BBWK 2

Question 1

Laboratory procedure that detects the presence of an

Answer 1

ANTIBODY DETECTION

Question 2

modernn method in ab detection

Answer 2

Modern Method: ELISA, Western Blot, Rapid Test Kitts, etc

Question 3

The process of removing antibodies from the surface of Red Blood Cells. It is performed when Coombs Test is Positive

Answer 3

ELUTION

Question 4

Primarily performed when in-vivo sensitization of RBC is suspected

Answer 4

ELUTION

Question 5

How can Elution be accomplished?

Answer 5

1. Disrupting the linkage between the antigen and the antibody

2.. Changing conditions that favor dissociation of antibodies from antigen

Question 6

The purpose of _____ is to detach the antibodies from the surface of the RBC in order to be tested against reagent red blood cells to determine if there is an immune antibody or not

Answer 6

The purpose of an elution is to detach the antibodies from the surface of the RBS in order to be tested against reagent red blood cells to determine if there is an immune antibody or not

Question 7

Used for eluting warm-reacting auto or allo antibodies

Answer 7

Acid Elution (glycine-HCI)

Question 8

Lowers pH, causing antibody to dissociate

Answer 8

Lowers pH, causing antibody to dissociate

Question 9

will acidify solution causing dissociation of antibody. Then _____will neutralize the reaction in order for antibodies to be tested

Answer 9

Glycine hydrochloric acid

phosphate buffer

Question 10

Organic Solvents examPLE

Answer 10

(ether, chloroform)

Question 11

Dissolves the lipid bilayer of erythrocytes causing dissociation of antibodies from binding

Answer 11

Organic Solvents

Question 12

Used for investigation of HDN caused by ABO incompatibility

Answer 12

Heat Elution

Question 13

reverse the agglutination process

Answer 13

Heat Elution

Question 14

Cells are lysed in the process causing elution

Answer 14

Lui Freeze thaw

Question 15

GENERAL STEPS BEFOR ELUTION

Answer 15

Confirm positive DAT on patient or donor
Wash DAT-Positive Cells 3-4 times to remove unbound antibodies
Decant the supernatant
Proceed to elution

Question 16

ACID ELUTION

Answer 16

Add equal amount Acid Elution reagent to washed, sensitized red cells

Concentration of glycine hcl is 0.1M. Glycine titrated to hcl to bring down rxn to PH 3

Mix gently and incubate for 1 minute

Add buffering solution (phosphate buffer) to neutralize the eluate

Centrifuge the solution

Transfer the eluate to another container

test the eluate against reagent panel

Question 17

HEAT ELUTION

Answer 17

Add 20 drops of 6% albumin/other potentiator to the washed RBC
Place the tubes at 56 degree Celsius for 10 minutes.
Agitate periodically to have constant temp through incubation
Centrifuge the solution at 3400 rpm for 5 minutes
Immediately transfer the supernatant in a clean test tube
Test the eluate against panel of cells

Question 18

ORGANIC SOLVENT ELUTION - CHLOROFORM

Answer 18

Add 1 ml of washed sensitized to 1ml of 6% BSA
Add 2ml of Chloroform
Shake vigorously for 10-15 seconds
Mix by gentle inversion for 1 minute
Incubate at 56 degrees Celsius for 5 minutes
Centrifuge at 3400 RPM for 5 minutes
Discard the top two layers (Chloroform)
Transfer the eluate to another tube

Question 19

ORGANIC SOLVENT ELUTION - XYLENE

Answer 19

Add 1 ml of washed sensitized to 1ml of NSS
Add 4 ml of Xylene
Shake vigorously for 10-15 seconds
Mix by gentle inversion for 1 minute
Incubate at 56 degrees Celsius for 5 minutes
Centrifuge at 3400 RPM for 5 minutes
Discard the top two layers
Transfer the eluate to another tube

Question 20

LUI-FREEZE THAW

Answer 20

Suspend cells in NSS
Freeze the suspension completely
Thaw at RT or 37 deg C
Repeat freeze-thaw cycle
Centrifuge to remove cell debris
Harvest the eluate

Question 21

If eluate shows agglutination with reagent cells, it indicates

Answer 21

presence of specific antibodies against the antigens present on the test cells

Question 22

HOW TO CHECK ELUTION

Answer 22

mix patient sample with RCS. if there will is macroscopic agglutination it means that the antibodies have been successfully eluted

Question 23

Pan reactive eluate is commonly associated with

Answer 23

warm autoantibodies

Question 24

Lack of agglutination indicates absence of specific antibodies against antigens on the reagent cell. this could be caused by

Answer 24

Drug-induced
Complement coating
Low- affinity antibodies

Question 25

clinical significance of elution

Answer 25

CLINICAL SIGNIFICANCE Investigation of HTR Diagnosis of AIHA Diagnosis of ABO HDFN Identification of specificity when multiple antibodies exist in a patient's serum Phenotyping red cells in patient with a positive DAT

Question 26

is removal of antibody from a plasma/serum by incubating it with red cells positive for the corresponding antigen

Answer 26

Adsorption

Question 27

procedures can be used to investigate

Answer 27

Adsorption

Question 28

mostly used to remove autoantibodies because it interferes with antibody identification.

Answer 28

ADSORPTION

Question 29

= reacts to all red cells.

Answer 29

Pan reactivity

Question 30

Uses patient's RCS to remove patient's antibodies from serum

Answer 30

·Autoadsorption

Question 31

Performed for patients that has not been transfused or pregnant in the last 13 weeks

Answer 31

·Autoadsorption

Question 32

Used for patient with recent transfusion

Answer 32

Alloadsorption

Question 33

Uses RBC with known phenotypes to selectively remove alloantibodies from patient's serum

Answer 33

Alloadsorption

Question 34

AUTOADSORPTION

Answer 34

To 0.5 ml of test serum, add an equal amount of the corresponding adsorbing cells to be used. Shake gently and spin down. Remove supernatant and then add an equal amount of the corresponding adsorbing cells again. Shake gently and spin down. Repeat the addition and removal of supernatant until all antibodies present in the serum have been removed. The final solution should show no agglutination or hemolysis. Record number of washings performed.

Question 35

autoadsorption interpretation

Answer 35

If the reactivity remains, the antibody has not been completely removed. Repeat the adsorption process If there is no reactivity, that means the antibody has been completely absorbed. Adsorbed plasma can now be tested against panel cells to identify alloantibodies

Question 36

significance of adsorption

Answer 36

Removing autoantibody permits detection and identification of coexisting alloantibodies. Reagent preparation Separating multiple antibodies present in plasma or eluate to aid in identification Can only be done if you have reagent red cells Confirmation of the presence of a weak antigen on red cells esp ABO discrepancies Confirmation of antibody specificity

Question 37

is the determination of the level of antibodies present in a patient's blood,

Answer 37

Antibody Titration

Question 38

antibody taitration used

Answer 38

useful are: Estimating antibody in Allo-immunized pregnant women Elucidating Autoantibody specificity Characterizing HTLA.

Question 39

ANTIBODY TITRATION process

Answer 39

Prepare 5% RCS of reagent cells. Serially dilute serum in 12 tubes. (1:1 to 1:2048) Add appropriate cells into each tube Centrifuge for 1 minute at 3400 rpm. Agitate tubes and evaluate for agglutination Set aside the positive tubes Add 22% BSA to the negative tubes then incubate for an hour Wash the incubated tubes for 3 times. Decant the last wash Add 2 drops of AHG then resuspend Centrifuge for 1 minute at 3400 rpm. Then check for agglutination

Question 40

The reciprocal of the highest dilution of plasma or serum that gives a 1+ reaction is referred to as the

Answer 40

titer

Question 41

anti-D titer

Answer 41

<32;

Question 42

Anti-A or Anti-B titer

Answer 42

<256

Question 43

antibody titration application

Answer 43

APPLICATION Monitoring the risk of HDFN in alloimmunized mothers Isohemagglutinin titration (in donor or patient) HTLA reactivity

Question 44

Put scores in agglutination. If sum of all the scores taken is 10 it means patient has HDFN

Answer 44

SCORE METHOD

Question 45

may be monitored to determine eligibility to receive a non-ABO identical organ

Answer 45

Isohemaglutinnin titer of transplant patients

Question 46

NORMALLY, Antibody reactivity weakens as the dilution increases however, some antibodies initially presents a weak reaction that persist in higher dilutions

Answer 46

HTLA

Question 47

HTLA in bgs

Answer 47

Usually directed against Knops, Ch/Rg, Cost, JMH

Question 48

htla

Answer 48

High Titer, Low Avidity

Question 49

EXPERIMENT FLOW

Answer 49

ADSORPTION ELUTION TITRATION eat