Haemoglobinopathy diagnosis
Diagnosis Full blood count
Blood film
Beta globin chain disorder – glutamine to valine substitution at the 6th residue of beta globin
Sickle solubility test § Should be performed whenever the electrophoretic or HPLC is characteristic of HbS § Also whenever there is a variant haemoglobin of uncertain significance as there are a number of variants that the HbS glutamic acid for valine substitution and others that change its electrophortic properties but still mean that the patient is prone to sickling. § Not useful when there are low levels of the variant haemoglobin (will only detect down to 20%) – need to correct the HCT to 50% when the patient is anaemic to avoid false negatives. Technique § Packed red cells reconstitute to a haematocrit of 50%. § + PO4 buffer containing reducing and lysis agents § Sickle Hb is induced to sickle by the reducing agent and gets trapped in red cells § Normal Hb is lysed § Centrifuge and read § Any sickle Hb results in turbidity § False negatives (limit of detection 20%) § Babies <6months § Massively transfused patients § False positives occur with § Hyperlipidaemia § Paraprotein § Heinz body haemolytic anaemia § Very high white count or nucleated red cell count
Haemoglobin electrophoresis § Best performed on lysed packed red cells § Very common technique for initial detection of variant haemoglobin though HPLC is increasingly taking its place. § Alkaline electrophoresis – pH 8.2-8.6 § Provisional ID of (in this order away from the origin (ie. C migrates the least distance): § H (and Barts, N-Baltimore, J-Baltimore, J-Toronto, Detroit, Tacoma run faster than the control – wich runs AFSC) § A § F § S / G (Philedelphia, Ferrara) /D (Iran, Punjab, Norfolk) / Lepore (less than A) § C (C-Harlem) /E / O-Arab / A2 § Agarose gel at an acidic pH will show the difference between S and D/G and C from E, C-Harlem and O-arab § Here the gel runs FASC § F – nothing else runs here § A – runs with D/G/lepore, E and A2 and H + ALL the things that run fast in the alkaline gel – (Barts, N-Baltimore, J-Baltimore, J-Toronto, Detroit, Tacoma) § S – runs with O-Arab, Q India, C-Harlem § C – runs only with Siriraj (which runs between S and C on alkaline gel)
Iso-electric focusing § Suitable for neonates § More expensive, but separates more variants than electrophoresis
HPLC § Separates by cation exchange chromatography § Identifies variant Hbs by change in electrical charge § Change in gradient of buffer means Hb attached to column will elute at different times § Enables provisional identification and quantification § Advantages over electrophoresis § Less labour intensive (offset by higher capital and reagent costs) § Can use very small samples § Quantification of normal and variant haemoglobins is easier § HbA2 is quantified – allows b-thal to be diagnosed with a single test (instead of a combination of electrophoresis and microcolumn chromatography) § Larger range of variant haemoglobins can be identified § HbA2 variants can be identified
Newborn screening § Heel prick – dried spot of blood will be stable at room temperature for 1 week § HbSS and HbSBo-thal – FS pattern § HbSBo-thal - low MCV and increased A2 (A2 may be not be elevated until 2 years) § HbAS – FAS pattern § HbSB+-thal –FSA pattern § HbSC – FSC pattern § Prenatal screening
Unstable haemoglobin Detected using heat or isopropanol test – positive if there is a precipitate at the end of the test that is not present in the normal control Old blood can give a false positive due to the presence of methaemaglobin Old fetal haemoglobin with be unstable (positive control) – Fetal haemoglobin is less stable than HbA – makes testing more difficult in featal samples
Heinz body detection Cresyl blue (same stain as for HbH) or methyl violet May appear after incubation for 24h at 37oC
Oxygen dissociation curve – with determination of P50 § Used for detecting high affinity haemoglobins
Globin chain analysis § Useful for the diagnosis of thalassaemias § Has to be done on fresh blood or bone marrow samples (<6h old) § Determines the proportion of alpha / beta chains by the incorporation of radioactivity § If the globin chain is very unstable the ratio may be abnormal because of very rapid destruction
DNA analysis Useful for: 1. alpha0 thalassaemia – particularly for genetic counseling 2. D-Punjab rather than any other D or G group haemoglobin 3. Prenatal diagnosis
Haemoglobin F (a2g2) § Predominant Hb in fetal life – increased affinity for O2 as has reduced binding of 2,3 DPG § Falls quickly during first year of life – then has a second slower fall and it may take until puberty for it to reach adult levels § In normal adults F is distributed heterogeneously, being found in a subset of erythrocytes – designated F cells (very variable 0.6-22%) § 85% of people have a HbF level of <0.6% and the remainder have a slightly increased percentage of F cells, which is inherited as X linked dominant (10% men 20% women) § Markedly increased in defects of b globin synthesis
Haemoglobin A2 (a2d2) § Normal range 2-3.5% in adult life (rises from 0.2% at birth until around the age of 3) § 4-5% usually indicates heterozygosity for B0 or severe B+ thal (usually 3.6-4.2% if heterozygous for B+ thal)
Causes of a reduced HbA2 § Infancy § Iron deficiency § a thalassaemia § d thalassaemia § db thalassaemia § (gdb thalassaemia reduces the amount of HbA2 but not the proportion of A2 synthesised)
Causes of a raised Hb A2 § b thalassaemia trait (almost all cases) § Some unstable haemoglobins § Hereditary high HbA2 (autosomal dominant) § Sickle cell trait § Sickle cell anaemia – particularly if there is coexisting alpha-thal § Sickle cell B0thal § Some forms of CDA § HIV / drugs
HbA1C § Can be quantified by HPLC or ion-exchange microcolumchromatography § Determined by age of red cells and serum glucose
Increased in § Poorly controlled diabetes § Iron deficiency § Transient erythroblastopenia
Reduced in Haemolytic anaemia
Haemoglobin H (b4) § Cresyl blue stain – causes precipitation of haemoglobin H
Haemoglobin Barts (g4)
Antenatal screening § Should be able to identify § B thalassaemia major and intermedia § B thal trait, deltabeta thal trait, E trait, E disease, Lepore trait in parents § Hb Barts and hydrops fetalis § Alpha o trait in parents § MCH <25, Chinese, S E Asia, Greece, Turkey, Cyprus, Sardinia § Sickle cell disease § Sickle trait or diease, B thal trait, C trait, D Punjab trait, O arab trait in parents
§ May be selective or universal § Universal preferred where there is a high ethnic mix | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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