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1. Antibody Identification Mohammed Jaber
3. The Basics?.. As we said in the previous lecture,
Antibody Screens use 2 or 3 Screening Cells to ?detect? if antibodies are present in the serum
If antibodies are detected, then they should be identified?
4. Why do we need to identify? Antibody identification 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 (i.e. transfusion reactions).
5. Key Concepts In blood banking, we test ?knowns? with ?unknowns?:-
When detecting/screening and/or identifying antibodies, we test patient serum (unknown that contain blood bank antibodies) with reagent RBCs (known)
6. Reagent RBCs Screening Red Cells and Panel Red Cells are the same with minor differences:
Screening red cells
Sets of only 2 or 3 vials
Panel red cells
At least 10 vials/set
7. Antibody Panel vs. Screen An antibody panel is just an extended version of an antibody screen
The screen only uses 2-3 red cells:
8. Antibody Panel While antibody panel usually includes at least 10 panel cells: (8-16 group O RBCs)
9. Panel Red Cells Group O red blood cells obtained from donors
10. Panel Red Cells 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
11. Panel Red Cells An autocontrol (AC) should also be run with ALL panels
12. Panel Red Cells The same phases used in an antibody screen are used in a panel
13. Antibody ID Testing A tube is labeled for each of the panel cells plus one tube for AC:
14. IS Phase Perform immediate spin (IS) and grade agglutination; inspect for hemolysis
Record the results in the appropriate space as shown:
15. (LISS) 37?C Phase 2 drops of LISS are added, mixed and the mixture is incubated for 10-15 minutes
Centrifuge and check for agglutination
Record results as previous but now fill the 37?C lane.
18. (LISS) 37?C Phase
19. 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)
20. IAT (AHG) Phase Wash red cells 3X with saline (manual or automated (cell washer))
Add 2 drops of AHG and gently mix
Read for agglutination
21. IAT (AHG) Phase
22. And don?t forget?.
24. You have agglutination?now what?
25. Interpreting Antibody Panels There are a few basic steps to follow when interpreting panels
?Ruling out? means crossing out antigens that did not react
Circle the antigens that are not crossed out
Consider antibody?s (from the circled) usual reactivity
Look for a matching pattern
26. Always remember:
27. Here?s an example:
28. 1. Ruling Out
29. 2. Circle antigens not crossed out
30. 3. Consider antibody?s usual reactivity
31. 4. Look for a matching pattern
33. Guidelines Again, it?s important to look at:
Negative - alloantibody
Positive ? autoantibody or DTR (i.e. alloantibodies)
IS ? cold (IgM)
37? - cold (some have higher thermal range) or warm reacting
AHG ? warm (IgG)?significant!!
1 consistent strength ? one antibody
Different strengths ? multiple antibodies or dosage
DTR ? delayed transfusion reaction (donor cells are sensitized with recipient patient?s antibody)
AshrafDTR ? delayed transfusion reaction (donor cells are sensitized with recipient patient?s antibody)
34. 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 for an antigen should not be crossed out because antibody may be too weak to react (see previous example)
35. 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
36. 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 panel cells with the antigen, +ve reaction.
Negative with 3 cells without the antigen and should not be reacting.
37. Our previous example fulfills the ?rule of three?
38. 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 good (I do not know if we are good or not!!) labs carry different lot numbers of panel cells
39. Patient Antigen Typing (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 (chimera)).
If reagent antisera (of the suspected antibody) is added to the patient RBCs, a negative reaction should result?Why?
40. Remember Landsteiner?s Rule
41. Multiple antibodies Multiple antibodies may be more of a challenge than a single antibody
Reaction strengths can vary
Matching the pattern is difficult
42. So what we have to do? Several procedures can be performed to identify multiple antibodies
43. 1- Selected Red 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.
44. Selected Red Cells ?.. Cont?d Every cell should be positive only for each of the antibodies and negative for the remaining suspicious antibodies
Let?s say you ran a panel and identified 3 different antibodies (you cannot rule out): anti-S, anti-Jka, and anti-P1
Selected cells could help?
45. Selected Red Cells ?.. Cont?d These are panel cells that are being used, I just didn?t include the rest of the antigram because it is not necessary.These are panel cells that are being used, I just didn?t include the rest of the antigram because it is not necessary.
46. 2- 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)
47. Neutralization ?.. Cont?d Manufacturer?s directions should be followed and a positive dilutional control should always be used
The positive dilutional control contains saline and serum (no substance is added) and should remain positive with panel cells.
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
48. Neutralization ?.. Cont?d Common substances
P1 substance (derived from hydatid cyst fluid)
Lea and Leb substance (soluble antigen found in plasma and saliva)
I substance can be found in breast milk
**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
49. 3- Again: Proteolytic Enzymes Can be used to enhance or destroy certain blood group antigens
Several enzymes exist:
In addition, enzyme procedures may be
50. 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
51. Enzyme techniques One-stage
Enzyme is added directly to the serum/panel cell mixture
Panel cells are pre-treated with an enzyme, and washed
Patient serum is added to treated panel cells and tested
52. Enzyme techniques If there is no agglutination after treatment, then it is assumed the enzymes destroyed the antigen
53. Enzyme treatment
54. 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
55. 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
56. Autoantibodies?. Warm & Cold Reacting
57. 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
58. 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
59. 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
60. Identifying autoantibodies Auto-antibodies can sometimes ?mask? clinically significant allo-antibodies, so it?s important to differentiate between auto- and allo-antibodies
61. 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
62. 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
63. 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
64. 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
65. 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
66. Warm autoantibodies Cause warm autoimmune hemolytic anemia (WAIHA)??H&H
How do you get a warm autoantibody?
Known disorder (SLE, RA, leukemias, UC, pregnancy, infectious diseases, etc)
Several techniques are used when warm autoantibodies are suspected?
67. Elution (whenever DAT is positive) Elution techniques ?free? antibodies from the sensitized red cells so that the antibodies can be identified
68. Elution The eluate is a term used for the removed antibodies
Testing the eluate is useful in investigations of positive DATs
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
69. Elution Methods Acid elutions (glycine acid)
Lowers pH, causing antibody to dissociate
Organic solvents (ether, chloroform)
Dissolve bilipid layer of RBC
Heat (conformational change)
Freeze-Thaw (lyses cells)
70. 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)
71. Adsorption Two types:
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
73. 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
74. 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
75. Summary If an unexpected antibody is detected in a patient?s serum or plasma it must be identified.
Once identified the clinical significance must be determined.
76. Summary If the antibody is clinically significant antigen negative donors must be found and crossmatched for the patient, a Coomb?s crossmatch must be done.
If the antibody is not clinically significant it is not necessary to provide antigen negative blood, but the donors must be compatible by the Coomb?s crossmatch.
77. Providing Compatible Donor Units Once an antibody has been identified, the next task is to provide appropriate units of RBCs for transfusion.
When clinically insignificant antibodies are detected, use of crossmatch-compatible RBCs is appropriate.
78. No further testing is needed to confirm compatibility when the antibody is anti-M, anti-N, anti-Pi. Lea, or Leb.
However, when a clinically significant antibody is identified, the blood must be cross-match compatible and confirmed as antigen-negative with reagent antisera. Providing Compatible Donor Units
79. Example Knowledge of the incidence of antigens is useful for determining how many units of blood to screen or cross?match for patients with antibodies.
If a patient with an anti-Jk(a-) needed 4 units of blood, how many units would need to be tested to find them?
Jk (a+)= 0.77
Jk (a-) = 0.23
4 units Jk(a-) blood needed = 17.4 units 0.23 incidence of Jk(a-)
In this case, testing 17 or 18 random units should yield 4 Jk(a-) units.
80. Example 2 The same calculations can be used when multiple antibodies are present if the antigen frequencies are first multiplied together.
E.g. a patient with an anti-K and anti-Jka, 10 random units would need to be tested to find 2 that are compatible.
Jk(a+) = 0.77 K positive = 0.09
Jk(a-) = 0.23 K negative = 0.91
Jk(a-) (0.23) X K negative (0.91) = 0.20 Jk(a-) and K negative
2 units needed = 10 units 0.20 Jk(a-) and Kell negative
81. THE END!!