ANTIBODY SCREENING

DR NAGLAA MAKRAM

CONSULTANT CLIN ICAL PATHOLOGY

BGH

Antibody screens are used for:Patients needing a transfusionCases of transfusion reactionsBlood and plasma donorsPregnant women

Purpose

To ensure optimal survival of transfused red cells in recipients.

Routine pretransfusion testing

ABO Group Rh (D) type Antibody screen for irregular antibodies IAT with donor’s RBC’s

Antibody screen

Principle: Antibody screen test is done using patient’s serum and antibody screen cells to try to detect unexpected antibodies that are capable of destroying transfused donor cells in vivo.

These antibodies are called “unexpected” because only 0.3 to 2 % of the general population have positive antibody screen.

Unexpected antibodies are a result of red cell stimulation (transfusion, HDN)

Unexpected antibodies may be:Clinically significant (IgG)

Not clinically significant (IgM)

Clinically Significant antibodies defined as:

One that shortens the survival of transfused red cells

Cause Hemolytic disease of the newborn (HDN)Hemolytic Transfusion reaction

Key Concepts

In blood banking, we test “knowns” with “unknowns”

When detecting and/or identifying antibodies, we test patient serum (unknown) with reagent RBCs (known)

Known:Unknown:Reagent RBCs+patient serum

Reagent antisera+patient RBCs

Characteristics of screen cells

Group O cells (so that naturally occurring anti-A or anti-B will not interfere with detection of unexpected antibodies. )

Known antigenic content They should be positive for all common blood group

antigens They should come from donors who are

homozygous for genes that produce antigens showing dosage.

There is no requirement that screening cells contain red cells with homozygous expression of antigens, however, the most workers prefer that such red cells are included in screening cells sets because many antibodies, especially JK and M antibodies, show dosage effect and give stronger reactions when tested against cells with homozygous expression of their corresponding antigen.

As a result of dosage, weakly reacting antibody may not be detected if serum samples are not tested against red cells with homozygous expression of the their corresponding antigen. (Rh, Duffy, Kidd).

Examples

Fya Fyb

SCI + + 2+SCII 0 + 4+

Fya Fyb

SCI + 0 4+

SCII 0 + 0

If patient’s serum contains anti-Fya, there will be a

stronger reaction because SCI is

homozygous for the Duffy antigen

In this case, the person has anti-Fyb. The antibody

reacts weaker with SCI (heterozygous) and

stronger with SCII (homozygous)

Detection of very low levels of antibody in a recipient’s serum is important because transfusion of antigen-positive red cells may result in a secondary immune response with rapid production of antibody

and subsequent destruction of transfused red blood cells.

The cells are selected so that the following antigens are present on at least one of the cell sample;

D, C, E, c, e, M N, S, s, P, Lea, Leb, K, k, Fya, Fyb, and Jkb.

Screening cells may also contain low incidence antigens like V, Cw, and Kpa

The presence of these antigens is not required for screening cells

D

Fyb

Lea

Jkb

Jka

s

SNM

k

K

e

E

c

C

BA

Lua FybFya

Leb

Screening cells come with a sheet of paper called an antigram.

Screening cells are an already prepared 2-5% RBC suspension

An antigram (2 or 3 cells) will list the antigens present in each vial A reaction to one or more cells indicates the presence of an unexpected antibody

It is important that the lot number on the screening cells matches the lot number printed-On the anti-gram

because antigen make up will vary with each lot.

Reagent Red Blood Cell Screening CellSectional Listing of Antigens Present

Nocell

D C E c e K k Fya

Fyb

Jka

Jkb

Lea

Leb S s M N P

1IS

37 AHG CC

I + + 0 + + + 0 0 + + + 0 + + + + 0 + 0 0 2+

II + 0 + + 0 0 + + 0 + 0 0 + 0 + + 0 + 0 0 0 2+

Screening Cells:

The screening cells are available in three form1- A single vial of no more than two donors pooled together in

one vial.2- Two vials each with a different donor.3- 3 vials representing three different donors.

single-donor vials offer increased sensitivity

Two or three cells screening sets are required for detection of antibodies in pre-transfusion testing.

Antibody ID Testing

A tube is labeled for each of the panel cells plus one tube for AC:

AC

1 2 3 4 5 6 7 8 9 10 11

1 drop of each panel cell

+

2 drops of the patients serum

IS Phase

Perform immediate spin (IS) and grade agglutination; inspect for hemolysis

Record the results in the appropriate space as shown:

2+

00

Last tube

Auto-logous Control.

Autologous control is considered as part of the Ab screening, it can be performed in parallel with the Ab screen and involves testing the patient’s serum against the patient’s red blood cells.

A positive auto-logous control is an abnormal finding and usually means that patient has a positive direct antiglobulin test (DAT).

Autocontrol

Screen AntibodyPanel (w/AC)

If Positive

If Po

sitiv

e

DAT

The AC and DAT can help in determining whether the antibodies are directed against the patient’s cells or transfused cells (allo or autoantibody).

Interpretation

Remember Landsteiner’s Rule

Individuals DO NOT make allo-antibodies against antigens they

have

Multiple antibodies

Multiple antibodies may be more of a challenge than a single antibody

Why?–Reaction strengths can vary

–Matching the pattern is difficult

Grading Reactions

Grading Reactions.Aggregation or hemolysis of test red blood cells is the visible end point of an Ab-Ag interaction.

Test results should be read immediately after centrifugation as delays in reading may cause elution of antibody and false- negative test results

The first step in reading hem-agglutination reactions is inspection of the supernatant for signs of hemolysis (red or pink coloration).

Red blood cells should be re-suspended by gentle shaking or tilting the tube until the cells no longer adhere to the sides. Agglutination is graded once the red blood cells are re-suspended.

Agglutination reactions are routinely graded as negative (no agglutination).

Weakly positive, and 1+ through 4+. The degree of the positive reaction generally indicates the amount of Ab present not its significance.

Interpretation

Agglutination or hemolysis at any stage of testing is a positive test result, indicating the need for antibody identification studies.

However, evaluation of the antibody screen and autologous control results can provide clues and give direction for the identification and resolution of the antibody or antibodies.

The investigator should consider the following questions: 1. In what phase(s) did the reaction(s) occur? 2. Is the autologous control negative or positive? 3. Did more than one screening cell sample react, and, if so, did

they react at the same strength and phase? 4. Is hemolysis or mixed-field agglutination present? 5. Are the cells truly agglutinated, or is rouleaux present?

Interpretation

1. In what phase(s) did the reaction(s) occur?

Antibodies of the IgM class react best at low temperatures and are capable of causing agglutination of saline-suspended RBCs (immediate spin reading).

Antibodies of the IgG class react best at the AHG phase. Of the commonly encountered antibodies,

anti-N. anti-I, and anti-PI are frequently IgM, whereas those directed against Rh. Kell. Kidd, and Duffy

antigens are usually IgG. Lewis and M antibodies may be IgG, IgM, or a mixture of

both.

Colder reacting antibodies are therefore considered insignificant and just cause interference when performing lab testingThe only important thing to remember concerning cold antibodies is that they may bind complement if a persons body temperature becomes low

Open-heart surgery Hypothermia If a lab uses an AC with the screen and it is

positive, they may run a DAT (patient cells + AHG) to detect in vivo coating

Positive Coombs

Negative Coombs

2. Is the autologous control negative or positive?

A positive antibody screen and a negative autologous control indicate that an alloantibody has been detected.

A positive autologous control may indicate the presence of autoantibodies or antibodies to medications.

If the patient has been recently transfused, the positive autologous control may be caused by alloanti body coating circulating donor RBCs.

Evaluation of samples with positive autologous control or DAT re sults is often complex and may require a lot of time and experience on the part of the investigator.

3.Did more than one screening cell sample react, and, if so, did they react at the same strength and phase?

A single antibody specificity should be suspected when all cells react at the same phase and strength.

Multiple antibodies are most likely when cells react at different phases and strengths.

autoantibodies are suspected when the autologous control is positive.

4. Is hemolysis or mixed-field agglutination present?

Certain antibodies-such as anti-Lea, anti-Leb, anti P+P1+Pk, and anti-Vel-are known to cause in vitro hemolysis.

Mixed-field agglutination is associated with anti-Sda and Lutheran antibodies.

5. Are the cells truly agglutinated, or is rouleaux present?

Serum from patients with altered albumin-to-globulin ratios (e.g., patients with multiple myeloma) or who have received high-molecular-weight plasma expanders (e.g.. dextran) may cause nonspecific aggregation of RBCs, known as rouleaux.

Rouleaux is not a significant finding in antibody screening tests, but it is easily confused with antibody-mediated agglutination.

Knowledge of the following characteristics of rouleaux helps in differentiation between rouleaux and agglutination:

a. Cells have a "stacked coin" appearance when viewed microscopically.

b. Rouleaux is observed in all tests containing the patient's serum, including the autologous control and the reverse ABO typing.

c. Rouleaux does not interfere with the AHG phase of testing because the patient's serum is washed away prior to the addition of the AHG reagent.

d. Unlike agglutination, rouleaux is dispersed by the addition of 1 to 3 drops of saline to the test tube.

Limitations of pretransfusion testing

Patient’s antibody is too week to be detected. Patient misidentification Hemolysis before entering the patient Non-hemolytic reactions Adverse reactions Transfusion Transmissible diseases cannot detect all such antibodiescannot detect all such antibodies.

Limitations Antigens with frequencies of less than 10 percent

(e.g., Cw. Lu-, Kpa) are not usually represented on screening cells, and, as a result, their corresponding antibodies are not detected in routine screening tests.

antibody levels decrease over time when the individual is no longer exposed to the corresponding antigen.

If the level of an RBC antibody drops too low, results of antibody screening tests and crossmatches will appear negative and may lead to transfusion of donor units that carry the corresponding antigen.

Re exposure to the RBC antigen will elicit a secondary immune response, resulting in a dramatic increase in the antibody titer and possible immunologic destruction of the transfused RBCs.

this is called a delayed hemolytic transfusion reaction (DHTR) because it occurs days or weeks after the transfusion.

Patient History

GET THE HISTORY!!– Mixed red cell populations from a previous transfusion

can remain for up to 3 months– Patient may have come from another hospital– Some diseases are associated with antibodies– Some antibodies occur at a higher frequency in some

races– Get diagnosis, age, race, etc…

Potentiators

Used in antibody detection and identification to enhance antigen-antibody reaction– Saline (may only enhance if incubated long time)– Low-ionic strength solution (LISS)…common– Bovine serum albumin (BSA)– Polyethylene glycol (PEG)– Proteolytic enzymes (can destroy some antigens)

Potentiators

Albumin Serum/cell mixture should incubate at least 20 - 30 minutes; doesn’t enhance warm autoantibodies

LISS Incubation time of 10 minutes;lowers ionic strength allowing better reaction; sensitive and quick!

PEGPolyethylene

glycol

Enhances warm autoantibodies; does not react well with insignificant antibodies (IgM)

Advantages

Screen cells can detect antibodies more effectively than can donor cells because they come from donors who are homozygous.

Specimen requirements for Pretransfusion testing

Special Identification ProceduresIf the patient was transfused or pregnant in the

past three months, the blood specimen used for pretransfusion testing must be no older than 3 days.

Selecting Blood for Transfusion

For a patient with clinically significant Antibodies (37oC and IAT)

Red cell should be tested and be negative for the appropriate antigen

Even if Ab. Is no longer detectable to prevent a secondary immune response

An antiglobulin cross-match is requiredThe absence of Ag should be confirmed with a potent

commercial antiseraFDA requires use of licensed (commercial) reagents

Selecting Blood for Transfusion

When rare type is needed –High- Incidence

–Multiple antibodies frequency of random donors negative for each antigen should be used ,

–Example: serum contains anti-C, Fya and s among random donors

18%CNeg34%FyaNeg

45%SNeg

Selecting Blood for Transfusion

The Frequency of compatible units would be 0.18×0.34 ×0.45= 0.028If patient is group O then 45% of random donors are group O then 0.028 ×0.45=1.3% of random donors would be compatible with the patient serum.

Requisitions

Signatures of blood collector & nurse Patient identification Name of the attending physician Birth date Past history Diagnosis Blood products required

Type and Screen

Cross-match and Savevs

Type and Screen

Once upon a time (in bloodland) all donors were crossmathed by IAT, even if the antibody screen was negative.

In addition, for many types of surgeries a high percentage of the donor blood that was crossmatched and held for patients was never used.

This is wasteful to both the time and money.

Advantages of Type and Screen

Better use of blood donor, as it is not tied up by being cross-matched and held

More efficient service of patients

Right blood

To the

Right patient

At the

Right time

savesblood livesSafe

Example 1Screening

CellIS 37°C AHG CC*

I 0 0 0

II 0 0 2+ ND

• IgG antibody

• Single specificity

• CC: Coombs Control Red Blood Cells• ND: Not Done

Example 2Screening Cell IS 37°C AHG CC

I 0 0 3+II 0 2+ 3+

• IgG antibody

• Multiple specificities

Example 3Screening Cell IS 37°C AHG CC

I 1+ 0 0 II 3+ 0 0

• IgM antibody

• Single specificity showing dosage

Neg AHG, add CC

Example 4Screening Cell IS 37°C AHG CC

I 0 0 2+II 0 0 2+

• IgG antibody

• Allo or autoantibody ?

( don’t know without further testing)

Possible results

All antibody screen cells are negative

One or more screen cells are positive

Screen cells negative and donor positive in IAT phase