Dr Sangeeta PahujaAssociate Professor,

Lady Hardinge Medical College and Assoc. Hospitals,New Delhi

Demonstration of red cell antigen-antibody reactions is key to immunohematology.

AABB Technical Manual

RED CELL Antibodies:

Alloantibodies

Reacts with foreign Ag not present on patient’s own RBC

Most produced as result of immune stimulation via transfusion or pregnancy (usually during delivery)

Autoantibodies

Reacts with an Ag on patient’s own cells & with that same Ag on the cells of other individuals

Characteristics of Antibodies

Immune Vs. Non-Immune red cell Antibodies: RBC Immune Antibody: antibody that

results from exposure to foreign red cell antigen either by transfusion or pregnancy

Non-RBC Immune Antibody: antibody that is present without any evidence of exposure to foreign red cell antigen

UNEXPECTED ANTIBODIES MAY BE DETECTED DURING-

1. In ABO cell & serumgrouping

2. In antibody screening3.Positve autocontrol4.Positive DAT5. In major cross matching

PROPER DETECTION & IDENTIFICATION OF ANTIBODIES IS IMPORTANT FOR-1. SELECTION OF APPROPRIATE

BLOOD FOR TRANSFUSION2. IN INVESTIGATION OF HAEMOLYTIC

ANAEMIAS3. IN INVESTIGATION OF HDN4. TRANSFUSION REACTION5. SEROLOGICAL CHANGES IN

ANTENATAL PERIOD

ANTIBODY SCREEN

The goals of antibody detection testing are as follows:

To detect as many clinically significant antibodies as possible.

To detect as few clinically insignificant antibodies as possible.

To complete the procedure in a timely manner.

A low ionic strength solution (LISS) IAT is considered to be the most suitable for the detection of clinically significant antibodies because of its speed, sensitivity and specificity.

Each negative AHG tube test must be followed by a control system of IgG-sensitized red cells (check cells)

British Committee for Standards in Haematology

SCREENING CELLS Cells used for antibody screening should comply with

the recommendations of the guidelines for compatibility procedures in blood transfusion laboratories

Antigens should be expressed on screening cells: C,c,D,E,e,K,k,Fya, Fyb, Jka, Jkb,S, s, M, N, P 1, Lea ,Leb.

It is recommended that one of the screening cells should be R1R1 and another should be R2R2 and that the Fya, Fyb, Jka, Jkb, S and s antigens should be represented on reagent cells with homozygous expression.

Screening cells must not be pooled.

Antibody Screening

D C E C E Cw

K k Kpa

Kpb

Jsa

Jsb

Fya

Fyb

Jka

Jkb

Lea

Leb

P1

M N S s Lua

Lub

Xg

R1R1

+ + 0 0 + + 0 + 0 + 0 + + 0 + 0 0 + + + 0 0 + 0 + +

R2R2

+ 0 + + 0 0 0 + 0 + 0 + 0 + 0 + 0 + + + 0 + 0 0 + +

rr 0 0 0 + + 0 + + + + 0 + 0 + + 0 + 0 + 0 + 0 + 0 + n

Rh-hr Kell Duffy Kidd Lewis P MNS Luth Xg

Each of the panel cells has been antigen typed (shown on antigram)

+ refers to the presence of the antigen0 refers to the absence of the antigen

If antibodies are detected, they must be identified…

present

Not present

Continued…

Positive antibody Antibodyscreening identification

Antibody specificity

Antibody significance

Ab IDENTIFICATION

Identification of an antibody to red cell antigen(s) by testing the serum against a panel of selected red cell samples(8-14) withknown antigenic composition for major blood groups.

A reagent red cell panel must make it possible to identify with confidence most commonly encountered clinically significant alloantibodies.

Antibody Identification

D C E c e Cw

K k Kpa Kpb Jsa

Jsb

Fya

Fyb

Jka

Jkb

Lea

Leb

P1

M N S s Lua

Lub

Xg

1 R1wR1

+ + 0 0 + + 0 + 0 + 0 + + 0 + 0 + 0 + + 0 0 + 0 + +

2 R1R1

+ + 0 0 + 0 + + 0 + 0 + 0 + + + 0 + + + 0 + 0 0 + 0

3 R2R2

+ 0 + + 0 0 0 + 0 + 0 + 0 + 0 + 0 0 + + + 0 + 0 + +

4 R’r 0 + 0 + + 0 0 + 0 + 0 + + + + 0 + 0 + + 0 + 0 0 + +

5 r”r 0 0 + + + 0 0 + 0 + 0 + + 0 + + 0 + 0 0 + + + 0 + +

6 rr 0 0 0 + + 0 + + 0 + 0 + + + 0 + 0 + + 0 + 0 + 0 + +

7 rr 0 0 0 + + 0 0 + + + 0 + + + + + 0 + 0 + + 0 + 0 + +

8 R0r + 0 0 + + 0 0 + 0 + 0 + 0 0 + 0 0 0 + + + 0 + 0 + +

9 rr 0 0 0 + + 0 0 + 0 + 0 + + 0 + + + 0 + 0 + 0 + + 0 +

10

rr 0 0 0 + + 0 0 + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 + + +

11

rr 0 0 0 + + 0 0 + 0 + 0 + + + + 0 0 0 + 0 + 0 + + + 0

Rh-hr Kell Duffy Kidd Lewis P MNS Luth Xg

Interpreting Antibody Panels

There are a few basic steps to follow when interpreting panels-

1. “Ruling out” means crossing out antigens that did not react

2. Circle the antigens that are not crossed out

3. Consider antibody’s usual reactivity

4. Look for a matching pattern

Guidelines

Again, it’s important to look at: Autocontrol

○ Negative - alloantibody○ Positive – autoantibody or DHTR (i.e.,alloantibodies)

Phases○ IS – cold (IgM)○ 37° - warm reacting or cold (some have higher thermal

range) ○ AHG – warm (IgG)…significant!!

Reaction strength○ 1 consistent strength – one antibody○ Different strengths – multiple antibodies or dosage

About reaction strengths……

Strength of reaction may be due to “dosage” If panel cells are homozygous, a strong

reaction may be seen If panel cells are heterozygous, reaction

may be weak or even non-reactive

Panel cells that are heterozygous should not be crossed out because antibody may be too weak to react

Rule of three

The rule of three must be met to confirmthe presence of the antibody

How is it demonstrated?Patient serum MUST be:

○ Positive with 3 cells with the antigen○ Negative with 3 cells without the antigen

Phenotyping

In addition to the rule of three, antigen typing the patient red cells can also confirm an antibody

How is this done? Only perform this if the patient has NOT

been recently transfused (donor cells could react)

If reagent antisera (of the suspected antibody) is added to the patient RBCs, a negative reaction should result…

What to do if all RBCs are

incompatible??

What to do if all RBCs are incompatible??

Autocontrol: Negative Autocontrol: Positive

Multiple antibodies Antibody to high frequency antigen Autoantibodies

Recent transfusion

Multiple antibodies

Can be discerned by the differences seen in serologic phases or strengths of reactivity with panel. Depending upon the configuration of the panel and the antibodies present

nonreactive cells may be obtained Rh and Kell phenotyping of red cells Enzyme treated red cells Select cells Adsorption/Elution studies

“Custom” panel

Custom panel configuration is facilitated with computer software ; allows the user to select the RBCs (by phenotype) for testing.

This program utilizes electronic information from the panel manufacturer, which allows mistake-free entry.

Only RBCs that are of the needed phenotype are tested, thus conserving serum (and the reagent RBCs);

The phenotype information on the selected RBCs is made into a panel worksheet by the software, thus preventing handwriting and associated errors. The information is presented in a panel format with space for recording test results

When antibodies to antigens of

high frequency the resolution can be time- and resource

consuming. Usually uniform reactivity with all reagent

RBCs tested Autologous RBCs are generally

nonreactive. Approaches to resolution of the antibody’s

specificity testing RBCs that have been treated with

different agents, e.g., enzyme or DTT looking for enhancement or diminution of

reactivity.

In cases of Autoimmune

hemolytic anemias

Always look for alloantibody underlying autoantibody (12%-40%).

In cases where a clinically significant alloantibody has been detected, an IAT crossmatch using absorbed plasma should be performed.

“How clinically significant is this RBC

antibody?”

“What will happen to the RBCs if

transfused—will they be destroyed intravascularly?extravascularly? how rapidly?”

Clinically Significant Antibody:

•DECREASES RED CELL SURVIVAL

•HTR

•HDFN

SPECIFICATION SPN214/3

The Clinical Significance of Blood Group Alloantibodies and the

Supply of Blood for Transfusion: Dr Geoff Daniels

This document outlines current knowledge on the clinical significance of blood group alloantibodies.

Its prime purpose is to enable clinical decisions to be made regarding the management and blood transfusion support of patients with blood group antibodies that are

○ not commonly encountered and○ for which antigen-negative blood is not available in the

routine stock.

Other factors

How urgently blood is required; the clinical diagnosis and the patient’s bone

marrow function; whether the patient is immunologically

compromised and unlikely to respond; strength and thermal amplitude of the

antibody; class and subclass of the immunoglobulin;

Other factors

In vitro functional assays : the antibody dependent cellular cytotoxicity

(ADCC) the chemiluminescence test (CLT) Monocyte monolayer assay (MMA)

.

In vivo red cell survival

1-hour 51Cr RBC survival study A radiolabeled 0.5 mL sample of incompatible RBCs is injected into

the patient and samples are obtained at 10 and 60 minutes. If radioactivity in the plasma at 10 and 60 minutes is < 3% of the

total injected and the RBC survival at 60 minutes is at least 70%, then transfusion of

the incompatible blood carries minimal risk.

In Vivo test / “in vivo crossmatch” or “biological crossmatch.”

10 to 50 mL of unlabeled incompatible RBCs is transfused and a sample is collected from the patient posttransfusion to check for hemoglobinemia.

This test only detects intravascular hemolysis. Extravascular RBC destruction cannot be predicted.

How to transfuse??

Where BCSH guidelines are provided for an antibody specificity these should be followed.

When an antibody is identified and considered to be of no clinical significance,particular care must be taken to ensure that it is not masking the presence of another, clinically significant, antibody.

How to transfuse??

Antigen negative red cells should also be selected when a clinically significant antibody has previously been identified, but cannot be detected or identified in the current sample.

Anti-A1 is rarely active at 37oC and not considered clinically significant. IAT-compatible blood should be selected (BCSH guidelines).

If anti-M is active by IAT at 37oC, M negblood must be selected (BCSH guidelines).

If anti-N is active by IAT at 37oC, IAT-compatible blood unselected for N may be used

Anti-S and -s can cause HTRs. Antigen-negative blood must be selected (BCSH guidelines).

Rh system

All Rh antibodies :considered to be potentially clinically significant, capable of causing both HTRs and HDN.

BCSH guidelines recommend that when an Rh antibody reactive in IAT (the majority of Rh antibodies) is present, antigen-negative blood must be selected.

IAT-compatible blood may be used when anti-Cw is detected.

Rh system

Antibodies to high frequency Rh antigens are rare. They include anti-Rh29, the antibody characteristically made by immunised Rh null individuals, and anti-Hro(-Rh17) and related antibodies that detect epitopes on the RhCcEe protein.

Anti-Rh29 :Only Rh null blood, which is extremely rare, is suitable.

Anti-Hr0 (-Rh17). Only Rh null or D-- (or related phenotype) blood is suitable.

Give antigen-negative red cells Anti-A, -B, -A,B Anti-M (active at 37oC), -S, -s, -U All Rh antibodies (except anti-Cw) Anti-Lub, -Lu3 Kell antibodies (including anti-K, -k, -Kpb, -Jsa, -Jsb, -Ku (but not anti-Kpa ,

-Ula and -K17) All Duffy antibodies (anti-Fya, -Fyb, -Fy3, -Fy5) All Kidd antibodies (anti-Jka, -Jkb, -Jk3) Anti-Dib, -Wrb Anti-Sc1 Anti-Coa Anti-H (in Oh individuals) Anti-Kx Alloanti-I (active at 37oC) Anti-P, -PP1Pk Anti-Duclos, -DSLK (Rh null cells suitable) Anti-Vel Anti-AnWj

Give red cells compatible by IAT at

37oC Anti-A1 Anti-N (active at 37oC), -Ena, antibodies to low frequency MNS antigens (anti-

Mia ) Anti-P1 (active at 37oC) Anti-Lua Anti-Cw Anti-Lea, -Leb, -Lea+b Anti-Kpa, Ula, -K17 Anti-Wra Anti-Ytb Anti-Xga Anti-Doa, -Dob Anti-Dia Anti-Cob Anti-H/HI in para-Bombay, use ABO identical Anti-HI (in patients with common ABO phenotypes) Anti-Ina Autoanti-I

Give serologically least incompatible red cells,

but antigen-negative red cells for strong

examples of the antibody(reaction strength ≥3

by IAT.

Antibodies to other (not anti-Lub or -Lu3) high frequency Lutheran antigens

Anti-Yta Anti-Gya, -Hy, -Joa and other Dombrock

antibodies Cromer antibodies Anti-Inb Anti-Jra Anti-Lan Anti-Ata

Give ABO/D compatible, least

incompatible red cells Anti-LWa, -LWab Chido/Rodgers antibodies Gerbich antibodies Knops antibodies Anti-JMH Anti-MER2 Anti-Csa Anti-Era Anti-LKE Anti-Emm, -PEL, -ABTI Anti-Sda (avoid Sd(a+++) donors)

Ideally antigen-negative red cells should be

given, but, due to their extreme rarity, ABO/D

compatible, least incompatible red cells should

be used with extra caution

Anti-Sc3, -SC5, -SC6, -SC7 Anti-Co3 Anti-Oka Anti-Gil Anti-CD59 Anti-MAM

1. Correction of anaemia wherever possible.2. Autologous pre-deposit (for surgery).3. Cell salvage (for surgery).4. Calling up donors of known phenotype.5. Testing members of the patient s family, especially

siblings5. Consultation of the National and International Frozen

Blood Banks.6. Mass screening of donations.7. Consultation of the International Rare Donor Panel.8. Transfusing serologically incompatible (or least

incompatible ) blood, with or without immunosuppression.

When compatible blood is not readily available

When antigen-positive blood is to be

transfused

Antibodies that show strong reactivity by IAT may be more active in vivo than if the same antibody showed weaker reactions.

Some of the antibodies listed are extremely rare and little or nothing is known about their clinical significance: Absence of evidence of clinical significance does not mean that a transfusion of incompatible blood will be uneventful.

The decision to issue ABO, compatible RhD matched, least incompatible blood should be made on the balance of risk of severe haemorrhage (anaemia, urgent requirement), versus a haemolytic transfusion reaction with potential complications including renal failure.

The patient should be monitored closely and reviewed.

When antigen-positive blood is to

be transfused

ABO compatible, RhD matched, serologically least incompatible blood, should be transfused with extra caution.

Where appropriate, use of intravenous immunoglobulin and high dose steroids, should be considered.

The transfusion should be given at the slowest rate consistent with the clinical condition and the patient observed closely throughout.

CASE 1

6 years old thalessmic male child History of multiple transfusions IAT

Antibody Screen

D C E C E Cw

K k Kpa

Kpb

Jsa

Jsb

Fya

Fyb

Jka

Jkb

Lea

Leb

P1

M N S s Lua

Lub

Xg

R1R1

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

R2R2

+ 0 + + 0 0 0 + 0 + 0 + 0 + 0 + 0 + + + 0 + 0 0 + + neg

rr 0 0 0 + + 0 + + + + 0 + 0 + + 0 + 0 + 0 + 0 + 0 + n 3+

Rh-hr Kell Duffy Kidd Lewis P MNS Luth Xg

Antibody identification

D C E c e Cw

K k Kpa Kpb

Jsa

Jsb

Fya

Fyb

Jka

Jkb

Lea

Leb

P1

M N S s Lua

Lub

Xg

1 R1wR1

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

2 R1R1

+ + 0 0 + 0 + + 0 + 0 + 0 + + + 0 + + + 0 + 0 0 + 0 N

3 R2R2

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

4 R’r 0 + 0 + + 0 0 + 0 + 0 + + + + 0 + 0 + + 0 + 0 0 + + N

5 r”r 0 0 + + + 0 0 + 0 + 0 + + 0 + + 0 + 0 0 + + + 0 + + 3+

6 rr 0 0 0 + + 0 + + 0 + 0 + + + 0 + 0 + + 0 + 0 + 0 + + N

7 rr 0 0 0 + + 0 0 + + + 0 + + + + + 0 + 0 + + 0 + 0 + + N

8 R0r + 0 0 + + 0 0 + 0 + 0 + 0 0 + 0 0 0 + + + 0 + 0 + + N

9 rr 0 0 0 + + 0 0 + 0 + 0 + + 0 + + + 0 + 0 + 0 + + 0 + N

10

rr 0 0 0 + + 0 0 + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 + + + N

1 rr 0 0 0 + + 0 0 + 0 + 0 + + + + 0 0 0 + 0 + 0 + + + 0 N

Rh-hr Kell Duffy Kidd Lewis P MNS Luth Xg

Anti- E & Cw

Adsorptiondone with E+Cw-cells

D C E c e Cw

K k Kpa Kpb

Jsa

Jsb

Fya

Fyb

Jka

Jkb

Lea

Leb

P1

M N S s Lua

Lub

Xg eluate

Adsorbedserum

1 R1wR1

+ + 0 0 + + 0 + 0 + 0 + + 0 + 0 + 0 + + 0 0 + 0 + + N 3+

2 R1R1

+ + 0 0 + 0 + + 0 + 0 + 0 + + + 0 + + + 0 + 0 0 + 0 N N

3 R2R2

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

N

4 R’r 0 + 0 + + 0 0 + 0 + 0 + + + + 0 + 0 + + 0 + 0 0 + + N N

5 r”r 0 0 + + + 0 0 + 0 + 0 + + 0 + + 0 + 0 0 + + + 0 + + 3+

N

6 rr 0 0 0 + + 0 + + 0 + 0 + + + 0 + 0 + + 0 + 0 + 0 + + N N

7 rr 0 0 0 + + 0 0 + + + 0 + + + + + 0 + 0 + + 0 + 0 + + N N

8 R0r + 0 0 + + 0 0 + 0 + 0 + 0 0 + 0 0 0 + + + 0 + 0 + + N N

9 rr 0 0 0 + + 0 0 + 0 + 0 + + 0 + + + 0 + 0 + 0 + + 0 + N N

10

rr 0 0 0 + + 0 0 + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 + + + N N

11

rr 0 0 0 + + 0 0 + 0 + 0 + + + + 0 0 0 + 0 + 0 + + + 0 N N

Rh-hr Kell Duffy Kidd Lewis P MNS Luth Xg

Anti-Cw

Anti-E

CASE 3

17 year old female DCT +ve (3+) Blood group A+

Antibody Screen

D C E C E Cw

K k Kpa

Kpb

Jsa

Jsb

Fya

Fyb

Jka

Jkb

Lea

Leb

P1

M N S s Lua

Lub

Xg

R1R1

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

R2R2

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

rr 0 0 0 + + 0 + + + + 0 + 0 + + 0 + 0 + 0 + 0 + 0 + n 1+

Rh-hr Kell Duffy Kidd Lewis P MNS Luth Xg

Antibody identification

D C E c e Cw

K k Kpa Kpb

Jsa

Jsb

Fya

Fyb

Jka

Jkb

Lea

Leb

P1

M N S s Lua

Lub

Xg

1 R1wR1

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

2 R1R1

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

3 R2R2

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

4 R’r 0 + 0 + + 0 0 + 0 + 0 + + + + 0 + 0 + + 0 + 0 0 + + 2+

5 r”r 0 0 + + + 0 0 + 0 + 0 + + 0 + + 0 + 0 0 + + + 0 + + 3+

6 rr 0 0 0 + + 0 + + 0 + 0 + + + 0 + 0 + + 0 + 0 + 0 + + 2+

7 rr 0 0 0 + + 0 0 + + + 0 + + + + + 0 + 0 + + 0 + 0 + + 2+

8 R0r + 0 0 + + 0 0 + 0 + 0 + 0 0 + 0 0 0 + + + 0 + 0 + + 2+

9 rr 0 0 0 + + 0 0 + 0 + 0 + + 0 + + + 0 + 0 + 0 + + 0 + 2+

10

rr 0 0 0 + + 0 0 + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 + + + 2+

Rh-hr Kell Duffy Kidd Lewis P MNS Luth Xg

AUTOADSORPTION done with ZZAP

treated autologouscells

D C E c e Cw

K k Kpa Kpb

Jsa

Jsb

Fya

Fyb

Jka

Jkb

Lea

Leb

P1

M N S s Lua

Lub

Xg eluate

Adsorbedserum

1 R1wR1

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

N

2 R1R1

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

N

3 R2R2

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

N

4 R’r 0 + 0 + + 0 0 + 0 + 0 + + + + 0 + 0 + + 0 + 0 0 + + 3+

N

5 r”r 0 0 + + + 0 0 + 0 + 0 + + 0 + + 0 + 0 0 + + + 0 + + 3+

N

6 rr 0 0 0 + + 0 + + 0 + 0 + + + 0 + 0 + + 0 + 0 + 0 + + 3+

N

7 rr 0 0 0 + + 0 0 + + + 0 + + + + + 0 + 0 + + 0 + 0 + + 3+

N

8 R0r + 0 0 + + 0 0 + 0 + 0 + 0 0 + 0 0 0 + + + 0 + 0 + + 3+

N

9 rr 0 0 0 + + 0 0 + 0 + 0 + + 0 + + + 0 + 0 + 0 + + 0 + 3+

N

10

rr 0 0 0 + + 0 0 + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 + + + 3+

N

11

rr 0 0 0 + + 0 0 + 0 + 0 + + + + 0 0 0 + 0 + 0 + + + 0 3+

N

Rh-hr Kell Duffy Kidd Lewis P MNS Luth Xg

No underlying allo antibody

Autoantibody

sangeeta.pahuja.s@gmail.com