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Results of the Survey of Hospital Coagulation Laboratory Practices, United States-2001
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Laboratory Practices, United States-2001

Shahram Shahangian, Ph.D.; Ana K. Stanković, M.D., Ph.D.; Ira M. Lubin, Ph.D.;James H. Handsfield, M.P.H.; Mark D. White, B.S.
 

Also available in a print-friendly PDF version

 
bullet Executive Summary
bullet Introduction
bullet Methods
bullet Results and Discussion
bullet Concluding Remarks
bullet References
bullet Appendix (PDF)
 

RESULTS AND DISCUSSION

Several surveys addressing specific areas in hospital coagulation laboratory practices have been conducted.112 However, none has dealt with a broad cross section of this specialty in clinical laboratory medicine. Considering that no survey of coagulation laboratory practices can cover all areas of coagulation testing, we tailored the present survey to capture 2 types of information. One set of questions related to general laboratory practices that can also be posed for other areas of laboratory medicine. The other set dealt with specific tests and coagulation disorders with substantial public health significance. In doing so, we hoped to gain a better understanding of the state of coagulation laboratory practices and the extent of their variability across a random sample of hospital laboratories. Our objective was not to formulate testing recommendations. Rather, we undertook to document the extent of variability in laboratory practices so that laboratory and health systems researchers could further evaluate its impact on patient and population health outcome. The result of these outcome-based studies can then be used to formulate recommendations and guidelines for more uniform laboratory testing practices.1317

Response and Sampling Rates

We received 632 responses corresponding to a response rate of 79% (including 20 responses submitted electronically). Response rates of large and small hospitals were as follow:

Survey Response

Number (Percent) of large hospitals

Number (Percent) of small hospitals

P

321 (76%)

311 (83%)

0.010

Due to the degree of participation (79% response rate) and sampling (26% of large and 9% of small hospitals), we believe that the results of this survey accurately reflect the state of coagulation laboratory practices as viewed by the respondents in 2001. This response rate may be compared to those for written surveys obtained for other more specific (and shorter) surveys: 51% for a survey of activated partial thromboplastin (aPTT) reporting in Canadian medical laboratories,1 77%5 and 70%11 for a survey of INR reporting in Canada, and 85% for a survey of prothrombin time (PT) monitoring of anticoagulation therapy in Massachusetts.12

Performance of Coagulation Testing

Of those responding, 98% of the large hospitals and 97% of the small hospitals stated that they performed coagulation testing.

Test Requisition and Specimen Management

Use of test requisition forms and information items requested on them. Fifty-six percent stated that they used test requisition forms. Responses of large and small hospital laboratories were as follows:

Use of Test Requisition Forms

Number (Percent) of large hospitals

Number (Percent) of small hospitals

P

135 (46%)

188 (67%)

< 0.001

It is conceivable that some participants negatively responding to this question may have done so because they ordered coagulation tests electronically without using a paper-based requisition form. There was a wide variation in the proportion of respondents requesting specific information items on test requisition forms:

Information item

Number (Percent) of large hospitals

Number (Percent) of small hospitals

P

Diagnosis

105 (78%)

156 (82%)

0.401

Coumadin use

74 (57%)

86 (51%)

0.276

Unfractionated heparin use

59 (49%)

49 (31%)

0.003

Heparinoid use

47 (39%)

43 (28%)

0.044

LMWH* use

32 (29%)

28 (18%)

0.045

Salicylate (Aspirin) use

18 (17%)

25 (16%)

0.882

Oral contraceptive use

10 (9%)

6 (4%)

0.087

*LMWH is low molecular weight heparin.

Rejection of coagulation specimens. The respondents noted the following reasons for rejecting coagulation specimens based on their policies and procedures:

Reasons for specimen rejection

Number (Percent) of large hospitals

Number (Percent) of small hospitals

P

Clotted specimen

312 (100%)

300 (100%)

1.000

Improperly anticoagulated specimen

308 (99%)

296 (99%)

0.961

Insufficiently labeled specimen containers

306 (99%)

297 (99%)

0.678

Excessive specimen transport time

279 (92%)

270 (92%)

0.915

Conflicting patient information

273 (91%)

274 (93%)

0.399

Hemolyzed specimen

266 (86%)

252 (85%)

0.739

Specimen stored at inappropriate temperature

251 (83%)

251 (87%)

0.282

Lack of hospital medical record number

175 (59%)

88 (31%)

< 0.001

Specimen collected via indwelling catheter

96 (33%)

90 (32%)

0.896

These findings indicate that a minority of both large and small hospital respondents noted their policy against use of indwelling catheters for specimen collection. It has been recommended that, due to the presence of anticoagulants at such collection sites, specimens used for monitoring heparin therapy should be collected from a different extremity than the one used for heparin infusion.16

Practices Relating to Prothrombin Time (PT) Assay

Performance of PT assay. Of the respondents that provided valid responses, 100% noted performing PT assay.

Anticoagulant concentration. The respondents used the following sodium citrate concentrations:

Concentration

Number (Percent*) of large hospitals

Number (Percent*) of small hospitals

3.2% (109 mmol/L)

244 (81%)

193 (68%)

3.8% (129 mmol/L)

60 (20%)

96 (34%)

*Percentages total >100% due to 8 respondents (4 large and 4 small hospitals) noting that they used both concentrations of sodium citrate.

The respondents reported exclusive use of 3.2% sodium citrate as follows:

ExclusiveUse of 3.2% Sodium Citrate

Number (Percent) of large hospitals

Number (Percent) of small hospitals

P

240 (80%)

189 (66%)

< 0.001

Based on the WHO recommendations and the NCCLS guidelines, 3.2% citrate is the anticoagulant of choice for coagulation testing.14 In a recent study,18 the recommendation to use 3.2%, as opposed to 3.8%, sodium citrate was supported by noting that the concentration of sodium citrate had a significant effect on PT and aPTT assay results. In another study,19 underfilling of specimen tubes containing 3.8% sodium citrate prolonged PT and especially aPTT compared to 3.2% sodium citrate. When responsive PT and aPTT reagents are used, concentration of sodium citrate has a significant effect on assay results, with 19% of patients receiving intravenous heparin therapy having a greater than 7-second difference when aPTT results were compared.20 In a survey of aPTT reporting in Canadian medical laboratories,1 46% of laboratories were still using 3.8% citrate. In this survey, 27% of the respondents noted that they used 3.8% citrate (5% of these respondents noted that they used both 3.2% and 3.8% citrate), potentially resulting in falsely elevated PT or aPTT results.

Reporting of PT results. Virtually all (99.8%) respondents used international normalized ratio (INR) to report PT; 97% also reported PT in seconds and/or as therapeutic PT ratio:

Results reported in

Number (Percent)

International normalized ratio (INR)

601 (100%)

Seconds

577 (97%)

Therapeutic PT ratio

77 (16%)

Reporting PT results in seconds may lead clinicians to inappropriately compare results between institutions.5 Reliance on therapeutic PT ratio has been documented to cause errors in anticoagulant therapy.5,21 Reporting of PT results in INR only is, therefore, the preferred method. Reporting of PT results in INR has increased over the years from 557% in 199211,12 to 98100% in 19965,10 and 99.8% in the current survey. Reporting of PT results in INR only in Canada increased from 15% of all licensed medical laboratories in 1992 to 36% of these laboratories in 1996. This survey shows that 3% of the U.S. hospital laboratories reported PT results in INR only. Reporting PT results in both seconds and INR increased steadily over the years from 36% in 1992 (Canada) to 60% in 1996 (Canada) and 80% in 2001 (U.S.). Finally, the practice of reporting PT results in seconds only decreased from 36% in 1992 to <1% in 19962001.

Ninety-seven percent of the respondents provided PT results in seconds. This may be compared to the rates of 99% reported in 1992,12 95% reported in 1996,10 72% in a 1992 Canadian survey reported in 1995,11 and 60% in a follow-up 1996 Canadian survey reported in 1998.5

Sixteen percent provided therapeutic PT ratio. This rate may be compared to a rate of 43% in a survey of anticoagulant therapy monitoring reported in 1996,10 and rates of 13% and 2.5% in the 199211 and 19965 surveys of Canadian medical laboratories, respectively. The rate of 43% in the former survey is less generalizable since it involved only 58 U.S. health centers, which were all academic institutions. Furthermore, those conducting the survey of these centers themselves have noted that wide variation existed in the reporting of coagulation tests (seconds and INR) and of patient therapeutic status.10

The following table shows how PT results were reported in our survey compared to the results of the 1992 and 1996 Canadian surveys:

Format Used to Report PT Result by the U.S. Hospitals and Canadian Medical Laboratories

Reporting format

U.S., 2001 (n = 626)

Canada, 1996 (n = 649)5

Canada, 1992 (n = 857)10

Seconds and INR

60%*

60%

36%

Seconds, INR and PT ratio

12%

Not specified

4%

INR only

3%

36%

15%

INR and PT ratio

0.5%

1.5%

6%

Seconds only

0%

<1%

36%

PT ratio only

0%

1%

7%

*An additional 20% noted that they reported PT results in both seconds and INR but had failed to indicate whether or not they used therapeutic PT ratio.

This proportion may be as high as 32% since an additional 20% noted that they reported PT results in seconds and INR while failing to note whether they used therapeutic PT ratio.

Reference interval for PT assay. Ninety-two percent of the respondents conducted in-house evaluations to establish reference intervals for PT assay. Responses from large and small hospitals were significantly different:

Number (Percent) of large hospitals

Number (Percent) of small hospitals

P

291 (97%)

277 (87%)

< 0.001

The respondents not conducting in-house evaluations to establish reference intervals for PT did so as follows:

Other method used to establish PT reference interval

Number (Percent)

Manufacturer's instructions

31 (57%)

Published values

16 (30%)

Others

10 (19%)

*Percentages total >100% due to 3 small hospital respondents noting that they used both published values and manufacturers instruction to establish their PT reference intervals.

In-house determination of the PT reference interval was based on the following minimum number of subjects:

Minimum number of subjects used

Number (Percent *) of large hospitals

Number (Percent*) of small hospitals

20 or fewer

24 (8%)

62 (25%)

21-39

112 (38%)

123 (49%)

40-59

88 (30%)

40 (16%)

60-119

56 (19%)

18 (7%)

120-199

11 (4%)

4 (2%)

200 or more

5 (2%)

4 (2%)

*Percentages do not equal 100% because of rounding to the nearest 1%. The response patterns of the large and small hospital respondents were significantly different (P < 0.001).

Most respondents (46% of the large hospitals and 74% of the small hospitals, P < 0.001) used less than 40 subjects to establish their PT reference intervals. To establish a reference interval, the NCCLS has recommended a minimum of 120 subjects for each reference population or subclass as the smallest number allowing determination of a 90% confidence interval around reference limits.22 In this survey, 5% of the respondents noted using at least 120 subjects to establish their reference ranges for PT assay.

Methods and reagents used to assay for PT. The respondents used the following methods to assay for PT:

Method Used to Assay for PT

Method

Number (Percent)

Optical

527 (88%)

Mechanical

71 (12%)

Manual

1 (0.2%)

None of the above

1 (0.2%)

In the 1996 survey of Canadian medical laboratories, 1% used a manual method to assay for PT.5

The respondents used the following reagents for PT assay:

Reagents Used for PT Assay*

Reagent

Large hospital (percent) response

Small hospital (percent) response

Dade Behring Thromboplastin C Plus

76 (25%)

106 (36%)

Innovin

72 (23%)

46 (16%)

Hemoliance Brain Thromboplastin

37 (12%)

23 (8%)

OTC Simplastin L

26 (8%)

11 (4%)

Pacific Hemostasis D

4 (1%)

10 (3%)

Others

98 (32%)

97 (33%)

*The use of trade names is for identification purposes only and does not constitute endorsement by the CDC or the U.S. Department of Health and Human Services. There were 4 large hospitals that responded by stating that they used 2 different reagentsresulting in percentages totaling >100%.

Among thromboplastins listed, Innovin is the only recombinant protein (human tissue factor or factor VIIa).23 Results obtained with recombinant thromboplastin on an optical analyzer even after prolonged storage of the plasma samples at room temperature were considered suitable for oral anticoagulation control.24 Advantages of recombinant reagents such as Innovin and Recombiplastin are purity and consistency of reagents. Innovin and Recombiplastin have been shown to yield different INR values when compared to traditional reagents purified from tissue extracts.23,25,26 Of all the respondents, 20% used Innovin. Of the large hospital respondents, 23% reported using Innovin compared to 16% of the small hospital laboratory respondents (P = 0.019). Although use of Recombiplastin as the other major recombinant thromboplastin was not assessed, larger facilities may use this and other recombinant reagents in preference to traditional reagents prepared from tissue extracts with their inherent variability from lot to lot or from reagent to reagent.

Sensitivity of PT assay to heparin

>Determining sensitivity of PT assay to heparin. Seventeen percent of the respondents determined the sensitivity of their PT assays to heparin.

Selecting a PT-thromoboplastin reagent insensitive to heparin in the therapeutic range. Fifty percent of the respondents selected a PT-thromboplastin reagent that was insensitive to heparin in the heparin therapeutic range. Significantly different proportions of the large and small hospitals selected an insensitive PT-thromboplastin reagent:

Number (Percent) of large hospitals

Number (Percent) of small hospitals

P

170 (59%)

101 (40%)

< 0.001

According to consensus guidelines developed at the 1997 CAP conference, laboratories should determine the sensitivity of their PT assay to heparin and, where possible, select a thromboplastin that is insensitive to heparin in the therapeutic range.14

International sensitivity index (ISI) of thromboplastin lot currently used. The ISI of the respondents current thromboplastin lot was 0.892.63 (average, 1.60; median, 1.81). The large hospital respondents reported an average ISI of 1.52 (median, 1.56), while the small hospitals reported an average of 1.70 (median, 1.89).

Due to increased variability in INR resulting from ISI values deviating significantly from unity, various professional organizations have recommended using thromboplastin reagents with ISI values closer to 1. The CAP recommends thromboplastins with a manual ISI between 0.9 and 1.7 and toward the lower end of this scale.14 The ISI of the respondents current reagent lots ranged between 0.89 and 2.63. Forty-four percent reported ISI values of < 1.70. Of the large hospital respondents, 50% reported ISI values of < 1.70 compared to 36% of the small hospital respondents (P = 0.001).

Two published articles have reported ISI values of survey participants. A 1992 report involving 88 acute care hospitals in Massachusetts reported ISI values of 1.892.74.10 The second involved a survey of all licensed Canadian medical laboratories in 1996 and 1992.5 This latter report noted that average ISI value had decreased from 2.07 in 1992 to 1.63 in 1996.11 Of the respondents to the 1996 survey of Canadian medical laboratories, 35% had reported ISI values of < 1.2 as recommended by the American College of Chest Physicians.27 In this survey, 34% of the respondents used an ISI of < 1.20. Forty-two percent of the large hospital respondents reported ISI values of < 1.20 compared to 24% of the small hospital respondents (P < 0.001).

In a survey of 58 academic institution in the U.S., 79% of the hospitals did not confirm accuracy of the ISI for their own analyzers.10 Coagulation instruments may affect ISI, which can differ from the assigned value by a clinically significant degree.14,28 We did not capture in this survey the proportion of the respondents confirming accuracy of the ISI value for their own analyzers.

Practices Relating to Activated Partial Thromboplastin Time (aPTT) Assay

Performance of aPTT assay. Ninety-nine percent of the respondents performed aPTT assay.

Therapeutic range. The respondents noted having an aPTT therapeutic range for heparin as follows:

Number (Percent) of large hospitals

Number (Percent) of small hospitals

P

213 (73%)

142 (53%)

< 0.001

While 64% of the respondents stated they reported the aPTT therapeutic range for heparin when monitoring heparin therapy, 9% included the corresponding heparin concentration with aPTT results.

It has been recommended that each laboratory establish an individual therapeutic range for heparin specific to its own reagent and instrument system.29,30 Significant variations between heparin sensitivity of aPTT reagents produced under the same name by the same supplier have been observed.29 Variations were such that, using the recommended aPTT ratio or prolongation of aPTT for monitoring heparin therapy, one would have achieved significantly different degree of heparinization from year to year. Also, the latest CAP consensus guideline notes that therapeutic range of unfractionated heparin for the aPTT reagent-instrument system should be determined with each change in reagent (lot number or manufacturer) or instrument.16

In a 19981999 survey of aPTT reporting in Canadian medical laboratories,1 66% of institutions had established an individual therapeutic range for aPTT testing. The current 2001 U.S. survey of hospital coagulation laboratories produced similar results (64%).

How the aPTT therapeutic range for heparin was determined. The respondents did the following to determine the aPTT therapeutic range for heparin:

Practices to determine the aPTT therapeutic range for heparin

Large hospitals

Small hospitals

P

Using samples from patients on heparin therapy to compare a new
reagent lot to an old reagent lot

116 (66%)

57 (50%)

0.007

Using heparin spiked samples to compare a new reagent lot to an
old reagent lot

80 (47%)

50 (46%)

0.881

Performing anti-Xa assay

76 (47%)

17 (18%)

< 0.001

Using heparin spiked samples to compare a new heparin lot to an
old heparin lot

19 (12%)

22 (21%)

0.038

Using samples from patients on heparin therapy to compare a new
heparin lot to an old heparin lot

19 (11%)

14 (14%)

0.602

Performing protamine sulfate titration

17 (11%)

5 (5%)

0.134

The number of practices used to determine the aPTT therapeutic range for heparin was as follows:

 

Number of practices*

Number (Percent) of large hospitals

Number (Percent) of small hospitals

0

121 (38%)

192 (62%)

1

103 (32%)

74 (24%)

2

65 (20%)

33 (11%)

3

25 (8%)

9 (3%)

4

5 (2%)

2 (0.6%)

5

1 (0.3%)

1 (0.3%)

6

1 (0.3%)

0

*The number of practices were significantly different for the large and small hospital respondents (P < 0.001).

Percentages do not total 100% due to rounding to the nearest 1%.

Reagent sensitivity in ex vivo samples has been reported to be substantially different to that in in vitro samples.30 Specific therapeutic ranges may be necessary. Samples prepared by adding heparin to normal plasma in vitro can be misleading and should not be used. In vitro sensitivity curves using different reagents are varied at therapeutic heparin levels.25 In contrast, aPTT reagents reportedly did not differ ex vivo. Studies have shown that in vitro curves demonstrate poor performance. In one study,31 60% of patients did not adequately compare by aPTT estimation of plasma heparin levels. The aPTT therapeutic range for heparin should be determined by comparing (1) ex vivo specimens with an appropriately validated heparin assay (preferably) or (2) ex vivo specimens to a previously calibrated aPTT using a method to control for reagent drift.16 Equivalence should be determined by using ex vivo plasma samples obtained from patients treated with unfractionated heparin rather than spiked in vitro heparinized plasma samples.30,31 Forty-six percent of all respondents reported using heparin-spiked samples to determine their aPTT therapeutic range for heparin, while 59% used ex vivo specimens from heparinized patients. These may be compared to the rates of 67% and 33% for heparin spiked and ex vivo specimens, respectively, obtained in the 19981999 Canadian survey.1 As part of a 1995 CAP comprehensive coagulation survey, 23% of laboratories reported using in vitro heparin spiking to establish an aPTT therapeutic range for heparin.32

In this survey, 9% (n = 22) used protamine sulfate titration to assay for heparin in ex vivo specimens, while 37% (n = 93) used anti-Xa assay for heparin assay. Other methods were used by 27 respondents. Sixty-five percent of the respondents assaying for heparin did so using an anti-Xa assay, 15% used protamine sulfate titration and 19% used other methods. In the 19981999 Canadian survey, 90% used an anti-Xa assay and 10% used protamine sulfate titration. In this Canadian survey, however, no option was provided for other methods.1

When the aPTT therapeutic range for heparin was reconfirmed. The respondents reconfirmed the aPTT therapeutic range for heparin under the following circumstances:

Circumstances to reconfirm the aPTT therapeutic range for heparin*

Number (Percent) of hospitals

When new instrumentation is used

282 (79%)

When new reagent lots are used

269 (75%)

When new reagents are used

181 (51%)

After a specified time period

77 (22%)

None of the above

29 (8%)

Current consensus maintains that therapeutic ranges should be recalculated after the introduction of a new reagent or a new lot of the same reagent or a change in instrument.1,16,33 Ninety percent of the respondents reconfirmed the aPTT therapeutic range for heparin when either new reagents, new reagent lots or new instrumentation was implemented; and 47% did so in all 3 circumstances. The response patterns obtained from large and small hospitals were significantly different. Fifty-two percent of the large hospitals noting adherence to at least 1 of the 3 practices adhered to all of the 3 practices compared to 39% of the small hospitals (P = 0.021).

Specimen management for aPTT assay. The respondents indicated they adhered to the following practices to manage specimens before aPTT analysis:

Practices used for the aPTT assay specimen management

Large hospitals

Small hospitals

P

Specimens were assayed within 4 hours after phlebotomy

276 (96%)

259 (97%)

0.490

Specimens were centrifuged within 1 hour of collection

229 (84%)

238 (92%)

0.007

Specimens were kept at room temperature prior to testing

223 (84%)

196 (80%)

0.188

Specimens were kept at 4 C prior to testing

47 (20%)

54 (24%)

0.335

Specimens for aPTT assay are stable for up to 8 hours at room temperature except for patients receiving unfractionated heparin therapy.34 Heparinized samples, when stored uncentrifuged at room temperature, demonstrate a clinically significant shortening of aPTT and individual samples demonstrate a more than 50% decrease in ex vivo heparin levels at 4 hours. According to an approved NCCLS guideline,35 samples can be assayed up to 4 hours after phlebotomy if centrifuged within 1 hour of collection.

Ninety-six percent of all respondents stated that specimens were assayed within 4 hours after phlebotomy, and 88% noted that specimens were centrifuged within 1 hour of collection. Also, 22% of the respondents noted that specimens were kept at 4 C prior to testing, and 82% of the respondents stated that specimens were kept at room temperature before testing. In the 19981999 survey of Canadian medical laboratories,1 90% of responding laboratories reported analyzing specimens within 4 hours of specimen collection. Of the participants responding to 1 or both questions relating to aPTT assay within 4 hours after collection and tube centrifugation within 1 hour of specimen collection, 99% adhered to at least 1 practice and 74% adhered to both. In this survey, of the large hospital respondents, 69% assayed for aPTT within 4 hours of phlebotomy and centrifuged specimens within 1 hour of collection compared to 79% of the small hospital respondents (P = 0.005).

Practices Relating to Assays for von Willebrand Disease (vWD)

Performance of von Willebrand factor antigen (vWF Ag) assay. Six percent of the respondents performed vWF Ag assay. Responses from large and small hospitals were significantly different:

Number (Percent) of large hospitals

Number (Percent) of small hospitals

P

35 (12%)

1 (0.4%)

< 0.001

Reporting of ABO specific reference interval for vWF Ag assay. Nineteen percent of the respondents that performed vWF Ag assay, reported an ABO specific reference interval for this assay.

Significantly higher vWF Ag levels have been found in individuals homozygous for the Se allele than in those heterozygous for this allele,36 and lower levels of factor VIII and vWF Ag have been reported in individuals with blood type O compared to individuals with other ABO blood types.37 Data clearly show significant linkage between the ABO locus and vWF Ag, with levels of vWF Ag exhibiting significant differences between O heterozygotes and non-OO homozygotes. However, use of ABO adjusted ranges for vWF Ag levels might not be essential for the diagnosis of this disorder; bleeding symptoms may depend on vWF Ag regardless of the ABO type.38

Methodology used for vWF Ag assay. The respondents used the following methodologies to assay for vWF Ag:

Methodology used for vWF Ag assay

Number (Percent)

Latex immunoassay (LIA)

13 (37%)

ELISA

10 (29%)

Electrophoresis

6 (17%)

Others*

6 (17%)

*Five of the respondents used immunoturbidimetric assays and 1 used Laurel rocket immunoassay. No respondent reported using more than 1 methodology for vWF Ag assay.

The electroimmunodiffusion (EID) method, also known as Laurel rocket immunoassay is associated with the greatest variability in vWF Ag test results, and both the EID and LIA methods show poorer sensitivity at low vWF Ag levels compared to ELISA methods.4 Forty percent of the respondents used LIA or EID methods.

Performance of assay for von Willebrand factor (Ristocetin cofactor) activity. Seven percent of the respondents performed assay for Ristocetin cofactor activity. Responses from the large and small hospital respondents were significantly different:

Number (Percent) of large hospitals

Number (Percent) of small hospitals

P

41 (14%)

1 (0.4%)

< 0.001

Methodology used for assay of Ristocetin cofactor activity. The respondents used the following methodologies to assay for Ristocetin cofactor activity:

Methodology for assay of Ristocetin cofactor activity

Number (Percent)

Platelet aggregometry

31 (76%)

ELISA

2 (5%)

Collagen binding assays

1 (2%)

Others*

7 (17%)

*One respondent stated using a platelet function analyzer while the other 6 noted using platelet agglutination/aggregation methodswhich is the same as platelet aggregometrywith 2 of these specifying use of the Dade Behring method. No respondent reported using more than 1 methodology for Ristocetin cofactor activity assay.

Collagen binding assays for vWF activity (Ristocetin cofactor activity) have been shown to perform better than other assays in terms of their ability to detect functional von Willebrand factor (vWF) discordance, i.e., Type 2 vWD.4 However, these assays are less sensitive than vWF Ag assay in terms of their ability to measure overall level of vWF since they detect only highly adhesive vWF. For assays of vWF activity, the greatest variability in results and the poorest sensitivity to low vWF Ag levels was obtained using the platelet agglutination/aggregation methods; however, these methods showed better performance in identifying Type 2 vWD.4 In our survey, 5% performed ELISA to assay for vWF activity and 2% used a collagen binding assay. The remaining 93% used platelet function tests based on platelet agglutination/aggregation as functional tests for vWF activity. Of the respondents noting that they performed 1 or more vWF assays (antigen, activity or multimers assays), 38% performed both vWF Ag assay and a vWF activity assay, 25% performed only vWF activity assays, 15% performed only vWF Ag assays, 15% performed all 3 vWF assays, and 6% provided results for vWF multimers.

Sixty-nine percent of the respondents performed vWF Ag test. In a 1999 Australasian multi-laboratory survey of diagnostic practice and efficacy of laboratory tests for vWD, all 25 laboratories performed tests for vWF Ag, 60% of the respondents performed both vWF Ag assay and a vWF activity assay, and 40% performed all 3 vWF assays.4 In our 2001 U.S. survey, 46% performed a single vWF test (vWF activity, 25%; vWF Ag, 15%; vWF multimers, 6%) to evaluate vWD, while no respondents in the 1999 Australasian survey did so. Assaying for vWF Ag will not detect many qualitative defects; so, use of this assay alone will lead to many Type 2 vWD patients being missed by the laboratory.4 A survey conducted in Australasia pointed to considerable variation among laboratories in the tests and methods as well as the composite test panels used to diagnose vWD.3 What we have observed here implies a laboratory practice pattern not consonant with an adequate assessment of vWD.39 Performance of vWF Ag assay by only 69% of the respondents and performance/use of only a single test by 46%, including 15% using vWF Ag alone, were somewhat surprising. Caution is indicated against diagnosis of vWD made based on single vWF assay results.40 In our survey, 79% of the respondents assayed for vWF activity; and they did so either alone (25%), in combination with vWF Ag assay only (38%), and in combination with both vWF Ag and vWF multimers assays (15%). These results should be viewed in light of the fact that the questions on vWF Ag and vWF activity dealt with actual performance of these assays; some participants responding negatively to these questions may provide results for vWF Ag and vWF activity tests by sending their specimens to outside laboratories.

Provision of vWF multimers results. Two percent of the respondents provided results for vWF multimers. The responses from large and small hospitals were significantly different:

Number (Percent) of large hospitals

Number (Percent) of small hospitals

P

10 (3%)

1 (0.4%)

0.007

The respondents noted the following circumstances for the assay of vWF multimers:

Circumstance for the assay of vWF multimers

Number (Percent)

Only when ordered by a clinician

9 (82%)

When Ristocetin cofactor is decreased

3 (38%)

When Ristocetin cofactor is disproportionately decreased relative to vWF Ag

2 (29%)

When antigen and activity are both low

2 (25%)

Only if Ristocetin induced platelet aggregation indicates a Type II B vWD

1 (13%)

This assay is used to confirm or subtype vWD that has been previously diagnosed.40 It has been suggested that it would not be appropriate to assay for vWF multimers during initial vWD investigation process. Although 8 of the 11 the respondents assayed for vWF multimers in combination with assays for vWF Ag and vWF activity, 3 respondents assayed for vWF multimers without noting that they assayed for either vWF Ag or vWF activity. In 2 recent Australasian surveys of vWF testing laboratories, 1216% of the respondents assayed for vWF multimers.3,4 This may be compared to 21% of the U.S. hospital laboratory respondents doing the same as reported in this survey. The result of this survey should be considered in light of the observation that the value of vWF multimers assay is diminishing in the face of better test systems and diagnostic processes.40 Assays for vWF multimers are used to imply probable Type II A or Type II M vWD and not Type II B vWD.40 Interestingly, 9 of the 11 respondents stated that they performed vWF multimers assays only when ordered by a clinician.

The following shows vWF testing patterns in this 2001 U.S. survey and a 1999 Australasian survey:

Test Patterns Used by the U.S. Hospitals and the Australasian Medical Laboratories for Diagnosis of vWD

Test(s)

U.S., 2001 (n = 52)

Australasia, 1992 (n = 25)4

vWF Ag and vWF activity

20 (38%)

15 (60%)

vWF Ag, vWF activity and vWF multimers

8 (15%)

10 (40%)

vWF activity only

13 (25%)

vWF Ag only

8 (15%)

vWF multimers only

3 (6%)

Practices Relating to Thrombosis/Hypercoagulability Workup

Protein S assays. Five percent of the respondents usually performed the assay for protein S activity (functional test) before the antigenic assay. The responses from large and small hospitals were significantly different:

Number (Percent) of large hospitals

Number (Percent) of small hospitals

P

31 (10%)

1 (0.3%)

< 0.001

If the results of the functional test were decreased, 17% performed antigenic assay to differentiate Type I deficiency from Type II while 20% performed free and total protein S antigen assay.

Performance of activated protein C (APC) resistance and factor V Leiden mutation assays. Six percent of the respondents performed activated protein C (APC) resistance assay. The responses from large and small hospitals were significantly different:

Number (Percent) of large hospitals

Number (Percent) of small hospitals

P

32 (11%)

3 (1%)

< 0.001

If after performing the APC resistance assay, results indicated resistance to APC, 61% obtained results for factor V Leiden mutation.

More than 11 million Americans are factor V Leiden carriers, and 143,000 are homozygous for this mutation.41 Homozygosity or heterozygosity for factor V Leiden in the absence of symptoms does not necessarily indicate that preventive treatment is required.42 Furthermore, there is no established intervention to reduce thrombotic risk. Thrombotic risk can be modified by various acquired and environmental conditions such as pregnancy, oral contraceptives, estrogen therapy, malignancy, stroke with extremity paresis, trauma, surgery or immobility.43

Depending on the APC resistance functional assay used and the cut-off values for defining an abnormal result, factor V Leiden mutation may account for 8595% of patients with APC resistance.43 Factor V Leiden mutation is believed to produce a relative risk of venous thrombosis of 7-fold in the heterozygous state and 80-fold in the homozygous state. Even in the homozygous state, however, this mutation does not appear to cause disease early in life, as seen with protein S and protein C homozygosity. Activated protein C resistance with normal factor V genotype is a risk factor for venous thrombosis.44 However, there is no major effect of APC resistance on life expectancy.45 Consequently, long-term anticoagulation in carriers of factor V Leiden, on the basis of the carrier state alone, is not indicated. In view of all these, it was interesting to note that 61% of the respondents obtained results for factor V Leiden mutation if results indicated resistance to APC.

Algorithm for Diagnosing a Lupus Anticoagulant (LA)

Offering an LA profile. Eighteen percent of the respondents offered an LA profile. The responses from large and small hospitals were significantly different:

Number (Percent) of large hospitals

Number (Percent) of small hospitals

P

92 (30%)

18 (6%)

< 0.001

Our obtained rate of 18% for offering an LA profile may be compared to the rate of 34% obtained in the 19981999 Canadian survey.1

Practices leading to mixing studies. Participants noted the following circumstances for performing a mixing study when PT result was prolonged:

When mixing studies are performed with prolonged PT*

Number (Percent) of large hospitals

Number (Percent) of small hospitals

Only if there is an additional order for the mixing study

227 (78%)

98 (34%)

Our laboratory does not offer mixing studies for PT

36 (12%)

175 (61%)

Always when PT is prolonged

10 (3%)

5 (2%)

Only if PT was ordered as part of the LA Profile

10 (3%)

0

Others

9 (3%)

7 (2%)

*The response patterns from large and small hospitals were significantly different (P < 0.001).

Percentages do not total 100% due to rounding to the nearest 1%.

Participants noted the following circumstances for performing a mixing study when aPTT result was prolonged:

When mixing studies are performed with prolonged aPTT*

Number (Percent) of large hospitals

Number (Percent) of small hospitals

Only if there is an additional order for the mixing study

228 (78%)

98 (37%)

Our laboratory does not offer mixing studies for aPTT

30 (10%)

153 (58%)

Always when aPTT is prolonged

12 (4%)

5 (2%)

Only if aPTT was ordered as part of the LA Profile

13 (4%)

0

Others

8 (3%)

8 (3%)

*The response patterns from large and small hospitals were significantly different (P < 0.001).

Percentages do not total 100% due to rounding to the nearest 1%.

Workup to diagnose an LA. Seventeen percent of the respondents routinely initiated a workup to diagnose an LA if results of the mixing study for aPTT did not correct to normal. The responses from large and small hospitals were significantly different:

Number (Percent) of large hospitals

Number (Percent) of small hospitals

P

54 (21%)

9 (8%)

0.004

If results of the mixing study for aPTT did not correct to normal, the respondents indicated routinely performing the following assays to diagnose an LA:

Tests performed

Number (Percent)

Dilute Russell viper venom time (1)

46 (79%)

Hexagonal phase phospholipid assay (Staclot LA) (2)

24 (51%)

Lupus sensitive aPTT (3)

19 (40%)

Platelet neutralization procedure (4)

17 (35%)

Tissue thromboplastin inhibition test (5)

4 (9%)

Kaolin clotting time (6)

2 (5%)

Of those using 1 or more of the above tests, 58% used more than 1 test. The respondents noted the following test combinations more than once:

Test combinations performed*

Number (Percent)

1 and 2

8 (23%)

1 and 4

6 (17%)

1, 2 and 3

6 (17%)

3 and 4

3 (9%)

1 and 3

2 (6%)

2 and 3

2 (6%)

1, 2, 3 and 4

2 (6%)

*There were a total of 13 test combinations. The above combinations constituted 83% of all those using more than 1 test. The numbers refer to the designated tests noted in the previous table.

Percentages refer to the proportion of respondents noting the use of more than 1 test.

Practices Relating to Monitoring of Low Molecular Weight Heparin (LMWH) Therapy

Fourteen percent of the respondents monitored LMWH therapy. The responses from large and small hospitals were significantly different:

Number (Percent) of large hospitals

Number (Percent) of small hospitals

P

55 (19%)

27 (10%)

0.002

Since we did not ask whether their institutions used LMWH therapy, we do not know what proportion of the respondents using LMWH actually monitored for it. In the 19981999 survey of Canadian medical laboratories, 71% of institutions used LMWH, thus obviating many of the issues surrounding aPTT testing.1 Some form of monitoring for LMWH therapy was used by 25% of all Canadian medical laboratories (35% of those using LMWH therapy). This rate of 25% may be compared to the overall rate of 14% in our survey.

Assays used. Those monitoring LMWH therapy used the following assays:

Assay used to monitor LMWH therapy

Number (Percent) of large hospitals

Number (Percent) of small hospitals

P

aPTT

23 (58%)

24 (96%)

0.001

Anti-Xa

32 (65%)

3 (18%)

0.001

Factor Xa (inhibitor assay)

3 (8%)

1 (6%)

0.795

Thrombin inhibitor assay (HEP test)

0

0

The chromogenic antifactor Xa method is recommended for monitoring LMWH, while aPTT is not.15 Of all the respondents, 53% used an anti-Xa assay to monitor LMWH therapy, while 72% used aPTT assay to do so. While a significantly smaller proportion of the large hospital respondents used aPTT assay to monitor LMWH therapy compared to the small hospital respondents (P = 0.001), a significantly greater proportion of the large hospitals respondents used anti-Xa assay to do so (P = 0.001). In the 19981999 Canadian survey, 71% of those monitoring LMWH did so by a chromogenic anti-Xa assay, and the remaining 29% used an anti-Xa clotting assay. In agreement with our findings, none of the Canadian respondents used thrombin inhibitor assay (HEP test).

Calibrators used. The respondents indicated they used the following calibrators for anti-Xa assay:

Calibrator

Number (Percent)

LMWH supplied by pharmacy

19 (53%)

Internal standard LMWH

8 (22%)

Unfractionated heparin

5 (14%)

Internal standard unfractionated heparin

4 (11%)

Heparinoid

1 (3%)

Others

8 (22%)

Seventy-four percent of the respondents used different calibration curves for LMWH and unfractionated heparin, while 42% used different calibration curves for each type of LMWH. The CAP has recommended that laboratories use different calibrations for LMWH and unfractionated heparin,15 and establish calibration curves with each lot and type of LMWH.16 The CAP has also recommended that pharmaceutical heparin be calibrated against an international (preferably the WHO) standard using an anti-factor Xa assay.16

Four of the 19 the respondents reporting use of a LMWH supplied by pharmacy also stated that they used unfractionated heparin as a calibrator for the anti-Xa assay. One of these 4 respondents also used heparinoid as a calibrator. Finally, 1 of the 8 respondents using an internal LMWH standard also used an internal standard for unfractionated heparin. A calibrated LMWH has been recommended for establishing the standard curve for assay of LMWH, and unfractionated heparin cannot be used for this purpose.15 Nevertheless, 28% of the respondents reported using reagents other than LMWH as a calibrator for the anti-Xa assay. Nine of these 10 respondents used unfractionated heparin while 1 used heparinoid.

Timing of anti-Xa assay. We obtained the following responses regarding how long after subcutaneous administration of LMWH the laboratory recommended specimen collection for anti-Xa testing:

Time of specimen collection after subcutaneous administration of LMWH

Number (Percent)

Our coagulation laboratory does not recommend a time for testing

17 (46%)

4 hours after injection

12 (32%)

Between 2 and 4 hours after injection

5 (14%)

Do not know

2 (5%)

5 hours or more after injection

1 (3%)

Others

0

When LMWH is monitored, the sample should be obtained 4 hours after subcutaneous injection.15

Availability of Specific Coagulation Tests

The respondents performed the 29 tests examined in this survey as follows:

Test

Number (Percent) of large hospitals

Number (Percent) of small hospitals

P

PT

310 (100%)

295 (100%)

1.000

aPTT

309 (99%)

292 (98%)

0.229

Bleeding time

277 (89%)

270 (90%)

0.699

Fibrinogen

295 (95%)

163 (59%)

< 0.001

D-dimer

252 (83%)

124 (46%)

< 0.001

Fibrin(ogen) degredation products

200 (67%)

92 (35%)

< 0.001

Activated clotting time

170 (58%)

70 (27%)

< 0.001

Thrombin time

159 (54%)

50 (19%)

< 0.001

Factor VIII activity

105 (36%)

5 (2%)

< 0.001

Factor IX activity

92 (32%)

3 (1%)

< 0.001

Factor VII activity

70 (25%)

3 (1%)

< 0.001

Platelet aggregation study

70 (24%)

2 (0.8%)

< 0.001

Factor V activity

67 (24%)

2 (0.8%)

< 0.001

Factor X activity

64 (23%)

2 (0.8%)

< 0.001

Factor II activity

54 (19%)

2 (0.8%)

< 0.001

Heparin assay (Anti-Xa)

48 (17%)

4 (2%)

< 0.001

vWF (Ristocetin cofactor) activity

41 (14%)

1 (0.3%)

< 0.001

Bethesda assay-inhibitor titer

40 (14%)

0

< 0.001

Ristocetin titration of platelet aggregation

36 (13%)

1 (0.4%)

< 0.001

vWF Ag

35 (12%)

1 (0.4%)

< 0.001

Factor VIII antigen

33 (12%)

2 (0.8%)

< 0.001

Activated protein C resistance

32 (11%)

3 (1%)

< 0.001

Euglobulin clot lysis time

25 (9%)

2 (0.8%)

< 0.001

Factor V Leiden

27 (10%)

0

< 0.001

Plasminogen (functional) assay

23 (8.2%)

1 (0.4%)

< 0.001

Factor X antigen

17 (6.0%)

0

< 0.001

Platelet antibody

14 (5.0%)

0

< 0.001

vWF multimers

5 (1.8%)

0

0.035

Plasminogen antigen

2 (0.7%)

0

0.184

Except for the hospitals performing tests for PT, aPTT and bleeding time, and the 2 large hospitals assaying for plasminogen antigen, a significantly greater proportion of large hospitals performed any of the above tests in-house compared to small hospitals (P = 0.035 for 5 large hospitals performing vWF multimers and P < 0.001 for all other tests.). Only 4 small hospitals performed an in-house anti-Xa assay for heparin. This is in agreement with our finding (previously discussed) noting a significantly lower uptake of anti-Xa assay for LMWH therapy compared to the large hospital respondents (P = 0.001). It also shows that the apparent reason the small hospitals mostly use aPTT assay in lieu of anti-Xa assay to monitor LMWH therapy may be that few of them even perform an in-house anti-Xa assay.

  
RESULTS and DISCUSSION - CONTINUED

This page last reviewed: 7/12/2004
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