Haemolytic Anaemia

Question 1

What are FBCs measured by?

Answer 1

Automated counting machines

Question 2

How is cellulose acetate electrophoresis used to diagnose B-thalassaemia?

Answer 2

Hb electrophoresis at pH 8.4-8.6 using a cellulose acetate membrane.

This procedure is simple, reliable and cheap.

It enables the provisional identification of haemoglobin A, F, S/G/D, C/E/O-Arab, H and a number of less common variants.

Differentiation between haemoglobins migrating to a similar position can be used by electrophoresis on acid (agarose) gels, HPLC or IEF.

Using this method to determine which Hb types are present in a sample can diagnose a disease, eg no A but only F indicates major B- thalassaemia.

Question 3

What are the analytical features of B-Thalassaemia major?

Answer 3

In Hb electrophoresis:

• cord blood <2% Hb A
• Adult:
  
  • B^0/B^0: no HbA, 90% HbF, variable HbA2
  • B^0/B+ and B+/B+: some HbA, mostly HbF, increased HbA2

Question 4

What are the lab tests used to diagnose haemolytic anaemia?

Answer 4

FBC

Peripheral blood smear:

• polychromasia (inc reticulocytes = blue tinge)
• schistocytes (red cell fragments)
• spherocytes
• aniso/poikilocytosis

Evidence of haemolysis:

• Increased LDH levels
• Decreased serum haptoglobin levels
• Increased direct and indirect bilirubin levels
• Increased stercobilinogen

Evidence of membrane abnormalities:
- osmotic fragility, eg spherocytosis

Evidence of antibody involvement:
- direct antiglobulin test (positive)

Question 5

What are the clinical indications of HA?

Answer 5

Shortened RBC survival time
Evidence of RBC increased production
- reticulocytes count (Bone marrow is compensated - trying so hard, that they are being released prematurely = uncleared RBCs in circulation. First sign of HA)
- erythrocytes hyperplasia in bone marrow. Normal myeloid:erythrocytes ratio of 2:1 reduced or reversed.
- nucleated RBCs
- Red cell fragments

Question 6

What is the Direct Antibody Test (Coombs test)?

Answer 6

Reflects in vivo sensitisation of erythrocytes. Determines whether RBCs have been coated in IGg, complement or both.

Mum has produced IgGs coating the RBCs of newborn. RBCs have become sensitised in the baby.

IgGs are small, so needs to crosslink to agglutinate, so in the lab we add anti-IgG AHG reagent. This will only agglutinate if the RBCs have then sensitised in vivo.

It is used for:
- haemolytic transfusion reactions
- haemolytic disease of the foetus and the newborn (HDFN) and 
   autoimmune HA
- drug induced immune haemolysis

Question 7

What is the Indirect Antibody Test and what are the uses?

Answer 7

Used to detect the presence of IGg antibodies in the patient’s blood sample (detects in vitro sensitisation).

Patient plasma sample is added to reagent red cells
Incubated at 37 degrees C
Antibody coats (sensitises) red cells that express the corresponding antigen
Was to remove free IGg (not required using gel techniques)
Addition of AHG
Centrifugation
In positive sample AHG causes agglutination of red cells so IGg can be predicted.

Uses:

• whether there are antibodies to the Rh factor in the mother’s blood
• compatibility testing
• antibody identification (can detect very low concentrations in a patient’s plasma/serum before a transfusion)

Question 8

What is the purpose of Eosin-5-malemide in HS diagnosis?

Answer 8

Staining agent the shows a left shift and decreased intensity in HS. Shows a mean channel fluorescence due to membrane band 3 deficiency.