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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.