Practical 4: Haemoglobin Investigation Flashcards
What test is used for the investigation of HbF in maternal circulation?
The Kleihauer-Betke test (Kleihauer test)
What is the principle behind the Kleihauer test
(5)
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
Why is it important to determine the volume o f fetomaternal haemorrhage
To determine how much anti-D should be given to the mother during RhD incompatibility
Describe the structure of haemoglobin
(2)
Composed of two pairs of polypeptide chains
Each chain is linked to the haem, a tetrapyrrolic nucleus (poryphrin) which chelates an iron atom
What determines the type of haem molecule
The globin determines the type
What polypeptide chains constitute normal human Hb?
a, B, gamma and delta,
List some Hb variants
Hb S
Hb C
Hb E
Hb D
What causes the formation of abnormal haemoglobins
Substitution of amino acids by mutation
Why are abnormal haemoglobins detrimental to health
The Hb will have different surface charges
Hb will have different electrophoretic mobilities
What mutation causes Hb S
B6 Glu> Val
What mutation causes Hb C
B6 Glu>Lys
What percentage of sickle cell disease patients have HbS/C
20-50%
What causes Hb E
B26 Glu>Lys
What causes Hb D
B121 Glu>Gln
What does decreased synthesis of one of the Hb chains lead to
Quantitative abnormalities of Hb known as thalassaemia
What methods do we use to investigate thalassaemia
(6)
Clinical history and/or physical examination
Family history
Full blood count and blood film
Primary technique
Secondary technique
Supplementary tests
What primary techniques are used to investigate haemoglobinopathies
(3)
High performance liquid chromatography
Isoelectric focusing (IEF)
Hb electrophoresis
What secondary techniques are used to investigate Haemoglobinopathies
(3)
Hb electrophoresis
HPLC
Sickle solubility test
What supplementary tests can be used to investigate haemoglobinopathies
Reticulocyte count
HbH inclusions
DNA analysis
Mass spectrometry
Write a note on high performance liquid chromatography
(3)
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
What is the principle behind HPLC
(2)
Different variants have different retention times
Hb is absorbed into column and eluted by means of a gradient of increasing ionic strength
How does HPLC work
(4)
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
What are the benefits of HPLC?
(2)
Separates S from Hb Lepore, Hb D-Punjab and Hb G-Philadelphia
Separate Hb E from Hb C and Hb O-Arab
What is the downfall of HPLC
Cannot discriminate between HbA2, HbE and Hb Lepore
What type of electrophoresis is Hb electrophoresis
An alkaline/acid gel electrophoresis
What is the principle behind Hb electrophoresis
HBE utilises the charged properties of normal Hb and its variants to replicate characteristic mobility patterns on both alkaline and acidic gels
Why might Alkaline HBE be used
(3)
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
Why might an acid HBE be used
(3)
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
What do we more commonly use other than gel elctrophoresis
Capillary electrophoresis
Give an example of a capillary electrophoresis we use
Sebia
What is the Sebia assay based on
(3)
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
What is the screening test for HbS
The sickle solubility test
What is the principle of the sickle solubility test
(2)
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
Give the name of a commercial test we can use for sickle cell
Sickle scan
What is sickle scan?
A rapid, qualitative lateral flow immunoassay kit for the identification of SCD of HbA, HbS and HbC
How do you use a sickle scan test?
(4)
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
When carrying out the Kleihauer test, how many cells do you need to count
6000 adult cells
(about 650 counted x9
How do you determine the amount of FM bleed in mls from your HbF count
(Number of foetal cells/ number of adult cells) x 2400 = mls
How much anti-D should be given for a 1ml foetal bleed
125 iu per ml of foetal cells
What should you do if the FMH shows a bleed over 2.5mls
The size of the bleed should be reassessed using flow cytometry which is more accurate for the assessment of large bleeds
You need to be able to interpret Alkaline Haemoglobin Electrophoresis
You need to be able to interpret Alkaline Haemoglobin Electrophoresis
Look in notes and lab manual for practice
What tests would you carry out to investigate haemoglobin
HPLC
Hb electrophoresis or Sebia assay (capillary electrophoresis)
Sickle scan -> HbS
Sickle solubility -> HbS
