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UK NEQAS for H&I Scheme 2A - Cytotoxic Crossmatching PBL/T-CELL AND B-CELL CROSSMATCHING METHOD OF SAMPLE 2A05+2A06/2015 DESPATCHED ON 19TH MAY 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 62 101 112 114 116 117 119 120 126 128 136 139 143 145 147 149 154 157 159 162 163 169 174 181 186 189 190 193 194 195 197 204 206 209 210 212 216 223 227 229 232 235 238 243 245 252 259 260 262 268 284 293 294 297 301 311 315
20-May 20-May 20-May 20-May 20-May 20-May 20-May 20-May 20-May 20-May 20-May 20-May 20-May 20-May 20-May 20-May
21-May 21-May 20-May 20-May 20-May 22-May 20-May 21-May 21-May 20-May 20-May 21-May 20-May 20-May 20-May 21-May
20-May 20-May 20-May 20-May 20-May 28-May 20-May 20-May 21-May 20-May 20-May 20-May 20-May 20-May 21-May 22-May 20-May 20-May 20-May 20-May 20-May 20-May 20-May 20-May 20-May
21-May 21-May 20-May 20-May 21-May NT 21-May 21-May 21-May 21-May 21-May 21-May 21-May 21-May 22-May 22-May 20-May 21-May 21-May 21-May 21-May 21-May 20-May 21-May 21-May
20-May 20-May 20-May 20-May 20-May 20-May
21-May 20-May 21-May 21-May 20-May 21-May
21-May 20-May 20-May 20-May 21-May
22-May 20-May 21-May 20-May 21-May
21-May 20-May 20-May 21-May 22-May 20-May 20-May 20-May 19-May 21-May 25-May 21-May 20-May 20-May 20-May 21-May 20-May 20-May 21-May 20-May 20-May
21-May 22-May 29-May 21-May 25-May 20-May 29-May 21-May 20-May 21-May 25-May 21-May 20-May 21-May 21-May 21-May 21-May 22-May 20-May 21-May
PBL
Immunobeaded Cells used cells used? T-cells B-cells T-cells B-cells 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
Cedarlane Cedarlane Cedarlane Cedarlane TCS Biosciences Cedarlane Cedarlane Cedarlane Cedarlane Cedarlane Cedarlane Cedarlane Cedarlane Cedarlane Cedarlane TCS Biosciences
0.01M 0.01M 10% 0.02M 0.01M
Yes Yes Yes Yes Yes Yes Yes Yes
0.05 0.01M 0.01M 0.1M 0.1M 0.1M 0.1M
Incubation Times DTT Pre Comp Post Comp Comp T-cell B-cell T-cell B-cell T-cell B-cell
1 1 0.1 1 1 0 4 * 1 1 10 0.1 0.1 2
1 1 0.9 2 1 1 36 1 1 1 70 0.9 1 36 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 5 5
1 NA 1 NA 5
1 1 2 NA 45
1 1 1 NA
5 5 5 NA
5
Yes
Yes Yes
Yes Yes
Yes
Yes
Yes
Yes Yes
0.05M 0.05M
0.1 4
1 36
1
Yes Yes
Yes Yes
Yes Yes
0.1M
1 2
1 38 20
1 1 10
5 6 20
1 45
1 1
5 5
Yes Yes
One Lambda Cedarlane Pel-Freeze Biorad Servibio Cedarlane One Lambda In-House Cedarlane Cedarlane Invitrogen In-House
Yes Yes Yes Yes Yes
Cedarlane Cedarlane In-House Cedarlane Cedarlane One Lambda
Yes Yes Yes Yes Yes Yes
Cedarlane Invitrogen Innotrain Cedarlane Cedarlane In-House
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
Volumes Sera Cells
Yes NA Yes NA Yes
Yes
Yes
DTT
Cedarlane Cedarlane Cedarlane Cedarlane
Yes
Yes
DTT Concentration
Yes Yes Yes Yes Yes
Yes
Yes Yes Yes Yes Yes Yes
DTT addition Sera Cells
Yes Yes Yes Yes Yes
Yes Yes
Yes
Complement manufacturer
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
NA NA
0.003M NA
Yes
5
Yes Yes
2 0.5
NA 45 NA
30 5
10
1 1 1 1 1 1 1 1
3 5 5 5 5 5 5 5
1 2 1 2 5
1 19 2 19 38 95
1 1 2 1 1
5 6 5 5 5 6
0.01M 0.015M
1/20 0.5 1 100
1 0.5 1 1
1 0.5 1 1
5 3.3 3 5
30
1/10
1
1
1
5
30
95 90 0.5
1 0.5
Yes
20%
1 2
Yes
0.1M
38 1 0.95 1
1 1 1 1
6 5 2.5 5 5 5 5 5
15
1/10
5 10
1 1 27 19 450 19 19 1 1 76
1 1 1 1 2400 1 1 1 1
Yes Yes Yes Yes
Yes
0.001M 0.05M 0.01M 5% 5mM
Yes Yes
0.005M 5mM Yes 0.1mg/L
One Lambda Cedarlane Cedarlane One Lambda
Yes Yes
1/20
Biorad Biorad Immucor
Yes
Yes Yes Yes
Yes Yes
Yes Yes Yes Yes Yes Yes Yes
1/2 1/10
0.1M Yes
Yes Yes
1M 0.1M 1%
10 0.1
0.05 1 1 1 3 1 50 1 1 1 4
30 30 10 30 30 NA 30 NA 30 30 NA 5 30
NA 45 NA
30
38 0.5 1 or 2 1 1 0.9 1 90
Yes Yes Yes
Biotest Biorad Invitrogen Biorad BAG Innotrain Biorad Innotrain Life Technologies Gentrak Cedarlane Invitrogen Pel-Freeze Biochem Koninchen Servibio Servibio
30 30 10 30 30 NA 30 NA 30 30 10 NA 5 30
5 5 5 5 5 3 6 5 5 5
30 30
5
30
30 45 30 45 60 30 40 45 45 30 30 45 60 45 60 60
30 90 60 75 60 60 40 90 60 45 90
60 45 45 60
30 90 90 75 60 60 40 90 60 45 90 90 60 90 60 120
60 90 45 60
EB/AO Fluoroquench EB/AO EB/AO EB/AO EB EB/AO Fluoroquench Fluoroquench EB/AO EB/AO Fluoroquench EB/AO EB/AO EB/AO EB/AO
60 60 60 60 30
60 60 60 60 30
60 60 90 120 60
60 60 90 120 60
Fluoroquench PI/AO Fluoroquench EB/AO Fluoroquench
30 20 60 30 30 30
30
60 60 120 60 60 30
60
60 30 30 45 30 60 30 60 30 60 30 35
45 45 30 35
5 45 45 30 75
30 40 30
30 40 30
15
15
30 30 40 30 30 30
15 35
15 35 10 60
30 45 30 30 30
15
20
15 30
15 30
30
30
45 35 60 15 30 30
45 35 60 15 30 30
15
15
30 45 30 30 45 30 45 30 30 30 30 30 30 40 60 30 30 30 30 30
30 45 30 45 60 30 40 45 45 30 30
30 60 30 60 30 75
60 60 60 45 60 60 60 120 60 60 60 35
60 120 60 60 60 75
Fluoroquench EB/AO Fluoroquench Eosin Fluoroquench 7AAD Stain Fix EB/AO Eosin Eosin EB/AO Fluoroquench Eosin Eosin Fluoroquench Fluoroquench Fluoroquench Fluoroquench Fluoroquench
30 30 40 30 30 30
45 60 40 60 60 45
45 60 40 60 60 45
EB/AO Fluoroquench EB/AO Fluoroquench Stainfix Fluoroquench
30 45 30 30 30
60 90 45 60 60
60 90 45 60 60
Fluoroquench Fluoroquench Fluoroquench Fluoroquench
30 30
30 45 30
45 30 30
60 60 60 60 60 60 90 60 60
30 30 60 30 40 60 30 30 30 30 30
60 60 60 60 60 120 30 60 60 60 50
45 30 30
60 30
60 45 60
90 60 45
PI EB/AO Fluoroquench EB/AO Fluoroquench Eosin Fluoroquench Fluoroquench EB/AO
60 60 90 60 60 120 30 60 60 60 50
PI/AO Fluoroquench Eosin EB/AO EB/AO Eosin Fluoroquench Fluoroquench EB/AO Fluoroquench Fluoroquench
60 60 60
Modifications to the standard NIH CDCXM method
Visualisation of dead cells
* 81µl of patient sera treated with 9µl 0.1M DTT -> mixed -> add 1µl of treated sera on tray -> incubate @ 37°C for 30 min prior to adding cells.
* cells are resuspended in 10%DTT (in RPMI) solution and added to sera.
Extended incubation time: 60 and 120 minutes pre- & post-complement addition respectively. AHG Flow CDC (FCTOX)
Washes
AHG
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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