manual Manual Manual
PBS PBS PBS PBS-BSA1% PBS 0.02% NaN3 2% FBS BD cell wash
3 2.5 3 1 2 1 2
Manual Manual
TBS PBS + FBS 8%
3 2
Automated Manual Manual
PBS NH4CL
5 2 2
2 1 2
Manual
PBS+FCS
0.2
2
Manual
PBS
5
2
Manual
PBS
No
1
2
3
2
1 1 1 2
5 2
Manual Manual Manual Manual
Cell wash BD PBS Cell wash BD
1 3 0.5 2
Manual Manual
Cold PBS Azide PBS+1%BSA
1 1
Yes Yes No Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes No Yes
Yes Yes No Yes No Yes No Yes Yes Yes No Yes
Diluted
ECD Phycoerythrin-cyanine 5 Phycoerythrin-cyanine 5 A750 PE Phycoerythrin PerCp Phycoerythrin PE Phycoerythrin-PE
PerCp PE - Phycoerythrin APC APC APC-CD3 PE PE Phycoerythrin Phycoerythrin PerCp Phycoerythrin PE
PE
APC
Yes No Yes Yes No Yes No No No No No No Yes No Yes No Yes Yes
No No No No No Yes No Yes No PC7 No PerCp Yes No Yes Phycoerythrin Yes Yes No No CD2 No Yes No Yes No No Yes PE No Yes Yes Yes No Yes PE No Yes No Yes PE No Yes Yes Yes PC7 No Yes Yes Yes No No
Yes Yes Yes Yes
No No No No No No No
Yes Yes
APC Phycoerythrin-cyanine 5
Yes Y
No Yes
Yes Yes Yes 4 No Yes 22-24 Yes Yes Yes 22 Yes 20-25 Yes RT Yes 22 Yes
APC 450nm ECD Phycoerythrin Phycoerythrin APC PerCp Phycoerythrin-cyanine 5 Phycoerythrin Phycoerythrin PerCp APC
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
No Yes No No No No No No No No Yes No
Yes Yes Yes Yes
PerCp Phycoerythrin-cyanine 5 PerCP-Vio700
Yes PC7 Yes Yes Yes
No Yes No Yes
4
Dilution
PBS
1:30
FACS diluent
1/11.5 1:60
Detection cocktail
1:2
PBS azide
1/20
2% FBS/PBS
1:20
PBA
1/10
PBS
1:10
PE
Yes Yes
RT
Diluent
PE
Phycoerythrin PE Phycoerythrin-cyanine 5 PerCP Phycoerythrin PE V450 PE
RT 4 22
Yes
Other
Yes Yes Yes Yes Yes Yes
No
Manual
RPE
RT
PBSCS
Manual Manual
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
RT
Manual
Yes
Yes Yes Yes Yes
Phycoerythrin-cyanine 5 PE Phycoerythrin
No
No No No
Phycoerythrin-cyanine 5 PerCp PE
Yes No Yes Yes
4 4 RT
CD3
Vol / (μl)
Inc time (mins)
(OC)
50 3 115 100 10 5 5 5 5 5 5 10 100 4 4.75 5 50 4 5 5 5 5 5 5 20 20 10 200 10 10 50 2.5 25 15 5 5 10 5 20 25 10 10 10 15 20 10 50 4 5
30 20 20 15 30 15 30 30 15 30 30 20 30 20 10 15 15 30 30 15 30 15 30 20 20 30 30 15 30 30 30 20 20 40 20 15 30 20 15 30 20 30 20 30 20 30 30 30 30
4 4 4 4 4 2-8 4 2-8 4 4 4 2-8 4 4-8 22 22 Ice 4 4 4 4 22 4 4 4 4 4 RT RT 4 4 4-8 Ice 4 4 4 4 4 2-4 4 4 4 4 20 4 4 4 4 4
10 10 5 5 20 5 5 5
20 30 30 30 15 30 30 30
20 4 4 RT 4 5 22 22
10 100
30 20
4 4
5 50 10 3 5 5 5 5 5 5 20 10
15 30 30 30 20 30 10 30 30 20 20 30
RT 4 4 4 22 22-24 20 RT 22 4 4 22
5 5 4 50
30 30 30 30
4 4 4 RT
tube
RT
No Yes
Labelled with
Yes Yes No
1% FCS/PBS 1
Anti-T cell reagent added with the anti-B cell reagent
PBS+2% FBS
1:4
PBS
1/20
PBS+2% FCS
1:16
Washing buffer
1/4
PBS
1:25
PBS
1:10
BD cell wash
1:10
PBS
3/100
2% FCS0, 5% NaN3-PBS
512
PBS 2% FBS
1/4
PBS+2%FCS
10
PBS+1%BSA
1:10
Inc temp
SCHEME 2B - CROSSMATCHING BY FLOW CYTOMETRY - METHODOLOGY 2017 5. Anti-B cell reagent Lab Code
9 11 14 15 19 20 23 24 25 28 34 35 38 39 41 42 45 48 51 54 58 62 101 112 114 115 117 118 119 120 122 126 130 133 136 138 139 142 143 144 145 147 149 154 157 159 160 163 167 169 176 186 190 191 193 194 195 201 202 204 206 209 218 220 227 235 238 245 246 252 260 262 271 284 293 297 341 351 358
Manufacturer
DAKO
BD BD BD BD BD
IgG added
Wash step before adding
cell reagent
anti-B cell reag
No Yes No
Yes
Manual
Yes
Automated
Yes Yes Yes No Yes Yes
Beckman Coulter DAKO BD DAKO
No No Yes
DAKO Beckman Coulter BD Beckman Coulter Beckman Coulter Iotest BD BD BD DAKO Beckman Coulter Beckman Coulter Beckman Coulter
Yes Yes No Yes Yes Yes No Yes Yes No Yes Yes Yes Yes
BD BD
Yes Yes
Coulter
Yes Yes
Wash medium
Cold PBS/Azide
Automated
1% PBSA
Yes Yes No
Manual Manual
PBS azide 1% FBS in PBS buffer
No
Vol /
Number of
Temp
tube
washes
(OC)
Manual Manual
PBS
4
1
22
3
1
RT
4 4
3
1 1
2
4 RT
4
No No Manual Manual
No No Yes No
BD Beckman Coulter
No
Yes
Beckman Coulter
Yes Yes No Yes Yes Yes
Beckman Coulter BD
Yes Yes
No
Beckman Coulter eBioscience Beckman Coulter BD Beckman Coulter Beckman Coulter
Yes
BD Beckman Coulter BD BD
No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
BD BD Miltenyi
Yes Yes Yes Yes
Yes
Yes Yes Yes
Phycoerythrin Phycoerythrin Allophycocyanin APC Phycoerythrin Phycoerythrin
Yes Yes Yes Yes Yes
Phycoerythrin Phycoerythrin-cyanine 5 BV241 Phycoerythrin
Yes Yes Yes Yes
Diluent
Dilution
Phycoerythrin-cyanine 5 Phycoerythrin Phycoerythrin Phycoerythrin Phycoerythrin-cyanine 5 ECD Phycoerythrin-cyanine 5 Phycoerythrin-cyanine 5 PE Phycoerythrin-cyanine 5 Phycoerythrin Phycoerythrin-cyanine 5 Phycoerythrin Phycoerythrin
Yes Yes
PBS
1:10
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
PBS+2% FBS
1:4
PBS
1/200
PBS+2% FCS
1:16
Phycoerythrin Phycoerythrin
Yes Yes
Yes No
Washing buffer
1/2
Phycoerythrin Phycoerythrin
Yes Yes
No No
Yes Yes Yes Yes Yes Yes Yes Yes Yes
Yes No No No No Yes No Yes No No
PBS
Manual
Washing buffer
2
2
RT
0.2
2
4
1 3 1 2 1 3 2
RT 22 RT 20 4 4 RT
APC Phycoerythrin APC Phycoerythrin-cyanine 5 Phycoerythrin-cyanine 5 Phycoerythrin Phycoerythrin Phycoerythrin Phycoerythrin-cyanine 5
PerCp Phycoerythrin
PBS PBS-BSA1% PBS 0.02% NaN3 2% FBS BD cell wash
Manual Manual
TBS PBS + FBS 8%
3 2
1 1
18 4
Automated Manual Manual
PBS NH4CL
5 2 2
2 1 2
RT 4 22
Manual
PBS PBS+FCS
0.2
2
20
Manual
PBS
5
2
RT
No No No No No
Phycoerythrin Phycoerythrin
Yes Yes
No Yes
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
No Yes No No No No No No
Yes Yes Yes Yes
No Yes No Yes
Cell wash BD PBS PBS 2% FBS
1 3 0.5 2
1 1 1 2
Manual Manual
Cold PBS Azide PBS+1% BSA
1 1
5 2
4 RT
PBS
1
2
3
2
APC Phycoerythrin R-Phycoerythrin Phycoerythrin
PBS
1:25
PBS BD cell wash
1:10
Yes
Phycoerythrin-cyanine 5 Phycoerythrin-cyanine 5 Per-Cp Cy5.5 Phycoerythrin-cyanine 5
Manual Manual Manual Manual
Manual
Yes
Yes Yes Yes Yes Yes Yes Yes
Phycoerythrin APC Phycoerythrin 4 Phycoerythrin APC 22-24 PC7 Phycoerythrin Phycoerythrin 22 APC 4 APC 4 Phycoerythrin 22 BV421
Manual
1:2
Yes No No No No No No Yes No Yes No No Yes
RT RT RT
3 2.5 3 1 2 1 2
Detaction cocktail
1/20
1 3 3
PBS
1/23 1:50
1:80
3 2 2
Yes No Yes No
PBS/BSA/Azide FACS diluent
PBS azide
Manual Manual Manual
Manual Manual Manual Manual Manual Manual Manual
Yes
No Yes Yes No Yes No No No No No
2% FBS/PBS
4 4
No
No
Phycoerythrin Phycoerythrin-cyanine 5
150 1 0.4 1
Manual
Yes Yes Yes Yes Yes No Yes No No
CD19 CD20 Other Diluted
Vol /
Inc time (mins)
Inc temp
(μl)
3 115 100 5 5 5 5 5 5 5
20 20 15 30 15 30 30 15 30 30
4 4 4 4 2-8 4 2-8 4 4 4
100 4 0.25 5
30 20 10 15
4 4-8 22 22
4 5 10 5 5 5 7 5 20 10 200 10 10 50 2.5 25 15
30 30 15 30 15 30 20 20 30 30 15 30 30 30 20 20 40
4 4 4 4 22 4 4 4 4 4 RT RT 4 4 4-8 Ice 4
10 10
30 20
4 4
25 10 10 10 10 20 20 50 2 5
30 20 30 20 30 20 30 30 30 30
4 4 4 4 20 4 4 4 4 4
10 10 5 5 20 5 20 5
20 30 30 30 15 30 30 30
20 4 4 RT 4 5 22 22
10 100
30 20
4 4
10 50 10 3 5 5 5 5 5 5 20 2.5
15 30 30 30 20 30 10 30 30 20 20 30
RT 4 4 4 22 22-24 20 RT 22 4 4 4
5 5 3 50
30 30 30 30
4 4 4 RT
tube
DPB without Ca++ PBS+2% FBS
No No
Labelled with
(ml)
Yes
BD Immunotech BD Beckman Coulter Beckman Coulter BD Beckman Coulter BD Beckman Coulter
BD BD Beckman Coulter
Wash step
with anti-B
No
PBS
2/100
PBS-CD3-IgG
512
No Yes No
1/4
PBS+2%FCS
10
PBS+1%BSA
1:10
(OC)
SCHEME 2B - CROSSMATCHING BY FLOW CYTOMETRY - METHODOLOGY 2017 6. Final wash Lab
Wash step
Code
Wash
Vol /
Number of
Temp
medium
tube
washes
( C)
O
Resuspension of
Vol /
cells in
tube
(ml)
9 11 14 15 19 20 23 24 25 28 34 35 38 39 41 42 45 48 51 54 58 62 101 112 114 115 117 118 119 120 122 126 130 133 136 138 139 142 143 144 145 147 149 154 157 159 160 163 167 169 176 186 190 191 193 194 195 201 202 204 206 209 218 220 227 235 238 245 246 252 260 262 271 284 293 297 341 351 358
Manual Manual Manual Automated
PBS PBS/Azide
Automated Automated Automated Automated Manual Automated Manual Manual Manual Automated Manual Manual Manual
PBS PBS/FBS BD cell wash
Manual Manual Manual Manual Automated Manual Manual Manual Manual Manual Manual Manual
Manual Manual Manual Manual Manual Manual Manual Manual Manual Manual Manual Manual Manual Manual Manual
100 4 2 3
3 1 2 1
RT 22 RT RT
Phosphate buffered saline Phosphate buffered saline PBS/BSA/Azide FACS diluent
400 400 300 200
2 1 BD LWA 1 2.5 5 2 4 4 4 2 1 4
1 3 4ml x1 3 2 1 3 1 1 1 2 1 1
2-8 4 RT RT 4 4 4 4 RT 22 22 RT 21
Phosphate buffered saline Fixative BD cell wash 1% PBSA Sheath fluid TBS Phosphate buffered saline PBS azide 1% FBS in PBS buffer 2% FBS/PBS 1% FCS/PBS PBA Local flow diluent
300 300 300 500 250 280 150 500 250 300 280 220 500
1 1 1 2 2 1 1 1 1 3 1 2
4 4 22 Ambient Ambient 4 4 24 RT RT RT 22
Sheath fluid Phosphate buffered saline PBSCS Phosphate buffered saline Fixative Fixative Fixative Phosphate buffered saline Phosphate buffered saline Phosphate buffered saline PBS1x - BSA1% Fixative
100
PBS+2% FCS
4 4 4 2-3 0.8 150 0.4 2 3 2 2 1
PBS/azide/FCS 3% PBS PBS
2 200 4 0
2 1 1 3
RT RT 4 22
1.5 4 3 2.5 3 1 2 1 2 2
1 2 1 3 1 2 1 3 2 2
2-4 4 RT 22 RT 20 4 4 RT 20
Fixative Phosphate buffered saline Phosphate buffered saline Phosphate buffered saline Phosphate buffered saline Phosphate buffered saline Stain buffer Fixative Fixative Phosphate buffered saline Fixative Fixative Phosphate buffered saline Phosphate buffered saline BD cell wash Phosphate buffered saline
3 2 0.2 5 2 2 4
1 1 4 2 1 2 1
18 4 20 RT 4 22 22
Sheath fluid Phosphate buffered saline Phosphate buffered saline Phosphate buffered saline Phosphate buffered saline Fixative Phosphate buffered saline
500 300 300 100 250 300 250
0.2 0.2
4 2
22 20
Phosphate buffered saline Phosphate buffered saline
450 400
5 200 0.2 1 4 3 10 1 1 3 0.5 2
1 2 3 2 1 3 3 2 1 1 1 2
RT 4 22 4 22 22-24 20 RT 22 20-25 20-25 22
Phosphate buffered saline 2% FCS0, 5% NaN3-PBS+7-AAD Phosphate buffered saline Phosphate buffered saline Phosphate buffered saline Phosphate buffered saline Sheath fluid Phosphate buffered saline Fixative Phosphate buffered saline PBS 2% FBS Cell wash BD
300 120 450 500 200 300 300 500 500 250 300 500
1 1 1
3 2 5 2
22 22-25 4 RT
Phosphate buffered saline Phosphate buffered saline Fixative Fixative
500 300
PBS
PBA
PBS azide 1% FBS in PBS buffer 1% FCS/PBS
PBS 0.1% azide PBSCS PBS PBS DPBS without Ca++ PBS+2% FBS PBS PBS PBS
BD phatmingen stain buffer PBS PBS PBS 0.02% NaN3 2% FBS BD cell wash PBS 1X/BSA 0.5%
Manual Manual Manual Automated Manual Manual Manual Manual
TBS PBS + FBS 8%
Manual Manual
PBS PBS+FCS
Manual Manual Manual Manual Manual Manual Manual Manual Manual Manual Manual Manual Manual Manual Manual Manual
(μl)
PBS PBS-BSA1% PBS PBS
2% FCS0, 5% NaN3-PBS PBS PBS PBS + SVF FACS Flow PBS x1 Cell wash BD PBS 2% FBS
PBS PBS PBS+1%BSA
0.5 450 150 300 650 200 500 400 350 100 400 500 450 400 600 100 500 250 500 100 400 350 100/150
100
SCHEME 2B - CROSSMATCHING BY FLOW CYTOMETRY - METHODOLOGY 2017 7. Data Acquisition Lab Code
9 11 14 15 19 20 23 24 25 28 34 35 38
39 41 42 45 48 51 54 58 62 101 112 114 115 117 118 119 120 122 126 130 133 136 138 139 142 143 144 145 147 149 154 157 159 160 163 167 169 176 186 190 191 193 194 195 201 202 204 206 209 218 220 227 235 238 245 246 252 260 262 271 284 293 297 341 351 358
Flow cytometer
Live gated
Calibration beads used
Used for
Type of beads Min
Beckman Coulter BD FACS Canto II FACS Canto II BD FACS Canto II
Yes Yes Yes Yes
Yes Yes Yes Yes
Alignment check, Fluorescence check Alignment check, Laser check, Fluorescence check Alignment check, Laser check, Fluorescence check, Fluorescence quantitation Alignment check, Laser check, Fluorescence check
Flowcheck, Flow set CST 7 colour set-up CST
Beckman Coulter FC500 BD FACS Calibur BD FACS Canto BD FACS Canto II BD FACS Calibur Beckman Coulter BD FACS Canto Beckman Coulter 500 MPL
No No
Yes Yes Yes Yes Yes Yes Yes Yes
Alignment check, Laser check, Fluorescence check Alignment check, Laser check, Fluorescence check Alignment check, Laser check, Fluorescence check Alignment check, Laser check, Fluorescence check, Fluorescence quantitation Alignment check, Laser check, Fluorescence check Alignment check, Fluorescence check Laser check, Fluorescence check Alignment check, Laser check, Fluorescence check, Fluorescence quantitation
Flowcheck, Flow set, Immunobrite Rainbow 8 peak, Calibrite CST, 7 colour set-up CST, 7 colour set-up BD Calibrite-3 colour kit Flowcheck, Immunobrite CST, 7 colour set-up Flowcheck, Flow set
Yes Yes Yes Yes Yes
Alignment check, Laser check, Fluorescence check, Fluorescence quantitation Alignment check, Laser check, Fluorescence check, Fluorescence quantitation Alignment check, Laser check, Fluorescence check, Fluorescence quantitation Alignment check, Laser check, Fluorescence check, Fluorescence quantitation Alignment check, Laser check, Fluorescence check, Fluorescence quantitation
Flowcheck, Flow set, Immunobrite CST 7 colour set-up Flow check, Flow set
Yes Yes Yes No Yes Yes Yes Yes
Alignment check, Laser check, Fluorescence check, Fluorescence quantitation Alignment check, Laser check, Fluorescence check, Fluorescence quantitation Alignment check, Laser check
CST, DAKO fluorospheres Flowcheck, Flow set, Immunobrite Fluorospheres
Alignment check, Laser check, Fluorescence check, Fluorescence quantitation Alignment check, Laser check, Fluorescence check Alignment check, Laser check, Fluorescence check Alignment check, Laser check, Fluorescence check, Fluorescence quantitation Alignment check, Laser check, Fluorescence check Alignment check, Laser check, Fluorescence check, Fluorescence quantitation Alignment check, Laser check, Fluorescence check Alignment check, Laser check, Fluorescence check
Flowcheck, Flow set, Quantum-MESF Calibrite 3 Calibrite + Rainbow Cal. Part. Calibrite 3
Yes Yes No Yes Yes
Beckman Coulter - FC500/Navios BD BD FACS Canto I BD FACS Canto II Beckman Coulter
Yes
BD FACS Canto II Beckman Coulter FC500 Beckman Coulter Beckman Coulter Beckman Coulter FC500 FACS Calibur FACS Calibur BD Calibur Coulter Epics-XL FACS Canto BD FACS Canto II
No Yes Yes Yes Yes Yes Yes Yes No Yes Yes
FACS Scan
Yes
FACS Calibur
No No Yes
Beckman Coulter BD FACS Canto II FACS Calibur BD Beckman Coulter FC500 Beckman Coulter FACS Canto II FACS Calibur BD BD FACS Calibur BD FACS Canto II
CD Canto II Beckman Coulter Navios BD FACS Calibur FACS Calibur
Yes
Yes Yes Yes No Yes No No
Yes Yes Yes Yes No
Yes Yes Yes Yes Yes Yes
Yes Yes Yes Yes Yes Yes No Yes Yes
Yes Yes Yes Yes Yes Yes Yes Yes
Alignment check, Laser check, Fluorescence check, Fluorescence quantitation Alignment check, Laser check, Fluorescence check Alignment check, Laser check, Fluorescence check, Fluorescence quantitation Alignment check, Laser check, Fluorescence check, Fluorescence quantitation Alignment check, Laser check, Fluorescence check Alignment check, Laser check, Fluorescence check Alignment check, Laser check, Fluorescence check Laser check
CST, Rainbow Flowcheck, Flow set
Beckman Coulter FC500 MACSQuant/Miltenyi
Yes Yes
Yes Yes
Alignment check, Laser check, Fluorescence check Alignment check, Laser check, Fluorescence check, Fluorescence quantitation
Flowcheck, Flow set Miltenyi Biotec
LSR II BD FACS Canto II Beckman Coulter
No Yes Yes Yes Yes No
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
Alignment check, Laser check, Fluorescence check Alignment check, Laser check, Fluorescence check Alignment check, Laser check, Fluorescence check Alignment check, Laser check, Fluorescence check, Fluorescence quantitation Alignment check, Fluorescence check Alignment check, Laser check, Fluorescence check, Fluorescence quantitation
CST CST Flow set BD Calibrite Immuno-brite Flowcheck, Flow set
Alignment check, Laser check, Fluorescence check Alignment check, Laser check, Fluorescence check, Fluorescence quantitation Alignment check, Laser check, Fluorescence check, Fluorescence quantitation Alignment check, Laser check, Fluorescence check Alignment check, Laser check, Fluorescence check, Fluorescence quantitation
BD Calibrite bead Unlabeled, FITC, PE, APC CST, Immunobrite CST CST
Yes Yes Yes
Alignment check, Laser check, Fluorescence check, Fluorescence quantitation Alignment check, Laser check, Fluorescence check Fluorescence check, Fluorescence quantitation
CST CST, 7 colour set-up Fluorospheres
FACS Canto II BD FACS Canto II Cube 6
Yes No Yes No
Yes Yes Yes
3000
5000
200000 250 10000
7500 6000 10000
500
10000 500
Min
B-cells Max
3000 300 100
7500 5000 10000
200 1000 1000 100
1000 100 300
500
500 50 1000 3000 5000
5000 5000 >1000 4000
5000 5000
5000
500 50 1000
5000 500 >1000
1500 20000
20000 1000
10000 1000 1000
5000 20000 or 120sec
350
Flowcheck, Flow set, Immunobrite Fluorocheck, Fluoroset Flowcheck, Flow set, Immunobrite CST Calibrite Calibrite 3 Calibrite CST
Calibrite Calibrite Flowcheck, Flow set Rainbow Flow check
Events T-cells Max
1000
2000 20000
Yes
3000 events on B-cells FACS Canto II BD FACS Canto II
5000
Min
3000 300
Sphero rainbow calibration Calibrite 3 beads Calibrite CST, Rainbow Flow check pro, Flow set pro CST Calibrite 3 and APC
BD FC500
Beckman Coulter Navios BD FACS Canto
3000
Flowcheck, Flow set, Quick-comb, Immunobrite Calibrite 3 7 colour set-up
Fluorescence check, Fluorescence quantitation Alignment check, Laser check, Fluorescence check Alignment check, Laser check, Fluorescence check, Fluorescence quantitation Alignment check, Laser check, Fluorescence check Alignment check, Laser check, Fluorescence check Alignment check, Laser check, Fluorescence check, Fluorescence quantitation Alignment check, Laser check, Fluorescence check Alignment check, Laser check, Fluorescence check Alignment check, Fluorescence check, Fluorescence quantitation Alignment check, Laser check, Fluorescence check Alignment check, Fluorescence check, Fluorescence quantitation Alignment check, Laser check, Fluorescence check Alignment check, Laser check, Fluorescence check, Fluorescence quantitation Alignment check, Laser check, Fluorescence check Alignment check, Laser check, Fluorescence check Alignment check, Laser check, Fluorescence check
Yes Yes
Total events Max
5000
5000 15000-20000
6000
10000
5000
10000
5000 50000 3000 1000 5000
10000 100000 5000
15000
5000
10000
10000
15000 100000 15000 5000 >2000 10000 10000 30000 1000 20000
12000 3000
12000 2000 10000 3000 20000 10000
1500 1000 1000 1000
50000
30000 10000
10000 ≥2000
400 3000 800
10000 Unlimited 10000
5000
1000 50000 500 500 500 >2000 1000 1000 300
200 3500
200 1500
15000 250
2000-5000 250
1000
10000 100000
1000 2000 1000
1000
5000
10000
Log channel Log channel Log channel Log channel Log channel Log channel Log channel Linear channel Log channel Log channel Log channel Log channel
Median Median Median Median Median Median Median The median fluorescence value is converted in to a linear channel value. The mean linear channel value of the negative controls are subtracted from the mean linear channel of each sample to give a linear channel shift. Median Log channel Median Log channel Log channel Median Log channel Median Log channel Median Median Median Mean Median Median Median Median Median Mean Median Geomean Mean Median Geomean Kolmogorov Mean Median Median Median, Mean Median Median Median Median Median Mean Mean Geometric mean
Log channel Log channel Log channel Log channel Log channel Linear channel Log channel Log channel Linear channel Log channel Log channel Linear channel Linear channel Linear channel Log channel Linear channel Log channel Log channel Log channel Linear channel Log channel Log channel Linear channel Log channel Log channel Linear channel Linear channel Log channel Linear channel Log channel
100
Median Median Mean
Log channel Log channel Log channel
>1000
Median/Mean Median
Linear channel, Log channel Log channel
Mean Median Mean Median Median Median Median Median Mean Median Median
Log channel Linear scale Log channel Log channel Log channel Linear channel Log channel Log channel Log channel Log channel Log channel Log channel
Median Geomean Median Median
Log channel Linear channel Log channel Log channel
3000
20000 500 100000 10000 100000 2500 100
Median Median Median Median
Log channel Log channel Linear channel, Log channel Log channel
300000 1000 30000 40000
Scale used
Median Mean Mean Mean
50000
10000 <10000
Parameter used to assess peak performance
2000 500 1000 5000 1000
500 100
2000 1000
5000
10000
500 500 250 1000
2000 3000 1000
1000 2000 5000
10000 10000 10000
300 500 5000
10000 1000 10000
SCHEME 2B - CROSSMATCHING BY FLOW CYTOMETRY - METHODOLOGY 2017 8. Data Analysis
9. Use of XM by flow cytometry
Lab Code
Parameter used to assess difference in fluorescence of anti-human IgG reagent
9
Median difference
pos/neg trimmed mean value >3
>1.5 x trimmed mean
Pre-transplant crossmatching
11 14
Median difference Median difference
>Mean AB +2SD 4x neg
>1.6 RMF 4x neg
1.5x mean neg
Clinical/Diagnostic, Pre-transplant crossmatching Clinical/Diagnostic, Pre-transplant crossmatching
15
Median difference
20x neg
10x neg
NEQAS RMF >1.5
19 20 23 24 25 28
Median difference Median difference Median difference Median difference Ratio of median fluorescence of test sample to negative control
60 +3SD for mean of AB 1.5x neg
60 +2SD 2x neg
ratio ≥3.1
ratio ≥3.6
34 35 38
Mean difference Median difference Linear channel shift
2x neg
2x neg
>46 channels
>63 channels
40 T: +3SD for mean of AB, B: +2SD T: 1.5x neg, B: 2x neg RMF >1.3 T-cell ≥1.6 B-cell ≥2.0 +2SD Mean +3SD T-cells: >46 channels B-cells: >63 channels
39 41 42 45 48 51 54 58 62 101 112
Peak shift, Median difference Mean difference Mena difference
>1.5 RMF 10x neg MCS ~30, RMF >1.3 40 channel shift
>2.0 RMF 10x neg MCS ~45, RMF >1.5
Test of neg control MESF ratio Median difference Median difference Mean difference Ratio
114 115 117 118 119
Median difference Median difference Median difference Median difference Mean difference
ratio >1.285
ratio >1.7
120
Median difference
ratio >13
ratio >12
122 126 130 133 136 138 139 142 143 144 145 147 149 154 157 159 160 163 167 169 176 186
Fluorescence index
FI >1.5
FI>2.0
T-cell
Minimum value for valid positive control B-cell
Relative median fluorescence
Mean difference Peak shift, Median difference, Mode difference
Mean difference Peak shift Mean difference, Median difference Mean difference, Median difference Ratio median log channel test/Median log channel neg % shift Ratio median log channel /median log channel neg control Mean difference Median difference Mean difference Mean difference Ratio of geometric mean
MFI of test serum/MFI of neg control serum Mean difference
4x neg a test/neg MESF ratio of >10 1.3x mean RMF >1.5
1.3x mean RMF >3.0
>1.5 ratio
>2.0 ratio
40 channel >40
70 channel >60 RMF >4
Discriminator value taken to distinguish negative from positive
Discriminator value to distinguish clinically significant binding
T-cells: 2.4x mean neg B-cells: 2.45x mean neg OTX RMF >4 Prev. Tx RMF >2.3
Clinical/Diagnostic, Clinical/Diagnostic, Clinical/Diagnostic, Clinical/Diagnostic, Clinical/Diagnostic,
T: 2SD, B:3SD T: RMF <1.3, B: RMF<1.5 40 channel shift 1.3x neg
40 T: +3SD, B: +2SD T: 1.5x neg, B: 2x neg RMF >1.3 T-cell ≥1.6 B-cell ≥2.0 +2SD Mean +3SD T-cells: >46 channels B-cells: >63 channels >1.5 RMF/ >2.0 RMF T: 2SD, B:3SD T: RMF <1.3, B: RMF<1.5 40 channel shift 2.3x neg
≥1.2 MESF ratio 1.3x mean
≥1.5 MESF ratio 1.3x mean
T-cell: >1.5 ratio B-cell: >2.0 ratio 50 >40 channel shift T: RMF >1.3, B: RMF >1.5 T: Med neg +66 ch, B: Med neg +25.5 ch T-cells: ratio >1.285 B-cells: ratio >1.7 T-cells: ratio >1.6 B-cells: ratio >2 3SD
Not determined
Pre-transplant crossmatching Pre-transplant crossmatching Pre-transplant crossmatching Pre-transplant crossmatching Clinical/Diagnostic, Pre-transplant crossmatching, Post-transplant monitoring
T: 60 channel, B: 90 channel T: RMF >1.3, B: RMF 1.5-1.7 T: Med neg +66 ch, B: Med neg +25.5 ch T-cells: ratio >1.285 B-cells: ratio >1.7
>x4 of NC
2.5 Mean ratio +2SD Mean ratio +2SD
150 channel shift 100 channel shift 100 mean log channel 2.3
250 channel shift 100 channel shift 170 mean log channel 1.7
0.5
0.22
100
300
ratio >0.7
ratio >1
2
2.5
1.5x
2x
190
Mean difference
191 193 194 195 201 202 204
Mean difference Geometric mean Median difference % shift Ratio x-mean sample/NC
206 209 218
4 Ratio MFI (sample/neg control)
Ratio >2
Ratio >3
Mean difference
CTL x1.2
CTL x1.7
220 227 235 238 245 246 252 260 262 271 284
8000 - mean of neg control Mean difference Median difference MESF Serum/MESF neg control Median difference Median log. Fl ratio Median ratio Mean difference % shift Median channel shift Median difference
8000 - mean of neg control 1.5x neg
8000 - mean of neg control 2x neg
7 1.75 Median ratio ±2SD 2x neg control MFI >12% 64 ratio >1.2
15 2.5 Median ratio ±3SD 2x neg control MFI >12% 90 ratio >1.2
>500 Ratio Mean ratio +2SD t: >40 channel, B: >60 channel Mean ratio +2SD 150% of neg control and shape of the curve 2SD from neg control T: 40 channel shift, B: 100 channel shift >Mean of neg sera +2SD T: 2.3, B: 1.7
T: 0.50, B:0.22 T-cells: ratio >1.5 x the local neg B-cells: ratio >2 x the local neg
T-cells: 1.5x the local neg B-cells: 2x the local neg RatioT>1.07, Ratio B>1.17
Pre-transplant crossmatching Pre-transplant crossmatching Pre-transplant crossmatching Pre-transplant crossmatching Clinical/Diagnostic, Research/Development, Pre-transplant crossmatching, Post-transplant monitoring
Clinical/Diagnostic, Clinical/Diagnostic, Clinical/Diagnostic, Clinical/Diagnostic, Clinical/Diagnostic,
Pre-transplant crossmatching, Post-transplant monitoring Pre-transplant crossmatching, Post-transplant monitoring Post-transplant monitoring Research/Development, Pre-transplant crossmatching, Post-transplant monitoring Pre-transplant crossmatching, Post-transplant monitoring
Clinical/Diagnostic, Research/Development, Antibody screening, Pre-transplant crossmatching, Post-transplant monitoring
>+50% of neg, >+100% of neg NR >200
Clinical/Diagnostic, Pre-transplant crossmatching, Post-transplant monitoring Pre-transplant crossmatching Pre-transplant crossmatching, Post-transplant monitoring
150% of neg control and shape of the curve >10 channel shift over cutoff T: 40 channel shift, B: 100 channel shift >Mean of neg sera +2SD plus anti-HLA antibody T: 2.3, B: 1.7
Clinical/Diagnostic, Research/Development, Pre-transplant crossmatching Clinical/Diagnostic, Pre-transplant crossmatching, Post-transplant monitoring Clinical/Diagnostic, Research/Development, Pre-transplant crossmatching, Post-transplant monitoring Clinical/Diagnostic, Pre-transplant crossmatching, Post-transplant monitoring Clinical/Diagnostic, Pre-transplant crossmatching Pre-transplant crossmatching, Post-transplant monitoring Clinical/Diagnostic, Pre-transplant crossmatching Clinical/Diagnostic, Pre-transplant crossmatching Pre-transplant crossmatching, Post-transplant monitoring Pre-transplant crossmatching, Post-transplant monitoring Clinical/Diagnostic, Pre-transplant crossmatching, Post-transplant monitoring Clinical/Diagnostic, Pre-transplant crossmatching, Post-transplant monitoring
T: 0.50, B:0.22 NA
Clinical/Diagnostic, Research/Development, Pre-transplant crossmatching, Post-transplant monitoring Pre-transplant crossmatching
T <1.2Neg >1.5Pos B <1.2Neg >2.5Pos T: >30, B: >100
Clinical/Diagnostic, Pre-transplant crossmatching
Mean +3SD 40 Ratio Mean ratio +2SD None
NA
Clinical/Diagnostic, Clinical/Diagnostic, Clinical/Diagnostic, Clinical/Diagnostic, Clinical/Diagnostic,
Depend on autocrossmatch done in parallel
Clinical/Diagnostic, Research/Development, Pre-transplant crossmatching, Post-transplant monitoring
Neg geometric mean x2.5 ratio sample/neg control, T:1.3, B: 1.6 NA
Clinical/Diagnostic, Pre-transplant crossmatching Clinical/Diagnostic, Pre-transplant crossmatching Clinical/Diagnostic, Research/Development, Pre-transplant crossmatching, Post-transplant monitoring
Clinical/Diagnostic, Pre-transplant crossmatching, Post-transplant monitoring
1.5 or 2 (T or B cells) of neg mean log channel 40 1.76 Mean ratio +2SD Tpos-Tneg=200 Mean ratio +2SD
Research/Development, Pre-transplant crossmatching Pre-transplant crossmatching, Post-transplant monitoring, Renal transplant Pre-transplant crossmatching Pre-transplant crossmatching, Post-transplant monitoring Research/Development, Pre-transplant crossmatching, Post-transplant monitoring (if antibody screening is not possible)
3SD
T: >40 channels, B: >80 channels >x4 of NC
Clinical/Diagnostic, Research/Development, Pre-transplant crossmatching
Pre-transplant Pre-transplant Pre-transplant Pre-transplant Pre-transplant
crossmatching, crossmatching, crossmatching, crossmatching crossmatching,
Post-transplant monitoring Post-transplant monitoring Post-transplant monitoring Post-transplant monitoring
RatioT>1.2, RatioB>1.3
T-cell: CTL x1.2 B-cell: CTL x1.7 8000 - mean of neg control T: 1.5x neg, B: 2x neg >2 of neg median log channel T: ratio >1.8, B: >2.2
T-cell: CTL x1.2 B-cell: CTL x1.7 8000 - mean of neg control T: 1.5x neg, B: 2x neg
2x neg control MFI Median RFU sample/Median RFU neg >2
2x neg control MFI
ratio >1.2
ratio >1.2
Clinical/Diagnostic, Pre-transplant crossmatching Pre-transplant crossmatching Clinical/Diagnostic, Pre-transplant crossmatching Research/Development, Pre-transplant crossmatching, Post-transplant monitoring Pre-transplant crossmatching, Post-transplant monitoring Clinical/Diagnostic, Pre-transplant crossmatching Clinical/Diagnostic Clinical/Diagnostic, Antibody Screening, Pre-transplant crossmatching Clinical/Diagnostic, Pre-transplant crossmatching, Post-transplant monitoring Pre-transplant crossmatching, Post-transplant monitoring Pre-transplant crossmatching, Post-transplant monitoring Clinical/Diagnostic, Pre-transplant crossmatching
SCHEME 2B - CROSSMATCHING BY FLOW CYTOMETRY - METHODOLOGY 2017 10. Reporting results Lab Code
Is Positive or Negative adequate?
9 11 14 15 19 20 23 24 25 28 34 35 38 39 41 42 45 48 51 54 58 62 101 112 114 115 117 118 119 120 122 126 130 133 136 138 139 142 143 144 145 147 149 154 157 159 160 163 167 169 176 186 190 191 193 194 195 201 202 204 206 209 218 220 227 235 238 245 246 252 260 262 271 284
Yes No Yes Yes Yes Yes No Yes No Yes Yes Yes Yes Yes No Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
Reason
"Ok to proceed" - If positivem discussion with clinician considering patient history, sensitisation and clinical need.
Immunological risk. Would need to know HLA mismatch and antibody levels. An equivocal range is a good idea.
The positive result should be explained in relation to the prescence of antibodies. Clinically significant? We also use the term weakly positive if the test/neg MESF value is between 1.2 and 1.5. As no other clinical data is available to review with the flow crossmatch results then pos/neg along with equivocal is adequate.
Yes Yes Yes Yes No Yes Yes Yes No Yes Yes Yes Yes No Yes
No Yes Yes Yes Yes Yes Yes Yes
Low positive would also be useful
The results seen to be equivocal because they are close to the positive threshold. In general our clinical rules consist of investigating the whole historical medical patient file.
A high proportion of samples sent are marginally positive. In practice such results are evaluated after consideration of other laboratory and clinical sata.
It would be useful to include a "Weakly reactive/Borderline" result option. In clinical terms, there is a big difference between "definitely no reaciton" and "probably something"
The option "Equivocal result" is interesting.
Yes
No Yes Yes Yes Yes No Yes Yes Yes Yes Yes Yes
Sometimes results are indeterminare or borderline. Insufficient samples is sent so they cannot be tested in duplicates as the patient samples are.
SCHEME 2B - CROSSMATCHING BY FLOW CYTOMETRY - METHODOLOGY 2017 Other comments Lab Code
9 11 14 15 19 20 23 24 25 28 34 35 38 39 41 42 45 48 51 54 58 62 101 112 114 115 117 118 119 120 122 126 130 133 136 138 139 142 143 144 145 147 149 154 157 159 160 163 167 169 176 186 190 191 193 194 195 201 202 204 206 209 218 220 227 235 238 245 246 252 260 262 271 284
With the introduction of uncertainty of measurement by UKAS, how does NEQAS plan to assess potential weak positive/negative results? Will you be taking MoU into account? It might be relevant to show replication of samples and controls -> How the result was obtained as this may show the integrity of the test and the ability of the scientist.
I think for the purpose of an EQA scheme you can only apply a positive/negative result to achieve meaningful results. To reflect the clinical situation is not possible. Perhaps measurement uncertainty somehow needs to be included in the future.
Please provide more cells for crossmatching. IS it possible to have HLA typing of cells before results?
For labs that perform T and B-cell crossmatch test in separate tubes, the amount of total cell provided is often too low. Doesn't reflect a real clinical lab situation where you have knowledge of patients historic antibody status and donor/recipient HLA typing.
Samples are degenerate when they arrive. Insufficient samples is sent so they cannot be tested in duplicates as the patient samples are.
Add the possibility of equivocal
