Results of a Worldwide Survey on the Assessment …...Introduction Since it was first introduced almost 50 years ago,[1;2] light transmission aggregometry (LTA) has remained the most

Posted on Website May 1, 2009

Correspondance to: Marco Cattaneo MD, Unità di Medicina III, Ospedale San Paolo, Università di Milano, Via di Rudinì 8, 20142 Milano, Italy, Tel: (39)0250323095, Fax: (39)0250323090, Email: [email protected] *Equal authorship contributions

†Unità di Medicina III, Ospedale San Paolo, Dipartimento di Medicina, Chirurgia e Odontoiatria, Università di Milano, Milano, Italy, ‡Department of Pathology and Molecular Medicine and §Department of Medicine, McMaster University, Hamilton, Ontario, Canada; ¶Hamilton Regional Laboratory Medicine Program, Hamilton, Ontario, Canada; ††Center for Platelet Research Studies, Division of Hematology/Oncology, Children’s Hospital Boston, Harvard Medical School, Boston, MA, U.S.A.

Results of a Worldwide Survey on the Assessment of Platelet Function by Light Transmission Aggregometry: a Report from the Platelet Physiology

Subcommittee of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis

M. Cattaneo,*† C.P.M. Hayward,*‡§¶ K.A. Moffat,§¶ M.T. Pugliano,† Y. Liu,§ A.D. Michelson††

Abstract

Background: Light transmission aggregometry (LTA) is the most common method used in clinical and research laboratories to assess platelet function. However, the method has never been standardized. Objectives: As the first step towards development of methodological guidelines, the Platelet Physiology Subcommittee of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (ISTH) undertook a large, detailed, global survey of LTA practices. Methods: Members of ISTH and of External Quality Assurance in Thrombosis and Haemostasis organizations were invited to complete a 129 item, online questionnaire. Results were analyzed anonymously to participant identities. Results: 359 (244 clinical, 115 research) laboratories from 48 countries participated in the survey. LTA was widely used to assess inherited or acquired bleeding disorders. Common practices were identified in sample collection, processing and analysis and although some are generally considered acceptable, others are not ideal. The agonist concentrations used for LTA varied, and many laboratories used ADP, collagen, epinephrine and Ristocetin, at more than one concentration, in addition to arachidonic acid. The parameters commonly used to assess LTA responses were maximal amplitude or % aggregation, which was considered particularly important, in addition to the presence of a “secondary wave”, deaggregation, shape change and a measure of the lag phase. However, many laboratories did not have appropriate reference intervals. Conclusions: This is the largest and most detailed survey of LTA practices ever undertaken. It shows a very high variability in LTA practices worldwide, and, as a consequence, methodological standardization is necessary. The information gathered in this survey will be helpful in the development of ISTH methodological guidelines for LTA.

Keywords: light transmission aggregometry, platelet aggregation, platelet disorders, platelet function, standardization, platelets

Introduction

Since it was first introduced almost 50 years ago,[1;2] light transmission aggregometry (LTA) has remained the most common method used to assess platelet function. In this method, platelet aggregation in response to an agonist is measured in platelet-rich plasma (PRP) by turbidometry. The fundamental advantage of LTA is that it measures, albeit in an in vitro system, a very important function of platelets: the kinetics of their aggregation in a glycoprotein (GP) IIb/IIIa (integrin IIb3)-dependent manner.

However, LTA methodology has never been standardized. Recent surveys by proficiency testing organizations have identified variations in LTA practices and the need for guidelines to standardize LTA.[3-5] As a first step in LTA guideline development on behalf of the International Society on Thrombosis and Haemostasis (ISTH), we report here on a large, detailed, global survey on LTA practices undertaken by the Platelet Physiology Subcommittee of the Scientific and Standardization Committee (SSC) of the ISTH.

Methods A 129 item, online questionnaire survey was prepared by the ISTH SSC Working Party on LTA. Invitations to participate were distributed in 2007 to members of the ISTH and to members of the programs of External Quality Assurance in Thrombosis and Haemostasis (EQATH) [6]. Each participant was required to self-identify as being from a “clinical” or “research” laboratory; this distinction was not based on pre-specified, standardized criteria. Data were collected on the type of aggregometry performed by participant laboratories. However, only the methodological details of turbidometric LTA were assessed, not whole blood impedance platelet aggregation (WBA), or other platelet aggregation tests, such as the point-of-care VerifyNow method (Accumetrics, San Diego, CA). The collected information was exported into a database and checked for duplicate entries before making participant identities anonymous. Analyses were performed, with correction for skipped responses, to describe practices for all participants, clinical laboratories and research laboratories. Quantitative data were analyzed to determine means, medians, modes, percentiles and ranges. Box plots were generated to show, and distinguish, commonly used agonist concentrations from outliers [7]. The concentrations of ADP and arachidonic acid used by all clinical laboratories that did, or did not, monitor antiplatelet therapy were compared by mixed models that take into account the multiple concentrations used by laboratories.

Results

Survey participants

A total of 359 (244 clinical, 115 research) laboratories participated in the survey. Tables 1 and 2 summarize information on the countries of participants and the aggregation studies that their laboratories performed, including the number of aggregation tests performed annually (which varied considerably). LTA was the most commonly performed aggregation test (Table 2). Some laboratories also performed WBA, albeit less frequently than LTA in most cases (Table 2).

Subjects evaluated by LTA Table 3 summarizes the types of subjects evaluated by LTA. Most clinical laboratories used aggregometry to study individuals with inherited or acquired bleeding disorders, whereas research laboratories used LTA mainly for other purposes. Although some sites tested when specific criteria were met (e.g., if abnormal bleeding manifestations, especially if typical of defects of primary hemostasis, were present after exclusion of von Willebrand disease, thrombocytopenia and/or clotting factor deficiencies), most (54%) had no criteria and performed LTA whenever requested by a physician (Table 3). Very few sites restricted testing to individuals with abnormal screening assays of primary hemostasis (e.g. bleeding time, platelet function analyzer 100 (PFA-100®, Siemens, Newark, Delaware)) (Table 3). About 40% of clinical and research laboratories performed LTA to monitor patients on antiplatelet therapy with aspirin or clopidogrel (Table 3).

Blood sampling for LTA testing Most clinical and research laboratories did not require subjects to fast, rest, refrain from smoking, or abstain from caffeine prior to sample collection (Table 4). About 70-75% recorded information on prescription and non-prescription drugs taken by subjects (Table 4); 95% did not require subjects to be free of all drugs, and only about 30% required subjects to be free of drugs known to impair platelet function (unless LTA was used to assess antiplatelet therapy) (Table 4). Subjects who were studied with LTA for suspected abnormalities of platelet function were required to abstain from aspirin for 2-14 days (most commonly, 7-14 days), and from other NSAIDs for 1-14 days (most commonly, 3-14 days) prior to LTA. Only 54% of laboratories drew samples from controls to test in parallel with patients and more clinical than research laboratories (53% compared to 32%) redrew another control if the first had abnormal aggregation findings.

Samples were commonly collected using 21 gauge needles (range, 16-23) and a tourniquet (released soon after needle insertion) into vacuum containers (mainly from Becton Dickinson and Company, Franklin Lakes, NJ, USA); some laboratories collected blood into anticoagulant-containing plastic tubes, either drawn directly through a butterfly needle (about 40%) or into an empty syringe (about 20%). Some laboratories (48%) routinely collected samples after a discard volume or draw for other tests (commonly 5-10 mL). The anticoagulant used to prepare aggregation samples was sodium citrate in more than 99% laboratories: most clinical laboratories (73%) used 3.2% (with or without buffer), while most research laboratories (64%) used 3.8% (with or without buffer). Almost all (≥98%) laboratories used a 9:1 ratio of blood to anticoagulant and gently mixed samples with anticoagulants. Most (90%) did not allow sample transport by pneumatic tube.

Preparation of platelet-rich and platelet-poor plasma for LTA Approximately 50% of laboratories (43% clinical, 56% research) purposely rested the collected blood before centrifugation, most frequently for 15-30 minutes. PRP was most frequently prepared by centrifuging blood in 5-10 mL volumes at 150 g for 10 minutes, at ambient temperature, without a brake (78%); some laboratories (4%) prepared PRP

at temperatures ≤18°C (range of temperatures: 4-18°C). Plastic pipettes were commonly used to transfer PRP into new tubes (75%), while keeping samples upright in a rack or in one hand (59%). Most (75%) clinical laboratories did not have a special procedure for harvesting PRP from subjects with macrothrombocytopenia, although some allowed samples to sediment, with or without tilting the tube, and others modified the centrifugation force and duration. Almost all (97%) laboratories checked sample platelet counts, mainly using automated cell counters (88%).

Commonly, platelet-poor plasma (PPP) was prepared by centrifuging the residual sample in 5-10 mL collection tubes after removing PRP (79%), most frequently at 1500–2000 g for 10 min at ambient temperature. Some laboratories tested PRP samples without adjustment of the sample platelet count (15% clinical, 26% research); however, more laboratories adjusted all samples (40% clinical, 50% laboratory) or the samples with platelet counts above their acceptable limit (45% clinical, 24% research), which was frequently >300 x 109 platelets L–1. Most laboratories (95%) adjusted PRP platelet counts by adding autologous PPP, with 55% of laboratories adjusting the sample to 250 x 109 platelets L–1 final. Some laboratories (42% clinical, 25% research) adjusted the control PRP to the same platelet count as the patient’s sample when the platelet count was low. Some laboratories (43% clinical, 30% research) did not test PRP with <100 x 109 platelets L–1.

Many laboratories did not have criteria to discard specimens if clotted (28% of labs), hemolyzed (38%), delayed in processing or testing (45%), under-filled (51%), lipemic (55%) or showing loss of swirling (81%), which is indicative of platelets in a resting state.

LTA instrumentation and release assays

Most laboratories (83% clinical, 69% research) had only one type of instrument for LTA, commonly from either Bio/Data Corporation (Horsham, PA, USA), Chrono-Log Corporation (Haverston, PA, USA) or Helena Laboratories Corp. (Beaumont, TX, USA). Most instruments had an arithmetic scale for recording LTA, and adjustable settings for temperature, rpm and light transmittance (LT), but not for sensitivity. Most instruments used siliconized glass or plastic cuvettes, and disposable (35%) or nondisposable (25%), plastic-coated, iron stir bars.

Laboratories with more than one type of instrument (n=36) selected the instruments to use based on: whether testing included an assessment of platelet secretion (56%); the number of subjects being tested (39%); or whether the testing was for clinical or research purposes (39%). Most laboratories (64% clinical, 55% research) did not evaluate platelet secretion simultaneously with platelet aggregation. A minority tested aggregation and secretion simultaneously on all samples (15% clinical, 7% research). Other laboratories tested secretion on selected (18% clinical, 30% research) patients, to evaluate abnormal aggregation (88% clinical, 82% research) or suspected platelet-type bleeding with normal aggregation (62% clinical, 59% research). Among sites that tested secretion in the same cuvette as aggregation responses (n=87; 54 clinical, 33 research), most measured ATP release by the luciferase method (79%); others used

14C serotonin assays (17%), biochemical assays for nucleotides (8%), biochemical assays for serotonin (3%) or other methods (11%).

Analytical aspects of LTA

Only about 60% of the laboratories (59% clinical, 63% research) routinely let the PRP samples rest at room temperature prior to testing, most frequently for 15 (45%) or 30 (38%) minutes. About 50% of the laboratories did not use any method to maintain the pH of the PRP sample constant during the procedure, such as keeping the samples capped until testing (used by 48% of clinical and 28% of research laboratories). Most laboratories (94%) performed LTA studies at 37°C; in addition, 58% of laboratories pre-incubated the PRP samples at the test temperature in the aggregometer for 0-2 minutes, which is likely insufficient to bring the sample to the desired temperature. Therefore, the majority of laboratories most likely perform LTA studies at a sample temperature that is lower than 37°C. LT limits were commonly (87%) set automatically by the instrument. Most laboratories set the lower limit at 0% (76%), and the upper limit at 100% (80%) with autologous PPP (94%). Most laboratories (64%) tested 250, 450 or 500 L of PRP (most frequently 450 L). Most laboratories (66%) added the same agonist volume for all tests, which was typically ≤ 50 L, and ≤10% of the final volume. Only about 70% of laboratories inspected tracings for baseline oscillations and stability in LT before adding agonists (61% clinical, 73% research; typically for 1-2 minutes). Aggregation was most frequently monitored for 5, 6 or 10 minutes after adding each agonist.

Many laboratories (76% clinical, 43% research) had a set order of testing agonist responses. Almost all laboratories (96%) had a maximal time frame of ≤ 4 hours for completing LTA studies after blood collection (most frequently: 4 hours for clinical laboratories; 2 hours for research laboratories).

Agonists used for LTA

Tables 5-8 and Figures 1-7 summarize data on agonists used for LTA in both clinical and research laboratories. In both clinical and research laboratories, ADP, collagen, epinephrine, arachidonic acid and Ristocetin were used more often than the agonists thromboxane analogue U46619, platelet activating factor, thrombin receptor activating peptide (SFLLRN) and gamma thrombin. The box plots in Figs. 1-3 illustrate the range and variability in concentrations of different agonists used by laboratories, and the most commonly used concentrations (boxes). The majority of laboratories tested some agonists at single concentrations whereas ADP, collagen, epinephrine and Ristocetin were often tested at multiple concentrations. Figs. 4-7 show frequency plots that illustrate the commonly used concentrations of ADP, collagen, epinephrine and Ristocetin. The clinical laboratories that did or did not monitor antiplatelet therapy used similar concentrations of ADP (P = 0.81) and arachidonic acid (P = 0.68).

Post analytical evaluation of LTA

Table 8 summarizes the LTA parameters used to interpret results, and which parameters laboratories considered most important for test interpretation. The majority evaluated maximal amplitude or % aggregation, the presence of a “secondary wave”, deaggregation, shape change and a measure of the lag phase. Among these parameters, maximal amplitude or % aggregation were considered to be particularly important for interpreting findings. The parameters included in standard reports were most commonly an overall interpretative comment (71% clinical, 37% research) and the maximal amplitude or % aggregation (60% clinical, 70% research), with ≤35% reporting other parameters. A minority commented that they determined the threshold concentration for aggregation with different agonists. Most laboratories did not have different evaluation criteria for different agonists (59% clinical, 72% research).

A minority of laboratories (39% clinical, 31% research) had determined reference intervals for their instrument and reagent combinations, which was most frequently done with 20 controls (ranges: clinical 10-299; research: 11-1000). Reference intervals were more commonly evaluated for maximal amplitude or % aggregation (99% clinical, 97% research) than for slope (36% clinical, 45% research), lag phase (35% clinical, 26% research) or other parameters (10% clinical, 6% research). Those laboratories that had determined reference intervals used mean ± 2 standard deviations, without (81% clinical, 72% research) or with log transformation of some data to normalize the distribution (11% clinical, 21% research) or nonparametric statistical tests (17% clinical, 21% research). Few laboratories used statistical tests that accommodate the inclusion of repeat values for controls tested on multiple occasions (2% clinical, 7% research).

Discussion

LTA, which was independently developed by Born [1] and O’Brien [2] in the early 1960s, is the gold standard for the in vitro study of platelet function in both clinical and research laboratories. However, the method has never been standardized. In 2005, the Platelet Physiology SSC decided to undertake the task of developing guidelines for LTA practices. An SSC Working Party on LTA was formed, and decided to approach this task using a two-step strategy. In the first step, a global survey on LTA practices worldwide was organized to provide information on common practices which, given the lack of experimental data in the field, will be of considerable help for the second step, during which specific guidelines will be developed. The first step, reported in this manuscript, resulted in the largest survey of LTA practices ever undertaken, and gathered more information on LTA uses, sample preparation, and the analytical and post analytical evaluation of LTA than previous surveys. Another positive feature of this survey was the participation by both clinical and research laboratories. A very high variability in LTA practices emerged from this survey, but with many similarities between clinical and research laboratories. However, clinical laboratories mainly use LTA to study individuals with bleeding disorders while research laboratories often use LTA for other purposes. Consistent with ISTH recommendations, most laboratories do not restrict LTA testing to individuals with prolonged PFA-100® closure times [8]. But, contrary to other ISTH recommendations [9], many laboratories use LTA

to assess responses to antiplatelet therapy outside of research studies, and, not surprisingly, these laboratories did more LTA testing than other sites. We found that the majority of laboratories evaluate LTA using ADP, collagen, epinephrine, Ristocetin and arachidonic acid. The majority assess the maximal amplitude or % aggregation, the presence of a “secondary wave”, deaggregation, shape change and a measure of the lag phase, and most consider the maximal amplitude or % aggregation particularly important for interpreting findings. However, the lack of standardization in many aspects of laboratory LTA practices emphasizes the need for international guidelines on LTA. Worldwide, most laboratories use procedures for LTA sample collection and processing that are generally considered to be acceptable but some common practices are not ideal. For example, many laboratories do not take care to inspect the quality of the samples. Additionally, many laboratories (46%) perform LTA on patient samples without evaluating a control sample. Furthermore, the majority either have not determined reference intervals for each agonist (to aid interpretation of findings) or they have used statistical approaches that are not optimal for the unusual distribution of LTA data [10]. The Clinical and Laboratory Standards Institute (CLSI) guidelines on platelet function testing [11], which were published after our survey findings were presented, recommend that each laboratory run healthy control samples with patient tests, and establish and report reference intervals for each agonist with patient results. Our data indicate that implementation of such recommendations would require the majority of laboratories worldwide to change their practices. Some laboratories do not adjust PRP to a standardized platelet count, consistent with recent reports that indicate this is an acceptable practice [12;13]. For thrombocytopenic samples, some laboratories compare results to an adjusted control sample, tested at the same platelet count, similar to a reported method, validated for testing PRP containing <100 x 109 platelets L–1 for Bernard-Soulier syndrome and type 2B von Willebrand disease [12]. Presently, there are limited data on the number of agonists, and agonist concentrations, that are needed to detect common platelet function disorders. Like other surveys of LTA [3-5], we found that ADP, collagen, epinephrine, Ristocetin and arachidonic acid are the most commonly used agonists. A recent study found that collagen, epinephrine, arachidonic acid and thromboxane analogue U46619 are the agonists that are most helpful for detecting common platelet function disorders [14]. Although many laboratories test LTA with multiple concentrations of some agonists, single concentrations of each agonist (selected based on sensitivity to platelet function disorders) have been reported to have similar diagnostic utility to testing multiple concentrations [14]. Our survey provides information on the most commonly used concentrations of ADP, collagen, epinephrine, Ristocetin, arachidonic acid and thromboxane analogue U46619, and these values are within the ranges that other organizations have recommended [11, 15]. We used box plot analysis [6] to describe the most commonly used concentrations, as there were some striking outlier values that could be due to a minority of laboratories mistakenly reporting initial rather than final

concentrations or the wrong concentration units. Interestingly, the ADP and arachidonic acid concentrations used by clinical laboratories that do or do not monitor antiplatelet therapy were not significantly different. However, some recommended agonist concentrations have greater sensitivity to common platelet function disorders than others [14], and the detection of LTA abnormalities due to type 2B von Willebrand disease may require more than 0.6 mg mL-1 Ristocetin [16]. It will be important for the next step in development of ISTH guidelines on LTA to consider the evidence that has emerged on LTA methodologies that optimize sensitivity and specificity. Now that the worldwide LTA practices are defined, ISTH guidelines are under development with the aim of standardizing LTA methodologies. Acknowledgments

The statistical analysis was supported by funding from a Canada Research Chair from the Government of Canada (CPMH). The authors thank ISTH and EQATH for contacting participants, the North American Specialized Coagulation Laboratory Association for supporting the on-line questionnaire, and the 359 participants for their essential input. The following individuals, from the ISTH SSC Working Group on LTA, are acknowledged for their input into the questionnaire content: Paul Harrison (Oxford, UK), Steve Kitchen (Sheffield, UK); A. Koneti Rao (Philadelphia, PA); Judith Lahav (Petah Tikva, Israel); Steve Watson (Birmingham, UK); Harvey J. Weiss (New York, NY).

Disclosure of Conflict of Interests

The authors state that they have no conflict of interests.

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10 Hayward CP, Moffat KA, Pai M, Liu Y, Seecharan J, McKay H, Webert KE, Cook RJ, Heddle NM: An evaluation of methods for determining reference intervals for light transmission platelet aggregation tests on samples with normal or reduced platelet counts. Thromb Haemost 2008; 100: 134-145.

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15 Guidelines on platelet function testing. The British Society for Haematology BCSH Haemostasis and Thrombosis Task Force. J Clin Path 1988; 41:1322-1330.

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Table 1. Information on the countries of LTA survey participants. Results summarize findings for all participants and for clinical laboratories and research laboratories.

Country All

n=359 from 48 countries

Clinical


Research


Not provided 13 10 3

USA 106 69 37

Italy 31 19 12

UK 30 22 8

Germany 22 9 13

Canada 21 14 7

France 18 14 4

Australia 17 15 2

The Netherlands 10 6 4

Japan 7 5 2

Switzerland 7 5 2

Belgium 6 5 1

Ireland 5 2 3

Israel 5 4 1

Russia 5 4 1

Spain 5 3 2

Argentina 4 4 0

Brazil 4 2 2

Thailand 4 3 1

Austria 2 0 2

Czech Republic 2 2 0

Denmark 2 1 1

Indonesia 2 2 0

Poland 2 1 1

Slovenia 2 0 2

Taiwan 2 1 1

Uruguay 2 2 0

Table 1, continued.

Country All Clinical Research

Bulgaria 1 1 0

Chile 1 1 0

China 1 1 0

Croatia 1 1 0

Egypt 1 1 0

Finland 1 1 0

Greece 1 0 1

India 1 1 0

Korea 1 1 0

Lithuania 1 1 0

Macedonia 1 1 0

Malaysia 1 1 0

New Zealand 1 1 0

Nigeria 1 1 0

Norway 1 1 0

Romania 1 1 0

Saudi Arabia 1 0 1

Serbia 1 1 0

Singapore 1 1 0

South Africa 1 1 0

Sweden 1 0 1

Turkey 1 1 0

Table 2. Information on LTA survey participants. Results summarize findings for all participants and for clinical laboratories and research laboratories.

Laboratories: All

% (n/total)

Clinical

% (n/total)

Research

% (n/total)

ISTH member provided responses 67 (241/359) 63 (153/244) 77 (88/115)

Laboratory director provided responses

66 (237/359) 60 (146/244) 79 (91/115)

Number of aggregation tests performed per year

median: 150

range: 0-12,000

median: 105

range: 0-12,000

median: 155

range: 0-10,000

Aggregometry performed:

LTA, No WBA

LTA and WBA, but LTA more frequently

LTA and WBA but WBA more frequently

Only WBA

LTA and WBA, similar frequency

Other (e.g. VerifyNow)

70 (242/347)

19 (67/347)

5 (16/347)

3 (12/347)

1 (4/347)

2 (6/347)

72 (170/236)

15 (36/236)

6 (13/236)

5 (12/236)

<1 (1/236)

2 (4/236)

65 (72/111)

28 (31/111)

3 (3/111)

0 (0/111)

3 (3/111)

2 (2/111)

Data were collected on the type of aggregometry performed by participant laboratories. However, only the methodological details of turbidometric LTA were assessed, not whole blood impedance platelet aggregation (WBA), or other platelet aggregation tests, such as the point-of-care VerifyNow method.

Table 3. Uses of LTA.

Laboratories: All % (n/total)

Clinical % (n/total)

Research % (n/total)

Perform LTA to study patients with/on: inherited bleeding disorders 77 (254/331) 94 (213/227) 39 (41/104) acquired bleeding disorders 70 (233/331) 90 (205/227) 27 (28/104) antiplatelet therapy 44 (144/331) 45 (103/227) 39 (41/104) other reasons 32 (107/331) 15 (35/227) 69 (72/104) LTA test criteria for assessment of bleeding disorders:None; test all subjects referred by physicians 54 (151/281) 54 (116/216) 54 (35/65) if bleeding typical of defective primary hemostasis

33 (93/281) 38 (81/216) 18 (12/65)

if bleeding manifestations, any type 21 (58/281) 20 (44/216) 22 (14/65) after exclusion of von Willebrand disease 25 (71/281) 27 (58/216) 20 (13/65) after exclusion of thrombocytopenia 35 (98/281) 38 (81/216) 26 (17/65) after exclusion of clotting factor deficiencies 23 (64/281) 24 (51/216) 20 (13/65) only if bleeding time prolonged 5 (13/281) 6 (13/216) 0 (0/65) only if abnormal screening assay of primary hemostasis, e.g. PFA-100

7 (19/281) 6 (13/216) 9 (6/65)

LTA test criteria for monitoring antiplatelet therapy:subjects on aspirin 68 (102/150) 66 (71/107) 72 (31/43) subjects on clopidogrel 61 (92/150) 59 (63/107) 67 (29/43) any subject referred by treating physicians 47 (71/150) 55 (59/107) 30 (13/43) only subjects suspected to be noncompliant 14 (21/150) 18 (18/107) 7 (3/43) only subjects with thrombotic events on treatment

19 (29/150) 23 (25/107) 9 (4/43)

other subjects 15 (22/150) 10 (11/107) 26 (11/43)

Table 4. Preanalytical requirements for LTA.

Laboratories: All

% (n/total)

Clinical

% (n/total)

Research

% (n/total)

Require subjects to:

Fast 29 (72/246) 32 (53/163) 24 (20/83)

Rest 22 (54/246) 17 (28/163) 31 (26/83)

Refrain from smoking 9 (22/246) 6 (9/163) 16 (13/83)

Abstain from caffeine 13 (33/246) 12 (19/163) 17 (14/83)

Information collected on drugs:

Prescription 74 (214/291) 77 (156/203) 66 (58/88)

Non-prescription 66 (192/291) 69 (141/203) 58 (51/88)

Homeopathic/alternative 46 (133/291) 51 (104/203) 33 (29/88)

Checklist of drugs that interfere with platelet function 36 (106/291) 34 (68/203) 43 (38/88)

None of the above 12 (34/291) 13 (26/203) 9 (8/88)

Require subjects to be:

Free of all drugs 5 (14/291) 3 (7/201) 8 (7/90)

Free of antiplatelet drugs (unless LTA used to monitor antiplatelet therapy)

72 (211/291) 73 (147/201) 71 (64/90)

Free of homeopathic/alternative medicines known to affect platelet function

21 (62/291) 23 (46/201) 18 (16/90)

None of the above 23 (68/291) 24 (49/201) 21 (19/90)

Table 5. Information on ADP, collagen and epinephrine concentrations used for LTA. This information was provided by 148 clinical laboratories and 63 research laboratories (all concentrations are final concentrations). Results summarize data for all laboratories, clinical laboratories and research laboratories. Data for agonists obtained from different suppliers were analyzed together with the exception that the data for type I collagen, and for collagen from Bio/Data Corporation (Horsham, PA), were analyzed separately.

All/Clinical/Research

Agonist: ADP Collagen – Type 1 Collagen – Bio/Data Epinephrine

n (% ) using the agonist 211 (100%)

148 (100%)

63 (100%)

128 (61%)

115 (78%)

13 (21%)

25 (12%)

23 (16%)

2 (3%)

151 (72%)

122 (82%)

29 (46%)

Median concentration 5.0 M

5.0 M

5.0 M

2.0 µg/mL

2.0 µg/mL

2.0 µg/mL

190 µg/mL

190 µg/mL

190 µg/mL

7.3 M

7.0 M

10 M

Range of concentrations tested

0.1-200 M

0.2-200 M

0.1-100 M

0.08-200 µg/mL

0.08-100 µg/mL

0.1-200 µg/mL

38-200 µg/mL

50-200 µg/mL

38-190 µg/mL

0.08-1500 M

0.1-1500 M

0.08-110 M

25% Percentile 2.0 M

2.0 M

2.0 M

1.0 µg/mL

1.0 µg/mL

1.0 µg/mL

100 µg/mL

100 µg/mL

190 µg/mL

2.5 M

3.0 M

1.0 M


10.0 M

10.0 M

5.0 µg/mL

5.0 µg/mL

5.0 µg/mL

190 µg/mL

190 µg/mL

190 µg/mL

20 M

20 M

10 M

Test one concentration 31 (15%)

21 (14%)

10 (16%)

54 (42%)

50 (43%)

4 (31%)

22 (88%)

20 (87%)

2 (100%)

71 (47%)

58 (47%)

13 (45%)

Test two concentrations 75 (36%)

54 (36%)

21 (33%)

40 (31%)

38 (33%)

2 (15%)

2 (8%)

2 (9%)

-

50 (33%)

40 (33%)

10 (53%)

Test additional concentrations

105 (50%)

73 (49%)

32 (51%)

34 (26%)

27 (23%)

7 (54%)

1 (4%)

1 (4%)

-

30 (20%)

24 (20%)

6 (21%)

Table 6. Information on arachidonic acid and Ristocetin concentrations used for LTA. Other concentrations of Ristocetin were only used by clinical laboratories. Results are shown as for Table 5.


Agonist: Arachidonic acid Low dose Ristocetin High dose Ristocetin Other Ristocetin*

n (% ) using the agonist

162 (77%)

121 (82%)

41 (65%)

131 (62%)

119 (80%)

12 (19%)

145 (69%)

122 (82%)

23 (37%)

47 (32%)

Median concentration

1.0 mM

1.36 mM

1.0 mM

0.5 mg mL-1

0.5 mg mL-1

0.5 mg mL-1

1.50 mg mL-1

1.50 mg mL-1

1.23 mg mL-1

1.0 mg mL-1


0.01-60 mM

0.125-16 mM

0.01-60 mM

0.2-2.5 mg mL-1

0.2-2.5 mg mL-1

0.4-1.2 mg mL-1

0.6-5.0 mg mL-1

0.6-5.0 mg mL-1

0.6-5.0 mg mL-1

0.5-2.0 mg mL-1

25% Percentile 1.0 mM

1.0 mM

0.5 mM

0.5 mg mL-1

0.5 mg mL-1

0.5 mg mL-1

1.2 mg mL-1

1.2 mg mL-1

1.1 mg mL-1

0.8 mg mL-1

75% Percentile 1.6 mM

1.6 mM

1.6 mM

0.6 mg mL-1

0.6 mg mL-1

0.6 mg mL-1

1.5 mg mL-1

1.5 mg mL-1

1.5 mg mL-1

1.2 mg mL-1

Test one concentration

147 (91%)

115 (95%)

32 (78%)

130 (99%)

119 (100%)

11 (92%)

142 (98%)

122 (100%)

20 (87%)

35 (74%)

Test two concentrations

14 (22%)

6 (5%)

8 (20%)

1 (<1%)

0 (0%)

1 (8%)

1 (<1%)

0 (0%)

1 (4%)

6 (13%)


1 (<1%)

0 (0%)

1 (2%)

0 (0%)

0 (0%)

0 (0%)

2 (1%)

0 (0%)

2 (9%)

6 (13%)

Table 7. Information on thromboxane analogue U46619, TRAP, -thrombin and PAF used for LTA. Results are shown as for Table 5.


Agonist: U46619 TRAP -thrombin PAF

n (% ) using the agonist

41 (19%)

22 (15%)

19 (30%)

55 (26%)

26 (17%)

29 (46%)

24 (11%)

11 (7%)

13 (21%)

6 (3%)

3 (2%)

3 (5%)

Median concentration 1.0 M

1.0 M

1.0 M

10.0 M

10.0 M

10.0 M

0.50 U mL-1

0.45 U mL-1

0.50 U mL-1

1.0 M

5.0 M

1.0 M


0.05-40 M

0.05-40 M

0.1-10 M

0.25-500 M

1.0-100 M

0.25-500 M

0.01-10 U mL-1

0.05-10 U mL-1

0.01-5.0 U mL-1

0.03-145 M

0.2-145 M

0.03-10 M


1.0 M

1.0 M

5.0 M

4.5 M

5.0 M

0.50 U mL-1

0.45 U mL-1

0.50 U mL-1

0.1 M

0.2 M

0.1 M


2.0 M

2.0 M

20.0 M

22.5 M

20.0 M

1.0 U mL-1

1.0 U mL-1

1.0 U mL-1

5.0 M

145 M

2.0 M

Test one concentration

33 (80%)

18 (95%)

15 (79%)

48 (87%)

24 (92%)

24 (83%)

20 (83%)

9 (82%)

11 (85%)

3 (50%)

3 (100%)

0 (0%)

Test two concentrations

6 (15%)

3 (14%)

3 (16%)

4 (7%)

0 (0%)

4 (14%)

2 (8%)

1 (9%)

1 (8%)

2 (67%)

0 (0%)

2 (67%)


2 (5%)

1 (5%)

1 (5%)

3 (5%)

2 (8%)

1 (3%)

2 (8%)

1 (9%)

1 (8%)

1 (33%)

0 (0%)

1 (33%)

Table 8. Parameters evaluated for LTA responses and their perceived importance.

Parameter evaluated

% (n/total)

Most important parameters*

% (n/total)

Laboratories: All Clinical Research All Clinical Research

Maximal amplitude or % aggregation

93 (203/219) 90 (134/149) 99 (69/70) 85 (182/215) 83 (122/147) 88 (60/68)

Presence of “secondary wave”

73 (160/219) 80 (119/149) 59 (41/70) 53 (115/215) 61 (89/147) 38 (26/68)

Deaggregation 70 (153/219) 79 (118/149) 50 (35/70) 53 (113/215) 57 (84/147) 43 (29/68)

Visual inspection of the tracing

63 (139/219) 75 (111/149) 40 (28/70) 51 (113/215) 63 (84/147) 25 (29/68)

Slope of aggregation 62 (135/219) 58 (87/149) 69 (48/70) 47 (102/215) 45 (66/147) 53 (36/68)

Presence of a shape change

54 (119/219) 58 (87/149) 46 (32/70) 32 (69/215) 35 (52/147) 25 (17/68)

Measure of lag phase 52 (114/219) 55 (82/149) 46 (32/70) 32(69/215) 31 (46/147) 34 (23/68)

Amplitude of % aggregation at end of observation

38 (83/219) 36 (54/149) 41 (29/70) 24 (52/215) 27 (39/147) 19 (13/68)

Amplitude of % aggregation after standardized number of minutes

11 (23/219) 8 (12/149) 16 (11/70) 6 (12/215) 4 (6/147) 9 (6/68)

* more than one choice was allowed.

Fig. 1. Concentrations of ADP, collagen and epinephrine used for LTA. Box plots show data for all participants, clinical laboratories and research laboratories as medians (lines within boxes, or → if at box limits), means (+), 25th and 75th percentiles (top and bottom of boxes), ranges for smallest and largest non-outlier values (whiskers) and outlier values (□) that were beyond the first and third quartiles by 1.5 times the interquartile range. Data with extreme outlier values are indicated (●; indicated by ■ if there were outliers indicated by □ at the axis limit; numbers indicate how many of the three boxes were clipped). Data are shown separately for type I and Bio/Data collagen (boxes not shown for research group due to the limited numbers of observations, see Table 5).

Fig. 2. Concentrations of low and high dose Ristocetin used for LTA. Data are displayed as in Fig. 1.

Fig. 3. Concentrations of arachidonic acid, thromboxane analogue U46619, TRAP and -thrombin used for LTA. Data are displayed as in Fig. 1.

0

20

40

60

80

100

120

0.1

0.2

0.25 0.3

0.33 0.4

0.5

0.65

0.75 0.8 1

1.1

1.2

1.25 1.3

1.6

1.7 2

2.2

2.5 3

3.2

3.3

3.7 4

4.4

4.5 5 6

6.3

6.4

6.5 7

7.5 8

8.4 9 10 11 16 20 22 30 50 75 100

200

ADP Concentration (μM)

# o

f la

bo

rato

ries

All Clinical Research

Fig. 4. ADP concentrations used for LTA. The frequency plot shows concentrations used by all laboratories, clinical laboratories and research laboratories.

0

10

20

30

40

50

60

0.08 0.1

0.5

0.6 1

1.25 1.5 2

2.5

2.75 3 4 55.

5 6 77.

3 8 10 11 12 15 16 20 25 30 40 50 55 60 75 100

110

150

200

250

300

1000

1500

Epinephrine Concentration (μM)

# o

f la

bo

rato

ries


Fig. 5. Epinephrine concentrations used for LTA. The frequency plot shows concentrations used by all laboratories, clinical laboratories and research laboratories.

Fig. 6. Collagen concentrations used for LTA. The frequency plot shows concentrations of type I collagen, and Bio/Data collagen, used by all laboratories, clinical laboratories and research laboratories.

0

20

40

60

80

0.2

0.25 0.3

0.35

0.38 0.4

0.5

0.6

0.63

0.65 0.7

0.75 0.8

0.9 1

1.1

1.2

1.25 1.3

1.4

1.5

1.6 2

2.5 5

Ristocetin Concentration (mg/mL)

# o

f la

bo

rato

ries


Fig. 7. Ristocetin concentrations used for LTA. The frequency plot shows all concentrations (low dose, high dose and others) used by all laboratories, clinical laboratories and research laboratories.

Documents

Results of a Worldwide Survey on the Assessment …...Introduction Since it was first introduced almost 50 years ago,[1;2] light transmission aggregometry (LTA) has remained the most