1. Antibody Identification
Mohammed Jaber

2. Antibody Presence
Presence of an antibody may be indicated by the following serological tests:
A discrepancy in the results of cell and serum ABH grouping.
A positive test for unexpected antibodies.
A positive direct Coomb’s test.
An incompatible major cross match.

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…
present
Not present

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)
Known: Unknown:
Reagent RBCs + patient serum
Reagent antisera + patient RBCs

6. Reagent RBCs
Screening Red Cells and Panel Red Cells are the same with minor differences:
Screening red cells
Antibody detection/screening
Sets of only 2 or 3 vials
Panel red cells
Antibody identification
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
Example: Panel Cell #10 has 9 antigens present: c, e, f, M, s, Leb, k, Fya, and Jka

11. Patient RBCs + Patient serum
Panel Red Cells
An autocontrol (AC) should also be run with ALL panels
Autocontrol
Patient RBCs Patient serum

12. Panel Red Cells
The same phases used in an antibody screen are used in a panel IS
37°C
AHG

13. 2 drops of the patients serum
Antibody ID Testing
A tube is labeled for each of the panel cells plus one tube for AC:     1 2 3 4 5 6 7 8 9 10 11 AC 
1 drop of each panel cell +
2 drops of the patients serum
Do not forget to write patient name and ID, Lab No.
Patient cells 
Patient serum

14. IS Phase
Perform immediate spin (IS) and grade agglutination; inspect for hemolysis
Record the results in the appropriate space as shown: 2+
Up to the Last tube

15. (LISS) 37°C Phase
2 drops of LISS are added, mixed and the mixture is incubated for minutes
Centrifuge and check for agglutination
Record results as previous but now fill the 37°C lane.

16. Agglutination Viewer

18. (LISS) 37°C Phase 2+ 2+ 2+ 2+

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)
Polyspecific AHG
Monospecific Anti-IgG
Monospecific Anti-Complement

20. IAT (AHG) Phase
Wash red cells 3X with saline (manual or automated (cell washer))
Add 2 drops of AHG and gently mix
Centrifuge
Read for agglutination
Record reactions

21. IAT (AHG) Phase 2+ 2+ 2+ 2+

22. ….add “check” cells to any negative AHG !
And don’t forget….
….add “check” cells to any negative AHG !

23. All cells are negative at AHG, so add “Check” CellsIS
LISS 37°
AHG CC 2+ 
All cells are negative at AHG, so add “Check” Cells

24. You have agglutination…now what?CC 2+    2+    2+    2+    ??

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:
An antibody will only react with cells that have the corresponding antigen; antibodies will not react with cells that do not have the antigen

27. Here’s an example:

28. 1. Ruling Out 2+    2+    2+   2+    
Cross out antigens that show NO REACTION in any phase; do NOT cross out heterozygous antigens that show dosage.

29. 2. Circle antigens not crossed out2+    2+    2+   2+    

30. 3. Consider antibody’s usual reactivity2+    2+    2+   2+    
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

31. 4. Look for a matching pattern
E doesn’t match and it’s a warmer rxn Ab 2+    2+    2+   2+    
…Yes, there is a matching pattern!

32. Interpretation:
anti-Lea

33. Guidelines Again, it’s important to look at: Autocontrol Phases
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
DTR – delayed transfusion reaction (donor cells are sensitized with recipient patient’s antibody)
Ashraf

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”2+  
3 Positive cells  2+    2+  
3 Negative cells 2+    
Panel Cells 1, 4, and 7 are positive for the antigen and gave a reaction at IS
Panel Cells 8, 10, and 11 are negative for the antigen and did not give a reaction at IS

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
Individuals DO NOT make allo-antibodies against antigens they have

41. Multiple antibodies
Multiple antibodies may be more of a challenge than a single antibody
Why?
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
Selected Cells
Neutralization
Chemical treatment
Proteolytic enzymes
Sulfhydryl reagents
ZZAP

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
For example:
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
Ag they have
Reaction pattern
Selected cells S
Jka P1 IS
37°C
AHG #1 + 2+ #5 3+ #8
These are panel cells that are being used, I just didn’t include the rest of the antigram because it is not necessary. 
These results show that instead of 3 antibodies, there are actually 2: anti-S and anti-Jka

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:
Ficin (figs)
Bromelin (pineapple)
Papain (papaya)
In addition, enzyme procedures may be
One-step
Two-step

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 Two-stage
Enzyme is added directly to the serum/panel cell mixture
Two-stage
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 Duffy antigens destroyed Kell antigens not affected
Anti-K
Perfect match for anti-Fya
Duffy antigens destroyed
Kell antigens not affected

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
2-mercaptoethanol (2-ME)

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
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)

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?
Idiopathic
Known disorder (SLE, RA, leukemias, UC, pregnancy, infectious diseases, etc)
Medications
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 Y
Elution Y Y
Sensitized RBC Y Y Y Y Y Y Y
Positive DAT
Frees antibody
Antibody ID

68. 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

69. 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

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: 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

72. 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

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. Providing Compatible Donor Units
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.

79. Example
Knowledge of the incidence of antigens is useful for determining how many units of blood to screen or cross­match 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+) = K positive = 0.09
Jk(a-) = 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!!