Haematology Flashcards
Name three genes that are associated with myeloproliferative disorders
JAK2 (V617F)
Calreticulin
MPL
Outline the treatment options for essential thrombocythaemia.
Aspirin
Hydroxycarbamide
Anagrelide (specifically inhibits platelet function but rarely used because of side-effects)
NOTE: hydroxycarbamide is an antimetabolite that suppresses cell turnover)
What might you expect to see in the blood film of a patient with primary myelofibrosis?
Leucoerythroblastic picture
Tear drop poikilocytes (dacrocytes)
Giant platelets
Circulating megakaryocytes
NOTE: can cause Budd-Chiari syndrome
What might you see on histological analysis of a trephine biopsy in primary myelofibrosis?
Increased reticulin and collagen fibrosis
Prominent megakaryocyte hyperplasia and clustering
New bone formation
Outline the treatment options for primary myelofibrosis.
Supportive – RBC and platelet transfusions (usually ineffective because of splenomegaly)
Hydroxycarbamide (may worsen anaemia)
Ruxolitinib – JAK2 inhibitor
Allogeneic stem cell transplantation
Splenectomy – dangerous operation but may provide symptomatic relief
List some diagnostic techniques used to identify the disease (CML) and monitor response to treatment.
FBC and leucocyte count
Cytogenetics and detection of Philadelphia chromosome (FISH)
RT-PCR to detect and quantify the number of copies of Bcr-Abl1 fusion transcript
NOTE: RT-PCR transcript % is the most sensitive
Bcr = breakpoint cluster region; Abl = ableson tyrosine kinase
What are the next steps in treatment if the first-line treatment of CML fails?
1st line fails (no complete cytogenetic response at 1 year or initial response is followed by resistance) switch to 2nd or 3rd generation
2nd line fails (inadequate response or disease progresses to accelerated or blast phase) allogeneic stem cell transplantation
How much more folate is needed in pregnancy than when non-pregnant?
200 µg/day more
What proportion of pre-eclampsia patients will develop thrombocytopaenia?
50% - probably due to increased activation and consumption
NOTE: also associated with coagulation activation
How is ITP in pregnancy treated?
IVIG
Steroids
Anti-D (if RhD-negative)
List the main changes in coagulation factors that occurs in pregnancy.
Factor 8 and vWF – increase 3-5 fold Fibrinogen – increase 2 fold Factor 7 – increase 0.5 fold Protein S – falls by 0.5 PAI-1 – increase 5 fold PAI-2 – produced by the placenta NOTE: all of these changes lead to a procoagulant state
List some other conditions in pregnancy that can increase the risk of thromboembolic disease.
Hyperemesis/dehydration Pre-eclampsia Obesity Thrombophilia Age (> 35 years) Parity Multiple pregnancy Ovarian hyperstimulation (IVF)
In women receiving anticoagulant therapy because of high VTE risk, when should it be stopped?
When labour begins or at the time of a planned delivery
Which type of haemoglobin variant cannot be identified using HPLC?
Alpha thalassemia
What are the possible complications that a mother with sickle cell disease may encounter during pregnancy?
More frequent vaso-occlusive crises Foetal growth restriction Miscarriage Preterm labour Pre-eclampsia Venous thrombosis
Which antigens do myeloma cells test positive for on immunohistochemistry?
CD138 CD38 CD56/CD58 Monotypic cytoplasmic immunoglobulin Light chain restriction
NOTE: CD38, CD138 and cytoplasmic Ig are present on normal plasma cells
Which antigens do myeloma cells test negative for on immunohistochemistry?
CD19
CD20 (unlike B cell lymphomas and CLL)
Surface immunoglobulin
Describe how the antibody screen of a patient’s plasma works.
Conducted using the indirect antiglobulin test (IAT)
2 or 3 reagent red blood cells are used which contain all the important red cell antigens
The patient’s serum is incubated with these screening cells
Anti-human immunoglobulin is added to the solution which allows bridging of red cells that are coated with IgG
This results in the formation of a visible clump
This is a group and screen
What is a full crossmatch?
Uses indirect antiglobulin test
Patients plasma is incubated with DONOR red cells at 37 degrees for 30-40 mins
Anti-human immunoglobulin is added to allow cross-linking of antibodies
Formation of a clump would suggest that antibodies against donor red cell antigens are present in the patient’s plasma
What is the adult dose of FFP?
15 mL/kg
List some causes of delayed transfusion reactions.
Delayed haemolytic transfusion reaction Infection (viral, malaria, vCJD) TA-GvHD Post-transfusion purpura Iron overload
Outline the mechanism of TRALI.
Anti-WBC antibodies in donor blood interact with WBC in the patient
Aggregates of WBCs get stuck to pulmonary capillaries resulting in the release of neutrophil proteolytic enzymes and toxic oxygen metabolites
This leads to lung damage
Which patient group tends to be affected by post-transfusion purpura?
HPA-1a negative patients who have previously been immunised by pregnancy or transfusion
How is post-transfusion purpura treated?
IVIG
If anti-D antibodies are detected in a pregnant women, what further steps should be taken?
Check if the father has the antigen
Monitor the level of antibody
Check cffDNA
Monitor foetus for signs of anaemia (MCA Doppler ultrasound)
Deliver the baby early because it gets a lot worse around term
What factor (related to stem cell harvesting) does the success of a bone marrow transplant depend on?
Number of CD34 cells per kg of weight of the recipient
List some clinical features of poorly controlled beta thalassemia major.
Anaemia heart failure, growth retardation
Erythropoietic drive bone expansion, hepatomegaly, splenomegaly
Iron overload heart failure, gonadal failure
How is a diagnosis of acute promyelocytic leukaemia confirmed?
Cytogenetic/FISH/molecular genetic analysis
15;17 translocation forming the PML: RARA fusion gene
How is acute promyelocytic leukaemia treated?
Platelets
Chemotherapy
All-trans-retinoic acid (ATRA)
NOTE: 90% 5-year survival
Outline the mechanisms by which anaemia of chronic disease causes anaemia.
Reduced red cell lifespan
Cytokine release (IFN-gamma, IL1, TNF) leads to reduced EPO receptor production and EPO synthesis
IL6 and LPS stimulate hepcidin production in the liver, which decreases intestinal iron absorption
Accumulation of iron in macrophages
Which HLA genotype is associated with Hodgkin lymphoma?
HLA-DPB1
List some investigations for thrombocytopaenia.
Coagulation screen
Blood film
Bone marrow aspirate
ANA/RAPA/anti-platelet antibodies (check for rheumatoid arthritis, SLE)
HIV (common cause of isolated platelet destruction worldwide)
List some investigations for suspected DIC.
Blood film D-dimer Fibrinogen Septic screen LFTs
What is paroxysmal nocturnal haemoglobinuria caused by?
An acquired defect in the GPI anchor which is one of two mechanisms by which cells attach proteins to their surface
Leads to increased risk of VTE and haemolysis
List some proteins that link the lipid bilayer to the cytoskeleton.
Band 3
Ankyrin-1
GPI
What is the difference between vertical interaction and horizontal interaction with regards to the red cell membrane?
Vertical – between proteins that link the lipid bilayer to the cytoskeleton (band 3, protein 4.2, Ankyrin, beta spectrin)
Horizontal – mainly involving alpha and beta spectrin (also protein 4.1)
What is the hall mark of red cells in hereditary spherocytosis?
Osmotic fragility – red cells show increases sensitivity to lysis in hypotonic saline
What is another test for hereditary spherocytosis?
Positive eosin 5-maleimide (dye) test (most sensitive)
This is shown by flow cytometry
Describe the appearance of the blood film in hereditary pyropoikilocytosis.
Fragmentation of red cells and a lot of variation in the shape of red cells (poikilocytosis)
It can cause severe haemolytic anaemia
Outline the importance of G6PD in the red cell.
G6PD catalyses the first step in the pentose phosphate pathway
This reaction generates NADPH which is required to maintain intracellular glutathione
Glutathione protects red cells against oxidative stress
A lack of G6PD, means that red cells are at increased risk of oxidative damage
Describe the appearance of the blood film in G6PD deficiency during acute haemolysis.
Contracted cells
Nucleated red cells
Bite cells
Hemighosts (Hb retracted to one side of the cell)
NOTE: high bilirubin, low haptoglobins and haemoglobinuria will be seen with all intravascular haemolysis
Which stain is used to look for Heinz bodies?
Methylviolet
What is a characteristic blood film feature of pyruvate kinase deficiency?
Echinocytes (aka Burr cells) – red cells with a lot of short projections
NOTE: as the cells decrease in size due to dehydration, the cells will resemble spherocytes. The number of echinocytes usually increases post-splenectomy
Describe how pyrimidine 5-nucleotidase deficiency leads to haemolytic anaemia.
Defect in nucleotide metabolism
Pyrimidine nucleotides are toxic to the cell but the cell must recycle purines
This means that red cells have a mechanism for selectively eliminating pyrimidines – this is dependent on pyrimidine 5-nucleotidase
Deficiency of pyrimidine 5-nucleotidase leads to an accumulation of toxic pyrimidines
What is a characteristic blood film feature of pyrimidine 5-nucleotidase deficiency?
Basophilic stippling
NOTE: this is also seen in lead poisoning because lead inhibits pyrimidine 5-nucleotidase
What are Ham’s test and flow cytometry for GPI-linked proteins used for?
Paroxysmal nocturnal haemoglobinuria
NOTE: Ham’s test looks at the sensitivity of red cells to lysis by acidified serum
List some specific criteria for splenectomy.
Transfusion dependence Growth delay Physical limitation Hypersplenism (where it causes pooling and physical symptoms) Age > 3 years and < 10 years
Which chromosomal duplications are most commonly associated with AML?
8 and 21 (there is a predisposition seen in Down syndrome)
Give an example of how disruption of a transcription factor can lead to leukaemogenesis.
Core binding factor (CBF) is the master controller of haemopoiesis
Translocation 8;21 fuses RUNX1 with RUNX1T1 leading to the formation of a fusion gene that drives leukaemia
The fusion transcription factor binds to co-repressors leading to a differentiation block
Inversion of chromosome 16 also affects CBF in a similar way
Which chromosomal aberration causes APML?
Translocation 15;17
PML-RARA fusion gene
What is a characteristic feature of APML? Why does this occur?
Haemorrhage – this is because APML is associated with DIC and hyperactive fibrinolysis
Give a type 1 and type 2 mutation for APML.
Type 1: FLT3-ITD
Type 2: PML-RARA
Outline the tests that may be used to diagnose AML.
Blood film
Bone marrow aspirate
Cytogenetic studies (done in EVERY patient)
Molecular studies and FISH
What do dense granules contain?
Energy stores (ATP and ADP)
Outline the initiation phase of the clotting cascade.
Damage to the endothelium results in exposure of tissue factor which binds to factor 7 and activates it to factor 7a
The tissue factor-factor 7a complex then activates factors 9 and 10
Factor 10a binds to factor 5a resulting in the first step of the coagulation cascade
Outline the amplification phase of the clotting cascade.
Activated factors 5 and 10 will result in the production of a small amount of thrombin
This thrombin will activate platelets
Thrombin will also activate factor 11 which activates factor 9
Thrombin also activates factor 8 and recruits more factor 5a
Factors 5a, 8a and 9a will bind to the activated platelet
Outline the propagation phase of the clotting cascade.
Activated factors 5, 8 and 9 will recruit factor 10a
This results in the generation of a large amount of thrombin (thrombin burst)
This enables the formation of a stable fibrin clot
Name two factors that convert plasminogen to plasmin.
Tissue plasminogen activator
Urokinase
Name two factors that directly inhibit plasmin.
Alpha-2 antiplasmin
Alpha-2 macroglobulin
What is the role of thrombin-activatable fibrinolysis inhibitor (TAFI)?
Inhibitor of fibrin breakdown
Outline the role of protein C and protein S.
Trace amounts of thrombin generated at the start of the clotting cascade activate thrombomodulin
This allows protein C to bind to thrombomodulin through the endothelial protein C receptor
Protein C is then fully activated in the presence of protein S
Fully activated protein C will inactivate factors 5a and 8a
When is treatment for platelet disorders required?
Platelet count < 30 x 109/L (this is associated with spontaneous haemorrhage)
What can cause immune-mediated thrombocytopaenia?
Idiopathic Drug-induced (e.g. quinine, rifampicin) Connective tissue disorder (e.g. SLE) Lymphoproliferative disease Sarcoidosis
List two non-immune mediated conditions that cause thrombocytopaenia.
DIC
MAHA
What is a typical lesion seen in coagulation factor disorders?
Ecchymoses
Outline the classification of von Willebrand disease.
Type 1 – partial quantitative deficiency
Type 2 – qualitative deficiency
Type 3 – complete quantitative deficiency
NOTE: encoded on chromosome 12
Describe the expected laboratory test results for the three types of von Willebrand disease.
Type 1 – low antigen, low activity, normal multimer
Type 2 – normal antigen, low activity, normal multimer
Type 3 – very low antigen, very low activity, absent multimer
List some causes of DIC.
Sepsis (MOST COMMON)
Trauma (e.g. fat embolism)
Obstetric complications (e.g. amniotic fluid embolism)
Malignancy
Vascular disorders
Reaction to toxin
Immunological (e.g. transplant rejection)
Describe the typical clotting study results in DIC.
Prolonged APTT and PT Prolonged TT Decreased fibrinogen Increased FDP Decreased platelets Schistocytes (due to shearing of red blood cells as it passes through a fibrin mesh)
Describe how liver disease leads to bleeding disorders.
Decreased synthesis of clotting factors 2, 7, 9, 10, 11 and fibrinogen
Dietary vitamin K deficiency
Dysfibrogenaemia
Enhanced haemolysis (decreased alpha-2 antiplasmin)
DIC
Thrombocytopaenia due to hypersplenism
Outline the treatment of:
a. Prolonged PT/APTT
b. Low fibrinogen
c. DIC
a. Prolonged PT/APTT Oral vitamin K FFP infusion b. Low fibrinogen Cryoprecipitate c. DIC Replacement therapy
In which conditions are acanthocytes/spur cells seen?
Abetalipoproteinaemia
Liver disease
Hyposplenism
In which conditions are echinocytes seen?
Pyruvate kinase deficiency
Uraemia
Gastric cancer
Which membrane proteins are defective in many cases of hereditary spherocytosis?
Spectrin (most common)
Also ankyrin, band 2 and band 3
Which proteins are defective in hereditary eliptocytosis?
Spectrin
Band 4.1
NOTE: all are autosomal dominant except hereditary pyropoikilocytosis which is autosomal recessive
Which substance accumulates in pyruvate kinase deficiency?
2,3-bisphosphoglyceric acid (2,3-BPG)
This shifts the oxygen dissociation curve to the right thereby releasing oxygen more readily
NOTE: PK normally converts phosphoenolpyruvate to pyruvate, but its absence means that an intermediate (2,3-BPG) collects instead
Which types of gallstones form in patients with hereditary spherocytosis?
Calcium bilirubinate
Where do the start and end steps of haem synthesis take place within the cell?
Mitrochondrion
What is the inheritance pattern of hereditary spherocytosis?
Autosomal dominant (75%) De novo mutations (25%)
What is the inheritance pattern of pyruvate kinase deficiency?
Autosomal recessive
What causes hyponatraemia in acute intermittent porphyria?
SIADH
Describe the clinical presentation of polycythaemia vera.
Incidental finding
Symptoms of hyperviscosity (headaches, visual disturbances, fatigue, dyspnoea)
Increased histamine release (Aquagenic pruritus, peptic ulceration)
Splenomegaly
Plethora
Erythromelalgia (red painful extremities)
Thrombosis
Retinal vein engorgement
Gout
Describe the appearance of a bone marrow biopsy in polycythaemia vera.
Increased cellularity (mainly erythroid cells) Slight reticulin fibrosis and megakaryocyte abnormalities
What investigation finding is considered diagnostic of polycythaemia vera?
Presence of JAK 2 V617F mutation
On chromosome 9
How is red cell mass and plasma volume measured?
Isotope dilution
Red cells are incubated with radioactive chromium
Plasma is incubated with radioactive iodine
On which exon is the JAK2 V617F mutation found?
Exon 14
Which other JAK mutation is a significant finding and which condition is it associated with?
Exon 12 mutation
It is associated with idiopathic erythrocytosis
What is idiopathic erythrocytosis?
Isolated erythrocytosis with low EPO
Treated with venesection only
NO JAK 2 V617F mutation, but some cases will have an exon 12 mutation
Define chronic idiopathic myelofibrosis.
A clonal myeloproliferative disease with proliferation mainly of megakaryocytes and granulocytic cells, associated with reactive bone marrow fibrosis and extramedullary haemopoiesis
Describe the structure of janus kinases.
They have a kinase domain and a catalytically inactive pseudokinase domain with regulatory function
What effect does the JAK 2 V617F mutation have on janus kinases?
It inactivates the pseudokinase domain thereby removing inhibition of activation so it becomes constitutively activated
What are the morphological features of leucoerythroblastic anaemia seen on blood film?
Tear drop red blood cells (aniso- and poikilocytosis)
Nucleated RBCs
Immature myeloid cells
What are the two main groups of haemolytic anaemia? List some examples.
Inherited (defects with the cell)
• Hereditary spherocytosis (membrane problem)
• G6PD deficiency (enzyme problem)
• Sickle cell disease, thalassemia (haemoglobin problem)
Acquired (defects with the environment)
• Immune-mediated
• Non-immune mediated
List some systemic diseases that can cause autoimmune haemolytic anaemia.
Cancer involving the immune system (e.g. lymphoma)
Disease of the immune system (e.g. SLE)
Infections (disturbs the immune system)
List some key features of MAHA.
Usually caused by underlying adenocarcinoma
Red cell fragments
Low platelets
DIC/bleeding
List some causes of neutrophilia.
Corticosteroids (due to demargination) Underlying neoplasia Tissue inflammation (e.g. colitis, pancreatitis) Myeloproliferative/leukaemia disorder Infection
List some infections that characteristically do not cause neutrophilia.
Brucella
Typhoid
Many viral diseases
List some key features of a reactive neutrophilia on a blood film.
Band cells (presence of immature neutrophils (band cells) show that the bone marrow has been signalled to release more WBCs)
Toxic granulation
Clinical signs of infection/inflammation
List some causes of monocytosis.
Bacteria: TB, Brucella, typhoid
Viral: CMV, VZV
Sarcoidosis
Chronic myelomonocytic leukaemia
Which gene mutation causes chronic eosinophilic leukaemia?
FIP1L1-PDGFRa fusion gene
Which type of virus typically causes basophilia?
Pox viruses
List some anticoagulant molecules produced by the endothelium.
Thrombomodulin Endothelial protein C receptor Tissue factor pathway inhibitor Heparans NOTE: it does not normally produce tissue factor
List some disadvantages of heparin.
Administered by injection
Risk of osteoporosis
Variable renal dependence
Risk of heparin-induced thrombocytopaenia
How is unfractionated heparin monitored?
It has variable pharmacokinetics and a variable dose-response
Must be monitored with APTT or anti-Xa levels
List some properties of DOACs.
Oral administration Immediate action (peak = 3-4 hours) Useful in long-term Short half-life No monitoring needed
How can the action of warfarin be reversed?
Administering vitamin K – takes 12 hours
Giving factors 2, 7, 9 and 10 – immediate
Outline the treatment of DVT/PE.
Start LMWH (e.g. tinzaparin 175 u/kg) + warfarin
Stop LMWH when INR > 2 for 2 days
ALTERNATIVE: start a doac
These should be continued for:
- Known cause: 3 months
- unknown cause or cancer VTE: 3-6 months
- thrombophilic/recurrent: lifelong
What should all patients > 60 years old with idiopathic thromboembolic disease be offered?
CT scan to check for an underlying cause
What are the key characteristics of myelodysplastic syndromes?
Development of a clone of marrow stem cell with abnormal maturation resulting in functionally defective blood cells and a reduction in cell counts
This leads to cytopaenia, functional abnormalities of cell maturation and an increased risk of transformation to leukaemia
Which staging systems are used for multiple myeloma?
Durie-Salmon Staging
International Staging System
What type of anaemia are all haemolytic anaemias?
Normocytic
How is severe hereditary spherocytosis treated?
Splenectomy
Vaccination
What bone abnormality may result from hereditary spherocytosis?
Osteoporosis
What is the pentad of thrombotic thrombocytopaenic purpura?
Fever Change in mental status Thrombocytopaenia Reduced renal function Haemolytic anaemia
NOTE: may require plasma exchange
List and describe some blood and bone marrow features of myelodysplastic syndromes.
Pelger-Huet anomaly (bilobed neutrophils)
Dysgranulopoiesis of neutrophils (failure of granulation)
Dyserythropoiesis of red blood cells (lack of separation between red cell precursors, presence of abnormal ring of cytoplasm around the nucleus of precursor red cells)
Dysplastic megakaryocytes (micro-megakaryocytes)
Increased proportion of blast cells in the bone marrow (normally < 5%)
What might you see if you stained for iron in the bone marrow of a patient with a myelodysplastic syndrome?
Ringed sideroblasts (accumulation of iron around the nuclei of red blood cell precursors)
List some factors that are taken into account when classifying types of myelodysplastic syndrome.
Cell lineage affected Blast cell proportions Cytogenetics Presence of ringed sideroblasts Cytopaenias
What are the five prognostic variables that are used to calculate prognostic risk using the Revised International Prognostic Scoring System (IPSS-R) for Myelodysplastic Syndromes?
Bone marrow blast percentage Karyotype Haemoglobin Platelets Neutrophils NOTE: high risk is considered a score > 6, low risk < 1.5
List some causes of primary bone marrow failure.
Fanconi anaemia (multipotent stem cell)
Diamond-Blackfan syndrome (red cell aplasia)
Kostmann syndrome (neutrophil progenitor)
Acquired: idiopathic aplastic anaemia (multipotent stem cell)
List some drugs that can cause bone marrow failure.
Cytotoxic drugs (predictable, dose-dependent)
Phenylbutazone, Gold salts (idiosyncratic, rare)
Antibiotics – chloramphenicol, sulphonamides
Diuretics – thiazide
Antithyroid drugs – carbimazole