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1 Laboratory Medicine Newsletter for clinicians, pathologists & clinical laboratory technologists. A Initiative. HEMOSTASIS AND THE LABORATORY This issue highlights: Primary Hemostasis Screening Tests Case Studies Next issue: Disorders of thrombosis Pre-analytical variables and coagulation assays G-3, Ground Floor, Chaand Towers, 128 Lattice Bridge Road, Behind Jayanthi Theatre, Thiruvanmiyur, Chennai Tel: , Mobile:
2 HEMOSTASIS AND THE LABORATORY Hemostasis is derived from the Ancient Greek word that literally means to stop the flow of blood. Hemostasis is the body s normal physiological response to the prevention and stopping of bleeding/hemorrhage after tissue injury. It helps ensure fluidity of blood and blood vessel integrity. Abnormalities in hemostasis can result in bleeding/hemorrhage and or clotting/thrombosis Hemostasis involves 3 main processes/mechanisms Primary Hemostasis: o Local vascular constriction o Primary platelet plug formation Secondary Hemostasis: o Formation of stable clot by interaction between various Coagulation factors and inhibitors in a sequential manner resulting in a coagulation cascade Fibrinolysis: o Breakdown or lysis if the formed clot once vascular integrity is restored. How to investigate a case of bleeding? Clinical history: History and description of bleeds o Frequency o Severity o Anatomic location (superficial/deep) o Spontaneous/provoked bleeds Complete medication history (including OTC drugs) History of prolonged bleeding after trauma/surgery including dental extraction, circumcision etc Unexplained menorrhagia and post-partum bleeds Family history of bleeding, history of consanguinity History of transfusion of blood/blood products if any and indication for transfusion History of any underlying systemic disorder like liver disease, malignancy, auto immune disorders Anaemia of blood loss 2
3 Clinically significant mucocutaneous bleeds are defined as: Spontaneous or provoked bleeding from 2 or more distinct mucocutaneous sites Bleeding from a single site warranting blood transfusion Bleeding from a single site on 3 or more occasions Physical examination: Signs or evidence of bleeding Jaundice Hepatosplenomegaly Joint swelling/hyper mobility How do we investigate these in the laboratory? The initial laboratory tests that investigate the efficiency of Primary hemostasis form the Screening tests or first line tests. They are: Bleeding time: Usually performed by Ivy s method or Template method. A prolonged bleeding time indicates qualitative platelet defect, which may be hereditary (n Willebrand Disease, Glanzmanns thrombasthenia, Bernard Soulier syndrome) or acquired (Uremia, liver disease, Myelodysplastic syndrome, Leukemias, amyloidosis) Prothrombin time: This measures the efficacy of the extrinsic pathway of coagulation. It is reported in INR-International Normalized ratio. A prolonged PT is seen in coagulation factor deficiencies(factor, II, VII), oral anticoagulant therapy, Vitamin K deficiency Activated partial thromboplastin time: APTT measures the performance and efficiency of the intrinsic pathway. It is reported in seconds. A prolonged APTT is seen in coagulation factor deficiencies (Factor VIII, IX, XI, XII), heparin therapy. Thrombin time: Is a test designed to assess the fibrin formation from fibrinogen in plasma. A prolonged Thrombin time signifies Fibrinogen abnormality (hypofibrinogenemia, dysfibrinogenemia, afibrinogenemia), Impairment of fibrin Screening Tests: Bleeding time Prothrombin time Activated partial thromboplastin time Thrombin time Fibrinogen Platelet count 3
4 formation due to DIC, Amyloidosis, Multiple myeloma and Thrombin inhibitory effects (due to heparin, hirudin, DTI or circulating antibodies to thrombin) Fibrinogen: It is a soluble plasma protein that is converted to fibrin. Decreased levels are seen in hypo/afibrinogenemia, end stage liver disease, acute fibrinolysis, massive blood transfusions, DIC Platelet count: The normal platelet count in an adult is between to /µL. Thrombocytopenia as in ITP, TTP/HUS, and viral infections like dengue may result in bleeding. Algorithm for testing History of bleeding Screening tests PT APTT TT Normal Prolonged Normal Prolonged Normal Prolonged Mixing studies with normal plasma 50:50mix. Perform the test that is prolonged Complete correction of test result No correction, test result still prolonged Perform appropriate factor assays Check for presence of circulating 4
5 Clinical Applications and interpretation: CASE 1: 2y/M with history of easy bruising and prolonged bleeding after trivial injuries. First child of consanguinous parents with a maternal uncle with similar history Test Patient result Units PT 13 Seconds APTT 105 Seconds TT 17 Seconds Reference range Fibrinogen 270 Mg/dL :50 mix with normal plasma-aptt 32 Seconds INTERPRETATION: Probable Intrinsic pathway Coagulation factor deficiency, Factor VIII assay: < 1% (Reference range: %) DIAGNOSIS: SEVERE HEMOPHILIA A (FACTOR VIII DEFICIENCY) CASE 2: 23y/F presented with severe menorrhagia, nil gynaec history, second born to first degree consanguinous parents. PT 96 Seconds APTT 34 Seconds TT 17 Seconds Fibrinogen 235 Mg/dL :50 mix with normal plasma-pt 14 Seconds INTERPRETATION: Probable Extrinsic pathway Coagulation factor deficiency, Factor VII assay: 1% (Reference range: %) DIAGNOSIS: SEVERE FACTOR VII DEFICIENCY 5
6 CASE 3: 10 day old baby, previously well, breast fed. Found unconscious with bleeds from mouth. PT 100 Seconds APTT >180 Seconds TT 17 Seconds Fibrinogen 300 Mg/dL :50 mix with normal plasma-pt 14 Seconds 50:50 mix with normal plasma-aptt 30 Seconds INTERPRETATION: Possibilities: Vitamin K deficiency Factor V, X deficiency The gross prolongation of PT and APTT in the absence of birth complications like umbilical stump, bleeds suggests an acquired disorder PT and APTT were normalized after 3 days of Vitamin K injection DIAGNOSIS: VITAMIN K deficiency Possible causes: Low levels of Vitamin K at birth/preterm Poor placental transfer of vitamin K Low levels of vitamin K in breast milk [but not in cow s milk] Sterility of the fetal 6
7 CASE 4: 27y/F with no significant bleeding history visited an infertility clinic for a consult PT 14 Seconds APTT 86 Seconds TT 16 Seconds Fibrinogen 325 Mg/dL :50 mix with normal plasma-aptt 83 Seconds INTERPRETATION: APTT not correcting with a 50:50 mix with NP suggests presence of circulating inhibitor. Follow up with a DRVV (Dilute Russell viper venom) test to detect Lupus anticoagulants done DRVV Screen 108 Seconds Screen ratio DRVV confirm 32 Seconds Confirm ratio NORMALISED RATIO DIAGNOSIS: Lupus anticoagulants detected Next issue highlights: Disorders of thrombosis Pre-analytical variables and coagulation assays Please share The LaboratoryMatters with your colleagues or write to us for a copy in your mail box. You can access this Newsletter at our website Questions or comments to newsletter@crestlaboratories.com, G3-Ground Floor, CHAAND TOWERS, 128, LATTICE BRIDGE ROAD, THIRUVANMIYUR, CHENNAI Phone: Mobile info@crestlaboratpories.com / crestlbaoratories@gmail.com 7
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