antibody detection lecture presentation handout

Antibody Detection dand

IdentificationIdentification

Michelle BrownMichelle Brown

f dFrequency of unexpected antibodies in antibodies in hospitalized ppatients…2%

Purpose of Antibody Detection and IdentificationIdentification…

To detect and identify antibodies that are clinically significantclinically significant

What is a Clinically Significant y gAntibody?

An antibody that reacts at 37°C which can cause in vivo red cell which can cause in vivo red cell

destruction

What if you Have a Clinically S f b d ?Significant Antibody?

1 Identify the antibody1. Identify the antibody2. Find antigen negative blood3. Crossmatch compatible

What’s Clinically Significant?

Always Significant Sometimes SignificantAlways SignificantABORh

Sometimes SignificantLea

MN RhKellDuffy

MN P1

LutheranDuffyKiddSs

Lutheran

Rarely SignificantLeb

Xga

How do we Know if the Patient h b d ?has an Antibody?

They have a positive antibody screenscreen.

ABO Discrepancy – AB with anti-MAA B A1 B cell

M + M -

How do we know the Patient has an Antibody?

I tibl t hIncompatible crossmatchTransfusion reactionHDNPositive DATPositive auto control

What do we need for Antibody Identification?

ABO d RhABO and RhAntibody screenResults of previous testingHistory of transfusion or pregnancyDiagnosisMedicationsMedications

Reagent Red Cells - ABSCReagent Red Cells ABSC

G OGroup O

2-cell or 3-cell screens2 cell or 3 cell screens

Typed for all common antigens in j bl d tmajor blood group systems

Match lot # with antigen profile sheet Match lot # with antigen profile sheet (antigram)

Required Testing ParametersRequired Testing Parameters

M lMay use serum or plasmaMethods that will demonstrate li i ll i ifi t tib diclinically significant antibodies

Must include 37°C incubation and AHGAHGPolyspecific or anti-IgGIgG presensitized cells (check cells)

Antibody Panels

10 20 O t d ll10-20 group O reagent red cellsAntigram – indicates the phenotypes f th t d llof the reagent red cells

Pattern of reaction of patient serum i h ll i h d h f with cells is matched to the pattern of

antigen typing to determine the antibody specificityantibody specificity

D C E c e M N S s K k Fya

Fyb

Jka

Jkb

IS 37 AGT

1 0 + 0 + + + 0 + + 0 + + 0 + 0 0 0 0

2 + + 0 0 + + + + 0 0 + + + + 0 0 0 0

3 + + + 0 + 0 + 0 + 0 + + + 0 + 0 0 +3 0 0 0 0 0 0 0 +

4 + 0 + + 0 0 + + 0 + 0 0 + + + 0 0 +

5 0 0 0 + + + + 0 + 0 + + 0 + + 0 0 05 0 0 0 + + + + 0 + 0 + + 0 + + 0 0 0

6 0 0 + + + + + + + 0 + 0 + 0 + 0 0 +

7 + 0 0 + + 0 + 0 0 + + 0 0 + 0 0 0 0

8 0 0 0 + + + 0 0 + 0 + + + 0 + 0 0 0Pt 0 0 0

D C E c e M N S s K k Fya

Fyb

Jka

Jkb

IS 37 AGT

1 0 + 0 + + + 0 + + 0 + + 0 + 0 0 0 0

/ / / / / / / / // / / / /

2 + + 0 0 + + + + 0 0 + + + + 0 0 0 0

3 + + + 0 + 0 + 0 + 0 + + + 0 + 0 0 +3 0 0 0 0 0 0 0 +

4 + 0 + + 0 0 + + 0 + 0 0 + + + 0 0 +

5 0 0 0 + + + + 0 + 0 + + 0 + + 0 0 05 0 0 0 + + + + 0 + 0 + + 0 + + 0 0 0

6 0 0 + + + + + + + 0 + 0 + 0 + 0 0 +

7 + 0 0 + + 0 + 0 0 + + 0 0 + 0 0 0 0

8 0 0 0 + + + 0 0 + 0 + + + 0 + 0 0 0Pt 0 0 0

D C E c e M N S s K k Fya

Fyb

Jka

Jkb

IS 37 AGT

1 0 + 0 + + + 0 + + 0 + + 0 + 0 0 0 0

/ / / / / / / / /

2 + + 0 0 + + + + 0 0 + + + + 0 0 0 0

3 + + + 0 + 0 + 0 + 0 + + + 0 + 0 0 +3 0 0 0 0 0 0 0 +

4 + 0 + + 0 0 + + 0 + 0 0 + + + 0 0 +

5 0 0 0 + + + + 0 + 0 + + 0 + + 0 0 05 0 0 0 + + + + 0 + 0 + + 0 + + 0 0 0

6 0 0 + + + + + + + 0 + 0 + 0 + 0 0 +

7 + 0 0 + + 0 + 0 0 + + 0 0 + 0 0 0 0

8 0 0 0 + + + 0 0 + 0 + + + 0 + 0 0 0Pt 0 0 0

Evaluating Antibody Specificity

V i t th f ti itVarying strength of reactivityDosageM lti l tib diMultiple antibodiesVarying strength of antigen on red cells (P Le I Fy Jk D)(P, Le, I, Fy, Jk, D)

If difficult pattern, may phenotype the patients cells to see which the patients cells to see which antibodies they could make

Panel Interpretation

Some cells pos, some neg, auto neg

D C E c e M N S s K k Fya

Fyb

Jka

Jkb

IS 37 AGT

1 0 + 0 + + + 0 + + 0 + + 0 + 0 0 0 0

2 + + 0 0 + + + + 0 0 + + + + 0 0 0 0

3 + + + 0 + 0 + 0 + 0 + + + 0 + 0 0 +3 0 0 0 0 0 0 0 +

4 + 0 + + 0 0 + + 0 + 0 0 + + + 0 0 +

5 0 0 0 + + + + 0 + 0 + + 0 + + 0 0 05 0 0 0 + + + + 0 + 0 + + 0 + + 0 0 0

6 0 0 + + + + + + + 0 + 0 + 0 + 0 0 +

7 + 0 0 + + 0 + 0 0 + + 0 0 + 0 0 0 0

8 0 0 0 + + + 0 0 + 0 + + + 0 + 0 0 0Pt 0 0 0

Interpretation of Typical Panel Results

Some cells pos some neg auto negSome cells pos, some neg, auto negSingle alloantibody

Many or all cells pos, auto neg

D C E c e M N S s K k Fya

Fyb

Jka

Jkb

IS 37 AGT

1 0 + 0 + + + 0 + + 0 + + 0 + 0 0 0 +

2 + + 0 0 + + + + 0 0 + + + + 0 0 0 +

3 + + + 0 + 0 + 0 + 0 + + + 0 + 0 0 +3 0 0 0 0 0 0 0 +

4 + 0 + + 0 0 + + 0 + 0 0 + + + 0 0 +

5 0 0 0 + + + + 0 + 0 + + 0 + + 0 0 +5 0 0 0 + + + + 0 + 0 + + 0 + + 0 0 +

6 0 0 + + + + + + + 0 + 0 + 0 + 0 0 +

7 + 0 0 + + 0 + 0 0 + + 0 0 + 0 0 0 +

8 0 0 0 + + + 0 0 + 0 + + + 0 + 0 0 +Pt 0 0 0

Interpretation of Typical Panel Results

Some cells pos some neg auto negSome cells pos, some neg, auto negSingle alloantibody

Many or all cells pos, auto negy p , gMultiple antibodiesAntibody to high incidence antigen

Few cells pos, no evident patternD C E c e M N S s K k Fy

aFyb

Jka

Jkb

IS 37 AGT

1 0 + 0 + + + 0 + + 0 + + 0 + 0 + 0 0

2 + + 0 0 + + + + 0 0 + + + + 0 0 0 0

3 + + + 0 + 0 + 0 + 0 + + + 0 + 0 0 03 0 0 0 0 0 0 0 0

4 + 0 + + 0 0 + + 0 + 0 0 + + + 0 0 0

5 0 0 0 + + + + 0 + 0 + + 0 + + 0 0 05 0 0 0 + + + + 0 + 0 + + 0 + + 0 0 0

6 0 0 + + + + + + + 0 + 0 + 0 + 0 0 0

7 + 0 0 + + 0 + 0 0 + + 0 0 + 0 0 0 0

8 0 0 0 + + + 0 0 + 0 + + + 0 + + 0 0Pt 0 0 0

Interpretation of Typical Panel Results

Some cells pos some neg auto negSome cells pos, some neg, auto negSingle alloantibody

Many or all cells pos, auto negy p , gMultiple antibodiesAntibody to high incidence antigen

Few cells pos no evident patternFew cells pos, no evident patternWeak antibodyAntibody showing dosageTechniques to enhance reactions?

All cells pos, including auto controlD C E c e M N S s K k Fy

aFyb

Jka

Jkb

1 0 + 0 + + + 0 + + 0 + + 0 + 0 +

2 + + 0 0 + + + + 0 0 + + + + 0 +

3 + + + 0 + 0 + 0 + 0 + + + 0 + +3 0 0 0 0 0 +

4 + 0 + + 0 0 + + 0 + 0 0 + + + +

5 0 0 0 + + + + 0 + 0 + + 0 + + +5 0 0 0 + + + + 0 + 0 + + 0 + + +

6 0 0 + + + + + + + 0 + 0 + 0 + +

7 + 0 0 + + 0 + 0 0 + + 0 0 + 0 +

8 0 0 0 + + + 0 0 + 0 + + + 0 + +Pt +

Interpretation of Typical Panel Results

Some cells pos some neg auto negSome cells pos, some neg, auto negSingle alloantibody

Many or all cells pos, auto negy p , gMultiple antibodiesAntibody to high incidence antigen

Few cells pos no evident patternFew cells pos, no evident patternWeak antibodyAntibody showing dosage

All cells pos, auto posCold autoantibodyWarm autoantibodyWarm autoantibodyRouleaux

Selected Cells

If d t h th ll If you don not have the cells on your panel to rule out an antibody, you need to run additional cellsneed to run additional cells

If d h h ll If you do not have the cells on your panel to prove an antibody, you need to run additional cellsto run additional cells

Methods of Testing for Antibody

Low Ionic Strength Solution (LISS)

+ ++

++

+

LISS

I iti itIncreases sensitivity

Don’t increase serum:cell ratio

Less sensitive method for detection of Kell antibodies

PEG

P l th l l lPolyethylene glycolRemoves water, concentrates the

tib d t tib d t k antibody, promotes antibody uptake, and enhances strength of reactionD ’ ifDon’t centrifugeUse anti-IgG for AGT

T t T bTest TubeGelSolid Phase Red Cell Adherence

Gel Test: Pos test Neg testPos test Neg test

Gel Test: Antibody Screeny

Gel Test: ABO Typing

Solid Phase Red Cell Adherence

Solid Phase Red Cell Adherence