• Secondary (to conditions such as syphilis, viral

infections).

¾ DAT negative autoimmune hemolytic anemia

• Primary (idiopathic)

• Secondary (to conditions such as lymphoma, SLE).

FIG. 11.4: Illustration of HDN

342 Concise Book of Medical Laboratory Technology: Methods and Interpretations Drug-induced Hemolytic Anemia

Also, certain drugs namely, penicillin, procainamide,

cephalosporins may also be associated with immune red

blood cell destruction thereby demonstrating a positive

DAT (Fig. 11.5).

Importance of Serological Studies in DAT Positive

Results (Table 11.5)

As per blood bankers’ technical manual, three investigation approaches are helpful in evaluation of positive

DAT:

¾ Test the DAT positive red blood cells with monospecific

anti-human IgG and monospecific anti-human C3d

reagent to characterize type of proteins sensitized with

red blood cell membrane,

¾ Test serum/plasma to detect and identify clinically

significant antibodies to red blood cell antigens.

TABLE 11.5: Probable serological findings with DAT positive—AIHA/drug induced hemolytic anemia

Parameter WAIHA CAS Mixed type AIHA PCH Drug-induced AIHA

DAT positive result IgG/IgG + C3/C3 Mostly C3 IgG + C3 Mostly C3 IgG/IgG + C3

Immunoglobulin

Type

IgG sometimes

IgA or IgM

rarely alone

IgM IgG, IgM IgG IgG

Eluate IgG Non reactive IgG Non-reactive IgG

Serum • May react

by IAT

• May hemolyze

enzyme

treated red

cells at 37°C

• Mostly

agglutinate

enzyme

treated red

cells at 37°C

• May

agglutinate

untreated

red cells at

20°C

• Rarely

agglutinate

untreated

cells at 37°C

• IgM

hemagglutinating

antibody

reactive at

4°C usually

react at

30°C in

albumin

• IgG IAT

reactive

antibody

• IgM hemagglutinating

antibody

usually react

at 30–37°C

in saline, also

may react at

4°C saline

• IgG biphasic

hemolysin

(Donath

Landsteiner

antibody)

• IgG antibody

similar to

WAIHA

Specificity Usually Rh

specificity

Usually Anti-I

but can be

Anti-I rarely

Anti-Pr

Usually

specificity

unclear, can

be Anti-I,

Anti-I or other

cold aggluthin

specificities

Anti-P (nonreactive with

p and Pk

red cells

Specificity

often Rh

related

FIG. 11.5: Illustration of drug-induced antibody reactions

¾ Test eluate prepared from sensitized red blood cells

with a panel of reagent red blood cells to define

whether the sensitized protein is immunoglobulin

or complement component. Elution frees antibody

from sensitized red blood cells and recovers antibody

Blood Banking (Immunohematology) 343

in usable form. When only complement is sensitized,

eluates are frequently non-reactive.

Indirect Anti-human Globulin Test (IAT)

In IAT procedures, serum or plasma is incubated with

red blood cells, washed to remove unbound globulins.

Agglutination that occurs after addition of Anti-human

globulin reagent indicates reaction between antibody

in the serum and antigen present on the red blood cell

membrane.

Applications of IAT

IAT determines in vitro sensitization of red blood cells and

is used in the following situations:

¾ Detection of incomplete antibodies to potential donor

red blood cells, pregnant women, blood donors

¾ Identification of antibody specificity using a panel of

red blood cells with known antigenic profile

¾ Determination of red blood cell phenotype using

known antisera (e.g. Du testing)

¾ Titration of incomplete antibodies.

Probable Sources of Error in Anti-human

Globulin Testing

False Negative Results

¾ Neutralization of anti-human globulin reagent

¾ Failure to wash cells adequately to remove all serum/

plasma. Fill tube at least three-fourth full of saline for

each wash

• If increased serum volumes are used, routine wash

may be inadequate. Wash additional times more

than three or four wash phases

• Contamination of Anti-human globulin reagent

by extraneous protein. Do not use finger or hand

to cover tube. Contaminated droppers or wrong

reagent dropper can neutralize entire vial of Antihuman globulin reagent

• High concentration of IgG paraproteins in test serum

(cryoglobulin). Wash additional times more than

three or four wash phases

¾ Interruption in testing

• Bound IgG may dissociate from red blood cells or leave

too little IgG to detect or may neutralize Anti-human

globulin reagent. Perform the test immediately

• Agglutination of IgG coated cells will weaken.

Centrifuge and read immediately

¾ Improper reagent storage

• Anti-human globulin reagent may lose reactivity

if frozen. Reagent may become bacterially

contaminated. Store at the recommended storage

condition

• Excess heat or repeated freeze/thaw cycles may cause

loss of reactivity of anti-human globulin reagent.

Replace the reagent back to the recommended

storage condition

¾ Improper procedure

• Overcentrifugation may pack cells so tightly that

agitation required to resuspend cells breaks up

agglutinates. Undercentrifugation may not be

optimal for agglutination. The optimum centrifugation speed should be ascertained for each centrifuge

• Failure to add test serum, enhancement medium or

Anti-human globulin reagent may lead to negative

test result. Follow the manufacturer’s instructions

meticulously