Haematology Flashcards
What is anaemia
Insufficient oxygen carrying
capacity is due to reduced haemoglobin concentration as seen with insufficient RBC
Haemoglobin concentration below the accepted normal range. The normal range for haemoglobin is affected by sex, age, ethnic group and altitude.
The clinical features of anaemia are largely caused by compensatory measures mobilised to counteract hypoxia. Anaemia can be classified according to red cell morphology or aetiology. Red cell indices and morphology correlate with the underlying cause of anaemia. Wherever possible the cause of anaemia should be determined before treatment is started. Blood transfusion is only required in a minority of cases.
Describe iron deficiency anaemia
Iron is a constituent of haemoglobin and is essential for erythropoiesis.
Iron deficiency is most often caused by long-term blood loss.
Iron deficiency causes a hypochromic microcytic anaemia.
The anaemia is usually easily corrected with oral iron supplements.
It is important to establish the cause of iron deficiency-it may be the presenting feature of Gastrointestinal malignancy.
What is Hb
Iron containing oxygen transport metalloprotein
Within RBCs
Reduction in haemoglobin = anaemia
(reduction in oxygen carrying capacity)
What do maturation of RBC require
Vitamin B12 & folic acid; DNA synthesis
Iron; Haemoglobin synthesis
Vitamins
Cytokines (erythropoeitin)
Healthy bone marrow environment
What can cause anaemia
Failure of Production: hypoproliferation
Reticulocytopenic
Ineffective Erythropoiesis
Decreased Survival
Blood loss, haemolysis, reticulocytosis
Microcytic
-low Hb levels
Iron deficiency (heme deficiency)
Thalassaemia (globin deficiency)
Anaemia of chronic disease
Reticulocyte count then adds further clue as to failure of production or increased losses
Normocytic anaemia
Anaemia chronic disease Aplastic anaemia Chronic renal failure Bone marrow infiltration Sickle cell disease Reticulocyte count then adds further clue as to failure of production or increased losses
Macrocytic anaemia
B12 deficiency, folate deficiency
Myelodysplasia, alcohol induced, drug induced, liver disease, myxoedema.
Reticulocyte count then adds further clue as to failure of production or increased losses
What is nutritional anaemia
Reticulocyte count then adds further clue as to failure of production or increased losses
Reticulocyte count then adds further clue as to failure of production or increased losses
Iron
Essential for O2 transport
Most abundant trace element in body.
Daily requirement for iron for erythropoeisis varies depending on gender and physiolgical needs.
Iron is an essential component of cytochromes, oxygen-binding molecules (i.e., haemoglobin and myoglobin), and many enzymes.
Dietary iron is absorbed predominantly in the duodenum.
Fe+++ ions circulate bound to plasma transferrin and accumulate within cells in the form of ferritin. Stored iron can be mobilized for reuse.
Adult men normally have 35 to 45 mg of iron per kilogram of body weight. Premenopausal women have lower iron stores as a result of their recurrent blood loss through menstruation.
More than two thirds of the body’s iron content is incorporated into haemoglobin in developing erythroid precursors and mature red cells.
Most of the remaining body iron is found in hepatocytes and reticuloendothelial macrophages, which serve as storage deposits.
Reticuloendothelial macrophages ingest senescent red cells, catabolise haemoglobin to scavenge iron, and load the iron onto transferrin for reuse.
Iron metabolism is unusual in that it is controlled by absorption rather than excretion. Iron is only lost through blood loss or loss of cells as they slough.
Men and nonmenstruating women lose about 1 mg of iron per day. Menstruating women lose from 0.6 to 2.5 percent more per day.
An average 60-kg woman might lose an extra 10 mg of iron per menstruation cycle, but the loss could be more than 42 mg per cycle depending on how heavily she menstruates.
Daily dietary requirements
Daily dietary iron requirements differ at various stages of development, between men and women, and between pregnant and nonpregnant women.
The data reported in this table assume an average dietary iron absorption of 10%.
Foods that are rich in iron include:
• Meats: Liver, Liverwurst, Beef, Lamb, Ham, Turkey, Chicken, Veal, Pork, Dried beef
• Seafood: Shrimp, Dried cod, Mackerel, Sardines, Oysters, Haddock, Clams, Scallops, Tuna
• Vegetables: Spinach, Beet greens, Dandelion greens, Sweet potatoes, Peas, Broccoli, String beans,Collards, Kale, Chard
• Breads & Cereals: White bread (enriched), Whole wheat bread, Enriched macaroni, Wheat products, Bran cereals, Corn meal, Oat cereal, Cream of Wheat, Rye bread, Enriched rice
• Fruits: Prunes, Watermelon, Dried apricots, Dried peaches, Strawberries, Prune juice, Raisins, Dates, Figs
• Other Foods: Eggs, Dried peas, Dried beans, Instant breakfast, Corn syrup, Maple Syrup, Lentils, Molasses
Iron metabolism
> 1 stable form of iron:
Ferric states (3+) and Ferrous states (2+)
Most iron is in body as circulating Hb
Hb: 4 haem groups, 4 globin chains able to bind 4 O2
Remainder as storage and transport proteins
ferritin and haemosiderin
Found in cells of liver, spleen and bone marrow
Iron absorption
Regulated by GI mucosal cells and hepcidin
Duodenum & proximal jejunum
Via ferroportin receptors on enterocytes
Transferred into plasma and binds to transferrin
Amount absorbed depends on type ingested
Heme, ferrous (red meat, > than non-heme, ferric forms Heme iron makes up 10-20% of dietary iron
Other foods, GI acidity, state of iron storage levels and bone marrow activity affect absorption
Iron Regulation: Hepcidin
“ the iron-regulatory hormone hepcidin and its receptor and iron channel ferroportin control the dietary absorption, storage, and tissue distribution of iron…
Hepcidin causes ferroportin internalization and degradation, thereby decreasing iron transfer into blood plasma from the duodenum, from macrophages involved in recycling senescent erythrocytes, and from iron-storing hepatocytes.
Hepcidin is feedback regulated by iron concentrations in plasma and the liver and by erythropoietic demand for iron.
Iron transport and storage
Iron transported from enterocytes and then either into plasma or if excess iron stored as ferritin
In plasma: attaches to transferrin
and then transported to bone marrow binds to transferrin receptors on RBC precursors
A state of iron deficiency will see reduced ferritin stores and then increased transferrin
Describe laboratory iron studies
Serum Fe-> hugely variable during the day
Ferritin->primary storage protein and providing reserve, water soluble.
Transferrin saturation->ratio of serum iron and total iron binding capacity-revealing % age of transferrin binding sites tat have been occupied by iron.
Describe transferrin
Made by liver, production inversely proportional to Fe stores. Vital for Fe transport.
Describe total iron binding capacity
Measurement of the capacity of transferrin to bind to iron. It is an indirect measurement of transferrin.
What are the laboratory results in deficiency anaemia
Ferritin-low
Saturation-low
TIBC-high
Serum iron-low/normal
Iron deficiency causes
NOT ENOUGH IN Poor Diet Malabsorption Increased physiological needs LOSING TOO MUCH Blood loss menstruation, GI tract loss, paraistes
Iron deficiency investigations
FBC: Hb, MCV, MCH, Reticulocyte count
Iron Studies: Ferritin, Transferrin Saturation
Blood film
?BMAT and Iron stores
Describe stages of IDA
Before anaemia develops, iron deficiency occurs in several stages.
Serum ferritin is the most sensitive laboratory indicators of mild iron deficiency. Stainable iron in tissue stores is equally sensitive, but is not performed in clinical practice.
The percentage saturation of transferrin with iron and free erythrocyte protoporphyrin values do not become abnormal until tissue stores are depleted of iron.
A decrease in the haemoglobin concentration occurs when iron is unavailable for haem synthesis.
MCV and MCH do not become abnormal for several months after tissue stores are depleted of iron
Iron deficiency anaemia-prevalence
World’s most common nutritional deficiency
2% in adult men (≤ 69 years old)
4% in adult men ≥ 70 years old*
10% in Caucasian, non-Hispanic women
19% in African-American women
Common cause of referral
Excessive menstrual losses 1st cause in premenopausal
women
Blood loss from the GI tract is the most common cause of IDA in adult men and postmenopausal women.
Iron deficiency is the most common nutritional deficiency as well as the most common cause of anaemia throughout the world and a common cause of referral.
Premenopausal women with excessive menstrual losses are particularly at risk of developing iron deficiency anaemia (IDA).
because of the
Iron deficiency anaemia symptoms and signs
Symptoms
fatigue, lethargy, and dizziness
Signs pallor of mucous membranes, Bounding pulse, systolic flow murmurs, Smooth tongue, koilonychias
B12 and Folate deficiency
Folate necessary for DNA Synthesis:
Adenosine, guanine and thymidine synthesis
Both have very similar laboratory finding and clinical symptoms
Can be found together or as isolated
pathologies
Macrocytic Anaemia
Low Hb and high MCV with normal MCHC
Macrocytic anaemia
Megaloblastic : Low reticulocyte count Vitamin B12/Folic acid deficiency Drug-related (interference with B12/FA metabolism) Nonmegaloblastic Alcoholism ++ Hypothyroidism Liver disease Myelodysplastic syndromes Reticulocytosis (haemolysis)
Megaloblastic vs non megaloblastic anaemia
Megaloblastic changes of blood cells are seen in B12 and Folic Acid deficiency. They are characterized on the peripheral smear by macroovalocytes and hypersegmented neutrophils.
What are the causes of folate deficiency
Increased demand:
- pregnancy/breast feeding
- infancy and growth spurts
- haemolysis and rapid cell turnover, eg Sickle Cell Disease
- Disseminated cancer
- Urinary losses (e.g heart failure)
Decreased intake:
- poor diet
- elderly
- chronic alcohol intake
Decreased absorption
- Medication (folate antagonists)
- Coeliac
- Jejunal resection
- Tropical Sprue
Folate comes from most foods with 60-90% lost in cooking. It is absorbed in the Jejunum and the body has enough stores usually for 3-5 months
What is Vitamin B12
Essential co-factor for methylation in DNA and cell metabolism
Intracellular conversion to 2 active coenzymes necessary for the homeostasis of methylmalonic acid (MMA) and homocysteine
Foods containing vit B12:
Animal sources: Fish, meat, dairy
UK intake recommendations are 1.5mcg/day
EU: 1mcg/day and USA: 2.4mcg/day
average western intake 5-30mcg/day
Body (liver) storage: 1-5mg so many years for deficiency.
Requires the presence of Intrinsic Factor for absoprtion in terminal ileum
IF made in Parietal Cells in stomach
Transcobalamin II and Transcobalamin I transport vitB12 to tissues
What are some of the causes of B12 deficiency
Impaired absorption:
- Pernicious anaemia
- Gastrectomy or ileal resection
- Zollinger-Ellison syndrome
- Parasites
Decreased intake:
- malnutrition
- Vegan diet
Congenital causes:
- intrinsic factor receptor deficiency
- Cobalamin mutation C-G-1 gene
Increased requirements
- Haemolysis
- HIV
- Pregnancy
- Growth spurts
Medication
- Alcohol
- NO
- PPI, H2 antagonists
- Metformin
Haematological consequences of B12 deficiency
MCV: normal or raised –>megaloblastic anaemia, ineffective erythropoiesis
Hb: Normal or low
Reticulocyte count: low
LDH: raised –>intramedullary haemolysis
Blood film: Macrocytes, ovalocytes, hypersegmented neutrophils
BMAT: Hypercellular, megaloblastic, giant metamyelocytes –>unusual to need.
MMA: increased, not standard lab test.
Clinical consequences of B12 deficiency
Brain: cognition, depression, psychosis Neurology: myelopathy, sensory changes, ataxia, spasticity (SACDC) Infertility Cardiac cardiomyopathy Tongue: glossitis, taste impairment Blood: Pancytopenia
What is pernicious anaemia
Autoimmune disorder
Lack of IF
Lack of
B12 absorption
Gastric Parietal cell antibodies
IF antibodies
What are the treatments for iron, folic acid, and B12 deficiency
Treat the underlying cause **
Iron – diet, oral, parenteral iron supplementatin, stopping the bleeding
Folic Acid – oral supplements
B12 – oral vs intramuscular treatment
What is included in a FBC
RED BLOOD CELL RESULTS Hb: concentration of Haemoglobin Hct: Percentage of blood volume as RBC MCV: Average size of RBC MCH: Average haemoglobin content of RBC RDW: Range of deviation around RBC size Reticulocyte count Blood film WHITE BLOOD CELL RESULTS Total WBC and differential Neutrophils, lymphocytes, monocytes, basophils, eosinophils
PLATELET RESULTS
Platelet count and size
OTHERS: WARNING FLAGS
What does a blood film show?
WHITE BLOOD CELL RESULTS
Total WBC and differential
Neutrophils, lymphocytes, monocytes,
basophils, eosinophils
PLATELET RESULTS
Platelet count and size
OTHERS: WARNING FLAGS
Red cells: -size/anisocytosis (big or small) -colour (Hb content) -shape (round, TDP, irregular, elliptocytes-poikylocytosis -Polychromasia -Inclusions White cells: -Numbers (too many/few) -Normal morphology -immature cells (myelocytes, precursors) -Abnormal cells (blasts, atypical lymphoid cells) Inclusions
What are the different colour coded tubes in laboratories and what are they used for?
Red lid=for serum= plain tube, no anticoagulant, so clot forms. Used generally.
Yellow lid=for serum=contains serum separator gel, known as Serum separator tube (SST)
Purple lid=for plasma=contains ethylene diamine tetra acetic acid (EDTA) anticoagulant, for whole blood analysis, red cell analysis and lipids and lipoproteins.
Green lid: lithium heparin anticoagulant, for plasma, general use.
Grey lid: for plasma, contains fluoride oxalate, for glucose, lactate and alcohol.
Blue lid: for serum, contains trace element, for copper and zinc
Heparinised syringe: for arterial blood sampling.
Symptoms of anaemia
Fatigue, weakness, dyspnoea, palpitations, headache, dizziness, tinnitus, and chest pains (due to exacerbation of angina)
Symptoms of leucopenia (particularly neutropenia) //low white cell count
Unusually severe or recurrent infections. It is usually a reduction in neutrophils (neutropenia) which will cause clinical problems. Serious blood diseases such as acute leukaemia can present as life threatening infections or as apparently trivial infections (eg sore throat) which are unusually refractory to normal treatment. Perineal sepsis can be a problem.
Symptoms of thrombocytopenia (low platelet count)
Thrombocytopenia leads to haemorrhagic tendency and common presentations include epistaxes (nose bleeds), bleeding from gums, menorrhagia, and excessive bleeding after trauma or surgery. Patients may also complain of easy bruising or petechial rash.
Symptoms attributable to abnormal coagulation
Patients with a defect in the coagulation cascade (low factor VIII in haemophilia A) bleed easily after surgery and trauma, but the pattern of spontaneous haemorrhage is normally different to that seen in platelet disoders. Excessive bleeding after trauma, spontaneous bleeds into joints and muscles.
symptoms of infiltration by malignancy
Malignant disorders of the blood such as leukaemia, and lumphomas, have the capacity to invade tissues. Lumps, caused by lymphadenopathy, pain, neurological symptoms.
Difference between anaemia and haemolytic anaemia
ANAEMIA = reduced haemoglobin level for the age and gender of the individual
HAEMOLYTIC ANAEMIA = anaemia due to shortened RBC survival
What is haemolysis
Shortened red cell survival 30 - 80 days
–>
Bone marrow compensates with increased red blood cell production
–>
Increased young cells in circulation = Reticulocytosis +/- nucleated RBC
–>
Compensated haemolysis: RBC production able to compensate for decreased RBC life span = normal Hb
Incompletely compensated haemolysis: RBC production
What are the clinical findings of haemolysis
Jaundice
Pallor/fatigue
Splenomegaly
Dark urine
Haemolytic crises-increased anaemia and jaundice with infections/ precipitants
Aplastic crises-anaemia, reticulocytopenia with parvovirus infection
Chronic clinical findings include:
Gallstones - pigment
Leg ulcers (NO scavenging)
Folate deficiency (increased use)
What are the haemolytic anaemia laboratory finings
Increased reticulocyte count Increased unconjugated bilirubin Increased LDH (lactate dehydrogenase) Low serum haptoglobin protein that binds free haemoglobin Increased urobilinogen Increased urinary haemosiderin Abnormal blood film
How do we classify haemolytic anaemias
- Inherited Hereditary spherocytosis
- Acquired Paroxysmal nocturnal haemoglobinuria
Site of RBC destruction - Intravascular Thrombotic thrombocytopenic purpura
- Extravascular Autoimmune haemolysis
Origin of RBC damage - Intrinsic G6PD deficiency
- Extrinsic Delayed haemolytic transfusion reaction
What is hereditary spherocytosis
Common hereditary haemolytic anemia
Inherited in autosomal dominant fashion (75%)
Defects in proteins involved in vertical interactions between the membrane skeleton and the lipid bilayer
Decreased membrane deformability
Bone marrow makes biconcave RBC, but as membrane is lost, the RBC become spherical.
Defects in vertical interaction Spectrin Band 3 Protein 4.2 Ankyrin
What is hereditary elliptocytosis
Defects in horizontal interaction
Protein 4.1
Glycophorin C
(Spectrin – HPP)
Clinical features of hereditary spherocytosis
Asymptomatic to severe haemolysis
Neonatal jaundice
Jaundice, splenomegaly, pigment gallstones
Reduced eosin-5-maleimide (EMA) binding – binds to band 3
Positive family history
Negative direct antibody test
What happens if you have glucose 6 phosphate deficiency
Role of the HMP shunt:
Generates reduced glutathione
Protects the cell from oxidative stress
Effects of oxidative stress:
Oxidation of Hb by oxidant radicals
Resulting denatured Hb aggregates & forms Heinz bodies – bind to membrane
Oxidised membrane proteins – reduced RBC deformability.
Hereditary, X-linked disorder
Common in African, Asian, Mediterranean and Middle Eastern populations
Mild in African (type A), more severe in Mediterraneans (type B)
Clinical features range from asymptomatic to acute episodes to chronic haemolysis
Features: Haemolysis Film: Bite cells Blister cells & ghost cells Heinz bodies (methylene blue) Reduced G6PD activity on enzyme assay May be falsely normal if reticulocytosis
Describe pyruvate kinase deficiency
PK required to generate ATP Essential for membrane cation pumps (deformability) Autosomal recessive Chronic anaemia Mild to transfusion dependent Improves with splenectomy
What is thalassaemias
-describe also the diagnosis of thalassaemia traits
Imbalanced alpha and beta chain production
Excess unpaired globin chains are unstable
precipitate and damage RBC and their precursors
Ineffective erythropoiesis in bone marrow
Haemolytic anaemia
Diagnosis of thalassaemia trait:
Asymptomatic
Microcytic hypochromic anaemia
Low Hb, MCV, MCH
Increased RBC
Often confused with Fe deficiency
HbA2 increased in b-thal trait –(diagnostic)
a-thal trait often by exclusion
globin chain synthesis (rarely done now)
DNA studies (expensive)
What is beta thalassaemia major
Transfusion dependent in 1st year of life
If not transfused:
Failure to thrive
Progressive hepatosplenomegaly
Bone marrow expansion – skeletal abnormalities
Death in 1st 5 years of life from anaemia
Side effects of transfusion:
Iron overload
Endocrinopathies
Heart failure
Liver cirrhosis
Features of SCD
Clinical: Painful crises Aplastic crises Infections Acute sickling: Chest syndrome Splenic sequestration Stroke Chronic sickling effects: Renal failure Avascular necrosis bone
Laboratory: Anaemia Hb often 65-85 Reticulocytosis Increased NRBC Raised bilirubin Low creatinine
Describe immune haemolysis
Autoimmune: Idiopathic Usually warm IgG, IgM Drug-mediated Cancer associated LPDs
Alloimmune: Transplacental transfer: Haemolytic disease of the newborn: D, c, L ABO incompatability Transfusion related Acute haemolytic transfusion reaction ABO Delayed haemolytic transfusion reaction E.g Rh groups, Duffy
Describe non-immune acquired haemolysis
Paroxysmal nocturnal haemoglobinuria Fragmentation haemolysis: Mechanical Microangiopathic haemolysis Disseminated intravascular coagulation Thrombotic thrombocytopenic purpura Other: Severe burns Some infections: e.g. malaria
What is hereditary spherocytosis and hereditary elliptocytosis an example of?
Inherited red cell membrane disorder of haemolytic anaemias. They are caused by a deficiency in red cell membrane.
What are glucose-6-phosphate dehydrogenase and pyruvate kinase?
They are key enxymes in red cell metabolism, whose inherited deficiency leads to haemolysis.
What is haemostasis
Protective process evolved in order to maintain a stable physiology. Normal haemostasis is in a state of equilibrium between fibrinolytic factors, anticoagulant proteins and coagulation factors and platelets.
What is thrombosis
Thrombosis is a decrease in fibrinolytic factors, anticoagulant properties, and an increase in coagulation factors and platelets. This can lead to chronic venous insuffiency:
-atrophic changes, hyperpigmentation and ulceration and infection.
What is the term for easy bruising?
Ecchymosis: virtually all bleeding disorders and often in normals.
How do we get hemostatic plug formation?
Primary aggregation and secondary coagulation, which lead to a hemostatic clot.
There will be vessel injury, leading to local vasoconstriction. Then there will be platelet adhesion and platelet aggregation, but also activation of coagulation cascade and fibrin formation. This all then leads to primary haemostatic plug formation, which then leads to fibrinolytic activity and repair of vessel damage.
What are the 3 phases of the haemostatic system
Primary haemostasis: -Vasoconstriction (immediate) -Platelet adhesion (within seconds) -Platelet aggregation and contraction (within minutes) Secondary haemostasis -Activation of coagulation factors (within seconds) -Formation of fibrin (within minutes) -Lysis of the plug (within hours)
Describe the vessel wall events in primary and secondary haemostasis
- Damage to the blood vessel wall, exposure of collage.
- Platelet aggregation, secretion of vWF, fibrinogen etc.
- Exposure of tissue factor
- Activation of coagulation cascade, thrombin generation
- Formation of haemostatic plug:-platelets, fibrin, leucocytes.
Describe platelets
Adhesion: VWF binds to extracellular collagen and GpIb-IX-V comples.
ACTIVATION: GpIIb-III undergoes conformational change
-various agonists bind to specific surface receptors.
Structural platelet shape changes and RELEASE reaction.
Exposure negatively charged phospholipids and provide pro-coagulant surface.
Describe the coagulation pathway
Initiation is the activation or exposure of TF/Factor VIIa
TF/VIIa then activate factor 9
Factor Factor 9 then turns into factor 9a by
What are the pre-analytical errors
Problems with blue top tube: Partial fill tubes, vacuum leak and citrate evaporation.
Problems with phlebotomy:
-Heparin contamination, wrong label, slow fill, underfill, vigorous shaking, difficult venepuncture.
Biological effects:
-Hct greater than or equal to 55 or less than or equal to 15.
Lipaemia, hyperbilirubinaemia, haemolysis.
Laboratory errors:
-delay in testing, prolonged incubation at 37
-freeze/thaw deterioration.
What factor causes blood coagulation cascade to occur?
Tissue factor
What causes platelet adhesion to occur?
Collagen exposure
What factor causes vasoconstriction at the site of vessel injury?
Serotonin, thromboxane A2, ADP
What is a natural inhibitor of platelet function?
Nitric oxide
- released from endothelial cells
- inhibits platelet activation and promotes vasodilation.
Describe the coagulation cascade
Blood coagulation in vivo, involves a biological amplification system, in which relatively few initiation substances sequentially activate by proteolysis a cascade of circulation precursor proteins (factors).
Initiation:
In initiation, the coagulation is initiated after vascular injury by the interaction of the membrane bound tissue factor (exposed and activated by vascular injury) with plasmin factor VII.
The tissue factor-VIIa complex then activates factor IX and factor X.
Meanwhile factor Xa is forming small amounts of thrombin from prothrombin.
Intrinsic pathway
Factor XII is activated by exposed collagen and other negatively charged components of the endothelium.
Activation of factor XII leads to the sequential activation of factors XI, IX, VIII (as cofactor), X and prothrombin.
Factor VIII and factor V are converted to factor VIIIa and Va by the small amounts of thrombin that were generated during initiation.
In the extrinsic pathway tissue factor complexes with factor VII with sequential activation of factors VII, X and prothrombin.
Both intrinsic and extrinsic pathway then terminate in the final common pathway, where activated factor X, in addition with cofactor factor Va, convert prothrombin into thrombin.
Thrombin in turn, then converts fibrinogen to fibrin to form fibrin monomers. The fibrin monomers then combine to form the fibrin polymer. Factor XIII crosslinks the fibrin polymer to consolidate the thrombus.
Prothrombin time
measure of the function of the extrinsic pathway
Activated partial thromboplastin time
APTT, monitors the intrinsic pathway.
Prolongation is caused by deficiency or inhibition of one or more of the following coagulation factors: XII, XI, IX, VIII, X, V, II, fibrinogen. Most common cause of coagulation disorder is haemophilia, christmas disease.
Simply, describe the classic coagulation cascade
Intrinsic pathway:
-Factor XII -> Factor XIIa
Factor XiIa activates factor XI into factor XIa
This then activates factor IX into IXA.
Thrombin time test
sensitive to deficiency of fibronogen or inhibition of thrombin.
Diluted bovine is added to citrated plasma at a concentration giving a clotting time of 14-16 seconds with normal subjects.
Prolongation suggests Deficiency or abnormality of fibrinogen or inhibition of thrombin by heparin or FDPs.
Most common cause of coagulation disorder is DIC, or Heparin therapy.
Prothrombin time (PT)
Measures factos VII, X,V, prothrombin and fibrinogen. Tissue thromboplastin or synthetic tissue factorwith lipids and calcium is added to citrated plasma. Normal time for clotting is 14-16s.
Prolongation due to Deficiency or inhibition of one or more of the following coagulation factors: VII, X, V,II, fibrinogen.
The most common cause is Liver disease, warfarin therapy and DIC.
How to differentiate between inhibitor or deficiency?
Prolonged clotting times in the PT and APTT because of factor deficiency are corrected by the addition of normal plasma to the test plasma (50:50 mix). If there is no correction, or incomplete correction with normal plasma, the presence of an inhibitor of coagulation is suspected.
Neutrophilia/neutrophil leucocytosis
Increase in neutrophils in blood
Sign of infection and tissue injury
Causes of neutrophil leucocytosis
Physiological (pregnancy)
Bacterial infections inflammatory diseases (vasculitis, inflammatory bowel disease)
Trauma/surgery
Malignancy
Acute haemorrhage
Severe metabolic disorders (diabetic ketoacidosis)
Myeloproliferative diseases ( chronic myeloid leukaemia)
Iatrogenic (treatment with growth factors, corticosteroids)
Neutropenia
Reduced neutrophils in blood
Seen in a wide range of inherited and acquired disorders
What are the common causes of lymphocytosis
Infections:
-Acute anti viral infections (eg pertussis, infectious mononucleosis, rubella)
-Chronic infections (eg tuberculosis, toxoplasmosis)
Malignancy:
-Chronic lymphocytic leukaemia and variants -common
-Non-Hodgkin’s lymphoma (minority)
-Acute lymphoblastic leukaemia
B-lymphocytes
Respond to an appropriate antigen by transforming into plasma cells and secreting specific antibody (humoral immunity)
T-lymphocytes
Cooperate with antigen-presenting cells in the recognition of antigen; recognition triggers a clonal proliferation of activated T cells (cell mediated immunity).
what are the factors predisposing to leukaemia?
Radiation exposure
Previous chemotherapy (particularly analytic agents)
Occupational chemical exposure (eg benzene)
Some genetically determined disorders (Down syndrome)
Viral infection
Myelodysplastic and myeloproliferative disorders
Other possibilities (cigarette smoking)
Acute myeloid leukaemia
AML arises out of the malignant transformation of a myeloid precursor.
Symptoms mainly result from anaemia, neutropenia and thrombocytopenia.
Prognosis largely depends on age, initial response to treatment and genetic abnormalities.
Acute lymphoblastic leukaemia
Clonal malignancy of lymphoid precursor cells.
There is a peak incidence in childhood and a gradual rise in later years.
Accumulation of lymphoblasts in the bone marrow often leads to anaemia, infection and haemorrhage. CNS involvement is more common than in acute myeloid leukaemia.
The majority of children are curable with standard chemotherapy regimens and CNS prophylaxis.
In adults, cure by chemotherapy alone is much less frequent. Autologous or allogenic stem cell transplantation may be considered in ‘high’ risk cases.
Chronic myeloid leukaemia
CML is a clonal myeloproliferative disorder arising from an acquired genetic change in a pluripotent stem cell.
The hallmark of CML cells is the philaldeplphia chromosome, and the resultant chimeric BCR-ABL gene.
There is gross overproduction of neutrophils and their precursors.
cml HAS AN INDOLENT CHRONIC PHASE FOLLOWED BY A PERIOD OF acceleration and a final, generally fatal, acute leukaemic phase.
Tyrosine kinase inhibitors (imatinib) have much improved the prognosis of CP-CML.
Allogenic stem cell transplantation is the only well proven curative treatment but is associated with significant mortality.
Chronic lymphocytic leukaemia
CLL is the most commonest form of leukaemia in the western world. It is a disease of the elderly.
There is a clonal proliferation of B-lymphocytes.
Symptoms/signs include anaemia, recurrent infections, weight loss, lymphadenopathy and hepatosplenomegaly.
The clinical course is often indolent but it can be more aggressive in advanced stages. Chemotherapy is often not immediately needed in early cll.
The combination of fludarabine, cyclophosphamide, and rituximab (FCR) is the initial treatment of choice in most cases.
Define acute leukaemia
Defined as the presence of >20% of blast cells in the bone marrow at clinical presentation. Need to differentiate between myeloid or lymphoid lineage.
What chromosome is the philadelphia chromosome
chromosome 22
What are lymphomas
Group of diseases caused by malignant lymphocytes that accumulate in lymph nodes and other lymphoid tissue and cause the characteristic clinical feature of lymphadenopathy.
Hodgkin’s lymphoma
The term hodgkins lymphoma describes a group of lymphomas distinct drom the non Hodgkins lymphoma.
The presumed malignant cells, Reed Sternberg and mononuclear Hodgkin’s cells, compose a minority of tumour cells.
Common clinical presentations are palpable lymphadenopathy and constitutional symptoms.
Prognosis is largely determined by the stage of the disease.
Chemotherapy leads to high cure rates even in advanced disease. The late side effects of such treatment (eg secondary malignancy) are significant.
Non-Hodgkin’s lymphoma
The term NHL encompasses solid tumors of lymphoid tissue which are not Hodgkin’s lymphoma.
Histological classification is complex. There is great clinical heterogeneity with indolent and aggressive types of disease.
Indolent (eg follicular) NHL often initially responds well to chemotherapy but cure is elusive.
Aggressive (e.g diffuse large B-cell) NHL may be cured with conventional chemotherapy combined with rituximab; autologous stem cell transplants are increasingly used for ‘high risk’ and relapsed disease.
Myeloma
A malignant proliferation of plasma cells.
Diagnostic features include paraproteins in the serum and/or urine, osteolytic bone lesions and infiltration of the bone marrow by malignant plasma cells.
Bone pain is the most common presenting symptom.
Complications include renal failure, hypercalcaemia and amyloidosis.
Commonly used agents include thalidomide, lenalidomide and bortezomib. Autologous stem cell transplantation is performed in younger fitter patients.
Good palliative care, especially pain relief is crucial.
TDP
tear drop RBC, seen in patients with myelofibrosis or hereditary elliptocytosis
elliptocytes
epithelial shaped RBC , seen in various anaemias
poikylocytosis
variation in shape of RBC.
G6PD features and oxidative precipitants
Oxidative precipitants (what can cause oxidative stress) Infections Fava/ broad beans Many drugs e.g.: Dapsone Nitrofurantoin Ciprofloxacin Primaquine
Features Haemolysis Film: Bite cells Blister cells & ghost cells Heinz bodies (methylene blue) Reduced G6PD activity on enzyme assay May be falsely normal if reticulocytosis
-As if given blood transfusion, then measure enxyme activity, you are measuring enzyme activity in transfused cells, not patient cells. If good haemolysis, then there will be high reticulocyte count. Reticulocytes have an increase in number of enzymes as they are the younger RBC. Therefore will show high level of G6PD enzyme. If you measure in months time, then G6PD is normal level. Therefore can be seen as falsely normal if reticulocytosis (high reticulocytes).
How do we confirm the diagnosis of sickle cell anaemia
Solubility test
Expose blood to reducing agent
Hb S precipitated
Positive in trait and disease
Electrophoresis- structure
hplc -BUT IS NOT DEFINITIVE, need sickle solubility test.
Acquired anaemias
-Immune haemolysis vs non immune haemolysis
Immune haemolysis
Alloimmune vs autoimmune.
auto immune -children and aduluts -transient get then go away Idiopathic Usually warm Makes an antibody that reacts with your selves, IgG, IgM Drug-mediated (antibiotics) Cancer associated LPDs
Alloimmune Transplacental transfer: -Haemolytic disease of the newborn: D, c, L --ABO incompatability Transfusion related -Acute haemolytic transfusion reaction -ABO -Delayed haemolytic transfusion reaction E.g Rh groups, Duffy
Non-immune acquired haemlysis
Non immune acquired haemolysis
Paroxysmal nocturnal haemoglobinuria -Bone marrow failure Fragmentation haemolysis: Mechanical Microangiopathic haemolysis Disseminated intravascular coagulation Thrombotic thrombocytopenic purpura Other: Severe burns Some infections: e.g. malaria
SCD complications-chronic and acute
Acute: -stroke: ischaemic and haemorrhagic -cholecystitis -hepatic sequestration -Dactylitis -Bone pain and infarcts -Osteomyelitis -Retinal detachment -Vitreous haemorrhage -Chest syndrome -Splenic sequestration -Haematuria: papillary necrosis -Priapism -Aplastic crisis -Leg ulcers Chronic complications: -silent infarcts -Pulmonary hypertension -Chronic lung disease, bronchiectasis -Erectile dysfunction -Azoospermia -Chronic pain syndromes -Delayed puberty -Moya-moya -Retinopathy, visual loss -Chronic renal failure -Avascular necrosis -leg ulcers
Describe the diagnosis of thalassaemia trait
Asymptomatic Microcytic hypochromic anaemia Low Hb, MCV, MCH Increased RBC Often confused with Fe deficiency HbA2 increased in β-thal trait –(diagnostic) a-thal trait often by exclusion globin chain synthesis (rarely done now) DNA studies (expensive)
What are thalassaemias
Imbalanced alpha and beta chain production
Excess unpaired globin chains are unstable
precipitate and damage RBC and their precursors
Ineffective erythropoiesis in bone marrow
Haemolytic anaemia
What can go wrong? Quantitative - thalassaemias:
Production increased/ decreased amount of a globin chain (structurally normal)
Qualitative – variant haemoglobins:
Production of a structurally abnormal globin chain
HbS, HB Koln (heinz body formation), HbC (decrease solubility and crystallisation).
Why is it wrong to believe that coagulation is of intrinsic, extrinsic, and then common pathway
classical concept of distinct intrinsic, extrinsic then common pathway is NOT physiologically correct
BUT useful to interpret routine coagulation screen
FAILS to explain
FXII deficient patients do not bleed
FXI deficiency only usually a mild disorder
FVIII/IX deficiency causes severe bleeding disorder but should be bypassed by FVII activity
thrombin generation has a lag phase then rapid production
Describe the coagulation pathway
Initiation of coagulation occurs when sub-endothelial tissue is exposed to the circulation at a site of injury. These tissues express tissue factor at their surface, which binds to endogenous activated FVII
This complex binds small amounts of FX and FV to the exposed endothelial surface, which produce small quantities of thrombin
The thrombin activates platelets that are attracted to the site by the process, as well as other plasma-borne clotting factors
The activated factors (among them FVIII and FIX) enable the binding of activated FX and FV to the surface of platelets whose activation has produce conformational changes in their surface membranes to expose the ‘reaction sites’ necessary for continuation of the process
This leads to the ‘thrombin burst’ that is necessary for the large-scale production of fibrin and so the development of an effective clot
These three stages are called the initiation, amplification and propagation phases of coagulation
Examples of anticoagulants
TFPI inhibits factor 7a
Antithrombin inhibits factor 10 a and factor 2a (thrombin)
Activated protein C inhibits factor 8a from activating factor 10.
PT (prothrombin Time)
Sensitive to extrinsic pathway and to a lesser extent common pathway
TF driven
Need:
1. Thromboplastin
Tissue factor
Phospholipid
2. Calcium
APTT
Sensitive to intrinsic pathway and to a lesser extent common pathway
Contact activated
Need:
1. Surface activating agent
(e.g. kaolin)
2. Phospholipid
3. Calcium
Deficiency test
32s/28s
normal APTT = 24-34s
If time after mixing test with control still elongated, then it s an antibody in excess that is inhibiting coagulation in intrinsic pathway occurring.
TT
Sensitive to defects in conversion of fibrinogen to fibrin
Need:
Thrombin
Limitations of clotting time
CT is at the very beginning, initiation stage.
Clot strength is great.
mixing studies
Mix patient and normal plasma in equal volumes (50:50 mix)
Repeat abnormal coagulation test
Test normalises – factor deficiencies
Test remains abnormal – inhibitor (usually antibody)
D-dimer testing
D-dimer Testing
A measure of the D-dimer, a fibrin degradation product
found elevated in the situation of enhanced fibrinolysis (Thrombosis, DIC)
Not specific for thrombosis also elevated as an acute phase reactant
A Negative result is useful if clinical suspicion of VTE is low
describe the what blood types people of different blood groups can receive
If you have type A blood, you can only receive types A and O blood.
If you have type B blood, you can only receive types B and O blood.
If you have type AB blood, you can receive types A, B, AB, and O blood.
If you have type O blood, you can only receive type O blood.
If you are Rh+, you can receive Rh+ or Rh- blood.
If you are Rh-, you can only receive Rh- blood.
Type O blood can be given to anyone with any blood type. That is why people with type O blood are called universal blood donors.
What are the types of leucocytes we find in our blood
White blood cells (leucocytes) protect the body against infections, either by preventing or fighting invading organisms. There are two main types of leucocytes: the blood phagocytes provide the first line of defence by ingesting and killing microorganisms, whilst lymphocytes are responsible for antibody production and cell-mediated immunity.
Describe numbers of a normal full blood count
Haemoglobin Normal Male 130 - 180 g/L
(Hb) Normal Female 120 – 160 g/L
White Cell (Blood) Count (WBC) Normal Adult 4.0 - 11.0 x 109 / L
Platelet Count Normal Adult 150 - 400 x 109 / L (Plt)
Describe the numbers of a white blood cell count
Normal Range x 109 / L
Adult
Neutrophils 3.5 x 109 / L 2.0 - 7.5
Lymphocytes 1.7 x 109 / L 1.5 - 4.0
Monocytes 0.3 x 109 / L 0.2 - 0.8
Eosinophils 0.2 x 109 / L 0.04 - 0.4
Basophils 0.0 x 109 / L <0.01 - 0.1
Basophils
If raised, the most common cause is a haematological malignancy of some sort. Not involved in infection.
Eosinophils
Involved in parasytic infection.
If eosinophilia, do a stool sample in case of infecction by parasites. §
Monocytes
Involved in infections like TB (tuberculosis), and also in raised haematological malignancies.
Neutrophils
Involved mainly for bacterial infections.
Lymphocytes
Mainly for viral infections .
Components of a Romanowsky stain
Azure B or methylene Blue -> basic dye
Eosin Y->acidic dye
3 types of lymphocytes and what they are involved in
T cells -cel mediated immunity B cells, in humoral immunity eg antibody production.
NK cells are part of the innate immune system.
What are the terminology related to neutrophilias
Toxic Granulation
Shift to the LEFT
Shift to the RIGHT
Döhle Bodies
Difference between leukaemia and leukaemioid infection
A leukemoid reaction is an increase in the white blood cell count, which can mimic leukemia. The reaction is actually due to an infection or another disease and is not a sign of cancer. Blood counts often return to normal when the underlying condition is treated
Acute leukaemia
Will produce blasts
cml, will produce more mature cells.
2 features of haematopoietic stem cells
Pluripotent and self maintaining
Incidence of leukaemia
higher incidence rate in older people
higher in men than in woman.
symptoms of leukaemia
varies between diff leukaemias
3 typical symptoms:
aCUTE LEUKAEMIA diagnosis
Peripheral blood blasts test (PB): to check for presence of blasts and cytopenia. >30% blasts are suspected of acute leukaemia.
Bone marrow test/biopsy (BM): taken from pelvic bone and results compared with PB.
Lumbar puncture: to determine if the leukemia has spread to the cerebral spinal fluid (CSF).
describe the molecular and pathophysiological characterisation of acute laukaemia
Many chromosome translocations
Involve genes for transcription factors
Which control cell differentiation
Chromosome abnormalities also help determine prognosis and response to treatment
What are the therapies for CLL Leukaemias
Chemotherapy
Targeted therapy: tyrosine kinase inhibitors, immunotherapy-monoconal antobodies
Stem cells and bone marrow transplants
Presentation of leukaemias
Varies between types of leukaemia Typically first presents with symptoms due to loss of normal blood cell production -Abnormal bruising-commonest -Repeating abnormal infection -Sometimes anaemia
diagnosis of acute leukaemia and symptoms
DIAGNOSIS
Peripheral blood blasts test (PB): to check for presence of blasts and cytopenia. >30% blasts are suspected of acute leukaemia.
Bone marrow test/biopsy (BM): taken from pelvic bone and results compared with PB.
Lumbar puncture: to determine if the leukemia has spread to the cerebral spinal fluid (CSF).
SYMTPOMS
Typical symptoms due to bone marrow suppression:
Thrombocytopenia: purpura (bruising), epistaxis (nosebleed), bleeding from gums.
Neutropenia: Recurrent infections, fever.
Anaemia: lassitude, weakness, tiredness, shortness of breath.
DESCRIBE the molecular and pathophysiological characterisation of acute leukaemi
Molecular and pathophysiological characterisation
Many chromosome translocations
Involve genes for transcription factors
Which control cell differentiation
Chromosome abnormalities also help determine prognosis and response to treatment
IN Chronic Leukaemia , what does the BCR ABL oncogene cause
Unregulated BCR-ABL= tyrosine kinase activity that causes:
Proliferation of progenitor cells in the absence of growth factors
Decreased apoptosis
Decreased adhesion to bone marrow stroma
Myeloma symtpoms and clinical features
Three aspects of myeloma give rise to different clinical features
Suppression of normal bone marrow, blood cell and immune cell function
Bone resorption and release of calcium
Pathological effects of the paraprotein –(single monoclonal Ig in the serum- high levels – malignancy))
Rouleaux
blood cells stacked on top of each other
Diagnosis of myeloma
Diagnosis
Serum electrophoresis for paraprotein
Urine electrophoresis
Bence-Jones protein represents free monoclonal light chains
Increased plasma cells in bone marrow
ESR (very high due to rouleaux formation)
Radiological investigation of skeleton for lytic lesions
Treatments for myeloma
Treatment Chemotherapy (not curative) Cytotoxic drugs Glucocorticoids Thalidomide analogues Bortezomib
Allogeneic bone marrow transplant
Only available for a small number of younger patients (<45yo, not too ill)
Need to find an HLA (MHC) matched donor
But potentially curative
Diagnosis of CLL
Presentation Most often as incidental finding on fbc Persistent infection(s) due to immunosuppression Low IgG, suppression of normal B cells Lymph node enlargement Symptoms of bone marrow suppression
Epidemiology
85% of cases >50yo
Diagnosis FBC: Lymphocytosis Immunophenotyping Cell surface markers Light chain restriction Cytogenetics
EBV driven lymphoma
EBV or Human Herpes Virus 4 (HHV4) directly transforms B-lymphocytes in culture
Due to viral oncogene LMP-1
Over half of all normal individuals carry latent EBV infection
Do not develop lymphomas due to effective immune surveillance by cytotoxic T-cells
In highly immunosuppressed individuals
the endogenous latent EBV may transform B-cells
No longer eliminated by cytotoxic T-cells
Develop high grade lymphoma
Transplant patients on cyclosporine
Lymphoma usually regresses on withdrawal of immunosuppression
But patient may lose the graft
AIDS patients
Lymphoma may regress on successful HAART
Diff between leukaemia, lymohoma and myeloma
Leukemia, lymphoma, and multiple myeloma are all cancers of the blood-forming
organs, or hematopoietic neoplasms. They arise due to errors in the genetic information
of an immature blood cell. As a consequence of these errors, the cell’s development is
arrested so that it does not mature further, but is instead replicated over and over again,
resulting in a proliferation of abnormal blood cells.
Leukemia can arise in either of two main groups of white blood cell types -lymphocytes
or myelocytes. Either type of leukemia can be acute, a rapidly progressing form of the
disease in which the affected cells are very immature and unable to serve their proper
purpose, or chronic, which progresses more slowly and is distinguished by cells that are
relatively well differentiated but still function poorly. Lymphoma involves lymphocytes
and can also be subclassified
Agglutination importance
Agglutination is the clumping together of red cells into visible agglutinates by antigen-antibody reactions
Agglutination results from antibody cross-linking with the antigens
As the antigen-antibody reaction is specific, agglutination can identify:-
The presence of a red cell antigen
i.e. blood grouping
The presence of an antibody in the plasma
i.e. antibody screening/identification
