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2.2 Haematology > Practical 4: Exam Notes > Flashcards

Practical 4: Exam Notes Flashcards

1
Q

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

A

7 bands can be seen

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2
Q

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

A

Origin

Slow bands

C band

S band

F band

A band

Fast bands

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3
Q

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

A

Constant spring

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4
Q

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

A

C
E
O-Arab
A2
C-Harlem

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5
Q

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

A

S
G
D
Lepore

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6
Q

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

A

H
Barts
J

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7
Q

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

A

Hb-S

Hb-C-Harlem

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8
Q

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

A

Carry out a H prep

Stain with new methylene blue

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9
Q

What is a normal Hb composition for an adult

A

Mostly HbA with a small percentage of HbA2

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10
Q

What would a high HbA2 indicate

A

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

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11
Q

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

A

This is a patient with sickle cell anaemia

But they have just been transfused with HbA

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12
Q

What does a band at S mean (alkaline)

A

Could be HbS

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

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13
Q

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

A

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

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14
Q

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

A

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)

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15
Q

What is HbH?
(4)

A

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

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16
Q

What are some tell tale signs of HbH
(3)

A

Teardrop cells

Anisopoikilocytosis

Target cells

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17
Q

What are some tell tale signs of HbE
(3)

A

Microcytic

Hypochromic

Abundance of target cell

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18
Q

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

A

Marked anisopoikilocytosis
Target cells
Basophilic stippling
Pappenheimer bodies
Numerous nRBCs

19
Q

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

A

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

20
Q

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

A

B- thalassaemia trait

Mild microcytic anaemia

-> megaloblastic anaemia

21
Q

What indicates B- thalassaemia trait?

A

High A2 only

Mild microcytic anaemia

Megaloblastic anaemia

22
Q

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

A

B-thalassemia intermedia

B- thalassemia major post transfusion

23
Q

What indicates B-thalassemia intermedia
(3)

A

High HbF

Low A0

Slightly raised A2

24
Q

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

A

Hereditary persistence of foetal haemoglobin

25
Q

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

A

B-thalassaemia major

26
Q

What indicates B-thalassaemia major on a HPLC?

A

Very high HbF

No normal HbA

Small increase in HbA2

27
Q

What indicates HbH disease?

A

Fast eluting Haemoglobin

HbH peaks before A

28
Q

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

A

HbS peak after A2

HbF is increased

No HbA

HbA2 is increased

29
Q

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

A

Sickle cell disease

30
Q

How do you know its HbA2 in the peak?

A

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

31
Q

What would indicate HbE?
(2)

A

No HbA

Very large peak at A2 = HbE

32
Q

What does a very large peak at A2 indicate?

A

HbE

33
Q

What is the principle behind the Kleihauer test
(5)

A

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

34
Q

What is the equation for a corrected bleed

A

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

35
Q

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

A

125 iu per ml of foetal cells

36
Q

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

A

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

37
Q

What is the principle behind HPLC
(4)

A

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

38
Q

What are the benefits of HPLC

A

Separates S from Hb Lepore, Hb D-Punjab and Hb G-Philadelphia

Separate Hb E from Hb C and Hb O-Arab

39
Q

What is the main downfall of HPLC

A

Cannot discriminate between HbA2, HbE and Hb Lepore

40
Q

What is the principle behind Hb electrophoresis
(3)

A

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

41
Q

When might you use an acid gel electrophoresis

A

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

42
Q

What is the principle behind capillary electrophoresis?

A

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

43
Q

What is the principle behind the sickle solubility test

A

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

2.2 Haematology (38 decks)

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