TM Methods

Question 1

Define primary vs secondary sensitization

Answer 1

Primary: Ab binds antigen on cells (can’t see this)

Secondary: Ab coated cells bind each other (lattice/cross-linking); requires slight Ab excess; results in visible hemagglutination

Question 2

factors that affect sensitization

Answer 2

• concentration
  > antibody
  > antigen (dosage, individual variation)
• temperature
  > 4C - room temp for IgM
  > 37C for IgG
• time (longer time = more time for rxn but too long = popping off so rxn starts to decrease)
• pH (used to our advantage to elute Ab)
  > react best at 7.0 - 7.5
  > less reactive <6 or >8

Question 3

zeta potential

Answer 3

• force of repulsion between red cells
• red cells carry a net negative charge at their surface (sialic acid)
• when suspended in saline, Na+ form ionic cloud around cells (like charges repel)

Question 4

enhancement media (additives to decrease ZP)

Answer 4

1. Low Protein (6%) Albumin
   - similar to plasma concentration
   - IgM will bind at 6% but not IgG
   - used as a control instead of autocontrol
2. LISS*:
   - reduces positive charge bc sodium is decreased

*NOTE: Low Ionic Strength Solution

Question 5

PEG

Answer 5

• ideal charge helps Ab uptake
• dehydrates cell (removes ionic water from cell surface)
• this assists with hydrophobic interactions of Ab/Ag

Question 6

disadvantages of PEG

Answer 6

• must wash away really well once Ab:Ag complex is attained
• can cause BINDING OF CLINICALLY INSIGNIFICANT Abs

Question 7

T or F: PEG is more sensitive than LISS

Answer 7

TRUE; PEG is more sensitive than LISS

Question 7

enzymes increase sensitivity of TM testing by..

Answer 7

• removes sialic acid (decreasing Z potential) AND steric hindrance to allow Ab to bind

Question 8

3 natural enzyme treatments in TM

Answer 8

• Ficin from figs
• Papain from papayas
• Bromelin from pineapples

Question 9

method to detect IgG antibodies

Answer 9

Antiglobulin test
- uses LISS, PEG, enzymes for Ab uptake at 37°C
- anti-IgG Ab (IgM) as 2° Ab
- increases sensitivity

Question 10

IAT principle

Answer 10

• IgG antibody (plasma or antisera) mixed with cells (screen cell, panel cell donor cell or patient cell) and enhancement media
• 37°C incubation: IgG binds cell
• WASH removes unbound Ab (in plasma)
• anti-IgG binds sensitized cells = agglutination
• if no agglutination is present = add Coombs cells to ensure AHG is working

Question 11

DAT vs IAT

Answer 11

Direct Antiglobulin Test:
- used to detect immunoglobulin, complement, or both on the surface of red blood cells (RBCs)

Indirect Antiglobulin Test:
- used to detect RBC antibodies in patient serum

NOTE: immunoglobulin = antibodies

Question 12

DAT procedure

Answer 12

• add one drop PATIENT RED CELLS (3%)
• WASH three times
• 2 drops poly AHG + spin
• If negative: add 1 drop COOMBS control

Question 13

causes of a positive DAT

Answer 13

• AIHA (warm, cold)
• hemolytic TRANSFUSION RXNS
• HDFN
• DRUG-related hemolytic anemia

NOTE: sensitized cells = pos DAT

Question 14

what are the Coombs control cells?

Answer 14

• IgG sensitized cells
• added to negative IAT and DAT tests
• ensures that AHS was added; is working; adequate washing to remove unbound Abs
• expected = 1-2+
• neg result = invalid test and must be repeated

Question 15

causes of false negatives for antiglobulin tests

Answer 15

• poor washing; unbound IgG neutralized AHG
• reagent deterioration
• failure to decant wash saline (dilutes AHG)
• under-centrifugation
• overshaking tubes
• plasma not added
• incorrect incubation time/ temperature
• contaminated glassware

Question 16

causes of false positives for antiglobulin tests

Answer 16

• failure to wash before adding AHG (PEG)
• overcentrifugaiton
• undershaking
• IAT performed using - DAT positive RBC
• clotted, old, refrigerated specimen
• Wharton’s jelly (if doing DAT on cord cells)

Question 17

How much of an increase in Ab titre is significant ?

Answer 17

x²; by two powers

Question 18

Method used to identify an Ab

Answer 18

Acid elution; decreases pH to pop off Ab

Question 19

Method used to obtain DAT neg cells for Ag typing. What is its limitation ?

Answer 19

ZZAP, WARM
- destroys Duffy, MNS, Kell