Lab testing of haemoglobinopathies Flashcards
(24 cards)
What are the tests for structural haemoglobin variants?
Sickle cell solubility test
Haemoglobin electrophoresis at acidic pH (when high HbF)
Iso-electric focusing
High performance liquid chromatography (HPLC)
What are the tests for the rare structural haemoglobin variants?
DNA sequencing
Mass spectrometry
What are the DNA tests for beta thalassaemia?
ARMS
Beta-globin gene sequencing
What are the tests for alpha thalassaemia
DNA testing for definitive diagnosis
Gap PCR
MLPA
Southern blotting (but less so nowadays)
Sickle cell solubility test
HbS precipitates in the presence of the reducing agent sodium metabisulphate
When is the sickle cell solubility test used?
Pre-op as it only shows the presence of HbS, not if they are a carrier/if they have the disorder
Haemoglobin electrophoresis
Used to detect Hb variants as they have a changed electrical charge
Variants separate from HB A and show up as an extra band
Can either be run at alkaline of acidic pH
Electrophoresis at a alkaline pH
Uses cellulose acetate
Allows rapid separation of Hb
However may miss Hb variants in low concentrations (HbA2, HbH, Hb Bart’s)
Also sees some Hb variant co-migrate so unable to determine exactly which one present
Electrophoresis at acidic pH
Uses citrate agar
Separation determined by the relative affinity for agaropectin by certain Hb surface groups
Doesn’t allow co-migration (delineates them)
Can be used to diagnose sickle cell with high HbF
Iso-electric focusing (IEF)
Separates Hb variants in a gel with a pH gradient according to their iso-electric point (pI)
Hb molecules migrate until they reach a point where the pH corresponds to their pI
Which is better out of electrophoresis and iso-electric focusing and why?
Iso-electric focusing
Offers superior resolution where the bands are sharper and easier to distinguish between
High performance liquid chromatography (HPLC)
Separates variants based on their net charge
Hb molecules absorbed into a column
Identified optically in eluate and identified by elution time
Quantified by computing area under the peak
What are the tests for beta thalassaemia trait?
DNA techniques:
ARMS
Beta-globin gene sequencing
What are the tests for beta thalassaemia major?
Full blood count using an automated blood cell counter
HPLC/electrophoresis
What results from a full blood count indicates beta thalassaemia major?
Low to normal Hb
High RBC count
Low mean cell volume (MCV)
Low mean cell Hb (MCH)
What result from HPLC or electrophoresis indicates beta thalassaemia major?
Only HbF and HbA2 present
ARMS (amplification refractory mutation systems)
Targeted to specific common mutations
PCR primers designed so amplification only happens if mutation is present
Can then be run on electrophoresis to check
Beta-globin gene sequencing
Will detect majority of beta-globin mutations that could lead to loss of function
What mutations cause no beta globin production (βO)?
Nonsense
Frameshift
Splicing
Large deletion
What mutations causes low levels of beta globin (β+)?
Promoter mutations
Mutations that reduce splicing efficiency
Why can’t alpha thalassaemia be diagnosed using simple haematology?
Can mimic iron deficiency
Gap PCR
When normal fragment of chromosome 16 run there is no PCR product as too large
If alpha genes have been deleted then the fragment will return a product
MLPA
Uses oligonucleotide probes which hybridise to adjacent target sequences
All probe products amplified by PCR using only one primer pair
This product has a unique length
Products run on electrophoresis with a control to see if less DNA present
What tests are carried out on antenatal screens?
FBC
HPLC
IEF
