Lab Hemostasis

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

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3Introduction

Hem

ost

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)

Platelet Count

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-

4

Fig. 1. Photomicrograph of a normal peripheral blood smear showing several platelets with normal morphology (Arrows).

Platelet Count, Bleeding Time


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 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.

gicut (International Technidyne Corp, Edison, NJ) and the Triplett and Tip Tripper Bleeding Time Devices (Helena Laboratories, Beaumont, TX). Blood is wicked

bleeding time has been entirely discontinued at some medical institutions without a measurable adverse affect on patient care.(13)

5

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.

Bleeding Time


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).

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-

Platelet Aggregometry

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)

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.

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,

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

6

Platelet Aggregometry

PrimaryAggregation

MaximalAggregation

ShapeChange

Dilution

SecondaryAggregation

Time

Lig

ht T

rans

mis

sio

n

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.


Heparin-induced, immune-mediated thrombocy-topenia (HIT type II) is an unfortunate, but relatively common complication of heparin therapy arising from autoantibodies specific for a complex of heparin and platelet factor 4 (PF4). The IgG/heparin/PF4 im-mune complexes bind to the FcyRIIA (CD32) receptor on the platelet membrane, resulting in platelet activa-tion, the release of additional PF4, new immune com-plexes, and rapid platelet consumption. The excess PF4 also binds to glycosaminoglycans on endothelial cells, leading to antibody-mediated endothelial dam-age, thrombosis, and disseminated intravascular co-agulation. Since serum from patients with HIT can aggregate normal platelets in the presence of heparin, platelet aggregometry with heparin is often used to confirm the clinical suspicion of HIT.(20; 21) How-ever, due to the operational complexity of this assay and its relatively low sensitivity, this assay has been

Bernard-Soulier syndrome is also characterized by thrombocytopenia and large platelets, while the plate-let count and morphology are normal in Glanzmann thrombasthenia but clot retraction is absent. These two separate but specific defects in essential platelet surface components have provided valuable informa-tion on the role(s) of platelets in formation of the initial hemostatic plug.

largely replaced by enzyme immunoassay and flow cytometry. As a combinatorial strategy, the immuno-assay can be used as a screening tool, with the aggre-gometry test for confirmation in patients that are antibody-positive.

The ability of vWF to aggregate platelets in the pres-ence of the antibiotic ristocetin is the basis for the ristocetin cofactor assay, the most common labora-tory method to measure vWF activity for the diagno-sis and monitoring of von Willebrand disease.(22) This assay is performed by incubating formalin-fixed platelets with test plasma, adding ristocetin, and then performing platelet aggregation. The results are in-terpolated from a standard curve prepared from ag-gregation slopes obtained with testing of dilutions of normal pooled plasma. Due to the time consuming manual nature of the classic ristocetin cofactor assay,

7

Disorder CollagenEpinephrine

ADP ArachidonicAcid

Ristocetin

Bernard-SoulierDisease

Normal Normal Normal Absent

Glanzmannthromboasthenia

Absent Absent Absent Normal

Aspirin, many drugs

Reduced or absent Variable Reduced or absent Normal

Storage pooldisease

Reduced or absent Variable Variable Normal

vWD, Type I Normal Normal Normal Reduced or absent

vWD, Type IIb Normal Normal Normal Increased

Fig. 5. Effect of aspirin on platelet function. Diagram shows aggregation tracings (% aggregation vs. time) for platelet-rich plasma from a donor who had recently ingested aspirin. The aggregation response to aspirin is markedly decreased to arachidonic acid (10% final aggregation). Epinephrine (76%), ADP (79%), and col-lagen (103%) show essentially normal responses.

0 1 2 3 4 5Minutes

-20

0

20

100

40

60

80

% A

gg

reg

atio

n

Arachidonic acid

EpinephrineADP

Collagen

Table IPlatelet Aggregometry - Characteristic Findings in Different Diseases


tion with clopidogrel or NSAISs in elective cardiac surgery patients., monitoring the efficacy of therapy with platelet GpIIb-IIIa antagonists in patients un-dergoing percutaneous coronary intervention or re-ceiving medical therapy for non-ST elevation acute coronary syndromes., and predicting post-operative blooding and blood product utilization in patients undergoing cardiac surgery with cardiopulmonary bypass.

Platelet Aggregationunder Flow Conditions

The PFA-100 (DADE-Behring, Miami FL, USA) is a rapid, automated laboratory instrument that is sensi-tive to quantitative and qualitative abnormalities of platelets and von Willebrand factor (vWF). In the PFA-100, citrated whole blood is aspirated from a res-ervoir under constant vacuum conditions through a microscopic 150 um aperture.(31-36) This aperture is cut into a biologically active nitrocellulose membrane in a disposable cartridge device coated with a combi-nation of platelet agonists. These agonists are either collagen (fibrillar Type I equine tendon) and epi-nephrine (C/Epi) or collagen and adenosine-5-diphosphate (C/ADP). The blood is forced through the aperture at a high shear rate (5000-6000 seconds-1) that roughly corresponds to the flow conditions present in small arteries.(32; 33) As the blood is forced through the aperture, platelets undergo adher-ence, activation and aggregation on the membrane surrounding the aperture and progressively form a plug that finally occludes the aperture. The closure time (CT) is the time required for the complete occlu-sion to occur.

The PFA-100TM is more rapid and less expensive than the bleeding time for the evaluation of platelet function.(35; 37) Since there is a good correlation between the bleeding time and the PFA-100 in certain patient populations, there, there is a trend to replace

automated agglutination techniques are under evalu-ation(23; 24), as well as techniques using enzyme immunoassay.(25-28) The aggregation test as cur-rently performed has a large standard deviation, which is unfortunate considering that von Willebrand disease is the most common hemostatic disorder en-countered in the hematology clinic.

Automated PlateletFunction Analysis

The manual, laborious nature of conventional platelet aggregometry is unsuitable for many applications where point of care and/or rapid testing is indicated. Therefore, there is increasing interest in non-complex, automated techniques of platelet function analysis particularly suitable for the cardiovascular suite, cardiovascular laboratory, dialysis, or intensive care unit.(29) A number of innovative techniques are presently available, and more are likely forthcoming in the near future.

Platelet Aggregationwith Impedance Platelet Counting

Plateletworks (Helena Laboratories, Beaumont, Texas) is a rapid in vitro point of care platelet aggregation screening technique based on impedance platelet counting and specifically developed for cardiopul-monary bypass and cardiac catheterization settings.(30) The technique uses anticoagulated blood to measure the change in platelet count due to plate-let aggregation. Two separate samples of blood are taken, including one containing ADP and collagen platelet agonists. The platelet count is measured in each tube using a small impedance hematology ana-lyzer, and the percent aggregation is calculated. An eight-profile hematology profile is provided at the same time.(30) The Plateletworks assay has been re-cently used to monitor the reversal of platelet inhibi-

the bleeding time with the PFA-100TM for a first-line screening test for platelet dysfunction in patients undergoing preoperative evaluation. Other clinical applications of the PFA-100 include the following:

The non-specific identification of patients with inherited platelet dysfunction, including Bernard-Soulier syndrome, Glanzmanns thrombasthenia, and other diseases.(38)

The evaluation of women with menorrhagia to exclude platelet dysfunction.

The determination of aspirin resistance, aspirin hyperresponsiveness, and the assessment of

8

Automated Platelet Function Analysis

Fig. 6. Schematic diagram of PFA-100 instrument. Citrated blood is forced through a small mem-brane at high shear rate meant to simulate physi-ologic conditions. Platelet agonists on the mem-brane initiate platelet adhesion and aggregation that eventually occlude the membrane and stop the flow of blood (Closure time). Diagram from DADE-Behring.


the end point (collagen-induced thrombosis forma-tion). At the time of this writing, the CSA is no longer being commercially developed but has important features which are not found on other available in-struments.

The Platelet-Stat (Precision Haemostatics, Inc., Clovis, CA) is a physiologic in vitro simulation of the tem-plate bleeding time, using blood anticoagulated with acid-citrate-dextrose (ACD). The device consists of a membrane with a slit, similar to the template-induced injury. Blood is forced at constant pressure from a syringe through the slit, resulting in occlusion of the slit as a platelet plug is formed. The time from the start of blood flow through the slit until blood clot-ting at the slit is termed the bleeding time. Phase I studies show that the in vitro bleeding time (Platelet-Stat) is successful in predicting dysfunctional plate-lets. The Platelet-Stat has been successfully used to diagnose TTP and monitor therapy with plasma exchange.(48)

Acceleration of Kaolin ActivatedClotting Time by Platelet-Activating Factor

The hemoSTATUS (Medtronic, Minneapolis, MN) is an automated system designed for whole blood point-of-care platelet function testing, especially in cardiovascular surgery. The assay principle is a com-parison of the activated clotting time quantitated in cartridges containing different concentrations of kao-lin or kaolin and platelet-activating factor.

The system also provides quantitative analysis of heparin concentration by heparin/protamine titra-tion, as well as a base-line clotting time (platelet-activated clotting time). Clinical evaluation of the instrument has been controversial, with several stud-ies failing to demonstrate a correlation of results with perioperative blood loss or an adequate sensitivity to drugs affecting platelet function.(49-52)

patient compliance with aspirin and other anti-platelet receptor agents during therapy.(39-41)

Monitoring deamino-D-arginine (DDAVP) ther-apy in vWD patients belonging to subsets of vWD that are responsive to DDAVP including most type 1 and some type 2 patients.

There are several cavets in the clinical utilization of the PFA-100. Strict adherence to specimen require-ments, specimen transportation, and specimen proc-essing is required, since the PFA-100 is affected by critical pre-analytical variables such as hematocrit or platelet count, blood collection technique, and trans-portation through pneumatic tube systems.(42) Since the PFA-10 has been reported as insensitive to some patients with platelet function defects, clinical corre-lation is critical, with follow-up with a different screening technique in cases of high clinical suspicion.(16; 38) The PFA-100TM is insensitive to al-terations in the quantity or quality of fibrinogen and therefore has not been shown to be useful in evaluat-ing patients for the presence of dysfibrinogenemia or hypofibrinogenemia. It is not sensitive to defects or deficiencies in the classic coagulation factors and appears to have little if any significant utility in as-sessing Hemophilia A and B.

The Clot Signature Analyzer (CSA, Xylum Corpora-tion, Scarsdale, NY) is an automated in vitro instru-ment designed to simulate in vivo clotting and plate-let function under physiological conditions using unanticoagulated whole blood.(43-47) In the CSA, blood flow is passed through two channels. In the punch channel, shear-induced platelet activation is simulated by two small (0.015 cm) holes punched in a blood conduit, causing a pressure drop in the lumen until closure of the punch holes occurs (platelet he-mostasis time). The collagen channel incorporates a small aperture with a collagen fiber immobilized at the center of the aperture. Platelets adhere to the col-lagen and eventually close the aperture, representing

Automated Optical PlateletAggregometry

A recent innovation is the development of optical platelet aggregometry for point of care analysis using microbead agglutination technology. The VerifyNow System (Accumetrics, San Diego, CA) consists of a small optical analyzer and disposable, single-use as-say cartridges that contain all necessary reagents, including fibrinogen-coated microbeads. The patient sample of 3.2% citrated whole blood is automatically dispensed from the blood collection tube into the assay cartridge without operator intervention. Assay devices for the monitoring of aspirin and anti-GP Iib/IIIa receptor antagonists (i.e., abciximab and eptifiba-tide) are commercially available, and an assay to monitor Clopidogrel (Plavix) therapy is under devel-opment. To date, the VerifyNow assay has been pri-marily used to measure aspirin resistance in patients with coronary artery disease.(53; 54)

One instrument is especially marketed for the detec-tion of GPIIb/IIIa receptor blockade in patients treated with the platelet antagonist abciximab. The Ultegra Accumetrics RPFA uses a turbidimetric opti-cal detection system to measure the agglutination of fibrinogen-coated microparticles in in anticoagulated whole blood. In the assay, platelets with unblocked GPIIbIIIa receptors are activated and cause micropar-ticle agglutination with a change in optical light transmission.(55; 56) However, a recent study did not confirm the sensitivity of the Accumetrics RPFA in comparison to conventional platelet aggregometry of the Platelets assay.(57)

Whole Blood Hemostatometry

Thromboelastography, measurement of platelet con-tractile force, and related procedures are analytical techniques to measure the global process of coagula-

9

Automated Platelet Function Analysis


sample is placed in a shallow cup and is trapped be-tween parallel surfaces when an upper plate is low-ered onto the upper surface of the forming clot (Fig. 1). The upper surface is attached to a strain gauge transducer. As the clot forms and the platelets pull within the network, a downward force is transmitted to the upper plate and transducer. The downward force stresses the transducer and a voltage propor-tional to the distance moved is generated. Since the transducer actually measures distance moved, a cali-bration constant relating distance moved to force is used to convert distance to force. Early work with this device confirmed that the forces produced by plate-lets (platelet contractile force, PCF) in platelet rich plasma or whole blood clots were significant (several kilodynes in magnitude) and easily measured.(68) The onset of force development occurred as soon as the fibrin network was in place. Utilizing this new technique, PCF was found to be directly dependent on platelet count, to be sensitive to temperature and cal-cium concentration, but to be relatively independent of fibrinogen concentration over the normal fibrino-gen range of 100 to 400 gm/dL.(69) PCF is also a very stable parameter, that persists in whole blood stored at room temperature for as long as ten days. In con-trast, platelet function by conventional aggregometry must be performed within four to six hours. The ro-bust nature of the parameter and its absolute de-pendence on platelet viability have led some groups to examine the use of the PCF parameter as a marker of platelet survival in stored and modified platelet preparations.(70)

The thrombin generation time is another parameter measurable by the Hemodyne. This is performed by the use of Batroxobis, a snake venom proteolytic en-zyme from the fer-de-lance that directly clots fibrino-gen via cleavage of fibrinopeptide A. The addition of batroxobin to citrated whole blood results in rapid clot formation, but no initial PCF development. Al-though batroxobin does not activate platelets, after a

tion (i.e., primary hemostasis to fibrinolysis) using whole blood. Although this technology was originally developed decades ago, there has been a recent resur-gence of interest due to the increasing need for im-mediate information in critically ill patients and those undergoing liver transplantation, cardiovascu-lar surgery, and other procedures where rapid hemo-static changes occur.(58-63)

Thromboelastography

The conventional (rotational) thromboelastograph uses a sample cuvette cup filled with native (unanti-coagulated) whole blood to measure clot formation/dissolution kinetics and the tensile strength of the clot. A pin suspended from a torsion wire is lowered into the cuvette and the cup is rotated through a 45o angle over a period of time. Torque from the rotating cup is transmitted from the pin and suspending rod to a recorder. There is no initial torque, but this in-creases as the clot forms and decreases as fibrinolysis occurs. More recent thromboelastographs use optical detection systems to measure the movement of the rotating pin, as well as computer hardware and soft-ware for data collection and analysis.(64) Commercial thromboelastographs include the TEG system Hae-moscope Corporation (Niles, IL), and the ROTEG (Pentapharm GmbH, Munich, Germany). Thromboe-lastography has been extensively used for interopera-tive cardiopulmonary and near-patient coagulation monitoring to guide blood product utilization.(64) Although thromboelastography can be measured in citrated blood, the results are not compariable to whole blood.(65)

Clot Retraction

A technology recently developed by Hemodyne, Inc. (Richmond, VA) the Hemostasis Analysis System permits direct measurement of the forces produced in the sample during clot formation.(66; 67) The

variable lag phase PCF development is noted. During the lag phase, thrombin is generated as a conse-quence of sample re-calcification. Since the fibrin network is in place prior to the generation of throm-bin, PCF becomes apparent as soon as a small amount of thrombin is generated. Thus, the inflection or take off point in the PCF curve serves as a marker of thrombin generation in the batroxobin mediated assay. Assays of prothrombin fragment 1+2, reveal a concurrent burst of activation fragment generation at the moment of PCF upswing.(71) The lag phase is thus the thrombin generation time (TGT). In normal individuals, PCF developed by the addition of ba-troxobin differs only in the time of onset. However, if thrombin generation is inhibited by the addition of anticoagulants or by the presence of clotting factor

10

Whole Blood Hemostatometry

Fig. 7. A schematic illustration of the Hemodyne he-mostasis analyzer used to measure platelet contrac-tile force and clot elastic modulus. The test specimen is placed in a sample space between a thermostated cup and a parallel upper surface. During blood clot-ting, platelets pull fibrin strands inward, generating a force that is detected by a displacement transducer and converted to a voltage proportional to the amount of force generated. Diagram used with per-mission of Hemodyne, Inc.


pathways of the coagulation system (Fig. 8). Similar functional assays have been developed to measure fibrinolysis and other coagulation pathways.

The clinical coagulation laboratory uses clotting as-says (prothrombin time, activated partial thrombo-plastin time) in which tissue phospholipids are added to platelet-poor plasma as full or partial thrombo-plastins to to initiate clotting for screening of hemo-philiac defects or for specific factor assays (Fig. 9). Instruments for automated performance of clot-based assays are available from several manufactur-ers, including Beckman Coulter, Inc. (Fullerton, CA), Dade Behring (Deerfield, IL), Diagnostica Stago, Inc. (Parsippany NJ), Global Medical Instrumentation, Inc. (St. Paul, Minnesota), and Sysmex Corporation (Kobe, Japan). Several similar assays using whole blood are available for near-patient testing. The most widely used of these assays is the activated clotting time used to monitor clotting during cardiopulmonary bypass.

Activated Clotting Time (ACT)

The ACT was developed in 1966 as a modification of the Lee-White whole blood clotting time to monitor coagulation status and heparinization in immediate need situations.(52) The ACT uses tubes containing a negatively-charged particulate activator of coagula-tion, such as kaolin, celite of diatomaceous earth. When whole blood is drawn into the tube, the contact system is activated and clotting occurs. The assay is useful at high levels of heparin such as used in open-heart surgery, but is also affected by platelets.(85-88)

The manual ACT has been replaced in recent years by an increasingly sophisticated variety of microprocessor-controlled instruments, exemplified by those manufactured by Helena Laboratories Corp. (Beaumont, Texas), ITC (Edison, NJ), Medtronics (Minneapolis, MN), and Roche Diagnostics Corpora-

deficiencies, PCF in the batroxobin clots is dramati-cally delayed and deficient. TGT is sensitive to the effects of heparin(72; 73), low molecular weight heparins(74), dermatan sulfate(75), non-heparin anti-thrombins(76), inherited clotting factor deficienci-es(77) and clotting factor deficiencies induced by warfarin. In vitro studies indicate the potential for documentation of the correction of deficient throm-bin generation by hemostatic agents such as recom-binant FVIIa.(78)

The Sonoclot Coagulation and Platelet Function Ana-lyzer (Sienco Inc., Wheat Ridge, Colorado) is a versa-tile, whole blood point of care system that uses a vis-coelastic clot detection mechanism to analyze the global process of hemostasis, including coagulation, fibrin gel formation, clot retraction (platelet func-tion) and fibrinolysis.(79) The Sonoclot uses the os-cillation of a tubular probe within a blood sample to generate an analog electronic signal that reflects re-sistance to motion during clot formation and fibri-nolysis. Data processing by a microcomputer gener-ates a qualitative graph (Sonoclot Signature) as well as quantitative results on clot formation kinetics and the rate of fibrin polymerization. A variety of differ-ent reagent kits are available for general coagulation monitoring, as well as more specific purposes, includ-ing heparin monitoring, hyperfibrinolysis screening, hypercoagulable screening and platelet function assessment.(80-84)

Each of these instruments has its own distinct fea-tures and advantages for the diagnostic laboratory, but a full specific assessment of global hemostasis defects requires multiple approaches.

Clot-Based Assays

Functional assays based on clot formation as the endpoint are widely used in the clinical laboratory to determine the integrity of the intrinsic or extrinsic

tion (Indianapolis, IN). Many of these instruments perform the PT, aPTT, thrombin time, fibrinogen level, and other hemostatic assays in addition to the activated clotting time. Some manufacturers also provide ACT reagents containing heparinase so that a patients baseline value can be established in the presence of heparin. These instruments are increas-ingly being applied to the near-patient monitoring of direct thrombin inhibitors and low molecular weight heparins in critical situations.(89-91)

11

Clot-Based Assays

PLT

PLT

Prothrombin

Fibrinogen

XIIa

XIa

X

Thrombin

Fibrin

XII

XI

IX

VIII

VIIIa

V

Va

VII/VIIa

TF

XIII

XIII

X-LinkedFibrin

Xa

IXa

ContactSystem

Fig. 8. A color-coded schematic illustration of the coagulation system. The diagram shows components of the contact system (orange), extrinsic pathway (blue), intrinsic pathway (magenta), and common pathway (green). In vivo, platelets (yellow) are essen-tial for contributing phospholipid and providing a surface for the tenase and prothrombinase reactions to occur.


one of a variety of techniques (photo-optical, elec-tromechanical, etc.)(Fig. 8). The result is reported in seconds (prothrombin time), or as a ratio compared to the laboratory mean normal control (prothrombin ratio, PTR). The PT is critically dependent on the characteristics of the thromboplastin used in the as-say, as well as manner of blood coagulation, the type of container, the type of anticoagulant, specimen transport and storage conditions, incubation time and temperature, assay reagents, and the method of end point detection. This means that patients on coumadin will have different clotting times when tested in different laboratories, so a means of stan-dardization of results must be employed.

The International Normalized Ratio (INR) was intro-duced by the World Health Organization (WHO) in the early 1980s as a means of standardizing PT results.(94) For this purpose, a very responsive batch of human brain extract was designated as the first International Reference Preparation (IRP), and a cor-

Prothrombin Time (PT, Protime,Quicks time, Partial Prothrombin Time)

The PT provides a functional determination of the integrity of the extrinsic (tissue factor) pathway of coagulation and is sensitive to the vitamin-K depend-ent clotting factors (factors II, VII, IX, and X) as well as to factors of the common pathway (fibrinogen, prothrombin, factor V, factor X). The PT is a widely used laboratory assay for the detection of inherited or acquired coagulation defects related to the extrin-sic pathway of coagulation, and is the standard test for monitoring oral anticoagulation therapy (coumadin).(92; 93)

In the PT an aliquot of test platelet-poor plasma is incubated at 37oC with a reagent containing a tissue factor, phospholipid (thromboplastin), and CaCl2. The time required for clot formation is then measured by

rection factor (International Sensitivity Index, ISI) was developed to correlate the sensitivity of commer-cial thromboplastin preparations to the IRP. By defi-nition, the ISI of the first IRP was 1.0. An additional term, the INR, was introduced to compare a given prothrombin ratio measurement to the IRP. Commer-cial vendors of thromboplastin preparations supply the ISI with each reagent lot. If the ISI is known, the INR for each clotting time is easily calculated. How-ever, the ISI can be affected by instrumentation and other laboratory factors and thus must be verified by each testing site according to standards of the College of American Pathologists. Unfortunately, even with the INR, current prothrombin reagent/instrument calibration techniques are insufficient to provide good intralaboratory agreement.(95; 96)

There is great interest in point of care and patient self-testing of oral anticoagulation status are popular for patient convenience and to improve the efficiency of medical care. Considering the 600,000 to 900,000 patients in the United States with heart valves, and the millions requiring oral anticoagulation for hyper-coagulability states, it is not surprising that several small, user-friendly instruments are presently avail-able for home testing by prescription from blood ob-tained by fingerstick. These instruments include the AvocetPT-Pro (Avocet Medical, Inc. San-Jose, CA), the CoaguChek (Roche Diagnostics, Basal, Switzerland), the Harmony INR Monitoring System (LifeScan, Inc., Milpitas, CA) , the INRatio Meter. (HemoSense, Inc. San Jose, CA),and the HemosProTime Microco-agulation System (ITC, Edison, NJ), Presently, these assays are CLIA waived and have been covered by Medicare since late 2001. Point of care monitoring of the PT and INR has been the subject of several recent reviews.(97-103)

12

Clot-Based Assays

Platelet-poor plasma Incubation

Fibrin clot

Clotting agent, Ca ++

Fig. 8. Basic principle of clot-based assays of coagulation. A clotting activator, cal-cium, and a source of phospholipids is incubated with platelet-poor plasma, re-sulting in activation of the extrinsic clot-ting system. The endpoint of the reaction is the formation of a fibrin clot that can be measured by visual, photo-optical, elec-tromechanical means. The result is usually reported as the time required for clot for-mation. Common clot-based assays used in the clinical hemostasis laboratory include the PT, aPTT, thrombin time, reptilase time, dilute Russell Viper venom time, and activated protein C resistance assay. Clot-based assays are also used for factor analysis and to determine the presence of factor deficiencies and anti-factor inhibi-tors.


failure to promptly mix the blood with the citrate anticoagulant, improper transport or storage, or a prolonged interval between specimen collection and analysis. The sensitivity of the assay to factor defi-ciencies, inhibitors, and heparin also varies with the reagents used in the assay. Because of these variables, a normal aPTT result does not exclude a mild coagu-lation factor deficiency or the presence of a low-titer or slow-reacting inhibitor. However, a significant pro-longation of the aPTT indicates the presence of a fac-tor deficiency (VIII, IX, XI, XII, prekallikrein, HMWK), while prolongation of both the PT and aPTT suggests a deficiency of factor I, II, V, or X. The aPTT is not affected by deficiencies of factor VII or XIII.

Numerous modifications of the aPTT have been de-scribed for the functional analysis of specific coagulation-related substances. Those routinely util-ized in the coagulation laboratory at the present time include the reptilase time, the Bethesda assay, protein C and protein S activity, and several assays for lupus anticoagulants (dilute Russell viper venom time [dRVVT], platelet neutralization test, and hexagonal phospholipid assay.

Specific anti-factor VIII antibodies (inhibitors) are a serious medical problem for patients with hemo-philia. Mixing studies can detect the presence of in-hibitors, but other assays are required for the precise measurement of antibody activity necessary for pa-tient care.(121) The Bethesda assay is a modified aPTT based on the ability of factor VIII inhibitors to neutralize factor VIII activity in normal plasma. A series of dilutions of patient plasma are added to a standard amount of normal plasma and assayed for factor VIII levels after two hours incubation at 37C: the titer at which half of the FVIII activity remains is used to calculate the Betheda units of inhibition. Several modifications of the Bethesda assay have been developed to improve its sensitivity.(122-124) The new Oxford assay is similar, but uses factor VIII

Activated Partial Thromboplastin Time(aPTT, Activated Prothrombin Time)

The partial thromboplastin time (PTT) is the clotting time obtained when partial thromboplastin is added to plasma. Partial thromboplastin is the phos-pholipid fraction of a tissue extract, and differs from a complete tissue extract (i.e., thromboplastin) by the lack of tissue factor. The PTT is sensitive to the intrinsic pathway of coagulation, but is most sensitive to the contact factors (i.e., factor XII, prekallikrein, high molecular weight kininogen) when a particulate activating agent (i.e., silica, celite, kaolin, mi-cronized silica, ellagic acid) is added to the reaction (activated PTT, aPTT). Many different phosophlipid reagents animal and plant origin, such as cephalin, have been used as partial thromboplastins, and a va-riety of activating substances are in use.(104-110)

In the aPTT an aliquot of undiluted, platelet-poor plasma is incubated at 37oC with an activator and phospholipid (partial thromboplastin). CaCl2. is then added, and the time required for clot formation is measured by one of a variety of techniques (photo-optical, electromechanical, etc.). The aPTT result is reported as the time required for clot formation after the addition of CaCl2. The aPTT is functional deter-mination of the intrinsic (factors XII, XI, IX, VIII, V, II, I,) and common pathways of coagulation.(111; 112) The aPTT is utilized to detect congenital and ac-quired abnormalities of the intrinsic coagulation pathway, monitor patients receiving heparin or co-agulation factor replacement therapy, and to detect inhibitors of the intrinsic and common pathways.(113-120)

The aPTT clotting time may be influenced by many pre-analytical and analytical variables and caution must be used in the interpretation of the result. Pre-analytical variables include slow or difficult specimen collection, an improper blood:anticoagulant ratio,

concentrate as the source of factor VIII. Enzyme im-munoassay, gel techniques, and other methods have been also used to detect inhibitors.

The direct thrombin inhibitors are among the latest form of anticoagulant drugs developed with the goal of eliminating the side effects and improving the therapeutic efficacy of anticoagulants which exert an indirect antithrombin effect, including warfarin, heparin, and low molecular weight heparin.(125) The present generation of direct thrombin inhibitors in-cludes recombinant hirudin (lepirudin), bivalirudin, argatroban, and melagatran. Unfortunately, the direct thrombin inhibitors present a problem for the hemo-stasis laboratory, since conventional coagulation as-says such as the aPTT, thrombin time, and activated clotting time show poor reproducibility and linearity in the presence of these drugs.(126) Two modifica-tions of the aPTT, the ecarin clotting time (ECT) and prothrombinase-induced clotting time (PiCT) have been developed for monitoring the direct thrombin inhibitors, as well as chromogenic and enzyme immunoassays.(127-129) There is presently no clear concensus on the most optimal laboratory method for direct thrombin inhibitor monitoring, although the automated chromogenic assays and chromogenic-based point of care assays appear to offer adequate sensitivity and precision and avoid interference prob-lems by heparin and other substances.(126; 130-132)

Thrombin Time (ThrombinClotting Time, TCT, TT)

The thrombin time measures the thrombin-induced conversion of fibrinogen to fibrin directly in patient plasma, bypassing all other clotting factors. The thrombin time is performed by the addition of a low concentration of thrombin (usually bovine thrombin) directly to the citrated plasma and measuring the time required for the formation of fibrin monomers by visual, mechanical, or opto-electronic

13

Clot-Based Assays


human plasma deficient (

monoclonal paraproteins, and drugs such as heparin. Clinical and other laboratory clues are necessary to identify the inhibitor. For example, lupus anticoagu-lants are usually not associated with clinical bleeding, while specific factor inhibitors frequently cause bleeding. Generally, factor deficiencies produce a complete correction of the prolonged clotting time (i.e., corrected to within the normal range), specific antibodies show very little, if any correction, and non-specific may show a partial correction, (i.e., shortened clotting time but not to within the normal range). The presence of heparin and other nonspe-cific inhibitors can be confirmed by other coagula-tion tests such as the thrombin clotting time and rep-tilase time, while lupus anticoagulants are identified by a phospholipid-sensitive test such as the dilute Russell Venom time (dRVVT). The last clue is pro-vided by the effect of incubation on the activity of the inhibitor. An immediate mixing study is performed by mixing equal amounts of the "test" plasma with NPP (1:1 mix) and immediately performing a clotting time (i.e., PT, aPTT, or TT) on the mixed plasma specimen.(147-149) Most factor inhibitors (except factor VIII) and a most lupus anticoagulants (fast reacting inhibitors) produce an immediate clotting time inhibition and do not require incubation. In contrast, most factor VIII inhibitors and some lupus anticoagulants (15%) are weak and/or time depend-ent (slow reacting inhibitors), and require incuba-tion of the 1:1 plasma mixture at room temperature or 37oC for one or two hours (incubated mix) to cause prolongation of the clotting time.(150-152) A false diagnosis of a factor deficiency can result with-out incubation, since slow-reacting inhibitors may correct the immediate mix. Some laboratories also include a 4:1 aPTT mix (i.e., 4 parts patient plasma, 1 part NPP) to improve the detection of weak inhibitors that minimally prolong the aPTT (usually 3-5 seconds above baseline). The markedly prolonged aPTT of

tinin or other plasmin inhibitor. Many studies have shown that fibrin degradation products cause an overestimation of the fibrinogen level by the washed clot and immunologic assays, and an underestima-tion by the clot-based techniques.(141) The kinetic assay has also been reported to yield higher fibrino-gen levels in patients receiving oral anticoagulation than the von Clauss technique.

Plasma Mixing Studies (Clotting FactorInhibitor Screen, Circulating Anticoagulant Screen)

A prolonged clotting test (i.e., PT, aPTT, and/or thrombin time) indicates the presence of a factor de-ficiency or inhibitor of coagulation. The plasma mix-ing study is the initial step in the evaluation of a pro-longed clotting time. The goal of a mixing study is to determine if the prolonged clotting time is shortened or corrected by mixing the test plasma with equal volume of normal pooled plasma (NPP; also called citrated normal plasma, CNP). Even a profound defi-ciency of a clotting factor, such as the 1% factor VIII level encountered in severe hemophilia, will be cor-rected to the normal range by mixing with NPP, since a 50% level of any factor will still yield a normal clot-ting time. Factor assays are then performed to iden-tify the deficient clotting factor.

The failure of a prolonged clotting test to correct in the mixing study indicates the presence of a inhibi-tory substance that is preventing clotting from oc-curring. Unfortunately, this is somewhat difficult to accomplish since there are several different types of inhibitors (also called circulating anticoagulants). Specific inhibitors are immunoglobulins with speci-ficity for phospholipid ("lupus anticoagulants") or a specific clotting factor ("factor inhibitors"). Global or non-specific inhibitors affect more than part of the clotting process and include fibrin(ogen) degra-dation products, some pathologic antibodies such as

plasma from a patient with hereditary prekallikrein deficiency is normalized by prolonged preincubation (i.e., 10 minutes) of the plasma with aPTT reagent before the assay is performed. This unique feature of prekallikrein deficiency is reportedly due to the au t o a c t i v a t i o n o f f a c t o r X I I du r i n g preincubation.(153)

Mixing studies are simple in principle, but can be difficult to interpret. For example, if the laboratory range for the aPTT is 24-35 seconds, and the patient aPTT is 70 seconds, a 1:1 mixing study result of 34 seconds would clearly indicate a factor deficiency, while a value of 69 seconds would indicate an inhibi-tor. However, what if the mixing study produced val-ues of 39 seconds, 51 seconds, or 63 seconds? The situation is made even more difficult because there are no official criteria for determining if a correc-tion has occurred. Furthermore, a number of patient-specific and laboratory-specific variable can affect the result and are difficult to compensate for. These include the biological heterogeneity of anti-factor antibodies, the presence of drugs and other sub-stances in the patient specimen, reagent and instru-ment sensitivity, the source of NPP, the validity of the laboratory reference range, pre-analytical variables, and other factors. Therefore, each laboratory pres-ently establishes their own criteria for interpreting mixing studies. As summarized by Ledford-Kraemer (2004), these criteria generally fall into three catego-ries:

The use of the upper limit of the laboratory ref-erence range as the correction target. A value, such as 2SD, 3SD, or within 5 seconds of the upper limit of the reference range is chosen as the criteria for correction. A failure of correc-tion is assumed if this value is not reached.

The use of NPP tested in conjunction with the patient 1:1 mix. This is particularly valuable to correct for the decreased activity of the labile

15

Clot-Based Assays


gen molecule, reptilase cleaves only fibrinopeptides A and AP. The resulting fibrin monomers polymerize end-to-end to form a fibrin clot. Reptilase has no fi-brinolytic activity, does not activate plasminogen, and is not inhibited by antifibrinolytics, thrombin inhibitors (heparin, hirudin, anti-thrombin antibod-ies) or antithrombin III.

The reptilase time is used in the evaluation of a pro-longed aPTT, specifically to exclude the presence of dysfibrinogenemia. Hypofibrinogenemia and dysfi-brinogenemia are the usual causes of a prolonged reptilase time. Prolongation of both the thrombin time and reptilase time suggests hyopfibrinogenemia or dysfibrinogenemia. A prolonged aPTT and normal reptilase time indicates that heparin or other anti-thrombins is the cause of the prolonged aPTT. Mye-loma proteins reactive with thrombin may prolong the reptilase time. Fibrin degradation products (FDPs) may slightly prolong the reptilase time.

Dilute Russell Viper Venom Assay (dRVVT)

The dRVVT is used to detect lupus anticoagulants (LA), one type of autoantibody characteristic of pa-tients with the antiphospholipid antibody syndrome.(155-157) LA are autoantibodies of the IgG

coagulation factors, V and VIII, during incu-bated studies. Common criteria for correction of the patient sample include to within 5 sec-onds, or to within 10% or 15% of the NPP value. The Rosner index for the aPTT mixing study quantitates the amount of correction to the pa-tient plasma aPTT. A correction is assumed if the Rosner index is 15.

The Chang percentage, a formula that incorpo-rates the degree of correction in relation to the initial aPTT prolongation.

Chang and co-workers performed a detailed evalua-tion of the sensitivity and specificity of different methods to define correction of the 1:1 mix.(148; 149) They found that the three most widely used criteria for a correction of the aPTT 1:1 mix (upper limit of normal, NPP aPTT + 5 seconds, Rosner index 15) all had high sensitivity (88-100%) but low specificity (7-13%) for detecting a factor deficiency, and low sensi-tivity (7-15%) and high specificity (88-100%) for de-tecting an anticoagulant. Using their correction for-mula and a % correction cutoff at 50%, the immediate aPTT 4:1 mix had a 75% sensitivity for a factor defi-ciency and a 91% sensitivity for an anticoagulant. The corresponding specificies were 91% and 75%. Using an incubated aPTT 4:1 mix with a cutoff value of > 10% gave sensitivities and specificities of 100% for both factor deficiencies and anticoagulants. There-fore, the authors recommend performing immediate and incubated 1:1 aPTT mixes, with the interpretation as follows:

Reptilase Time

The reptilase time measures the conversion of fi-brinogen to fibrin clot by reptilase (Batroxobin, Atroxin), a thrombin-like enzyme derived from the venom of the fer-de-lance (barba amarilla, Bothrops atrox).(135; 136; 154) In contrast to thrombin, which cleaves fibrinopeptides A, AP, and B from the fibrino-

and IgM classes that interfere with the function of anionic phospholipids and prolong phospholipid-dependent clotting tests such as the aPTT and dRVVT.(158-162) The dRVVT is more specific for LA than the aPTT since it is not influenced by deficien-cies of the contact or intrinsic pathway factors or an-tibodies to factors VIII, IX, or XI.(159; 163; 164) The coagulant protein in Russells viper venom (RVV) is a serine protease that directly activates factor X in the presence of Ca++, bypassing the intrinsic and extrin-sic pathways. The activated factor X then activates prothrombin (factor II) in the presence of factor V and phospholipid. In the dilute Russells viper venom time (dRVVT), phospholipid is diluted to the point that the clotting time becomes very sensitive to the presence of substances blocking availability of the phospholipid surface. The DVVtest is a commercial reagent (American Diagnostica, Inc., Greenwich, CT) developed to standardize the dRVVT. Similar reagents are available from Precision Biologic (Dartmouth, Nova Scotia) and other vendors. The DVVtest reagent combines RVV, plant phospholipid, and calcium into a single reagent. A second reagent, DVVconfirm, con-tains RVV, extra plant phospholipid, and calcium. The extra phospholipid in the DVVconfirm reagent is provided to see if it corrects a prolonged DVVtest time (by overwhelming the LA). The finding of a pro-

16

Clot-Based Assays

% Correction = PP PT (or aPTT) - 1:1 (or 4:1) Mix PT (or aPTT)

PP PT (or aPTT) - CNP PT (or aPTT)X 100

Index = 1:1 Mix aPTT - CNP aPTT

PP aPTTX 100

Chang Percentage

Rosner Index

Fig. 9. Formu-las for calcula-tion of Chang Percentage and Rosner Index.


Chromogenic Analysis

Chromogenic analysis is a technique of enzyme analysis developed in the early 1970s. Chromogenic assays utilize synthetic substrates comprised of a colored chemical substance (chromphore, chroma-gen) linked to a short amino acid residue specific for the enzyme of interest.(172-174) Enzymatic action releases the chromophore, which is quantitated by spectrophotometry. The selectivity of chromogenic substrates is similar to the native enzyme substrate, but they are often more sensitive. Other advantages of chromogenic assays include reagent stability and the adaptability to a wide range of automated laboratory instruments, including those used in the chemistry and immunology laboratories. The selectivity of a chromogenic substrate to the desired enzyme is af-fected by the relative concentrations of sample and reagents, reaction conditions (i.e. pH, temperature, buffer type and concentration, ionic strength, etc.), the presence of inhibitors, substrate solubility and stability, and other factors.(175) The best substrates have high affinity for the enzyme and a high turnover rate. The most common substrate is para-nitroaniline (pNA), which has a maximum absorption spectrum at 405 nm.

longed dRVVT with patient plasma is presumptive evidence for the presence of a lupus anticoagulant. This presumption is confirmed if the dRVVT is not corrected with a mixture of normal and platelet plasma, but is corrected by the substitution of plate-lets for phospholipid. With the DVVtest and DVVcon-firm reagents, a DVVtest/DVVconfirm ratio >1.2 is confirmatory for the presence of LA.

Activated Protein C ResistanceAssay (APCR)

The rapid screening assay for activated protein C re-sistance for (APCR) is another widely used modifica-tion of the aPTT. In 1993, Dahlback and coworkers discovered a mutant clotting Factor V (Factor V Lei-den) which results in the failure of Activated Protein C to inactivate Va.(165-168) This defect in the protein C pathway is associated with a significantly increased risk of thromboembolic disease. The laboratory di-agnosis of APCR begins with the rapid screening test, followed by confirmation with a molecular assay if the screening assay is positive. In the presently used modification of Dahlbacks original aPTT-based screening assay, the test plasma is first diluted with factor Vdeficient plasma to inactivate therapeutic concentrations of heparin, correct for coagulation factor deficiencies, and counteract the effect of some lupus inhibitors. aPTT assays are then performed with and without the addition of exogenous activated protein C (APC).(169-171) The added APC signifi-cantly prolongs the aPTT in normal individuals, while patients with APCR show less of an increase. The re-sults are usually expressed as the ratio of the aPTT with and without added APC. The modified APCR screening assay is highly sensitive to factor V Leiden and most other less common mutations of factor V, can differentiate heterozygotes from homozygotes, and is not influenced by heparin or warfarin at therapeutic concentrations.(170)

The analysis of many coagulation factors utilize chromogenic substrates for factor X.(176; 177) For example, factor VIII is an enzymatic cofactor for fac-tor IX. Activated factor IX causes the activation of factor X, which then hydrolyzes the chromogenic sub-strate and releases the pNA chromophore that is read spectrophotometrically at 405 nm (Fig. 10). If the as-say conditions are properly controlled, the color in-tensity reflects the amount of factor VIII. In one com-parative study of chromogenic analysis, an antigenic assay, and the one-stage clotting assay for factor VIII, the chromogenic factor VIII technique was the opti-mal method, with good precision and freedom from interference by lupus inhibitors, heparin, or other anticoagulant drugs.(178)

Chromogenic substrates for thrombin, tissue-type plasminogen activator, urokinase, coagulation factors IX, X, and XII, and other substances are commercially available from Chromogenix (Orangeburg, NY), (Trinity Biotech Plc, Wicklow, Ireland) and other companies.

17

Chromogenic Analysis

Factor XFactor IXa, Ca++, Phospholipid

Factor VIII

Chromogenic

substratePeptide + pNA

Factor Xa

Fig. 10. A chromogenic method for the determination of factor VIII activity. Test plasma is incubated with calcium, phospholipid, and excess amounts of purified factors IX and X. The activated factor X generated by the reaction hydro-lyzes a chromogenic substrate, generating a colored reaction product that is measured by a spectrophotometer. The amount of generated factor Xa is directly proportional to the concentration of factor VIII activity.


Turbidimetry and the related technique of nephelo-metry are extensively utilized in the clinical immu-nology for the quantitation of a large number of medically important substances since they are pre-cise, rapid, and automated. In the coagulation labora-tory the use of these techniques is more limited. Light scattering by particles in solution is the basic princi-ple of turbidimetry and nephelometry. When an im-mune complex is formed under carefully controlled conditions, measurement of light scatter can provide information regarding the quantity of analyte present.(181) Turbidimetric techniques determine the reduction in the intensity of incident light from all interactions of an immune complex with a light beam, while nephelometric techniques measure light scattered at a specific angle to the incident beam. Nephelometry is more sensitive to small particles than turbidimetry, while turbidimetry more accu-rately measures large complexes.(182)

Turbidimetry has been used since the early 1950's as a method of quantitative analysis, particularly for large immune complexes. In this technique, the change in light intensity caused by interaction of a light beam with a suspension of particles is deter-mined spectrophotometrically. The major use of latex particle agglutination and turbidimetry in the clinical coagulation laboratory is the detection and semi-quantitation of fibrin degradation products (FDPs) and D-Dimers. Fibrin degradation products (FDPs) are the result of plasmin degradation of fibrinogen, fibrin monomers, fibrin polymers or cross-linked fibrin, while D-dimers are degradation products that arise specifically from the plasmin degradation of fibrin crosslinked by Factor XIIIa activity.(183) Thus, the measurement of cross-linked degradation prod-ucts (XDPs), unlike total FDPs, is a specific measure of fibrinolysis. Most turbidimetric assays for D-dimers utilize latex beads or other microparticles coated with monoclonal antibody specific for fibrin D-dimer or the fragment D of fibrin but not with in-

Latex Agglutinationand Turbidimetry

Agglutination is a secondary immune phenomenon that occurs when insoluble or particulate antigens (cells or other particles) are cross-linked by an im-mune reaction. Agglutination occurs because anti-bodies have two or more antigen recognition sites (bi- or multivalency). If multiple antigenic recogni-tion sites are present on a particle, lattices can be formed that grow in size and eventually become a mass that is macroscopically visible. The major fac-tors affecting the agglutination reaction include the class, affinity and avidity of the antibody, the proxim-ity and number of binding sites on the particle, the relative concentrations of antibody and particles, electrostatic interactions ("zeta potential"), and the viscosity of the medium. Antibodies of the IgM class, with ten antigen combining sites, are usually the best agglutinins, and are more efficient than IgG in ag-glutination. Agglutination assays are classified as direct or indi-rect, depending on whether the analyte is present in its native state, or linked to a particle (carrier) to al-low detection of the antigen-antibody reaction. Carri-ers vary in size from about 0.05 micron to 7 micron, and may be red blood cells, latex particles, liposomes, microcapsules, or other particles.(179)

The use of latex particles for immunoassay was first reported by Singer and Plotz in 1956.(180) Latex par-ticles are usually coated by passive means, with the quantity of the adsorbed protein adjusted to provide agglutination of the analyte in its biological range. In addition, the use of latex particles avoids much of the variability encountered with red blood cells. Even so, the prozone phenomenon can still be significant, and careful adherence to the manufacturer's instructions is necessary during the performance of clinical assays utilizing coated microspheres.

tact fibrinogen, permitting the analysis of whole hu-man plasma. Elevated D dimers are seen in DIC, pul-monary embolism, arterial and venous thrombosis, septicemia, cirrhosis, carcinoma, sickle cell crisis, and following operative procedures. However, D-dimer analysis is principally used in the evaluation of pa-tients with suspected thromboembolic disease, espe-cially pulmonary embolism and deep vein thrombosis.(184-188) Both FDPs and XDPs are pre-sent during late pregnancy and for approximately 48 hours post-surgery. During fibrinolytic therapy the FDP test is positive, while the D-dimer test is negative in the absence of thrombolysis. Enzyme immunoas-say has also been utilized for the detection of D-dimers.(184; 189-192) Other turbidimetric reagents are available for the analysis of von Willebrand factor, free protein S, and other substances.

Nephelometric techniques have been applied very successfully to the immunochemical measurement of specific proteins, drugs and other substances.(182; 193) In nephelometry, a known amount of specific antibody is added to a solution containing the anti-gen being measured. The intensity of light scattered from the large antigen-antibody complexes formed during the reaction is measured, and the rate signal is transmitted to a microcomputer, where concentration units are determined. Nephelometry is used by some clinical laboratories for the quantitation of fibrino-gen or factor VIII-related antigen.

Enzyme Immunoassay

The enzyme immunoassay (EIA) is a type of non-isotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used as labels.(194; 195) The major prerequisite for an EIA is that an antigen or antibody must be linked to an enzyme without destroying the immunologic or en-zymatic activity of the antigen-antibody complex. In solid-phase EIA techniques, the antigen or antibody

18

Latex Agglutination


1970s and rapidly became an essential instrument for the biologic sciences. Spurred by the HIV pandemic and a plethora of discoveries in hematology, special-ized flow cytometers for use in the clinical laboratory were developed by several manufacturers. The major clinical application of flow cytometry is diagnosis of hematologic malignancy, but a wide variety of other applications exist, such as reticulocyte enumeration and cell function analysis. Presently, more than 40,000 journal articles referencing flow cytometry have been published. This brief review of the princi-ples and major clinical applications of flow cytome-try may be supplemented by several recent review articles and books. (199-203)

Prepared single cell or particle suspensions are nec-essary for flow cytometric analysis. Various immuno-flurescent dyes or antibodies can be attached to the antigen or protein of interest. The suspension of cells or particles is aspirated into a flow cell where, sur-rounded by a narrow fluid stream, they pass one at a time through a light beam. Light and/or fluorescence scatter signals are detected and amplified. The result-ing electrical pulses are digitized, and the data is stored, analyzed, and displayed through a computer system.(203; 204) The end result is quantitative in-formation about every cell analyzed. Since large numbers of cells are analyzed in a short period of time (>1,000/sec), statistically valid information about cell populations is quickly obtained.

The flow cytometer has been essential for the analy-sis of platelet structure and function in the research laboratory. Although the small physical size and bio-variability of the platelet creates inherent difficulties for flow cytometric analysis, several clinical assays are performed by specialized flow cytometry labora-tories. These assays will achieve more widespread practice in the near future as standardized techniques and controls become available. These assays have been classified by Bode and Hickerson to include

must be bound to a polystyrene test tubes or micro-titer tray, a particle of polystyrene, latex, or agarose, a magnetized bead, or another physical support.

Enzymes utilized in immunoassay systems must also be stable, available in a highly purified state, have a high turnover rate, and undergo minimal interference by substances likely to be in the test solution, and be specific for the substrate. The final reaction product should be detected by a convenient means with a low detection limit. The most widely utilized enzyme in enzyme immunoassay is horseradish peroxidase (HRP). The substrate of HRP is hydrogen peroxide (H202) and the product is oxygen. This oxygen pro-duced during the reaction is used to oxidize a re-duced, colorless chromagen (usually reduced ortho-phenylenediamine, OPD). The final product, oxidized OPD, has a brown color. Most EIA's utilize horserad-ish peroxidase or alkaline phosphatase as labels, al-though glucose oxidase, beta-D-galactosidase, and a wide variety of other enzymes have also been used. Utilizing fluorimetric techniques, the respective de-tection limits for HRP, beta-galactosidase, and alka-line phosphatase are 5, 0.2, and 10 attomol.(196) The practical detection limit of the EIA is approximately 0.01 to 0.02 attomol of ligand.(197; 198) The enzyme-Linked Immunosorbent Assay (ELISA) is the most widely utilized type of enzyme immunoassay.

Enzyme immunoassay is a critical technique in the clinical laboratory for a wide variety of analytes, in-cluding both antigens and antibodies. In the hemo-stasis laboratory, enzyme immunoassay is used for the quantitation of antigen levels of most clotting factors, fibrinolytic components, and regulatory sub-stances.

Flow Cytometry

Flow cytometry is a technique of quantitative single cell analysis. The flow cytometer was developed in the

platelet surface receptor quantitation and distribu-tion for the diagnosis of congenital platelet function disorders, platelet-associated IgG quantitation for the diagnosis of immune thrombocytopenias and for platelet cross-matching in transfusion, reticulated platelet assay to detect stress platelets, fibrinogen receptor occupancy studies for monitoring the clini-cal efficacy of platelet-directed anticoagulation in thrombosis, and the detection of activated platelet surface markers, cytoplasmic calcium ion measure-ments, and platelet microparticles for the assessment of hypercoagulable states.(205)

Flow cytometry is a critical research technique for the study of diseases of platelet surface receptors, and has been applied to clinical diagnosis by larger labo-ratories. The identification of Glanzmann thrombas-thenia, the Bernard-Soulier syndrome, and even rarer platelet receptor disorders is performed with panels of monoclonal antibodies specific for the receptor antigens under consideration. The use of monoclonal antibody panels specific for different epitopes is par-ticularly information in defining the heterogeneity and extent of disease expression.

Flow cytometry has been utilized to detect both platelet-associated immunoglobulins of autoimmune and alloimmune origin. In general, this is performed by incubating washed platelets with fluorochrome-labeled antihuman immunoglobulin and quantitating platelet surface fluorescence. Gating procedures are used to exclude irrelevant cells and particles. The differentiation of positive and negative results is de-pendent upon an adequate negative control, usually platelets from normal individuals. Flow cytometry is ideal for the study of low numbers of patients in thrombocytopenic patients, since it is sensitive and avoids the platelet activation, with the release of en-dogenous immunoglobulin molecules.(206) Although procedural standardization and non-specificity have limited the use of flow cytometric analysis for platelet

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ELISA, Flow Cytometry


The recent wealth of new discoveries in the field of hemostasis and thrombosis has included a number of antiplatelet agents of value in the therapy of patients with inherited or acquired hypercoagulability and/or those undergoing vascular interventional procedures. As the most prevalent and functionally important platelet surface receptor, the GPIIbIIIa complex is the target of several of these agents. Abciximab is a murine monoclonal antibody that blocks the GPII-bIIIa receptor to prevent platelet activation. Others, such as Integrilin and Aggrastat are small peptides that saturate the receptor. A variety of receptor oc-cupancy assays have been developed to monitor the clinical efficacy of these agents. Flow cytometry is the most sensitive and versatile of these techniques, al-though it is not a point of care assay at the present time.(219)

Electrophoresis

Historically, the Laurell rocket assay, radial immuno-diffusion, and other gel-based procedures were widely used in the coagulation laboratory for quanti-tative analysis of certain clotting factor protein levels, especially the FVIII/vWF complex.. Today, these pro-cedures have been largely replaced by enzyme immu-noassay, and other more accurate and efficient meth-ods, with the exception of von Willebrand factor mul-timer analysis. This is performed by electrophoresis on agarose gel containing some acrylamide. The pro-tein bands are then transferred to nitrocellulose for Western blot with polyclonal anti-vWF antibody and finally visualized by radiolabeling, enzymatic detec-tion, or chemiluminescence.(220; 221) The pattern of multimeric units and satellite bands differentiates von Willebrand disease into type I, type IIA, B, M, and N, and type III. Multimer analysis is a specialized procedure only performed by a few coagulation refer-ence laboratories, but it is sometimes critically im-portant to subtype von Willebrand disease to avoid

associated immunoglobulin in idiopathic thrombo-cytopenia purpura (ITP), the technique appears more clinically promising for the detection of alloimmune antibodies arising from the transfusion of non-autologous platelets (Post-transfusion Purpura) or the entrance of fetal cells into the maternal circula-tion (Neonatal Alloimmune Thrombocytopenia Pur-pura). The antibodies are typically formed against alleles of the human platelet alloantigen system (HPA), notably HPA-1a (Pl-A1) and HPA1b (Pl-A2).(207-209) These antibodies can be specifically detected by flow cytometry, and flow cytometric crossmatching can be performed to reveal HLA in-compatibilities between donor platelets and sensi-tized recipients.(210-212)

Reticulated platelets are recently released into the circulation, larger than average, and contain small amounts of RNA remaining from the megakarocytic process.(213) The quantitation of reticulated platelets is valuable for the differentiation of accelerated de-struction from impaired production in patients with thrombocytopenia of unknown etiology(214-216) Reticulated platelets can be detected by the utiliza-tion of thiazole orange, a brightly fluorescent dye that binds to nucleic acid.(217; 218) The assay is some-what difficult to perform, since the platelets must be permeabilized and positive controls are difficult to obtain.

Hypercoagulability is one of the most common medi-cal problems, and it is not surprising that a wealth of new discoveries have arisen from the application of modern technology. Flow cytometry has been an es-sential technique for the elaboration of platelet func-tion and understanding the contribution of platelet activation to hypercoagulability. To date, many flow cytometric studies have involved the detection of platelet surface markers, the study of cytosolic cal-cium ion levels, and the detection of circulating plate-let microparticles.(205)

inappropriate treatment (i.e., DDAVP is contraindi-cated in vWD Type IIb).

Genetic and Molecular Assays

The advances of genetic and molecular methods of study during the past two decades have had a pro-found impact upon our understanding of hemostasis, as well as other fields of medicine. The molecular origin and function of many substances involved in hemostasis are now understood, and the genetics and molecular of the hemophilias and many diseases is much clearer.(222-225) A diversity of new diagnostic assays resulting from these discoveries is expected in the near future, but at present the major clinical role for molecular analysis is in the diagnosis of inherited thrombophilia.

The majority of patients who develop recurrent ve-nous thromboemboli (inherited thrombophilia) have discernable abnormalities of the coagulation system, including factor V Leiden, deficiencies of protein C, protein S, antithrombin III, the prothrombin G20210A gene mutation, homocysteinemia, elevated factor lev-els, dysfibrinogenemia, or abnormalities of the fibri-nolytic system.(226) Most of these abnormalities cause deficiencies of the regulatory substances of clotting. Genetic abnormalities are especially com-mon in individuals who develop thrombi at an early age (< 40 years) and in those with a family history of thrombosis. Although no genetic abnormality is de-tectable in about 15 percent to 20 percent of indi-viduals with recurrent thromboembolic disease, re-search in this area is rapidly proceeding and new ge-netic abnormalities may be described in the near fu-ture.

Factor V Leiden, the major cause of APCR, first iden-tified in February 1993, is the most common inher-ited cause of thrombosis known at this time.(167) It is found in about 5 percent of the general population

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Electrophoresis, Genetic & Molecular Assays


the tR2 haplotype and factor V Cambridge (Arg306Thr).

A mutation in the prothrombin gene that produces elevated levels of prothrombin was discovered in 1996.(227-231) The mutation involves a single amino acid substitution (20210G>A) in the 3-UTR untrans-lated region of the prothrombin gene on chromo-some 11, leading to more effective mRNA translation and elevated plasma prothrombin. There is increas-ing evidence that the G20210A mutation is an impor-tant risk factor for deep venous thrombosis, myocar-dial infarction and stroke. The use of estrogen or oral contraceptives increases the risk of thrombosis even further in patients with the prothrombin 20210 muta-tion.

and is responsible for 20 percent to 50 percent cases of inherited thrombosis. Approximately 50,000 indi-viduals die yearly in the United States from complica-tions caused by this abnormality.(154; 168; 170) Het-erozygous individuals are at five to 10 times greater risk of thrombosis than the general population, while homozygotes are at 50-100 times greater risk. The use of estrogen or oral contraceptives increases the risk of thrombosis even further. In 90 percent to 95 percent of cases, APCR is a result of a single point mutation (Arg506Gln) in the gene for factor V on chromosome 1q23, inherited as an autosomal domi-nant trait. This mutation renders activated factor V (Va) more resistant to inactivation by APC. The re-maining five to 10 percent of APCR is due to other genetic abnormalities in the factor V gene, including

Hyperhomocysteinemia and homocysteinemia are inherited abnormalities of homocysteine metabo-lism. Homocysteine is a naturally occurring sub-stance involved in the metabolism of certain amino acids, including cysteine and methionine. Abnormali-ties in at least three enzymes, methylenetetrahydrofo-late reductase (MTHFR), cystathionine beta-synthase (CBS) and methionine synthase (MS) associated with homocysteine metabolism in the body can lead to increased homocysteine levels in the body (hyper-homocysteinemia). Genetic abnormalities in these enzymes, particularly homozygous defects in MTHFR, are the common risk factors for thrombotic disease, including heart disease and stroke.(232; 233) Hyperhomocysteinemia also may be associated with vitamin deficiency, advanced age, hypothyroidism, impaired kidney function, systemic lupus erythema-tosus and the use of certain medications, including nicotinic acid, theophylline, methotrexate and L-dopa.

Inherited abnormalities in antithrombin, protein C, protein S, other regulatory of the coagulation system are less common and more complex genetically. In-herited abnormalities in antithrombin, protein C, protein S occur in two forms, leading to either low plasma concentrations (Type I deficiency) or fun-tionally abnormal but quantitatively normal (Type II deficiency) of the involved proteins. To date, more than 250 mutations have been described in the anti-thrombin gene, together with more than 100 each in the protein C and protein S genes. The likelihood of clinically significant thrombotic disease or crises in any one patient is greatly elevated when more than one of these traits is present.

The molecular evaluation of the hypercoagulable states was the subject of a recent review by Nagy, Schrijver, and Zehnder (2004).(234) A variety of mo-lecular techniques have been employed, but the field is in a state of rapid development. The molecular techniques presently employed in the evaluation of

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Genetic & Molecular Assays

Assay Accuracy Throughput Present Clinical Applications

PCR/RFLP Good Limited Factor V Leiden (1691G>A), prothrombin 20210AG>A, MTHFR 677C>T

PCR/ARMS Excellent Intermediate

Light Cycler Excellent Intermediate Factor V Leiden (1691G>A), prothrombin 20210AG>A, MTHFR 677C>T

Array technology Excellent Very high Under development

Invader assays Excellent Limited Under development

Ligand-based technologies

Excellent Very high Under development

Table IIMolecular Techniques for the Evaluation of Hypercoaguable States*

*Modified from Nagy PL, Schrijver I, Zehnder JL. Molecular diagnosis of hypercoagulable states. Lab Med. 2004;35:214-221.


platelets labelled with 14C-serotonin are incubated with patient's serum in the presence and absence of therapeutic and high concentrations of heparin. If > 20% of the 14C is released at a heparin concentration of 0.1 U/ml heparin and < 20% is released by 100 U/mL heparin, the test is positive for heparin antibod-ies. This was the original assay used by Sheridan et al. to establish laboratory diagnosis of the HIT syn-drome after observing spontaneous aggregation of platelets incubated in HIT patient plasma with hea-prin, and has remained the gold standard for valida-tion of other diagnostic techniques.(240) Now, how-ever, even more sensitive assays based on flow cy-tometry detection of circulating platelet microparti-cles are under development.(241)

References

1. Bates SM, Weitz JI. 2005. Coagulation assays. Cir-culation 112(4):e53-60.

2. Bain BJ, Arnold JA, Jowzi Z. 2004. Spurious auto-mated platelet count. Am J Clin Pathol 122(2):316; author reply 316.

3. Kakkar N. 2004. Spurious rise in the automated platelet count because of bacteria. J Clin Pathol 57(10):1096-1097.

4. Kakkar N, Garg G. 2004. Spuriously elevated auto-mated platelet count in severe burns--a report of two cases. Indian J Pathol Microbiol 47(3):408-410.

5. Segal HC, Briggs C, Kunka S, Casbard A, Harrison P, Machin SJ, Murphy MF. 2005. Accuracy of plate-let counting haematology analysers in severe thrombocytopenia and potential impact on plate-let transfusion. Br J Haematol 128(4):520-525.

6. Gulati GL, Hyun BH. 1994. Blood smear examina-tion. Hematol Oncol Clin North Am 8(4):631-650.

7. Moreno A, Menke D. 2002. Assessment of platelet numbers and morphology in the peripheral blood smear. Clin Lab Med 22(1):193-213, vii.

8. Sutor AH, Grohmann A, Kaufmehl K, Wundisch T. 2001. Problems with platelet counting in throm-bocytopenia. A rapid manual method to measure

the inherited thrombophilia disorders are summa-rized in Table II.

Electron Microscopy

Ultrastructural examination of the platelet (platelet electron microscopy) is performed in research stud-ies of platelets, and to confirm the diagnosis

Lab Hemostasis

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