Serological techniques

Question 1

Explain the steps & principle of the INDIRECT antiglobulin (IAT) test w/ the use of anti-human globulin (AHG)

Answer 1

1. incubate patient plasma w/ Ag-known RBC @ 37ºC (optimal temp of IgG) => sensitised RBC
2. Wash 4x (remove unbound IgG = not create false neg result ≠ agglutination)
3. Add AHG => binds to IgG => agglutination (+ve)
   * detect if IgG is present in plasma

Question 2

Explain the steps & principle of the DIRECT antiglobulin test (DAT) w/ the use of anti-human globulin (AHG)

Answer 2

1. Wash patient sample 4x (remove unbound IgG)
2. Add AHG => bindsto IgG => agglutination (+ve)
   * detect IgG already bound to RBC surface

Question 3

Why is the wash step important when the AHG test is performed in tubes?

Answer 3

To remove unbound IgG so that it doesn’t cause a false negative result ie. AHG bound to excess IgG ≠ agglutination = false neg.

Question 4

When are AHG control cells added to the AHG test? Why?

Answer 4

a) AHG control cells are added to if sample have a negative rxn after adding AHG
b) checks that AHG is working (see agglutination) = valid ie. no agglutination means that IgG is not bound to RBC & not due to dysfunctional AHG reagent

Question 5

Explain the difference between the DAT(/DCT) and IAT(/DCT).
*Coombs’

Answer 5

• DAT: detects Ab &/or C3d bound to patient RBC in vivo

- IAT: detects if Ab in plasma binds to RBC in vitro

Question 6

When are DAT & IAT used?

Answer 6

• IAT: Ab screening & identification, cross-matching, phenotyping
• DAT: Haemolytic transfusion reactions (HTR), Haemolytic disease of new born (HDNB)

Question 7

Why are potentiators used in the AHG test?

Answer 7

to decrease the SETA potential => dec. distance b/w RBC = inc. Ab binding (required since IgG is small = indirect agglutination)

Question 8

Explain the mechanism of action for low ionic strength saline as a potentiators.

Answer 8

dec. ionic strength = dec. ZETA potential = inc. Ab binding

Question 9

Explain the mechanism of action for polyethylene glycol (PEG) as a potentiators.

Answer 9

remove water in b/w cells = concentrates Ab around RBC

Question 10

Name 3 potentiators & explain their mechanism of action

Answer 10

• low ionic strength saline: dec. ionic strength = dec. ZETA potential = inc. Ab binding
• polyethylene glycol (PEG): remove water in b/w cells = concentrates Ab around RBC
• Albumin: inc. dielectric constant (Epotential) of medium = dec. ZETA potentail

Question 11

When performing the AHG test using cards, why do the samples not require washing at any point of the test?

Answer 11

bc a high density solution sits on top of the beads > traps plasma proteins

Question 12

What are the benefits of performing haemagglutination reactions in cards?*I

Answer 12

• Because you don’t need to wash

- Quick & easy bc all reagents prepared in card

Question 13

Describe the appearance of each reaction grades when using the 0-4+ scale when using the card method

Answer 13

0: dense agglutinate sits on bottom
1+: small smudge of agglutinate sitting 1/4 to bottom
2+: large smudge of agglutinate 2/3 from the bottom
3+: slight dense agglutinate sitting 3/4 to 1/2 of tube
4+: dense agglutinate sitting just below the surface of the beads

Question 14

What is identified @ each of the 3 phases of IAT?

Answer 14

1. immediate spin: detect IgM = agglutination or haemolysis
2. 37ºC: detect strong IgG = agglutination or haemolysis (complement)
3. AHG: detect IgG = agglutination (bound to RBC)

Question 15

Why are the IS & 37ºC phase not commonly included when performing Ab screen?

Answer 15

bc most significant Ab are only detected at the AHG phase

Question 16

Can a long cell panel provide 100% surety that Ab are not present in patient’s serum?

Answer 16

No bc there are some Ab that can be missed if @ low frequency or lo titre Ab

Question 17

What is an autocontrol set up Ab ID procedure 7 what is the significance of a positive result?

Answer 17

• Detect auto-Ab

- positive result means Ab are present

Question 18

Causes of false pos in control tube - supposed to be negative

Answer 18

• very Hi [RBC] - must be 1% cell suspension
• expired card
• Fibrin in sample
• not centrifuged long enough
• Wharton’s jelly

Question 19

Are you required to do RVS ABO grouping on baby’s plasma?

Answer 19

No if <4months bc Ab will come from mum

Question 20

Principle of CAT/card method

Answer 20

• CAT: column agglutination tech.
• High density solution over sephadex beads/matrix
• reagent Ab can be incorporated in the beads

Question 21

Describe the types of cards & the uses/purpose of each

Answer 21

a) Anti -A, -B, -D (DVI-), ctl, buffer bc can add patient’s plasma
b) know ABO/Rh group of transfusion recipients

Question 22

a) What’s in the columns of a ABO/Rh for newborns DVI+ card

b) What’s its uses/purpose

Answer 22

a) Anti- A, -B, -A,B, -D (DVI), ctl, DAT

b) determine ABO/Rh group of newborns & DAT

Question 23

a) What’s in the columns of a Coombs DVI+ card

b) What’s its uses/purpose

Answer 23

a) has buffer bc Can add patient plasma & Screening cells
b) Ab screening, Ab ID, X-match & phenotyping? (all things that use IAT)