1. Detection and Identification of alloantibodies to Red Cell Antigens

2. Unexpected Alloantibodies:
Any Red Cell
Alloantibodies other than naturally occurring anti-A or anti-B
Detected by: Performing an antibody screen test
Found in: % 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

3. 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 Test
Antibody Screen Test
Cross-match Test
Eluate

4. Once an antibody is detected:
Determine Specificity
Asses Clinical significance
Clinically Significant antibodies defined as:
One that shortens the survival of transfused red cells
Cause 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

5. Preparing Compatible blood for a recipient : Goals
NOTE: If no data exists on certain antibody ,decision must be based on the temp. that one antibody is active
37C
And Or
Reactive 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

6. Techniques:
Techniques for Antibody detection Antibody identification are similar, but methods for:
Antibody detection is broad to detect antibodies with differing patterns of reactivity
Antibody Identification is more focused based on reactivity patterns identified in the antibody detection
Use of flowcharts as a guide to
- expediting process
- minimizing unnecessary tests.

7. Specimen Requirements: Either 1- SerumOr
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

8. commercially available - Available as sets of two or three
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

9. 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

10. - Use panel of selected red cell with known antigen composition
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 weeks

11. - 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

12. 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

13. Antiglobulin Reagents
Antiglobulin phase to detect clinically significant antibodies
Use antiglobulin reagent as
Polyspecific to detect antibodies that bind complement kidd antibodies
IgG- specific

14. Enhancement Media & Enhancement 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)

15. Enhancement Media & Enhancement 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

16. Autologous Control To determine alloantibody from Autoantibody
Not performed in antibody screening
Used with panel cells

17. Basic Antibody Identification Techniques
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

18. Basic Antibody Identification Techniques
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

19. 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 required
The absence of Ag should be confirmed with a potent commercial antisera
FDA requires use of licensed (commercial) reagents

20. 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% C Neg
34% Fya Neg
45% S Neg

21. Selecting Blood for Transfusion
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 ×0.45=1.3% of random donors would be compatible with the patient serum.