Division of Laboratory Medicine Flashcards
What are normal are Normal Haemoglobin?
- Haemoglobin A - α2β2 - Major haemoglobin in adults
- Haemoglobin A2 - α2δ2 - Minor haemoglobin in adults
- Haemoglobin F - α2γ2 - Major haemoglobin in foetal life
What are Haemoglobinopathies?
- Haemoglobin variants: Alteration in the globin protein structure
- Thalassaemias: Inadequate production of structurally normal globin protein.
- Thalassaemic haemoglobinopathies: Structurally abnormal haemoglobin synthesised at a reduced rate e.g. HbE.
What are features of Haemoglobinopathies?
- Frequency of different haemoglobinopathies varies in different ethnic groups
- May inherit more than one haemoglobin abnormality
- Many haemoglobin mutations of no clinical significance
- Severe morbidity and mortality in sickle cell disease and beta thalassaemia major
- Carriers usually asymptomatic.
What are features Sickle Cell Disease?
- Sickle haemoglobin (HbS). Occurs mainly in people of African origin
- Valine replaces glutamic acid at sixth amino acid in the β globin chain: α2β26Glu→Val (missense mutation)
- Deoxygenated HbS has reduced solubility leading to polymerisation
- Polymers distort red cell into crescent/ sickle shape
What is the Pathogenesis of Sickle Cell Disease?
Sickling leads to:
- Shortened red cell survival
- Impaired passage of cells through capillaries leading to obstruction and tissue infarction
Increased adhesion to endothelium contributes to vascular occlusion
What are some acute complication of Sickle Cell disease?
- Infections
- Anaemia
- Vaso-occlusive problems (Acute painful episodes, Stroke, Renal infarction, Dactylitis or bone infarction)
What are Chronic Complications of Sickle Cell Disease?
- Avascular necrosis of bone
- Pulmonary hypertension
- Organ damage
- Blindness
- Leg ulcers
- Gall stones
- Chronic Renal Disease
What are Benefits of Screening
- Early administration of prophylactic penicillin reduces incidence of pneumococcal sepsis.
- Pneumococcal vaccines increase immunity to pneumococcal infections
- Close clinical monitoring to detect acute splenic sequestration. Reduces morbidity and mortality. Pooling of blood in the spleen can lead to to hypovolaemic shock
- Parental education
What is Newborn Screening Results?
- FS: Hb SS, Hb S/ β0 thalassaemia, Hb S/ HPFH
- FSA or FS: Hb S/ β+ thalassaemia
- FSC: Hb SC
- FSD: Hb S/DPunjab
- FSE: Hb S/E
- FSOArab: Hb S/OArab
- FSV: Unidentified Hb variant in combination with sickle Hb
What are forms of β-thalassaemia?
- β0 mutations: absent or very low transcription of β globin gene
- β+ mutations: reduced expression of β globin gene
- β- thalassaemia major (die at 4 yrs age)
What are symptoms of β-thalassaemia major (die at 4 yrs age)?
- Severe anaemia
- Dependent on blood transfusions
- Bone deformity if untreated
- Can cause gross hepatomegaly
What are features of β-thalassaemia major?
- No HbA may indicate no β-chain synthesis
- HbA concentration <1.5% of total haemoglobin
- Newborn screening result: ‘F-only’
What are conditions for newborn screening results?
- FC: Hb CC, C/β-thalassaemia
- FD: Hb DD, D/β-thalassaemia
- FE: Hb EE. Hb E/ β-thalassaemia also has an FE pattern but these children often become transfusion dependent so the condition is not clinically benign.
What are conditions for newborn screening results?
- FAS: HbAS. This is a Sickle cell carrier/trait
- FAC: HbAC. This is a Carrier of Hb C
- FAD: HbAD. This is a Carrier of Hb DPunjab
- FAE: HbAE. This is a Carrier of Hb C
- FAOArab: HbAOArab. This is a Carrier of Hb OArab
What are the linked programmes for Sickle cell screening?
- Screening laboratory should be informed of antenatal screening results
- If parents are carriers then ‘at risk’ form sent to lab.
- Antenatal screening results written on newborn screening card
What are methods for screening Sickle Cell Anaemia?
- High performance liquid chromatography (HPLC)
- Isoelectric focusing (IEF)
- Capillary electrophoresis (CE)
- Tandem mass spectrometry (MS/MS)
How is High performance liquid chromatography (HPLC) used to investigate Sickle Cell Anaemia?
- Ion exchange and photometric detection at 415nm.
- Buffer gradient used to displace the haemoglobins bound to the charged particle matrix in the cartridge (column).
- As the haemoglobins have differing charges some are bound more strongly to the matrix.
- Retention times are reproducible for a particular column, buffer, exchange resin and temperature
How is Isoelectric Focusing (IEF) used to investigate Sickle Cell Anaemia?
- Sample applied to agarose gel with a pH gradient consisting of ampholytes,
- On application of a high voltage, narrow buffered zones with slightly different pHs
- Haemoglobin variants migrate through the zones until reach individual isoelectric point
- Thin discrete bands formed
How is Capillary Electrophoresis (CE) used to investigate Sickle Cell Anaemia?
- Uses combination of ion migration and electro-osmotic flow to separate protein molecules
- Automated analyser by SEBIA
How is Tandem Mass Spectrometry used to investigate Sickle Cell Anaemia?
Hb variants characterised
- Positions of mutations and amino acid sequences known
Trypsin digestion of globin chains predictable
- Cleavage of peptide bonds. β-chain (T1-15)
- Mutations alter cleavage sites & generate peptides specific to variant present
Specific MRM transitions for each peptide
- HbS, C, DPunjab, E & OArab, γ (HbF)
What happens with sickle cell screening in Prematurity?
- If Hb A less than 2% and baby born ≤30 weeks which cccurs rarely then repeat sickle cell screen on day 28 sample
What is done when transfusion is in a patient who needs to be screened for Sickle Cell Anaemia?
- Original protocol. Repeat sample 4 months post-transfusion
- Now, pre-transfusion sample collected on day 0 for babies on NICU
- If no pre-transfusion sample then DNA test for HbS
What are Newborn Screening Standards?
- All screen positive results are given to parents by a trained healthcare professional face-to-face by 28 days of age.
- The baby must enter the care of a specialist haemoglobinopathy centre by 90 days of age
