Practical 1-tests for Hb Flashcards
ideal Hb
HbA
How does HbS come About
Point mutation of nucleotides on beta chain at position 6 of HbA from glutamic acid to valine changing it from hydrophilic to hydrophobic rendering the newly formed HbS unsoluble.
Tests for sickling status
Hb electrophoresis
2% sodium metabisulphite method
solubility test
what does 2% metabisulphite do?
it sucks the oxygen out of the RBC, rendering those with HbS the tendency to sickle. HbS sickles under low oxygen tension
2% sodium metabisulphite method
- put blood in an anticoagulant
- put 2% metabisulphite on a glass slide and add a drop of patients blood
- cover and keep like that for an hour
- if HbS is present all or some cells will sickle
this shows there is at least an S present
precautions for 2% metabisulphite method
it is important to cover the slide so no oxygen enters the prepared solution. If oxygen is allowed, sickling status cannot be detected
Limitations of 2% sodium metabisulphite method
It could not detect if one was fully a sickler or not.
Principle of electrophoresis
Electrophoresis is the migration of charged solutes in a liquid medium/buffer under the influence of an electric field.
1.Hb has a net negative charge in alkaline medium of pH 8.2-8.6
2. Hb will migrate towards the anode in an electric field
3 The electric current applied seperates the Hb into normal or abnormal types
How many types of electrophoresis do we have
4
- cellulose acetate paper
2.Agarose gel electrophoresis - polyacrylamide gel electrophoresis
4starch gel electrophoresis
How is haemolysate prepared
wash blood 3-4 times with normal saline centrifuge to collect RBC freeze RBC or lyse with saponin thaw and add a few drops of CCl4 CCL4 will extract pure Hb vortex and centrifuge to collect Hb solution
In what sequence do the different Hb move?
HbA > HbF > HbS >HbC
AFSC
How to detect S beta thallasemia from Hb electrophoresis
A widened band between A and S with more of the band toward S
What conclusion can you derive when 3 concrete bands are present in an adult
It means person has been recently transfused. i.e if you see ASC
i.e AC dude has recently been transfused with AS blood
What does ASF in an adult mean
hereditary persistence of fetal Hb
Principle of solubility test
RBC lysed by saponin to release Hb
Hb is reduced by sodium dithionite /hydrosulfite in a concentrated PO4 buffer of pH 7.1
solution is mixed for about 5 mins
if end solution is clear, patient is neither SS OR SC ( AA AC AS)
If end solution is turbid characterized by low solubility and formation of crystals patient is SS OR SC
USUALLY DONE IN REMOTE AREAS
IMPORTANCE OF Hb F determination
It is important to determine rhesus state. that is is the rhesus state of mother and child is compatible
Precursor of HbF
Hb Barts
How is HbF Test used to determine compatibility between mother and child
Lack of compatibility between mother and child rhesus factors will cause mothers antibodies to break down the RBCs of child causing them to mix up - hemolytic dx of newborn
HbF is resistant to denaturation by alkaline solution
NaOH is added to the mother child blood mix FOR 1 MINUTE
After 1 minute add acidified ammonium sulphate
Mothers RBC isnt resistant and will be lysed and precipitated
Whatever color is left will be for HbF
HbF determination tests include
kleihauer betke test
mixture of mother and child blood plus alkaline solution
Principle of kleihauer betke test
Freshly air dried film of rbc is fixed with 80% ethanol
slide is rinsed with water repeatedly
Formula for finding HbF percentage in kleihauer betke test
count number of HBF cells
……………………………………………… * 100
total number of cells
How to calculate total volume of fetal bleed
%HBF * 50
Number of injections of rhogam
total volume of fetal bleed
………………………………………………
30
Mothers cells on fixed slide are known as
Ghost cells. They appear pale.
