This lecture includes information about ABO Incompatible titres ,its methos and how to interpret it and useful in clinical application.

1. Dr. Trupti Barot
Prathama Blood Centre
Ahmedabad

2. INTRODUCTION
 Organ transplant( live or deceased) is the therapy of
choice for a variety of end stage solid organ diseases
 Advances in HLA typing and cross-matching,
immunosuppressive medications & procedures for
patient management have dramatically improved
allograft & recipient outcome since the 1st renal
transplant performed > 50 yrs. ago

3.  Severe donor shortage limit this treatment.
 Immunologic barriers(ABO & DSA) considered as
absolute contraindications to transplantation are
being re-evaluated.
 UNOS wait list-107,337 patients for all organs
 >84,000 for renal transplant

4. GROWING NEED OF KIDNEY ALLOGRAFT!
 The prevalence of CKD is 17.2% & ~1.6% have CKD
stage 4-5 in India.
 Incidence of ESRD is 170,000/year
 Only 10,000 patient started on
Hemodialysis/Peritoneal dialysis and 5,000 renal
transplant/year
 Need of RT as this is the most effective treatment
of ESRD, being inherently most cost effective,
better pt survival and QLI in comparison to HD.
Singh et al. BMC Nephrology 2013, for SEEK study

5.  86,500 patients on the deceased donor waiting list
addition of ~28,000/ annually
 ~ 3,500 potential live donors are excluded from
donation each year as a result of blood group
incompatibility
 4,619 patients died while waiting - 12.6 deaths per
day
OPTN data as of May 6, 2011

6. STRATEGIES TO EXPAND DONOR POOL
 Extended criteria donor/marginal donor
 Kidney Paired Donation (KPD)
 Deceased donor Programme
 ABO incompatible protocols
 HLA Abs Desensitization protocols

7. ABO Blood Group System
Landsteiner’s Law Whichever ABO
antigens are lacking on a given person’s RBCs,
that person will always have the corresponding
antibody or isohemagglutinin

9.  ABO antigens are composed of sugar chains and
exist not only on red cells but also on many other
cells including endothelial cells and epithelial cells
of various organs such as kidney, heart, bowel,
lung, and pancreas (Marionneau et al., 2001)
 Blood group A consists of two subtypes, A1 (80%)
& A2
 Immunogenic risk A1>B>A2O recipients have a
higher incidence of ABMR

10. Development ofABOisoagglutinins
• In most infants, anti-A and anti-B agglutinins (presumably IgM)
produced by the infant canfirst bedemonstratedat 3–6 months.
• The titre of anti-A and anti-B agglutinins reaches its
maximumat the age of 5–10 years.
• It maybe wholly IgM or partlyIgM andpartlyIgG, partly IgM
and partly IgAor may be madeof all three immunoglobulins

11. MECHANISM OF HYPERACUTE REJECTION
(ABO-Incompatible Kidney Transplantation-
Mina Hur et al)

13. TITER MONITORING
 Baseline (pre-PP)
 Before/after each PP treatment
 Daily for first 2 weeks
 Weekly for next one month
 At 2, 3, 6, 12 months

14. Antibody Titration
 Titration is a semiquantitative method used to
determine the concentration of antibody in a given
sample
 The titer is the highest dilution of serum at which 1+
agglutination is observed macroscopically .
 The titer is reported as the reciprocal of the dilution
level (eg, 32—not 1 in 32 or 1:32).

15. Methods
• CTT
• CAT
(Fully automated & Semi
automated)
• SPRCA assays
• Flow Cytometry

16. Controversies in ABO
titration
Thepreferred method ?
Standardization ?
Endpoints ?
Critical titre levels(specially for
ABOi-KT) ?

17. Tubetechnique(TT)
It isthemostcommonlyperformedmethodinlaboratories.
Theroomtemperature(RT)incubationtechniqueand theindirect antiglobulin
test(IAT)have
beeninterpreted asthemethodsdetectingIgM and IgG, respectively.
Both IgM and IgG of ABO Ab can agglutinate RBCs at RT
(20-24°C) or below and efficiently activate thecomplement
at 37°C.
Thereforetiters usingRTtechniquesor IATondithiothreitol (DTT)untreated
samplesmaybe morereflective of themixedconcentrationof IgM and IgG
of ABO Ab.

18. Making Doubling Dilutions(Master Dilution)
100 ul serum
in tube 1 & 2
100ul saline in each tube
Mix and Transfer
Not to be Discarded
1.Label 10 test tubes according to the serum dilution (eg, 1 in 1, 1 in 2, etc).
2.Deliver one volume of saline to all test tubes except the first (undiluted, 1 in
1) tube.
3. Add an equal volume of serum to each of the first two tubes (undiluted and
1 in 2).
4. Using a clean pipette, mix the contents of the 1-in-2 dilution several times,
and transfer one volume into the next tube (the 1-in-4 dilution).
5. Continue the same process for all dilutions,

19. TubeAgglutinationGrading

20. Disadvantages
Variation in cell suspension
Cell lossduring washing
Alteration in cell:serum ratio
Inter-observer variation (1+ and wk+ are
subjective:End-point ??)
Stability of test results
Variation in repeat testing

21. Column agglutination technology(gel)
Conceptualizedby Lapierre (1985)
Principle: Controlled centrifugation of red cell with/ without serum
through a porous dextran or
polyacrylamide gel columnof defined pore sizeunderdefined setsof incubation.
Gel actsasa sievesothat unagglutinated cellssettle at bottom and cellsforming
lattice get trapped at
variouszonesacrossthecolumn.

22. Advantages
morequalitative in grading
the strength of agglutination
reaction
the inter-observer variation isminimal
less time-consuming
usessmaller volumesof serumand
RBCs
Limitation: COST
??
0

23. Microplatetechnique
MICROPLATES
Smalltray with 96 small wells
Holds200-300microlitres of reagent
Threetypes: V-type, flat-bottom, U-type
Advantage
More sensitive –very weak cell suspensioncanbe used
Very small amountof reagents are needed
Titrations are easier with multi channel pipettes
Grades of reaction canbe compared
Disadvantage: high viscosityin serum/plasma causesred
cellsto adhere to side
of wells

24. Solid phase redcelladherence assay(SPRCA)
Componentsof antigen-antibody reaction isimmobilized onto a solid medium.
On centrifugation antigen positive cells spread outwhile antigen negative
cells form a buttonat the
bottomof thewell.
Excessplasma is blotted out and anti IgG bound indicator red cells are added to
give visible reaction.
SPRCA:Available inautomated
platforms

25. SPRCA
(Contd.)
Antigen
coat
+ Testserumor plasma, incubation
at 37oC
Antibody attached
to RBC’santigen
Washto remove
unboundantibody
+Indicator RBC
PositiNegati
Indirect
test

26. Flowcytometry
 FLOW: Inmotion.
 CYTO: Pertainingto cells.
 METRY:Measurement.
 It isa technology that measuresvariousproperties of cells/particles of
interest ina samplebased on
markerspresentby passingthemin a fluid streamundera beamof light.
 It deals
with,
 Anyparticle that can be suspendedina fluid: cells,chromosomes, and
individual
molecules,
canbe characterized by flow cytometry.
IDENTIFICATION ANALYSIS
PURIFICATION (SORTING)
PHYSICO-CHEMICAL
PROPERTIES

28. Acceptable Variation
 The variation of no more than one standard dilution is
within acceptable limits for titration methods.
Roback JD, Combs MR, Grossman BJ, Hillyer CD. Technical manual.
16th ed. Bethesda (MD): American Association of Blood Banks; 2008.
 CAP: Variation upto two dilutions on either side of the
mean value is acceptable

29. Is Reporting of Both IgG & IgM titers essential ??
US working Group recommends Both
(Montogomery et al 2004)
IgG Titers decide :-
 Patient eligibility,
 Rejection risks &
 TPE Protocols

30. What is the best way to minimize such
Titer Variations??
End Point Titer is always 1+ though the corresponding
Dilution/Titer For the same sample May Vary Depending
on technique used

31. Desensitization
Immunosuppression
To reduce Formation
of New Antibodies
O
Definition
Treatment protocol to
eliminate or reduce
antibodies to Donor HLA
and/or ABO antigens to
permit successful
Transplant & prevent AMR
– No Optimal Protocol
– Varies From Centre To Centre
r reduce antibodies to Donor
HLA and/or ABO antigens to
permit successful Transplant &
prevent AMR
EART
To Remove
Existing
Antibodies
Modalities
Plasmapheresis
1. Conventional (Non Specific)
2. Cascade(Semi Selective)
3. Immunoadsorption (Most
specific)

32. ANTIBODY DEPLETION:PLASMAPHERESIS
 The simplest and most common method to remove
antibody
 Eliminates approximately 20% of the anti-ABO
antibodies
 Not sufficiently selective-removes volume and plasma
proteins including coagulation factors
 Stegall et al. recommended initiating PPx if titer increases
to 1 : 16 in the first two weeks after transplantation.
 Gloor et al. showed that humoral rejection was rare
when the antibody level was maintained less than 1 : 8 in
the first week and 1 : 16 in the second week after
transplantation.
G. Tyd´en, G. Kumlien, and M. Efvergren, “Present techniques for
antibody removal,” Transplantation, vol. 84, supplement12, pp. S27–
S29, 2007

33. DOUBLE FILTRATION PLASMAPHERESIS
(ABO-Incompatible Kidney
Transplantation-

34. Technology Conventional Plasma
Exchange
Cascade Filtration &
DFFP
Immunoadsorption
Column
Constituent
Removal
Removes plasma along with non
selective removal of constituents
Semi-Selective depletion of
plasma constituents based on
the molecular size
Highly Selective
Immunoadsorption of ABO
antibodies
Substitution
Fluid
Required (Albumin/FFP/Saline) Reduced No requirement
Monitoring
(Coagulation
profile)
Required Not required Not required
Advantage
Simultaneously remove different
anti-bodies such as ABO antibodies,
HLA antibodies, and vascular
endothelial cell antibodies.
Pore Size based filtration of
immunoglobulins
Specific adsorption of Anti A &
Anti B
Least expensive There Is reduced Need For
Replacement Fluids, Lower
Risk Of Contamination And
Allergic Reactions
Greater Anti A/B Titer
reduction per treatment
session there by offsetting the
High cost

35. TRANSFUSION THERAPY
RBCs:
 ABO identical with recipient
 Passenger lymphocyte syndrome: Group O
Plasma:
Compatible with recipient RBC & donor kidney
 A/B/AB  O AB or donor type
 AB  A/B ---AB
 A B or vice versa -AB
Platelets: express ABO ag & plasma has ABO ab
 O group with reduced plasma
 Recipient type with reduced plasma

36. CHOICE OF BLOOD PRODUCT
• CMV seronegative patients receiving seronegative
organs should receive CMV safe blood products
(leukoreduced, or from CMV seronegative donors)
•TA-GVHD is rare despite immune suppression,
routine irradiation is not considered necessary
•Products should be leukoreduced

37. Pros ABOi-KT Cons ABOi-KT
 Reducing waiting list time
 Expanding living donor pool
 Improvement of patient's
prognosis
 Excellent graft survival
(comparable with ABOc-KT)
 Higher immunological risk
 Higher incidence of acute AMR
 Intensified immunosuppression
 Antibody depletion therapy
 Increasing expenditure
 Higher incidence of viral
infection

38. What is the cut off Titer for entry into ABOi
Kidney Transplant Program ??
1:256/1:512/1:1024
Very High Anti ABO Antibody titers are difficult to desensitize

39. What if the antibody Removal Fails Despite
Extensive TPE & Immunosuppressants?
Kidney Paired Donor Exchange Program

40. What should be the choice of Induction &
Maintenance Immunosuppressive agents in a
given Transplant??

41. What is the likely number of TPE procedures for
any given IgG titer??

42. Conclusion
Interlaboratoryvariationsinthetechnicalprocedures
and resultsdooccurinmeasurementof theABOAbtiter.
CATsignificantlydecreasesvariationascomparedtothetube test.
A periodically conducted assessment could help in
continued improvement of the resultsof
ABOAbtitermeasurement.

43. “Challenges are what make life
interesting;
Overcoming them is what makes life
meaningful.”
Joshua J. Marine
Thank You