lesson 3

Question 1

Antiglobulin test aka Coomb’s Test was introduced by

Answer 1

Robert Royston and Amos Coombs in 1945

Question 2

The antiglobulin test can be used to detect Red cell sensitization with IgG alloantibodies, IgG autoantibodies or complement proteins

Answer 2

antiglobulin test

Question 3

Moreschi reaction =

Answer 3

there is an apparent agglutination with the use of AHG

Question 4

can be a really good enhancer or activator of complement activation

Answer 4

IgM

Question 6

High tittered, High Avidity

Answer 6

Polyclonal AHG

Question 8

Polyclonal AHG contains

Answer 8

Contains anti-IgG and anti-C3d

Question 9

problem in polyclonal AHG

Answer 9

Problem: Prone to false positive reaction

Question 10

ADVANTAGE INPOLYCLONAL AHG

Answer 10

Advantage in detecting ab not usually detected at low titers such as kidd, kell, duffy which causes delayed HTR

Question 11

DISADVANTAGE IN MONOCLONAL AHG

Answer 11

Dis:false negative

Question 12

Detects circulating antibodies against RBC.

Answer 12

INDIRECT COOMBS TEST

Question 13

Determines if the patient has antibodies against a blood product.

Answer 13

IAT

Question 14

Applications of IAT

Answer 14

Antibody Detection and Identification
Crossmatching
Antibody Titration
Weak D Typing

Question 15

IAT PROCEDURE

Answer 15

PROCEDURE
Place 2-4 gtts of the patient’s serum in a test tube

Add 1 gt of 2-5% Reagent RCS

Mix and incubate for 60 minutes

Add 1-2 gtts of AHG serum

Centrifuge at 3400 for 1 minute

Gently agitate to dislodge and examine for agglutination\

Add Check cells if negative.

Question 16

REMOVE WHEN WASHING Between incubation and addition of AHG serum, in IAT

Answer 16

emove unbound immunoglobulins

Question 17

could react to AHG reagent, causing false negative.

Answer 17

unbound immunoglobulins

Question 18

INTERPRETATION OF IAT

Answer 18

INTERPRETATION

(+) AGGLUTINATION
The patient has antibodies to the antigens in the reagent cells

(-) AGGLUTINATION
The patient has no antibodies to the antigens present on the reagent cells

Question 19

FACTORS AFFECTING IAT

Answer 19

Temperature
Incubation Time
Ratio of Cell to serum
Washing of Cells
Addition of AHG reagent
Centrifugation
Quality of AHG reagent
Perform quality check of reagent cell before doing test - it should have agglutination pattern
Reaction medium used

Question 20

HOW DOES TEMP AFFECT AHG

Answer 20

Cold = decrease reactivity of immunoglobulin

Question 21

HOW DOES incubation time affect AHG

Answer 21

More incubation = more agglutination
Shortened incubation = weakened agglutination

Question 22

HOW DOES centrifugation affect AHG

Answer 22

Increased rpm = false positive
Decreased rpm = false negative

Question 23

CLINICAL SIGNIFICANCE
IAT

Answer 23

Presence of Maternal Antibodies against fetus
Detection of antibodies not detected by other techniques (Le, Fy, Jka)
Compatibility/Incompatibility of Patient and Blood Unit
Presence of Unexpected antibodies

Question 24

Detects sensitized cells

Answer 24

DIRECT COOMBS TEST

Question 25

Applications of DAT

Answer 25

Diagnosis of HDFN Diagnosis of Acquired Immune Hemolytic Anemia investigation of Drug Induced Red Cell sensitization Investigation of Hemolytic Transfusion reaction

Question 26

dat procedure

Answer 26

Prepare 3-5% RCS of the patient's blood Transfer 2-3 gtts of 5% RCS in another test tube Add 2-3 gtts of AHG Incubate for 15 to 30 minutes at 370C Centrifuge under 3400 rpm for 1 minute Gently agitate to dislodge the cells Check for agglutination Add Coomb's Check Cells to negative Tubes

Question 27

DAT INTERPRETATION

Answer 27

(+) AGGLUTINATION The patient's has bound IgG or complement proteins The patients' RBC is coated//sensitized (-) AGGLUTINATION The patient has no bound IgG or complement proteins

Question 28

For IgG coated CC: Use

Answer 28

RH antibodies

Question 29

For C3b coated CC:

Answer 29

Incubate cells in LISS or anti-Lea or anti-l

Question 30

For C3d coated CC:

Answer 30

Incubate C3b sensitized cells with Fresh serum or Tryps

Question 31

Preparation of Check cells

Answer 31

Prepare 5% suspension of a Blood Type O RhD positive sample Mix equal amounts of Anti-D and the Freshly prepared RCC, Incubate for 15 minutes at 37oC Wash 3 times with isotonic saline 3-5 times. Decant the last wash Resuspend using NSS to make 5% suspension of Coombs Control Cells

Question 32

Check cells are added to negative tubes in order to confirm true negative reaction\

Answer 32

POSITIVE AGGLUTINATION True negative AHG reagent was not neutralized during process NEGATIVE AGGLUTINATION False negative AHG reagent was neutralized during process

Question 33

FALSE + IN ahg

Answer 33

Technical Error - most common 1. Residual enzyme treated cells may react with AHG rgt 2. Overcentrifugation 3. Usage of agglutinated RBC 4. Improper use of PEG Nontechnical Error 1. Bacterial contamination of Test cells (septicemia) 2. Presence of strong cold agglutinins 3. Rouleaux formation Add saline to remove excess protein

Question 34

FALSE NEGATIVE REACTION in AHG

Answer 34

Technical error Inadequate washing Delayed processing Zone phenomenon ; Improper cell:AHG ratio Insufficient incubation Failure to add AHG Microscopic agglutination Improper storage of reagent n inactivation/loss of sensitivity Use of Plasma