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2.2 Haematology > Practical 4: Haemoglobin Investigation > Flashcards

Practical 4: Haemoglobin Investigation Flashcards

1
Q

What test is used for the investigation of HbF in maternal circulation?

A

The Kleihauer-Betke test (Kleihauer test)

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2
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

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3
Q

Why is it important to determine the volume o f fetomaternal haemorrhage

A

To determine how much anti-D should be given to the mother during RhD incompatibility

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4
Q

Describe the structure of haemoglobin
(2)

A

Composed of two pairs of polypeptide chains

Each chain is linked to the haem, a tetrapyrrolic nucleus (poryphrin) which chelates an iron atom

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5
Q

What determines the type of haem molecule

A

The globin determines the type

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6
Q

What polypeptide chains constitute normal human Hb?

A

a, B, gamma and delta,

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7
Q

List some Hb variants

A

Hb S

Hb C

Hb E

Hb D

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8
Q

What causes the formation of abnormal haemoglobins

A

Substitution of amino acids by mutation

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9
Q

Why are abnormal haemoglobins detrimental to health

A

The Hb will have different surface charges

Hb will have different electrophoretic mobilities

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10
Q

What mutation causes Hb S

A

B6 Glu> Val

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11
Q

What mutation causes Hb C

A

B6 Glu>Lys

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12
Q

What percentage of sickle cell disease patients have HbS/C

A

20-50%

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13
Q

What causes Hb E

A

B26 Glu>Lys

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14
Q

What causes Hb D

A

B121 Glu>Gln

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15
Q

What does decreased synthesis of one of the Hb chains lead to

A

Quantitative abnormalities of Hb known as thalassaemia

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16
Q

What methods do we use to investigate thalassaemia
(6)

A

Clinical history and/or physical examination

Family history

Full blood count and blood film

Primary technique

Secondary technique

Supplementary tests

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17
Q

What primary techniques are used to investigate haemoglobinopathies
(3)

A

High performance liquid chromatography

Isoelectric focusing (IEF)

Hb electrophoresis

18
Q

What secondary techniques are used to investigate Haemoglobinopathies
(3)

A

Hb electrophoresis

HPLC

Sickle solubility test

19
Q

What supplementary tests can be used to investigate haemoglobinopathies

A

Reticulocyte count

HbH inclusions

DNA analysis

Mass spectrometry

20
Q

Write a note on high performance liquid chromatography
(3)

A

Method of choice for haemoglobin variants

Able to detect and resolve a large number of Hb variants especially when used in conjunction with other methods of ID

Able to quantify Hb

21
Q

What is the principle behind HPLC
(2)

A

Different variants have different retention times

Hb is absorbed into column and eluted by means of a gradient of increasing ionic strength

22
Q

How does HPLC work
(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

23
Q

What are the benefits of HPLC?
(2)

A

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

Separate Hb E from Hb C and Hb O-Arab

24
Q

What is the downfall of HPLC

A

Cannot discriminate between HbA2, HbE and Hb Lepore

25
Q

What type of electrophoresis is Hb electrophoresis

A

An alkaline/acid gel electrophoresis

26
Q

What is the principle behind Hb electrophoresis

A

HBE utilises the charged properties of normal Hb and its variants to replicate characteristic mobility patterns on both alkaline and acidic gels

27
Q

Why might Alkaline HBE be used
(3)

A

At alkaline pH Hb is negatively charged and hence migrates towards the anode

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

28
Q

Why might an acid HBE be used
(3)

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

29
Q

What do we more commonly use other than gel elctrophoresis

A

Capillary electrophoresis

30
Q

Give an example of a capillary electrophoresis we use

A

Sebia

31
Q

What is the Sebia assay based on
(3)

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

32
Q

What is the screening test for HbS

A

The sickle solubility test

33
Q

What is the principle of the sickle solubility test
(2)

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

34
Q

Give the name of a commercial test we can use for sickle cell

A

Sickle scan

35
Q

What is sickle scan?

A

A rapid, qualitative lateral flow immunoassay kit for the identification of SCD of HbA, HbS and HbC

36
Q

How do you use a sickle scan test?
(4)

A

A small amount of blood is taken and placed into buffer to release Hb by lysing erythrocytes

Three drops of the treated sample are added to the sample inlet of the test

Results are red in five minutes

Presence of Hb variants A, S and C will be indicated by blue lines

37
Q

When carrying out the Kleihauer test, how many cells do you need to count

A

6000 adult cells

(about 650 counted x9

38
Q

How do you determine the amount of FM bleed in mls from your HbF count

A

(Number of foetal cells/ number of adult cells) x 2400 = mls

39
Q

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

A

125 iu per ml of foetal cells

40
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

41
Q

You need to be able to interpret Alkaline Haemoglobin Electrophoresis

A

You need to be able to interpret Alkaline Haemoglobin Electrophoresis

Look in notes and lab manual for practice

42
Q

What tests would you carry out to investigate haemoglobin

A

HPLC

Hb electrophoresis or Sebia assay (capillary electrophoresis)

Sickle scan -> HbS

Sickle solubility -> HbS

2.2 Haematology (38 decks)

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