Detecting AntibodiesDetecting Antibodies

The Antibody Screen

CLS 422

Clinical Immunohematology I

ObjectivesObjectives

Explain the purpose of performing an antibody screen.

Discuss the antigen characteristics important in the composition of screening cells.

Describe the phases of antibody detection. Describe the types of antibodies that can be

encountered in each of the phases of antibody detection.

ObjectivesObjectives State the difference between an alloantibody and an

autoantibody. List factors affecting the antigen-antibody reaction in

the indirect antiglobulin test. Discuss the action of potentiators. Compare and contrast methods for performing an

antibody screen. Assess sources of error affecting the indirect

antiglobulin test. Interpret the results of an antibody screen.

The Antibody ScreenThe Antibody Screen

Used to detect “irregular” antibodies. Maximize detection of clinically significant

antibodies Minimize detection of insignificant

antibodies Patient’s serum or plasma is tested

against reagent red blood cells (RBCs) with known antigens (screen cells).

Allo vs. AutoAllo vs. Auto

Alloantibody – antibody directed against antigens that the individual does not possess Immune Naturally-occurring

Autoantibody – antibody directed against one’s own antigens Auto control – patient’s RBCs tested against

patient’s plasma

What makes an antibody What makes an antibody clinically significant?clinically significant?

The ability to cause decreased RBC survival. If the antibody activates complement, there may be

intravascular RBC lysis. There may be extravascular destruction of

antibody-coated RBCs by the macrophages of the RES.

Hemolytic Transfusion Reaction (HTR) Hemolytic Disease of the Fetus & Newborn

(HDFN)

Who needs to be tested?Who needs to be tested?

OB patients – looking for antibodies that may cause HDFN in the fetus.

Patients who need an RBC transfusion – looking for antibodies in the recipient that could destroy the donor RBCs (HTR).

Blood, organ and tissue donors – avoid passive antibody transfer; evaluate as source of anti-serum and rare RBCs.

An Application of the An Application of the Indirect Antiglobulin TestIndirect Antiglobulin Test

Patient’s plasma (unknown antibody) is tested against reagent RBCs (known antigen). Y

YY Incubated at 37oC to allow antibody to sensitize RBCs. Antiglobulin phase to allow sensitized RBCs to agglutinate.

Screen Cell CompositionScreen Cell Composition

Sets of 2 to 4 different cells Group O Rh Positive and Rh

Negative cells Other major blood group

antigens are represented

Antigen Profile Antigen Profile (Antigram)(Antigram)

D C c E e Cw K k Fya Fyb Jka Jkb Lea Leb P1 M N S s Lua Lub Xg

I + + 0 0 + 0 + + 0 + + 0 0 + 0 + 0 + 0 0 + +

II + 0 + + 0 0 0 + + 0 + + 0 + + 0 + 0 + 0 + 0

III 0 0 + 0 + 0 0 + + 0 0 + + 0 + + 0 + + 0 + +

• If the patient’s serum/plasma contains an antibody directed against one of the antigens on the screen cell, the RBCs will agglutinate (or hemolyze)= positive reaction.

• We can not be certain which antigen is the target of the antibody.

Phases of TestingPhases of Testing

Immediate spin (I.S.)

(optional phase)

37oC

AHG

Lewis, M, N, P1, cold autoantibodies (I, H, IH)

May see D, E, K, strong cold antibodies

Rh, Kell, Kidd, Duffy, Ss

SpecimenSpecimen

Plasma Serum

Antibody Screen Antibody Screen Tube MethodTube Method

37C inc

IDII

IDIIIID

I

√√

√

Antibody ScreenAntibody ScreenGel MethodGel Method

Antibody Screen Antibody Screen Gel MethodGel Method

Ortho ProvueOrtho Provue

Courtesy Ortho-Clinical Diagnostics Raritan, NJ

Antibody Screen Antibody Screen Solid Phase Adherence MethodSolid Phase Adherence Method

RBC antigens are bound to the sides of a microtiter well.

Different wells possess different antigens, and thus are different “screen cells”.

Antibody Screen Antibody Screen Solid Phase Adherence MethodSolid Phase Adherence Method

YY

Immucor AutomationImmucor Automation

ABO and Rh are performed using direct agglutination in a microtiter well.

Screens, panels and DATs are performed using solid phase adherence.

Reporting ResultsReporting Results

Reporting is “all or nothing”, rather than reporting the results of each cell at each phase.

If all cells are nonreactive at all phases, and the Coombs Control Cells are positive, the screen is reported as negative.

IS 37 AHG CC

I 0 0 0 3+

II 0 0 0 3+

III 0 0 0 3+

Reporting ResultsReporting Results

If one or more cells react at any phase of testing, the screen is reported as positive.

An antibody panel should be performed to determine the specificity of the antibody present.

IS 37 AHG CC

I 0 0 0 2+

II 0 0 2+

III 0 0 0 2+

Are We Done Now?Are We Done Now?

NO – all cells should be tested through all phases, even if a positive reaction has already been observed.

IS 37 AHG CC

I 2+

II 0

III 1+

How Would you Report This How Would you Report This Screen?Screen?

IS 37 AHG CC

I 0 1+ 3+

II 0 0 0 2+

III 0 0 0 2+

How Would you Report This How Would you Report This Screen?Screen?

IS 37 AHG CC

I 0 0 2+

II 0 0 2+

III 0 0 2+

How Would you Report This How Would you Report This Screen?Screen?

IS 37 AHG CC

I 2+ 0 0 2+

II 1+ 0 0 2+

III 0 0 0 2+

How Would you Report This How Would you Report This Screen?Screen?

IS 37 AHG CC

I 0 0 0 0

II 0 0 0 2+

III 0 0 0 2+

How Would you Report This How Would you Report This Screen?Screen?

(Performed Using Gel)(Performed Using Gel)

IS 37 AHG CC

I 0

II 0

III 0

How Would you Report This How Would you Report This Screen?Screen?

(Performed Using Solid Phase (Performed Using Solid Phase Adherence)Adherence)

IS 37 AHG CC

I 0

II 2+

III

Factors Affecting Factors Affecting the Antibody the Antibody

ScreenScreenThese factors will affect ANY

application of the indirect antiglobulin test!

Antigen/Antibody Ratio Antigen/Antibody Ratio

Y

Y

Y

YY

Y Y

Y

Y

Y

Y

Y

Y

YY

YY

YY

Y

Y

Y

Y

YY

Y

Y

Prozone PostzoneEquivalenceRatio is usually 2 drops of serum or plasma to 1 drop of RBC suspension.

Reaction TemperatureReaction Temperature

Temp oC ABO Rh Kell Duffy Kidd Ss MN Lewis P1 Ii

45

40

35

30

25

20

15

10

5

0

IG Class

IgM Some IgG

IgG IgG IgG IgG IgG IgM IgM IgM IgM

Reaction TimeReaction Time

IgG antibodies are incomplete Require incubation at 37oC to react Length of incubation dependant on test

medium

Test MediumTest Medium

May add potentiators to overcome the effects of shielding and the zeta potential Albumin LISS PEG Enzymes

Quantity and location of Quantity and location of AntigenAntigen

pHpH

Optimal 6.5 to 7.5

Sources of Error Sources of Error in the Antibody in the Antibody

ScreenScreenFalse Negative Results

Improper WashImproper Wash

Y

Y

Y

Y

Y

Y

YY

Y

Y

YY

Failure to…Failure to…

Add plasma (the antibody source) Make it a habit to add plasma to

tube before adding RBCs. Add reagents Follow manufacturer’s directions Recognize hemolysis as a positive

reaction

Work QuicklyWork Quickly

Y

Y

Y

Y

Y

Y

Y

Other causes of false Other causes of false negative resultsnegative results

AHG reagent neutralized Using expired reagents Under-centrifugation Complement-dependant antibody/plasma

specimen

Sources of Error Sources of Error in the Antibody in the Antibody

ScreenScreenFalse Positive Results

False PositivesFalse Positives

Over-centrifugation Contaminated reagents Debris Rouleaux

Limitations of the Antibody Limitations of the Antibody ScreenScreen

Will not detect ABO incompatibility Will not detect antibodies to antigens that are

not present on the screen cells May not detect antibodies exhibiting dosage May not detect antibodies that are low in titer

Primary vs. Secondary Primary vs. Secondary Humoral ResponseHumoral Response

First exposure

Second exposure

IgM

IgM

IgG

IgG

Today’s Lab…Today’s Lab…

The Type & ScreenThe Type & Screen(ABO, Rh, and Antibody Screen)(ABO, Rh, and Antibody Screen)