1. DAT Negative AIHA Dr. Sudipta Sekhar Das
TRANSMEDCON 2018
DAT Negative AIHA
Dr. Sudipta Sekhar Das
MD (Transfusion Medicine), SGPGIMS
PDCC (Aphaeresis & Component Therapy), SGPGIMS
Senior Consultant & Head, Transfusion Medicine
Apollo Gleneagles Hospital, Kolkata
Professor (Apollo Hospitals, India)

2. AIHA in General
Characterized by increased RBC destruction and / or decreased red cell survival d/t autoantibodies directed against self RBC antigens
Incidence 1 in 80,000 to 100,000 given population/year
DAT remains the crucial serological assay in AIHA diagnosis
A positive DAT almost always is associated with AIHA & forms hallmark of diagnosis
However, positive DAT does not indicate overt disease & combination of clinical and laboratory evidences establish the diagnosis

3. Pathophysiology Trigger 80% extravascular hemolysis; Spherocytes
Genetic (HLA-DQ6)
Infections
Inflam. Disorders
Malignancies
Drugs
80% extravascular hemolysis; Spherocytes
Less frequently intravascular

4. Autoimmune Hemolytic anemia
Diagnosis is made
in the reverse way
Anemia
Hemolytic
Immune
Auto

5. Laboratory Investigations
An approach to the probable diagnosis
Hematology
Hb: 5.4 g/dL
Hct: 20%
Retic: 8%
Plt: 167x103/mm3
TLC: 5.5x103/mm3
MCV: 86fL
MCH: 30pg
MCHC: 34%
RDW
Blood smear
NCNC
Spherocytes +
Biochemistry
S.Bilirubin: 5.2mg/dL
LDH: 1625U/L
SGOT: 35U/L
SGPT: 30U/L
Alk. Phosphate: 350U/L

6. Probable diagnosis A patient with hemolytic anemia (HA) +
Clinical features
Weakness, lethargy
Pallor
Tachycardia
Splenomegaly
Markers of invivo hemolysis
Hb: g/dL
Hct: 20%
Retic: 8%
S.Bilirubin: 5.2mg/dL
LDH: 1625U/L +
A patient with hemolytic anemia (HA)

7. How to diagnose AIHA Whether immune or non-immune etiology ?
Probable diagnosis is hemolytic anemia
Detailed blood bank work-up can only diagnose immune HA
Detection of RBC antibody by DAT is the hallmark of diagnosis
Whether immune or non-immune etiology ? Y Y
RBC Y Y
2-4 % cases
RBC Y Y
Antibody Y Y
DAT negative by tube technique
DAT positive by tube technique –
gold standard

8. Sensitive technique (CAT) is the demand of the day
Various methods of DCT
Methods of DAT
Sensitivity
(molecules / red cell)
Conventional tube technique
300 – 500
CAT / Microplate / SPRCA
120 – 180
Enzyme linked anti globulin test
80 – 120
Flow cytometry
Gold standard
Most sensitive
Sensitive technique (CAT) is the demand of the day

9. Classification of AIHA
Warm AIHA (48-70%)
Primary (Idiopathic)
Secondary (LPD, AI Dis, immune deficiency states)
Cold AIHA
Cold Agglutinin Syndrome (16-32%)
Secondary (Infections, LPD)
PCH (32% in children)
Secondary (Syphilis, Viral inf.)
Mixed AIHA (7-8%)
Primary (Idiopathic)
Secondary (Lymphoma, SLE)
Drug induced AIHA (12-18%)
DAT negative AIHA (2-4%)

10. DAT Negative AIHA Reasons for DAT negativity
Incidence: % 0f all AIHA
Reasons for DAT negativity
IgG mol / RBC necessary for accelerated in-vivo destruction is lower than number necessary to yield a positive DAT by CTT (Gold standard)
RBC coated with IgA autoantibodies or monomeric IgM.
Low affinity Ab. causing hemolysis easily dissociate from RBC while washing: Strong IgG in vivo appear to have little / no IgG in vitro.

11. Techniques implemented
Cold wash DAT: retain low affinity IgG
CAT / SPRCA / Microplate < 200 IgG/RBC
Techniques detecting < 100 IgG / RBC
Enzyme linked antiglobulin test (ELAT)
Complement-fixation antibody consumption test
Immunoradiometric assay (IRMA)
Mitogen stimulated – DAT
MMA
Flow cytometry (Most sensitive 35 IgG /RBC)

13. Flow cytometric analysis of AIHA patients (N=12)
Age/ Sex
Hb g%
Retic %
S.Bil mg%
LDH
IU/L
CTT GT
Flow cytomtery
Poly
IgG
C3d
MFI
Result
22/F
6.8
8.5
2.8
2124 3+ 2+ 1+ 4+
19.7
Pos
35/F
5.1 25
3.8
1817
21.63
27/F
7.5 16
4.9
1618
17.15
30/F
9.9
2.9
2.3
690
Neg
30.5
18/F
3.2 20
1374
25.7
17/F
7.1 3
937
15.02
31/F
6.5 2
2.4
834
12.9
54/F
9.2
2.5
3.3
695 W+
17.1
15/F 5
4.8
1013
11.9
62/F
8.3 9
4.1
678
8.9
41/F
6.9 6
0.9
680
8.4
24/m
0.7
710
9.3
Hematology 2006

14. Flow cytometry in DAT negative AIHA
Hematology 2006
Neg control
MFI 4..8
Pos control
MFI 31.8
DAT Neg AIHA
MFI 8.6

15. Unpublished data, Das et al 2018 DAT positive by polyspecific
Under peer review
True AIHA suspects
N = 353
No conclusion
No follow up
N = 9
DAT positive by CTT
N = 326
DAT negative by CTT
N = 27 18
DAT positive by polyspecific
CAT
N = 8
DAT positive
by monospecific CAT
N = 2
DAT positive
by IgG
subclass CAT
N = 1
Positive
cold wash
DAT
N = 2
DAT positivity
confirmed
by elution
N = 1
DAT negative by available methods but suspected AIHA responded to therapy
N = 4
Figure : Simple methodologies to diagnose DAT negative AIHA

16. Conclusion
All conventional DAT negative patients with strong clinical suspicion of AIHA should be further evaluated
Alternate sensitive methods which are otherwise less practiced may be implemented
We observed that such techniques are less cumbersome and do not lead to financial constraints
Blood bank may establish these useful simple techniques and stick to the defined protocols to diagnose DAT negative AIHA.