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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