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.
Table of ContentsCBC/Platelet Count/Blood Smear Examination ________________________In Vivo Evaluation of Primary Hemostasis _____________________________
Platelet Aggregometry _____________________________________________Automated Platelet Function Analysis ________________________________
Platelet Aggregation with Impedance Platelet Counting _________
Platelet Aggregation Under Flow Condition ____________________Acceleration of Kaolin Activated Clotting Time by
Platelet-Activating Factor ________________________________Automated Optical Platelet Aggregometry
Whole Blood Hemostatometry ______________________________________
Thromboelastography _____________________________________Clot Retraction ___________________________________________
Clot-Based Assays _______________________________________________ Activated Clotting Time (ACT) ______________________________Prothrombin Time (PT) _____________________________________
Activated Partial Thromboplastin Time _______________________Thrombin Time ___________________________________________
Clotting Factor Assays ____________________________________Fibrinogen Analysis _______________________________________Plasma Mixing Studies ___________________________________
Reptilase Time __________________________________________Dilute Russell Viper Venom Assay __________________________
Activated Protein C Resistance ____________________________Chromogenic Analysis ___________________________________________Latex Agglutination/Turbidimetry __________________________________
Enzyme Immunoassay ___________________________________________Flow Cytometry _________________________________________________
Electrophoresis _________________________________________________Genetic and Molecular Assays ____________________________________Electron Microscopy _____________________________________________
Radioimmunoassay ______________________________________________References ______________________________________________________
Table of Contents
asis Medical evaluation of the hemostasis sys-
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
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.
Laboratory Evaluation of Hemostasis
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)
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%.
In Vivo Evaluation ofPrimary Hemostasis
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-
CBC/Platelet Count/PeripheralBlood Smear Examination
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.
Peripheral Blood Smear Examination
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)
Acquired thrombocytopenia secondary to leukemia, myeloproliferative disorders, or other hematologic diseases is more common than congenital platelet disorders. In addition, peripheral smear examination
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-
Fig. 1. Photomicrograph of a normal peripheral blood smear showing several platelets with normal morphology (Arrows).
Platelet Count, Bleeding Time
Laboratory Evaluation of Hemostasis
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)
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 & 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
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 i