BB Lab Practical

Question 1

Making a 3-5% RBC suspension

Answer 1

serum to cell ration is key
-transfer 2gtts of an EDTA whole blood sample to test tube
-Add 0.9% saline until you visually equate it to commercial grade red cell 3-5% sus provided
-label test tube
- Add 1gtt of saline into each
-Add 1 gtt of Red cell sus to T
-Centrifuge both tubes for 30s
-observe and compare the size of button

Question 2

Why is serum to cell ratio important?

Answer 2

agglutination of Ab-Ag complex requires Ab and Ag be present in optimal portions. Excess of either can lead to false positives or false-negatives.

Question 3

List 3 causes of possible false-negatives in antiglobulin tests

Answer 3

Saline left in the tube after washes, over/under centrifugation, failure to add AHG, serum, or enhancement media, excessive heat, etc…

Question 4

Why are Red Cells thoroughly washed prior to the performance of the Antiglobulin test?

Answer 4

To remove unbound protein (free globulin)

Question 5

What does a small button result indicates?

Answer 5

-not enough cells- then add another drop of red cells mix and retest.
-too much saline-remove some saline by spinning test tube with the cell sus for 60s. Remove 1-2 gtts of saline from tube. Resus and retest

Question 6

What does a large button result indicate?

Answer 6

-Too many cells- add more saline, mix and retest

Question 7

Why do we incubate RBCs with Antisera?

Answer 7

allows time for Ab molecule attachment to RBC antigen

Question 8

What are the steps involved in DAT?

Answer 8

-Ab/Ag sensitization IN VIVO- directly from pt
-wash 3-4 tmes
- AHG
-Read/interpret
-Auto/alloantibodies
- IgG or C3

Question 9

What are the steps involved in DAT?

Answer 9

-Ab/Ag sensitization IN VIVO- directly from pt
-wash 3-4 tmes
- AHG
-Read/interpret
-Auto/alloantibodies
- IgG or C3

Question 10

What are the steps involved in IAT?

Question 11

After addition of Ig-G sensitized Red cells to a negative antiglobulin test (eg. IAT), the test is invalid if there is …

Answer 11

no agglutination. Check cells are cells coated with IgG and should react positively with the AHG in the Tube. Check cells bridge desensitized cells and confirms validity of negative result). If check cells are negative (no agglutination), the procedure was not performed correctly and should be repeated. Goal is positive rxn with AHG.the test result is considered invalid if there is no agglutination. No agglutination -This is because the absence of agglutination indicates that the antiglobulin reagent was unable to detect the sensitized cells.

Question 12

If the Ig-G sensitized red cells fail to react appropriately, what is the next step?

Answer 12

Restart

Question 13

For the weak D test to be valid, what is the result of the Rh (D) control?

Answer 13

Negative.

Question 14

Why is Albumin used in Antibody detection?

Answer 14

22% albumin allows Ab-coated RBCs to come into closer contact with each other. 30 min incubation. disadvantage long incubation, cost.

Question 15

What is the purpose of Autocontrol?

Answer 15

Provide Useful information in determining whether patient antibody is directed against his or her a red blood cell.

Question 16

LISS

Answer 16

Low Ionic Strength solution media- enhances Ab uptake- 10-15min incubation-shorter incubation- potentiator by decreased zeta potential.

Question 17

Forward

Answer 17

red cell typing-using KNOWN reagent antisera (Ab) to detect UNKNOWN Ag on RBC membrane

Question 18

Reverse

Answer 18

serum typing-using reagent cells with KNOWN ABO Ag and testing the UNKNOWN serum of the pt for ABO group Ab

Question 19

Does hemolysis indicate antigen-antibody reaction?

Answer 19

Yes- hemolysis is normally cauused by complement activation. You may observe small button with pink to red supernatant is observed after centrifugation.

Question 20

The antiglobulin test involves __ to patient_.

Answer 20

AHG reagent (contains monospecific to IgG) to plasma. Plasma contains Antibody.