Detection and Identification Detection and Identification of alloantibodies to Red Cell of alloantibodies to Red Cell

AntigensAntigens

Unexpected Alloantibodies:

Any Red Cell

Alloantibodies other than naturally occurring anti-A or anti-B

Detected by: Performing an antibody screen test

Found in: 0.3-38% of the population

- Depending on group of patient or Donor Studied

- Sensitivity of the test methods used

Reaction: Only with allogenic red cells VS. Auto- antibodies that reacts with red cells from

other individuals

Immunization to red cell Ag:

Results from pregnancy-Transfusion-Transplantation- Injection with

immunogenic material-No specific- Immunizing event

Initial detection of alloantibodies

In tests that use serum of Plasma including:ABO TestAntibody Screen TestCross-match TestEluate

Once an antibody is detected:Determine SpecificityAsses Clinical significance

Clinically Significant antibodies defined as:One that shortens the survival of transfused red

cellsCause Hemolytic disease of the newborn

(HDN)Hemolytic Transfusion reaction

* Reported experience with other examples of antibody with the same specificity can be used in assessing clinical significance

NOTE: If no data exists on certain antibody ,decision must be based on the temp. that one antibody is active 37C

And OrReactive by the Indirect Antiglobulin Test (IAT)

Preparing Compatible blood for a recipient :Goals• Detect as many clinically significant Antibodies• Detect as few clinically insignificant Antibodies• Complete the procedure in a timely manner Patients with clinically significant antibodies should,

receive red cells that are negative for the corresponding antigen.

In prenatal testing, the specificity and immunoglobulin class of an antibody influence the likelihood of HDN

Techniques:Techniques for Antibody detection Antibody identification are

similar, but methods for:o Antibody detection is broad to detect antibodies with

differing patterns of reactivityo Antibody Identification is more focused based on reactivity

patterns identified in the antibody detectiono Use of flowcharts as a guide to

- expediting process

- minimizing unnecessary tests.

Specimen Requirements:

Either 1- Serum

Or

2- Plasma

10-ml aliquot is enough for identifying simple antibody specificities

Medical History:When performing an antibody identification it is useful to

know:

1- Patient’s clinical diagnosis

2- History of Transfusion

3- History of pregnancies

4- Recent drug therapy

5- Ethnic background

6- Sex M.F.

7- Age

Reagents:

Screening Cells:

- Group O red cells

commercially available

- Available as sets of two or three

vials of single-donor red cells

- Pooled antibody screening cells are used for donor serum and not for recipients’ specimens

Reagents:

- Reagent cells licensed by the Food and Drug administration (FDA) must Express the following antigens D,C,E, c, e, M,N,S, s, P1,Lea ,Leb,K,K, Fya, Fyb, Jka, Jkb

-weakly reactive antibodies react only with screening red cells from donors who are homozygous , a serologic phenomenon called dosage effect .

- When not in use reagent should be refrigerated at 2-8ºС

Red cell Panels:- Use panel of selected red cell with known antigen composition- Obtained commercially or institutionally (Horne made) from local- Group O cells except in special circumstances- Each cell from different individual- The pattern of reactivity should not overlap with

any other, Example 0 Not all K+ should also be E+- Expiration for commercially prepared cells is every 2-4 weeks

- Use only reagent phenotype listing sheet for the specific kit

- Suspension of 2-5% Red Cells in a Preservative medium

Saline – Suspended Red cells Technique: Simplest serologic Incubation of saline suspended cell with serum at

at: Immediate spin Room Temperature 37oC

Antibodies reacting below 37 oC are Anti-M, N, P1, Lea, Leb

This phase reading can be omitted to avoid finding little clinically significant antibodies.

37 oC antibody detection anti – D, K, E also some

Some antibodies like anti- Lea, Jka can be detected in this phase by Lysing antigen- incompatible red cells

Antiglobulin Reagents Antiglobulin phase to detect clinically significant

antibodies Use antiglobulin reagent as

Polyspecific to detect antibodies that bind complement kidd antibodies

IgG- specific

Enhancement Media & Enhancement Media & Enhancement TechniquesEnhancement Techniques

Techniques Temperature reduction Increased serum to cell ratio Increased incubation time Alteration of PH Inhibition tests

Lewis substances (saliva) P1 substance (pigeon egg whites) Sola substance (body fluids-urine) Pooled plasma (Chido & Rodgers)

Enhancement Media & Enhancement Media & Enhancement TechniquesEnhancement Techniques

Include incubating Serum/Plasma and reagent red cells in such media as: Albumin 22% or 30% LISS (low-ionic-strength saline) PEG (polyethylene glycol) Enzyme techniques Polybrene

Autologous ControlAutologous Control

To determine alloantibody from Autoantibody

Not performed in antibody screening Used with panel cells

Basic Antibody Identification Basic Antibody Identification TechniquesTechniques

Traditional serologic method in the united states are based agglutination performed in tubes or micro-plates

Other methods are not dependent on agglutination– Solid- Phase– Flocytometry– Agglutination test (gel tests) column techniques– Automated systems

Initial observation

Basic Antibody Identification Basic Antibody Identification TechniquesTechniques

Interpreting results– Positive and Negative– Exclusion or “crossing out”

Antibodies to High- Incidence Antigens anti-H, I, P, LW , Sda, Vel , Ch, Rg, U, Jsb

Antibodies to low-Incidence Antigens anti-Wra

Selecting Blood for Selecting Blood for TransfusionTransfusion

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 required The absence of Ag should be confirmed with a potent

commercial antisera FDA requires use of licensed (commercial) reagents

Selecting Blood for Selecting Blood for TransfusionTransfusion

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 donors18% C Neg34% Fya Neg

45% S Neg

Selecting Blood for Selecting Blood for TransfusionTransfusion

The Frequency of compatible units would be 0.18×0.34 ×0.45= 0.028

If 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.