Practical 4: Exam Notes

Question 1

During alkaline gel electrophoresis to investigate haemoglobin, how many bands are produced?

Answer 1

7 bands can be seen

Question 2

During alkaline gel electrophoresis to investigate haemoglobin, what 7 bands are produced?

Answer 2

Origin

Slow bands

C band

S band

F band

A band

Fast bands

Question 3

During alkaline gel electrophoresis to investigate haemoglobin, what Hb types can be found in the slow band?

Answer 3

Constant spring

Question 4

During alkaline gel electrophoresis to investigate haemoglobin, what five Hb types can be found in the C band?

Answer 4

C
E
O-Arab
A2
C-Harlem

Question 5

During alkaline gel electrophoresis to investigate haemoglobin, what three Hb types can be found in the S band?

Answer 5

S
G
D
Lepore

Question 6

During alkaline gel electrophoresis to investigate haemoglobin, what three Hb types can be found in the fast band?

Answer 6

H
Barts
J

Question 7

What two Hb types can result in a positive sickle solubility test?

Answer 7

Hb-S

Hb-C-Harlem

Question 8

What would you do if you found there to be Hb H from gel electrophoresis

Answer 8

Carry out a H prep

Stain with new methylene blue

Question 9

What is a normal Hb composition for an adult

Answer 9

Mostly HbA with a small percentage of HbA2

Question 10

What would a high HbA2 indicate

Answer 10

Beta thalassaemia
-> lack B chain needed to form HbA1
-> HbA2 made of alpha chain and delta chain

Question 11

What would a band at S and A indicate (alkaline gel)?

Answer 11

This is a patient with sickle cell anaemia

But they have just been transfused with HbA

Question 12

What does a band at S mean (alkaline)

Answer 12

Could be HbS

Need to confirm not Hb G, D or Lepore which would be in the same band

Question 13

What does a band at S and C mean (alkaline)
(4)

Answer 13

Heterozygous -> two types of Hb

One type in S band -> Hb S, G, D, Lepore

Other type in C band -> Hb C, E, O-Arab, A2, C-Harlem

Need to carry out other tests

Question 14

What does a thick band at A and a weak band at C mean?

Answer 14

Not B thalassaemia as there is the correct amount of HbA1

Must be a carrier of a Hb from band C (C, E, O-Arab, A2, C-Harlem)

Question 15

What is HbH?
(4)

Answer 15

alpha-thalassemia intermedia

It is a form of Alpha-thalassemia which most commonly occurs due to deletion of 3 out of 4 of the α-globin genes

Because of this alpha deficiency, HbA1 cannot be formed, excess beta chains build up

Tetramers of beta chains form (B4-tetramers) -> called HbH

Question 16

What are some tell tale signs of HbH
(3)

Answer 16

Teardrop cells

Anisopoikilocytosis

Target cells

Question 17

What are some tell tale signs of HbE
(3)

Answer 17

Microcytic

Hypochromic

Abundance of target cell

Question 18

What are some tell tale signs of B-thalassaemia major
(5)

Answer 18

Marked anisopoikilocytosis
Target cells
Basophilic stippling
Pappenheimer bodies
Numerous nRBCs

Question 19

What are some tell tale signs of B-thalassaemia minor
(6)

Answer 19

Mild anaemia
Microcytic, hypochromic
Occasional target cells
Anisopoikilocytosis range from marked to mild
Basophilic stippling
Polychromasia

Question 20

What does a slightly high A2 only on a HPLC indicate?

Answer 20

B- thalassaemia trait

Mild microcytic anaemia

-> megaloblastic anaemia

Question 21

What indicates B- thalassaemia trait?

Answer 21

High A2 only

Mild microcytic anaemia

Megaloblastic anaemia

Question 22

What does a high HbF, low A0 and raised A2 indicate
(2)

Answer 22

B-thalassemia intermedia

B- thalassemia major post transfusion

Question 23

What indicates B-thalassemia intermedia
(3)

Answer 23

High HbF

Low A0

Slightly raised A2

Question 24

What does a high HbF only indicate, no other clinical symptoms?

Answer 24

Hereditary persistence of foetal haemoglobin

Question 25

What does very high HbF, no normal Hb A and a small increase in HbA2 indicate?

Answer 25

B-thalassaemia major

Question 26

What indicates B-thalassaemia major on a HPLC?

Answer 26

Very high HbF No normal HbA Small increase in HbA2

Question 27

What indicates HbH disease?

Answer 27

Fast eluting Haemoglobin HbH peaks before A

Question 28

What indicates HbS -> sickle cell disease? (4)

Answer 28

HbS peak after A2 HbF is increased No HbA HbA2 is increased

Question 29

What does a HbS peak, HbF increase, no HbA and increased HbA2 indicate?

Answer 29

Sickle cell disease

Question 30

How do you know its HbA2 in the peak?

Answer 30

HbE can only be found in more than 30% concentration -> so small quantities has to be HbA2

Question 31

What would indicate HbE? (2)

Answer 31

No HbA Very large peak at A2 = HbE

Question 32

What does a very large peak at A2 indicate?

Answer 32

HbE

Question 33

What is the principle behind the Kleihauer test (5)

Answer 33

The fact that foetal rbcs contain mostly HbF and that these cells are resistant to acid elution Whereas HbA is acid-sensitive Therefore when exposed to acid buffer HbA will elute from rbcs and HbF will be retained After staining with H&E the foetal cells will stain dark pink and the maternal cells will be very pale (only outlines of cells really) The % of Foetal cells identified can be then used to estimate the volume of fetomaternal haemorrhage

Question 34

What is the equation for a corrected bleed

Answer 34

multiply the ratio of foetal cells/mothers cells by 2400 to give your answer in mls

Question 35

How much anti-D should be given for a 1ml foetal bleed

Answer 35

125 iu per ml of foetal cells

Question 36

What should you do if the FMH shows a bleed over 2.5mls

Answer 36

The size of the bleed should be reassessed using flow cytometry which is more accurate for the assessment of large bleeds

Question 37

What is the principle behind HPLC (4)

Answer 37

Exchange column contains small spheres of silica which are modified to be weakly cationic An anticoagulated specimen is lysed and diluted in buffer and injected into column Hb is absorbed into column and eluted by means of a gradient of increasing ionic strength Eluate passes through a photometer which measures changes in absorbance

Question 38

What are the benefits of HPLC

Answer 38

Separates S from Hb Lepore, Hb D-Punjab and Hb G-Philadelphia Separate Hb E from Hb C and Hb O-Arab

Question 39

What is the main downfall of HPLC

Answer 39

Cannot discriminate between HbA2, HbE and Hb Lepore

Question 40

What is the principle behind Hb electrophoresis (3)

Answer 40

HBE utilises the charged properties of normal Hb and its variants to replicate characteristic mobility patterns on both alkaline and acidic gels Variant haemoglobins have alterations in their surface charge due to changes in surface amino acids This hence changes the speed of its migration and causes characteristic separation based on set mobility patterns

Question 41

When might you use an acid gel electrophoresis

Answer 41

Acid agarolse gel is available commercially and can complement alkaline gel electrophoresis It is able to separate Hb C from the other Hb variants with the same mobility at alkaline pH It is also able to do the same for Hb S

Question 42

What is the principle behind capillary electrophoresis?

Answer 42

The principle of capillary electrophoresis in free solution Hb fractions are separated in silica capillaries by their electrophoretic mobility and electroosmotic flow at a high voltage in an alkaline buffer Hb fractions are directly detected at the specific absorbance of 415nm

Question 43

What is the principle behind the sickle solubility test

Answer 43

HbS is insoluble in the deoxygenated state in a high molarity phosphate buffer (sodium hydrosulfite) The crystals that form refract light and cause the solution to be turbid, can't see through