Red Cell Disorders Flashcards
List the different causes/ mechanisms for haemolysis
Intrinsic causes
1) Enzymes
- G6PD
- PK deficiency
- P5N deficiency
2) Haemoglobin
- Haemoglobinopathies/ unstable haemoglobin (HbS, HbCS etc…)
- Thalassaemia
3) Membrane/ cytoskeleton
- Hereditary spherocytosis
- Hereditary elliptocytosis/ Hereditary pyropoikilocytosis
- South East Asian Ovalocytosis
- Hereditary Stomatocytosis
- PNH
Extrinsic causes
1) Immune
- Warm AIHA
- Cold AIHA
- PCH
- Alloimmune
- Drug induced
2) Non-immune
- Fragmentation haemolysis (TTP/HUS, DIC, HELLP, APLS, malignancy, medications, cardiac)
- Spur cell haemolysis
- Malaria
List the basic tests performed to assess for haemolysis and provide a brief description of how they are performed
1) Reticulocyte count (>250 highly specific)
- Manual method: supravital staining with new methylene blue or brilliant cresyl blue. 1000 RBCs counted to determine the %.
- Automated: fluorochromes bind to RNA. Fluorescent cells enumerated on flow cytometer
- Can be reported as reticulocytes %, absolute and corrected
2) Haptoglobins (acute phase reactant)
- Samples dispensed into wells on agarose gel that contain an antibody to haptoglobin.
- Precipitation rings form.
- The diameter of the ring is proportional to the concentration of haptoglobins.
3) LDH
4) DAT
- Antiglobulin reagent mixed with patient red cells.
- Antiglobulin bind to the immunoglobulin (or complement) on the sensitised cells causes agglutination
5) Urinary haemosiderin
- Urine centrifuged for 10-15 minutes
- Transferred onto a slide and air dried
- Slide fixed and stained with Perl’s stain and examined under the microscope.
List the tests that could be performed to diagnose hereditary spherocytosis?
1) Full blood count with reticulocyte count: anaemia, increased MCHC, increased reticulocytes.
2) Blood film: spherocytes, polychromasia.
3) Tests to confirm haemolysis: LDH (high), haptoglobins (low).
4) Exclude AIHA: DAT.
5) Osmotic fragility test:
- Spherocytes= SA:V at baseline, take on less volume before lysis.
- increasingly hypotonic solutions of saline are used: more hypotonic, more lysis.
- HS cells will lyse at NACL concentrations ≥5g/L.
- Curve shifts to the right.
6) Acidified glycerol test:
- Glycerol in a hypotonic saline solution slows RBC lysis enabling a more convenient measurement of lysis
- Lysis measured in a spectrometer at 625nm.
- In HS, range of AGLT50 is seconds to 2-3 minutes (normal >30 min).
7) EMA binding test
- EMA binds to band 3.
- Deficiency or abnormality in the band 3 protein will lead to reduced fluorescence.
- HS cells= decreased fluorescence.
Discuss hereditary elliptocytosis and HPP
- Inherited disorder caused by mutations in spectrin.
- Autosomal dominant inheritance.
- Heterozygotes usually asymptomatic with mild compensated or no haemolysis.
- Homozygotes and compound heterozygotes often have severe, symptomatic haemolytic anaemia (haemolytic HE or HPP)
- HPP= most severe form of HE
- HPP morphology: poikilocytes, spherocytes, microspherocytes, fragmentation. Elliptocytes uncommon. Extreme microcytosis, (MCV 30-50 fL), high MCHC.
- Most often diagnosed by morphology
- EMA binding can be used. Borderline reduced fluorescence in HE, markedly reduced fluorescence in HPP
Briefly describe the glycolytic, pentose phosphate and glutathione pathways and how they interact.
- In order to function, red blood cells need an energy source in the form of ATP and a source of reducing power
- ATP is generated via the glycolytic pathway
- The reducing agent NADH and NADPH are generated via the glycolytic and pentose phosphate pathway respectively
- NADH reduces metHb
- NADPH reduces and detoxifies free radicals and hydrogen peroxide
- NADPH also drives the glutathione pathway leading to the formation of the antioxidant GSSH
- The pathways are linked: glucose is converted to G6P by hexokinase. G6P can be used in the glycolytic pathway to generate NAD and ATP or in the PPP to generate NADPH and activate the glutathione cycle
- The glycolytic pathway is also linked to the Rapoport-Luebering shunt which produces 2,3-DPG which regulates the oxygen affinity of haemoglobin
Discuss PK deficiency
- Pyruvate kinase converts phosphoenolpyruvate (PEP) to pyruvate which can then be converted to lactate by lactate dehydrogenase.
- This reaction generates ATP from ADP and reduces NADH to NAD
- PK deficiency is the most common enzymopathy of the glycolytic pathway.
- Deficiencies in PK lead to a moderate to severe chronic non-spherocytic haemolytic anaemia.
- FBC shows a moderate to severe normocytic anaemia with reticulocytosis +/- mild macrocytosis.
- Blood film shows anisopoikilocytosis and reticulocytosis. Post splenectomy ‘prickle’ cells or spheroacanthocytes are seen.
- Deficient activity of PK leads to an accumulation of upstream metabolites including 2,3-DPG.
- This decreases oxygen affinity.
- Can be tested for by the PK activity assay:
- Get rid of WCC first: tests only in RBCs
- PEP, ADP, NADH and LDH added so that test is dependent on PK
- Absorbance measured at 340nm: absorbance will fall as NADH is converted to NAD.
- Rate of fall of absorbance is proportional to the amount of PK present
- Can also measure 2,3-DPG levels (which increase in PK deficiency) using a similar principle.
Discuss the tests used in the diagnosis of G6PD
Screening tests:
1) Supravital staining
- Using methyl violet, new methylene blue or brilliant cresyl blue.
2) Fluorescent screening/ fluorescent spot test
- NADPH produced by G6PD fluoresces under UV light: NADP does not
- G6P and NADP added to patients blood sample and placed on Whatman No1 filter.
- incubated for 10mins then examined under UV light
- No fluorescence: no G6PD
3) Methaemoglobin reduction test
- Sodium nitrate converts Hb to Hi
- Reduced back to Hb when methylene blue is added
- Colour change observed: clear red= normal; brown= deficient.
Diagnostic tests:
1) G6PD assay
- NADPH has a peak UV light absorption at 340nm whereas NADP does not
- Reaction kits containing NADP added to patients blood. The change in absorbance is then measured over the first 5 minutes of the reaction and the G6PD activity is determined by a formula.
2) Gene sequencing
Discuss some of the issues with G6PD testing
- Gene for G6PD is on the X chromosome.
- Males can be normal or deficient.
- Females can be homozygous or heterozygous.
- Due to inactivation of one of the X chromosomes, heterozygous female can have 20- 80% functioning G6PD. Can therefore be difficult to detect female heterozygotes.
- Red cells are likely to haemolyse only if they have < 20% of the normal enzyme.
- During an acute haemolytic episode the G6PD cells in a heterozygote may have undergone haemolysis leaving the G6PD sufficient cells when testing is undertaken
- G6PD levels are highest in reticulocytes, samples taken during an acute haemolytic episode can have higher G6PD activity- even within the normal range.
Describe the changes to haemoglobin synthesis and morphological appearances of red cells in lead poisoning
- Lead inhibits most enzymes in the haem biosynthetic pathway: especially ALAD.
- Lead also limits iron delivery to ferrochelatase (FECH) in erythroid cells. As a result, zinc is inserted into protoporphyrin, similar to the process that occurs in iron deficiency.
- Lead is an inhibitor of pryimidine-5’-nucleotidase leading to an acquired deficiency of P5N.
- Lead poisoning manifests with ineffective haematopoiesis and haemolysis as well as other manifestations in multiple organ systems.
- Blood film features:
• Normocytic and normochromic anaemia
• Increased polychromasia
• Nucleated red cells
• Basophilic stippling
• Bone marrow hypoplasia
Discuss how P5N deficiency is diagnosed
- Normal nucleotide pool of RBCs is >95% purines with low levels of the pyrimidines (cytidine and uridine).
- In P5N deficient cells >50% of this pool consists of pyrimidines.
- In acidic solutions, cytidine has a maximum absorbance at 280nm whereas purines have a maximum absorbance at 260nm.
- The ratio of absorbance at 260nm to 280mn is used to determine the amount of purines present compared to cytidine.
What haemoglobin variants run together on alkaline electrophoresis (cellulose acetate)?
- SGD
- A2 CEO
What haemoglobin variants run together on acid electrophoresis (citrate agar)?
- AGED Lepore
What should a good thalassaemia antenatal screening programme achieve?
1) Detect couples at risk of a major thalassaemia disorder
- Identifies all couples with beta globin gene abnormalities
- Identifies all couples at risk of HbH disease and/ or Hb Barts hydrops (2 gene alpha thal, non-deletional alpha thal (CS))
2) Provides genetic counselling regarding phenotypes of thalassaemia and prenatal diagnosis
3) Identifies and counsels other family members at risk
4) Identifies and treats iron deficiency
How does antenatal screening for thalassaemia work?
- FBC performed at time of booking
- If thalassaemic indices present (particularly low MCV, low MCHC); iron studies and HbEp should be performed
- If the female has any thing abnormal on this screen- the partner should also be screened with FBC, ferritin and HbEp
- If the male and female are at risk of a major thalassaemic disorder based on the above tests then DNA confirmation is required.
Why are at babies at risk of thalassaemia/ haemoglobinopathy screened at delivery?
- Neonatal screening can be performed on a cord blood, capillary or venous sample.
- Absent HbA= major thalassaemic disorder
- HbA <10% possible beta thal trait
- Variant haemoglobins may be detected
- Hb Barts detected: alpha thalassaemia
