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BASIC ANTIBODY
IDENTIFICATION TECHNIQUES
Edda Rodriguez
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Enhancement Techniques
• Investigation of ABO discrepancies
• Enhancement reagents
• Serum techniques
• Red cell techniques
• Elutions
• Titrations
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Investigation of ABO
Discrepancies
• Incubation at room temperature
• Decreasing the temperature
–18oC / 4oC
– Include control cells
• Group O cells
• Auto control
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Investigation of ABO
Discrepancies
• Use of lectins
–Made from plant seeds
–React with specific carbohydrate
antigens
–Control cells
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Use of Lectins
• Ulex europaeus
–Anti-H specificity
– Identifies Bombay phenotype
• hh genotype
• Produce potent anti-H
–Reacts with all red cells
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Use of Lectins /
Additional Reagent Red Cells
• Dolichos biflorus
–Anti-A1 specificity
– Identifies A1 phenotype
• Reagent A2 red cells
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Enhancement Reagents
LISS Albumin PEG
• May see nonspecific reactivity
• Clinically significant antibodies are
excluded
• Repeat with different reagent
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Cold Autoantibodies
• Cold autoadsorption techniques
– Adsorb autoantibody onto autologous red
cells
– Re-test absorbed serum for underlying
clinically significant alloantibodies
– Cannot be performed on recently
transfused recipient
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Autoadsorption of Recently
Transfused Recipient
Recipient Donor
Autoantibody
Alloantibody
Serum Absorbed Serum
Red Cells Adsorbed Red Cells
RecipientDonor
Autoantibody
Alloantibody
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Prewarmed Techniques
• Can be performed on recently
transfused recipient
• Results used to characterize antibody
• Results may be used to exclude
alloantibodies
• May be only method to find compatible
donor red cell units
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Prewarmed-IAT
• Warm serum / cells
• No enhancement
reagent
• Extended
incubation
• Wash / warm saline
• Anti-IgG
• Warm serum / cells
• Enhancement
reagent
• Incubation
• Wash / warm saline
• Anti-IgG
No reactivity at AHG--no underlying clinically
significant antibodies
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Settle Technique
• Warm reagent cells and serum
• Incubate at 37oC until red cells settle
• Do not centrifuge
• No AHG reagent
• No reactivity at 37oC
– Antibody is probably not clinically
significant
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Inhibition / Neutralization
• Performed on specific antibodies
– Lewis, P1, Sda, Chido/Rogers
• Specific substance can neutralize
specific antibody
• Re-test neutralized serum
– Look for underlying clinically significant
antibodies
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Inhibition / Neutralization Test
• Add neutralizing substance to serum
aliquot
• Allow to incubate
• Re-test neutralized serum
• Need for control
– Dilutional effect of technique
– Saline and serum aliquot
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Inhibition / Neutralization
InterpretationTest Control Interpretation
0 + No additional antibodies
+ + Additional antibodies orhigh-titered antibody
0 0 Invalid results (weak Ab)
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Rouleaux
• Due to abnormal serum proteins
• May cause pseudoagglutination
• May be detected at room temp / 37oC
• Not observed at AHG phase
– Saline disperses rouleaux
• Observe serum:cells microscopically
– Stacked coin appearance
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Saline Replacement
• Centrifuge serum:cell mixture
• Remove serum
• Add 2 drops of saline
• Re-centrifuge
• If negative----rouleaux
• If positive-----true agglutination
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Enzyme Treatment
Untreatedcells
Enzyme-treatedcells
Interpretation(presumptive)
Fy(a+)Jk(a+)
Fy(a+)Jk(a+)
2+ 0 Anti-Fya
2+ 3+ Anti-Jka
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DTT / AET / ZZAP
• Can dissolve disulfide bonds
• Can modify various high incidence
antigens
• Procedure
–Treat reagent cells and re-test with
serum
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Red Cell Treatment for
Antigen Typing
• Use of specific reagents
– DTT / AET / CDP / EDTA/acid
• Can dissociate antibodies from coated
red cells
– Positive DAT
• Frees antigens for antigen typing--IAT
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Indications for Elution
• Removes coating IgG antibody
–Warm autoantibody
–Alloantibody in recently transfused
recipient
• Delayed or immediate transfusion
reaction
• Allows identification of coating IgG
antibody
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Eluting Mediums
• Coating IgG antibody is removed
–Thermal conditions
• Heat, cold, freeze-thaw
• ABO antibodies
–Organic solvents
• Acid, ether
• IgG antibodies
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Elution Procedure
• Wash the red cells
– Removes unbound antibody
• Treat red cells with eluting medium
– Antibody is released into eluate
• May require phosphate buffer
– Stabilize pH for further testing
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Elution Control
• Wash steps are critical
– Ensures antibody identified in eluate is the
coating antibody
• Test aliquot of last wash step along with
eluate
• If control negative--valid eluate results
• If control positive--invalid eluate results
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Antibody Titration Procedure
• Prepare Master Dilution
– Prepare 2-fold dilutions of serum
• Add 2 drops of each dilution
• Add 1 drop of appropriate red cells
• Incubate all tubes / centrifuge
• Observe for agglutination beginning with
largest dilution
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Titration Terms
• Titer
– Reciprocal of the last dilution that exhibits
1+ agglutination
• Endpoint
– Reciprocal of last dilution that exhibits any
agglutination
• Score
– Addition of assigned values to each
agglutination grade
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Titration Score
12 4+ agglutination
10 3+ agglutination
8 2+ agglutination
5 1+ agglutination
2 w+ agglutination
0 No agglutination
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Titration Example #1
1:1 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256
2+ 1+ 1+ w+ 0 0 0 0 0
8 5 5 2 0 0 0 0 0
Titer 4
Endpoint 8
Score 20
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Titration Example #2
1:1 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256
3+ 2+ 2+ 1+ 1+ 1+ w+ w+ 0
10 8 8 5 5 5 2 2 0
Titer 32
Endpoint 128
Score 45
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Antibody Titration
Interpretation
Baseline Secondsample
Titer 4 32
Score 20 45
Titer > 3 dilutions
Score >10
Are these results clinically significant?
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Indications for Antibody
Titrations
• Monitoring maternal IgG antibody
– HDN
• Assessing thermal amplitude of cold
autoantibody
• Identifying antibodies with HTLA
characteristics
• Determining ABO secretor status