Practical 1-tests for Hb

Question 1

ideal Hb

Answer 1

HbA

Question 2

How does HbS come About

Answer 2

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.

Question 3

Tests for sickling status

Answer 3

Hb electrophoresis
2% sodium metabisulphite method
solubility test

Question 4

what does 2% metabisulphite do?

Answer 4

it sucks the oxygen out of the RBC, rendering those with HbS the tendency to sickle. HbS sickles under low oxygen tension

Question 5

2% sodium metabisulphite method

Answer 5

1. put blood in an anticoagulant
2. put 2% metabisulphite on a glass slide and add a drop of patients blood
3. cover and keep like that for an hour
4. if HbS is present all or some cells will sickle

this shows there is at least an S present

Question 6

precautions for 2% metabisulphite method

Answer 6

it is important to cover the slide so no oxygen enters the prepared solution. If oxygen is allowed, sickling status cannot be detected

Question 7

Limitations of 2% sodium metabisulphite method

Answer 7

It could not detect if one was fully a sickler or not.

Question 8

Principle of electrophoresis

Answer 8

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

Question 9

How many types of electrophoresis do we have

Answer 9

4

1. cellulose acetate paper
   2.Agarose gel electrophoresis
2. polyacrylamide gel electrophoresis
   4starch gel electrophoresis

Question 10

How is haemolysate prepared

Answer 10

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

Question 11

In what sequence do the different Hb move?

Answer 11

HbA > HbF > HbS >HbC

AFSC

Question 12

How to detect S beta thallasemia from Hb electrophoresis

Answer 12

A widened band between A and S with more of the band toward S

Question 13

What conclusion can you derive when 3 concrete bands are present in an adult

Answer 13

It means person has been recently transfused. i.e if you see ASC

i.e AC dude has recently been transfused with AS blood

Question 14

What does ASF in an adult mean

Answer 14

hereditary persistence of fetal Hb

Question 15

Principle of solubility test

Answer 15

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

Question 16

IMPORTANCE OF Hb F determination

Answer 16

It is important to determine rhesus state. that is is the rhesus state of mother and child is compatible

Question 17

Precursor of HbF

Answer 17

Hb Barts

Question 18

How is HbF Test used to determine compatibility between mother and child

Answer 18

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

Question 19

HbF determination tests include

Answer 19

kleihauer betke test

mixture of mother and child blood plus alkaline solution

Question 20

Principle of kleihauer betke test

Answer 20

Freshly air dried film of rbc is fixed with 80% ethanol

slide is rinsed with water repeatedly

Question 21

Formula for finding HbF percentage in kleihauer betke test

Answer 21

count number of HBF cells
……………………………………………… * 100
total number of cells

Question 22

How to calculate total volume of fetal bleed

Answer 22

%HBF * 50

Question 23

Number of injections of rhogam

Answer 23

total volume of fetal bleed
………………………………………………
30

Question 24

Mothers cells on fixed slide are known as

Answer 24

Ghost cells. They appear pale.