Keywords blood coagulation - hemostasis - diagnosis - laboratory - instrumentation - analyzer
Hemostasis is an intricate and multifaceted biological pathway, whose appropriate
functioning is essential for survival of humans and of many other animal species.[1 ] Physiological hemostasis is conventionally divided into three parts: primary and
secondary hemostasis, which are mainly aimed at allowing the generation of a stable
blood clot once the integrity of blood vessels has been jeopardized, and which are
accompanied by fibrinolysis, a biological process aimed to further control and dissolve
blood clots once hemorrhage has been arrested.[1 ]
Hemostasis disturbances can be conventionally classified as hemorrhagic, when blood
coagulation cannot efficiently prevent the leakage of blood from arterial or venous
vessels, or as thrombotic, when blood clot generation is disproportionate or apparently
unnecessary (i.e., not associated with disruption of blood vessels integrity).[2 ] Both these conditions represent important causes of morbidity and mortality around
the world.[3 ] The current diagnostic approach to patients with hemostasis disturbances encompasses
an integrated strategy, combining clinical history and physical examination, alongside
results of laboratory testing.[4 ] The many available hemostasis tests are usually divided into sequential priority
classes, the first being represented by the so-called first-line (screening) analyses,
mostly entailing prothrombin time (PT), activated partial thromboplastin time (APTT),
fibrinogen, D-dimer, and potentially platelet function screening tests (i.e., by platelet
analyzer [PFA] 100/200), followed by second-line tests, mainly aimed at identifying
the underlying source of the hemorrhagic or thrombotic disease, which are then followed
by third-line tests, mostly represented by molecular biology and other highly specialized
analyses aimed at recognizing the precise molecular or biochemical defect.[1 ]
[5 ]
[6 ]
Since laboratory diagnostics has now become a mainstay for the diagnostic and therapeutic
approach of hemostasis disorders, the availability of rapid, accurate, precise, automated,
and relatively inexpensive, laboratory tests is becoming increasingly important.[4 ]
[7 ] The new generation of coagulation analyzers is designed to meet most of these essential
aspects, thus encompassing fully automated functioning, cap piercing, random analysis
of samples (for both routine and urgent testing), large test menus (including a vast
array of clotting, chromogenic, and immunoturbidimetric assays), advanced software
enabling automatic retesting or reflex testing, suitability for laboratory automation,
and, with some analyzers, automatic check of sample quality.[8 ]
[9 ]
[10 ] Therefore, the present study was aimed to perform a preliminary analytical assessment
of three first-line coagulation tests (i.e., PT, APTT, and fibrinogen) on the new
Roche cobas t 711 fully automated coagulation analyzer.
Materials and Methods
Analyzer Description
The Roche cobas t 711 analyzer (Roche Diagnostics GmbH) is a new fully automated, random continuous-access
coagulation analyzer, which has broadened the hemostasis testing concept to use reagent
cassettes, as characterizing other clinical chemistry and immunochemistry analyzers
of the Roche cobas series. The cassettes can contain both liquid and lyophilized reagents.
Regarding the latter, the instrument performs an automatic resuspension of lyophilized
material, thus potentially overcoming the inherent imprecision and potential inaccuracy
of manual pipetting.[11 ] Reconstitution of reagents can be automatically scheduled, thus improving walk-away
reagents management. The test menu encompasses a variety of clotting (optical clot
detection), chromogenic, and immunoturbidimetric assays. The analyzer has a capacity
of 225 samples and is capable of performing up to 390 tests per hour, along with automatic
checking of sample tube pressure and quality, to address the presence of relevant
concentrations of interfering substances such as cell-free hemoglobin, bilirubin,
and lipids.[12 ] The analyzer has hence different capabilities to detect clotted and/or preactivated
specimens. Sample pipetting is constantly monitored by pressure sensors so that pipetting
will not be performed and test results will be flagged accordingly when the delta
pressure is compatible with clot aspiration. When the sample is partially clotted,
or serum is aspirated (even without clots), the measurements will be performed. In
such cases, however, clotting will not take place in an established timeframe, and
results will be then flagged. The reagents used in this study were PT, clotting assay
with human recombinant thromboplastin as activator (cobas PT Rec; Roche Diagnostics
GmbH), APTT, clotting assay with ellagic acid as activator (cobas aPTT; Roche Diagnostics
GmbH), fibrinogen, Clauss clotting assay (cobas Fibrinogen; Roche Diagnostics GmbH;
[Table 1 ]). The reference ranges declared by the manufacturer are comprised between 8.4 and
10.6 seconds for PT, 23.6 and 30.6 seconds for APTT, and between 1.9 and 4.1 g/L for
fibrinogen, respectively.
Table 1
Description of analyzers, reagents, and methods used for this study
Test
Analyzer
Reagent
Method
Reference range[a ]
PT
Roche cobas t 711
Cobas PT Rec (Lyophilized; ISI, 0.91)
Clotting assay with human recombinant thromboplastin as activator
8.4–10.6 s
PT
IL ACL TOP
HemosIL RecombiPlasTin (ISI, 0.99)
Clotting assay with human recombinant thromboplastin as activator
9.4–12.5 s
PT
Stago STA-R MAX
STA-NeoPTitimal (ISI, 1.00)
Clotting assay with rabbit brain thromboplastin as activator
11.7–15.3 s
APTT
Roche cobas t 711
Cobas aPTT
Clotting assay with ellagic acid as activator
23.6–30.6 s
APTT
IL ACL TOP
HemosIL SynthASil
Clotting assay with colloidal silica activator
25.1–36.5 s
APTT
Stago STA-R MAX
STA-Cephascreen
Clotting assay with polyphenolic activator
26.4–32.0 s
Fibrinogen
Roche cobas t 711
Cobas Fibrinogen (Lyoholized)
Clauss clotting assay
1.9–4.1 g/L
Fibrinogen
IL ACL TOP
HemosIL Fibrinogen-C XL
Clauss clotting assay
2.4–5.0 g/L
Fibrinogen
Stago STA-R MAX
STA-Liquid Fib
Clauss clotting assay
2.0–4.0 g/L
Abbreviations: APTT, activated partial thromboplastin time; ISI, international sensitivity
index; PT, prothrombin time.
a As quoted by manufacturers.
Lyophilized Reagents Reconstitution Studies
This preliminary aspect of our study was planned to evaluate accuracy and imprecision
of manual and automatic resuspension of the two methods based on cassettes containing
lyophilized material (i.e., PT and fibrinogen). More specifically, 10 PT empty cassettes
and 10 fibrinogen empty cassettes were weighted on a precision balance (AG135 Dual
Range; Mettler Toledo; linearity range: 101–0.0001 g; imprecision: ± 0.0003 g), and
were then loaded into the analyzer for automatic resuspension with distilled water
(i.e., 33 mL for PT and 14.4 mL for fibrinogen, respectively, as declared by the manufacturer).
Immediately after resuspension, the cassettes were unloaded, weighed on the same precision
balance, and the weight difference after and before automatic resuspension was finally
calculated for each cassette (1 mL of distilled water = 1 g). Successively, 10 PT
empty cassettes and 10 fibrinogen empty cassettes were also weighed on a precision
balance and manually resuspended by pipetting the nominal amount of distilled water,
as for automatic resuspension. The cassettes were then weighed on the same precision
balance and the weight difference after and before manual resuspension was finally
calculated for each cassette. The ensuing analysis encompassed the calculation of
accuracy (i.e., mean percent difference from nominal filling volume of the cassettes)
and imprecision (i.e., coefficient of variation; CV%) for both automatic and manual
resuspension.
Two automatically resuspended PT cassettes and two automatically resuspended fibrinogen
cassettes were then used for measuring PT and fibrinogen on 200 routine plasma samples,
randomly selected from those referred to the local laboratory for routine coagulation
testing. Test results were then compared with those obtained on the same set of plasma
samples using two other manually resuspended PT and fibrinogen cassettes. Data comparison
was performed using Spearman's correlation and Bland and Altman plots (mean values
and 95% confidence interval [95% CI]).
Imprecision Studies
This part of our study planned to evaluate the within-run, between-run, and total
imprecision of cobas t 711 analyzer, using automatically resuspended cassettes. Two plasma pools each for PT
and APTT (labeled as “low” and “high”) and three plasma pools for fibrinogen (labeled
as “low,” “medium,” and “high”) were prepared from plasma samples referred to the
local laboratory for routine coagulation testing. The pools were then divided into
11 matched plasma aliquots each. The first aliquot was used for within-run imprecision
studies, which were carried out by performing 20 consecutive measurements on the same
plasma aliquot. The remaining 10 plasma aliquots of each pool were frozen at −80°C;
one plasma aliquot of each of the seven pools was then thawed throughout each of the
10 following working days for analyzing PT, APTT, and fibrinogen. The within-run,
between-run, and total imprecision were finally expressed as CV%.
Linearity Studies
This part of our study planned to evaluate the linearity of cobas t 711 analyzer, using automatically resuspended cassettes. Two plasma pools each for PT,
APTT, and fibrinogen, displaying high and low values of these tests, were prepared
using plasma samples referred to the local laboratory for routine coagulation testing.
The “high” and “low” plasma pools were serially mixed at fixed ratios (10 + 0, 1 + 9,
8 + 2, 7 + 3, 6 + 4, 5 + 5, 4 + 6, 3 + 7, 2 + 8, 1 + 9, 0 + 10), to obtain scalar
values of all tests covering the most clinically significant ranges. The resulting
dilutions were then tested in duplicate on cobas t 711 analyzer; the mean values of the duplicate tests were calculated and the linearity
of PT, APTT, and fibrinogen were finally assessed with Pearson's correlation, by plotting
theoretical versus measured values.
Methods Comparison Studies
This part of our study planned to evaluate the comparability of PT, APTT, and fibrinogen
test results obtained using cobas t 711 analyzer versus those measured in paired plasma samples with Instrumentation Laboratory
ACL TOP 700 (Instrumentation Laboratory [IL]) and Stago STA-R MAX (Diagnostica Stago
SAS), as otherwise representing established coagulation systems. For this purpose,
120 routine plasma samples were randomly selected (40 from outpatients, 30 from emergency
department patients, and 40 from patients on warfarin therapy) and divided in three
identical aliquots, which were frozen at −80°C until measurement. All the three aliquots
of each plasma sample were then thawed during the same day; the first aliquot was
tested on cobas t 711 analyzer, the second on IL ACL TOP 700, and the third on STA-R MAX (description of
reagents is provided in [Table 1 ]). Comparison of data generated by the three different coagulation analyzers was
performed using Spearman's correlation and Passing and Bablok regression analysis.
Sample Collection and Ethical Committee Approval
All the samples used in this study were collected by straight needle venipuncture,
directly into evacuated blood tubes containing 0.105 mmol/L buffered sodium citrate
(Vacutest Kima). The plasma was separated by centrifugation at 1,300 × g for 15 minutes
at room temperature. The entire study was based on preexisting and anonymized samples
referred to the local laboratory for routine coagulation testing, and representing
excess material otherwise destined for discarding, so that patient-informed consent
was unnecessary. Test results obtained in this study were used only for this analytical
evaluation and were not reported. The study was approved by the local ethical committee
(University Hospital of Verona, Protocol n. 971CESC; July 7, 2016).
Results
Lyophilized Reagents Reconstitution Studies
The results of reagent reconstitution studies for PT and fibrinogen cassettes are
shown in [Table 2 ]. As predictable, the precision of the automatic resuspension was consistently better
than that of manual resuspension for both PT (respective imprecision 0.04 vs. 0.27%;
p < 0.001) and fibrinogen (0.09 vs. 0.26%; p < 0.001) cassettes. The difference between theoretical and measured filling volume
was comparable for both PT (automatic: −0.4 ± 0.1%; manual: 0.4 ± 0.3%) and fibrinogen
(automatic: −0.6 ± 0.1%; manual: 0.5 ± 0.3%) cassettes. The difference of values obtained
measuring 200 routine plasma samples with automatically or reconstituted reagent cassettes
was neither statistically nor clinically significant for both PT (r = 0.989 and p < 0.001; mean bias, −0.4% and 95% CI: −0.8% to −0.0%; p = 0.05) and fibrinogen (r = 0.985 and p < 0.001; mean bias: −0.8% and 95% CI: −2.8 to 1.1%; p = 0.408).
Table 2
Accuracy and imprecision of manual and automatic lyophilized cassettes resuspension
on Roche cobas t 711 analyzer (1 mL of distilled water = 1 g)
Automatic resuspension
Manual resuspension
Test
Theoretical filling weight[a ]
Filling weight[b ]
CV%
Difference vs. theoretical filling weight
Filling weight[b ]
CV%
Difference vs. theoretical filling weight
PT
33 g
32.86 ± 0.01 g
0.04%
−0.4 ± 0.1%
33.12 ± 0.09 g
0.27%
0.4 ± 0.3%
Fibrinogen
14.4 g
14.32 ± 0.01 g
0.09%
−0.6 ± 0.1%
14.47 ± 0.04 g
0.26%
0.5 ± 0.3%
Abbreviations: APTT, activated partial thromboplastin time; CV%, coefficient of variation;
PT, prothrombin time.
Notes: Data are calculated on 10 cassettes each of both PT and fibrinogen reagents.
Source: Data are shown as mean ± standard deviation (SD).
a As declared by the manufacturer.
b Weight difference of the cassettes after and before resuspension.
Imprecision Studies
The results of imprecision studies are shown in [Table 3 ]. Briefly, within-run (n = 20), between-run (n = 10), and total imprecision were 0.4 to 0.5%, 0.5 to 0.6%, and 0.7% for PT; 0.6
to 0.8%, 1.5 to 1.7%, and 1.7 to 1.8% for APTT; 0.8 to 1.7%, 1.7 to 2.7%, and 1.9
to 3.2% for fibrinogen, respectively.
Table 3
Results of imprecision studies on Roche cobas t 711 analyzer
Test
Within run (n = 20)
Between run (n = 10)
Total CV%
Values (mean ± SD)
CV%
Values (mean ± SD)
CV%
PT (s)
Low
7.99 ± 0.03
0.4%
7.97 ± 0.05
0.6%
0.7%
High
28.34 ± 0.14
0.5%
27.82 ± 0.13
0.5%
0.7%
APTT (s)
Low
27.07 ± 0.16
0.6%
25.77 ± 0.43
1.7%
1.8%
High
43.36 ± 0.33
0.8%
40.85 ± 0.63
1.5%
1.7%
Fibrinogen (g/L)
Low
1.22 ± 0.01
0.8%
1.21 ± 0.02
1.7%
1.9%
Medium
2.54 ± 0.04
1.7%
2.56 ± 0.05
2.0%
2.6%
High
6.01 ± 0.10
1.7%
6.06 ± 0.16
2.7%
3.2%
Abbreviations: APTT, activated partial thromboplastin time; CV%, coefficient of variation;
PT, prothrombin time; SD, standard deviation.
Linearity Studies
The linearity studies showed excellent performance of the three reagents tested, over
a clinically significant range of PT, APTT, and fibrinogen values. More specifically,
PT was found to be highly linear (r = 0.992; p < 0.001) between 7.6 and 47.3 seconds, APTT (r = 0.984; p < 0.001) between 24.5 and 131.7 seconds, and fibrinogen (r = 0.999; p < 0.001) between 0.08 and 7.48 g/L.
Methods Comparison Studies
The results of the method comparison studies are shown in [Table 4 ] and [Fig. 1 ]. The correlations of values (n = 120) between cobas t 711 and ACL TOP or STA-R MAX were 0.97 for PT, 0.88 and 0.81 for APTT, and 0.97 for fibrinogen,
respectively. In general, these correlations were similar of even better than those
between ACL TOP and STA-R MAX ([Table 4 ]). Due to the use of different reference values (and reagents) of both PT and APTT
across the three analyzers, a substantial difference of absolute values was unsurprisingly
observed for these tests, especially for PT (i.e., slopes comprised between 0.61 and
1.25, intercepts between −0.54 and 0.12), while a much better agreement was observed
for fibrinogen (slopes comprised between 0.90 and 0.96, intercepts between 0.0 and
0.39).
Fig. 1 Results of method comparison studies. The dotted lines are drawn at the 95% prediction
interval. APTT, activated partial thromboplastin time; PT, prothrombin time.
Table 4
Results of methods comparison studies
Test
Cobas t 711 vs. ACL TOP
Cobas t 711 vs. STA-R MAX
ACL TOP vs. STA-R MAX
PT
y = 0.78x −0.51
r = 0.97 (95% CI, 0.96–0.98; p < 0.001)
y = 0.61x + 0.12
r = 0.97 (95% CI, 0.9ss5–0.98; p < 0.001)
y = 1.25x −0.54
r = 0.98 (95% CI, 0.97–0.98; p < 0.001)
APTT
y = 1.47x − 13.39
r = 0.88 (95% CI, 0.83–0.91; p < 0.001)
y = 1.03x − 2.84
r = 0.81 ( 95% CI, 0.74–0.87; p < 0.001)
y = 0.97x + 2.75
r = 0.74 (95% CI, 0.65–0.81; p < 0.001)
Fibrinogen
y = 0.90x + 0.39
r = 0.97 (95% CI, 0.96–0.98; p < 0.001)
y = 0.94x + 0.02
r = 0.97 ( 95% CI, 0.96–0.98; p < 0.001)
y = 0.96x + 0.38
r = 0.95 (95% CI, 0.93–0.97; p < 0.001)
Abbreviations: 95% CI, 95% confidence interval; APTT, activated partial thromboplastin
time; PT, prothrombin time.
Discussion
The availability of fully automated, rapid, accurate, precise, and versatile laboratory
instrumentation has now become increasingly important for the efficient diagnostics
of hemostasis disorders. Compared with the many other marketed coagulation analyzers,
cobas t 711 analyzer presents several interesting features, such as full compatibility with total
laboratory automation and, especially, availability of lyophilized reagents in barcoded
cassettes, which can be automatically resuspended by the analyzer itself. This represents
several interesting advantages for the total quality of hemostasis testing, as it
ensures high on-board reagent capacity, up to 24 months unopened and up to 2 weeks
on-board stability of reagents; eliminates the inherent risk of manual lyophilized
reagent reconstitution; and improves walkaway time. Inside the analyzer, the reagents
are kept in a specific area, and are then automatically moved to the disposal section
once needed. This theoretical advantage seems to be coupled with excellent analytical
performance, at least for the three reagents/tests that we have assessed in this analytical
evaluation.
Overall, our data demonstrate that automatic resuspension is indeed more precise than,
and equally accurate as, manual reconstitution, as shown in [Table 2 ]. Notably, the imprecision of automatic resuspension of lyophilized reagents was
three- to sixfold lower than manual reconstitution. Albeit this finding is somehow
predictable, due to the virtually unavoidable intra- and, especially, interoperator
imprecision of manual pipetting;[11 ] our results clearly show that automatic resuspension would help improve consistency
and comparability of values generated after replacing empty vials of lyophilized reagents
of the same lot. Overall, the analytical imprecision of cobas t 711 was also found to be optimally low, as attested by total CV% of 0.7% for PT, 1.7
to 1.8% for APTT, and 1.9 to 3.2% for fibrinogen, respectively ([Table 3 ]). These data are aligned with, or even better than, those of other commercial coagulation
analyzers. For example, earlier published studies reported that between-run imprecision
was comprised between 3.0 and 3.1% for PT, 2.7 to 3.3% for APTT, and 3.4 to 6.5% for
fibrinogen on ACL TOP,[13 ] and total imprecision was comprised between 1.7 and 2.7% for PT, 0.9 and 2.2% for
APTT, and 1.8 to 3.8% for fibrinogen on Stago STA.[14 ] Nevertheless, linearity of cobas t 711 was excellent over clinically relevant ranges of PT, APTT, and fibrinogen values,
displaying correlation coefficients comprised between 0.994 and 0.999. It is noted
that linearity is not usually required in clinical laboratories for validating clotting-time
assays. Methods comparison studies revealed that results of PT, APTT, and fibrinogen
on cobas t 711 were globally aligned with those obtained using identical plasma samples on IL ACL
TOP 700 and Stago STA-R MAX ([Table 4 ]), with correlation coefficients always greater than 0.81. Predictably, better correlation
coefficients were indeed observed for PT (i.e., 0.97) and fibrinogen (i.e., 0.90 and
0.94), while the correlation of APTT values with those obtained on the other two analyzers
was less satisfactory (i.e., 0.81 and 0.88), though it was still better than that
between IL ACL TOP and Stago STA-R MAX. These findings are not really surprising,
as the APTT reagents of the three manufacturers are based on different contact activators
(i.e., ellagic acid for Roche, colloidal silica for IL, and polyphenols for Stago;
[Table 1 ]), thus mirroring recent evidence showing that harmonization in hemostasis testing
is still an unmet target, even for longstanding and widely used tests such as the
APTT.[15 ]
[16 ]
[17 ] The bias may also be explained by differences in the phospholipid sources and/or
concentration of different APTT reagents, the use of different mathematical algorithms
for detection of clot endpoint, the use of different detection methods between Stago
(mechanical endpoint) and IL ACL TOP and cobas t 711 (both optical endpoints), as well as by the possibility that some specimens might
have had lupus anticoagulant activity, with different potential sensitivity among
the three APTT reagents. Further study could hence be conducted to assess other aspects
of this new analyzer, including normal reference ranges and heparin therapeutic range,
sensitivity to factor deficiencies and lupus anticoagulants, responsiveness to nonheparin
anticoagulants, along with instruments comparability for APTT values greater than
60 seconds.
In conclusion, the results of this preliminary evaluation of PT, APTT, and fibrinogen
reagents on cobas t 711 analyzer demonstrate that this innovative coagulation instrumentation displays excellent
performance for routine use in clinical laboratories.
