Haemostasis and Disorders of Haemostasis PDF

HAEMOSTASIS AND DISORDERS OF HAEMOSTASIS COURSE TITLE: Medical Laboratory Haematology I COURSE CODE: MLS402 [email protected] 1  LEARNING OUTCOMES At the end of the lecture, you should be able to: ❑Identify th...

HAEMOSTASIS AND DISORDERS OF HAEMOSTASIS COURSE TITLE: Medical Laboratory Haematology I COURSE CODE: MLS402 [email protected] 1  LEARNING OUTCOMES At the end of the lecture, you should be able to: ❑Identify the main components involved in haemostasis. ❑Understand the mechanisms of haemostasis and identify the roles of blood vessel, platelet and coagulation factors in haemostasis. ❑Evaluate the roles of inhibitors of the coagulation system. ❑Describe the classification of haemostatic and thrombotic disorders. 2  INTRODUCTION Blood is tissue in a fluid state. Keeping blood in a fluid state, confined to the circulatory system? Haemostasis can be defined as the process of arresting bleeding from an injured blood vessel, which comes from Greek, haeme meaning blood and stasis meaning to stop. 3 AN OVERVIEW OF HAEMOSTASIS BV Injury Tissue Factor Neural Blood Vessel Platelet Coagulation Constriction Aggregation Cascade Primary haemostatic plug Platelet Reduced Activation Fibrin Blood flow formation Stable Haemostatic Plug 4  NORMAL CLOTTING Response to Blood Vessel (BV) Injury: ❑Vasoconstriction to reduce blood flow ❑Platelet plug formation (von Willebrand factor binds damaged vessel and platelets) ❑Activation of clotting cascades with the generation of fibrin clot formation. ❑Fibrinolysis (clot breakdown). 5  THE VASCULAR PHASE WHEN A BLOOD VESSEL IS DAMAGED, VASOCONSTRICTION RESULTS. Vascular spasm Reduces the flow of blood from an injured vessel. Cause: ❑Sympathetic reflex ❑Release of vasoconstrictors (TXA2 and serotonin) from platelets that adhere to the walls of damaged vessels. 6  PLATELET PHASE PLATELETS ADHERE TO THE DAMAGED SURFACE AND FORM A TEMPORARY PLUG. Mechanism: ❑Platelet adhesion ❑Platelet activation ❑Platelet aggregation 7  PLATELET ADHESION When a blood vessel is injured, platelets adhere to the exposed collagen and von Willebrand factor in the wall via platelet receptors platelet activation. Swell, becomes irregular & protrudes pseudopodia. Platelets become sticky & adhere to the collage. Activated platelets release the contents of their granules, including ADP, and secrete TXA2 → activates nearby platelets to produce further accumulation of more platelets (platelet aggregation) and form a platelet plug. 8 PLATELET ADHESION 9  PLATELET ACTIVATION Platelets secrete ADP and Thromboxane A2. Activates other platelets, and the cycle continues. 10 PLATELET RELEASE ACTION 11  PLATELET AGGREGATION Activated sticky platelets stick to each other and form aggregation. It also increases by Platelet Activating Factor (PAF) released by neutrophils, monocytes and platelet cell membrane lipids. 12 PLATELET AGGREGATION 13 PLATELET TEMPORARY PLUG Platelet Adhesion Platelet Aggregation Fairy Loose Plug 14  INHIBITION OF FURTHER PLUG FORMATION Prostacyclin from membrane phospholipids Inhibit Thromboxane formation. Terminates the cycle 15  FORMATION OF DEFINITIVE HAEMOSTATIC PLUG Temporary plug converted to definitive (secondary) plug by blood coagulation. Formation of a tight, unyielding seal. 16  BLOOD COAGULATION Throughout life, blood is in a fluid state, but when removed or shed or collected in a container it becomes a jelly-like (clot). Fluidity – necessary for circulation. Coagulation – protect from excessive bleeding. 17  SECONDARY HAEMOSTATIC PLUG “Cascade of reactions” by Macfarlane, R.G.,1967. It states that ‘inactive’ enzymes are activated, and the ‘activated’ enzymes in turn activate other inactive enzymes until the final step is reached. 18  COAGULATION PHASE BLOOD COAGULATION THEORIES: 1. Morawitz 2. Macfarlane or Waterfall, or enzyme cascade 3. Modern Concept - THROUGH TWO SEPARATE PATHWAYS, THE CONVERSION OF FIBRINOGEN TO FIBRIN IS COMPLETE. 19  MORAWITZ THEORY Paul Oskar Morawitz The classical theory of coagulation was proposed by Paul Morawitz in 1905. Many of the coagulation factors are not represented in this theory - factors VIII, IX and XI are absent from this model – haemophiliacs. 20  CASCADE THEORY The enzyme cascade model of coagulation. This cascade does not explain why factor XII deficiency is not associated with a bleeding disorder. In addition, factors VIII and V are actually co-factors for factors IX and X, respectively. The model also lacks calcium and phospholipids, essential coagulation The concept of blood coagulation dates back to 1960's when Davie, Ratnoff and Macfarlane described the “waterfall” and “cascade” requirements. 21 MODERN CONCEPT 22  ACTIVATION OF CLOTTING CASCADE Normally, the ingredients, called clotting factors, act like a row of dominoes toppling against each other to create a chain reaction. If one of the factors is missing, this chain reaction cannot proceed. 23 FIBRINOLYTIC PHASE 24 THE COAGULATION SYSTEM 25  CAUSES OF BLEEDING DISORDERS VESSEL DEFECTS PLATELET DISORDERS FACTOR DEFICIENCIES OTHER DISORDERS 26  VESSEL DEFECTS VITAMIN C DEFICIENCY BACTERIAL & VIRAL INFECTIONS ACQUIRED & HEREDITARY CONDITIONS 27  PLATELET DISORDERS THROMBOCYTOPENIA THROMBOCYTOPATHY 28  THROMBOCYTOPENIA INADEQUATE NUMBER OF PLATELETS 29  THROMBOCYTOPENIA DRUG INDUCED BONE MARROW FAILURE HYPERSPLENISM OTHER CAUSES 30  OTHER CAUSES Lymphoma HIV Virus Idiopathic Thrombocytopenia Purpura (ITP) 31  THROMBOCYTOPATHY ADEQUATE NUMBER BUT ABNORMAL FUNCTION 32  THROMBOCYTOPATHY URAEMIA INHERITED DISORDERS MYELOPROLIFERATIVE DISORDERS DRUG INDUCED 33  FACTOR DEFICIENCIES (CONGENITAL) HAEMOPHILIA A HAEMOPHILIA B von Willebrand’s DISEASE 34  FACTOR DEFICIENCIES HAEMOPHILIA A (Classic Hemophilia) ❑80-85% of all Haemophiliacs ❑Deficiency of Factor VIII ❑Lab Results - Prolonged PTT 35  FACTOR DEFICIENCIES HAEMOPHILIA B (Christmas Disease) ❑10-15% of all Hemophiliacs ❑Deficiency of Factor IX ❑Lab Test - Prolonged aPTT 36  FACTOR DEFICIENCIES VON WILLEBRAND’S DISEASE ❑Deficiency of VWF & amount of Factor VIII ❑Lab Results - Prolonged BT, PTT 37  OTHER DISORDERS (ACQUIRED) ORAL ANTICOAGULANTS ❑COUMARIN ❑HEPARIN LIVER DISEASE MALABSORPTION BROAD-SPECTRUM ANTIBIOTICS 38  INHIBITORS 30% of people with haemophilia develop an antibody to the clotting factor they receive for treatment. These antibodies are known as inhibitors. These patients are treated with high doses of FVIIa for bleeds or surgery. This overrides defects in FVIII or FIX deficiency. Long-term management involves attempting to eradicate inhibitors by administering high-dose FVIII (or FIX) in an immune tolerance process. 39 COAGULATION DISORDERS 40  HAEMOSTATIC DISORDERS Disorders of primary haemostasis Inherited disorders of the connective tissue Disorders of primary haemostasis o Disorders of the vascular system Hereditary disorders of the vascular system Acquired disorders of the vascular system Adapted from Tables 122-2. In: Haematology Basic Principles and Practice, by o Platelet disorders Hoffman R, et al., 7th edition (2018). Elsevier Classification of acquired disorders of the vascular system Bleeding sites in disorders of primary haemostasis Modified from Tables 33.2 (upper panel), 33.3 (left lower panel) and 33.4 (right lower panel). In: Clinical Laboratory Hematology, by McKenzie SB, et al. 4th edition (2020). 41 Pearson  HAEMOSTATIC DISORDERS Disorders of secondary haemostasis o Disorders of the proteins of fibrin formation: hereditary or acquired Disorders of secondary haemostasis Bleeding characteristics in disorders of secondary haemostasis Adapted from Table 34.1. In: Clinical Laboratory Hematology, by McKenzie SB, Adapted from Tables 122-3. In: Haematology Basic Principles and Practice, by et al. 4th edition (2020). Pearson Hoffman R, et al., 7th edition (2018). Elsevier 42 HAEMOSTATIC DISORDERS Disorders of secondary haemostasis o Disorders of fibrinolysis Adapted from Table 122-4. In: Haematology Basic Principles and Practice, by Hoffman R, et al., 7th edition (2018). Elsevier 43  THROMBOTIC DISORDERS - THROMBOPHILIA Physiologic alterations contributing to thrombosis Thrombus formation o Arterial thrombi (white thrombi) o Venous thrombi Thrombophilia: increased risk of thrombosis. Any disorder either inherited or acquired associated with an increased risk of venous thrombosis (VTE) o Hereditary thrombophilia Abbreviations: AT, antithrombin; TFPI, tissue factor pathway inhibitor; HCII, heparin cofactor II; APC, activated protein C. Adapted from Table 35-1. In: Clinical Laboratory Hematology, by McKenzie SB, et al. 4th edition (2020). Pearson 44  THROMBOTIC DISORDERS - THROMBOPHILIA Hereditary thrombophilia: individuals with a genetic predisposition resulting in an increased risk of thrombosis Hereditary conditions associated with thrombosis Adapted from Table 35-3. In: Clinical Laboratory Hematology, by McKenzie SB, et al. 4th edition (2020). Pearson Abbreviations: HCII, heparin cofactor II; PAI-I, plasminogen activator inhibitor; PC, protein C; PS, protein S 45 BLEEDING DISORDERS 46  CLINICAL FEATURES OF BLEEDING DISORDERS Platelet Coagulation disorders factor disorders Site of bleeding Skin Deep in soft tissues Mucous membranes (joints, muscles) (epistaxis, gum, vaginal, GI tract) Petechiae Yes No Ecchymoses (“bruises”) Small, superficial Large, deep Hemarthrosis / muscle bleeding Extremely rare Common Bleeding after cuts & scratches Yes No Bleeding after surgery or trauma Immediate, Delayed (1-2 days), usually mild often severe 47 PLATELET COAGULATION 48 Petechiae, Purpura Hematoma, Joint bl. PETECHIAE (typical of platelet disorders) Do not blanch with pressure (cf. angiomas) Not palpable (cf. vasculitis) 49 HAEMARTHROSIS 50 HAEMATOMA 51 PETECHIAE 52 PURPURA 53 ECCHYMOSIS 54 SENILE PURPURA 55 Petechiae in a patient with Rocky Mountain Spotted Fever 56 HENOCH-SCHONLEIN PURPURA 57 ECCHYMOSES (typical of coagulation factor disorders) 58 CT scan showing large hematoma of the right psoas muscle 59  COAGULATION FACTOR DISORDERS Inherited bleeding Acquired bleeding disorders disorders Hemophilia A and B Liver disease Vitamin K vonWillebrands disease deficiency/warfarin Other factor deficiencies overdose DIC 60 THANK YOU 61  INVESTIGATION OF BLEEDING DISORDERS COURSE TITLE: Medical Laboratory Haematology I COURSE CODE: MLS402 [email protected] 1  LEARNING OUTCOMES At the end of the lecture, you should be able to: ❑Provide a background on normal haemostasis. ❑Understand the role of CBC in the investigation of bleeding disorders ❑Describe the principles and procedures for bleeding time and whole blood clotting time. ❑Describe the principles of screening tests (PT, APTT, TT and Fibrinogen concentration assay (Clauss technique)). ❑Carry out manual APTT, PT and TT determination using test tubes. ❑Discuss confirmatory tests in haemostasis. 2  HAEMOSTASIS - OVERVIEW Mechanisms o Vessel wall contraction o Platelet adhesion and aggregation Platelet plug formation o Plasmatic coagulation Fibrin clot formation, initiated by tissue factor binding with FVIIa Fibrinolysis: dissolution of the clot and restoration of normal blood flow Components involved in haemostasis o Blood vessels; platelets; coagulation factors; Adapted from Figure 24.1, In: Hoffbrand Essential haematology, by coagulation inhibitors; fibrinolytic system Hoffbrand AV and Steensma DP.,8th edition (2020). Wiley Blackwell 3 N.B. Abnormality in any of the 5 components may lead to bleeding or thrombophilia  THE COAGULATION CASCADE Plasma-based in vitro coagulation: extrinsic, intrinsic and common pathways o Interactions among pathways PK, HMWK Tissue factor FVIIa/TF also activates FIX (TF) TF/ Coagulation factors in intrinsic, extrinsic, and common pathways PT measures Modified from Table 32.1. In: Clinical Laboratory Hematology, by McKenzie SB, et al. 4th edition (2020). Pearson Coagulation cascades Abbreviations: APTT, activated partial throboplastin Modified from Figure 24.12, In: Hoffbrand Essential haematology, by Hoffbrand AV and time; HMWK (HK), high molecular weight kininogen; Steensma DP.,8th edition (2020). Wiley Blackwell 4 PT, prothrombin time; TT, thrombin time  LABORATORY INVESTIGATIONS OF BLEEDING DISORDERS PK, HMWK Full blood count and blood film Tissue factor (TF) o Platelet count Screening tests of blood coagulation TF/ o Prothrombin time (PT) PT measures o Thrombin time (TT) o Activated partial thromboplastin time (APTT) o Fibrinogen concentration Mixing studies (50:50 mix) Specific assays of coagulation factors Coagulation cascades Modified from Figure 24.12, In: Hoffbrand Essential haematology, by 5 Hoffbrand AV and Steensma DP.,8th edition (2020). Wiley Blackwell  FULL BLOOD COUNT Also known as CBC, provides a screen: Blood film examination and reporting 6  PLATELET COUNT PLATELET COUNT: ❑NORMAL 150,000 - 400,000 CELLS/mm3 ❑< 100,000 Thrombocytopenia ❑50,000 - 100,000 Mild Thrombocytopenia ❑< 50,000 Severe Thrombocytopenia REFER TO LECTURES ON CELL COUNTING METHODS. 7  BLEEDING TIME (BT) This refers to the time an injured blood vessel takes to stop bleeding. It is the time interval between the skin puncture and spontaneous, unassisted stoppage of bleeding. It measures how fast INJURED small blood vessels in the skin stop bleeding. The bleeding time test is used to evaluate how long it takes the injured vessels to constrict and how long it takes for platelets in the blood to seal off the opening. 8  BLEEDING TIME Principle: ❑The bleeding time test is a useful tool for testing platelet plug formation and capillary integrity. ❑The bleeding time is dependent upon: ▪ The efficiency of tissue fluid in accelerating the coagulation process ▪ The capillary function. ▪ The number of blood platelets present and their ability to form a plug. 9  BLEEDING TIME Prolonged bleeding times are generally found when: ❑The platelet count is below 50,000/µL, and ❑There is platelet dysfunction. It is one of the tests carried out on patients suspected of having a bleeding disorder (others are PT, APTT, PLT and Fib). 10 PATIENTS FOR BLEEDING TIME History of frequent, persistent or spontaneous bleeding. Before every minor and major surgery -(e.g. tooth extraction). Before a biopsy ( bone marrow, liver, kidney, etc.). Before and during anticoagulant therapy. Family history of bleeding disorder. 11 BLEEDING TIME - METHODS The Duke method - Less than 3 minutes. The Ivy Method – Less than 8 minutes. The Mielke Method -. The Simplate or Surgicutt - 3.65 +/- 1.22 minutes. General interpretations of bleeding time: o1-9 minutes: Normal. o9-15 minutes: Platelet dysfunction oMore than 15 minutes: Critical; test must be discontinued, and pressure should be applied. 12  BLEEDING TIME - METHODS Apparatus Required: ❑Sterile finger prick (lancet), clean filter paper, stopwatch 13  DUKE METHOD A standardised puncture of the ear lobe is made, and the time required for bleeding to cease while the blood is being blotted every 30 seconds is recorded. A lancet is used to make the puncture. No repeat testing is allowed due to space. Causes apprehension in the patient. This test method is the easiest to perform but is the least standardised and has the worst precision and accuracy. 14  IVY METHOD A blood pressure cuff maintains a constant pressure within the capillaries, helping to standardise the procedure. The cuff is inflated to 40 mm Hg on the upper arm to control the capillary tone and to improve the sensitivity and reproducibility. The forearm is the bleeding time site used. A sterile, disposable blood lancet is used and the length of time required for bleeding to cease is recorded. 15 16  IVY METHOD The greatest source of variation in this test is largely due to difficulty in performing a standardised puncture. This usually leads to erroneously low results. 17  MIELKE METHOD A modification of the Ivy Method. A Bard-Parker or similar disposable blade is used, along with a rectangular polystyrene or plastic template that contains a standardised slit. The blade is placed in a special handle containing a gauge to standardise the incision depth. The same procedure as described for the Surgicutt method is employed. 18  MIELKE METHOD … Advantages of this method include: – ❑That the surgical incision more closely approximates the patient’s haemostatic response to surgery, when compared to the puncture in the Ivy Method. ❑The depth of the incision can be controlled. Disadvantages of this method include: ❑Cost- The scalpel and template required sterilisation after each use. ❑Patient apprehension due to unconcealed scalpel. ❑Small scars might form. 19  SIMPLATE/SURGICUTT METHOS – PREFERRED METHOD A modification of the Ivy Method. The first bleeding time device introduced was the Simplate. The Simplate device has a trigger and spring method for the blade, which has a depth of 1.0 mm and a width of 5.0 mm. Another brand name is the Surgicutt. Advantages: ❑Instrument is a sterile, standardised, easy-to-use that makes a uniform incision. 20  SIMPLATE/SURGICUTT METHOS – PREFERRED METHOD … Instrument is a spring-activated surgical steel blade which is housed in a plastic unit. This eliminates the variability of blade incision. This method is the most standardised method of all the bleeding time procedures. – Inexpensive. Disadvantages: Slight scarring can occur, so the patient should be informed. 21  MATERIALS Blood pressure cuff. Sterile, disposable blood lancet, capable of a puncture 5 mm wide and 1 mm deep. Stopwatch. Whatman No. 1 circular filter paper. Alcohol pads. Gloves. Butterfly bandage. 22 23  PREPARATION OF THE PATIENT After proper greeting and identification of the patient, explain the test to the patient. It is critical that you ask the patient whether or not they have taken aspirin, aspirin-containing compounds (many over-the-counter medications contain aspirin) or blood thinners such as heparin or coumadin recently. These drugs will cause a falsely abnormal bleeding time, and the test should not be performed. 24  SIMPLATE/SURGICUTT PROCEDURE Greet and identify the patient. Explain the procedure to the patient. Obtain a history of aspirin or aspirin-containing compounds taken within the last 7 - 10 days. Select a site on the patient's forearm approximately three fingers wide below the elbow bend that is free of visible subcutaneous veins. Cleanse the outer surface of the patient's forearm by moving the alcohol pad in concentric circles from the incision site outward; allow it to air dry. 25  SIMPLATE/SURGICUTT PROCEDURE Place a blood pressure cuff on the patient's arm above the elbow. Turn the knob on the bulb of the sphygmomanometer until it stops. Squeeze the bulb to inflate the sphygmomanometer. Inflate the cuff and maintain pressure at 40 mm Hg. Remove the Surgicutt device from the blister pack, being careful not to contaminate or touch the blade-slot surface. Remove the safety clip. Holding the skin tight, depress the “trigger” on the bleeding time device. The puncture must be performed within 30 to 60 seconds of the blood pressure cuff being inflated. Simultaneously start the stopwatch. 26 27  SIMPLATE/SURGICUTT PROCEDURE After 30 seconds, blot (do not wipe) the blood with the filter paper. The filter paper must not touch the wound on the arm. Blot the site at regular thirty- second intervals. Rotate the filter paper after every 30 seconds. When bleeding ceases, and blood is no longer drawn to the filter paper, stop the watch and release the blood pressure cuff by turning the knob next to the bulb in the opposite direction used to inflate the cuff. Remove the blood pressure cuff. Record the bleeding time. Bleeding time is determined to the nearest 30 seconds. 28  SIMPLATE/SURGICUTT PROCEDURE If bleeding continues for more than 15 minutes, the procedure should be discontinued, and pressure applied to the wound sites. The bleeding time should be repeated on the other arm. If bleeding has again not ceased within 15 minutes, the results are reported as greater than 15 minutes. After ensuring that the bleeding has stopped, carefully bandage the site. Appropriately discard all used materials and wash hands. 29 30  RESULTS Normal Values: 1.0 – 9.0 minutes. NOTE: ▪ If bleeding continues for more than 15 minutes, the procedure should be discontinued, and pressure applied to the wound sites. The bleeding time should be repeated on the other arm. If bleeding has again not ceased within 15 minutes, the results are reported as greater than 15 minutes. 31  SOURCES OF ERROR The bleeding time may be prolonged if the patient has taken aspirin or aspirin-containing compounds 7 to 10 days before the procedure. Results may be affected by an improperly performed puncture. A puncture that is too shallow or too deep or in an inappropriate location will adversely affect test results. The alcohol must be completely dried before making the puncture. If residual alcohol is on a puncture site, the bleeding time will be erroneously prolonged. The results will be adversely affected if the Scientist does not initiate the timing of the procedure simultaneously with the puncture. 32  SOURCES OF ERROR … If the Scientist allows the filter paper to touch the wound, the platelet clot may be dislodged, causing falsely elevated results. If the stopwatch has not been appropriately calibrated, it may keep incorrect time. Stopwatches should be calibrated regularly as part of the quality assurance program. The direction of the incision should be consistent. A horizontal incision gives a longer bleeding time than a vertical incision. 33  SOURCES OF ERROR … The bleeding time is prolonged in thrombocytopenia, hereditary and acquired platelet dysfunctions, von Willebrand's disease, afibrinogenemia, severe hypofibrinogenemia, and some vascular bleeding disorders. 34 WHOLE BLOOD CLOTTING TIME (CT) It refers to the time interval between the entry of blood into a glass capillary tube or a syringe and the formation of fibrin threads. Methods: ❑Wright’s capillary glass tube. ❑Duke’s Drop method. ❑Lee and White test-tube method. Apparatus Required: 10-12 cm long, glass capillary tube with uniform diameter, stopwatch. Normal Clotting Time: ❑3 – 6 min (Wright’s). 35  THE SCREENING TESTS FOR BLOOD COAGULATION 36  SCREENING TESTS Prothrombin time (PT) Activated partial thromboplastin time (APTT) Thrombin time (TT) Fibrinogen concentration Mixing studies (50:50 mix) 37  SPECIMEN COLLECTION & PROCESSING Whole blood is aseptically collected by venepuncture. The anticoagulant of choice is 3.2% sodium citrate. The volume of anticoagulant necessary for a 5ml blood sample is adjusted according to the haematocrit value. One may keep the anticoagulant volume of 0.5ml constant and adjust the added blood volume according to the haematocrit. The volume of blood to be added (to 0.5ml of 0.109M citrate) is calculated from the formula: ❑(60 – haematocrit)/100 x 4.5 38 39  SPECIMEN PROCESSING Citrated whole blood is centrifuged to obtain plasma for coagulation testing. PLATELET-POOR PLASMA (PPP): The blood sample is centrifuged at a minimum of 1700 rpm for at least 10 minutes (2500 rpm for 15 minutes), preferably at 4°C, in a refrigerated centrifuge. Open the samples at RT, separate and store the plasma samples at 4°C as quickly as possible. When RT > 25 °C, labile coagulation proteins may be affected; a refrigerated (4°C) centrifuge should be used. Platelet poor plasma < 10 x 109/L. 40  SPECIMEN PROCESSING … SAMPLES FOR IMMEDIATE TESTING: Samples should be tested within 4 hours of sample collection when possible. Longer storage should be avoided for screening tests and clotting factor assays. Samples for factor assays should be stored at 4°C prior to testing. Samples for screening tests and the assay of factor VII should be maintained at RT to avoid the possibility of cold activation. 41  SPECIMEN PROCESSING … DEEP FREEZING OF PLASMA: Samples can be frozen for testing at a later stage. Storage at -70°C or lower is preferable (better), but at -35°C is adequate for most tests. Storage at -20°C is usually inadequate. Double centrifugation should be used if samples are frozen prior to analysis for lupus anticoagulant. Freezing and thawing are best avoided before APTT determinations since results obtained by some can be affected. 42  PROTHROMBIN TIME (PT) PK, HMWK Principle: measure the clotting Tissue factor (TF) time of recalcified plasma in the presence of thromboplastin. TF/ Reagents: plasma, recombinant PT thromboplastins (recombinant measures human tissue factor + synthetic phospholipids), CaCl2 Common causes of prolonged PT? Coagulation cascades Modified from Figure 24.12, In: Hoffbrand Essential haematology, by Hoffbrand AV and Steensma DP.,8th edition (2020). Wiley Blackwell 43  PROTHROMBIN TIME (PT) PT Measures the function of the Extrinsic coagulation Pathway. It is the time required for the plasma to clot after an excess of thromboplastin and an optimal calcium concentration has been added. The test is sensitive to Factors I, II, V, VII, and X. The PT evaluates patients suspected of having an inherited or acquired deficiency in these pathways. The test’s sensitivity is influenced by the reagent and technique used, and it is important to establish a reference range locally. 44 THE PRINCIPLE OF PT When a preformed tissue thromboplastin and calcium chloride are added to citrated plasma, the plasma clots. The time taken for the clot to appear is called Prothrombin time (PT). 45  PROTOCOL FOR PT REQUIREMENTS/REAGENTS – ▪ Patient’s platelet-poor plasma (PPP). ▪ Normal/ control plasma. ▪ Thromboplastin Reagents (this may contain calcium chloride). ▪ Calcium chloride - 0.025 mol/l (only required if thromboplastin reagent does not contain calcium chloride). 46 PROTOCOL FOR PT … REQUIREMENTS/REAGENTS – ▪ Glass Tubes ▪ Automatic Micropipettes of 100 μl. ▪ A Water bath set at 37°C. ▪ Stop watches. ▪ A Table Lamp. 47  PROCEDURE FOR PT 1. Add 100 µl of plasma to a glass test tube pre-warmed in the water bath. 2. Add 200 µl of thromboplastin with calcium, mix, and IMMEDIATELY start recording the time until a visible clot forms. 3. Repeat the procedure for the plasma sample (duplicate). 4. If PT is prolonged, mixing studies will be needed. To do mixing studies, mix 100 µl of abnormal plasma with 100 µl of normal plasma in a fresh tube, apply 100 µl of the mixture to a glass tube pre-warmed in the water bath as suggested in Step 1, and repeat the thromboplastin/calcium clot test. The mixing study should be repeated. 48 PT RESULTS 49  PT RESULTS RESULTS CAN BE EXPRESSED IN: ❑Mean of duplicate recordings in seconds. oNORMAL VALUE = 11-16 seconds ❑A ratio of the patient’s PT time compared to the laboratory mean of normal control (prothrombin ratio, PTR) ❑INR = (PT of patient/Control PT)ISI. NORMAL INR VALUE = 0.8 - 1.1, INR of 2.0 to 3.0 for those taking warfarin. 50  INTERPRETATION OF PT RESULTS Prothrombin Time is prolonged due to a deficiency of factors II, V, VII, and X and in the presence of Heparin. This can occur in the following conditions: ▪ Oral anticoagulant therapy (Vitamin K antagonists). ▪ Obstructive jaundice. ▪ Liver disease. ▪ DIC ▪ Malabsorption. ▪ Vitamin K deficiency. 51 ▪ Hereditary deficiency of concerned factors.  INTERNATIONAL NORMALISED RATIO (INR) PT test may also be called an INR test. INR stands for standardising the results of PT tests, no matter the testing method. It helps us understand the test results in the same way, even when they come from different labs and test methods. Using the INR system, treatment (with anticoagulant therapy) will be the same. In some labs, only the INR is reported and the PT is not reported. 52  THE INR VALUES An INR of 1.0 means that the patient’s PT is normal. An INR greater than 1.0 means the clotting time is elevated. INR of greater than 5 or 5.5 = unacceptable high risk of bleeding, whereas if the INR=0.5, there is a high chance of a clot. The normal range for a healthy person is 0.9–1.3, and for people on warfarin therapy, 2.0–3.0, although the target INR may be higher in particular situations, such as for those with a mechanical heart valve. 53  UNDERSTANDING THE INR It is needed for the standardisation of PT results. The International Normalized Ratio (INR) was introduced by WHO. A calibration system was developed to relate any PT ratio to a WHO standard. 54  ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT) PK, HMWK Ca2+ Tissue factor (TF) Principle: measures the clotting time of plasma after the activation of TF/ contact factors and the addition of PT phospholipid and CaCl2. measures o Reagents: platelet-poor plasma, Kaolin (add some glass beads), phospholipid, CaCl2 o Common causes of prolonged APTT? Coagulation cascades Modified from Figure 24.12, In: Hoffbrand Essential haematology, by Hoffbrand AV and 55 Steensma DP.,8th edition (2020). Wiley Blackwell  APTT Measures the clotting time of plasma after activating contact factors but without adding tissue thromboplastin. It evaluates the overall efficacy of the intrinsic coagulation pathway. It is also sensitive to circulating anticoagulants (inhibitors) and heparin. It depends on contact factors, factors VIII, IX, X, V, prothrombin, and fibrinogen. 56 APTT – PRINCIPLE Plasma is incubated with an activator (which initiates the intrinsic pathway of coagulation by contact activation). Phospholipids and calcium are then added, and clotting time is measured. 57  APTT TEST PROTOCOL REAGENTS: ❑ Platelet-poor plasma (PPP) - patient. ❑Kaolin 5 gm/l (0.5g in 100ml barbiton buffered saline, pH 7.4. ❑Silica, celite or ellagic acid can be used. ❑Phospholipid ❑Calcium chloride 0.025 mol/l 58  APTT PROCEDURE 1. Shake the reagent for APTT vigorously for approximately 15 seconds before use. 2. Add 100 µl of the reagent for APTT to a glass test tube pre-warmed in the water bath. 3. Add 100 µl of plasma to the tube, mix well and incubate in the water bath for 3 minutes. 4. Add 100 µl of calcium solution, mix, and start recording the time it takes for a visible clot to form immediately. 5. Repeat steps 1-4 for this plasma sample (duplicate). 6. In case of prolonged APTT, mixing studies will be needed. Mix 100 µl of the abnormal plasma with 100 µl of normal plasma in a fresh tube, and take 100 µl of the mixture for step 3. All the other steps are the same as above. Repeat steps 1-4 for this mixture of plasma samples. 59 60 APTT RESULTS 61  APTT PROCEDURE 2 1. Mix equal volumes of phospholipid reagent and the kaolin suspension and leave them in a glass tube in a water bath at 37°C. 2. Place 0.1ml of plasma into a new glass tube. 3. Add 0.2ml of the kaolin-phospholipid solution, mix the contents and start a stopwatch simultaneously. 4. Leave at 37°C for 10 minutes with occasional shaking. 5. At exactly 10 minutes, add 0.1ml of prewarmed CaCl2 and start a second stopwatch. 62  APTT PROCEDURE 2 … 6. Record the time taken for the mixture to clot. 7. Repeat the test on the patient and the control plasma at least once. 63  INTERPRETATION OF APTT Normal aPTT range: 30 – 40 seconds. A prolonged aPTT with Normal PT indicates a possible deficiency of factor VIII, IX, XI, XII, high molecular weight kininogen, prekallikrein, or the presence of an inhibitor. 64  CAUSES OF PROLONGED APTT Haemophilia A and B. Deficiencies of other coagulation factors in intrinsic and common pathways. Presence of coagulation inhibitors. Heparin therapy. DIC. Liver disease 65  CRITICAL LIMITS OF APTT Statistically, the aPTT is slightly lengthened in young individuals and slightly shortened in older populations. Premature infants have prolonged aPTT values, which return to normal by 6 months of age. Interferences: ❑Lipemia and hyperbilirubinemia interfere with the detection of clot formation by photo-optical methods. 66  THROMBIN TIME (TT) PK, HMWK Tissue factor (TF) Principle: measures the clotting time after thrombin is added to TF/ plasma PT measures Detect fibrinogen only Reagents: platelet-poor plasma, thrombin solution. Common causes of prolonged TT? Coagulation cascades Modified from Figure 24.12, In: Hoffbrand Essential haematology, by Hoffbrand AV and 67 Steensma DP.,8th edition (2020). Wiley Blackwell  THROMBIN TIME The thrombin time is the time required for thrombin to convert fibrinogen to an insoluble fibrin clot. Fibrin formation is triggered by the addition of thrombin to the specimen and, therefore, bypasses prior steps in the coagulation cascade. The TT does not measure defects in the intrinsic or extrinsic pathways. 68  THROMBIN TIME - PRINCIPLE Thrombin is added to the plasma, and the clotting time is measured. TT is affected by abnormal levels of fibrinogen and dysfibrinogenaemia and the presence of antithrombins such as heparin and direct thrombin inhibitors such as hirudin and FDPs. 69 THROMBIN TIME - PROTOCOL REAGENTS: Platelet-poor plasma A thrombin solution induces the clotting of normal plasma in about 15 seconds. 70  PROCEDURE FOR TT TIME 1. Add 100 µl of saline to a glass tube. 2. Place the tube in the 37ºC water bath for 3 - 5 minutes. 3. Add 100 µl of plasma to the tube and mix well. 4. Add 100 µl of diluted bovine thrombin and mix, then start the stopwatch to record the time immediately. Stop the stopwatch when a visible clot has formed. 5. Repeat steps 1-4 for this plasma sample. 6. If TT is prolonged, mixing studies should be carried out. To perform mixing studies, apply 100 µl of the abnormal plasma and 100 µl of the normal plasma (‘N’), respectively, into a fresh tube (50:50 mix), mix well and take 100 µl of the mixture for step 3. All the other steps are the same as above. The mixing study should be repeated. 71 72 TT RESULTS 73  CAUSES OF PROLONGED TT Hypofibrinogenaemia as in DIC and more rarely in a congenital defect or deficiency. Raised concentrations of FDP, as in DIC or liver disease. Extreme prolongation of the TT is nearly always a result of unfractionated heparin. Dysfibrinogenaemia, either inherited or acquired, in liver disease or neonates. Hypoalbuminaemia Paraproteinaemia. 74 75  MIXING STUDIES Also known as Correction Tests, Mixing Experiments or 50/50 using the prothrombin time (PT) or activated partial thromboplastin time (APTT). Principle: A 50:50 mix of the patient’s plasma with normal plasma, then detect PT or APTT. Reagents: used in PT and APTT, patient plasma, normal plasma. Interpretation: correction indicates a possible factor deficiency, whereas failure to correct suggests the presence of an inhibitor. 76  INTERPRETING MIXING STUDIES APTT EXAMPLE: APTT control = 35 Sec APTT test = 60 Sec If a 50:50 mix of the patient to control plasma gives 42 Sec – this is a good correction (i.e. factor deficiency): If a 50:50 mix of the patient to control plasma gives 52 Sec – this is a poor correction (i.e. probably, an inhibitor is present): 77  CONFIRMATORY TESTS Reptilase Time (RT) Mixing Tests Fibrinogen Assay (Modified Claus Assay) 78  MEASUREMENT OF FIBRINOGEN CONCENTRATION Principle of fibrinogen assay (Clauss technique): diluted plasma is clotted with a strong thrombin solution. o A standard curve is needed: record the clotting time for each serial dilution of the calibration plasma. o The plasma must be diluted to give a low level of any inhibitors (e.g. FDPs and heparin). o A strong thrombin solution must be used so that the clotting time over a wide range is independent of the thrombin concentration. Reagents: calibration plasma (with a known level of fibrinogen calibrated against an International Reference Standard), platelet-poor plasma, thrombin solution, and buffer. Interpretation: - Low fibrinogen levels: hypofibrinogenaemia/afibrinogenaemia, inherited dysfibrinogenaemia, others (e.g., DIC, liver disease). 79  FIBRINOGEN ASSAY - METHOD Make dilutions of the calibration plasma in veronal buffer to give a range of fibrinogen concentrations (i.e. 1 in 5, 1 in 10, 1 in 20, and 1 in 40). 0.2 ml of each dilution is warmed to 37°C. Add 0.1 ml of thrombin solution, Clotting time is measured. A calibration curve is prepared by plotting clotting time in seconds against the fibrinogen conc in g/l. The normal range is approximately 1.8–3.6 g/l. 80 FIB STD CURVE 81  MIXING STUDIES Also known as Correction studies or mixing experiments. It is used to distinguish between factor deficiencies and factor inhibitors. If APT, PT or TT is prolonged, the patient’s plasma is mixed with an equal volume of normal plasma. Plasma samples with abnormal screening tests (i.e. PT/APTT) may be further investigated. 82 83  INDIVIDUAL FACTOR ASSAYS One staged assay based on PT and APTT. Comparing the ability of dilutions of a standard or reference plasma and test plasma to correct the PT or APTT of a plasma known to be totally deficient in the measured clotting factor. Functional FVII activity is measured by a PT-based, FVII-deficient plasma clotting assay. Using r-human thromboplastin will yield results more likely to reflect in vivo FVII levels. Therapeutic options include FFP, prothrombin complex concentrates, and recombinant FVIIa. 84 For major surgery, plasma FVII levels of at least 20% are sufficient.  ONE-STAGE ASSAY OF FACTOR VII (BASED ON PT) Principle: ❑The assay of factor VII is based on the PT. Assay compares the ability of dilutions of the patient’s plasma and a standard plasma to correct the PT of a substrate plasma. It is easily adapted to the prothrombin assay, factor V or factor X. Reagents: ❑PPP from the patient. ❑Standard/reference plasma 85  ONE STAGE ASSAY OF FACTOR VII (BASED ON PT) Reagents … ❑Factor VII-deficient (substrate) plasma- Commercial or from a patient with known severe deficiency. ❑Barbitone buffered saline. ❑Thromboplastin 86  ONE-STAGE ASSAY OF FACTOR VII (BASED ON PT) Method: ❑Prepare 1 in 5, 1 in 10, 1 in 20 and 1 in 40 dilutions of the standard and test plas in buffered saline. ❑0.1 ml of each dilution +0.1 ml of deficient (substrate) plasma. ❑Mix and allow to warm to 37 °C. ❑Add 0.1 ml of dilute thromboplastin and start the stopwatch. ❑Record the clotting time. ❑Calculation of Results ❑Plot the clotting times of the test and standard against the concentration of factor VII on graph paper. 87 ❑The normal range is 50–150 iu/dl. 88  ONE-STAGE ASSAY OF FACTOR VIII (BASED ON APTT) Principle: Based on APTT according to the bioassay principle. Reagents: PPP from the patient, Standard/reference plasma, factor VIII deficient plasma (substrate), barbitone buffered saline, reagent for APTT, plastic tubes, and ice bath. Method-place the APTT reagent and CaCl2 37 °C and patient’s, standard and substrate plasma in the ice bath until used One- stage. 89 ONE-STAGE ASSAY OF FACTOR VIII (BASED ON APTT) METHOD: ❑Make 1 in 10 dilutions of the test and standard plasma in buffered saline in plastic tubes in the ice bath. ❑Using 0.2 ml volumes, make doubling dilutions in buffered saline to obtain 1 in 20 and 1 in 40 dilutions. ❑Place 0.1 ml of three dilutions in glass tubes. ❑Add 0.1 ml of freshly reconstituted or thawed substrate plasma to each dilution and warm up at 37°C. 90  ONE-STAGE ASSAY OF FACTOR VIII (BASED ON APTT) ❑Perform APTTs according to the laboratory protocol. ❑The dilutions should be tested at 2-minute intervals on the master watch. ❑ Normal range- 45- 158 IU/dL. 91  REDUCED FACTOR VIII CONCENTRATION IN: Reduced factor VIII concentration is found in: Haemophilia A VWD, types I and III and some cases of type II. Congenital combined deficiency of factors VIII and V. DIC 92  EXAMPLE In this example, the FVIII concentration in the test sample is 7 % of that in the standard. If the standard has a concentration of 85 IU/dL, the test sample has a concentration of 85 IU/dL × 7% = 6 IU/dL. 93  LAB INVESTIGATIONS OF FIBRINOLYTIC SYSTEM Detection of FDPs using latex agglutination method. D-dimer test. Euglobin clot lysis. 9 4  FDP ASSAYS FDPs are fragments produced by proteolytic digestion of fibrinogen or fibrin by plasmin. For the determination of FDPs, blood is collected in a tube containing thrombin (to remove all fibrinogen by converting it into a clot) and soybean trypsin inhibitor (to inhibit plasmin and thus prevent in vitro breakdown of fibrin). A suspension of latex particles linked to anti-fibrinogen antibodies is mixed with dilutions of patients’ serum on a glass slide. If FDPs are present, agglutination of latex particles occurs. Increased levels occur in DIC, DVT, severe pneumonia and recent MI. 95  D-DIMER ASSAY D-dimer is derived from the breakdown of fibrin by plasmin. The D-dimer test is used to evaluate fibrin degradation by plasmin. Blood samples can be either plasma or serum. Latex or polystyrene microparticles coated with monoclonal antibody to D-dimer are mixed with the patient’s sample and observed for microparticle agglutination. 96  D-DIMER IS RAISED IN DIC Intravascular thrombosis (MI, stroke, venous thrombosis, pulmonary embolism) and during the postoperative period or following trauma. 97  D-DIMER IS RAISED IN ❑D-dimers are raised in: ❑DIC, intravascular thrombosis (MI, stroke, venous thrombosis, pulmonary embolism) and during the postoperative period or following trauma. 98  REPTILASE TIME Assess the rate of fibrinogen → fibrin conversion in the presence of heparin. 99 AUTOMATED COAGULOMETER 100  CAVERON ALPHA ANALYZER It is a fully automatic coagulation measuring instrument used for in vitro diagnostic use. It performs all plasma coagulation tests, both routine and research. It can analyse samples using clotting, chromogenic and turbidimetric methods. The Caveron Alpha consists of an analyser, a personal computer and an optional printer. It operates on the photometric measurement principle. 101  CAVERON ALPHA ANALYZER This measurement method finds the coagulation time by optical detection of the change of turbidity caused by the formation of fibrin fibres. For chromogenic methods, the absorbance change is detected after adding a chromogenic substrate after plasma and reagent incubation. For immunological methods, the absorbance change is also detected during the antigen and antibody complex formation reaction. A calibration converts the results into concentration units. 102 THANK YOU 103  BONE MARROW - EXAMINATION - MODULE TITLE: MEDICAL LABORATORY HAEMATOLOGY I MODULE CODE: MLS402 MOSES D LUGOS; PHD [email protected] 1  LEARNING OBJECTIVES AT THE END OF THIS LECTURE, THE STUDENT WOULD BE ABLE TO: 1. DESCRIBE BONE MARROW STRUCTURE AND FUNCTIONS. 2. KNOW THE CONDITIONS FOR WHICH BONE MARROW EXAMINATION IS INDICATED. 3. INDICATE THE SITES OF BONE MARROW ASPIRATION IN THE DIFFERENT AGE GROUPS. 4. DISCUSS THE TECHNIQUES FOR THE PREPARATION AND EXAMINATION OF BONE MARROW SMEARS. 5. DISCUSS BONE MARROW CELLULARITY AND MYELOID TO ERYTHROID RATIO. 2  BONE MARROW BONE MARROW IS A HIGHLY CELLULAR, VISCOUS, AND HIGHLY VASCULAR TISSUE WITHIN THE HOLLOW CAVITIES OF HARD BONE. IT IS DESIGNED TO SUPPORT THE PROLIFERATION, DIFFERENTIATION, AND MATURATION OF HAEMATOPOIETIC CELLS. 3  BONE MARROW TYPES OF BONE MARROW: ❑MEDULLA OSSIUM RUBRA (RED MARROW - CONSISTING MAINLY OF HAEMATOPOIETIC TISSUE) ❑MEDULLA OSSIUM FLAVA (YELLOW MARROW - CONSISTING MAINLY OF FAT CELLS) 4  TYPES OF BONE MARROW RED MARROW: ❑ ACTIVE PORTION ❑ CONTAINS HAEMATOPOIETIC CELLS YELLOW MARROW: ❑INACTIVE PORTION ❑ CONTAINS FAT CELLS NORMAL VALUE – 3000 – 4000ML RED MARROW – 1500ML 5  NORMAL BONE MARROW ONE OF THE LARGEST ORGANS IN THE BODY. LOCATED WITHIN THE CAVITIES OF THE BONE. CONSISTS OF: ❑HAEMATOPOIETIC CELLS ❑VASCULAR SINUSOIDS ❑FIBROBLASTS ❑FAT CELLS ❑MACROPHAGES NO LYMPHATIC CHANNELS 6  BONE MARROW ON AVERAGE, BONE MARROW CONSTITUTES 4% OF THE TOTAL BODY MASS OF HUMANS. IN ADULTS WEIGHING 65 KG (143 LBS), BONE MARROW ACCOUNTS FOR APPROXIMATELY 2.6 KG (5.7 LBS). THE HAEMATOPOIETIC COMPARTMENT OF BONE MARROW PRODUCES APPROXIMATELY 500 BILLION BLOOD CELLS DAILY. 7 PATTERN OF DISTRIBUTION AT BIRTH, ALL BONE MARROW IS RED. WITH AGE, MORE AND MORE OF IT IS CONVERTED TO THE YELLOW TYPE; ONLY AROUND HALF OF ADULT BONE MARROW IS RED. IN ADULTS, RED MARROW IS FOUND MAINLY IN THE FLAT BONES AND THE EPIPHYSEAL ENDS OF LONG BONES SUCH AS THE FEMUR AND HUMERUS. YELLOW MARROW IS FOUND IN THE MEDULLARY CAVITY, THE HOLLOW INTERIOR OF THE MIDDLE PORTION OF LONG BONES. 8 http://cnx.org/content/col11496/1.6/, Jun 19, 2013. AMOUNT OF FATTY TISSUES THE AMOUNT OF FATTY TISSUES DEPENDS ON THE ACTIVITY OF THE HAEMATOPOIETIC CELLS. CHILDREN – 20 -20% OF FAT CELLS ADULTS – 50% OF FAT CELLS ELDERLY – 70% OF FAT CELLS 9 SITES OF HAEMATOPOIESIS 10  STRUCTURE OF BONE MARROW VASCULAR COMPARTMENT HAEMATOPOIETIC COMPARTMENT: ❑HAEMATOPOIETIC CELLS ❑STROMAL CELLS 11 HAEMATOPOIETIC CELLS 12 TYPES OF STEM CELLS 13  STROMA IT ISN’T DIRECTLY INVOLVED IN THE PRIMARY FUNCTION OF HAEMATOPOIESIS. THE YELLOW BONE MARROW BELONGS HERE AND MAKES MOST OF THE BONE MARROW STROMA AND STROMAL CELLS IN THE RED BONE MARROW. STILL, THE STROMA IS INDIRECTLY INVOLVED IN HAEMATOPOIESIS SINCE IT PROVIDES THE HAEMATOPOIETIC MICROENVIRONMENT, I.E. STRUCTURE SUPPORT. FOR INSTANCE, THEY GENERATE COLONY-STIMULATING FACTORS, AFFECTING HAEMATOPOIESIS. 14  BONE MARROW STROMA CELLS THAT CONSTITUTE THE BONE MARROW STROMA ARE: ❑FIBROBLASTS (RETICULAR CONNECTIVE TISSUE) ❑MACROPHAGES ❑ADIPOCYTES ❑OSTEOBLASTS ❑OSTEOCLASTS ❑ENDOTHELIAL CELLS, WHICH FORM THE SINUSOIDS. 15 16  FUNCTIONS OF BONE MARROW BONE MARROW MAKES STEM CELLS, WHICH PRODUCE PLATELETS, WHITE BLOOD CELLS AND RED BLOOD CELLS. BONE MARROW PRODUCES RED BLOOD CELLS THAT CARRY OXYGEN, WHITE BLOOD CELLS THAT PREVENT INFECTION AND PLATELETS THAT CONTROL BLEEDING. THE ABSENCE OF BONE MARROW CAN BE FATAL SINCE IT'S AN ESSENTIAL PART OF THE BODY. 17 HISTORICAL PERSPECTIVE OF BONE MARROW EXAMINATION 18  BONE MARROW EXAMINATION REFERS TO THE PATHOLOGIC ANALYSIS OF SAMPLES OF BONE MARROW OBTAINED BY BONE MARROW ASPIRATION AND BONE MARROW BIOPSY(OFTEN CALLED A TREPHINE BIOPSY). THE ASPIRATE YIELDS SEMI-LIQUID BONE MARROW, WHICH CAN BE EXAMINED ❑UNDER A LIGHT MICROSCOPE ❑ ANALYSED BY FLOW CYTOMETRY (IMMUNOPHENOTYPING) ❑CHROMOSOME ANALYSIS, OR ❑POLYMERASE CHAIN REACTION(PCR) 19  BONE MARROW EXAMINATION IT PROVIDES A SEMI-QUANTITATIVE AND QUALITATIVE ASSESSMENT OF THE STATE OF HAEMATOPOIESIS. IT CAN BE: 1. BONE MARROW ASPIRATION 2. BONE MARROW BIOPSY 3. CLOT SECTION 4. BIOPSY IMPRINT SMEARS Bone marrow biopsy and aspiration - Mayo Clinic 20  BONE MARROW EXAMINATION IT IS AN INDISPENSABLE ASPECT OF THE HAEMATOLOGICAL INVESTIGATION AND DIAGNOSIS. MARROW EXAMINATION IS NECESSARY FOR: ❑DISCOVERING OR CONFIRMING A VARIETY OF DIAGNOSES. ❑THE MONITORING OF HAEMATOLOGIC AND NON-HAEMATOLOGIC DISEASES (SOLID TUMOURS) AND LEUKAEMIC PATIENTS UNDERGOING INTENSIVE CHEMOTHERAPY. 21  BONE MARROW EXAMINATION CYTOLOGICAL AND HISTOLOGICAL EXAMINATION ARE THE MAJOR ASPECTS OF BONE MARROW INVESTIGATION, ALTHOUGH IN RECENT YEARS: ❑BONE MARROW CULTURE FOR CYTOGENETIC AND KINETIC STUDIES. ❑ISOTOPIC LABELLING. ❑PROCESSING FOR ELECTRON MICROSCOPY. ❑CLONAL STUDIES. ❑CULTURE AND OTHER METHODS HAVE BECOME ESTABLISHED. 22  APPLICATIONS OF BONE MARROW EXAMINATION DIAGNOSE AND STAGE HAEMATOLOGIC AND NONHAEMATOLOGIC NEOPLASIA. DETERMINE THE CAUSE OF CYTOPENIAS. CONFIRM OR EXCLUDE METABOLIC AND INFECTIOUS CONDITIONS. 23  REQUEST FOR BONE MARROW EXAMINATION EACH BONE MARROW PROCEDURE IS ORDERED AFTER CONSIDERATION OF CLINICAL SYMPTOMS AND PERIPHERAL BLOOD FINDINGS BONE MARROW PUNCTURE IS PROHIBITED IN PATIENTS WITH COAGULOPATHIES SUCH AS HAEMOPHILIA OR VITAMIN K DEFICIENCY, ALTHOUGH THROMBOCYTOPENIA IS NOT AN ABSOLUTE CONTRAINDICATION. 24  INDICATIONS FOR BONE MARROW EXAMINATION UNEXPLAINED ANAEMIA, CYTOPENIA OR CYTOSIS. ABNORMAL PERIPHERAL BLOOD SMEAR MORPHOLOGY DIAGNOSIS, STAGING AND FOLLOW-UP OF HAEMATOLOGICAL MALIGNANCIES: o ACUTE AND CHRONIC LEUKAEMIAS, o MYELODYSPLASTIC SYNDROMES, o CHRONIC MYELOPROLIFERATIVE DISORDERS, o LYMPHOMAS, o PLASMA CELL MYELOMA, o AMYLOIDOSIS, o MASTOCYTOSIS 25 o INVESTIGATION OF SUSPECTED BONE MARROW METASTASES INDICATIONS FOR BONE MARROW BIOPSY HYPERCALCEMIA, LYTIC LESIONS, HYPERGAMMAGLOBULINEMIA. LYMPHOMA STAGING (CLASSIFICATION). INFECTIONS (GRANULOMA). FEVER OF UNKNOWN ORIGIN METASTATIC (TRANSFER OF DISEASE FROM ONE ORGAN TO ANOTHER) WORKUP. SMALL CELL TUMOURS OF CHILDHOOD MAST CELL DISEASE IDIOPATHIC THROMBOCYTOPENIC PURPURA PRIMARY AMYLOIDOSIS 26 INDICATIONS FOR BONE MARROW BIOPSY METABOLIC BONE DISEASE THERAPEUTIC FOLLOW-UP: o CHEMOTHERAPY/BONE MARROW TRANSPLANTATION o TREATMENT OF ISOLATED CYTOPENIA 27  TYPES OF BONE MARROW COLLECTION BONE MARROW ASPIRATION oIS A PROCEDURE TO COLLECT A SAMPLE OF BONE MARROW FLUID. oTHIS FLUID IS THEN LOOKED AT UNDER THE MICROSCOPE. oIT IS CHECKED FOR ABNORMAL CELLS. IT MAY ALSO BE TESTED IN OTHER WAYS. oUSUALLY SIMPLE, SAFE AND RELATIVELY PAINLESS BONE MARROW BIOPSY oA TREPHINE BIOPSY YIELDS A NARROW, CYLINDRICALLY SHAPED SOLID PIECE OF BONE MARROW WHICH IS EXAMINED MICROSCOPICALLY (SOMETIMES WITH THE AID OF IMMUNOHISTOCHEMISTRY) FOR CELLULARITY AND INFILTRATIVE PROCESSES. 28 BONE MARROW ASPIRATION VS BIOPSY BONE MARROW ASPIRATION o FINE CYTOLOGICAL DETAILS o CYTOCHEMICAL STAINS o MICROBIOLOGICAL CULTURE o FLOW CYTOMETRY o CYTOGENETIC AND MOLECULAR STUDIES BONE MARROW BIOPSY o ASSESSMENT OF CELLULARITY AND ARCHITECTURE o DETECT LESIONS o ASSESSMENT OF APLASTIC ANAEMIA, METASTASIS o ARCHIVAL MATERIAL 29  BONE MARROW SPECIMENS SAMPLES OF BONE MARROW CAN BE OBTAINED BY: o ASPIRATION USING A SPECIAL NEEDLE AND SYRINGE, E.G., SALAH, KLIMA, AND ISLAM’S ASPIRATION NEEDLES. o PERCUTANEOUS TREPHINE, IN WHICH A SECTION OF BONE IS TAKEN FOR EXAMINATION. o OPEN SURGICAL BIOPSY OR OPEN TREPHINE THAT REQUIRES FULL OPERATING THEATRE PRACTICE. MOST BONE MARROW SAMPLES FOR HAEMATOLOGICAL PURPOSES ARE OBTAINED BY ASPIRATION, OFTEN COMBINED WITH NEEDLE OR TREPHINE BIOPSY: o THE ASPIRATION PROCEDURE IS USUALLY SIMPLE, SAFE AND RELATIVELY PAINLESS. 30  SITES OF BONE MARROW ASPIRATION AND BIOPSY THE SITE SELECTED FOR THE ASPIRATION DEPENDS ON: o THE AGE OF THE PATIENT o WHETHER OR NOT A NEEDLE OR TREPHINE BIOPSY IS REQUIRED THE APPROPRIATE SITES IN AN ADULT INCLUDE THE POSTERIOR ILIAC CREST (PREFERRED SITE), ANTERIOR ILIAC CREST, AND STERNUM. UNDER 12 YEARS – ILIAC CREST. THE TIBIA MAY BE USED IN INFANTS YOUNGER THAN 18 MONTHS OF AGE. 31  SITES OF BONE MARROW ASPIRATION AND BIOPSY THE STERNUM o THE BEST SITE WHEN ASPIRATION ONLY IS NEEDED o THE EASIEST TO PUNCTURE o CONSIDERED TO YIELD THE MOST CELLULAR SAMPLES o A DISADVANTAGE IS THAT THE PATIENT HAS A CLEAR VIEW OF THE PROCEDURE, WHICH MAY CAUSE DISTRESS. 32  SITES OF BONE MARROW ASPIRATION AND BIOPSY ANTERIOR OR POSTERIOR ILIAC SPINES o HAVE THE ADVANTAGE THAT IF NO MATERIAL IS ASPIRATED, A MICRO TREPHINE BIOPSY CAN BE PERFORMED IMMEDIATELY. IN DISORDERS ASSOCIATED WITH THE REPLACEMENT OF HAEMOPOIETIC MARROW BY OTHER TISSUES OR CELLS (E.G., MALIGNANCIES IN THE BONE MARROW) o MARROW ASPIRATION MAY BE DIFFICULT OR IMPOSSIBLE; THE SO-CALLED DRY TAP - IN SUCH CASES, A NEEDLE OR TREPHINE BIOPSY IS ESSENTIAL. 33 NEEDLES FOR ASPIRATION AND BIOPSY SAHLA TYPE KILMA JAMSHIDI 34 SWENT ASPIRATION AND BIOPSY BONE MARROW BIOPSY FROM THE SUPERIOR PART OF THE POSTERIOR ILIAC SPINE (BACK OF THE HIPBONE) 35  ASPIRATION AND BIOPSY INFANTS AND CHILDREN: THE STERNUM IS NATURALLY THIN, AND AN ALTERNATIVE SITE IS PREFERRED. o UNDER 12 YEARS – ILIAC CREST o UNDER 2 YEARS – ACTIVE MARROW IN THE LONG BONES MAKES THE PROXIMAL ANTERIOR PORTION OF THE TIBIA A POSSIBLE SITE. IN DISORDERS ASSOCIATED WITH THE REPLACEMENT OF HAEMOPOIETIC MARROW BY OTHER TISSUES OR CELLS (E.G., MALIGNANCIES IN THE BONE MARROW). o MARROW ASPIRATION MAY BE DIFFICULT OR IMPOSSIBLE; THE SO-CALLED DRY TAP ❑ IN SUCH CASES, A NEEDLE OR TREPHINE BIOPSY IS ESSENTIAL. 36  ASPIRATION AND BIOPSY A MINIMUM AMOUNT OF MARROW SHOULD BE ASPIRATED o VOLUMES OVER 0.5ML WILL ALMOST CERTAINLY BE DILUTED WITH BLOOD, MAKING PROCESSING AND INTERPRETATION MORE DIFFICULT. CAREFUL PREPARATION IS ESSENTIAL. IT IS DESIRABLE, IF POSSIBLE, TO CONCENTRATE THE MARROW CELLS AT THE EXPENSE OF THE BLOOD IN WHICH THEY ARE DILUTED. 37  PREPARATIONS FOR THE PROCEDURE THE PROCEDURE SHOULD BE EXPLAINED IN DETAIL TO THE PATIENT. THE PAST CLINICAL HISTORY OF THE PATIENT SHOULD BE OBTAINED INFORMED CONSENT SHOULD BE OBTAINED FROM THE PATIENT. A BLOOD COUNT AND SMEAR SHOULD BE OBTAINED ADEQUATE SEDATION AND ANALGESIA DETERMINED ASSESSMENT OF THROMBOCYTOPENIA OR COAGULOPATHIC RISKS CONSIDER THE SITE FOR BM EXAMINATION CAREFULLY 38  PROCEDURE FOR BONE MARROW ASPIRATION 1. PATIENT PREPARATIONS SHOULD BE ENSURED. 2. A NEEDLE IS INSERTED INTO THE ILIAC CREST OR SPINE 3. THE NEEDLE AND STYLET ARE PUSHED INTO THE BONE WITH A SLIGHT ROTARY MOTION. 4. WHEN IT IS FELT THAT THE NEEDLE IS FIRMLY IN PLACE, THE STYLET IS REMOVED, A SYRINGE WITHOUT ANTICOAGULANT IS ATTACHED, AND 5,000 ) and bone marrow (± spleen and lymph node). The most common adult leukaemia (~25%). The median age is ~55 to 65 years. ( rare < 40 years). 1.5 to 2 times more common in men than women. 17  FEATURES OF CLL 40% of patients are asymptomatic at diagnosis. Moderate lymphadenopathy and splenomegaly. Lymphocytosis (>5,000/µL): o Small mature appearing lymphocytes o Condensed (“soccer ball”) nuclear chromatin o Numerous “smudge cells” Predisposition in infarction Autoimmune phenomena, particularly autoimmune haemolytic anaemia. 18 CLL STAGING 19  IMMUNOPHENOTYPE OF SMALL B-CELL NEOPLASMS 20  LYMPHOMA ▪ Malignancies of developing lymphoid cells (predominantly B cells) restricted to the lymphatic system rather than the peripheral blood. ▪ 2 Main types: o Hodgkin lymphoma o Non-Hodgkin lymphoma ▪ more common than Hodgkin lymphoma ▪ several subtypes. 21  MOST COMMON SITES inguinal 22  CLINICAL FEATURES Lymphadenopathy, or swelling of lymph nodes – It is the primary presentation in lymphoma. “B symptoms” o Fever o Night sweats o Weight loss DAlcohol-induced pain (Hodgkin) Alcohol-induced pain (Hodgkin) Pruritis Loss of appetite or anorexia 23 Fatigue  HODGKIN LYMPHOMA (HODGKIN’S DISEASE) First Haematological Malignancy Described by Thomas Hodgkin 1832 In 1898, Sternberg described a unique cell in this tumour, further described by Reed - The Reed-Sternberg Cell Large, abnormal lymphocytes may contain more than one nucleus. Also called Hodgkin and Reed-Sternberg cells. Found in people with Hodgkin lymphoma. 24  PATHOGENESIS Aetiology unknown Possible infectious aetiology Up to 50% of cases associated with EBV Increased incidence with higher social status, better housing, and smaller families. Possible abnormal response to infection later in life. 25 A POSSIBLE MODEL OF PATHOGENESIS 26  CLASSICAL HODGKIN LYMPHOMA ▪ It is relatively aggressive malignant lymphoma characterised by: o presence of a few large binucleated cells (Reed-Sternberg ) surrounded by reactive cells (lymphocytes, plasma cells, eosinophils). o involving cervical lymph nodes in young adults (most often) 27  AGE-RELATED INCIDENCE Can occur at any age But normally, bi-modal distribution 20-30 years (commonest malignancy in young adults) >50 years Twice as common in males Incidence has not changed in 20 years 28  CLINICAL FEATURES Most cases present with painless, asymmetrically enlarged lymph nodes. o 60-70% cervical o 10-15% axillary o 5-12% inguinal 29  LOCATION OF LYMPHADENOPATHY ▪ 80% LN above the diaphragm oAntireior mediastinum oCervical oSupraclavicular 30  SYSTEMIC SYMPTOMS ▪ 40% of patients - systemic symptoms oB-symptoms - fever, night sweat, weight loss oChronic pruritus 31 EXTRANODAL INVOLVEMENT ▪ “E” lesion by direct invasion# ▪ Haematogenous metastasis (stage VI): Spleen, lungs 32 HODGKIN LYMPHOMA CLINICAL PRESENTATION 33  EPIDEMIOLOGY ▪ Age: Bimodal peak age oThird decade oAfter 50 years ▪ Gender: Male to 34  AETIOLOGY AND RISK FACTORS ▪ Unknown ▪ Possible aetiological factors oFamilial factor oViruses - EBV 35  SIGNS AND SYMPTOMS ▪ HD is a lymph node-based malignancy. ▪ Common = asymptomatic lymphadenopathy ▪ Systemic symptoms ▪ Extranodal involvement 36  DIAGNOSIS ▪ Biopsy ▪ Pathology: “Reed-Sternberg Cell” oDiagnostic tumour cell oMust be identified oLarge size, binucleated, large eosinophilic nucleoli 37  HISTOLOGY ▪ Lymphocyte predominant (LP) = few RS cells, good prognosis. ▪ Nodular sclerosis (NS) = the most common, young adult female. ▪ Mixed cellularity (MC) = generalised lymphadenopathy. ▪ Lymphocyte depletion (LD) = numerous RS cells, poor prognosis. 38  STAGING: THE COTSWOLDS CLASSIFICATION FOR HD ▪ I: A single LN region or lymphoid structure (eg, spleen, thymus, Waldeyers ring). ▪ II: Two or more LN regions on the same side of the diaphragm. ▪ III: LN regions or structures on both sides of the diaphragm. o1: With/without involvement of splenic hilar, celiac, or portal nodes 39  DESIGNATIONS APPLICABLE TO ANY DISEASE STAGE ▪ A: No symptoms ▪ B: Fever, drenching sweats, weight loss. ▪ X: Bulky disease o1/3 the width of the mediastinum o10cm maximal dimension of nodal mass. ▪ E: Involvement of a single 40  REED-STERNBERG CELL Large lobulated or multi-nucleate cells Also present are lymphocytes and eosinophils 41 REED-STERNBERG CELLS ARE CD15+ AND CD45- CD15 CD45 42  CLINICAL STAGING Spread by contiguity in the lymphoid system, i.e., it spreads sequentially from one site to the neighbouring nodes. o Stage I: tumour in one lymph node area o Stage II: tumour in 2+ sites on same side of diaphragm o Stage III: tumour in lymph nodes on both sides of the diaphragm o Stage IV: tumour outside the lymph nodes o Stage may be modified by the presence of “B symptoms” (B) or not (A) 43  TREATMENT - RADIOTHERAPY ▪ Mainly for Stage I/II disease mantle Inverted Y 44  CHEMOTHERAPY MOPP ABVD Mustine Adriamycin (Doxorubicin) Alkylating agent Anthracycline (intercalator) Oncovin Bleomycin Tubulin polymerisation inhibitor DNA Strand breaks Procarbazine Vinblastin (Oncovin) Alkylating agent Tubulin polymerisation inhibitor Prednisolone Dacarbazine corticosteroid Alkylating agent 45  PROGNOSIS Mortality has fallen dramatically over the last 30 years: ▪ Stage I + II 85% five-year survival ▪ Stage III - 70% ▪ Stage IV - 50% 46  NON-HODGKIN’S LYMPHOMA ▪ Malignancies of lymphoid tissue ▪ May occur in many different tissues ▪ Wide range of histological classification ▪ Variable clinical presentation ▪ Most cases involve B-lymphocytes 47  EPIDEMIOLOGY ▪ Incidence increases by 2-3% per year ▪ Rural areas > urban areas ▪ (increase not due to AIDS-related lymphoma alone) ▪ 57,000 new cases USA 1999 (HD 7,200) ▪ 26,000 deaths (HD 1,200) ▪ 5th leading cause of cancer death in males. 48  CLINICAL PRESENTATION ▪ Highly variable ▪ Asymmetric painless lymph nodes ▪ Liver and spleen often enlarged ▪ Many organs may be affected 50  DIAGNOSIS ▪ Cytology - lymph node biopsy ▪ PCR ▪ Immunophenotyping (Flow cytometry) ▪ FISH testing. 51  STAGING ▪ Stage 1 oIn one group of lymph nodes or oIn just one organ or area of the body outside the lymphatic system (extranodal lymphoma) ▪ Stage 2 oIn 2 or more groups of lymph nodes on the same side of your diaphragm or oIn 1 or more groups of lymph nodes and also in one nearby organ or area of the body, all on the same side of the diaphragm 52  STAGING ▪ Stage 3 oIn lymph nodes on both sides of the diaphragm or oIn lymph nodes on both sides of the diaphragm and a nearby organ or area of the body is affected ▪ Stage 4 oThroughout one or more organs that are not part of the lymphatic system or oIn an organ that is not part of the lymphatic system, and it has also spread to organs or lymph nodes far away from the organ or oIn your liver, bone marrow, cerebrospinal fluid (CSF) or lung (unless it has spread to your lung from nearby lymph nodes) 53  2 BROAD TYPES OF NHL ▪ Indolent Lymphomas – Slow growing, not invasive ▪ Aggressive Lymphomas – Rapidly growing, invasive 54  FOLLICULAR LYMPHOMA ▪ FL is a malignant proliferation of germinal centre B cells centrocyte with at least a partially follicular pattern. ▪ Due to overexpression o f Bcl2 caused by t(14;18). ▪ Most common type of “indolent” lymphoma (25% ). ▪ Presented as Lymphadenopathy (100%), splenomegaly (80%), BM involvement (60%) and blood involvement (40%). ▪ Indolent but incurable (some exceptions). 55  BCL-2 AND FOLLICULAR LYMPHOMA ▪ >90% of all cases have a chromosomal translocation - t (14;18). ▪ Joins Bcl-2 gene on chromosome 18 to Ig heavy chain gene on chromosome 14 ▪ Deregulation of bcl-2 ▪ Bcl-2 inhibits apoptosis 56  B SYMPTOMS ▪ Fever (greater than 38°C) ▪ Drenching night sweats which soak your nightclothes and bedding ▪ Unexplained weight loss in the last six months (10% or more of your previous weight). 57  DIAGNOSIS ▪ Immunophenotyping: oPostive for CD10, CD20 and Bcl2. oNegative for CD5 (in most cases) 58  MANAGEMENT ▪ Median survival is about 10 years. ▪ Transformation to aggressive lymphoma (DLBCL) can occur. 59  BURKITT’S LYMPHOMA ▪ 2 variants: oEndemic – African Children oLarge jaw mass oAssociated with EBV, malaria 60  BURKITT'S LYMPHOMA ▪ High-grade non-Hodgkin's B-cell lymphoma, which is rapidly growing and highly aggressive with extremely short doubling time (24 hrs). ▪ Types of Burkitt's lymphoma: oEndemic: It is associated with chronic malaria and EBV in equatorial Africa. It particularly affects the jaw, other facial bones, and breasts. oSporadic: occurs throughout the world and affects GIT. oImmunodeficiency-associated: associated with HIV infection or the use of immunosuppressive drugs. 61  GENETICS OF BURKITT'S LYMPHOMA ▪ There is a universal association between Burkitt’s lymphoma and translocation of the c-MYC proto- oncogene at ch 8 to the immunoglobulin gene at ch14 t(8;14). ▪ The c-MYC is a nuclear transcription factor. Burkitt’s lymphoma is the fastest-growing tumour in humans. 62  CLINICAL PRESENTATION ▪ CURE RATE: o90% at early phase After 25 D of intensive o70% at advanced disease. chemotherapy 63  BURKITT’S LYMPHOMA ▪ Both types show diffuse proliferation of small lymphocytes with lighter staining histiocytes – ‘starry sky’ 64  MOLECULAR BIOLOGY ▪ Chromosomal translocations involving: ▪ c-myc oncogene on chromosome 8 ▪ immunoglobulin genes o Heavy chain t(8;14) 75-85% 65  DLBCL CYTOLOGY H & E section of a DLBCL Touch prep smear of DLBCL 66  DLBCL - IMMUNOPHENOTYPE positive negative DLBCL is positive for expression of CD20 DLBCL shows a monoclonal light chain restriction - only expresses kappa OR lambda normal B-cell populations express both light chains. 67  TREATMENT: R-CHOP CHEMOTHERAPY R – Rituximab (anti-CD20 monoclonal Ab) C – Cyclophosphamide alkylating agent (damages DNA) H – Hydroxydaunorubicin - anthracycline antibiotic (intercalator) O – Oncovin (Vincristine) – inhibitor of tubulin polymerisation P – Prednisolone - corticosteroid 68 EFFECT OF RITUXIMAB THERAPY ON SURVIVAL IN NHL PATIENTS 48.5% 69  MULTIPLE MYELOMA ▪ Malignant B neoplasm characterised by a triad of abnormalities: oAccumulation of plasma cells in the bone marrow. oLytic Bone lesions oProduction of a monoclonal immunoglobulin (Ig) or Ig fragments. 70 PATHOGENESIS OF MM 71  SUMMARY ▪ Lymphoma is a malignancy of lymphoid cells (usually B cells) that is restricted to the lymphoid system rather than the peripheral blood ▪ Divided into 2 types: Hodgkin and Non-Hodgkin (NHL) ▪ Hodgkin is associated with the presence of RS cells and is linked with EBV infection ▪ Progresses sequentially to adjacent nodes ▪ Treated by chemo or radiation ▪ NHL indolent or aggressive ▪ A number of types of NHL - prognoses vary ▪ Burkitt is very aggressive – linked directly to EBV infection and malaria ▪ DLBCL – most common type ~55% ▪ Treated with chemotherapy and monoclonal antibody (rituximab) 72 THANK YOU 73  Classification and investigation of common haemoglobinopathies COURSE TITLE: MEDICAL LABORATORY HAEMATOLOGY I COURSE CODE: MLS402 1 MOSES D LUGOS; PhD [email protected] 2 Learning Outcomes ❑ At the end of this lecture, you should be able to: ▪ Define and state the functions of haemoglobin. ▪ Describe the normal structure of haemoglobin. ▪ Understand haemoglobinopathies ▪ Describe the diagnostic investigations of haemoglobinopathies. 3 Introduction  4 The Red Blood Cell Stained RBCs RBCs Haemoglobin in a RBC 5 Haemoglobin  Haemoglobin (Hb or Hgb) is the protein in RBCs that carries O2 from the lungs to the body's tissues and returns CO2 from the tissues to the lungs.  It is estimated that each RBC contains about 250 million Hb molecules.  Therefore, each RBC can carry one billion oxygen molecules.  Hb is 97% saturated when it leaves the lungs.  Deficiency of Hb can result in anaemia. 6 Haemoglobin Structure  Tetrameric (4 polypeptides) protein – 2 pairs of globin chains.  One pair is alpha-like, and the other beta- like – variants.  Each polypeptide chain has a haem (heme) group.  A haem group binds Fe2+  Each Fe2+ binds to a molecule of O2.  Therefore, each Hb molecule binds 4 O2 molecules. 7 Haemoglobin Variants Hb A (α2β2) Hb Gower 1 (ζ2ϵ2) Hb F (α2γ2) Hb A2 (α2δ2) Hb Portland (ζ2 γ2) Hb Gower 2 (α2 ϵ2) 8 Haemoglobin Variants  Haemoglobin A (α2β2) – normal haemoglobin after birth – adult Hb.  Haemoglobin A2 (α22); minor component of erythrocytes after birth ( GTG in codon 6 leading to amino acid substitution  Haemoglobin S is the resulting variant associated with SCA  Differences in the aa properties cause structural changes in the haemoglobin molecules.  13 Inheritance of SCD Autosomal recessive Adapted from Fig.154-3Ai. In: Goldman-Cecil Medicine, by Goldman Adapted from Fig.24.2. In: Rodak’s Hematology: Clinical Principles and L and Schafer AI, 26th edition (2020). Elsevier Applications, by Keohane EM, et al. 6th edition (2020). Elsevier N.B. Some authors describe SCD as autosomal codominant inheritance, with one  gene inherited from each parent 14 15 Sickle Cell Anaemia  Most common haemoglobinopathy  Single gene disorder following Mendelian Inheritance  Autosomal Recessive Inheritance  Frequency of mutated gene – 1:5 in West African; 1:10 in Afro-Carribean; 1:50 in Asian; and 1:100 in Mediterranean and Middle eastern ethic groups.  Hypothesised that the SCA has evolved as protection against malaria ❑Heterozygotes - some resistance to malaria and asymptomatic. ❑Homozygotes – more resistant but suffer from serious clinical manifestations. 16 Sickle Cells Formation  HbS forms crystals in a low-oxygen environment.  The sickle Hb polymerises into long fibres, leading to a change in the shape of erythrocytes.  Sickle cells are less flexible – which can lead to the blocking of tiny blood vessels and infarction of various organs. 17 Laboratory Investigations of Haemoglobinopathies 18 Laboratory Investigations  METHODS - 1. Full Blood Count or CBC and Peripheral Blood Film reading. 2. Sickling test 3. Sickle solubility test 4. Hb Electrophoresis 5. High-performance liquid chromatography (HPLC) 6. DNA analysis (Restriction fragment length polymorphism ( RFLP)) 7. Paternal testing 19 Full Blood Count  Also known as CBC, provides a screen:  Blood film examination and reporting 20 Hb Sickling Test  Also known as the dithionite solubility test.  Sodium dithionite (Na2S2O4), sodium hydrosulfite, is a potent reducing agent.  PRINCIPLE – ❑ When dithionite encounters haemoglobin, it readily removes an oxygen molecule from the iron atom within the heme group, converting it from oxyhaemoglobin (HbO2) to deoxyhaemoglobin (Hb). ❑ In a deoxygenated state, RBCs having HbS will form a typical sickle shape as dithionite oxygen depletion triggers HbS to polymerise. 21 Hb Sickling Test …  MATERIALS – ❑ Anticoagulated patient’s peripheral blood sample. ❑ Dithionite solution: 10% sodium dithionite (Na2S2O4) in phosphate buffer, pH 6.8. ❑ Pasteur pipette ❑ Microscope slides and coverslips. ❑ Petroleum jelly-paraffin wax or depex. 22 Hb Sickling Test …  REAGENT – ❑ Make a fresh dithionite solution by dissolving 10 mg of sodium dithionite in 1 mL of phosphate buffer, pH 6.8. Mix well and use immediately.  PROCEDURE – ❑ Place a drop of dithionite solution and a drop of anticoagulated blood into a test tube. ❑ Mix the blood and dithionite gently to ensure thorough mixing. ❑ Place a drop of the mixture on a clean microscope slide. ❑ Cover the slide with a coverslip and avoid trapping air bubbles. ❑ Cover all edges of the cover slip with petroleum jelly-paraffin wax or depex. ❑ Observe under the microscope. 23 Hb Sickling Test …  INTERPRETATION –  In HbS disease, sickling can manifest significantly faster than the HbS trait.  While HbS disease might exhibit immediate or near-immediate sickling, observing clear sickling in the HbS trait within an hour at 37°C might not be conclusive and might require additional time or altered conditions like lower oxygen tension. 24 Sickling Positive 25 SICKLE SOLUBILITY TEST  PRINCIPLE – ❑ Under deoxygenated conditions, HbS becomes insoluble, and the crystals refract light, causing the solution to be turbid.  BRIEF METHOD – ❑ In the sickle solubility test, a phosphate buffer supplemented with saponin and sodium dithionite is mixed with blood samples (saponin is used as a haemolyser and sodium dithionite is used as a reducing agent to induce deoxygenation). ❑ After incubation, the solution is placed in front of a white card with narrow black lines and read for turbidity. ❑ If the solution is opaque or turbid, as defined by an inability to see the lines through the test tube, then the result is deemed positive, indicating the presence of HbS.  SICKLE SOLUBILITY TEST RESULTS 26 1 2 1 2  FIRST PANEL – Tube 1 is positive for HbS  SECOND PANEL – Tube 2 is positive for HbS 27 Sickle Solubility Test X  In deoxygenated conditions, HbS is insoluble Y  Crystals are formed, which refract the light.  Blood sample is added to a phosphate buffer with saponin and sodium dithionite.  Saponin – causes haemolysis.  Sodium dithionite – induces deoxygenation.  Opaque/turbid solution after incubation Sickle Solubility test means positive result, i.e. presence of HbS. 28 Sickle Solubility Test … Patients A, B, and C are the same as the electrophoresis ones Make sure turbidity is recorded before you centrifuge Draw/ capture results before and after centrifugation After you have observed your findings for the solubility test, consider: What is the red band at the top of the tube? Why the solution is pink or colourless after centrifugation? 29 How to Read Sickle Solubility Test  From the previous slide, we have also run solubility for samples in lanes 1,2 and 5.  Based on gel data which is which in the images below? 5 2 5 1 2 30 Example X Y Z  Electrophoresis  Separation technique which can identify Hb molecule variants based on charge and mol. weight.  The medium is commonly agarose.  Common in molecular biology – can be used for DNA, RNA, and protein Today, we will do PROTEIN ELECTROPHORESIS Haemoglobin electrophoresis (Acidic pH)  Haemoglobin Electrophoresis  Variants of Hb have different aa – slightly different charges.  Haemoglobin standards are used to mark known variants’ migration.  Haemoglobin variants can be identified by cellulose acetate/agarose at alkaline pH or citric agar/agarose at acidic pH. At alkaline pH At acidic pH ❑Hb is negatively charged ❑Used as confirmation ❑Migrates towards anode(+) ❑Various Hb charges ❑Separation based on charge. ❑Interaction with components in agarose