Antibody Identification.ppt 6yr4n
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Antibody Identification ZEESHAN YOUSUF MEDICAL TECHNOLOGIST AGA KHAN UNIVERSITY HOSPITAL KARACHI, PAKISTAN
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” Known: Reagent RBCs + Reagent antisera +
Unknown: patient serum patient RBCs
When detecting and/or identifying antibodies, we test patient serum (unknown) with reagent RBCs (known)
Reagent RBCs
Screening Cells and Cells are the same with minor differences:
Screening cells Antibody detection Sets of 2 or 3 vials
cells Antibody identification At least 10 vials per set
Antibody vs. Screen
An antibody is just an extended version of an antibody screen The screen only uses 2-3 cells:
Antibody
An antibody usually includes at least 10 cells:
Group O red blood cells
Each of the cells has been antigen typed (shown on antigram)
+ refers to the presence of the antigen 0 refers to the absence of the antigen
Example: Cell #10 has 9 antigens present: c, e, f, M, s, Leb, k, Fya, and Jka
Autocontrol Patient RBCs + Patient serum
An autocontrol should also be run with ALL s
The same phases used in an antibody screen are used in a • IS
• 37° • AHG
Antibody ID Testing
1
2
A tube is labeled for each of the cells plus one tube for AC: 3
4
5
6
7
8
1 drop of each cell +
2 drops of the patients serum
9
10
11
AC
IS Phase
Perform immediate spin (IS) and grade agglutination; inspect for hemolysis Record the results in the appropriate space as shown: 2+ 0 0
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+ 0 0 0 0 0 2+ 0 0 2+ 0 2+ 0 0
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+ 0 0 2+ 0 0 2+ 0 2+ 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
And don’t forget….
….add “check” cells to any negative AHG !
IS 2+ 0 0 2+ 0 0 2+ 0 2+ 0 0
LISS AHG 37° 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0
CC
All cells are negative at AHG, so add “Check” Cells
You have agglutination…now what? CC
2+ 0 0 2+ 0 0 2+ 0 2+ 0 0 0
??
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
Interpreting Antibody s
There are a few basic steps to follow when interpreting s 1.
2.
3.
4.
“Ruling out” means crossing out antigens that did not react Circle the antigens that are not crossed out Consider antibody’s usual reactivity Look for a matching pattern
Always : An antibody will only react with cells that have the corresponding antigen; antibodies will not react with cells that do not have the antigen
Here’s an example:
1. Ruling Out 2+ 0 0 2+ 0 0 2+ 0 2+ 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
Cross out antigens that show NO REACTION in any phase; do NOT cross out heterozygous antigens that show dosage.
0 0 0 0 0 0 0 0 0 0 0 0
2. Circle antigens not crossed out 2+ 0 0 2+ 0 0 2+ 0 2+ 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
3. Consider antibody’s usual reactivity
2+ 0 0 2+ 0 0 2+ 0 2+ 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 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 E doesn’t match and it’s a warmer rx Ab
2+ 0 0 2+ 0 0 2+ 0 2+ 0 0 0
…Yes, there is a matching pattern!
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
Interpretation antiLea
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 cells are homozygous, a strong reaction may be seen If cells are heterozygous, reaction may be weak or even nonreactive
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 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”
3 Positive cells
3 Negative cells
2+ 0 0 2+ 0 0 2+ 0 2+ 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
Cells 1, 4, and 7 are positive for the antigen and gave a reaction at immediate spin 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 to fulfill the rule, then additional cells from another could be used Most labs carry different lot numbers of 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?
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 or screening cells to confirm or eliminate the antibody The cells are “selected” from other s 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 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 AHG 37°
#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 )
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
cells are pre-treated with enzyme, incubated and washed Patient serum is added to cells and tested
Enzyme techniques
If there is no agglutination after treatment, then it is assumed the enzymes destroyed the antigen
Enzyme treatment
Enzyme treament
Anti-K
Perfect match for anti-Fya
•Duffy antigens destroyed •Kell antigens not affected
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 alloantibodies, so it’s important to differentiate between auto- and allo-antibodies
Cold autoantibodies
React at room temperature with most (if not all) of the 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 s 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 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
Y Positive DAT
Y
Y
Sensitized RBC
Elution
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 cells, the eluate usually remains reactive with all cells if a warm autoantibody is present
Elution Methods
Acid elutions (glycine acid)
Organic solvents (ether, chloroform)
ABO antibodies
Most common Lowers pH, causing antibody to dissociate Dissolve bilipid layer of RBC
Heat (conformational change) Freeze-Thaw (lyses cells)
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 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
Remove serum and test for alloantibody
2 tubes Wash x3 after incubation
Centrifuge after incubating; and transfer serum to 2nd tube of treated cells; incubate and centrifuge again
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!! THANK YOU
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” Known: Reagent RBCs + Reagent antisera +
Unknown: patient serum patient RBCs
When detecting and/or identifying antibodies, we test patient serum (unknown) with reagent RBCs (known)
Reagent RBCs
Screening Cells and Cells are the same with minor differences:
Screening cells Antibody detection Sets of 2 or 3 vials
cells Antibody identification At least 10 vials per set
Antibody vs. Screen
An antibody is just an extended version of an antibody screen The screen only uses 2-3 cells:
Antibody
An antibody usually includes at least 10 cells:
Group O red blood cells
Each of the cells has been antigen typed (shown on antigram)
+ refers to the presence of the antigen 0 refers to the absence of the antigen
Example: Cell #10 has 9 antigens present: c, e, f, M, s, Leb, k, Fya, and Jka
Autocontrol Patient RBCs + Patient serum
An autocontrol should also be run with ALL s
The same phases used in an antibody screen are used in a • IS
• 37° • AHG
Antibody ID Testing
1
2
A tube is labeled for each of the cells plus one tube for AC: 3
4
5
6
7
8
1 drop of each cell +
2 drops of the patients serum
9
10
11
AC
IS Phase
Perform immediate spin (IS) and grade agglutination; inspect for hemolysis Record the results in the appropriate space as shown: 2+ 0 0
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+ 0 0 0 0 0 2+ 0 0 2+ 0 2+ 0 0
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+ 0 0 2+ 0 0 2+ 0 2+ 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
And don’t forget….
….add “check” cells to any negative AHG !
IS 2+ 0 0 2+ 0 0 2+ 0 2+ 0 0
LISS AHG 37° 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0
CC
All cells are negative at AHG, so add “Check” Cells
You have agglutination…now what? CC
2+ 0 0 2+ 0 0 2+ 0 2+ 0 0 0
??
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
Interpreting Antibody s
There are a few basic steps to follow when interpreting s 1.
2.
3.
4.
“Ruling out” means crossing out antigens that did not react Circle the antigens that are not crossed out Consider antibody’s usual reactivity Look for a matching pattern
Always : An antibody will only react with cells that have the corresponding antigen; antibodies will not react with cells that do not have the antigen
Here’s an example:
1. Ruling Out 2+ 0 0 2+ 0 0 2+ 0 2+ 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
Cross out antigens that show NO REACTION in any phase; do NOT cross out heterozygous antigens that show dosage.
0 0 0 0 0 0 0 0 0 0 0 0
2. Circle antigens not crossed out 2+ 0 0 2+ 0 0 2+ 0 2+ 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
3. Consider antibody’s usual reactivity
2+ 0 0 2+ 0 0 2+ 0 2+ 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 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 E doesn’t match and it’s a warmer rx Ab
2+ 0 0 2+ 0 0 2+ 0 2+ 0 0 0
…Yes, there is a matching pattern!
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
Interpretation antiLea
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 cells are homozygous, a strong reaction may be seen If cells are heterozygous, reaction may be weak or even nonreactive
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 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”
3 Positive cells
3 Negative cells
2+ 0 0 2+ 0 0 2+ 0 2+ 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
Cells 1, 4, and 7 are positive for the antigen and gave a reaction at immediate spin 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 to fulfill the rule, then additional cells from another could be used Most labs carry different lot numbers of 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?
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 or screening cells to confirm or eliminate the antibody The cells are “selected” from other s 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 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 AHG 37°
#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 )
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
cells are pre-treated with enzyme, incubated and washed Patient serum is added to cells and tested
Enzyme techniques
If there is no agglutination after treatment, then it is assumed the enzymes destroyed the antigen
Enzyme treatment
Enzyme treament
Anti-K
Perfect match for anti-Fya
•Duffy antigens destroyed •Kell antigens not affected
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 alloantibodies, so it’s important to differentiate between auto- and allo-antibodies
Cold autoantibodies
React at room temperature with most (if not all) of the 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 s 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 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
Y Positive DAT
Y
Y
Sensitized RBC
Elution
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 cells, the eluate usually remains reactive with all cells if a warm autoantibody is present
Elution Methods
Acid elutions (glycine acid)
Organic solvents (ether, chloroform)
ABO antibodies
Most common Lowers pH, causing antibody to dissociate Dissolve bilipid layer of RBC
Heat (conformational change) Freeze-Thaw (lyses cells)
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 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
Remove serum and test for alloantibody
2 tubes Wash x3 after incubation
Centrifuge after incubating; and transfer serum to 2nd tube of treated cells; incubate and centrifuge again
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!! THANK YOU
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