antibody screening and identification. antibody screening purpose purpose the purpose of the...
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Antibody Screening and Identification
Antibody Screening
Purpose The purposepurpose of the antibody screen is to detect red
blood cell antibodies other than anti-A or anti-B. These antibodies are called “unexpected”
because only 0.3 to 2 % of the general population have positive antibody screen.
Once an unexpected antibody is detected, antibody identification studies are performed to determine the antibodies specificity and clinical significance.
Antibody screening is usually undertaken at the same time as blood grouping and in advance of selecting blood for transfusion.
Antibody screening may be more reliable and sensitive than cross matching- most notably anti-Jka/Jkb but also anti-Fya, -Fyb, -S, and –s. Screening cells can be selected to reflect this, whereas donor cells are usually of unknown zygosity.
In addition, reagent red cells are easier to standardize than donor cells, and there is potentially less opportunity for procedural error, particularly in automated systems.
Clinically significant antibodies are those that are capable of causing patient morbidity as a result of accelerated destruction of a significant proportion of transfused red cells.
Antibody Screening
Antibody screening test involve testing patient’s serum against two or three reagent red blood cell samples called screening cells
Screening cells are commercially prepared group O group O cell suspensions obtained from individual donors that are phenotype for the most commonly encountered and clinically important red blood cell antigens.
Why group O ??
Antibody Screening
Group O cells are used so that naturally occurring anti-A or anti-B will not interfere with detection of unexpected antibodies.
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.
Antibody Screening
Screening Cells. The screening cells are available in three form
1- 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.
Two or three cells screening sets are required for detection of antibodies in pre-transfusion testing.
No single test will detect all blood group Abs, but an effective compromise is as :
1. Direct agglutination test in NISS or LISS 2. IAT after incubation at NISS or LISS 3. Enzyme tests; especially for detection of
Rh Abs. A positive result in Ab screen should be
followed by an Ab ID against a comprehensive cell panel.
Antibody Screening
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).
Antibody Screening
Grading Reactions. AggregationAggregation or hemolysishemolysis 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).
Why do we need to identify?
Antibody identification is needed for transfusion purposes and is an important component of compatibility testing
It will identify any unexpected antibodies in the patient’s serum
If a person with an antibody is exposed to donor cells with the corresponding antigen, serious side effects can occur .
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 serumReagent antisera + patient RBCs
Reagent RBCs
Screening Cells and Panel Cells are the same with minor differences: Screening cells
Antibody detectiondetection Sets of 2 or 3 vials
Panel cells Antibody identificationidentification At least 10 vials per set
Antibody Panel vs. Screen
An antibody panel is just an extended version of an antibody screen
The screen only uses 2-3 cells:
Antibody Panel
An antibody panel usually includes at least 10 panel cells:
Panel
Group O red blood cells
Panel
Each of the panel cells has been antigen typed (shown on antigram) + refers to the presence of the antigen 0 refers to the absence of the antigen
Example: Panel Cell #10 has 9 antigens present: c, e, f, M, s, Leb, k, Fya, and Jka
Panel
An autocontrol should also be run with ALL panels
Autocontrol
Patient RBCs +
Patient serum
Panel
The same phases used in an antibody screen are used in a panel
• IS
• 37°
• AHG
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
(LISS) 37°C Phase
2 drops of LISS are added, mixed and incubated for 10-15 minutes
Centrifuge and check for agglutination
Record results
(LISS) 37°C Phase
2+
00
2+
002+
0
02+
0
000
IAT Phase (or AHG)
Indirect Antiglobulin Test (IAT) – we’re testing whether or not possible antibodies in patient’s serum will react with RBCs in vitro
To do this we use the Anti-Human Globulin reagent (AHG) Polyspecific Anti-IgG Anti-complement
AHG Phase
Wash cells 3 times with saline (manual or automated)
Add 2 drops of AHG and gently mix Centrifuge Read Record reactions
AHG Phase
2+
00
2+
002+
0
02+
0
000
00
00
0
00
0
0000
00
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000
0 0 0
And don’t forget….
….add “check” cells to any negative AHG !
IS LISS 37°
AHG CC
2+ 0 0 0 0 0 0 0 0
2+ 0 0 0 0 0 0 0 0
2+ 0 0 0 0 0
2+ 0 0 0 0 0 0 0 0
All cells are negative at
AHG, so add
“Check” Cells
You have agglutination…now what?
2+
00
2+
002+
0
02+
0
000
00
00
0
00
0
0000
00
00
000
0 0 0
??
CC
Interpreting Antibody Panels
There are a few basic steps to follow when interpreting panels
1. “Ruling out” means crossing out antigens that did not react
2. Circle the antigens that are not crossed out
3. Consider antibody’s usual reactivity4. Look for a matching pattern
An antibody will only react with cells that have the corresponding antigen;
antibodies will not react with cells that do not have the
antigen
Always remember:
Here’s an example:
1. Ruling Out
2+
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2+
002+
0
02+
0
000
00
00
0
00
0
0000
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000
0 0 0
Cross out antigens that show NO REACTION in any phase; do NOT cross out heterozygous antigens that show dosage.
About reaction strengths……
Strength of reaction may be due to “dosage” If panel cells are homozygous, a strong
reaction may be seen If panel cells are heterozygous, reaction
may be weak or even non-reactive Panel cells that are heterozygous
should not be crossed out because antibody may be too weak to react
2. Circle antigens not crossed out
2+
00
2+
002+
0
02+
0
000
00
00
0
00
0
0000
00
00
000
0 0 0
3. Consider antibody’s usual reactivity
2+
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2+
002+
0
02+
0
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0
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0 0 0
Lea is normally a Cold-Reacting antibody (IgM), so it makes sense that we see the reaction in the IS phase of testing; The E antigen will usually react at warmer temperatures
4. Look for a matching pattern
2+
00
2+
002+
0
02+
0
000
00
00
0
00
0
0000
00
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000
0 0 0
…Yes, there is a matching pattern!
E doesn’t match and it’s a warmer rx Ab
Interpretation
anti-Leaa
Guidelines
Again, it’s important to look at: Autocontrol
Negative - alloantibody Positive – autoantibody or DTR (i.e.,alloantibodies)
Phases IS – cold (IgM) 37° - cold (some have higher thermal range) or
warm reacting AHG – warm (IgG)…significant!!
Reaction strength 1 consistent strength – one antibody Different strengths – multiple antibodies or dosage
Guidelines (continued)
Matching the pattern Single antibodies usually shows a
pattern that matches one of the antigens (see previous panel example)
Multiple antibodies are more difficult to match because they often show mixed reaction strengths
Rule of three
The rule of three must be met to confirm the presence of the antibody
How is it demonstrated? Patient serum MUST be:
Positive with 3 cells with the antigen Negative with 3 cells without the antigen
Our previous example fulfills the “rule of three”
2+
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2+
002+
0
02+
0
000
00
00
0
00
0
0000
00
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000
0 0 0
3 Negative cells
3 Positive cells
Panel Cells 1, 4, and 7 are positive for the antigen and gave a reaction at immediate spin
Panel Cells 8, 10, and 11 are negative for the antigen and did not give a reaction at immediate spin
What if the “rule of three” is not fulfilled?
If there are not enough cells in the panel to fulfill the rule, then additional cells from another panel could be used
Most labs carry different lot numbers of panel cells
THE END!!