Lab Hemostasis

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<ul><li><p>Laboratory Evaluationof HemostasisRoger S. Riley, M.D., Ph.D., Ann R. Tidwell, MT(ASCP) SH, David Williams, M.D., Ph.D., Arthur P. Bode, Ph.D., Marcus E. Carr, M.D., Ph.D.</p></li><li><p>Table of ContentsCBC/Platelet Count/Blood Smear Examination ________________________In Vivo Evaluation of Primary Hemostasis _____________________________</p><p>Platelet Aggregometry _____________________________________________Automated Platelet Function Analysis ________________________________</p><p>Platelet Aggregation with Impedance Platelet Counting _________</p><p>Platelet Aggregation Under Flow Condition ____________________Acceleration of Kaolin Activated Clotting Time by</p><p> Platelet-Activating Factor ________________________________Automated Optical Platelet Aggregometry</p><p>Whole Blood Hemostatometry ______________________________________</p><p>Thromboelastography _____________________________________Clot Retraction ___________________________________________</p><p>Clot-Based Assays _______________________________________________ Activated Clotting Time (ACT) ______________________________Prothrombin Time (PT) _____________________________________</p><p>Activated Partial Thromboplastin Time _______________________Thrombin Time ___________________________________________</p><p>Clotting Factor Assays ____________________________________Fibrinogen Analysis _______________________________________Plasma Mixing Studies ___________________________________</p><p>Reptilase Time __________________________________________Dilute Russell Viper Venom Assay __________________________</p><p>Activated Protein C Resistance ____________________________Chromogenic Analysis ___________________________________________Latex Agglutination/Turbidimetry __________________________________</p><p>Enzyme Immunoassay ___________________________________________Flow Cytometry _________________________________________________</p><p>Electrophoresis _________________________________________________Genetic and Molecular Assays ____________________________________Electron Microscopy _____________________________________________</p><p>Radioimmunoassay ______________________________________________References ______________________________________________________</p><p>Table of Contents</p><p>44</p><p>688</p><p>8</p><p>9</p><p>9</p><p>1010</p><p>111112</p><p>1313</p><p>141415</p><p>1616</p><p>171718</p><p>1819</p><p>202022</p><p>2222</p></li><li><p>3Introduction</p><p>Hem</p><p>ost</p><p>asis Medical evaluation of the hemostasis sys-</p><p>tem began with visual observation of the clotting process. During the time of medi-cal blood letting, observation of the size of the clot in a basin (clot retraction) was used to determine when blood letting had to be decreased. In the early 20th century, manual timing of whole blood clotting (i.e., Lee-White Whole Blood Clotting Time), and later plasma, in glass tubes permitted a more accurate measurement of blood clotting. Further discoveries about hemostasis in the 1930s and 1940s led to more sophisticated laboratory tests, including the prothrombin time, activated partial thromboplastin time, and specific assays of platelet function and fibrinolysis. The advent of the monoclonal antibody, molecular analysis, and the microcom-puter in the 1980s led to an explosion of knowledge about hemostasis and hemo-stasis testing that is still growing. In the </p><p>hemostasis laboratory, automated assays have replaced many of the manual proce-dure of the past, and there is increasing in-terest in rapid, point of care hemostasis as-says for perioperative and critical care, as well as self-testing to support the millions of patients now receiving oral anticoagula-tion for hypercoagulable diseases. Interest-ingly, measurement of clot retraction is still the focus of a variety of these tech-niques, a fact that would no doubt be ap-preciated by the early physicians. This pa-per presents a global overview of the tech-niques presently used in the hemostasis laboratory, with the realization that many of these may be quickly surpassed by new information, developments, and applica-tions in the near future. </p><p>Laboratory Evaluation of Hemostasis</p></li><li><p>may reveal evidence of liver, renal, or other causes of acquired platelet dysfunction. A predominance of large platelets may be the initial clue to the diagnosis of the Bernard-Soulier syndrome. The May-Hegglin anomaly, Chediak-Higashi syndrome, and other dis-eases affecting platelets may be discovered by periph-eral smear examination.(7)</p><p>Platelet Count</p><p>Modern hematology analyzers perform a platelet count by electrical impedance or light scattering techniques that are accurate to 5% in the range of 1000 - 3,000,000 platelets/L. A measurement of plate-let volume (mean platelet volume, MPV) is provided at the same time, as well as a platelet size distribution curve. Automated platelet counts can be affected by platelet aggregates due to spontaneous aggregation, cold agglutinins, EDTA anticoagulants ("spurious thrombocytopenia, pseudothrombocytopenia") or particulate debris, such as red or white cell fragments ("spurious thrombocytosis").(2-4) In addition, hema-tology analyzers may overestimate the platelet count in severe thrombocytopenia.(5) Therefore, confirma-tion of atypical platelet counts by manual inspection of a peripheral smear is essential. If necessary, plate-let counts can be performed in a hemocytometer by phase contrast microscopy to an accuracy of 10-20%.</p><p>In Vivo Evaluation ofPrimary Hemostasis</p><p>The Ivy skin bleeding time is an imprecise manual screening assay of primary hemostasis that was widely utilized in the past as a diagnostic assay for patients with suspected bruising and bleeding disor-ders, as a therapeutic guide in actively bleeding pa-tients, and as a predictor of hemorrhage in the gen-</p><p>CBC/Platelet Count/PeripheralBlood Smear Examination</p><p>The complete blood count (CBC), platelet count, and peripheral blood smear examination are the most fundamental assays of hemostasis and must be per-formed in all patients with suspected hemostatic ab-normalities.</p><p>Peripheral Blood Smear Examination</p><p>Peripheral smear examination is the critical first step in the investigation of any suspected hematologic disease.(6) Peripheral smear examination reveals in-formation about platelet size, gross morphology, and granularity, as well as associated abnormalities in red and white blood cells. It is also helpful for confirma-tion of the automated platelet count. An estimate of the platelet count can be obtained by routine light microscopy of a Wright's-stained peripheral smear by multiplying the number of platelets per 1000x oil magnification oil immersion field by 10,000, or more accurately, by multiplying the sum of the number of platelets counted in 8-10 fields under 1000 x oil mag-nification by 2000.(7) A visual platelet counting tech-nique based on the white blood cell count (PCW, platelet count based on WBC) has also been devel-oped for thrombocytopenic samples.(8) Every pe-ripheral blood smear should be carefully evaluated for the presence of platelet clumps that may falsely lower the platelet count. Platelet aggregates usually indicate a poorly collected or anticoagulated blood specimen of the presence of EDTA-induced autoantibodies.(7) </p><p>Acquired thrombocytopenia secondary to leukemia, myeloproliferative disorders, or other hematologic diseases is more common than congenital platelet disorders. In addition, peripheral smear examination </p><p>eral population of patients undergoing surgery or invasive procedures.(9) Bleeding times are performed directly on the patient by phlebotomists or technologists who are trained and experienced in this assay. A blood pressure cuff is placed on the upper arm and inflated to 40 mm Hg to provide uniform capillary pressure, and a standard-ized incision is made on the volar surface of the fore-arm with a standard cutting device, such as the Sur-</p><p>4</p><p>Fig. 1. Photomicrograph of a normal peripheral blood smear showing several platelets with normal morphology (Arrows).</p><p>Platelet Count, Bleeding Time</p><p>Laboratory Evaluation of Hemostasis</p></li><li><p>from the incision with filter paper at 30-second inter-vals until bleeding ceases. The result is reported in seconds as the bleeding time.(10; 11) </p><p>The bleeding time is determined by many physiologic factors, including skin resistance, vascular tone and integrity, and platelet adhesion and aggregation. Thus, a prolonged bleeding time may reflect an in-trinsic platelet function defect, von Willebrand dis-ease, vascular anomaly, or medications that affects platelet function, such as aspirin. If the actual bleed-ing time exceeds the expected bleeding time by five minutes, a platelet function defect may be suspected. Unfortunately, the precision, accuracy, and repro-ducibility of the bleeding time are severely impaired by factors such as the thickness and vascularity of the skin, the location of the incision, skin temperature, wound depth, and patient anxiety. Because of its im-precision, the bleeding time must be used with ex-treme caution in a patient care setting. The US Food &amp; Drug Administration no longer accepts bleeding time data in patients as a surrogate marker for the evaluation of new hemostatic drugs, and it is no longer indicated for the preoperative screening for hemostatic defects.(12-15) The routine utilization of the bleeding time for the diagnostic evaluation of patients with von Willebrand disease, storage pool disorder, and other hereditary mucocutaneous hem-orrhagic diseases has been questioned.(16) The </p><p>Fig 2. Example of optical and impedance platelet counts with an automated hematology analyzer (Cell-Dyne 4000). In the optical technique (upper histo-gram), platelets (arrow) are discriminated from other cells by light scatter at 7o and 90o. An upper volume threshold is used to separate platelets from micro-cytic red blood cells. In the impedance platelet count (bottom histogram), platelets are differentiated from other cells by electrical resistance. The mean platelet volume (MPV) is determined from the platelet vol-ume data provided by impedance measurements.</p><p>gicut (International Technidyne Corp, Edison, NJ) and the Triplett and Tip Tripper Bleeding Time Devices (Helena Laboratories, Beaumont, TX). Blood is wicked </p><p>bleeding time has been entirely discontinued at some medical institutions without a measurable adverse affect on patient care.(13)</p><p>5</p><p>Fig 3. Performing the bleeding time. Upper photo-graph: A bleed pressure cuff was placed over the up-per arm and the skin of the forearm cleaned with alcohol. Middle photograph: Picture of skin incision marks left after a template was applied. Blood is starting to ooze from the wound. Bottom photo-graph: Wicking the wound with filter paper to de-termine the bleeding time. </p><p>Bleeding Time</p><p>Laboratory Evaluation of Hemostasis</p></li><li><p>costly assay restricted to specific clinical circum-stances. A variety of commercial instruments and reagents for platelet aggregometry are available from Chrono-Log Corporation (Havertown, PA), Bio/Data Corporation (Horsham, PA), and Helena Laboratories (Beaumont, TX). </p><p>Glanzmann thrombasthenia and the Bernard-Soulier syndrome are the best known inherited anomalies of platelet surface receptors, although both diseases are very rare. Glanzmann thrombasthenia arises from an aberration in the most prevalent platelet surface re-ceptor, GPIIbIIIa (specific binding site for fibrino-gen), leading to moderate to severe bleeding prob-</p><p>Platelet Aggregometry</p><p>Conventional platelet aggregometry (light transmis-sion aggregometry, turbidimetric aggregometry) measures the in vitro response of platelets to various chemical agents (i.e., aggregating agents, platelet ago-nists) that induce platelet functional responses.(17) In the clinical laboratory, platelet aggregometry is utilized for the diagnosis of inherited and acquired platelet disorders, the assay of von Willebrand factor activity (ristocetin cofactor assay) and for the diag-nosis of heparin-induced thrombocytopenia.(18)</p><p>Conventional optical platelet aggregometers are modified spectrophotometers that measure light transmission through platelet-rich plasma (PRP). Although the turbidity of fresh PRP limits light transmission, transmission progressively increases as platelet aggregation causes the formation of larger and larger particles.(17) More recent innovations include whole blood aggregometers and lumi-aggregometers. Whole blood aggregometers require less patient blood and provide faster turn-around time than optical aggregometers. Lumi-aggregometers simultaneously measure platelet ag-gregation and ATP secretion to provide a more accu-rate diagnosis of platelet function defects. The plate-let agonists routinely used in the clinical laboratory to differentiate various platelet function defects in-clude adenosine diphosphate (ADP), epinephrine, collagen, ristocetin, and arachidonic acid. Other ago-nists, such as thrombin, vasopressin, serotonin, thromboxane A2 (TXA2), platelet activating factor, and other agents are used by research and specialized clinical laboratories.</p><p>Conventional platelet aggregation is a complex labo-ratory assay that is particularly sensitive to the assay conditions, as well as drugs and other substances in the blood.(19) Because of these influences, platelet aggregometry is an advanced, manually intense, </p><p>lems in affected individuals. Platelet aggregometry reveals a lack of response to agonists requiring fi-brinogen binding, including adenosine diphosphate (ADP), epinephrine, arachidonic acid, and collagen. In contrast, the aggregation response to ristocetin is within normal limits. The Bernard-Soulier syndrome is clinically similar, but arises from the absence of another functionally important platelet surface recep-tor, GPIb-V-IX. However, platelets from patients with the Bernard-Soulier syndrome show normal aggrega-tion to agonists requiring fibrinogen binding, but show a lack of response to agents requiring GPIb (i.e., thrombin, ristocetin plus von Willebrand factor). The </p><p>6</p><p>Platelet Aggregometry</p><p>PrimaryAggregation</p><p>MaximalAggregation</p><p>ShapeChange</p><p>Dilution</p><p>SecondaryAggregation</p><p>Time</p><p>Lig</p><p>ht T</p><p>rans</p><p>mis</p><p>sio</p><p>n</p><p>Fig 4. Platelet aggregometry. The curve shows the five stages of an ideal response of platelets to the addition of a platelet agonist. Fol-lowing addition of the agonist, the platelets undergo a shape change after a short delay. This is fol-lowed by the release of stored agents, resulting in primary ag-gregation. The synthesis and re-lease of new agonists occurs after another short delay, producing a second wave of aggregation. Eventually, maximal aggregation has occurred and light transmis-sion is at is lowest. In practice, aggregation studies are per-formed with platelet-rich plasma and a variety of agonists (i.e., ADP, epinephrine, arachidonic acid, collagen, ristocetin, throm-bin, etc.). A conventional com-mercial platelet aggregometer (PACKS-4, Platelet Aggregation Chromogenic Kinetics System-4) is shown in the upper right. </p><p>Laboratory Evaluation of Hemostasis</p></li><li><p>Heparin-induced, immune-mediated thrombocy-topenia (HIT type II) is an unfortunate, but relatively common complication of heparin therapy arising fro...</p></li></ul>