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UK NEQAS for H&I Scheme 2A - Cytotoxic Crossmatching PBL/T-CELL AND B-CELL CROSSMATCHING METHOD OF SAMPLE 2A07+2A08/2015 DESPATCHED ON 14TH JULY 2015
Lab No.
Date Received
Date Tested
9 11 12 14 15 20 23 24 25 28 34 35 38 39 41 42 45 51 54 58 62 101 112 114 116 117 119 120 126 128 136 139 143 145 147 149 154 157 159 162 163 169 174 181 186 189
15-Jul 15-Jul 15-Jul 15-Jul
16-Jul 16-Jul 15-Jul 15-Jul
15-Jul 15-Jul 15-Jul 15-Jul 15-Jul 15-Jul 15-Jul 15-Jul 15-Jul 15-Jul 15-Jul 15-Jul 15-Jul 15-Jul 15-Jul 15-Jul 15-Jul 21-Jul 15-Jul 15-Jul 16-Jul 15-Jul 15-Jul 15-Jul 16-Jul 15-Jul 17-Jul 21-Jul 15-Jul 16-Jul 15-Jul 15-Jul 15-Jul 15-Jul 15-Jul 15-Jul 15-Jul 18-Jul 15-Jul 15-Jul 15-Jul
16-Jul 15-Jul 16-Jul 15-Jul 16-Jul 15-Jul 16-Jul 15-Jul 15-Jul 15-Jul 16-Jul 16-Jul 16-Jul 16-Jul 15-Jul 15-Jul 16-Jul 21-Jul 16-Jul 15-Jul 16-Jul 15-Jul 16-Jul 15-Jul 17-Jul 16-Jul 17-Jul 24-Jul 15-Jul 16-Jul 16-Jul 16-Jul 15-Jul 16-Jul 15-Jul 24-Jul 16-Jul 18-Jul 16-Jul 15-Jul 16-Jul
190 193 194 195 197 204 206 209 210 212 216 223 227 229 232 235 238 243 245 252 259 260 262 268 284 293 294 297 301 311 315
15-Jul 15-Jul
15-Jul 15-Jul
22-Jul
NT
15-Jul NT 15-Jul 20-Jul 16-Jul 20-Jul 15-Jul 15-Jul 20-Jul 17-Jul 15-Jul 15-Jul 15-Jul 15-Jul 22-Jul 16-Jul 16-Jul 15-Jul 15-Jul 15-Jul 16-Jul 15-Jul 15-Jul
15-Jul NT 16-Jul 20-Jul 22-Jul 21-Jul 17-Jul 24-Jul 20-Jul 17-Jul 15-Jul 16-Jul 15-Jul 15-Jul 22-Jul 16-Jul 16-Jul 15-Jul 16-Jul 16-Jul 16-Jul 16-Jul 16-Jul
16-Jul 15-Jul
16-Jul 15-Jul
PBL
Yes
Yes Yes Yes
Immunobeaded Cells used cells used? T-cells B-cells T-cells B-cells
DTT addition Sera Cells
DTT Concentration DTT
Yes Yes Yes Yes
Cedarlane Cedarlane Cedarlane Cedarlane
Yes Yes Yes Yes
0.01M 0.01M
0.05 1 1 0.1
1 1 1 0.9
1 1 1 1
5 5 5 5
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 Yes Yes
Cedarlane Cedarlane Cedarlane Cedarlane Cedarlane Cedarlane Cedarlane Cedarlane Cedarlane Cedarlane TCS Biosciences Cedarlane Cedarlane Cedarlane Cedarlane Cedarlane
Yes
0.01M
1
Yes Yes Yes Yes Yes Yes Yes Yes
0.05M
4 9 1 1 10 1 0.1 0.05
1 1 36 81 1 1 70 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1
5 5 5 5 5 5 5 5 5 5 5 5 5
2 1 45 0.9 1 36
1 1 1 1
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 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 Yes Yes
Yes
Yes Yes
Yes Yes
Yes Yes
Yes
NT
NT
NT
Yes NT
Yes NT
NT
Yes
Yes
Yes Yes
Yes Yes Yes Yes Yes Yes Yes
Yes Yes
Yes Yes Yes
Cedarlane One Lambda Cedarlane Pel-Freeze Biorad Servibio Cedarlane One Lambda In-House Cedarlane Cedarlane Invitrogen In-House
Yes
Yes Yes Yes
0.01M 0.01M 0.1M 0.01M 0.1M 0.1M 0.01M 0.005M
Yes
1 5 0.1 0.1 4
0.05M 0.05M
Yes Yes
1 2
Yes
0.05M
Yes
Yes Yes
Cedarlane Cedarlane In-House Cedarlane Servibio One Lambda One Lambda Cedarlane Invitrogen Innotrain
Yes Yes Yes Yes Yes Yes Yes Yes
Yes Yes
Cedarlane Cedarlane
Yes Yes
NT
NT
NT
Yes
Yes
Yes Yes
Yes Yes
Cedarlane NT Ingen Innotrain Biorad Biorad Biorad GTI Diagnostic
Yes NT Yes
Yes Yes
Yes NT 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 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 Yes Yes
Yes Yes Yes
Yes Yes
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
Yes Yes
1 1
Yes Yes Yes Yes
Yes
2 0.5
0.05M 10% 0.01M 0.1M% 5mM
0.125 10% 0.1
0.005M 5mM
0.1M NT
NT
5
0.005M
Yes
NT Yes
Incubation Times DTT Pre Comp Post Comp Comp T-cell B-cell T-cell B-cell T-cell B-cell
Yes Yes Yes Yes
Yes
NT
Cells
Yes Yes Yes Yes
Yes Yes Yes Yes Yes Yes Yes
0.1M
Volumes Sera
Yes Yes Yes Yes
Yes
Yes Yes Yes
Complement manufacturer
NT
NT 0.015M
Yes Yes
30 45 30 45
30 90 90 75
30 90 60 75
EB/AO Fluoroquench EB/AO EB/AO
30 40 45 45 30 30 45 60 45 45 60 60 60 60 60 60 30 60 30 20
30 40 45 45 30 30
60 40 90 60 45 90
60 20
60 40 90 60 45 90 90 60 90 60 120 90 60 60 90 60 60 120 30 30
30 30
60 60 40
EB EB/AO Fluoroquench Fluoroquench EB/AO EB/AO Fluoroquench EB/AO EB/AO EB/AO EB/AO EB/AO Fluoroquench PI/AO Fluoroquench EB/AO Fluoroquench Stain Fix Fluoroquench EB/AO Fluoroquench Eosin Fluoroquench EB/AO Stain Fix EB/AO Eosin Eosin EB/AO Fluoroquench Eosin Eosin Fluoroquench Fluoroquench Fluoroquench Fluoroquench Fluoroquench Eosin EB Fluoroquench EB/AO
30 30 30 30 30
60 5 30
60 5 30
30
30
5 5
45
45
5 5
30 30
1 38 0.5
1 1 0.5
5 6 2
1 45
1 1
1 5
30
30 5
5
20
20
30 30 30
60 45 45 60 30 60 60 60 120 30
60 90 45 60 75 60 60 90 120 60 60 30
60 40
30
30
45 45
5 45 45
35 30
35 30
30
30
60 30 30 45 30 60 30 60 30 60 30 35 60 30 30
5 5
30 30
30 30
30 30
30 30
60 60
60 60
1 2
1 1 1 0.875 1 1 1 1 1 1 1 2
3 5 5 5 5 5 5 5 5 5 6 5
1 2
19 38
1
30 60 30 60 30 35 60 30 30
60 60 60 45 60 60 60 120 60 60 60 75 120 45 60
60 120 60 60 60 75 120 45 60
30 45 30
60 45 60
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
1 NT 1 1 4
1 NT 1 1 4
5 NT 3 5 5 5
15 NT 10 30 60 30
15 NT 10 60 60
45 NT 30 30 20
Fluoroquench NT Fluoroquench
1 1 0.5 1 1 1 1
6 5 2.5 5 5 5 5 5
45 30 30
90 NT 60 60 40 60 60 60 60 60 60 90 60 60
90 NT 60 60 40
1 (1/3 dilution), 1 and 2 90 0.5
45 NT 30 30 20 30 45 30 30 45 30 45 30 30
90 60 45
PI EB/AO Fluoroquench EB/AO Fluoroquench Eosin Fluoroquench Fluoroquench EB/AO
30 30 60 30 40 60 30 30
60 60 60 60 60 120 30 60
60 60 90 60 60 120 30 60
PI/AO Fluoroquench Eosin EB/AO EB/AO Eosin Fluoroquench Fluoroquench
30 30
60 50
60 50
Fluoroquench Fluoroquench
1/10 0.005M 10%
10
Yes
20%
1 2
Yes
0.1M
5 1
38 1 95 1
0.01% 1/2 1/10 0.1M
Servibio
Yes Yes Yes Yes Yes Yes Yes
1 1 3 1 50 1 1
1 1 27 19 450 19 19
1 1 1 1 2400 1 1
5 5 5 5 5 3 6
Servibio Servibio
Yes Yes
0.1M 1%
1 76
1
4
5 5
20 20
20
15 30
15 30
30 15
30 15
35 60 15 30
35 60 15 30
30 30 30 30 40 60 30 30
15
15
30 30
45 30 30
60 60 60
Extended incubation
Cells extracted with Rosettesep Extended incubation time: 60 and 120 minutes pre- & post-complement addition respectively.
Washes
AHG incubation during 30minutes
We prepared DDT solution 0.016g in 10ml RPMI. Then we incubated 1/1 DDT and sera 30'c. 37'c pre-complement addition and post complement addition times are 40'c
Fluoroquench Fluoroquench
1/20 NT 1 100 4
Yes
0.1M
30 45 30 45
30
Yes Yes
Biotest BAG Invitrogen Biorad BAG Innotrain Biorad Innotrain Life Technologies Gentrak Cedarlane Invitrogen
30 30 30 10
30 30 30 30
38 0.5 1 or 2 1 1 0.9 1 90 1 1 19 2
10 1
30 30 30 10
Modifications to the standard NIH CDCXM method
Visualisation of dead cells
Delay in sample delivery
AGH for T lymphocytes XM AHG treatment for T-cell XM AHG
EB - ethidium bromide AO - acridine orange PI - propidium iodide Hb - haemoglobin CFDA - carboxy-fluorescein diacetate
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