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.
|