Lab 4 Haemoglobin investigations Flashcards
How to perform HbF estimation
Count 6000 adult cells over 25 fields of view.
* Count at least 667!! (667x9 = 6003)
Count #adult RBCs in small square
Count #foetal RBCs in big square. (bright pink cells).
*Select area of film where cells are touching but not overlapping.
How to calculate HbF - formula
Foetal RBCs / Adult RBCs (at least 6000) x 2400
How to interpret Hb electrophoresis
Different Hb migrate at different positions based on their charge and size.
Variant Hb have alterations in surface charge and changes in surface AAs which changes the speed of its migration - characteristic separation based on set mobility patterns.
Which Hbs can be separated using acid gel electrophoresis but not alkaline?
Separate Hb C from HbE and HbA2.
Also separates HbS from Hb G, D, Lepore.
Methods to diagnose haemoglobinopathies
HPLC, Gel electrophoresis, and more commonly capillary electrophoresis.
Normal Hb varients and their typical %
HbA = 96-98%
HbA2 = 2-3.5%
HbF = <1%
What is a con of HPLC?
Cannot discriminate between HbA2, HbE and HbLepore.
requires electrophoresis (acid gel)
How is HbS screened for?
- Sickle solubility test (qualitative test)
HbS is insoluble in deoxygenated state in a high molarity phosphate buffer - turbid solution indicates presence of HbS but no information of genotype (hbSS, HbAS, HbSC) - Sickle SCAN
Rapid qualitative lateral flow immunoassay for identification of HbA, S, C.
What further testing to be done if sickle cell anaemia is assumed from blood film review?
- Blood film investigation.
- Sickle solubility test.
If positive. - HPLC
What further testing is to be done if mild beta-thalassaemia is indicated from blood film review?
- Blood film (peripheral smear) -
Shows target cells, anisopoikilocytosis, basophillic stipling, hypochromasia, microcytes. - HPLC -
To differentiate normal vs abnormal Hb variants.
b-thal minor -> Decreased HbA + Increased HbA2.
What further testing is to be done if beta-thalassaemia major is indicated from blood film review?
- Blood film shows
hypochromasia, target cells, schistocytes, howell joly bodies, NRBCs - HPLC -
No HbA and Increase HbF (90-100%), HbA2 increased also.
Can quantify HbA2 and HbF. - Hb electrophoresis too.
Confirms structural variants.
Can resolve cases where HPLC shows ambiguous peaks. - Genetic testing
To confirm mutation in B-globin genes.
What further testing is to be done if alpha-thalassaemia is indicated from blood film review?
HPLC can detect HbH (B4 tetramers).
Perform HPrep which should show stained B-chain tetramers within cells - look like golfballs.
Indicates HbH disease.
What to do when review chromatograms?
- Make table with following
Hb type, Normal range (%), reported %. - Give probably diagnosis
- Give confirmatory test.
B-thalassaemia Chromatogram results
Low HbA
High HbA2 or
High HbF
a-thalassaemia chromatogram results
HbH (B4) or Hb Barts (y4) in severe cases.
Seen in fast fraction of chromatogram.
Low HbA/HbA2/HbF
Sickle cell disease chromatogram results
Predominantly HbS
Low/No HbA.
When is HbF elevated?
B-thal major, Hereditary presence of foetal haemoglobin, sickle cell disease.
What does slightly high HbF indicate in chromatogram?
Patient may be a baby.
How to confirm alpha-thalassaemia?
HbH prep = RBCs show blue golf ball inclusion.
What does a high HbA2 and low HbA in chromatogram indicate?
Beta-thalassaemia minor or
HbE variant present.
The peak may be measuring both HbE and HbA2 as their retention time is the same.
Perform confirmatory test using capillary electrophoresis
What does high HbS indicate?
HbS present
Amount of HbA indicates whether genotype is HbSS or HbAS.
Confirm presence of HbS using
Sickle solubility test.
HbF may be high due to drugs SCD patients are on.
Why is HbF significantly elevated in b-thal major but HbA2 remains only slightly increased?
HbA2 levels remain only moderately increased bc delta-globin gene expression is naturally low and does not compensate effectively for b-globin loss.
Whereas HbF can be upregulated easily bc it is already designed for high production during fetal life.
Why is HbA2 elevated in b-thal minor whereas HbF remains normal or only slightly elevated?
Only one B-globin gene is mutated leading to reduced but not absent HbA production.
delta-globin gene is not functionally suppressed like the y-globin genes so it naturally increases its expression to compensate for mild b-globin deficiency.
