Algorithms in Immunohaematology

Dolly Daniel, Dept of Transfusion Medicine, CMC, Vellore

Patient AXX

Patient ID

Anti A Anti B Anti D A cells B cells O cells Auto O pooled Group

AXX 4+ 4+ 4+ 3+ 3+ 3+ 3+ 2+ Uninterpretable

Request for 2 units of red cells – 28 year old female, with a Hb of 4.5 gm%

Patient AXX

• 28 year old female

• H/O fever and joint pains.

• 6 months ago transfused 3 units for severe anaemia Hb 4 gm% – detected during a medical check

• Started on steroids – Referred for a work up

• Now feels unwell – anaemic again Hb4.5 gm%

• T C and DC – normal. Platelets 50,000/cmm

• Peripheral smear – autoagglutination/ spherocytes

Patient BXX

Patient ID

Anti A Anti B Anti D A cells B cells O cells Auto O pooled Group

BXX 4+ - 4+ 3+ 3+ 3+ 3+ 3+ Uninterpretable

35 year old female – Hb 10.5 gm%, referred for a work up

Patient BXX

• 35 year old female

• H/O fever and joint pains – few months. Some skin lesions

• Hb 10.5gm% TC DC – normal. Platelets 100000/cmm - referred

• Now comes for a complete work up

• Hb 10.5 gm%

• T C and DC – normal. Platelets 100,000/cmm

• Peripheral smear – mild autoagglutination / spherocytes

Variability??

28 year old – 3rd pregnancy

• B Rh Negative , Antibody screen and ID positive with Anti C (from 24 weeks)

• Baby born at 37 weeks

• B Positive – DCT 3 +

• Hb – 9.5gm%, Bilirubin- 18 mg% (indirect predominantly)

• Increased to 25 – exchange transfusion required

25 year old - 2nd pregnancy

• B Rh Negative, Antibody screen and ID positive with anti C (from 24 weeks)

• Delivers a baby at 37 weeks

• B Positive, DCT 3+

• Hb - 15.8 gm%, Mild indirect hyperbilirubinemia

• Phototherapy 2 days – normalised - discharged

Patient ID

Anti A

Anti B Anti D

A cells

B cells O cells

Auto O pooled Group

AXX 4+ 4+ 4+ 3+ 3+ 3+ 3+ 2+ Uninterpretable

Patient ID

Anti A

Anti B

Anti D

A cells

B cells

O cells

Auto O pooled

Group

BXX 4+ - 4+ 3+ 3+ 3+ 3+ 3+ Uninterpretable

Scenarios

What combination of tests are best for

• defining clinical significance of antibodies?

• accurate diagnosis

• Providing info for clinical decision making?

• providing info for guiding therapy?

• providing info for prognostication / referral needs?

Antibodies

Auto

Allo

Mix

Cold TherapyWarm

AIHA

Clinicalcourse

Mixed

DAT pos

Clinicalseverity

DAT Neg

observations concerning the number of IgG molecules per red blood cell

DAT

Red cell

Which is clinically

significant???

Manual DAT - Pos if 100-500 molecules of IgG/red cell and 400-1100 molecules of C3d/red cell present

More sensitive - Important - “DAT negative AIHA”, try techniques with greater sensitivity - flow cytometry (FC) / gel cards / Enzymes

Chaudhary R, Das SS, Gupta R, Khetan D. Application of flow cytometry in detection of red-cell-bound IgG in Coombs-negative AIHA. Hematology. 2006;11:295–300

I am DAT negativeNumbers and

the ability to be picked up

by DAT

DestructionWith low no of ab

Low affinity ab – washing

Other ab spec– not picked up by DAT

Suspected Cold agglutinin

• Warm Collection

• Repeat grouping

• May resolve

• Cold agglutinin titre

• May have a mix of warm and cold auto antibodies

• Antibodies with a wide thermal amplitude – worse prognosis

Cold agglutinin disease Paul L. Swiecicki, Livia T. Hegerova and Morie A. Gertz Blood 2013 122:1114-1121;

Paroxysmal Cold Haemoglobinuria

• Donath-Landsteiner antibody

• Biphasic - that fixes complement at low temperature but dissociates at a higher temperature

• DAT – anti C3 usually

• If DAT at lower temperature – IgG can be picked up

• Cause lysis at a temperature nearer normal body temperature.

Strength of DAT

M. Lai, G. Leone, and R. Landolfi, “Autoimmune hemolytic anemia with gel-based immunohematology tests,” American Journal of Clinical Pathology, vol. 139, no. 4, pp. 457–463, 2013

Strength of DAT – a study of 94 DAT positive patients with AIHA

P < 0.006

Trend of this correlation continues even in the non IgG1 / IgG3 group

DAT

IgG

Responsible for haemolysis

Ab too far apart to activate

complement

C3

Could be IgM/ IgA

Could be low affinity IgG

Both

IgG – fixing complement

Destruction potent

Monospecific DAT – clinically very important –treatment options

Anti-IgG Anti-C3 Occurrence Antibody Antigen Comment

+ - AIHA IgG Rh protein complex Not seen in SLE; aldomet-induced

+ - Drug-induced IgG Drug attached to Rh complex Penicillin and aldomet

+ + Drug-induced IgG ? Fludarabine and cogeners

+ + AIHA IgG Glycoprotein Antibody fixes complement √

+ + Drug-induced IgG Drug + proteinDrug affixed to protein; needed to detect in serum

- + Drug-induced IgM or IgG Drug + proteinDrug not firmly affixed to protein; drug needed to detect serum antibody

- + AIHA IgG ?Antibody of low affinity (may be detected with enzyme treated red cells)

+ + AIHA IgM or IgA ?Warm-reacting antibody detected with specific anti-isotype antisera

- + Cold agglutinin disease IgM Polysaccharide Cold agglutinins in high titre in serum √

- + Paroxysmal cold hemoglobinuria IgG P antigen (polysaccharide)

Antibody fixes complement in the cold; hemolysis occurs when sample/patient is warmed; Donath-Landsteiner test may be positive

Significance of the pattern of positive direct antiglobulin (Coombs) test in the diagnosis of autoimmune hemolytic anemia

• C3d only

8 patients

• IgG only

27 patients

• IgG + C3d

29 patients

• IgG + IgM +/- IgA

3 patients

• IgG + IgM +/- IgA

• + C3d27 patients

A study of 94 DAT positive patients with AIHA from CMC Vellore

Severity of haemolysis – Solitary IgG vs IgG in combo

Severe haemolysis

• IgG alone

• 5 of 46

Severe haemolysis

• IgG + Combo

• 41 of 46

Statistical significance

• P<0.001

• OR – 10.2

IgG subtype – IgG1 and IgG3

Moderate AIHA Severe AIHA

IgG1 alone 4 (44.4%) 5(55.6%) 9(100%)

IgG1 with IgG3 1 0 1

Total 5 5 10

Subtype of

IgG with other

autoantibodies Moderate Severe

Total

number

IgG115(36.6%) 26(63.4%) 41(100%)

IgG1 with IgG32(22.2%) 7(77.8%) 9(100%)

Das SS, Nityanand S, Chaudhary R. Clinical and serological characterization of autoimmune hemolytic anemia in a tertiary care hospital in North India. Ann Hematol. 2009;88:727–32.

Significance of IgG1 / IgG3 subtypes -haemolysis

P value Odds ratio

IgG1 alone 0.012 3.671

IgG1 with IgG3 0.041 5.250

Method of producing human igg antibodies with enhanced effector functions WO 2006114700 A2

Suspected mix - coexisting alloantibody

DAT – A Coating on the red cell

Elution –removing the

coat(Autoantibody)

Auto adsorb using cells after elution –perform DAT -Positive

Continue to Auto adsorb after elution –perform DAT (weak positive)

Mix of auto and allo antibody Mix of less auto and Predominantly allo antibody

Only Allo antibody remains

DAT Negative – No auto antibody remaining

With the remaining plasma- alloantibody testing

Compatibility testing

• On post adsorption (auto) plasma

• Possible if not recently transfused

• Ensure DAT negative cells are phenotyped

• If alloantibody present – Antigen negative red cells

If recently transfused

Review clinical features and lab findings

Suspect cold agglutinin – perform a warm collection and repeat . Titre if required

DAT – grade strength of reactivity

Do a Monospecific DAT

If positive for IgG Subtype (IGG1 / IgG3) to be done

If Suspected alloantibody – elute / autoadsorb – repeat till DAT negative

Compatibility testing / antigen neg cells if required

Suggested Algorithm - AIHA

In the context of HDFN – an alloimmunised mother

• Challenge has been the variability of access to clinical care

• Preparedness required for the baby

• Monitoring frequency – advice regarding

Data from CMC – 85 of 3900 antenatal women - alloimmunised

• Overall prevalence for IgG1 and /or IgG3 subclasses of alloimmunizedantenatal women - 48.23%.

• IgG1 only, 20%; IgG3 alone 4%, IgG1 + IgG3: 25% and 51% were negative for either of these two subclasses.

• Choudhury et al , ISBT science series March 2016

Correlation

A highly significant difference was observed using the

Pearson’s chi square test in severity of disease when comparing

mothers negative for IgG1 and IgG3 as against mothers with

one subtype IgG1/ IgG3 and a combination of IgG1+IgG3

(p<0.001).

Titre impacted irrespective of subtype

DAT strength – correlated with severity of HDFN

Choudhury et al , ISBT science series March 2016

HDFN

Alloantibody in the mother

Alert the clinician / assess clinical significance

Titre if possible

Phenotype parents

Perform an IgG1 / IgG3 subtype

Prepare for exchange / top up / exchange transfusion / liaise with neonatology

Baby born – Lab parameters + DAT – elute and document if possible

Watch out for differential transfer of antibodies

Is it important?

• Accessibility to medical care at the appropriate time is an issue in justice

28 year old lady

• History S/O an intravascular transfusion reaction

• Two episodes

• Haematological disorder

• Multiply transfused In the past

• Repeat groups – B Pos and B Pos

• Compatibility testing – compatible at IS and coombs phase

• Antibody screen – Negative

• Culture etc – neg

• PNH work up suggested - Negative

• Antibody screen at 4 deg –positive

• Anti Le a identified

• Crossmatch – incompatible with unit issued

Warm the patient and the blood!!

Antigen negative as far as possible

An occasional case report found in literature!