2015-05-06 Methods


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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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