Transcript Detection and Identification of alloantibodies to Red Cell

Detection and Identification
of alloantibodies to Red Cell
Antigens
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. Autoantibodies that reacts with red cells from other
individuals
Immunization to red cell Ag:
Results from pregnancy-TransfusionTransplantation- Injection with
immunogenic material-No specificImmunizing event
Initial detection of alloantibodies
In tests that use serum of Plasma including:
ABO Test
Antibody Screen Test
Cross-match Test
Eluate
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
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
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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 reactivity
o Antibody Identification is more focused based on reactivity
patterns identified in the antibody detection
o 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 Techniques
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Techniques
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Temperature
reduction
Increased serum to cell ratio
Increased incubation time
Alteration of PH
Inhibition tests
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Lewis substances (saliva)
P1 substance (pigeon egg whites)
Sola substance (body fluids-urine)
Pooled plasma (Chido & Rodgers)
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
Autologous Control
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To determine alloantibody from
Autoantibody
Not performed in antibody screening
Used with panel cells
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
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Solid- Phase
Flocytometry
Agglutination test (gel tests) column techniques
Automated systems
Initial observation
Basic Antibody Identification
Techniques
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Interpreting results
– Positive and Negative
– Exclusion or “crossing out”
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Antibodies to High- Incidence Antigens
anti-H, I, P, LW , Sda, Vel , Ch, Rg, U, Jsb
 Antibodies to low-Incidence Antigens antiWra
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
Selecting Blood for
Transfusion
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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
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 0.028 ×0.45=1.3%
of random donors would be compatible with
the patient serum.