Antibody titrations: To Pool or Not to Pool - blood · Antibody titrations: To Pool or Not to Pool...

Antibody titrations:

To Pool or Not to Pool

Anne-Marie Wilkes St Vincent’s Hospital Melbourne NICE Albury 2014

Day/Month/Year Footnote to go here Page 2

What am I talking about?

Antibody Titrations

- What are they?

- Why are they done?

- How are they done?

What are titrations?

Day/Month/Year Footnote to go here Page 3

• A semi-quantitative test performed in the laboratory

• A test performed on clinically significant antibodies

detected during pregnancy

• Performed on renal transplant recipients having an

ABO incompatible kidney

Why are they done?

Day/Month/Year Footnote to go here Page 4

In pregnancy:

• Antibody titrations are performed to identify and monitor the

concentration of antibody in an allo-immunised women

• Clinically significant antibodies have the potential to cause haemolytic

disease of the fetus and newborn (HDFN)

• A titre or quantitation of the antibody should be determined by a

standardised technique and repeated throughout the pregnancy.

• When a clinically significant rise in the titre/quantitation occurs, the results

of antibody monitoring aid the clinician in determining when to initiate fetal

monitoring such as ultrasound, amniocentesis, acolor doppler

ultrasonography or cordocentesis.

How are they done?

Day/Month/Year Footnote to go here Page 5

Dilution Neat 1:2 1:4 1:8 1:16 1:32 1:64

Result 4+ 3+ 2+ 1+ +/- 0 0

Titre=8

•Serial two-fold dilutions of patient plasma are prepared and

tested against red cells of known phenotype.

• The reciprocal of the highest dilution of plasma that gives a

1+ reaction is referred to as the titre ie 1 in 8 dilution; Titre = 8

Influences?

There are many factors which can

influence the reported titre:

Day/Month/Year Footnote to go here Page 6

• Choice of diluent

• Age, concentration and phenotype of red cell

• Time and temperature of incubation

• Time and force of centrifugation

• Pipetting technique

• Test method (CAT or Tube).

America: AABB Technical Manual

Day/Month/Year Footnote to go here Page 7

• Test in parallel with the most recent sample

• Diluent: isotonic saline

• Cells: Group O 2%, pool of donors of same phenotype

• Prepared using 500uL volumes

• Test 100uL dilution, 100uL 2% cells or 1 drop 3-4% cell suspension

• Saline AHG 60min incubation 37C and anti-IgG AHG

• Endpoint: Score 1+

•1000x g for 15s

• The technical manual also states:

The selection of the most suitable phenotype of red cells to use is

controversial. Many select red cells from heterozygous donors to mimic

the red cell of the foetus. Many use red cells from homozygous donors for

strongest antigen expression and increased sensitivity.

British:

Day/Month/Year Footnote to go here Page 8

• Guideline for Blood Grouping and Antibody Testing in

Pregnancy (2008):

- Where an antibody has been detected, testing should

include titration and testing by IAT against reagent cells

heterozygous for the corresponding antigen.

- Careful attention to technique is necessary to minimise the

variables in the method and titrating the national anti-D

standard [NIBSC,2003] in parallel, as an internal control (to

be within one doubling dilution) is recommended.

Day/Month/Year Footnote to go here Page 9

ANZSBT Recomendation- NICE

method

• The current ANZSBT guidelines (March 2007) suggest use of the ‘NICE’ method.

• Testing the previously frozen sample in parallel. Minimising the possibility that

changes in the titre result from differences in technique and reagent red cell

selection

• For years this resulted in a standardised method across Australia and NZ.

So what are Australians doing?

Results from the latest RCPA QAP Antibody titre module and

from Rhonda’s survey and presentation in 2011. (Comparable)

Day/Month/Year Footnote to go here Page 10

• Diluent: 5% Albumin in PBS (38%), PBS (19%), Albumin 6% (?), 4% (?),

Bio-Rad ID-Diluent (5%), 3% Albumin (3%), In-house (1%)

• Red cell phenotype: Homozygous (73%), Heterozygous(10%),

Homo/Hetero Pool (7%)

•Test method: Tube (64%), CAT Gel (24%), CAT Glass Beads (11%),

Microtitre plate (1%)

• Cells: Only 1 cell (57%), Pool of 3 cells (33%) , Pool of 2 cells(10%)

Only 26% follow the NICE method

57 % use only 1 cell

Day/Month/Year Footnote to go here Page 11

Both the ANZSBT and AABB recommend using a pool of donor cells

Why?

• There is variation in the number of antigen sites amongst donors of

even the same phenotype

Why do many laboratories only use 1 cell?

• It’s easier

• Commercially prepared cells are more convenient to use and sourcing

2 or 3 homozygous cells may be challenging.

Our story

Day/Month/Year Footnote to go here Page 12

• St Vincent’s followed the NICE Method with one exception, they only

used 1 cell too.

• In July 2014 we moved from performing titrations by tube to a partially

automated method.

• Performing titres on the automated analyser enabled minimisation of

manual pipetting, allowed for standardised reading, scoring and

determination of the antibody titre endpoint.

Method:

Day/Month/Year Footnote to go here Page 13

• Serial two-fold dilutions continued to be prepared according to the

NICE method and the dilutions then programmed as a “crossmatch” on

our Diamed Gel Station and IH-1000.

Red cells

Day/Month/Year Footnote to go here Page 14

• Cross-matches can only be performed using packed

donor cells.

• Preparing and phenotyping cells is labour intensive and

open to error.

• Currently there is limited phenotyped packed cells

commercially available.

• St Vincent’s had previously only used one cell for titrations

and this process continued with the new method.

Day/Month/Year Footnote to go here Page 15

Print-out from analyser

Day/Month/Year Footnote to go here Page 16

Patient AA, June 2014, 10/40

Day/Month/Year Footnote to go here Page 17

B Rh D (Pos)

R1r K-

11 cell panel: anti-E

Day/Month/Year Footnote to go here Page 18

Bromelin

LISS/

COOMBS

Titre June 2014

Tube Method: Titre=<2

Day/Month/Year Footnote to go here Page 19

N 2 4 8 16 32 64

1+ 0 0 0 0 0 0

August 2014

Day/Month/Year Footnote to go here Page 20

Performed on Gel Station: Titre <2

Neat 1:2 1:4 1:81 1:16

September 2014

Day/Month/Year Footnote to go here Page 21

Titre = 8

August in parallel, also Titre 8

Both specimens with a different cell

Day/Month/Year Footnote to go here Page 22

Titre = <2

October

Day/Month/Year Footnote to go here Page 23

Titre=8

Antibody Screens

Day/Month/Year Footnote to go here Page 24

19/08/14 27/06/14 23/09/14

21/10/14

11 cell panel

Day/Month/Year Footnote to go here Page 25