MM Year 2 MCD Part 2

1. MM Year 2 MCD Part 2 Maheen Ahsan sma114@ic.ac.uk

2. Haem 9-11: • Haemostasis • Abnormalities of haemostasis • Abnormal white cell count • Diagnostics: • Chemical Pathology • Virology • Bacteriology • Histopathology and Cytopathology • Immunology: Antibodies

3. Haem LOs 1. Haemostasis: Summarise the mechanisms of normal haemostasis including the interactions of vessel wall, platelets and clotting factors. Coagulation tests: explain laboratory tests for investigating coagulation, and explain how results can contribute to a clinical diagnosis. Anticoagulants: explain the mechanism of action of anticoagulants, including warfarin and heparin, recall indications for their use and how we measure their effects in the laboratory. Antiplatelet agents: explain the mechanism of action of antiplatelet agents, and list indications for their use. Coagulopathy: compare the clinical features of bleeding due to different causes, and explain the principles of management of disorders of haemostasis. 2. Abnormal WBC: Leucocytosis: explain how to analyse the cause of a leucocytosis (differential count / blood film), list the common causes of a neutrophilia, eosinophilia and lymphocytosis, explain how a reactive polyclonal response may be differentiated from a lymphoproliferative disorder.

4. 1. Haemostasis Is a process which causes bleeding to stop, meaning to keep blood within a damaged blood vessel  The first stage of wound healing

5. Response to vessel injury - Overview 1. Vessel constriction 2. Formation of an unstable platelet plug -platelet adhesion -platelet aggregation 3. Stabilisation of the plug with fibrin -blood coagulation 4. Dissolution of clot and vessel repair -fibrinolysis Primary haemostasis Secondary haemostasis

6. The Endothelium Smooth muscle cells Composition: vessel lumen endothelial cells basement membrane collagen, elastin, glycosaminoglycans; Tissue factor Functions: -maintain barrier between blood and procoagulant subendothelial structures -Synthesis of PGI2, thrombomodulin, vWF, plasminogen activators

7. Platelet adhesion Von Willebrand factor platelet Endothelial cells or GlpIb GlpIa collagen Release of ADP & thromboxane Coagulation activation thrombin Platelet aggregation GlpIIb/IIIa platelet platelet Fibrinogen

8. Prostaglandin Metabolism Endothelial Cells Platelets Membrane phospholipid Membrane phospholipid phospholipase phospholipase Arachidonic acid Arachidonic acid Cyclo oxygenase Cyclo oxygenase Endoperoxides (PGG2, PGH2) Endoperoxides (PGG2, PGH2) Thromboxane synthetase Prostacyclin synthetase Prostacyclin (PGI2) Thromboxane A2 Note: Endoperoxides and thromboxane are potent induces of platelet aggregation when secreted Note: PGI2 is a potent inhibitor of platelet function

9. Main Antiplatelet therapies COX-1 inhibitor Aspirin ADP receptor antagonist clopidogrel, prasugrel GPIIb/IIIa antagonists abciximab, tirofiban, eptifibatide

10. Coagulation Cascade Who’s going to Volunteer? Muwahahaha

11. Blood coagulation XII XIIa XI XIa Tissue factor (vessel damage) VIIa IXa IX EXTRINSIC PATHWAY VIIa INTRINSIC PATHWAY VIIIa Pl X Xa X Va Pl COMMON PATHWAY Prothrombin thrombin (IIa) Fibrinogen Fibrin thrombin XIIIa XIII Crosslinked fibrin

12. Fibrinolysis tissue plasminogen activator, tPA Plasmin Plasminogen Fibrin clot Fibrin degradation products, FDP -elevated in DIC tPA and a bacterial activator, streptokinase, are used in therapeutical thrombolysis for Myocardial Infarction (Clot busters)

13. Anticoagulation Pathways XII XIIa ATIII XI XIa Tissue factor (vessel damage) VIIa ATIII IXa IX EXTRINSIC PATHWAY VIIa APC/P.S INTRINSIC PATHWAY VIIIa Pl ATIII X Xa X APC/P.S Va Pl ATIII COMMON PATHWAY Prothrombin thrombin (IIa) Fibrinogen Fibrin thrombin XIIIa XIII Crosslinked fibrin

14. Anti-coagulants • Warfarin – long term therapy • Vitamin K antagonists, inhibits the post-translational modifications (carboxylation) of clotting factors II, VII, IX, X • Heparin – immediate anticoagulation • Venous thrombosis and pulmonary embolism (intravenous) • Accelerates the action of antithrombin • Low molecular weight heparin acts more on factor Xa

15. Haemostasis abnormalities • Too much (pro-coagulant) – thrombosis • Too little (anti-coagulant) – bleeding

16. Bleeding Minor: Easy bruising, Gum bleeding, Frequent nosebleeds, Bleeding after tooth extraction, Post operative bleeding, Menorrhagia Significant: • Epistaxis not stopped by 10 mins compression or requiring medical attention/transfusion. • Cutaneous haemorrhage or bruising without apparent trauma (esp. multiple/large). • Prolonged (>15 mins) bleeding from trivial wounds, or in oral cavity or recurring spontaneously in 7 days after wound. Spontaneous GI bleeding leading to anaemia. • Menorrhagia requiring treatment or leading to anaemia, not due to structural lesions of the uterus. • Heavy, prolonged or recurrent bleeding after surgery or dental extractions.

17. Defects of Primary Haemostasis Platelets: • Thrombocytopenia (low plt count) - Bone marrow failure (leukaemia, B12 deficiency), Accelerated clearance (ITP, DIC, auto-ITP), Pooling and destruction in splenomegaly • Impaired function - Glanzmann’s thomboasthenia (no GlpIIb/IIIa), Bernard Soullier syndrome (no GlpIb). Storage pool disease (affects dense + alpha granules), Acquired due to drugs, eg aspirin, NSAIDs, clopidogrel Von Willebrand Disease (autosomal dominant): Type 1 – deficiency, Type 2 – impaired function The vessel wall: hereditary vascular disorders (Hereditary haemorrhagic telangiectasia), scurvy, steroids, age

18. Defects of Secondary Haemostasis Coagulation factor deficiency • Hereditary: Haemophilia A (FVIII) & B (FIX) – X linked, • Acquired: liver disease (most cf made in liver), dilution, anticoagulation Increased consumption of cf • Disseminated intravascular coagulation (DIC) - Generalized activation of coagulation by Tissue Factor > consumption of platelets and consumption/depletion of coagulation factors. Activation of fibrinolysis depletes fibrinogen > excess deposition of fibrin in vessels > organ failure. Associated with sepsis, major tissue damage, inflammation • Autoimmune – antibodies

19. Differences in Bleeding Presentation Primary Defect Secondary Defect Spontaneous bleeding is deep, into muscles and joints (haemarthrosis) Bleeding after trauma may be delayed and is prolonged Superficial cuts do not bleed (platelets) Bruising is common, Nosebleeds are rare Frequently restarts after stopping • Immediate • Prolonged bleeding from cuts • Epistaxes • Gum bleeding (areas of high shear without functioning vWF) • Menorrhagia (heavy + prolonged menstrual bleeding) • Easy bruising • Prolonged bleeding after trauma or surgery • Thrombocytopenia > petechiae (small dotted bruising)

20. Haemarthrosis – hallmark of haemophilia

21. Fibrinolysis defects Hereditary • Antiplasmin deficiency Acquired • Drugs that enhance tPA (tissue plasminogen activator) • Disseminated intravascular coagulation

22. Clotting Tests • Prothrombin Time (PT), 9.6-11.6, tests extrinsic + common pathways. Warfarin monitoring • Activated partial thromboplastin time (APTT), 26-32, tests intrinsic + common pathways. Heparin monitoring • Thrombin Time (TT) – tests common pathway • Factor assays and inhibitors Platelet Function tests • Bleeding time, 2-9 minutes • Platelet count, 150-400 x10^9/L • Platelet aggregation

23. Treatments • Replace Platelets: • Pooled platelet concentrates available Replace Factors: • Plasma • Contains all coagulation factors • Cryoprecipitate • Rich in Fibrinogen, FVIII, VWF, Factor XIII • Factor concentrates • Concentrates available for all factors except factor V. • Prothrombin complex concentrates (PCCs) Factors II, VII, IX, X • Recombinant forms of FVIII and FIX are available. • Immune destruction: • Immunosuppression (eg prednisolone) • Splenectomy for ITP • Others: • DDAVP - vWF • Tranexamic acid – inhibits fibrinolysis • Fibrin glue/spray

24. 2. Abnormal White Cell Count What’s normal? 4-11 x10^9/L How do we distinguish abnormal? • All types or just one? • Blood film: maturity of cells, morphology • Further investigations

25. Increased phagocytes Neutrophilia (> 7.5x10^9/l in adults) • Acute bacterial infection – may see toxic granulation • Inflammation and tissue necrosis (MI, appendicitis) • Myeloproliferative disorders (chronic myeloid leukaemia) • Demargination (exercise, extreme stress) • Corticosteriods Eosinophilia (> 0.4 x109/l ) • Parasitic infections • Atopic allergy (asthma, eczema) • Pulmonary eosinophilia • Hodgkin’s lymphoma Monocytosis • Certain chronic bacterial infections (tuberculosis, brucellosis and typhoid) • Chronic myelomonocytic leukaemia.

26. Toxic granulation and left-shit

27. Reactive elevated phagocyte count

28. Increased lymphocytes A lymphocyte count > 4.0 x 109/l (adults) Primary lymphocytosis (malignant clonal proliferation) • Lymphocytic leukaemia, or lymphoma. Secondary reactive lymphocytosis (polyclonal reactive proliferation) • Infection or inflammation.

29. The difference • Clinical picture, age of patient • Abnormal forms such as smear cell or blast cells - lymphoproliferative disorder • Monoclonal or polyclonal - population of monoclonal B cells only present one immunoglobulin light chain type, either κ or λ on cell surface • Mature lymphocytes: either primary or reactive. Immature cells/blasts: primary e.g. Acute Lymphoblastic Leukaemia (ALL)

30. Smear cells, characteristic of CLL Atypical lymphocytes in mononucleosis infection

31. Mini- Quiz (+Quick Break) • What would you likely see on a blood film of a patient with acute leukaemia? • What is formed by the end of primary haemostasis? • Young boy presents with swollen elbow, history of prolonged bleeding. What condition do you suspect and what tests to prove?

32. Diagnostics LOs 3. Chemical pathology List common chemical pathology diagnostic tests (including cardiac enzymes, electrolytes, urea, glucose), and recall how to collect test specimens. Request forms: explain a typical chemical pathology request form. 4. Virology Explain the role of virology tests in the diagnosis of infectious disease, list the types of specimen that are commonly sent for virological diagnosis, and list laboratory procedures that may be used. 5. Bacteriology Microbiological diagnosis: explain the concept of best guess diagnosis and the contribution of the laboratory to it, recall how a microbiology laboratory works and what investigations may be performed. Microbiological tests: recall how microbiology samples are collected, explain what investigations may be requested, explain the limitations of microbiology investigations and recognise how to interpret results 6. Histopathology and cytopathology List situations where histopathology and cytopathology might be used as a diagnostic method, summarise the main steps involved in processing a specimen, and recall the types of specimen that may be tested. Frozen section diagnosis: list situations where frozen section diagnosis may be required and recall the advantages of this technique. Immunohistochemistry: explain the information available from immunohistochemistry and recall when this technique may be used. Autopsy: summarise the benefits of the autopsy. Cytology screening: list benefits of cytology screening. 7. Immunology Antibodies: explain how antibodies may be produced for diagnostic purposes, list the therapeutic and diagnostic uses of antibodies, and list examples of types of substance that are identified diagnostically by antibodies. Antibody diagnostic methods: explain the principles of detection methods and recall their advantages in different situations; explain how antibodies may be used to aid diagnosis in clinical practice.

33. 3. Chem Path • Electrolytes (including, sodium and potassium). • Urea and creatinine • Calcium and phosphate. • Markers of liver function (liver enzymes): • Albumin • Bilirubin • Alkaline phosphatase (ALP) • Aspartate amino-transferase (AST) • Alanine amino-transferase (ALT) • Gamma-glutamyl transferase (GGT) • Hormone assays (thyroxine, TSH and cortisol) • Glucose • Cardiac enzymes: • Troponins • Creatine kinase (CK) • Aspartate amino transferase (AST) • Lactate Dehydrogenase (LDH)

34. Blood Tubes • Red top – no anticoagulant • Yellow top – speeds up clotting – U&Es, TFTs, LFTs • Purple top – potassium EDTA – FBC, Hb1Ac • Grey top – fluoride oxalate (poison) to prevent RBCs from consuming glucose

35. 4. Virology Ideal virological test: high specificity, sensitive, rapid, non-invasive, cost-effective • Cell culture • Electron microscopy (EM) - • Antibody detection e.g. anti-HIV antibody (Enzyme Immuno Assays) • Antigen detection (IF, EIA) - HBsAg in hepatitis B infection or RSV antigen in respiratory sample • Genome detection –PCR to detect viral DNA or RNA • Quantification of antigens and genomes (now essential for diagnosis and monitoring of HIV, HBV and HCV) • Genome sequencing: Genotyping, Antiviral resistance testing

36. Sample sites • Throat swab - for virus isolation (in virus transport medium, VTM) - useful in the diagnosis of enteroviruses and respiratory viruses. • Stools - for EM and Rotavirus EIA (in sterile pot) - for the diagnosis of enteroviruses and viruses that cause diarrhoea such as rotavirus, astrovirus, adenovirus, noroviruses, etc. • CSF - PCR for herpes and enteroviruses (in sterile container, VTM (viur transport medium) not required) - for the diagnosis of viruses causing meningitis or encephalitis such as HSV, VZV, enteroviruses, mumps, etc. • Nasopharyngeal aspirate (NPA) - for respiratory viruses using Immunofluoresence (IF) or PCR, such as RSV, influenza A&B, adenovirus, parainfluenza viruses, SARS etc. • Urine - virus isolation or PCR depending on which viruses you are interested in (in sterile container), e.g. BK virus, CMV, etc. • Blood (clotted) - for antibody detection. • Blood (EDTA) - for PCR. Used for detection and quantification of HIV, HBV and HCV

37. 5. Bacteriology • Culture i) Sterile sites (blood/CSF) ii) Non sterile sites • Microscopy - Gram stain of CSF, joint fluid, purulent exudates, ZN/auramine stain of e.g. sputum, for TB. FTA (fluorescent treponemal antibody) for antibodies to T. pallidum • Serology/ antigen detection - Meningococcal antigen in CSF, C. difficile toxin in faeces, Legionella and Pneumococcal antigen in urine • Molecular Techniques – PCR Chlamydia in genital specimens, Rapid PCR for MRSA • Antimicrobial Susceptibility Testing

38. Specimen Collection • In the acute phase of illness and before staring antimicrobials • Collection from proper site, avoiding contamination by normal flora • Prompt transport to lab since micro-organisms multiply in transit • Adequate quantity and appropriate number of specimens • Acute sera and Convalescent sera (paired), for rising antibody titres

39. Urine • Bedside: Naked eye - clear, cloudy, haemorrhagic. • Dipstick tests - ketones may indicate infection. Nitrites suggest bacteriuria • Microscopy: WBC (pyuria suggests infection) • Culture on MacConkey agar (urine should be sterile so any microbial growth is potentially significant in an appropriately taken sample) • • Antibiotic sensitivity testing of bacteria that grow Faeces • Naked eye, consistency, blood stained, colour, presence of worms • Microscopy: ova, cysts, parasites • Culture on inhibitory media – e.g. deoxycholatecitrate agar (DCA), selenite • Toxin detection (Clostridium difficile) • Special stains, e.g. for cryptosporidia • Diarrhoea • Can be bacterial, viral or parasitic Bacteria: Salmonella, Shigella, Campylobacter, E coli O157, C difficile, Cholera

40. Gradient MICs – minimum inhibitory concentrations

41. 6. Histo/cytopatholgy Histpathology (tissues): • Biopsies • Resection specimens • Frozen sections • Post-mortems Cytopathology (cells) • Smears • Fine needle aspirations Specimens: Fix in formalin Embed in paraffin wax Cut sections Frozen section: 30 minutes For biopsies: 2-3 days For resection specimens: 5-7 days • Stains: • Haematoxylin and eosin (H&E). Haematoxylin stains nuclei blue and eosin the cytoplasm pink. • Silver nitrate – fungi • Gram – bacteria • Ziehl-Neelson – Tuberculosis bacilli • Papanicolaou – cervical smears

42. 7. Immunology – Antibodies Diagnostic • Blood group serology • Immunoassays • hormones • antibodies • antigens • Immunodiagnosis • Infectious diseases • Autoimmunity • Allergy (IgE) • Malignancy (myeloma) Therapeutic: • Prophylactic protection against microbial infection • Anti-cancer therapy • Removal of T-cells from bone marrow grafts • Block cytokine activity e.g. TNF-α

43. Immortal No Ab production HGPRT -ve Limited cell division Produce Ab HGPRT +ve Immortal Produce Ab HGPRT +ve Clone by limiting dilution

44. Production of antibodies using recombinant DNA technology

46. Mini Quiz • What colour blood tube do I need to test for glucose and why? • When must samples for microbiological testing be taken? • 3 types of markers you can detect with antibodies?

47. Past SAQ A man goes to hospital and is treated for a pulmonary embolism. Two days later he comes back for blood tests. His PT and TCT tests are normal, but his APTT is prolonged. • a) What are three causes of a prolonged APTT (in general)? [3] • b) In this case, what is the most likely cause? [1] • c) Name three physiological inhibitors of the clotting cascade. [3] • d) What is the mechanism of action of warfarin? [2] • e) Which plasma protein does heparin act on to have its anticoagulant effects?

48. Thanks for listening!

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