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WEAK AS!A review of weakly

reacting antibodies in RCPA Transfusion QAP

Surveys

Mr Tony GreenfieldNeville Hermann - RCPA

Transfusion QAPCathy Hughes – RCPA Transfusion

QAP

Transfusion Weak As!

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Significance and Antibody Detection Techniques

• Failure to detect weakly reactive allo‐antibodies can lead to DHTR due to a rapid rise in antibody production following transfusion of incompatible red cells.

• Antibody detection techniques each vary in their ability to detect weak antibodies and no technique will detect all clinically significant antibodies.

3Transfusion Weak As!

Analysis of AHG methods2009 – 2012

Transfusion Weak As! 4

AHG Crossmatchmethods ‐ analysis by method group 

Tube CAT‐Gel CAT‐GlassBead No method indicated

2009‐1 30 (9%) 206 (59%) 64 (18%) 51 (15%)

2010‐3 20 (6%) 180 (49%) 64 (18%) 102 (28%)

2011‐1 22 (6%) 182 (52%) 67 (19%) 78 (22%)

2012‐2 21 (6%) 186 (52%) 56 (16%) 94 (26%)

2009 2011 2012

Tube CAT Gel CAT Glass Not Specified

2010

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RCPA QAP Transfusion Surveys 2010 - 2012

2010 2011 2012

1 Anti‐Fya+S 1  Anti‐D +K 1  Anti‐C+D

2 Anti‐D 2  Anti‐Fya 2  Anti‐E+K

3 Anti‐c+K 3  Anti‐E+Fya+Cw 3  Anti‐c

4  Anti‐e+C 4  Anti‐D 4  Anti‐K

5  Anti‐C+D 5  Anti‐ A1

6  Anti‐Fya 6  Anti‐Fyb

Transfusion Weak As! 5

Analysis of 2010-3 Crossmatch Reaction –

Anti-c

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AHG Crossmatch Reaction Strength Unit 2 (c+)Reaction Strength 0 Weak 1+ 2+ 3+ 4+No. of 

Participants 8 17 113 182 28 0

0

20

40

60

80

100

120

140

160

180

200

0 Weak 1+ 2+ 3+ 4+

No. of Participants

Transfusion Weak As!

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Analysis of 2010 3 Crossmatch Reaction –

Anti-cDonor 2 (c+)

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Breakdown of Unit 2 AHG reaction strengths by methodReaction Strength 0 Weak 1+ 2+ 3+

Tube % 10% 5% 35% 45% 5%CAT‐Gel % 0.5% 1% 29% 59% 10%

CAT‐GlassBead % 1.5% 17% 47% 31% 3%

0

10

20

30

40

50

60

70

0 Weak 1+ 2+ 3+

Tube %

CAT‐Gel %

CAT‐GlassBead %

Transfusion Weak As!

Analysis of 2010-3 Negative AHG Crossmatch - anti-c

Donor 2 (c+)

• Unit 2 – 8 labs found this unit compatible 

8Transfusion Weak As!

Method Tube CAT‐Gel CAT‐Glass Bead No Method

Participants 2 1 1 4

% refers to number of negative results compared to the total number of users in that method group

Participants % 10% 0.5% 1.5% 5%

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Analysis of 2010-6 Crossmatch Reaction –

Anti-Fya

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AHG Crossmatch Reaction Strength Unit 1 Fy(a+b‐)

Reaction Strength 0 Weak 1+ 2+ 3+ 4+

No. of Participants 6 1 41 214 72 8

0

50

100

150

200

250

0 Weak 1+ 2+ 3+ 4+

No. of Participants

Transfusion Weak As!

Analysis of 2010-6 Crossmatch Reaction –

Anti-FyaDonor 1 Fy(a+b-)

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Breakdown of unit 1 reaction strengths by methodReaction Strength 0 Weak 1+ 2+ 3+ 4+

Tube % 8% 0 12% 48% 28% 4%CAT‐Gel % 2% 1% 13% 59% 19% 4%

CAT‐GlassBead % 0 0 3% 72% 24% 1%

0

10

20

30

40

50

60

70

80

0 Weak 1+ 2+ 3+ 4+

Tube %

CAT‐Gel %

CAT‐GlassBead %

Transfusion Weak As!

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Analysis of 2010-6 Negative AHG Crossmatch - anti-Fya

Donor 1 Fy(a+b-)

• Unit 1 – 6 labs found this unit compatible 

11Transfusion Weak As!

Method Tube CAT‐Gel CAT‐Glass Bead No Method

Participants 2 4 0 0

% refers to number of negative results compared to the total number of users in that method group

Participants % 8% 2%

Analysis of 2010-6 Crossmatch Reaction –

Anti-Fya

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Reaction Strength 0 Weak 1+ 2+ 3+ 4+No. of 

Participants 7 3 62 196 69 5

0

50

100

150

200

250

0 Weak 1+ 2+ 3+ 4+

No. of Participants

Transfusion Weak As!

AHG Crossmatch Reaction Strength Unit 3 Fy(a+b+)

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Analysis of 2010-6 Crossmatch Reaction –

Anti-FyaDonor 3 Fy(a+b+)

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0

10

20

30

40

50

60

70

80

0 Weak 1+ 2+ 3+ 4+

Tube %

CAT‐Gel %

CAT‐GlassBead %

Breakdown of unit 3 reaction strengths by method Reaction Strength 0 Weak 1+ 2+ 3+ 4+

Tube % 8% 0 20% 48% 24% 0CAT‐Gel % 2% 2% 22% 56% 15% 2%

CAT‐GlassBead % 0 0 9% 68% 24% 0

Transfusion Weak As!

Analysis of 2010-6 Negative AHG Crossmatch - anti-Fya

Donor 3 Fy(a+b+)

• Unit 3 – 7 labs found this unit compatible 

14Transfusion Weak As!

Method Tube CAT‐Gel CAT‐Glass Bead No Method

Participants 2 4 0 1

% refers to number of negative results compared to the total number of users in that method group

Participants % 8% 2%

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Analysis of 2011-1 Crossmatch Reaction -

Anti-D• Patient with anti‐K (strong) and anti‐D (weak)

AHG Crossmatch Reaction Strength Unit 2 (D+ K‐)

Reaction Strength Neg Weak 1+ 2+ 3+ 4+

No. of Participants 58 133 76 24 3 1

0

20

40

60

80

100

120

140

Neg Weak 1+ 2+ 3+ 4+

No. of Participants

Transfusion Weak As!

Analysis of 2011-1 Crossmatch Reaction –

Anti-D Donor 2 D+ K-

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Breakdown of unit 2 reaction strengths by method Reaction Strength 0 Weak 1+ 2+ 3+ 4+

Tube % 54% 16% 16% 10% 5% 0%CAT‐Gel % 18% 50% 25% 7% 0% 1%

CAT‐GlassBead % 12% 28% 35% 20% 4% 0%

Transfusion Weak As!

0%

10%

20%

30%

40%

50%

60%

0 Weak 1+ 2+ 3+ 4+

Tube %

CAT‐Gel %

CAT‐GlassBead %

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Analysis of 2011-1 Negative AHG Crossmatch - anti-D

Unit 2 (D+K-)

• Unit 2 – 58 labs found this unit compatible 

17Transfusion Weak As!

Method Tube CAT‐Gel CAT‐Glass Bead No Method

Participants 10 27 6 14

% refers to number of negative results compared to the total number of users in that method group

Participants % 54% 18% 12% 15%

Analysis of 2012-2 Crossmatch Reaction -

Anti-EAHG Crossmatch Reaction Strength Unit 4 (E+ K‐)

Reaction Strength Neg Weak 1+ 2+ 3+ 4+

No. of Participants 20 60 134 97 12 1

Transfusion Weak As!

0

20

40

60

80

100

120

140

160

Neg Weak 1+ 2+ 3+ 4+

No. of Participants

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Analysis of 2012 2 Crossmatch Reaction –

Anti-EDonor 4 E+K-

Transfusion 19

Breakdown of unit 4 reaction strengths by method Method 0 weak 1+ 2+ 3+ 4+Tube 18% 14% 41% 27% 0 0

CAT‐Gel 5% 17% 29% 44% 4% 0CAT‐Glass Bead 0% 22% 49% 22% 6% 0

Transfusion Weak As!

0

10

20

30

40

50

60

0 weak 1+ 2+ 3+ 4+

Tube

CAT ‐ Gel

CAT‐Glass Bead

2012 2 Analysis of negative

crossmatch reaction –anti-E

Unit 4 (E+K-)• Unit 4 – 20 labs found this E+ unit compatible 

20Transfusion Weak As!

Method Tube CAT‐Gel CAT‐Glass Bead No Method

Participants 4  9  0 7

Participants % 18% 5% *

% refers to the number of negative results compared to the total number of users in that method group* The ‘No method’ includes labs that excluded the unit by phenotype or ‘Deselected’ the unit from crossmatching.

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Analysis of 2012-2 Crossmatch Reaction

Strong Anti-K

Transfusion 21

AHG Crossmatch Reaction Strength Unit 1 (E‐ K+)Reaction Strength Neg Weak 1+ 2+ 3+ 4+

No. of Participants 2 3 1 45 231 43

0

50

100

150

200

250

Neg Weak 1+ 2+ 3+ 4+

No. of Participants

Weak As!

Analysis of a 2012-2 Strong Crossmatch Reaction

Strong anti-KDonor 1 E- K+

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Breakdown of unit 1 reaction strengths by method Method 0 weak 1+ 2+ 3+ 4+Tube 4% 0 0 26% 57% 13%

CAT‐Gel 0.5% 1% 0.5% 17% 73% 7%

CAT‐Glass Bead 0 0 0 2% 69% 29%

Transfusion Weak As!

0

20

40

60

80

0 weak 1+ 2+ 3+ 4+

Tube

CAT ‐ Gel

CAT‐Glass Bead

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Total of all WEAK AS survey failures

Transfusion 23

2010‐3, 2010‐6, 2011‐1, 2012‐2Tube CAT Gel CAT Glass 

Bead

Number false negatives 20 45 7

% of method users 18% 4.8% 2.3%

Proportion of method users 1 in 5 1 in 21 1 in 43

Weak As!

Factors leading to decreased detection of antibodies

Incubation Time, Temperature, pH, Ab: Ag Ratio, Ionic Strength• All these variables can affect sensitivity of test systems• Follow the published method using correct diluents, reagents  

and correctly calibrated equipmentScreening Cell reactivity• Variable antigen strength, deteriorate with improper storage, 

poorly resuspended, wrong concentration or wrong diluent, not at correct temperature. 

• Evaluate reagent screening cells, perform pre‐acceptance testing and regular QC, make sure they are correctly stored, brought to correct temperature and are adequately resuspended before use.

24Transfusion Weak As!

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Factors leading to decreased detection of antibodies

AHG  & LISS Reagent Reactivity• Variable quality, deterioration with improper transportation & 

storage, not at correct operating temperature• Evaluate and perform pre‐acceptance testing and regular QC, 

make sure correctly stored and brought to correct temperature before use

Pipetting Technique• CAT Gel must have air bubble separating plasma/cells from gel• Correct volumes must be dispensed• Pipettes must be checked to ensure correct volumes are 

dispensed

25Transfusion Weak As!

Factors leading to decreased detection of antibodies Wash efficiency

• Tube techniques must wash cells free of plasma to prevent neutralisation of AHG reagent, and leave enough red cells  to be able to interpret agglutination.

• pH of tube wash solution – use buffered saline pH7• Use sensitized cells  to confirm all negative AHG tubesAHG Reagent sensitivityVariable quality, deteriorate with improper transportation & storage, 

not at correct operating temperatureEvaluate and perform pre‐acceptance testing and regular QC, make 

sure correctly stored and brought to correct temperature before useReading• Incorrect centrifugation of cards or tubes • Overaggitation of tubes• Poor visualisation of agglutination• High level of training is required for tube techniques

26Transfusion Weak As!

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Possible Aids to Improve Staff Performance

• Staff Competency Assessment• Individual Surveys• Replicate Testing• Regular Practice

27Transfusion Weak As!

The End

28Transfusion Weak As!