Headlines

DNA Down Under

New section on RNA interference as a tool to block virus replication.

 

Plasma Membrane

Haemoglobin S (HbS) - Sickle Cell Anaemia

• HbS ß⁶(A3) (ß=chain affected, at the 6th amino acid in the A3 position of the helix). Abnormality is in a change from glutamine to valine.
• In the deoxy form, haemoglobin S starts to polymerise and precipitate put in solution. This distorts the RBC making it rigid and therefore more rigid. This produces the Sickle Cell
• The most common, symptomatic haemoglobinopathy worldwide
• Cell may return to normal shape upon reoxygenation of Hb, however repeated sickling events cause irreversible membrane changes (making the RBC leaky and rigid) until removal is carried out by phagocytosis in the spleen, liver and bone marrow
• Clumps of cells in the spleen, fingers, heads of the femur can lead to infarcted tissue
• More RBCs behind release their oxygen to the hypoxic tissues and the plug increases in size
• HbS has a decreased oxygen affinity and the concentration of 2,3-DPG in homozygotes is increased to facilitate the release of more oxygen to the tissues. This increases the amount of deoxy HbS producing sickled cells.
• Most common abnormal Hb disease - 1 in 8-12% carry the gene
• Most mutations cause clinical signs of disease because the mutation affects solubility, function and/or stability of the Hb molecule
• First signs appear at approx. 6 months manifesting as:
  
  

Acute Disease (Infections and...)	Chronic Disease (Anaemia, retarded growth and..)
Cerebral Thrombosis	Eye Problems
Acute Chest Syndrome	Pulmonary hypertension
Splenic Sequestration	Congestive Heart Failure
Haematuria (Kidney Infarction)	Splenic Atrophy
Hand/Foot Syndrome (Ischaemia)	Hyposthenuria (failure to concentrate urine)
Acute Right Upper Quadrant Syndrome	Ankle Ulcers

• Vaso-occlusive crises occur from blockage of micro-vasculature (may be due to infection. decreased pressure, dehydration or slow blood flow)
• Accompanied by fever (low grade), organ dysfunction and tissue necrosis
• HbAS - heterozygous form of HbS trait (less than 50% abnormal Hb)
• HbSS - homozygous form of HbS trait (greater than 50% abnormal Hb)
• Increases in temperature, acidosis and dehydration encourage deoxygenation of Hb
• Patient usually has quite a severe anaemia due to haemolysis of sickled RBC
• Repeated crises in the spleen in early life causes splenic atrophy due to necrosis caused by the hypoxic, hypoglycaemic environment
• Overwhelming bacterial infection can occur in the young
• Gallstones are a feature of this and any chronic haemolytic anaemia
• Cardiac hypertrophy occurs as a result of haemodynamic changes to compensate for the oxygen deficit
  
  

Hb	Dec (especially in crisis)
Hct	Dec
MCV	Normal to Inc
MCH	Normal to Inc
RDW	Greater than 14.5%
WCC	Normal to Inc
Plt	Normal to Inc

Normochromic (haemoglobin still stains)
Anisocytosis +++ (polychromatic macrocytes)
Poikilocytosis (Sickle cells, Target cells and Acanthocytes)
Inclusion Bodies (due to autosplenectomy of hypofunction)

Basophilic Stippling
Pappenheimer Bodies

Bone Marrow-normoblastic hyperplasia (to compensate for lost RBCs)

• Shows all the signs of a Normochromic / Normo-Macrocytic Anaemia
  

• Using a RBC lysate, bands can be quantitated by scanning densitometry
• HbS runs with HbD (which does not sickle)
• If HbSS, HbS will be the predominant band
• If another band is present it will be HbF not A₁ as the ß chain is that which is affected (sames as HbF)
• Trait (HbAS) is generally asymptomatic, carrying mostly HbA₁ (65%) and HbS (35-40%)
• Always less than 50% abnormal Hb if one normal gene
  

• Lysate from saponin lysed RBC is stabilised with KCN.
• Addition of dithionate deoxygenates the blood, causing HbS to polymerise and making the solution opaque
• Looks for HbAS and HbSS

• A drop of patients blood is mixed with sodium metabisulphate (a reducing agent that deoxygenates blood) and examined under a microscope after 30min
• See a result with HbAS and HbSS

• No known long range therapy
• Proactive treatment is best - prevent situations that precipitate vaso-occlusion such as infection and dehydration (which increases the MCHC and therefore the HbS in a cell)
• Transfusion of whole blood or packed RBCs in aplastic or splenic sequestration - may result in alloimmunization (development of antibodies to elements in transfused blood that make further transfusions dangerous to the patient.
• Switch on HbF gene - limits/cuts down sickling under lower oxygen tension
• Recent discoveries include:
  • Ribozymes were introduced (via liposomes) into RBC precursors isolated from umbilical cord blood to correct the faulty gene and introduce the corrected form of the gene at the RNA level. (Duke University Medical Center)
  • Only requires a 10-20% improvement
    
    

Haemoglobin C (HbC)

• HbC ß⁶(A3). Change is from glutamine to lysine
• Disease requires both abnormal genes (HbCC, trait=HbAC)
• Decreased solubility with deoxyHb
• Not as damaging - no autosplenectomy
• Do get splenomegaly but the Hb crystallisation is not as bad
• Chronic haemolytic anaemia is not as bad
  
  

  Hb	90-100g/L
  Hct	Dec.

  
  
• Large number of target cells with a slight to moderate increase in reticulocytes
• Crystals not detected in peripheral blood - unless splenectomised
  

• C migrates with E (more predominant) and A₂

Haemoglobin S/C

• Both ß chains are AN (one codes for ß^s chains, the other for ß^c chains - thus HbA₁ is absent
• Nearly as severe as homozygous HbS - concentration of Hb in individual RBC is increased. HbC tends to aggregate and potentiate the sickling og HbS
• Clinical signs and symptoms are similar to mild SS anaemia
  • Develop vaso-occlusive crises with complications
  • Splenomegaly is prominent
• Mild to moderate normocytic, normochromic anaemia
  
  

  Hb	Dec.
  Hct	Dec.

Normochromic
Anisocytosis + - ++++
Poikilocytosis + - ++++ (Sickle cells, Target cells and Acanthocytes)
Inclusion Bodies (occasional HbC crystals)

• HbS/C cells have higher oxygen affinity than HbS cells
  

• Nearly equal amounts of HbS and HbC. HbF may be increased with no HbA₁ (because of the absence of normal ß chains)

Haemoglobin D

• Several variants-most common is HbD ß¹²¹ (GH4), change is from Glu to Gln
• Heterozygous and homozygous states are asymptomatic
• Homozygous state may occasionally have an increase in target cells and osmotic fragility
  

• Homozygous form demonstrates 95% HbD with the same electrophoretic mobility as HbS however HbD is non-sickling and soluble
• Use of citrate agar allows separation of HbS and HbD
• HbS/D is a mild SS anaemia due to aggregation of deoxyHb
• Elevated risk from inhabitants of India, Pakistan and the Middle East

Haemoglobin E

• 3rd most prevalent haemoglobinopathy worldwide
• HbE ß²⁶ (B8), change is from Glu to Lys
• Slightly unstable (falsely lowering reported levels) with oxidant stress, oxygen dissociation curve suggests decreased oxygen affinity
• Homozygous HbE is characterised by a mild, hypochromic, microcytic anaemia with a decreased RBC survival

• Approximately 95% HbE at alkaline pH, the remainder is HbA₂ (migrates with E) and F
• Trait is asymptomatic and haematologic parameters (HbA₁, F and A₂ apparent) are normal except for a slight microcytosis and hypochromia
• Elevated risk from inhabitants of South East Asia

Unstable Haemoglobin Disorders

• Contain amino acid (aa) mutations in critical internal portions of the globin chains which effect the stability of the molecule, most usually in heterozygous form
• Characterised by precipitation of the abnormal Hb as Heinz bodies
• Causes cell rigidity, membrane damage and subsequent haemolysis as the Heinz bodies attach to the membrane resulting in "Bite Cells" after macrophages have removed the rigid areas
  Extravascular Haemolysis
• Symptoms usually occur with drug administration, infection or other events that change the normal environment of the Hb molecule
• Those variants that cause symptoms are known as congenital Heinz body haemolytic anaemias
• Hypochromic, microcytic haemolytic anaemia
• MCH and MCHC may be slightly decreased due to removal of Hb as Heinz bodies
• Reticulocyte count is increased - "Polychromasia"
• May show basophilic stippling, and small contracted cells (acanthocytes)
  

• Most unstable Hb has the same charge and mobility as normal Hb. Only approx 45% can be diagnosed by electrophoresis - diagnostically unsatisfactory

• Always positive with unstable Hb
• Presence of a fine precipitate in a lysate of RBCs heated to 50^oC for 60min is specific

• Normal Hb remains soluble for 30-40min in isopropanol
• Unstable Hb precipitates within 20min due to isopropanol's nonpolar nature weakening the internal bonds within Hb
• Splenectomy may be performed if haemolysis is severe (to remove the site of haemolysis), else no therapy is required
• Patients are required to avoid oxidising drugs

Hb Variants With Altered Oxygen Affinity

• Amino acid substitutions in chains close to the haem pocket may affect its ability to carry oxygen by preventing binding the of haem to the globin chain or by keeping the iron in an oxidised (Fe⁺⁺⁺)state
• Other substitutions include those near alpha-ß contacts, at the C terminal end of the ß-chain (most substitutions are here) and substitutions near the 2,3-DPG binding site.

• metHb is Hb with iron in an oxidised state (in which oxygen cannot be carried) resulting in cyanosis
• metHbaemia is a clinical condition occuring when metHb encompasses more than 1% of the total Hb
• May be acquired (through drugs or other toxic substances oxidising Hb in the circulation) or inherited as a dominant (usually attributed to HbM variant) or recessive characteristic.
  Most HbM are produced by substitution of Tyr for proximal/Distal His in the haem pocket of the alpha or ß-chains. The Tyr stabilises the iron in an oxidised state, imparting a brownish colour to the blood with no other haematologic abnormality