Lab testing of haemoglobinopathies

Question 1

What are the tests for structural haemoglobin variants?

Answer 1

Sickle cell solubility test
Haemoglobin electrophoresis at acidic pH (when high HbF)
Iso-electric focusing
High performance liquid chromatography (HPLC)

Question 2

What are the tests for the rare structural haemoglobin variants?

Answer 2

DNA sequencing

Mass spectrometry

Question 3

What are the DNA tests for beta thalassaemia?

Answer 3

ARMS

Beta-globin gene sequencing

Question 4

What are the tests for alpha thalassaemia

Answer 4

DNA testing for definitive diagnosis
Gap PCR
MLPA
Southern blotting (but less so nowadays)

Question 5

Sickle cell solubility test

Answer 5

HbS precipitates in the presence of the reducing agent sodium metabisulphate

Question 6

When is the sickle cell solubility test used?

Answer 6

Pre-op as it only shows the presence of HbS, not if they are a carrier/if they have the disorder

Question 7

Haemoglobin electrophoresis

Answer 7

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

Question 8

Electrophoresis at a alkaline pH

Answer 8

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

Question 9

Electrophoresis at acidic pH

Answer 9

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

Question 10

Iso-electric focusing (IEF)

Answer 10

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

Question 11

Which is better out of electrophoresis and iso-electric focusing and why?

Answer 11

Iso-electric focusing

Offers superior resolution where the bands are sharper and easier to distinguish between

Question 12

High performance liquid chromatography (HPLC)

Answer 12

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

Question 13

What are the tests for beta thalassaemia trait?

Answer 13

DNA techniques:
ARMS
Beta-globin gene sequencing

Question 14

What are the tests for beta thalassaemia major?

Answer 14

Full blood count using an automated blood cell counter

HPLC/electrophoresis

Question 15

What results from a full blood count indicates beta thalassaemia major?

Answer 15

Low to normal Hb
High RBC count
Low mean cell volume (MCV)
Low mean cell Hb (MCH)

Question 16

What result from HPLC or electrophoresis indicates beta thalassaemia major?

Answer 16

Only HbF and HbA2 present

Question 17

ARMS (amplification refractory mutation systems)

Answer 17

Targeted to specific common mutations
PCR primers designed so amplification only happens if mutation is present
Can then be run on electrophoresis to check

Question 18

Beta-globin gene sequencing

Answer 18

Will detect majority of beta-globin mutations that could lead to loss of function

Question 19

What mutations cause no beta globin production (βO)?

Answer 19

Nonsense
Frameshift
Splicing
Large deletion

Question 20

What mutations causes low levels of beta globin (β+)?

Answer 20

Promoter mutations

Mutations that reduce splicing efficiency

Question 21

Why can’t alpha thalassaemia be diagnosed using simple haematology?

Answer 21

Can mimic iron deficiency

Question 22

Gap PCR

Answer 22

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

Question 23

MLPA

Answer 23

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

Question 24

What tests are carried out on antenatal screens?

Answer 24

FBC
HPLC
IEF