Haematology Flashcards
Tell me the key facts about acute promyelocytic leukaemia.
A subtype of AML. Chromosomes 15 and 17 (exchange of material). Formation of PML-RAR alpha fusion gene. Cells formed commonly have Auer rods. Is a medical emergency you get coagulopathy (anticoagulation). Treatment is immediate all-transretinoic acid (ATRA) and then for 3 months after.
Which cell type produces platelets
Megakaryocyte
Where are blood cells made at the different stages of development?
Following conception - yolk sac
~12 weeks foetus - liver and spleen
Baby born- bone marrow primary responsible
In adults the bone marrow production is confined to the axial skeleton whereas in children the long bones also contribute
What is the classic site for bone marrow biopsy
Posterior superior iliac crest
What the ‘influencing’ factors for blood cell production i.e. production of granulocytes, platelets and red blood cells and where are they released from?
G-CSF which is most specific for neutrophils is released by the endothelium and macrophages
EPO- production of red blood cells- produced by kidneys in response to hypoxia or anaemia
TPO- production of platelets- produced by the liver
What are the values corresponding to microcytic, normocytic and macrocyctic anaemia?
Microcytic is <80
Normocytic is 80-100
Macrocytic is >100
If a blood film has lots of reticulocytes (young red cells) what is the term used to describe the blood film?
Polychromasia meaning ‘many colours’- reticulocytes are larger and more blue than normal cells
What is one of the first investigations done after discovering that a patient has an abnormal blood cell count?
Peripheral blood film
General features of acute leukaemia
Proliferation of immature cells. Differentiation block (stops the bone marrow from being able to produce normal healthy blood cells). Present with symptoms of marrow failure. Anaemia, thrombocytopenia (bleeding), neutropenia (infection). Patients have a very short history and are often unwell. An aggressive type of cancer.
General features of chronic leukaemia
Mature cells, less acute, often present with increased WCC. CML = chronic myeloid leukaemia (myeloproliferative): granulocytes. CLL = chronic lymphocytic leukaemia (lymphoproliferative): lymphocytes
Give the names of the other myeloproliferative neoplasms (other than CML).
Polycythaemia rubra Vera = raised red cells/ increased Hb and Hct
Essential thrombocythaemia = increased platelets only
Primary myelofibrosis = bone marrow fibrosis
What is the commonest mutation causing the myeloproliferative neoplasms?
JAK2
What age group does Hodgkin’s lymphoma more commonly occur?
Young adults
General difference between leukaemia and lymphoma?
Lymphoma is mainly in the lymph nodes, leukaemia is mainly in the blood and bone marrow
Which age group is ALL most common in?
Children
Which age group is CLL most common in?
The elderly
Myeloma can cause CRABI. Name the components
HyperCalcaemia Renal dysfunction Anaemia Bone (lytic lesions, osteoporosis, fractures) Infections
What is the classic bruising pattern seen in thrombocytopenia?
Petechiae (pin-prick bruises) is classic platelet type bleeding
What is the lifespan of platelets, neutrophils and RBCs?
Platelets= 7 days Neutrophils = 24 hrs RBCs= 120 days
What may be the only sign of neutropenia?
Fever (severely neutropenic patients cant form pus in the same way).
Which industrial chemical is an important cause of AML?
Benzene
Is AML or ALL more common and what age group does AML present in?
AML is More common than ALL.
AML is more common in adults- increasing incidence with age.
What findings aid a diagnosis of AML?
FBC- low Hb, low platelets, low granulocyte count, high blast count
Blood film- lots of blasts (high nuclear:cytoplasmic ratio)
Bone marrow histology- blasts must be >20% of nucleated cells
Flow cytometry- CD13+ and CD33+
Cytogenetics- t(15;17) prognosis = good unless you catch it too late, t(8;21), inv16 = good prognosis. Monosomy 7, abnormalities chromosome 5, chromosome 1 and complex cytogenetics = poor prognosis
What is the treatment for AML?
(Most recent addition in Tx is the antibody Mylotarg (Gemtuzumab ozogamicin) which is a CD33 antibody).
Combination chemotherapy- important drugs include cytarabine (aka. Cytosine arabinoside) and daunorubicin. Total of 4-6 months of Tx, Tx induces profound bone marrow suppression and pancytopenia so need supportive care incl: transfusion of RBCs and platelets, antiseptic mouthwashes, clean diet, oral prophylactic anti-fungal agents and quinolones.
What factors may affect prognosis in AML?
Better prognosis if <60 years and favourable cytogenetics.
FLT3-negative, NPM1 positive >60% survival
What are the clinical signs of ALL?
Similar to AML (low Hb, low platelets, high blast cells), but more frequently: lymphadenopathy, hepatosplenomegaly and CNS involvements. Commonly causes severe bone pain, sweats and weight loss.
What findings aid a diagnosis of ALL?
As for AML.
Lumbar puncture is important to determine if there is evidence of CNS disease (usually done when peripheral blasts cleared).
Flow cytometry: blasts of B cell (80%) which are CD10, CD19 and CD22 positiv or T cell lineage (20%)
Cytogenetics: Philadelphia chromosome t(9;22) seen in 20-25% of adults.
What is the treatment for ALL?
Chemotherapy with complex combinations of cytotoxic drugs e.g steroids, vincristine, daunorubicin, asparaginase (4 drug induction). Different drugs for second phase of induction.
What is meant by the term ‘minimal residual disease’?
MRD is the name given to small numbers of leukaemic cells that remain in the patient during remission. These cells can cause relapse. Therefore the less MRD a patient has, the fewer leukaemia cells they have and hence the deeper the remission and the better the prognosis.
The multi drug treatment in ALL is in 3 phases- what are these?
Induction phase- 3 months’ intense chemotherapy to induce remission
Consolidation phase- 4 months’ intense chemotherapy to consolidate the remission
Maintenance phase- 2 years less intense chemotherapy to maintain remission
What techniques are used to try and detect MRD in a patient in remission?
- Microscopy of a blood film
- FISH
- Immunophenotyping
- PCR
These go up in sensitivity (able to detect lower number of cells present)
What is complete remission (in the context of ALL lecture)
When the MRD gets so low that even the most sensitive PCR testing can’t pick up any cancer cells (hence they are MRD negative). This doesn’t mean the person doesn’t have ANY cells left, they may have <5% left but we can’t detect them
What targeted therapies are emerging for the future treatment of leukaemia?
CAR-T cells (chimeric antigen receptor T cells)- this is when the T cells are removed from a patient and modified so that they express receptors specific to the patient’s particular cancer, which can then recognise and kill the cancer cells are reintroduced into the patient. There are also some novel antibody therapies.
How does a bone marrow transplant work?
- You give very high dose (3-10x standard chemotherapy) to kill all of the patient’s bone marrow cells
- You then give the patient an injection of bone marrow stem cells
- The stem cells migrate to the bone marrow and set up camp.
- They proliferate, giving the patient a new bone marrow and hence a new immune system
- This new immune system is able to kill any remaining leukaemic cells that might have survived the initial chemo because it recognises the cancer as non-self (a graft versus leukaemia effect).
What is the mortality rate associated with bone marrow transplants and why is it high?
20% mortality, due to neutropenic sepsis and graft versus host disease
What are the long-term consequences of bone marrow transplant?
- Infertility- the high dose chemotherapy destroys the gonads
- Secondary cancers- the high dose chemotherapy causes a second cancer elsewhere later on in life (1% at 10 years and 10% at 20 years).
Where can bone marrow transplant cells come from?
Other people- allogenic transplant.
The patient themselves - autologous: before the high dose chemo you remove some healthy bone marrow cells from the patient, you freeze them, you inject them back into the patient once you’ve wiped out their bone marrow with the high dose chemotherapy. You then hope the healthy bone marrow cells repopulate the bone marrow to rescue it.
What is the definition of anaemia?
Anaemia is a condition in which the number of red blood cells (and consequently their oxygen carrying capacity) is insufficient to meet the body’s physiologic need.
How do the following factors influence haemoglobin concentration: age, gender, altitude, smoking and pregnancy.
Age: Hb concentrations fall slightly in the older populations. Neonates tend to have a high Hb when compared to the adult reference range which then falls off and during infancy they usually have a lower Hb which rises again throughout childhood until they reach adult levels.
Gender: men have a higher Hb concentration than women due to hormones.
Altitudes: higher altitudes = higher Hb concentration (due to sensing of the relative hypoxia)
Smoking- smokers have a slightly higher Hb concentration (increased Hb to account for the CO which binds some of the Hb up)
Pregnancy: in the 2nd and 3rd trimesters Hb concentration starts to fall (total number of RBCs increases but plasma volume increases to a greater extent, so the actual concentration of Hb actually decreases).
What is the structure of Hb?
A tetramer of globin monomers and a haem ring. Different globing have different oxygen carrying properties, which globins make up the Hb depends on the stage of life. Foetal Hb: 2 alpha and 2 gamma. Adult Hb= 2 alpha and 2 beta.
What is HbA2?
A type of adult haemoglobin which is present in very small amounts compared to HbA. It is composed of 2 alpha and 2 delta globulin subunits.
What factors shift the oxygen dissociation curve to the right (and hence causing O2 to be off-loaded)?
An increase in temperature
An increase in CO2 concentration
Decrease in pH
Presence of DPG
What other things can affect the oxygen carrying capacity of Hb?
Binding of CO = carboxyhaemoglobin (prevents carriage of O2)
Methaemoglobin= when the iron is in the Fe3+ state not the Fe2+ state (prevents carriage of O2)
Carbaminohaemoglobin- form of Hb complexed with CO2- accounts for ~10% of CO2 carriage.
Sulphhaemoglobin- O2 cannot be delivered or carried. May be produced by the action of sulphur containing drugs and cannot be converted back to Hb.
What are some of the general features of anaemia?
Pallor Peripheral oedema Tachycardia Flow murmur (when blood flows more rapidly than normal through the heart) Palpitations Shortness of breath (esp. on exertion) Confusion (esp. in the elderly)
What is the most useful parameter when considering the cause of anaemia? Tell me briefly about it
Mean cell volume (MCV). It is the mean size of the RBCs and is the normal range is the same in men and women.
What are the causes of a microcytic anaemia?
Iron deficiency, thalassemia, anaemia of chronic disease
What are the causes of a normocytic anaemia?
Acute blood loss, haemolysis, anaemia of chronic disease, bone marrow infiltration, combined haematinic deficiency
What are the causes of a macrocytic anaemia?
B12/folate deficiency Haemolysis Hypothyroidism Liver disease Alcohol excess Myelodysplasia
What are some of the causes of iron deficiency?
Dietary (80% from meat, 20% from vegetables)
Physiological (infancy, adolescence, pregnancy)- describes a state where requirements of iron for growth are increased but these requirements are not being met through the diet.
Blood loss (note: chronic blood loss, not acute where you get a normocytic anaemia)
Malabsorption (e.g. coeliac disease)
Iron deficiency anaemia- what are the clinical features if it is severe?
Angular stomatitis (sores around the mouth)
Sore mouth
Koilonychia (spoon shaped nails)
Pharyngeal and oesophageal webs (rarely)
What are the laboratory features of iron deficiency anaemia?
Microcytic hypochromic anaemia.
Hypochromic means the cells don’t stain so brightly.
Low serum ferritin (beware as it is an acute phase protein, so in patients with inflammation/infective process, their ferritin levels will be bumped up which can mask iron deficiency as a cause of anaemia). Ferritin is the main storage complex of iron.
Low serum iron
Absent iron stores in the bone marrow (but wouldn’t tend to look at this)
Elevated TIBC (total iron binding capacity) and serum transferrin saturation
What characteristic feature might you see on the blood film of someone who is iron deficient?
Microcytic hypochromic anaemia. Some cells may also appear elongated/ oblong- these are called ‘pencil cells’.
What are the principles behind further investigations in iron deficient patients?
If think it has a physiological cause- treat with oral iron (until the period of physiological need has settled down)
If pre-menopausal female and thought likely to be due to periods (no GI problems) also treat with oral iron. However, if any worrying GI signs/symptoms or the woman is not having periods for whatever reason (e.g. OCP) then they should be investigated more throughly (e.g. colonoscopy/OGD). Similarly, males and post-menopausal women should be investigated.
What is meant by the term ‘anaemia of chronic disease’?
Depression of erythropoiesis of multifactorial aetiology seen as a secondary manifestation in a wide variety of disorders (usually chronic inflammatory process). More commonly a normocytic normochromic anaemia but can be microcytic hypochromic. Serum ferritin may be normal or increased (increased esp. if inflammatory process).
What things start to go wrong in anaemia of chronic disease?
Iron sequestration in macrophages and failure of transport of iron from reticular endothelial system to developing cells.
Slightly shortened red cell survival (their circulating half-life is shorter).
What is the treatment of anaemia of chronic disease?
Correction of underlying cause. Erythropoietin (+iron)
What type of anaemia do you see in vitamin B12 deficiency?
Macrocytic megaloblatic anaemia. (Megaloblastic means that you can identify an abnormality in the developing RBCs in the bone marrow- it results from inhibition of DNA synthesis during red blood cell production).
How is vitamin B12 absorbed?
It combines with intrinsic factor (IF) secreted by gastric parietal cells and is absorbed in the terminal ileum.
What are the causes of vitamin B12 deficiency?
Dietary: vegan is
Intrinsic factor deficiency: pernicious anaemia, gastrectomy, congenital
Intestinal malabsorption: diseases of the terminal ileum e.g. Crohn’s disease, blind loops and small bowel diverticula.
What are the clinical features of vitamin B12 deficiency?
Anaemia, jaundice (because the RBCs that are produced are broken down more readily in the circulation), glossitis (sore tongue), neurological deficit (this can even be seen in the absence of anaemia).
What are the laboratory features of vitamin B12 deficiency?
Anaemia, neutropenia, thrombocytopenia (because vitamin B12 is required for DNA synthesis and production of many cell types). On a blood film vitamin B12 deficiency can actually look like a haematological malignancy because you see a pancytopenia and the cells look a bit abnormal. Low serum vitamin B12. Antibodies against parietal cells or IF. Megaloblastic change in bone marrow. Features of haemolysis.
What type of anaemia results from folate deficiency?
Macrocytic Megaloblastic anaemia
Where in the body is folate absorbed?
The jejunum
What are the causes of folic acid deficiency?
Dietary- common in elderly, poor diet related to alcohol misuse
Increased utilisation- pregnancy, malignancy, haematological disorders with rapid cell turnover
Malabsorption e.g. coeliac disease
Drugs e.g. anticonvulsants
Excessive loss e.g. renal dialysis
What are the clinical features of folic acid deficiency?
Similar to vitamin B12 deficiency but neurological problems do not occur.
What is the treatment for B12/folate deficiency?
Address underlying cause. If the problem (with regards to vit B12) is that you don’t have enough IF, then giving oral vit B12 won’t help because the patient still wont be able to absorb it, so in these cases vit B12 is given by IM injection. Vitamin B12 replacement as IM infection most common -3 times per week for 2 weeks then maintenance phase. Oral folic acid replacement (even in malabsorptive conditions if you give a high enough dose then it will start to be absorbed more readily).
What do you need to be careful about when treating vitamin B12 and folate deficiency and how do you avoid the potentially fatal complication?
Risk of subacute combined degeneration of the cord (with permanent neurological sequence) if folic acid is replaced in the absence of vitamin B12- so always remember to replace vitamin B12 before folate in people who are deficient in both vitamin B12 and folate (or at least do them at the same time).
What is meant by combined haematinic deficiency?
Remember that MCV is a MEAN value. Therefore a combination of large and small red cells can produce an MCV within the normal range (e.g. in a patient with a really poor diet who you suspect is iron, folate and vitamin B12 deficient despite a normal MCV).
What is haemolysis?
Reduced red cell survival (normal life span 120 days) - the problem isn’t with production. RBC breakdown can be: intravascular (within the vessels releasing free Hb) or extravascular (by the reticulo-endothelial system) - predominantly the spleen. Erythroid expansion and increased production (up to 7 fold) by the bone marrow may partly compensate.
What are the inherited causes of haemolytic anaemia?
Membrane: hereditary spherocytosis - autosomal dominant
Enzymes: G6PD deficiency - x linked recessive. And pyruvate kinase deficiency - autosomal recessive
Haemoglobin: sickle cell anaemia, thalassaemia (both autosomal recessive)
What are the acquired causes of haemolytic anaemia?
Immune: autoimmune (can be primary or secondary to lymphoproliferative autoimmune disorders, infections, drugs) OR alloimmune i.e. the red cells and the immune system don’t match (e.g. haemolytic disease of the newborn or incompatible blood transfusion).
Infections: many mechanisms
Drugs and chemicals: many mechanisms
Mechanical: (where red cells are sliced up): MAHA
Other rare types: e.g. paroxysmal nocturnal haemoglobinuria
What does MAHA stand for?
Microangiopathic haemolytic anaemia (the red cells are sliced up in the very small blood vessels/ microvasculature)- so it’s a type of intravascular haemolysis, usually in sick patients)
What are the clinical features of haemolysis?
Anaemia (symptoms and signs associated with anaemia)
Jaundice (unconjugated bilirubin i.e. pre-hepatic)
Splenomegaly (if extravascular haemolysis and the spleen is busy breaking down RBCs)
Skeletal abnormalities (congenital forms of haemolysis when the bone marrow is trying to compensate)
Gallstones (pigment stones (bilirubin) suggest chronic haemolysis)
Haemoglobinuria (denotes intravascular haemolysis)
What are the features for a laboratory diagnosis of haemolysis?
Increased RBC production: reticulocytosis, polychromasia on blood film (reticulocytes are slightly blue tinged).
Increased RBC destruction: unconjugated hyperbilirubinaemia, increased LDH (non-specific, as any type of increased cell turnover/ breakdown will give you an increased LDH). Absent haptoglobins (they bind and clear free Hb so may be reduced). Intravascular: haemoglobinaemia, haemoglobinuria, haemosiderinuria
What are the laboratory investigations you can conduct to determine the cause of haemolysis (once you’ve established its haemolysis)?
Blood film is the most useful investigation (further investigations are directed by this and the clinical features)
Coombs test is used to detect immune coating on RBC by Ig and complement - is positive suggests immune cause, but does not differentiate between autoimmune or alloimmune
Tell me about spherocytes
Each time the RBC goes through the spleen, a bit more membrane is haemolysed -> volume to surface area ratio is increased (end result is a sphere). On the blood film: lose their pallor and look like small dense circles. Spherocytes may be indicative of HS (hereditary spherocytosis) or autoimmune haemolytic anaemia (history and Coombs test will help distinguish).
What does the presence of red cell fragments indicate?
MAHA (microangiopathic haemolytic anaemia).
The RBCs look like they have been chopped with a knife.
What are some of the causes of MAHA?
TTP (thrombotic thrombocytopenic purpura) HUS (haemolytic uraemic syndrome) DIC Prosthetic valve haemolysis Vascularise HELLP syndrome Malignant hypertension Metastatic adenocarcinoma
On which chromosomes are the genes that encode the globulin proteins?
Chromosomes 11 and 16. Note: proteins produced from both chromosomes are needed to make normal Hb (usually 2 alpha globins combine with 2 non-alpha globins).
What are haemoglobinopathies?
Inherited genetic defects of globin. Sickling disorders and thalassaemias are the most clinically important (both are autosomal recessive and carriers are asymptomatic). Note that mutations of the alpha globin genes affect both foetal and adult life. Beta globin mutations only manifest after birth when HbA replaces HbF.
What is thalassemia?
Reduced or absent synthesis of globin. Beta thalassaemia (beta globin). Alpha thalassaemia (alpha globin).
What is sickle cell anaemia in brief terms?
Altered beta globin protein structure/ function
Where in the world are thalassaemias more frequently found?
In the Mediterranean, Africa, western and Southeast Asia, India and Burma
What are the various clinical types of beta thalassaemia?
Beta thalassaemia trait (carrier status)- genotype beta0, beta. Asymptomatic, normal life span.
Beta thalassaemia intermedia- variable genotype, phenotype and life span.
Beta thalassaemia major- genotype beta0, beta0. No beta globin and no HbA. Early death if untreated (because it’s the beta gene, newborn babies aren’t symptomatic but they are very soon in the months after birth).
What is the pathophysiology of beta thalassaemia major?
Complete absence of HbA (as no beta globin). Excess alpha chains accumulate and damage red blood cells. Ineffective erythopoiesis. Excessive RBC destruction (intramedullary and extramedullary). Iron overload. Extra-medullary haemtopoiesis.
What are the clinical features of beta thalassaemia major?
Asymptomatic at birth. Symptomatic anaemia in the first few months of life. Jaundice. Growth retardation, failure to thrive. Medullary hyperplasia- bony abnormalities especially of the facial bones. Extra-medullary haematopoiesis- enlarged liver and spleen. Increased risk of thrombosis. Pulmonary hypertension and congestive heart failure.
What x-ray appearance might you describe in a patient with beta thalassaemia major where there is a process of medullary hyperplasia?
Hair on end appearance due to the medullary hyperplasia
What would you see on a blood film from a patient with beta thalassaemia major?
Very few RBCs, pale RBCs (hypochromia), target cells (codocytes) they have a little dot in the centre (look like a bullseye). Nucleated red cells.
What is the treatment of beta thalassaemia major?
Regular blood transfusions (life long). 2 aims: prevent symptomatic anaemia allowing children to live, grow and develop and also to suppress marrow hyperplasia with skeletal consequences. Iron chelation (removal of excess iron from the body). Folic acid. Bone marrow transplantation in early life if suitable donor.
What is one of the problems with giving regular blood transfusions to a patient with beta thalassaemia major as part of treatment?
Problem of iron overload (currently the life limiting factor) especially heart, liver, endocrine.
What might you see in beta thalassaemia trait? (heterozygous Carrier status)- note: it is frequently misdiagnosed as iron deficiency because iron deficiency is the most common cause of a microcytic anaemia
Mild microcytic hypochromic anaemia
Hb 100-110
MCV 50-70, MCH 20-22.
Asymptomatic, often identified on routine blood count
Relative increase in HbA2 (useful diagnostically)
Important to identify for family screening and avoidance of inappropriate iron therapy.
Tell me about alpha thalassaemia
Alpha thalassaemia: deficient/ absent alpha subunits.
Excess beta subunits soon after birth. Excess gamma subunits in utero and in newborns. Main types: silent carrier (1 gene not working). Trait (carrier): 2 genes not working. Haemoglobin H disease: 3 genes not working. Major (Haemoglobin Bart’s): all 4 genes not working.
What might you see in haemoglobin H (HbH) disease (one of the alpha thalassaemias) which might be useful diagnostically?
People with HbH make some beta tetramers which we can stain for to diagnose HbH.
Tell me about Hb barts (a type of alpha thalassaemia)
All 4 genes not working. No alpha chains produced. Mainly gamma chains in utero: form tetramers. In Hb Barts, the babies die before it is possible to transfuse them in utero
What are the genotypes and phenotypes for sickle cell anaemia?
One normal beta globin gene and one HbS gene = sickle cell trait (carrier, asymptomatic)- sickle trait is only very rarely associated with illness (e.g. hypoxia), but important for family screening.
2 HbS genes = sickle cell anaemia
Beta0 and HbS = sickle cell anaemia
HbS and some other beta globin mutations e.g. C (HbSC) = sickle cell anaemia
What is the mutation in sickle cell anaemia?
Glutamic acid is substituted for valine at the 6th amino acid and this change in the sequence leads to polymerisation of the Hb when it becomes deoxygenated- so it makes sticky long chains. Sickle cell Hb forms long, inflexible chains. Sickle RBCs are stiff and angular, causing them to become stuck in small capillaries. They also have a reduced lifespan compared to normal Hb.
What is priapism?
A painful erection that doesn’t go away and needs urgent treatment
What are the features of sickle cell anaemia?
- Haemolysis: chronic anaemia. Increased reticulocytes, LDH and bilirubin. Usually well-adjusted to anaemia, HbS has low oxygen affinity. Risk of aplastic crises, gallstones.
- Vaso-occlusion: acute episodes and chronic deterioration
- Hyposplenism: risk of infection and encapsulated bacteria
Which virus may be associated with an aplastic crisis in sickle cells anemia?
Parvovirus
It has a tendency to switch off RBC production
What is hand-for syndrome in the context of sickle cell anaemia complications?
It is an acute complication of sickle cell anaemia that occurs in children. It is infarction of the bones of the hands.
What is the most common reason for individuals with sickle cell anaemia to seek health care?
PAIN
Which drug can reduce the frequency of painful crises and acute chest syndrome in sickle cell anaemia?
Hydroxyurea (aka. Hydroxycarbamide)
How do you diagnose haemoglobinopathies?
Clinical information (age, ethnicity, gender, pregnancy status) Laboratory information (FBC parameters, blood film) HPLC (high pressure liquid chromatography= method of separating and quantifying the different types of Hb) Hb variant confirmation (e.g. electrophoresis) DNA analysis, mass spectrometry (in minority)
The sickle cell solubility test is a rudimentary test which can be done in places where they don’t have access to the other diagnostic / laboratory tests. Describe how it works.
It is a screening test for sickle cell anaemia. HbS is relatively insoluble in concentrated phosphate buffers compared to HbA and other Hb variants. HbS precipitates causing a cloudy solution.
Recap of normal haemostasis
- Collagen and tissue factor exposed
- vWF binds collagen
- Platelets adhere to vWF-collagen
- Platelets activate and aggregate (completing primary haemostasis)
- TF initiates rapid thrombin generation on activated platelets
- Thrombin converts fibrinogen to fibrin and completes platelet activation
- Stable fibrin-platelet clot is formed
What are the important regulatory proteins which help to prevent abnormal clot formation?
vWF activity is regulated by ADAMTS13
Antithrombin and activated protein C are switched on during the coagulation cascade and help to limit the amount of thrombin that is generated.
Plasmin- a fibrinolytic enzyme that degrades fibrin by proteolysing it into fibrin degradation products (d-dimer)
What is the pattern of bleeding in primary haemostasis disorders?
Petechiae/ bruising Epistaxis and oral bleeds Menorrhagia GI /CNS bleeds Timing of bleeds- immediate
What is the pattern of bleeding in coagulation factor disorders?
Bleeds into joints.
Soft tissue bleeds
GI/ CNS bleeds
Timing of bleeds- delayed
What are the first line laboratory tests for investigation of bleeding?
- Platelet count and blood film (indicates platelet number not function)
- Coagulation screen (PT and aPTT) indicate function of coagulation pathway
What do PT and aPPT stand for?
PT = prothrombin time aPTT= activated partial thromboplastin time
What abnormal coagulation factors does PT detect?
FVII (extrinsic pathway)
FII, FV, FX, Fibrinogen (common pathway)
What abnormal coagulation factors does aPPT detect?
FVIII, FIX, FXI (intrinsic factors)
FII, FV, FX, Fibrinogen (common pathway)
What coagulation factors are problematic (deficient) in both haemophilia A and haemophilia B?
Haemophilia A = (FVIII deficiency) - problem with F8 gene
Haemophilia B= (FIX deficiency) - problem with F9 gene
Out of the genetic clotting factor disorders which is most common?
Von willebrand disease (vWF deficiency)
Can you distinguish between haemophilia A and B clinically?
No, they are clinically indistinguishable
What are the clinical features of haemophilia?
Mild provoked bleeding if factor level >5%.
Severe spontaneous bleeding if factor level <1%
NB the level of factor depends on how severe the mutation of the F8 or F9 genes are, and hence whether or not there is any factor being made.
Soft tissue and joint bleeds (haemarthrosis). Life-threatening CNS or GI tract bleeds. Chronic arthropathy (degenerative arthropathy caused by bleeding into joints). Treatment acquired HCV and HIV
What is the treatment for haemophilia?
Prophylaxis of regular injections of recombinant factor concentrate
Tell me about Von willebrand disease (VWD)
Deficiency of vWF. vWF mediates platelet adhesion to collagen AND stabilises coagulation factor VIII in the plasma. Mild VWD = defective primary haemostasis only, causes mild bleeding symptoms (mucocutaneous bleeding). Severe VWD = the above plus additional coagulation pathway defect due to low FVIII (caused by two different genetic variants affecting vWF). Can lead to severe mucocutaneous and soft tissue/ joint bleeds.
How will the PT and aPTT be in someone with VWD?
Normal because vWF is not one of the numbered coagulation factors and so is not tested in PT/aPTT. However, in severe VWD the aPTT will be long (and the PT normal) because of reduced FVIII levels.
What is the treatment of VWD?
Tranexamic acid (reduces clot breakdown- anti-fibrinolytic) Desmopressin - releases endogenous FVIII and vWF (this is good if you need a short term boost e.g. for people with mild VWD to undergo minor surgery). vWF/ FVIII concentrate (used in severe VWD- its effective but expensive)
What are the vitamin K dependent clotting factors?
Factors II, VII, IX, X
Tell me about liver disease as a form of acquired bleeding problem
Liver disease affects primary haemostasis and coagulation pathways. Reduced synthesis of most clotting factors, fibrinogen and coagulation regulators. Reduced platelet number (thrombocytopenia and platelet function).
Cirrhosis -> portal hypertension -> hypersplenism -> big spleen breaks down more platelets -> reduced platelet count
Damaged liver makes less thrombopoietin -> less TPO = less drive for platelet formation
Tell me about disseminated intravascular coagulation (DIC)
Rare acquired disorder with bleeding and thrombosis. Usually complicating other severe illness (DIC doesn’t happen on its own- it is a complication of some other underlying illness). Because the activation of the coagulation pathway is so widespread the clotting factors, platelets and fibrinogen etc. Tend to get consumed used up (so on testing you would find low levels of all of these things and long clotting times) which can lead to bleeding
What is a potent cause of DIC?
Meningococcal sepsis
How do you recognise DIC?
Appropriate clinical setting (patients are usually already ill on ITU wards).
Increased PT and aPTT
Decreased fibrinogen and platelet count
High d-dimer
Often anaemia and evidence of organ dysfunction
What is the management of DIC?
Full supportive care (to support failing organs)
Correct the stimulus for DIC (e.g. antibiotics for infection, management of trauma etc).
Aggressive support to restore coagulation homeostasis: FFP (which has clotting factors and the important negative regulators of homeostasis), platelet transfusion, fibrinogen concentrate or cryoprecipitate.
Tell me about thrombotic thrombocytopenic purpura (TTP)
TTP is caused by anti-ADAMTS13 antibodies (tends to be autoimmune). Their vWF then becomes too big and ‘sticky’ and causes platelets to stick together on their own in blood vessels forming abnormal micro platelet rich thrombi which causes end-organ damage and RBCs to be smashed up as they flow though the partly occluded blood vessels (mechanical haemolysis)
What are the investigation results seen in TTP?
Reduced platelets and reduced Hb
Blood film is diagnostic- thrombocytopenia and red cell fragments
Increased LDH, PT and aPTT normal, eGFR normal or reduced, liver function tests normal or reduced, increased troponin, ECG, CT head abnormal, ADAMTS13 level reduced.
What is the treatment for TTP?
Main treatment is plasma exchange (filter off plasma and restore with FFP. This is needed to remove the anti-ADAMTS13 antibodies).
Also: supportive care, RBC transfusion, aspirin and LMWH, immunosuppression, IV corticosteroids, Rituximab
What are the clinical features of a DVT?
Unilateral pain, swelling, tenderness, redness. Dilated superficial veins (often if severe and sudden blockage of a major vein draining the leg). Venous gangrene (very rare- if tissue pressure gets so high because you have occluded venous return that the tissue pressure exceeds the perfusion pressure form the femoral artery then you get tissue ischaemia)
How does Wells score work for assessing DVTs?
Score >1 = DVT likely = Doppler ultrasound
Score of 1 or less = DVT unlikely = D-dimer
Tell me about post-thrombotic syndrome as a sequelae of DVT
Chronic swelling and aching. Venous eczema and ulcers. 25-50% of all DVT. Risk factors: elderly, recurrent DVT, above knee DVT, poor early anticoagulation control.
Tell me about factor V Leiden
Sequence change in factor V gene (F5) -> reduces inactivation of factor V by activated protein C (so its not switched off as well and you are more likely to form clots). Increases risk 4 fold (which isn’t actually that much). Detection of FV Leiden seldom influences anticoagulation decisions (people get normal Tx and it doesn’t influence e.g. pregnancy management).
Tell me about antiphospholipid syndrome
Autoimmune blood disorder (can occur in young and older people). Circulating antiphospholipid (APL) antibodies -> bind glycoprotein-phospholipid complexes on cell membranes -> some APL antibodies ‘activate’ vascular endothelial cells and platelets and are potently pro-thrombotic.
What are the laboratory sub-classifications of antiphospholipid syndrome?
- Anti-cardiolipin
- Anti- beta2 glycoprotein 1
- Lupus anticoagulant
Can have one, two or all three (triple positive), and in general the more tests the APL antibody is abnormal in, the higher the thrombotic risk.
What is important to remember about lupus anticoagulant?
May prolong clotting times (remember how in PT and aPTT phospholipids are added because they are needed for the coagulation system to work. Some APL antibodies can actually bind to the artificial phospholipid which is used in the lab when conducting the aPTT test. As a result, you could see a prolonged clotting time. This is a completely artifactual prolongation of the clotting time that does not reflect a bleeding tendency.
What is an example of a disorder which can cause clotting problems in both sides of the circulation (arterial and venous)?
Antiphospholipid syndrome
Why is there a distinction of anti-platelets and anticoagulants being used in different clotting scenarios?
Clots in arteries tend to be platelet rich, so you treat then with an anti-platelet.
Clots in low pressure scenarios / venous thrombosis tend to be fibrin rich, so you treat with anticoagulants
What is the antidote to warfarin?
Vitamin K
What is the antidote to heparin?
Protamine sulphate
What are the risks associated with blood transfusion?
Unavailable blood. Transfusion transmitted infections. Immunological reactions. Overloading: iron, fluid.
What is apheresis donation and therapy?
Regular donors, selective removal of the component required. It is essentially a centrifuge-type machine. Particularly used for platelet donation. Up to 3 adult doses of platelets per donor visit. Also used for treatment of patients to remove: white cells (leukaemia), plasma (plasma exchange), red cells (e.g. sickle cell)
What product for transfusion is a source of clotting factors?
FFP (fresh frozen plasma)
What is contained in cryoprecipitate?
Fibrinogen
What is the shelf life/ storage temperature of RBCs for transfusion?
35 day shelf life at 4 degrees
Transfuse <4 hours after removing from fridge
What is the shelf life/ storage temperature for platelets?
7 day shelf life at 22 degrees (shorter shelf life than RBCs because bacteria can grow much more easily)
Tell me about fresh frozen plasma (FFP)
Plasma frozen to -30 degrees. 2 year storage. It takes up to 30 mins to defrost so can’t be given immediately. For clotting factors replacement if bleeding (particularly used in major haemorrhage)
What is the haemoglobin threshold in a stable patient with acute anaemia? (Note: use of the term stable)
70g/L
What is the dose of RBC and FFP for transfusion in a major haemorrhage?
1 unit FFP, 2 units RBC
What is minor and major incompatibility in terms of blood transfusions?
Major incompatibility = the recipient has antibodies against transfused blood e.g. group A blood given to a group O patient.
Minor incompatibility= the transfused blood contains antibodies against recipient cells e.g. group O blood transfused to a group A patient
What are the signs/ symptoms of an acute transfusion reaction?
Fever, chills, pruritus, tachycardia, sweating, dark urine, SOB, back pain (kidneys)
What are the most common blood groups in Caucasian populations?
Group O and group A
What blood group is the universal donor for plasma transfusions? (Note the use of the term plasma)
Group AB
Tell me about RhD and haemolytic disease of the newborn
Originally called ‘rhesus factor’. Antibodies against RhD blood group (blood group forms the +/- in O+ or O-). Antibodies formed by exposure to blood from foetus. Attack red cells in foetus -> haemolysis. Now preventable by transfusing RhD negative blood to women, and anti-D immunoglobulin to prevent antibody formation.
How can you try to prevent transfusion associated graft-versus host disease?
Irradiated blood for certain immunocompromised patients
What is transfusion associated graft versus host disease (TA-GVHD)?
Where by chance, the person who has donated the blood is a pretty close tissue match to the person receiving the blood, and lymphocytes which have managed to stay in the blood product could engraft like they would if the person was having a bone marrow transplant and then attack the patient. Often this happens months after the transfusion and can prove fatal.
Which health professionals are responsible for the greatest number of WBIT (wrong blood in tube) events?
Doctors
What happens in a ‘crossmatch’
We take the cells from the donor and the serum from the recipient and mix them together and see if they react. If they don’t react, we say it is crossmatch compatible and we can send out the blood. If you need the blood more quickly and its the first transfusion they are having, you can do an electronic crossmatch (where you don’t actually do the mixing- its quicker but there is a slightly increased risk of not picking up all the antibodies)
From SHOT (serious hazards of transfusion) what was the reason for nearly 90% of near miss ‘incorrect blood component transfused’?
Errors in the sample taking process (i.e. wrong blood in tube)
What does TACO stand for?
Transfusion associated circulatory overload
Of the viral transfusion associated infections which is most common? (Although in itself not particularly common)
Hepatitis B virus (HBV)
What were most deaths related to transfusion reported in 2015 due to?
TACO
What is TRALI?
Transfusion related acute lung injury. Is defined as acute dyspnoea with hypoxia and bilateral pulmonary infiltrates during or within 6 hours of transfusion, not due to circulatory overload or other likely causes. Patients with TRALI are very ill and require care in ITU
What is TAD?
Transfusion associated dyspnoea. Characterised by respiratory distress within 24 hours of transfusion that does not meet the criteria for TRALI, TACO or allergic reaction.
How can we define a major haemorrhage?
Loss of more than one blood volume within 24 hours (>5L in a 70kg adult)
50% of total blood volume lost in less than 3 hours
Bleeding in excess of 150ml/min
BLEEDING WHICH LEADS TO A SYSTOLIC BP OF <90 OR A HR OF >110
Tell me about chronic lymphocytic leukaemia (CLL)
Mean age at diagnosis = 72 years (so tends to affect the elderly). In 70% of cases is an incidental finding. CLL is a clonal B cell lymphocytosis in the blood (>5x10^9) for more than 3 months. CLL is a chronic indolent condition.
What are the characteristics of CLL on a blood film?
The lymphoid cells are all quite mature (about the same size as the red cells) and there isn’t much cytoplasm. You see SMUDGE CELLS / SMEAR CELLS.
There may be borderline cases in diagnosing CLL where you are still wondering whether it could be due to e.g. a viral infection so how can we confirm that the cells we are seeing are clonal?
Flow cytometry. CLL is usually a cancer of the B lymphocytes but they paradoxically express CD5 which is a T cell antigen.
What staging system may be used for CLL?
Binet staging. There is also another staging system called rai staging.
What is a marker of more advanced disease in CLL?
Once you start getting anaemia and thrombocytopenia this is a sign that the bone marrow is being infiltrated more by the cells, so it is a marker of more advanced disease.
Tell me about lymphocyte doubling time and prognosis in CLL
If the lymphocyte count is doubling in <6 months, it is a strong indicator that they are going to need treatment
What is a key cytogenetic feature of some types of CLL that gives a bad prognosis
A 17p deletion of the p53 tumour suppressor gene (causes the cells to be more inherently resistance to any treatment)
What is meant by mature/ immature CLL?
B cells that have gone through the germinal centre and undergone their immunoglobulin gene rearrangement have a better prognosis because they are more mature than the ones that haven’t
What are the indications for treatment in CLL?
Progressive and bulky lymphadenopathy (>10cm)
Progressive splenomegaly
Lymphocyte doubling time <6 months
Cytopenias not due to autoimmune phenomena
What is the ‘go-go’ treatment for CLL in the younger/ fitter population? I.e. the more intensive treatment
FCR (fludarabine, cyclophosphamide and rituximab). Rituximab is an antibody against CD20. Oral regime but makes the patient very unwell (have a 20% chance of hospitilisation with sepsis).
What is the treatment option in CLL for the ‘slow-go’ patients i.e. the elderly
Use more gentle drugs such as chlorambucil and other CD20 antibodies. They have more ‘gentle’ treatment but for longer.
What are some of the associated problems with CLL?
Infection (low immunoglobulins)
Auto-immune disorders (auto-immune haemolytic anaemia and ITP)
Secondary malignant- especially basal cell carcinomas
What is Richter’s transformation?
A rare occasion when the CLL changes to a high-grade lymphoma (very poor prognosis)
Any examples of new treatments for CLL?
Ibrutinib and Idelalisib which target the tyrosine kinase cascade downstream from the B cell receptor as a means or preventing replication. Ibrutinib works in patients who have the p53 mutations which are resistant to chemotherapy. It is a tablet which is really well tolerated. There is also venetoclax which is an oral selective BCL-2 bax receptor inhibitor, you have to be careful with its use, but it is a very effective treatment.
Tell me about chronic myeloid leukaemia (CML)
CML is a cancer of the granulocytes, leading to massive accumulation of white blood cells. This leads to massive splenomegaly and a thick layer of white cells if you centrifuge the blood.
How does CML present?
CML tends to present between 40-60 years. May present as: asymptomatic, hypermetabolic state (weight loss and sweats), leukostasis, sluggish movement of the blood in the blood vessels because there are so many cells around: SOB and priapism, symptoms from splenomegaly: discomfort and early satiety (as spleen pushes on stomach).
What do you seen on the blood film in CML?
Mixture of different types of myeloid cells (huge numbers of mature granulocytes but also more immature granulocytes such as promyelocytes and myelocytes). So it is not just primitive cells (like in acute leukaemia), everything is increased (the mature and some of the immature cells).
Tell me about the Philadelphia chromosome in CML
Translocation between chromosome 9 (where there is the abl oncogene which is a tyrosine kinase) and chromosome 22 (bcr gene) creating a new chromosome called the Philadelphia chromosome. This causes the tyrosine kinase to be constitutively expressed.
What drug was developed once we discovered the Philadelphia chromosome?
Imatinib - a synthetic specific inhibitor of the BCR-ABL fusion tyrosine kinase protein.
What is meant by the terms: haematological remission, cytogenetic remission, and molecular remission? (CML)
Haematological remission- you can no longer see any cancer in the blood count or bone marrow. However this still misses about 10% of cells so there could still be some disease present.
Cytogenetic remission- you can no longer see any cancer when you do the patient’s cytogenetics (see how many genetic abnormalities there are in the blood sample). However this still misses about 1% of cells so there could still be some disease present
Molecular remission- you can no longer see any cancer when looking for the BCR-ABL translocation when you do an NMR study. This is precise and only missed around 1 in a million cells.
What are the types of myeloproliferative neoplasms that I need to know about?
Polycythaemia rubra Vera
Essential thrombocythaemia (ET)
Primary myelofibrosis
What is a key mutation involved in the myeloproliferative neoplasms?
JAK2 (the commonest being JAK2 V617F)
Tell me about polycythaemia rubra Vera
Raised red cell mass (Hct >0.52 in males and >0.48 in females).
Exclude hypoxia, excess EPO etc. Check molecular markers.
Tell me about essential thrombocythaemia
Raised platelet count (>450x10^9/L). Exclude reactive process and iron deficiency. Check molecular markers.
What are both polycythaemia rubra Vera and ET associated with?
Increased risk of thrombosis.
Transformation to either myelofibrosis (progressive scarring in the marrow) or AML (about 5% transform to AML).
What is the international prognostic score for thrombosis in ET?
Risk factors and points:
Age >60 years (1), cardiovascular risk factors (1), previous thrombosis (2), JAK2 (V617F) (2).
What is the treatment algorithm for essential thrombocythaemia?
All ET patients should be treated with aspirin if microvascular disturbances are present. If low risk ET (i..e age <60 years presenting with no high risk feature)-> watch and wait. If patient becomes high risk put them on cytoreductive therapy (first line is HYDROXYUREA and second line is ANAGRELIDE). If high risk ET (i.e. age >60 years, previous thrombotic event, platelet count >1500) = cytoreductive therapy.
What is the management of polycythaemia Vera?
Venesection to maintain the Hct to <0.45 (removing blood to reduce the blood count). Aspirin 75mg unless contraindicated. Cytoreduction should be considered if: poor tolerance to venesection, symptomatic or progressive splenomegaly, other evidence of disease progression e.g. weight loss or night sweats, thrombocytosis.
Tell me about myelofibrosis
Symptomatic anaemia. Discomfort from splenomegaly. Hypermetabolic syndrome (weight loss, sweats), rarely bleeding problems and bone pain. In myelofibrosis you get progressing scarring of the bone marrow, so the bone marrow can no longer produce haemopoietic cells, so the liver and spleen try and take over but they aren’t as effective and enlarge as they try and produce blood cells.
What is the characteristic blood film seen in myelofibrosis?
Tend to have a leukoerythroblastic blood film, where you have TEARDROP CELLS, primitive cells e.g. myelocytes and nucleated red cells. A leukoerythroblastic blood film is a sign of marrow stress or infiltration.
What is the prognostic score for myelofibrosis?
Age >65 years (1), constitutional symptoms (1), Hb<100g/L (1), leukocyte count >25 (1), circulating blasts >1% (1).
What drug may give some symptomatic relief to patients with myelofibrosis?
Ruxolitinib (a JAK2 inhibitor that isn’t completely specific for JAK2 so also knocks off some of JAK1, JAK3 and TYK2 which are important for producing the inflammatory cytokines that make people feel ill with myelofibrosis).
Briefly tell me about lymphoma
Heterogenous group of lymphoproliferative malignancies
Results from clonal expansion from B, T or NK cells
85-90% derived from B cell
Variable clinical presentation (asymptomatic -> medical emergency)
How may lymphoma present?
Enlarged painless lymph nodes. With or without: B symptoms (drenching night sweats, unexplained fever >38 degrees, weight loss >10% in last 6 months), with or without: other symptoms (fatigue, itching, symptoms related to cytopenias).
What is needed for a diagnosis of lymphoma?
TISSUE- i.e. lymph node biopsy or biopsy from involved site (colon, tonsils etc). CORE BIOPSY not FNA and occasionally total lymph node extraction. Also for diagnosis: peripheral blood flow cytometry, scan (CT/PET scan) for staging or ensuring best site for biopsy. Bone marrow biopsy in some cases.
What are some specific CD markers to know about?
T cell- CD3, CD4, CD8 B cell- CD20 Hodgkin’s lymphoma- CD45 NK cells- CD16, CD56 Lymphoblast- terminal deoxynucleotidyl transferase All lymphocytes- CD34
Tell me about Ann Arbor staging of lymphoma (uses CT-PET +/- bone marrow)
Stage 1- involvement of a single lymph node or group of nodes. stage 1A if no B symptoms, stage 1B if patient has B symptoms. (A and B work like this for the other stages too).
Stage 2- Involvement of two or more sites on the same side of the diaphragm.
Stage 3- disease on both sides of the diaphragm. May include spleen or localised extra nodal disease.
Stage 4- widespread extra-lymphatic involvement (liver, bone marrow, lung, skin).
What is RCHOP chemotherapy for lymphoma?
Rituximab (anti-CD20) Cyclophosphamide Hydroxy daunorubicin Oncovin Prednisolone Given every 3 weeks.
Which drug from RCHOP is not given for non-B cell lymphoma and why?
Rituximab because B cell lymphomas express the CD20 marker, whereas non-B cell lymphomas do not and rituximab is a monoclonal antibody against CD20.
Tell me some facts about Hodgkin lymphoma
From the germinal centre. Characterised by Reed-Sternberg cells. Bimodal distribution in the age of diagnosis (a peak in young adults and a peak in the elderly).
What is the treatment of Hodgkin lymphoma?
The gold standard Tx after diagnosis (remember they are CD45 positive) and staging (Ann Arbor CT-PET staging) is ABVD +/- radiotherapy. Adriamycin (aka. Hydroxy daunorubicin), Bleomycin, Vinblastine, Dacarbazine. All of these drugs are chemotherapy agents.
What are some of the late effects of treatment of Hodgkin lymphoma? (ABVD)
Second malignancy e.g. lung/ breast cancer.
Heart or lung problems (bleomycin can cause side effects in the lungs).
Tell me some facts about myeloma (hint: talk about MGUS)
All patients with myeloma had another medical condition first called MGUS. First mutation -> MGUS -> key mutation -> myeloma. In MGUS serum monoclonal protein <30g/L. In MGUS clonal bone marrow plasma cells <10%. In MGUS absence of end-organ damage. MGUS requires active monitoring only.
How do we diagnose multiple myeloma?
Diagnosis is often based on bone marrow biopsy. To diagnose it we have to find >10% clonal plasma cells in the bone marrow. The clonal cells can produce intact immunoglobulins (heavy and light chains) or can also produce immunoglobulin free light chains (detectable in blood stream and urine).
In diagnosing multiple myeloma what test gives us information about the QUANTITY of serum monoclonal protein?
Immunofixation
What should you do if a patient has asymptomatic (smouldering) myeloma?
Active monitoring
How might hyperviscosity syndrome present? (In relation to myeloma)
Blurred vision, headaches, mucosal bleeding, dyspnoea due to heart failure. Symptoms occur when: plasma viscosity >4. IgM level >30, IgA level >40, IgG level >60.
Tell me about spinal cord compression in relation to myeloma
5% of patients with myeloma. Sensory loss, paraesthesia, limb weakness, walking difficulty, sphincter disturbance. Mx: dexamethasone 40mg daily for 4 days. Urgent MRI whole spine.
