Clinical Chemistry 43: 1215-1222, 1997;
(Clinical Chemistry. 1997;43:1215-1222.)
© 1997 American Association for Clinical Chemistry, Inc.
Preparation of lyophilized partial thromboplastin time reagent composed of synthetic phospholipids: usefulness for monitoring heparin therapy
Anton M. H. P. van den Besselaara,
Jacoline Neuteboom,
Joyce Meeuwisse-Braun and
Rogier M. Bertina
Hemostasis and Thrombosis Research Centre, Department of Hematology, Leiden University Hospital, Bldg. 1, C2-R, P.O. Box 9600, 2300 RC Leiden, The Netherlands.
a Author for correspondence. Fax +31 71 5266755.
 |
Abstract
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To contribute to the development of a reference reagent for monitoring
heparin therapy, a lyophilized partial thromboplastin time
(PTT) reagent was prepared from synthetic dioleoylphosphatidylcholine,
dioleoylphosphatidylserine, and dioleoylphosphatidylethanolamine, with
colloidal silica as activator. The reagent, coded 91/558, was contained
in sealed glass ampoules; it deteriorated in a heat degradation
experiment, but its activity remained constant for at least 4 years
when stored at -70 °C. Within- and between-run precision with this
reagent complied with the requirements proposed by the International
Committee for Standardization in Haematology (ICSH) Panel on PTT. The
response of this reagent and of two other reagents to heparin added to
pooled normal plasma was nonlinear. Citrated samples from 58 patients
receiving intravenous heparin and from 24 apparently healthy volunteers
were tested with reagent 91/558, with Automated APTT (Organon Teknika),
with Manchester APTT reagent, with an anti-factor Xa assay, and with an
anti-factor IIa assay. The correlation of APTT with anti-Xa and
anti-IIa activity was poor. The best correlation was observed between
reagent 91/558 and the Organon Teknika reagent. Correlations were
improved when individual patients' samples were replaced by pooled
plasmas from heparinized patients, in whom the effect of oral
anticoagulation was minimal. These results suggest that preparation of
a lyophilized synthetic phospholipid reagent is feasible for use in
monitoring heparin therapy.
Key Words: indexing terms: anticoagulants • standardization
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Introduction
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The first report on the usefulness of the partial
thromboplastin time for monitoring anticoagulant therapy appeared in
1962 (1). Ever since, the activated partial thromboplastin
time (APTT)1
has become the most popular laboratory test for monitoring
full-dose heparin therapy. Many different APTT reagents are available
and show considerable variation in their responses to heparin.
Consequently, a therapeutic ratio of 1.5 to 2.5 times the value for a
healthy ("normal") control may be appropriate for some reagents,
but not for all (2)(3)(4)(5). The need for progress in
standardization of the APTT monitoring of heparin administration is
urgent (6)(7)(8)(9)(10). Standardization of the APTT may be made
feasible by establishing a reference reagent; however, no international
reference preparation for the APTT is available.
Most APTT reagents are prepared from biological lipid sources, e.g.,
animal brain or soya bean extracts. The phospholipid class and fatty
acid composition of these reagents are highly variable and cannot be
controlled in a simple way. In recent years, the possibility of
replacing natural phospholipids with synthetic preparations has been
investigated (11). Indeed, the use of a synthetic
phospholipid material may be preferable because its chemical
composition is well defined. Synthetic phospholipids have also been
used to prepare a recombinant tissue factor reagent (12).
Here, we describe the preparation of a lyophilized APTT reagent
comprising synthetic phospholipids and colloidal silica. This reagent
was characterized with respect to precision of clotting times and
response to in vitro and ex vivo heparin. For comparison, the response
of two other widely used APTT reagents was assessed—one prepared from
rabbit brain phospholipids with silica as activator (Automated APTT;
Organon Teknika), the other from human brain phospholipids with kaolin
as activator ("Manchester reagent," which has been evaluated in
international collaborative trials (13)).
This synthetic reagent should be regarded as a first step towards a
reference material for standardization of the control of heparin
therapy.
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Materials and Methods
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materials
Chemicals.
1,2-Dioleoyl-sn-glycero-3-phospho-L-serine
sodium (DOPS); 1,2-dioleoyl-sn-glycero-3-phosphocholine
(DOPC); and
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)
were obtained from the Biochemical Laboratory, State University of
Utrecht, The Netherlands. Cholesterol, D-mannitol, and
butylated hydroxyanisole were from Sigma Chemical Co., St. Louis, MO.
Hepes was from Calbiochem, La Jolla, CA. Silica powder (Aerosil OX50,
40-nm particle diameter) was kindly provided by Degussa Nederland,
Amsterdam, The Netherlands. Porcine mucosal heparin (Thromboliquine)
was from Organon Teknika, Boxtel, The Netherlands. Light kaolin was
obtained from British Drug House (Poole, UK).
Reagents.
Automated APTT was kindly provided by Organon
Teknika, Turnhout, Belgium. Manchester APTT reagent (cephalin batch
343) was kindly provided by J.M. Thomson (UK Reference Laboratory for
Anticoagulant Reagents and Control, Manchester, UK). These reagents
were used as recommended by their respective suppliers. The incubation
times were 5 and 10 min, respectively.
Synthetic APTT reagent.
Synthetic phospholipids (16
µmol of DOPS, 32 µmol of DOPC, and 32 µmol of DOPE) were mixed
with 80 µmol of cholesterol in chloroform. The chloroform was
evaporated under a stream of nitrogen in a waterbath at 37 °C. The
dried lipid mixture was suspended in 50 mL of an aqueous solution of 25
mmol/L Hepes (pH 7.5) containing 18 mg/L butylated hydroxyanisole
(14), by mechanical agitation with glass beads. Next,
Hepes buffer containing mannitol and silica powder was added. The final
concentrations were, per liter: 0.067 mmol of phospholipid, 0.067 mmol
of cholesterol, 0.75 g of butylated hydroxyanisole, 50 g of
D-mannitol, and 3 g of silica. The mixture was stored
at 4 °C and was shipped to the National Institute for Biological
Standards and Control at Potters Bar, Herts, UK. At the Institute, the
preparation was dispensed in ampoules (mean ± SD mass of
suspension per ampoule, 1.0165 ± 0.0007 g) and lyophilized. After
secondary desiccation of the material over phosphorus pentoxide, the
ampoules were sealed under nitrogen. The mean (SD) dry mass per ampoule
was 54.63 (0.19) mg, and the residual moisture was 0.0629% (0.0156%).
The batch size was 900 ampoules. The lyophilized material was stored at
-70 °C. The reagent was coded 91/558. The contents of each ampoule
was reconstituted with 1.0 mL of water and was used between 20 and 90
min after reconstitution.
samples
Plasma samples.
Plasma samples were prepared from blood
collected from 24 apparently healthy volunteers and from 58 patients
receiving a continuous infusion of sodium heparin (Thromboliquine). The
58 patients were also treated with oral anticoagulants, starting on the
first day of heparin infusion. From each subject 4.5 mL of blood was
added to 0.5 mL of 0.11 mol/L citrate (in a Vacutainer Tube; Becton
Dickinson, Franklin Lakes, NJ) and centrifuged at 2200g for
10 min, and the decanted plasma was further centrifuged at
27 000g for 30 min. The processed plasma was frozen and
stored at -70 °C. Before assay, the samples were thawed in a
waterbath at 37 °C.
Plasma pools.
Plasmas from patients receiving a
continuous infusion of sodium heparin, collected as described above,
were pooled. Three pools of patients' plasmas were prepared, each with
a different amount of heparin activity. Labeled H1, H2, and H3, the
three pools comprised 86, 84, and 84 individual plasmas, respectively.
To minimize the effect of oral anticoagulation, only plasmas with
International Normalized Ratio (INR) <2.5 were included. INR values of
the pooled plasmas were determined with the Thrombotest reagent after
heparinase treatment (15).
A fourth pooled plasma was prepared from patients on long-term coumarin
treatment who did not receive heparin therapy. The INR of this pooled
plasma (labeled C1) was ~5. All pooled plasmas were stored at
-70 °C.
procedures
Coagulation time determinations.
Reconstituted reagent
91/558 was used as follows: 0.1 mL of reagent was mixed with 0.1 mL of
plasma in a polystyrene tube at 37 °C; the mixture was incubated for
5 min at 37 °C, unless indicated otherwise; and 0.1 mL of prewarmed
calcium chloride (0.025 mol/L) was added and mixed, at which time the
timer was started. Coagulation times were determined with a
coagulometer according to Schnitger & Gross (Amelung, Lemgo, Germany),
with a KC10 (also Amelung), with an ACL-300 (Instrumentation Laboratory
SpA, Milan, Italy), with an Elecra-900 (Medical Laboratory Automation,
Pleasantville, NY), or with a Sysmex CA 5000 (Toa Medical Electronic
Co., Kobe, Japan).
Heat degradation study.
Ampoules of reagent 91/558 were
stored at 4 °C, 37 °C, and 44 °C for a total of 12 weeks. At
1-week intervals, ampoules were tested with two plasma samples: pooled
normal plasma, and pooled normal plasma containing heparin, 0.2 IU/mL.
Clotting times were determined with the coagulometer according to
Schnitger & Gross. APTT ratios were calculated by dividing the APTT of
abnormal plasma by the APTT of the normal plasma determined with the
same reagent sample.
Heparin assay.
Heparin activity was measured by factor
Xa inhibition with the chromogenic peptide substrate S-2222
(Chromogenix AB, Mölndal, Sweden), and by factor IIa inhibition
with use of a chromogenic substrate (Instrumentation Laboratory). The
assays were performed with the ACL-300. Thromboliquine was used to
construct calibration curves.
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Results
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Effect of lyophilization.
Before lyophilization of
reagent 91/558, we determined a dose–response curve for this reagent,
using the coagulometer according to Schnitger & Gross (Fig. 1
). The dose–response curve of the lyophilized reagent was
shifted to longer coagulation times. Similar results were obtained with
the ACL-300 (not shown). Stability of reconstituted reagent at room
temperature was tested with pooled normal plasma and with plasma
containing heparin (0.4 IU/mL). No significant change of APTT was
observed between 20 and 90 min (not shown).
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Figure 1. Effect of lyophilization of reagent 91/558 on the heparin
dose–response curve.
APTT/s was determined with the coagulometer according to Schnitger &
Gross before ( ) and after (•) lyophilization of the reagent.
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Effect of incubation time.
Incubation time, i.e., the
interval between addition of reagent to the test plasma and
recalcification, influenced the coagulation times of pooled normal
plasma and pooled patients' plasmas differently (Fig. 2
). The pooled normal plasma showed a monotonic decrease in the
coagulation time as the incubation time increased from 1 to 10 min. In
contrast, pooled patients' plasmas H2 and H3 had minimum coagulation
times at incubation times of ~3 min. For all other experiments with
reagent 91/558, a fixed incubation time of 5 min was used.
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Figure 2. Effect on APTT of varying the incubation time for pooled
normal plasma (NP) and three pooled patient plasmas (H1, H2, and H3).
Reagent 91/558 was used with the coagulometer according to Schnitger &
Gross.
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Precision of APTT determinations.
Within-run precision
(CVp) for reagent 91/558 was assessed by making
20 APTT determinations with pooled normal plasma and with the same
plasma containing heparin, 0.5 IU/mL (Table 1
). In a second experiment, between-ampoule variation and
precision were assessed as follows. Twenty ampoules of reagent 91/558
were reconstituted and the contents of each ampoule were used for
testing pooled normal plasma. The 20 values were used to calculate the
between-ampoule-variation (CVa). Then, the
contents of 20 ampoules were pooled, and the within-run precision was
determined by assaying the same pooled normal plasma 20 times (Table 1
).
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Table 1. Within-run precision (CVp) and between-ampoule
variation (CVa) of APTT determinations with reagent
91/558 (n = 20).
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Between-run variation was calculated from determinations in 20 runs of
deep-frozen pooled normal plasma and deep-frozen pooled patients'
plasmas (Table 2
).
Response to in vitro heparin.
The dose–response of
reagent 91/558, Manchester reagent, and Automated APTT to heparin in
pooled normal plasma is shown in Fig. 3
. The dose–response was nonlinear for all three systems.
Nonlinearity with Manchester reagent was observed mainly in the
concentration range 0–0.1 IU/mL. In the range 0.1–0.75, the response
with Manchester reagent was approximately linear.
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Figure 3. APTT as a function of heparin added to pooled normal
plasma, as determined with three reagent/instrument systems: reagent
91/558-ACL (•); Manchester reagent–Schnitger & Gross (x); and
Automated APTT-ACL ( ).
Determinations were performed on 7 separate days. Between-day SDs are
indicated by vertical bars.
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Response to ex vivo heparin.
Plasma samples from
patients being treated with intravenous heparin and concomitant oral
anticoagulation were used to assess the relations between the three
APTT systems. Log-transformed APTT values were plotted (Fig. 4
), as was done in previous studies
(7)(8)(9)(16), which yielded a more homogeneous
scatter of data points of the original clotting times. The relation
between reagent 91/558 and Manchester reagent results determined with
these ex vivo heparin samples differed from that determined with the in
vitro samples (Fig. 4
, top). A similar difference was observed for the
relation between results by Automated APTT (Organon Teknika) and by
Manchester reagent (Fig. 4
, middle). In contrast, the relation between
reagent 91/558 and Automated APTT results was practically the same for
ex vivo and in vitro heparin samples (Fig. 4
, bottom), and the scatter
of ex vivo data points in the latter was less wide than in the first
two comparisons.
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Figure 4. Scatterplots of APTT/s, comparing results obtained with
(top) reagent 91/558-ACL (x) vs Manchester
reagent–Schnitger & Gross (y); (middle)
Automated APTT-ACL (x) vs Manchester reagent–Schnitger &
Gross (y); and (bottom) reagent 91/558-ACL
(x) vs Automated APTT-ACL (y), where x
and y represent the natural logarithms of APTT/s.
In each panel, the straight (dashed) line
represents the orthogonal regression line for the normal (n = 24)
plus ex-vivo heparin (n = 58) samples. The equation for this line
(n = 82) is: top, y = 1.34 +
0.68x (r = 0.95); middle,
y = 1.48 + 0.69x (r = 0.94);
and bottom, y = -0.20 + 0.98x
(r = 0.98). The regression equation for the ex-vivo
heparin samples alone (n = 58) is: top,
y = 1.24 + 0.70x (r = 0.83);
middle, y = 1.25 + 0.74x
(r = 0.79); and bottom, y =
0.02 + 0.94x (r = 0.94). For comparison, the
in-vitro heparin points (see Fig. 3
) are represented as well
(connected by the solid line).
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Pooled patients' plasmas.
Pooled patients' plasmas
were prepared from specimens with low INR (1.2–1.3), to minimize the
effect of oral anticoagulation on the APTT.
The APTTs were determined with 5 different instruments (Table 3
). The coagulation times determined with the mechanical instruments
(Schnitger & Gross, KC10) were longer than those with the photooptical
instruments. Coagulation times determined with reagent 91/558 were
plotted against those obtained with Automated APTT (Fig. 5
). For each instrument, the relationship was practically linear,
with all r >0.9993. However, the relation between reagent
91/558 or Automated APTT and Manchester reagent was not linear (not
shown).
Relation between APTT and ex vivo heparin activities.
Log APTT values of ex vivo heparin samples were plotted against the
anti-factor Xa activities in these samples (Fig. 6
, left) and showed very wide scatter. Some samples had
relatively long APTT with low anti-Xa activities. In some samples the
effect of concomitant oral anticoagulation was very high (INR >5),
which in part may account for the long APTT values. The relation
between APTT and anti-Xa, based on results for pooled normal plasma and
the pooled ex vivo heparin plasmas H1, H2, and H3, is also shown in
Fig. 6
. The line connecting the four pooled plasma points is at the
lower limit of the scatter of individual patients' points. Wide
scatter was also observed when APTT values (log scale) of the same
samples were plotted against the anti-IIa activities (Fig. 6
, right).
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Figure 6. Scatterplot of APTT (determined with reagent 91/558-ACL;
logarithmic scale) vs anti-factor Xa activity (left) and
anti-IIa activity (right) in 50 ex-vivo heparin samples:
(•), samples with INR <5; (), samples with INR > 5.
Correlation (r) = 0.42 (left), 0.37
(right). Mean values for pooled normal plasma and the pooled
patient plasmas H1, H2, and H3 are also shown ( ): r
= 0.995 (n = 4) for each panel.
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The correlation between anti-Xa and anti-IIa activities of these
samples was much better (r = 0.87, not shown), in
agreement with previous studies (16).
Heat degradation study.
The clotting times for assays
performed with reagent 91/558 that had been stored at 4 °C did not
change during 3 months (Fig. 7
). When reagent 91/558 was stored at 37 °C and 44 °C, the
clotting times were prolonged. The APTT ratio (abnormal APTT:normal
APTT) tended to increase with increasing storage temperature.
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Figure 7. Heat degradation experiment with reagent 91/558 in
ampoules stored at 4 °C (•), 37 °C (), and 44 °C ().
(Top) With each ampoule, APTT was determined in pooled
normal plasma and in plasma containing heparin, 0.2 IU/mL.
(Bottom) The APTT ratio is the APTT of the heparin-treated
plasma divided by the APTT of the normal plasma.
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Long-term stability.
A program for regular monitoring of
reagent 91/558 stored at low temperature (-70 °C) was not provided.
After 4 years' storage at -70 °C, reagent 91/558 was tested with
the same lot of pooled normal plasma (Table 4
). The coagulation times were practically the same as the
initial values.
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Table 4. APTT/s of pooled normal plasma determined with reagent
91/558 in 1992 and 1996 after plasma and reagent were stored at
-70 °C.
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Discussion
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Synthetic phospholipids can be used to prepare a PTT reagent with
adequate response to heparin. In the present study we investigated the
feasibility of preparing a lyophilized synthetic phospholipid reagent
with acceptable characteristics for monitoring heparin therapy. This
may be the first step towards development of a reference reagent for
standardization of heparin therapy. Precautions were taken to avoid
oxidation of the phospholipids during preparation of the reagent
(14). In previous studies we used kaolin as activator but
here we replaced this with colloidal silica (17), which
does not sediment as rapidly as kaolin and is compatible with
nephelometric instruments such as the ACL-300. The activity of the
silica depends on the particle diameter (18): Activity per
unit mass peaks at ~40 nm and then decreases as the surface
area decreases (18).
Lyophilization of the mixture of liposomes and silica induced a slight
prolongation of the coagulation times (Fig. 1
). The mechanism of the
prolongation is not known, but limited coalescence of the colloidal
silica particles or liposomes cannot be excluded. The reproducibility
of the reagent preparation should be investigated.
Coagulation times were also influenced by the duration of incubation of
plasma with reagent (Fig. 2
). Interestingly, the coagulation time of
normal plasma was shortened by increasing incubation time, but in the
presence of heparin a minimum coagulation time was observed after 2–4
min. Consequently, the response to heparin (i.e., APTT ratio) increased
with the incubation time. A fixed incubation time of 5 min was chosen
because use of longer incubation times would be less economical.
Storage of reagent 91/558 at 37 °C and 44 °C resulted in
prolonged APTT values and APTT ratios (Fig. 7
), suggesting that the
heat stability of the reagent may need improvement. No deterioration
was observed when the reagent was stored at 4 °C for 3 months.
Shipment of reagent 91/558 during hot weather may thus require cooling
bags to avoid deterioration. After 4 years of storage at -70 °C,
reagent 91/558 showed no evidence of significant deterioration (Table 4
).
Within-run precision (Table 1
) was in agreement with the goal of <3%
CV proposed by the ICSH Panel on the PTT (19). The
Panel's proposed goal for between-run precision (<4% CV) was
achieved with deep-frozen normal plasma (Table 2
). Between-run CVs
tended to increase with increasing clotting times, as shown with some
pooled patients' plasmas (Table 2
).
Despite proposals that a linear response of the APTT to heparin should
be the aim (20), it is hard to achieve this in practice
(2)(21). With the three reagents used in this
study, the dose–response curve for heparin between 0 and 0.1 IU/mL is
less steep than the curve at higher heparin concentrations (Fig. 3
).
The response of the APTT to heparin is related to the phospholipid
composition and concentration (11). We used a phospholipid
composition and concentration that could be expected to result in a
nearly linear response (11). The nonlinear dose–response
curve of reagent 91/558 may be induced partly by the replacement of
kaolin by colloidal silica and partly by the lyophilization.
The similar response of reagent 91/558 and Automated APTT to in vitro
and ex vivo heparin samples (Fig. 4
, bottom) may be related to the use
of the same activator, i.e., silica. This may also explain the
linearity of the relation between these reagents for analyses of pooled
patients' plasmas (Fig. 5
). There was an obvious difference between ex
vivo and in vitro heparin when Manchester reagent was correlated with
either reagent 91/558 or Automated APTT (Fig. 4
, top and middle). In
vitro and ex vivo heparin may be adsorbed differently to silica and
kaolin, which might account for the different responses of these
samples in the APTT systems.
Several studies have shown that the incidence of deep vein thrombosis
is reduced substantially in patients treated with heparin, the dose
being adjusted by APTT monitoring (22)(23)(24)(25). Each of these
studies used a single APTT reagent, so the clinical efficacy of the
different APTT reagents cannot be compared. Nevertheless, good
correlation between APTT reagents on the basis of determinations of ex
vivo heparin samples suggests that these reagents have similar clinical
efficacy. The use of correlation coefficients may be misleading when
samples from apparently healthy individuals are included with the ex
vivo heparin samples. For example, the correlation coefficient for the
ex vivo heparin samples shown in Fig. 4
(bottom) was 0.94 but could
have been increased to 0.98 by including the data from the 24 samples
from healthy volunteers. Apparently the correlation coefficient depends
on the relative numbers of samples from each group (normal subjects and
patients), but there are no generally accepted guidelines for the
numbers required.
The poor correlation between APTT values and anti-Xa or anti-IIa values
in individual patient specimens (Fig. 6
) is caused by the lack of
specificity of the APTT for heparin. In this hospital full-dose heparin
treatment of venous thrombosis is combined with oral anticoagulation
from the beginning. Oral anticoagulation by vitamin K antagonists
induces prolongation of the APTT but does not influence anti-Xa and
anti-IIa results.
Interindividual variation of other factors, e.g., factor VIII, may also
contribute to the dissociation between the APTT and heparin
concentrations measured by anti-Xa and anti-IIa assays. Correlations
were improved when individual specimens were replaced by pooled
patients' plasmas having different heparin contents but with minimal
INR (Fig. 6
). This suggests that the amounts of other factors
influencing the APTT were similar in the pooled plasmas, and very
likely the effect of interindividual variation of these factors is
reduced by pooling.
Only a few clinical studies compare APTT and heparin assay in
monitoring heparin treatment of deep venous thrombosis. Levine et al.
(26) found no significant difference of recurrent venous
thromboembolism and bleeding between patients monitored by APTT and
those monitored by anti-Xa determinations. Holm et al.
(27) suggested that both heparin assay and APTT may serve
to identify patients whose heparin concentration is too low. Holm et
al. (28) maintained that an anti-Xa assay seemed best
suited for identifying patients at risk of bleeding, but APTT and
thrombin time with recalcified plasma were also useful.
Some authors recommended the adoption of a reference APTT reagent for
calibrating working APTT reagents (9). At present, there
is no consensus in the International Society on Thrombosis and
Haemostasis Scientific and Standardization Committee and its
subcommittee on control of anticoagulation concerning adoption of a
reference APTT reagent for standardization of heparin monitoring. An
advantage of using a reference APTT reagent for calibration of other
APTT systems is the relatively high correlation coefficients that can
be obtained (Figs. 4
and 5
). Certification of clotting times of pooled
patients' plasmas by using certain instruments in relation to heparin
concentrations (see Table 3
) may be another step towards
standardization of monitoring heparin therapy (29). Such
certification may be performed by multicenter studies.
In conclusion, a lyophilized synthetic phospholipid preparation can
replace a natural phospholipid APTT reagent for monitoring heparin
therapy, a finding that has possible applications for standardization.
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Acknowledgments
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This work was supported by a grant from the Netherlands Heart
Foundation (Nederlandse Hartstichting), grant number 88.068. We thank
T.W. Barrowcliffe and the staff of the National Institute of Biological
Standards and Control (Potters Bar, UK) for their generous assistance
with the preparation of reagent 91/558. Excellent technical assistance
was provided by E. Witteveen and H. Schaefer-van Mansfeld. C.
Wiarda-Labee also provided assistance.
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Footnotes
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1 Nonstandard abbreviations: (A)PTT, (activated) partial thromboplastin time; DOPC, dioleoylphosphatidylcholine; DOPS, dioleoylphosphatidylserine; DOPE, dioleoylphosphatidylethanolamine; ICSH, International Committee for Standardization in Haematology; and INR, International Normalized Ratio. 
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C. Eby
Standardization of APTT Reagents for Heparin Therapy Monitoring: Urgent or Fading Priority?
Clin. Chem.,
July 1, 1997;
43(7):
1105 - 1107.
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Abstract
Introduction
), and Sysmex CA 5000 (