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Antibody Identification
Renee Wilkins, PhD, MLS(ASCP)cm
CLS 325/435School of Health Related ProfessionsUniversity of Mississippi Medical Center
The Basics…..
As you recall, Antibody Screens use 2 or 3 Screening
Cells to “detect” if antibodies are present in the serum
If antibodies are detected, they must be identified…
present
Not present
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 detection Sets of 2 or 3 vials
Panel cells Antibody identification 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
AutocontrolPatient 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
00
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+
00
2+
002+
0
02+
0
000
00
00
0
00
0
0000
00
00
000
0 0 0
Cross out antigens that show NO REACTION in any phase; do NOT cross out heterozygous antigens that show dosage.
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+
00
2+
002+
0
02+
0
000
00
00
0
00
0
0000
00
00
000
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
00
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
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 (see first example)
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
A p-value ≤ 0.05 must be observed This gives a 95% confidence interval 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+
00
2+
002+
0
02+
0
000
00
00
0
00
0
0000
00
00
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
Phenotyping
In addition to the rule of three, antigen typing the patient red cells can also confirm an antibody
How is this done? Only perform this if the patient has NOT
been recently transfused (donor cells could react)
If reagent antisera (of the suspected antibody) is added to the patient RBCs, a negative reaction should result…Why?
Remember Landsteiner’s Rule
Individuals DO NOT make allo-antibodies against
antigens they have
Multiple antibodies
Multiple antibodies may be more of a challenge than a single antibody
Why? Reaction strengths can vary Matching the pattern is difficult
So what is a tech to do?
Several procedures can be performed to identify multiple antibodies Selected Cells Neutralization Chemical treatment
Proteolytic enzymes Sulfhydryl reagents ZZAP
Selected Cells
Selected cells are chosen from other panel or screening cells to confirm or eliminate the antibody
The cells are “selected” from other panels because of their characteristics
The number of selected cells needed depends on how may antibodies are identified
Selected Cells
Every cell should be positive for each of the antibodies and negative for the remaining antibodies
For example: Let’s say you ran a panel and identified
3 different antibodies: anti-S, anti-Jka, and anti-P1
Selected cells could help…
Selected Cells
Selected cells
S Jka P1 IS LISS 37°
AHG
#1 + 0 0 0 0 2+
#5 0 + 0 0 0 3+
#8 0 0 + 0 0 0
These results show that instead of 3 antibodies, there are actually 2: anti-S and anti-Jka
Neutralization
Some antibodies may be neutralized as a way of confirmation
Commercial “substances” bind to the antibodies in the patient serum, causing them to show no reaction when tested with the corresponding antigen (in panel)
Neutralization
Manufacturer’s directions should be followed and a dilutional control should always be used The control contains saline and serum
(no substance) and should remain positive
A control shows that a loss of reactivity is due to the neutralization and not to the dilution of the antibody strength when the substance is added
Neutralization
Common substances P1 substance (sometimes derived from hydatid
cyst fluid) Lea and Leb substance (soluble antigen found
in plasma and saliva) I substance can be found in breast milk Sda substance derived from human or guinea
pig urine
**you should be aware that many of these substances neutralize COLD antibodies; Cold antibodies can sometimes mask more clinically significant antibodies (IgG), an important reason to use neutralization techniques
Enzymes (proteolytic)
Can be used to enhance or destroy certain blood group antigens
Several enzymes exist: Ficin (figs) Bromelin (pineapple) Papain (papaya)
In addition, enzyme procedures may be One-step Two-step
Enzymes
Enzymes remove the sialic acid from the RBC membrane, thus “destroying” it and allowing other antigens to be “enhanced”
Antigens destroyed: M, N, S, s, Duffy
Antigens enhanced: Rh, Kidd, Lewis, I, and P
Enzyme techniques
One-stage Enzyme is added directly to the
serum/cell mixture Two-stage
Panel cells are pre-treated with enzyme, incubated and washed
Patient serum is added to panel cells and tested
Enzyme techniques
If there is no agglutination after treatment, then it is assumed the enzymes destroyed the antigen
Enzyme treament
Anti-K
Perfect match for anti-Fya
•Duffy antigens destroyed•Kell antigens not affected
Enzyme treatment
Sulfhydryl Reagents
Cleave the disulfide bonds of IgM molecules and help differentiate between IgM and IgG antibodies
Good to use when you have both IgG and IgM antibodies (warm/cold) Dithiothreitol (DTT) is a thiol and will
denature Kell antigens 2-mercaptoethanol (2-ME)
ZZAP
A combination of proteolytic enzymes and DTT
Denatures Kell, M, N, S, Duffy and other less frequent blood group antigens
Does not denature the Kx antigen Good for adsorption techniques
“frees” autoantibody off patient’s cell, so that autoantibody can then be adsorbed onto another RBC
Autoantibodies….
Warm & Cold Reacting
Autoantibodies
Autoantibodies can be cold or warm reacting
A positive autocontrol or DAT may indicate that an auto-antibody is present
Sometimes the autocontrol may be positive, but the antibody screening may be negative, meaning something is coating the RBC
Getting a positive DAT
We have focused a lot on the IAT used in antibody screening and ID, but what about the DAT?
The direct antiglobulin test (DAT) tests for the in vivo coating of RBCs with antibody (in the body)
AHG is added to washed patient red cells to determine this
What can the DAT tell us?
Although not always performed in routine pretransfusion testing, a positive DAT can offer valuable information If the patient has been transfused, the
patient may have an alloantibody coating the transfused cells
If the patient has NOT been transfused, the patient may have an autoantibody coating their own cells
Identifying autoantibodies
Auto-antibodies can sometimes “mask” clinically significant allo-antibodies, so it’s important to differentiate between auto- and allo-antibodies
Cold autoantibodies
React at room temperature with most (if not all) of the panel cells and give a positive autocontrol
The DAT is usually positive with anti-C3 AHG (detects complement)
Could be due to Mycoplasma pneumoniae, infectious mono, or cold agglutinin disease
Cold autoantibodies
Mini-cold panels can be used to help identify cold autoantibodies
Since anti-I is a common autoantibody, cord blood cells (no I antigen) are usually included
Group O individual with cold autoanti-I
Group A individual with
cold autoanti-IH
Anti-IH is reacting weakly with the cord cells (some H antigen present)
Avoiding reactivity Cold autoantibodies can be a
nuisance at times. Here are a few ways to avoid a reaction: Use anti-IgG AHG instead of
polyspecific. Most cold antibodies react with polyspecific AHG and anti-C AHG because they fix complement
Skipping the IS phase avoids the attachment of cold autoantibodies to the red cells
Use 22% BSA instead of LISS
Other techniques
If the antibodies remain, then prewarmed techniques can be performed: Red cells, serum, and saline are
incubated at 37° before being combined Autoadsorption is another
technique in which the autoantibody is removed from the patients serum using their own red cells The serum can be used to identify any
underlying alloantibodies
Warm autoantibodies
More common that cold autoantibodies
Positive DAT due to IgG antibodies coating the red cell
Again, the majority of panel or screening cells will be positive
The Rh system (e antigen) seems to be the main target although others occur
Warm autoantibodies
Cause warm autoimmune hemolytic anemia (WAIHA)…H&H
How do you get a warm autoantibody? Idiopathic Known disorder (SLE, RA, leukemias, UC,
pregnancy, infectious diseases, etc) Medications
Several techniques are used when warm autoantibodies are suspected…
Elution (whenever DAT is positive)
Elution techniques “free” antibodies from the sensitized red cells so that the antibodies
can be identified
Y
Y
YYSensitized
RBC
Positive DAT
Elution YYY Y
YY
Frees antibody Antibody ID
Elution
The eluate is a term used for the removed antibodies
Testing the eluate is useful in investigations of positive DATs HDN Transfusion reactions Autoimmune disease
The red cells can also be used after elution for RBC phenotyping if needed
When tested with panel cells, the eluate usually remains reactive with all cells if a warm autoantibody is present
Elution Methods
Acid elutions (glycine acid) Most common Lowers pH, causing antibody to
dissociate Organic solvents (ether, chloroform)
Dissolve bilipid layer of RBC Heat (conformational change) Freeze-Thaw (lyses cells)
ABO antibodies
Adsorption
Adsorption procedures can be used to investigate underlying alloantibodies
ZZAP or chloroquine diphosphate can be used to dissociate IgG antibodies from the RBC (may take several repeats)
After the patient RBCs are incubated, the adsorbed serum is tested with panel cells to ID the alloantibody (if present)
Adsorption
Two types: Autoadsorption
No recent transfusion Autoantibodies are removed using patient
RBCs, so alloantibodies can be identified Allogenic (Differential) adsorption
If recently transfused Uses other cells with the patients serum
Wash x3 after incubation
Centrifuge after incubating; and
transfer serum to 2nd tube of treated cells;
incubate and centrifuge again
2 tubes
Remove serum and
test for alloantibody
More reagents….
Many of elution tests can damage the antigens on the RBC
Choroquine diphosphate (CDP) and glycine acid EDTA reagents can dissociate IgG from the RBC without damaging the antigens Very useful if the RBC needs to be
antigen typed
Chloroquine diphosphate
Quinilone derivative often used as an antimalarial
May not remove autoantibody completely from DAT positive cells
Partial removal may be enough to antigen type the cells or to be used for autoadsorption of warm autoantibodies
THE END!!
