Practical 4: Exam Notes Flashcards
During alkaline gel electrophoresis to investigate haemoglobin, how many bands are produced?
7 bands can be seen
During alkaline gel electrophoresis to investigate haemoglobin, what 7 bands are produced?
Origin
Slow bands
C band
S band
F band
A band
Fast bands
During alkaline gel electrophoresis to investigate haemoglobin, what Hb types can be found in the slow band?
Constant spring
During alkaline gel electrophoresis to investigate haemoglobin, what five Hb types can be found in the C band?
C
E
O-Arab
A2
C-Harlem
During alkaline gel electrophoresis to investigate haemoglobin, what three Hb types can be found in the S band?
S
G
D
Lepore
During alkaline gel electrophoresis to investigate haemoglobin, what three Hb types can be found in the fast band?
H
Barts
J
What two Hb types can result in a positive sickle solubility test?
Hb-S
Hb-C-Harlem
What would you do if you found there to be Hb H from gel electrophoresis
Carry out a H prep
Stain with new methylene blue
What is a normal Hb composition for an adult
Mostly HbA with a small percentage of HbA2
What would a high HbA2 indicate
Beta thalassaemia
-> lack B chain needed to form HbA1
-> HbA2 made of alpha chain and delta chain
What would a band at S and A indicate (alkaline gel)?
This is a patient with sickle cell anaemia
But they have just been transfused with HbA
What does a band at S mean (alkaline)
Could be HbS
Need to confirm not Hb G, D or Lepore which would be in the same band
What does a band at S and C mean (alkaline)
(4)
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
What does a thick band at A and a weak band at C mean?
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)
What is HbH?
(4)
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
What are some tell tale signs of HbH
(3)
Teardrop cells
Anisopoikilocytosis
Target cells
What are some tell tale signs of HbE
(3)
Microcytic
Hypochromic
Abundance of target cell
What are some tell tale signs of B-thalassaemia major
(5)
Marked anisopoikilocytosis
Target cells
Basophilic stippling
Pappenheimer bodies
Numerous nRBCs
What are some tell tale signs of B-thalassaemia minor
(6)
Mild anaemia
Microcytic, hypochromic
Occasional target cells
Anisopoikilocytosis range from marked to mild
Basophilic stippling
Polychromasia
What does a slightly high A2 only on a HPLC indicate?
B- thalassaemia trait
Mild microcytic anaemia
-> megaloblastic anaemia
What indicates B- thalassaemia trait?
High A2 only
Mild microcytic anaemia
Megaloblastic anaemia
What does a high HbF, low A0 and raised A2 indicate
(2)
B-thalassemia intermedia
B- thalassemia major post transfusion
What indicates B-thalassemia intermedia
(3)
High HbF
Low A0
Slightly raised A2
What does a high HbF only indicate, no other clinical symptoms?
Hereditary persistence of foetal haemoglobin
What does very high HbF, no normal Hb A and a small increase in HbA2 indicate?
B-thalassaemia major
What indicates B-thalassaemia major on a HPLC?
Very high HbF
No normal HbA
Small increase in HbA2
What indicates HbH disease?
Fast eluting Haemoglobin
HbH peaks before A
What indicates HbS -> sickle cell disease?
(4)
HbS peak after A2
HbF is increased
No HbA
HbA2 is increased
What does a HbS peak, HbF increase, no HbA and increased HbA2 indicate?
Sickle cell disease
How do you know its HbA2 in the peak?
HbE can only be found in more than 30% concentration -> so small quantities has to be HbA2
What would indicate HbE?
(2)
No HbA
Very large peak at A2 = HbE
What does a very large peak at A2 indicate?
HbE
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
What is the equation for a corrected bleed
multiply the ratio of foetal cells/mothers cells by 2400 to give your answer in 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
What is the principle behind HPLC
(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
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 main downfall of HPLC
Cannot discriminate between HbA2, HbE and Hb Lepore
What is the principle behind Hb electrophoresis
(3)
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
When might you use an acid gel electrophoresis
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 is the principle behind capillary electrophoresis?
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 principle behind the sickle solubility test
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
