Coombs Test

Question 1

what does the antiglobulin test used to detect

Answer 1

detect antibodies against RBC

Question 2

who developed Antiglobulin Test (Coombs’ Test)

Answer 2

by Robin Coombs and colleagues in 1945
- in vivo sensitization on babies suffering from HDFN

*some antibodies like IgG are too small to directly cause visible clumping (agglutination) of the RBCs
- so scientist add AHG, antihuman globulin which
binds to the antibodies attached to RBC, links RBC tgt = agglutination

Question 3

how did the AHG test work and where does it bind to

Answer 3

animals (like rabbits or mice) are injected with human antibodies

their immune system produces antibodies against human antibodies

= called Anti-Human Globulins
- bind to:
IgG antibodies
Complement proteins

attached to RBC

Question 4

DAT vs IAT

Answer 4

(notes)

Question 5

type of reagents used in Antiglobulin Tests

Answer 5

1. Anti-IgG
   - detect IgG antibodies on RBCs
   - ab that reacts against human IgG antibodies
2. Anti-complement
   - detect complement proteins on RBCs
3. Anti-C3b / Anti-C3d
   - specific complement components

*these animals produce ab against human ab, which bcm the AHG rgt

Question 6

who confirmed the need of anti-C3d activity in AHG for use in DAT

Answer 6

1976, Garatty and Petz
- he proved that the reactions is enhanced if the anti-IgG and anti-c3b
- this is why modern AHG reagents often contain both:
Anti-IgG
Anti-C3b / Anti-C3d

Question 7

where does anti-IgG bind

Answer 7

Anti-IgG binds to the Fc region of IgG
Fc is the heavy chain portion
= called gamma heavy chain specific

Question 8

Anti-IgG Specificity

Answer 8

1. Gamma heavy chain specific (Anti-IgG)
   - binds only to IgG antibodies
2. Anti-light chain specificity
   - can bind IgG, IgM, or IgA

*if the rgt is not gamma heavy chain specific, it may contain:
- Anti-IgG
- Anti-IgM
- Anti-IgA
= these react with the light chains (Fab region)

Question 9

what are the complement components detected in the Anti-complement reagents

Answer 9

C3b
- active complement fragment
C3d
- degraded complement fragment

= these fragments can attach to RBCs during immune rxn

Question 10

why is anti-C3c considered important

Answer 10

has low chance of non-specific reactions
a reaction occurs → usually clinically significant

however, C3c disappears quickly, esp aft 15min of incubation

= cold and warm autoimmune hemolytic anemia

Question 11

Polyspecific vs Monospecific AHG Reagents

Answer 11

(notes)

Polyspecific AHG Reagents:
- polyspecific AHG contains multiple types of antibodies.

Monospecific AHG Reagents:
- monospecific AHG contains only one type of antibody specificity.

Question 12

Polyclonal vs Monoclonal Antibodies

Answer 12

(notes)

Polyclonal Antibodies:
- come from many plasma cell clones

Monoclonal Antibodies:
- monoclonal antibodies come from one clone of plasma cells

Question 13

antibodies capable of binding complement

Answer 13

(trans)
[ab can activate the complement system when they bind to RBCs
when complement is activated, it can lead to:
- RBC destruction (hemolysis)
- complement proteins attaching to RBCs
= detected using the Coombs’ test]

most:
- ABO
[as they are usually IgM which strongly activates complement]

some:
- Xga
- LKE
- Lan

rare:
- Rh (D antigen)
[rarely bind to complements]

Question 14

First demonstrated the principle of the Direct Antiglobulin Test (DAT) using rabbit antigoat serum

Answer 14

Carlo Moreschi, 1908

Question 15

First used AHG to detect weak Rh antibodies

Answer 15

Robin Coombs and associates

Question 16

Applied AHG test to diagnose Hemolytic Disease of the Fetus and Newborn; Kell antibodies reported

Answer 16

Coombs and team

Question 17

Demonstrated anti-gammaglobulin activity in Rh typing

Answer 17

Robin Coombs and Arthur Mourant

Question 18

Studied reactions of warm vs cold antibodies

Answer 18

John V. Dacie et al.

Question 19

Distinguished anti-gammaglobulin (anti-IgG) from anti-nongammaglobulin (anti-complement) activity

Answer 19

John V. Dacie and coworkers
- he discovered that beta globulin is responsible for that and has specificity with the complement

Question 20

Alloantibodies vs Autoantibodies

Answer 20

Alloantibodies:
- form when the body recognizes transfused RBCs as foreign.
- example:
incompatible transfusion

Autoantibodies:
- the immune system attacks the body’s own RBCs.
- example:
autoimmune hemolytic anemia

Question 21

3 things that AHG test can detect on RBCs

Answer 21

1. IgG alloantibodies
   - formed bcs of transfusion
   - body recognized the foreign RBC
2. IgG autoantibodies
   - immune system of the body generates antibodies against self
3. Complement components
   [this is impt as complement can attach to RBCs = hemolysis]

Question 22

what does polyspecific AHG reagent contain

Answer 22

Anti-IgG and Anti-C3d

Question 23

1. which organization approves AHG reagents before manufacturing
2. which FDA center evaluates biologic reagents like AHG

Answer 23

1. U.S. Food and Drug Administration
2. Center for Biologics Evaluation and Research

Question 24

what is the recommended ratio of serum to cells

Answer 24

According to Harmening:
target ratio = 40 : 1
- 40 parts serum
- 1 part red cells

EXAMPLE PREP
1. 2 drops serum + 1 drop 5% RBC suspension
- 40:1
- recommended

2. 4 drops serum + 1 drop 3% RBC suspension
   - Higher ratio
   - acceptable
3. Saline-suspended cells
   - 133:1
   - not recommended

[amt of serum (antibodies) and red blood cells (antigens) must be balanced
if ratio is wrong, the antigen–antibody reaction may not occur properly]

Question 25

what happens if incorrect ration in serum to cells

Answer 25

1. Prozone - too many antibodies - no agglutination 2. Postzone - too many antigens - no agglutination

Question 26

it is an environment where RBCs and antibodies react.

Answer 26

reaction medium [affects how easily antibodies attach to antigens. enhancement media like: LISS PEG can help increase sensitivity and speed]

Question 27

at what temp does te miost clinically significant antibodies react best at

Answer 27

body temp 37°C → IgG antibodies react best - similar to human body temp - complement activation also works best near body temp

Question 28

what is the normal incubation time for AHG test

Answer 28

ab need time to bind to RBC antigens - without enough time, rxn may not occur 1. Standard method: 30 minutes 2. With LISS or PEG: 10–15 minutes *should NOT exceed 30 minutes - if LISS incubation is extended to 40mi, the ab may setach from RBCs = elution, which leads to - decreased test sensittivity - false -ve results

Question 29

This is considered the most important step in the AHG test.

Answer 29

Washing of RBCs - remove unbounded ab in the serum - if ab remain, they can neutralize the AHG rgt - AHG will not react with RBCs *after washing, check cells are added to confirm that AHG reagent is working

Question 30

what is added after washing RBCs to confirm that AHG reagent is working

Answer 30

Check cells contain: - group O RBCs - sensitized with IgG antibodies *if agglutination occurs → AHG reagent is still active

Question 31

what is the ideal ph of a saline for washing

Answer 31

7.2 – 7.4 if saline is stored too long (especially in plastic containers): - pH decreases - antibodies may detach from RBCs This leads to: ➡ Elution of antibodies ➡ False negative AHG test

Question 32

this is responsible for the final agglutination reaction

Answer 32

Addition of AHG Reagent - AHG contains antibodies that bind to IgG antibodies Complement proteins *RBC antigen – antibody – anti-IgG (AHG) = visible agglutination !! shld be added immediately after washing IF DELAYED antibodies may detach from RBCs serum antibodies may neutralize AHG = false -ve

Question 33

this helps RBCs come closer together, making agglutination easier to see

Answer 33

Centrifugation REFERENCE 1. Siber - 1000 RCF - 20 seconds 2. Harmening - 500 RCF - 15–20 seconds EFFECT 1. Higher RCF - mre sensitive reaction 2. Too strong or incorrect handling - clerical or reading errors

Question 34

it is a substance added to help antigens on RBCs and antibodies react more easily.

Answer 34

reaction media [brings RBC close tgt, increase sensitivity of the test, reduce incubation time] *normally, ab rxn: 30-60min w enhancement media = drop too 10-15min

Question 35

briefly explain the 3 types of reaction media

Answer 35

1. Albumin 2. Low Ionic Strength Solution (LISS) 3. Polyethylene Glycol (PEG)

Question 36

Agglutination vs Sensitization

Answer 36

Sensitization: - ab attached to RBC Agglutination: - ab connects multiple RBCs forming a lattice (clump)

Question 37

what are the diseases in DAT where RBCs are attacked by antibodies in the body

Answer 37

IN VIVO SENSITIZATION 1. Hemolytic Disease of the Fetus and Newborn (HDFN) - maternal IgG antibodies attack fetal RBCs 2. Hemolytic Transfusion Reaction (HTR) - recipient antibodies attack transfused RBCs 3. Autoimmune Hemolytic Anemia (AIHA) - patient antibodies attack their own RBCs

Question 38

principle of DAT

Answer 38

1.Patient RBCs are washed 3 times. 2. AHG (Coombs’ serum) is added. 3. If antibodies or complement are on RBCs → agglutination occurs. *agglutination = positive DAT

Question 39

DAT vs IAT Principle

Answer 39

DAT: 1. px RBCs are washed 3 times. 2. AHG (Coombs’ serum) is added. 3. if antibodies or complement are on RBCs → agglutination occurs. Agglutination = positive DAT IAT: 1. px serum is mixed with test RBCs. 2. incubation at 37°C allows antibodies to bind RBCs. 3. RBCs are washed + AHG reagent is added. 4. Agglutination indicates antibodies are present.

Question 40

true or false: positive DAT alone does confirm disease

Answer 40

false - does not Evidence of hemolysis Recent transfusion Drugs taken Presence of antibodies *DAT results are supporting evidence, not final diagnosis.

Question 41

briefly explain the causes of positive DAT

Answer 41

means RBC are alr coated with antibodies or complement inside the body *coating may come from transfusion, drugs, autoimmune diseases, or pregnancy 1. Transfusion reactions - antibodies attack transfused RBCs 2. Drug-induced hemolysis - drugs cause antibodies to attach to RBCs 3. Autoimmune hemolytic anemia - body attacks its own RBCs 4. Hemolytic disease of newborn - mother’s antibodies attack baby’s RBCs 5. Miscellaneous causes - certain proteins or therapies coat RBCs

Question 42

This occurs when antibodies from donor or recipient attack RBCs after transfusion.

Answer 42

transfusion - introduces foreign RBC antigen

Question 43

when is IAT used in the following situations

Answer 43

1. Detection of incomplete (nonagglutinating) - antibodies to potential donor RBCs (compatibility testing) or to screening cells (antibody screen) in serum 2. Determination of RBC phenotype - using known antisera (e.g., Kelltyping, weak D testing) 3. Titration of incomplete antibodies - it is performed to determine the degree of antibody in the serum if it is enough to cause hemolytic reactions

Question 44

positive rection in IAT

Answer 44

sensitized Red Cell and add the Coomb’s Sera

Question 45

procedure of IAT

Answer 45

(trans) 1. mix gently and cover with parafilm. incubate at 37C for 15 minutes. 2. at the end of incubation. centrifuge for 30 seconds at 3,400 rpm. Examine for agglutination. 3. if NO agglutination is observed, wash the unknown tube three times with NSS. Decant completely after the last washing. 4. add 2 drops of antihuman globulin serum and mix well. 5. centrifuge for 30 seconds at 3,400 rpm. 6. gently dislodge the cell button and observe for agglutination. This part should be recorded/graded! Note: Agglutination indicates the presence of atypical antibodies in the patient's serum. 7. NO agglutination indicates the absence of atypical antibodies in the patient's serum. 8. if the test does NOT show agglutination, add 1 drop of Coomb's check cells to the unknown tube and centrifuge for 30 seconds at 3,400 rpm 9. gently dislodge the cell button. Observe for agglutination or hemolysis 10. grade each reaction and record the results

Question 46

sources of error in AHG testing false-positive results: false-negative results:

Answer 46

(trans)