Analytical Techniques In Transfusion Flashcards
What factors impede agglutination?
Antibody-antigen complex formation is a reversible reaction, so in the lab the aim is to force the reaction to the right as much as possible so the complex stays together longer allowing it to be measured.
BUT antibodies are MUCH smaller than RBCs and RBCs repel each other due to like negative charges = ZETA POTENTIAL
The zeta potential, whether negative or positive (in NaCl soln) keeps RBCs apart by approx 14 nm = THE SIZE OF AN IgG!
At pH used in the lab antibodies have a slight positive charge = attracted to negative RBCs. But in a saline resuspension RBCs are coated with positive Na, so repellent antibodies. Antibodies may be coated too by negative Cl.
How can these antibody-antigen interactions be manipulated in the lab?
SUMMARY:
- size: centrifuge to bring RBCs closer together
- charge: use potentiators to reduce zeta potential and free ions (eg LISS)
- pH: use buffers at pH 7 to keep RBC- and antibodies+
- temperature: incubation at RT and 37 deg
- concentration: increase Ab:Ag ratio
Low ionic strength saline (LISS):
- Alternative solution to saline with fewer Na and Cl ions
- Potentiates antibody-antigen binding (minimises changes in charge -
less induced charge formation = better interaction allowed)
Albumin:
- Acts as a potentiator and also minimises zeta potential
Polyethylene Glycol (PEG):
- Excludes H20 from around the RBCs by forming a shell
- In this way also creates ‘universal antibodies’ by masking the A or B
antigens on the surface
pH:
- Body pH is around 7.4, lab pH is 7
- If pH falls too low it causes dissociation (elution) of antibodies
and antigens (though this can be useful if we want to assign
specificity to an antibody
Temperature:
- cold reacting antibodies: identify carbohydrates - A,B, O family
lots of cold reacting antibodies don’t work well in humans, but in the
elderly many extremities are cold and agglutination of these can occur
- work best at RT
- clinically insignificant in vivo (except ABO)
- usually IgM
- react against carbohydrate antigens
- warm reacting antibodies: Identify proteins - Rh family
- work best at 37 deg
- clinically significant in vivo
- usually IgG
- react against protein antigens
- agglutination is checked at both temperatures, cold using IMMEDIATE
SPIN and warm using anti-human globulin (AHG).
Concentration:
- increasing the ratio of antibody to antigen to enhance the reaction
- increasing the amount of antibody containing serum relative to RBCs
- But too much antibody can saturate all the binding sites on the RBCs,
so cross links cant form and you cant see agglutination
What are the advantages of haemagglutination over molecular testing?
Advantages:
- Cheap
- Can be automated with cassettes and an optical reader that eliminates
subjectiveness of human interpretation. Also has a built in check (eg
concordance of forward and reverse typing).
But limitations mean molecular testing may become preferable in follow up of results
Limitations:
- Difficult to phenotype a recently transfused patient (Blood
sample will be a mixture own own cells and donor cells. On gel
card will get populations of cells that appear positive and negative for
different things, don’t know which is patients! - must be sent to
reference lab to find out genotype of patient through molecular
testing)
- Difficult to phenotype RBCs coated with IgGs (Cells aren’t binding to
reagent due to autoantibodies. Results appear negative that are actually
positive)
- Gives only an indirect indication of a foetus at risk of HDFN
(Haemagglutination of mum and dad will only estimate baby. Working
on risk - so give mum antiD anyway. If we could screen every foetus we
wouldn’t have to waste anti-D)
- Subjective test
Why have we not moved over to a fully molecular system?
There are limitations:
- Genotype doesn’t always equal phenotype = eg Bombay phenotype,
this person’s A and B transferase genes would appear normal but
actually they don’t have H.
- must be able to test silencing genes as part of this test
- GATA for FY typing
- RHD pseudogene (Reagents behave different. This pseudogene
turns off the RHD gene so they appear +ve, but its not working!)
- changes in GYPD in S antigen typing (Binds to glycoproteins in
membrane, post translational modification in this isn’t
detected)
- BMT will alter genotype depending on which tissue is sampled
- not all blood group antigens are the result of a SNP
- there may be many alleles for a phenotype, requiring multiple assays to
predict a phenotype (Lot of genetics that can make the same
antigen so this can increase the cost)
- not all alleles in ethnic populations are known
- molecular basis of some blood groups is still unknown
What is immediate spin phase reverse testing involve?
- 22 degrees C
- reagent RBCs in tube with known antigens
- add patient serum (with potential Abs to ags in)
- tube centrifuged (immediate spin)
- agglutination? Graded based on strength of agglutination. 4+ is
strongest
COLD ANTIBODIES
LOOKING FOR DIRECT AGGLUTINATION
What does tube testing for warm antibodies involve?
- 37 degrees C
- reagent RBCs and patient serum added to tube together
- centrifuged
- potentiator added and incubation at 37 degrees C to allow warm
reacting antibodies to bind - tube is centrifuged again
- agglutination? Graded based on strength of agglutination. 4+ is
strongest
WARM ANTIBODIES
LOOKING FOR DIRECT AGGLUTINATION
What does tube testing using AHG phase involve?
LOOKING FOR INDIRECT AGGLUTINATION
Recognises complementary antigen - looking for weak reactions that need enhancing with AHG
- after 37 degrees C phase, cells are washed to remove any unbound Ab
- addition of AHG
- AHG binds to any antibodies that have coated the RBCs
- agglutination? Graded based on strength of agglutination. 4+ is
strongest
