MLS Review - Hematology - Laboratory Evaluation of Hemostasis, Coagulation, & Fibrinolysis

MEDICAL LABORATORY SCIENCE REVIEW HEMATOLOGY: LABORATORY EVALUATION OF HEMOSTASIS, COAGULATION, & FIBRINOLYSIS OUTLINE • Laboratory Evaluation of Hemostasis & Coagulation o Hemostasis Specimen Collection & Management Errors That Require Specimen Rejection • Laboratory Evaluation of Primary Hemostasis o Qualitative Assessment of Platelet o Platelet Function Tests o Platelet Aggregometry o vWF Activity Assays • Laboratory Evaluation of Secondary Hemostasis o Clot-Based Screening Tests o Venom Activated Assays o PT Test o APTT Test o TT Test o Mixing & Substitution Studies • Lab. Evaluation of Fibrinolysis o D-Dimer Immunoassay o FDP Immunoassay o Plasminogen Assay o TPA Assay Legend: ! Memorize |  Tip/Mnemonic | → Practical Note LABORATORY EVALUATION OF HEMOSTASIS & COAGULATION HEMOSTASIS SPECIMEN COLLECTION AND MANAGEMENT ERRORS THAT REQUIRE SPECIMEN REJECTION • • Light blue top tubes used for coagulation studies o Red top/glass tube - not used for coagulation studies due to activation of intrinsic coagulation pathway by the negative charge of the glass surface Use fresh sample, if possible HEMOSTASIS SPECIMEN STORAGE TIMES AND TEMPERATURES Application PT with no UFH PTT with no UFH Factor assays Whole-blood aggregometry PTT for monitoring Centrifuge to separate UFH plasma within 1 hour, test within 4 hr PT when UFH is present Optical platelet Wait 30 min after aggregometry using centrifugation, test within 4 PRP hr of collection Storage in 2 weeks -20 degC household freezer Storage for 6 months 6 months or indefinite -70 degC PRP, Platelet-rich plasma; PT, prothrombin time; PTT, partial thromboplastin time, UFH, unfractionated heparin LABORATORY EVALUATION OF PRIMARY HEMOSTASIS QUALITATIVE ASSESSMENT OF PLATELET • • ERRORS FOR SPECIMEN REJECTION Error Short draw Specimen clot Visible hemolysis Lipemia or icterus Tourniquet application >1 minute Specimen storage at 1–6° C Specimen storage at >25° C Comments Whole-blood volume less than 90% of required volume or less than manufacturer specified minimum. PT and PTT are falsely prolonged. 3.2% sodium citrate tube - blood to anticoagulant = 4:1 Each specimen is inspected visually before centrifugation or during analysis; even a small clot interferes with hemostasis test results. Clotting consumes fibrinogen Pink or red plasma indicates in vitro activation of platelets and coagulation; unpredictable hemostasis test interference. Further, hemolysis interferes with optical endpoint coagulometer results. Optical instruments may fail to measure clots in cloudy or highly colored specimens. Interferes with chromogenic substrate methods. The practitioner must employ an electromechanical detection method instrument Blood stasis activates endothelial cells and elevates the concentration of von Willebrand factor and fibrinogen, falsely shortening clotbased test results Storage at refrigerator temperatures causes precipitation of large von Willebrand factor multimers, activation of coagulation factor VII, activation of platelets, and destruction of platelet integrity. Storage at above standard room temperature causes coagulation factors V and VIII to deteriorate. Specimen Stability Temperature Test within 24 hr, maintain 15 - 25 degC upright and sealed; (room temp) Separate plasma & RBC via centrifuge Test within 4 hr, maintain, upright and sealed Performed if patient have normal plt count however, the patient has bleeding history or bleeding risk Platelet Count (N) o Suggestive of bleeding history → assessment of Platelet function test is conducted o Reference value: 150,000-400,000 plts/uL PLATELET FUNCTION TESTS BLEEDING TIME TEST o o o o o o o Obsolete method Reflects the platelet function An infrequently performed in vivo measurement of platelet adhesion (GP Ib receptor, vWF) and aggregation (GP IIb/IIIa receptor, Fibrinogen, Ca2+ ions) on locally injured vascular sub-endothelium Provides an estimate of integrity of the platelet plug Measures interaction between capillaries and platelets Performed beside w/ clotting time for pre-surgical procedures Duke’s Method ▪ Obsolete method • No standardized depth of puncture = causes inaccuracies • Deeper puncture = longer BT ▪ Procedure: • Phlebotomist’s uses a lancet to make a small, controlled puncture wound (earlobe or fingertip). o Fingertip - ring finger is recommended as it is used rarely and with few callous o Puncture at the lateral side o Massage finger to promote blood flow o Squeeze during puncturing to lessen pain o Puncture site is blotted with filter paper every 30 seconds until the bleeding stopped. ▪ Use the time where the blood stops (30s w/blood, 60s w/o blood = 60s BT) 1 • → Do not squeeze the finger during blotting → First drop may not be wiped off o Reference Interval: 2 – 9 minutes (universally accepted) Standard/Modified Ivy Method o A blood pressure cuff is inflated to 40 mmHg. o A calibrated spring-loaded lancet (3mm deep) was triggered on the volar surface of the forearm a few inches distal to the antecubital crease. ▪ Two 3mm deep puncture sites are made o Puncture site is blotted with filter paper every 30 seconds until the bleeding stopped. o Prone to hematoma due to use of pressure cuff o Reference Interval: 2 – 9 minutes (universally accepted) o Petechiae, purpura, ecchymosis ▪ Petechiae - <3mm in diameter ▪ Purpura - 3mm - 1cm in diameter ▪ Ecchymosis - >1cm in diameter • Tourniquet Test (CDC Procedure) 1. Take the patient's blood pressure and record it, for example, 100/70. 2. Inflate cuff to a point midway between SBP & DBP and maintain for minutes. (100+70)÷2=85 mmHg 3. Reduce and wait 2 minutes. 4. Count petechiae below antecubital fossa. See image at right. 5. A positive test is 10 or more petechiae per 1 sq. in. o Note: Count below the tourniquet CLOT RETRACTION PLATELET ADHESION TEST • • • • • • • • • • Obsolete Test Degree of clot retraction is directly proportional to the number of platelets and inversely proportional to the hematocrit and the level of fibrinogen. Evaluates how well platelets keep the clot adhered to the sides of specimen tube Uses whole blood in red top tube (3 mL) o Plain red top tube with no clot activators o Clot activators - hasten clot activation = short clot retraction Examine clot at 1, 2, 4 and 24 hours for clot retraction o 24 hour no clot = clotting disorder/ bleeding risk By 30 minutes: clot starts to shrink After clot forms, remaining 40 - 60% consists of serum and red blood cell "fall - out" from clot Must have normal fibrinogen and hematocrit for test to be accurate • • • • Adherence of platelets to glass surfaces Counting the number of platelets before and after exposure to glass beads. o 2 platelet counts: pre- & post- exposure Not a reliable method. Retention of platelets determine quality of platelets Reference value: >25% of platelets adhered/retained Salzmann Method o Platelet adhesiveness to glass beads (diagnosis of von Willebrand’s Disease) PLATELET AGGREGOMETRY • CAPILLARY FRAGILITY TEST • • • • • o a.k.a Rumpel–Leede Capillary Fragility Test or Tourniquet Test The test is defined by the World Health Organization as one of the necessary requisites for diagnosis of dengue fever. A blood pressure cuff is applied and inflated to a point between systolic and diastolic blood pressures for 5 mins Positive: 10 or more petechiae per square inch Dengue Hemorrhagic Fever: 20 or more petechiae per square inch o Causative agent: dengue virus o Capillary fragility is only screening for dengue Does not have high specificity. ▪ Increased petechiae may be caused by varying tourniquet technique Platelet Aggregation o Based on some variations of Born Method o Principle: Platelet rich plasma is treated with known aggregating agents (plasma + aggregating agent) o Aggregated: Cloudiness or Turbidity can be measured by spectrophotometer. (As aggregation proceeds, more light passes through the sample) ▪ Clumping = ↑ transmitted light = ↑ %transmittance ▪ No clumping = ↓ transmitted light = ↓ %transmittance o Aggregating Agents: (Aggregates platelets) ACESTR ▪ ADP ▪ Collagen ▪ Epinephrine ▪ Snake Venom ▪ Thrombin ▪ Ristocetin 2 • Platelet Aggregometry Using Platelet-Rich Plasma o Using a specialized photometer: Light-Transmittance Aggregometer o Sodium citrate-anticoagulated blood → centrifuged o Requires 9-12mL of WHOLE BLOOD: to produce a sufficient PRP o PRP: plasma with platelet count of 200,000-300,000/μL o Specimen must be test within 4 hours of collection to avoid in vitro platelet activation & loss of normal activity. • Whole-blood Platelet Aggregometry o Measured by electrical impedance o Increase impedance is directly proportional to platelet aggregation. o Measures impeded light/blocked light o Specimen is collected in 3.2% sodium citrate & held at 15° C to 25° C until testing. o Aggregometry should be started immediately and must be completed within 4 hours of specimen collection (for undiminished ex vivo platelet activity). o Most specimens for whole blood aggregometry are mixed 1:1 with normal saline before testing, although if the platelet count is less than 100,000/mL the specimen is tested undiluted. o In Addition of aggregating agents, platelets will clump and adhere to the rods/ electrodes • Platelet Lumiaggregometry o Uses PRP o Diagnosis of platelet dysfunction o Simultaneous measurement of platelet aggregation & secretion of ATP from activated platelet dense granules. • Platelet Agonists in Aggregometry o Agonists used to activate platelets: Thrombin or Synthetic TRAP (Thrombin-Receptor-Activating Peptide), ADP (Adenosine Diphosphate), Epinephrine, Collagen, Arachidonic Acid & Ristocetin o These platelet agonists are used to test for abnormalities in specific membrane binding sites. o Arachidonic Acid: used to check for deficiencies in the eicosanoid synthesis pathway. o Ristocetin: used to check for abnormalities of plasma VWF in VWD VON WILLEBRAND FACTOR (vWF) ACTIVITY ASSAYS • VWF Antigen (VWF:Ag) Immunoasay o Collagen-Binding Enzyme-Linked Immunosorbent Assay (ELISA) - Introduced as an alternative procedure for RCo Assay o vWF-Antigen – marker of generalized endothelial dysfunction. • Clot-Based Coagulation Factor VIII Assay o Measure the activity of Factor VIII Ristocetin VWF Ristocetin Cofactor Assay (VWF:RCo) o Measures vWF o Mediated agglutination of platelets in the presence of an antibiotic (Ristocetin as agonist). o Most commonly used assay for the measurement of the functional activity of vWF. • 3 • Dilute Ristocetin-Induced Platelet Aggregation Assay (RIPA) o Also called: Ristocetin Response Curve o Employed to diagnose VWD subtype 2B LABORATORY EVALUATION OF SECONDARY HEMOSTASIS MICRO METHOD • • CLOT-BASED SCREENING TESTS FOR COAGULATION DISORDERS MACRO METHOD • Lee-White Whole Blood Coagulation Time Test o Single-Tube Method ▪ Procedure: 1. 3mL of whole blood in a test tube 2. Tilt every 30 seconds. 3. Observed for clotting. 4. Record time of clotting. VENOM ACTIVATED ASSAYS • • o Three-Tube Method (Modified LWCT) ▪ Procedure: 1. Label three tubes (1, 2, 3). Place the clean dry tubes in a 37 degC water bath. o Water bath enhances procedure 2. Obtain a 3mL venous blood (syringe). 3. Place 1mL of blood into each tube starting with Tube 1. 4. Start Timing as the blood is delivered into Tube 1. 5. Gently tilt Tube 3 every 30 seconds until blood solidifies. Handle Tube 2 in the same way. 6. Finally, Tilt Tube 2 until blood forms a Solid Clot. 7. Stop timing and record the coagulation time. ▪ NV: 7-15 minutes Drop or Slide Method o Procedure: 1. Perform a skin puncture. Discard the 1st drop of blood. • 1st drop might contain tissue looses 2. Place a drop of blood on a clean glass slide. Start timing. 3. Draw the tip of the lancet across the drop of blood at 30-second interval until fibrin threads cling to the tip. 4. Stop timing and record the coagulation time. ▪ NV: 2-4 minutes Capillary Tube/Dale & Laidlaws Method o Procedure: 1. Make a skin puncture. Wipe off the 1st drop of blood. 2. Fill a non-heparinized capillary tube ¾ with blood. 3. Start timing as soon as blood enters the tube. 4. Set the capillary tube aside in a horizontal position for 2 minutes. 5. Break off about 1cm of the capillary tube at 30second interval until fibrin thread bridges the broken ends of the capillary tube. 6. Record the coagulation time. ▪ NV: 2-4 minutes Reptilase Time (Pefakit Reptilase Time; Pentapharm) o Reptilase - thrombin-like enzyme, batroxobin, isolated from venom of Bothrops atrox that catalyzes conversion of fibrinogen to fibrin (in-vitro conversion) ▪ FIIa - in vivo conversion Russell Viper Venom Test o Russell viper venom (RVV) from the Daboia risselii viper, which triggers coagulation at the level of factor X, was once used as an alternative to the PT. o The assay was named the Stypven Time, but is now obsolete PROTHROMBIN TIME (PT) TEST • • • • • • • Early coagulation test developed by Dr. A.J. Quick Evaluates the function of Extrinsic (VII, TF, VIIa) & Common Pathway (X, Va, II, I) Major Use: Monitor Warfarin (Coumadin) Anticoagulant Therapy Also used as pre-surgery coagulation screening test Warfarin - Vitamin K antagonist, which means it blocks the action of vitamin K and vitamin K-dependent CFs. Prothrombin (II) - Produced in liver Principle of PT o Uses pre-warmed citrated plasma ▪ Plasma + PT reagent → observe for clot o The hemostasis pathway is activated when damage occurs to blood vessel endothelium or to tissue. o The extrinsic pathway is activated by tissue thromboplastin (factor III) in presence of calcium ions. o Factor X, a proenzyme, is converted to the enzyme Xa, which in turn converts prothrombin to the enzyme thrombin. o Thrombin then acts on fibrinogen to form fibrin monomers that make up the initial clot. 4 • • • • • PT Reagent o Counterpart of in vivo coagulation pathway o Contains a commercial preparation of tissue thromboplastin and calcium chloride, substitutes for tissue thromboplastin and calcium ions (Ca++) that are coagulation activators of extrinsic pathway (VII). ▪ Tissue thromboplastin → Tissue factor in vivo ▪ Calcium chloride → Calcium ions in vivo • Activates VII → … o Anticoagulated (N) Plasma + PT Reagent → Fibrin Clot Deficiency in Extrinsic or Common Pathway o PT: Prolonged Reference Value 3.2% Citrated Plasma: 10-13 secs International Normalized Ratio (INR) o Calculated value that allows standardization of prothrombin time reporting among different laboratories. o INR = ( PT Patient )ISI PT Mean Normal Facility o International Sensitivity Index (ISI) - Assigned by the manufacturer to each reagent lot is used with the prothrombin time to calculate the INR ▪ Will depend on the manufacturer o Reference Value: INR 1.0 – 1.4 THROMBIN TIME (TT) TEST • • • ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT) TEST • • • Is a coagulation test used to monitor low-dose heparin therapy and to screen for function of the intrinsic and common pathways of hemostasis Partial Thromboplastin o Lipid portion of tissue thromboplastin, is the reagent used in performing the APTT. o Counterpart of PF3 in vivo Partial Thromboplastin Reagent o Manufactured from rabbit or bovine brain tissue as well as from vegetable sources such as soybeans. o Performs the function of platelet factor 3 (PF3) in the APTT test o Contains activators such as: kaolin or silica to activate the contact factors in the intrinsic pathway o Calcium chloride (CaCl2): second reagent used in the test and is added to supply the ionized calcium required to activate prothrombin in the common pathway. Principle of APTT o Performed by combining citrated patient plasma with two reagents: partial thromboplastin and calcium chloride (CaCl2) o Pre-warmed patient plasma is combined with partial thromboplastin and allowed to react → activation of factors in the intrinsic pathway begins (activation that would be initiated by PF3 in vivo) o CaCl2 → added to plasma-partial thromboplastin mixture to allow the reaction necessary for fibrin clot formation. o Citrated plasma → add reagent 1 (partial thromboplastin) → incubate (water bath) → add reagent 2 (CaCl2) → timing of gel-like clot o Reference Value: 20-35 Seconds Used to measure the availability of functional fibrinogen Prolonged in cases of: o Low levels of fibrinogen (hypofibrinogenemia) o Non-functional fibrinogen (dysfibrinogenemia) o Presence of heparin | fibrin/fibrinogen products | thrombolytic agents (streptokinase) Principle of TT o The thrombin time test determines the rate of thrombin-induce cleavage of fibrinogen to fibrin monomers and the subsequent polymerization of hydrogenbonded fibrin polymers. MIXING AND SUBSTITUTION STUDIES MIXING STUDIES • • Mixing Study o Aka Circulating Anticoagulant Screen, Screening for Circulating Inhibitor Principle of Mixing Study o PTT mixing study is indicated when the PTT is prolonged in the absence of heparin therapy. o The assay is preceded by a thrombin time to detect therapeutic heparin or a direct thrombin inhibitor. o A mixing study is performed by measuring the APTT in the patient’s plasma, then mixing an equal volume of the patient’s plasma and normal pooled plasma (NPP) and repeating the APTT tests immediately and after 37°C incubation. 5 • • • NPP - normal pooled plasma PTT/APTT after PTT/APTT after 2Inhibitor or Immediate Hours of Deficiency Mixing Incubation at 37°C Factor Deficiency C C Factor VIII Inhibitor C NC Factor V (Leiden) NC NC Inhibitor Lupus Anticoagulant NC NC C, corrected (normalized); NC, not corrected (prolonged) • 3 measured PTT/APTT - (1) before addition, (2) immediate mixing, (3) incubation • Factor VIII Inhibitor - common cause of prolonged PTT/APTT • SUBSTITUTION STUDIES • • Substitution Study o Substitution studies and factor assays can be used to identify specific coagulation factor deficiencies. Principle of Substitution Study o Substitution studies may be performed using adsorbed plasma and aged serum with the AP to identify deficiencies of blood coagulation. o Substitution studies may also be performed using adsorbed plasma with the PT to identify a factor VII deficiency REMEMBER THIS WHEN DOING MIXING/SUBSTITUTION TEST Reagent !DOES NOT CONTAIN: ABSENT! CONTAINS: PRESENT Fresh Plasma ALL COAGULATION FACTORS Aged Plasma V, VIII I, II, VII, IX, X, XI, XII FACTOR V & VIII – LABILE | ↓ UPON STORAGE  LV (Louis Vuitton) - Labile factor V Adsorbed II, VII, IX, X I, V, VIII, XI, XII, XIII Plasma Fresh Serum I, V, VIII, XIII II, VII, IX, X, XI, XII Factor II - <20% present in fresh serum/ 80% are consumed during coagulation Other Coagulation Factors – entirely consumed during coagulation Aged Serum I, II, V, VIII, XIII VII, IX, X, XI, XII Factor II - Entirely consumed during Coagulation • Tips: o Corrected: present in reagent o Not corrected: not present in reagent o APTT: measure Intrinsic & common pathway o PT: measures extrinsic & common pathway Example 1: o APTT – PROLONGED o PT – PROLONGED o Problem: Common Pathway (X, V, II, I) o Find the Common Factor: ▪ Fresh Plasma (C) – all CF (X, V, II, I) ▪ Adsorbed Plasma (NC) – II, VII, IX, X ▪ Aged Serum (C) – VII, IX, X, XI, XII o Result: Factor X Deficiency Example 2: o APTT – PROLONGED o PT – NORMAL o Problem: Intrinsic Pathway (XII, XI, IX, VIII) o Find the Common Factor: ▪ Fresh Plasma (C) – XII, XI, IX, VIII ▪ Adsorbed Plasma (NC) – II, VII, IX, X ▪ Aged Serum (C) – VI, IX, X, XI, XII o Result: Factor IX Deficiency (Christmas disease) Example 3: o APTT – NORMAL o PT – PROLONGED o Problem: Extrinsic Pathway (VII) o Result: Factor VII Deficiency LABORATORY EVALUATION OF FIBRINOLYSIS D-DIMER IMMUNOASSAY • • • Fibrinolysis generates: FDPs and D-dimer at concentrations greater than 200 ng/mL. o D-dimer - degradation product of stable fibrin ↑ FDP & D-Dimer: Acute/Chronic DIC, Systemic Fibrinolysis, Deep Vein Thrombosis (DVT), Pulmonary Embolism Principle of Quantitative D-Dimer Assay: o Microlatex particles in buffered saline are coated with monoclonal anti-D-dimer antibodies. o The coated particles are agglutinated by patient plasma D-dimer; the resultant turbidity is measured by turbidimetric or nephelometric technology. FDP IMMUNOASSAY • • FDPs may be detected using a qualitative visible agglutination immunoassay that detects FDPs in serum and urine. Thrombo-Wellcotest: o A slide agglutination method in which the practitioner mixes one drop of sample with one drop of latex suspension. The polystyrene latex particles in buffered saline are coated with polyclonal antibodies specific for D and E fragments calibrated to detect FDPs at a concentration of 2 mg/mL or greater by the appearance of visible agglutination. 6 PLASMINOGEN ASSAY • • • • • • • • • • Plasma levels of plasminogen are quantitated using a chromogenic substrate method Reagent streptokinase activates plasminogen to form plasmin R-pNA designates a chromogenic substrate, where R indicates a peptide sequence specific to the enzyme being measured and pNA (para-nitroaniline) is the chromophore In the case of plasminogen, the T represents the peptide sequence valine-leucine-lysine (Val-Leu-Lys) Plasmin recognizes the Val-Leu-Lys amide sequence as its enzymatic cleavage site, releasing the pNA, which generates a yellow color Plasminogen is activated to Plasmin by: Streptokinase or U-PA (Urokinase Plasminogen Activator) (Activated) Plasmin is measure by substrate paranitroaniline (R-pNA) → producing a Yellow Color. Chromogenic Plasminogen Assay: semi-functional assessment of plasminogen activity. Plasminogen + Streptokinase (U-PA) → Plasmin Plasmin + (R-pNA) substrate → Yellow Color TISSUE PLASMINOGEN ACTIVATOR (TPA) ASSAY • • • • • Plasma levels of TPA are quantitated using a chromogenic substrate method To assay, plasma that contains TPA is added to plasminogen to produce plasmin Plasmin activity is measured using the same chromogenic susbtrate as in the plasminogen assay The intensity of color is proportional to TPA activity pNA, para-nitroaniline; R, variable peptide sequence • 7