Determination of heparin levels in blood with activated partial thromboplastin time by a piezoelectric quartz crystal sensor

Analytica Chimica Acta 432 (2001) 101–111

Determination of heparin levels in blood with activated partial thromboplastin time by a piezoelectric quartz crystal sensor Tzong-Jih Cheng a , Tsun-Mei Lin b , Tsui-Hsun Wu a , Hsien-Chang Chang a,∗ a

b

Institute of Biomedical Engineering, National Cheng Kung University, No. 1, Ta Hsueh Road, Tainan 70101, Taiwan, ROC Department of Medical Technology, National Cheng Kung University, Tainan 70101, Taiwan, ROC

Received 25 August 2000; received in revised form 17 November 2000; accepted 24 November 2000

Abstract A piezoelectric quartz crystal (PQC) sensor, possessing a sensitive frequency response to viscoelastic variations of substance loading on an electrode surface, was used to determine both whole blood activated partial thromboplastin time (WBaPTT) and plasma activated partial thromboplastin time (PLaPTT) induced by anticoagulant heparin. The PQC sensor results showed a linear relationship between WBaPTT (or PLaPTT) and heparin levels in clinically relevant concentrations (0–0.4 unit/ml). Mean of individual R2 (= 0.9491) for a regressive curve between WBaPTT and heparin concentrations was adequately shown. The PQC method can be used to evaluate the effect of heparin through determination of WBaPTT owing to comparable sensitivity (P < 0.01) with that of aPTT by optical coagulometry (OCaPTT). Furthermore, we found the results of WBaPTT with various heparin concentrations (n = 9) to be well correlated with those of OCaPTT (correlation coefficient = 0.9441). Linear calibration plots were extended into 3 units/ml of heparin in PLaPTT and WBaPTT. It was suggested that the PQC method will provide a more convenient operation, may be useful in clinical situations for rapid monitoring heparin therapy using small volume (20 ␮l) of whole blood specimens. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Heparin; Whole blood; Activated partial thromboplastin time (aPTT); Piezoelectric quartz crystal (PQC)

1. Introduction Heparin is an effective antithrombotic agent that reduces morbidity and mortality among patients having deep vein thrombosis and pulmonary embolism. Patients are administered heparin to minimize the risks associated with surgical procedures. Excessive heparinization has been shown to increase the frequency of thrombosis, which may lead to serious hemorrhagic complication [1]. Such a high risk can ∗ Corresponding author. Tel.: +886-6-2757575/ext. 63426; fax: +886-6-2760697. E-mail address: [email protected] (H.-C. Chang).

be especially dangerous for patients with previously silent lesions (e.g. carcinoma, gastrointestinal ulcers, and trauma) that may be revealed for the first time during heparin therapy. The physician must adjust the dosage of anticoagulant for these patients, to maintain sufficient level to prevent thrombosis and avoid bleeding [2]. For these reasons, laboratory monitoring of heparin therapy is very desirable to assess the effectiveness of treatments and to reduce the risk of hemorrhage [3]. Currently, there is no standard therapeutic approach to heparin therapy. Indeed, the level of heparin regarded as safe and effective depends very much on the condition of an individual patient. Clinicians must evaluate each patient’s hemostatic ranking

0003-2670/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 0 ) 0 1 3 4 6 - 5

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before, during, and after the therapeutic procedure for ensuring that patients safely receive the correct dose of anticoagulant and antithrombotic agents. The activated partial thromboplastin time (aPTT) and the activated clotting time (ACT) are the most widely used measures to monitor heparin effect. Accurate assessment of the intensity of heparin is essential because of the high variability in heparin responses among individuals. It is essential to establish a frequent monitoring system for heparin therapy. Coagulation testing plays a central role in providing important information for the physician to minimize patient risk. In clinical situations, it is critical to get rapid verification of adequate anticoagulant therapy. Obtaining laboratory data of aPTT has limitations, however, due to time consuming to receive analytical results. A recent study demonstrated the use of on-site coagulation assay to provide rapid and accurate results in guiding specific therapy in cardiac patients [4,5]. This idea is supported by the practicability of patient self-testing of oral anticoagulant therapy by a portable whole blood monitor [6]. Therefore, a rapid and convenient method of monitoring low levels of heparin is essential. On the other hand, the ACT can be erroneously shortened by procoagulant activity of platelet membrane fragments in the presence of heparin [7]. The measurement of ACT can only be carried out in a relatively high level of heparin (1–10 units/ml) contained in a blood specimen. However, in the case of postsurgical therapy, the anticoagulant range is generally controlled from 0 to 0.4 unit/ml. It is required to develop a new analyzer suitable for more wide range of heparin concentration assay. Many new and improved coagulation analyzers (devices) have emerged on the market in recent years [8–12]. Most of these coagulation analyzers are quite heavily weighted (from 1.9 to 5.44 kg). The major disadvantage of these instruments is that large volume of blood sample is required. Currently, only two handheld coagulation monitors (Coagucheck, Boehriger Mannheim Corp. and Protime, International Technidyne Corp.) have received USA Federal Drug & Food Administration (FDA) approved for home-care use. They are still unpopular because of the expensive prices of single-use test strip (∼US$ 10 per test strip). Otherwise, handheld prototypes of small capillary coagulation analyzers have also recently been developed. They are based on blood or plasma draw into a

small capillary with coagulation detected using either a laser (Biotrack, Ciba-Corning) or pressure sensing system (Nyco Med). However, these devices are quite complex. Thus, it is necessary to develop new methods and devices for measurement of blood coagulation. They should fit some criteria to be sufficiently inexpensive, timely, efficient, convenient, durable, and reliable for point-of-care use by untrained individuals. It will be clear for researchers of medical devices that any methodology or instrument that can facilitate rapid determination of coagulation function, and can reduce the amount of whole blood required for heparin therapy in the future would appear to offer distinct advantages in these clinical situations. Recently, an increasing amount of attention has been paid to the technology of the PQC sensors in many fields such as environmental inspection, food industry, biotechnology, and clinical diagnosis [13,14]. In many studies, the dependence of the PQC frequency response on the interface viscosity was used to monitor various biochemical reactions. In hematological studies, the PQC sensor has also been applied to determine fibrinogen concentration [15] and activities of blood coagulation factor VIII and IX [16]. It was also reported that the PQC sensor was used to study fibrinolysis [17] and erythrocyte aggregation [18]. The changing viscosity of an absorbed fibrin clot, as it dissolved after the addition of plasminogen activator, was monitored by a PQC sensor [19]. We also reported on the application of PQC sensor in the determination of plasma prothrombin time (PT) and whole blood recalcification time [20]. Recently, we published a communication paper describing our investigation of clotting time in whole blood containing anticoagulant heparin by the PQC sensors [21]. Our results showed that whole blood clotting time obtained with PQC sensors had a good linear relationship for heparin concentrations from 0 to 1.0 unit/ml and agreed with plasma aPTT results measured by optical coagulometry. In this study, we keep on the study of PQC sensors in advanced clinical hematology. Monitoring the effect of anticoagulants on a blood coagulation system with PQC sensors is one of our study topics. The PQC sensor is an electromechanical transducer that converts electrical energy into mechanical energy by the piezoelectric effect. It is employed for thickness monitoring in a thin film deposition system, since the resonance frequency depends on the amount of

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deposited mass on the crystal. The transducer is also called a quartz crystal microbalance (QCM), which is widely used in the biosensing field. The PQC sensor consists of two electrodes attached to both sides of a thin disk of AT-cut quartz. A number of factors can influence the oscillation frequency, for example, the thickness, density, shear modulus of the quartz, and the physical parameters of the adjacent media (density and viscosity of air or liquid). Changes in the resonance frequency (1f) are simply related to the mass (1m) accumulated on the crystal in air phase by the following equation [22]: 1f =

−2f 2 1m A(ρq µq )1/2

(1)

where ρ q and µq are the density and the shear modulus of quartz, respectively, f the fundamental resonance frequency of the quartz, and A the reactive area of the coated crystal. Since it has been shown that 1f depends upon the viscosity and density of the contacting solution, Eq. (2) has been derived [23]:   ρl ηl 1/2 (2) 1f = −f 3/2 πρq µq

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with a basic resonance frequency of 10 MHz. The crystal consisted of an 8 mm diameter, quartz wafer on which a pair of gold electrodes, 4.5 mm in diameter, was attached. The PQC sensor was then set up in a home made oscillating circuit [20], which was connected with a universal counter (Hewlett-Packard 53131A) and transferred to a computer via a GPIB card for the acquisition of the frequency data. This arrangement enabled an evaluation of changes in density and viscosity of plasma and whole blood during the coagulating process. Data recording and analyses were achieved with a program constructed by the HP-VEE software. This PQC system can provide real-time monitoring information of blood coagulation, which is an advantage not yet provided by a commercial coagulometer. The optical coagulometer (OC), a STAGO-ST4 system (Diagnostics Stago, France), was used to compare the aPTT (OCaPTT) results of the plasma specimen with the plasma aPTT (PLaPTT) and whole blood aPTT (WBaPTT) results measured by a PQC sensor. Heparin was purchased from Green Cross, CaCl2 was purchased from Sigma Co. and activated partial thromboplastin (aPT) reagent was obtained from Diagnostics Stago Co.

The frequency is proportional to (ρl ηl )1/2 , where ρl and ηl are the absolute density and the viscosity of the liquid solution, respectively. The purposes of this study are: (1) to evaluate the possibility and performance of the PQC sensor as a coagulometer for aPTT examinations for plasma and whole blood containing low heparin levels (0–0.4 unit/ml) and (2) to investigate whether whole blood and plasma aPTT determined with a PQC sensor are comparable to plasma aPTT examined by an optical coagulometer (OC). The technique presented here may be useful for engineers and physicians attempting to develop a convenient blood coagulometer for anticoagulant assay and may help to improve the safety and life-quality of patients using anticoagulant therapy.

2.2. Preparation of blood sample

2. Materials and methods

2.3. Optical coagulometry

2.1. Apparatus and materials

The optical experiment was carried out in a reaction cuvette with a steel bead vibrating within an alternating magnetic field. An amount of 100 ␮l of citrated PPP specimen having various concentrations of

The piezoelectric quartz crystal (PQC; TAI TEIN, Taiwan, ROC) sensor used in this work was AT-cut

Blood samples were collected from nine healthy male volunteers who were between 22 and 28 years of age. No volunteer was under medication that might affect the blood coagulation time. Whole blood was collected in two tubes containing one-tenth volume of 3.8% sodium citrate. One tube of sodium citrate–anticoagulated blood mixture was used for WBaPTT, while another was centrifuged at 2500 rpm for 10 min to obtain platelet poor plasma (PPP) for the use in the OCaPTT and PLaPTT tests. Each specimen was spiked with different concentrations of a heparin stock solution to prepare the various heparin levels in patient’s blood before testing. The heparin levels performed in this study were specified at 0–0.4 unit/ml.

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heparin solution were poured into the reaction cuvette, and 100 ␮l of aPT reagent solution was then added and prior warmed to 37◦ C for 180 s. The time was counted when 100 ␮l of 25 mM CaCl2 solution was added into the cuvette. When the steel bead vibrations stopped due to fibrin formation, the time value named OCaPTT was recorded. 2.4. Coagulation test by PQC system Citrated blood samples were kept on ice and transported to the laboratory for immediate processing. A 45 ␮l sample of the produced mixture, as well as 5 ␮l of various heparin solutions, was put into a polypropylene test tube for 60 s, and then 50 ␮l of aPT reagent was added and incubated for 180 s at room temperature. A 50 ␮l sample of 25 mM of CaCl2 was finally added to induce the coagulation. After blending for 30 s, 20 ␮l of the mixture was taken and dripped onto the surface of the PQC sensor. The gate time for the oscillation frequency measurement was 1000 s at a sampling interval of one second. The measurement was achieved in a chamber controlled at 37◦ C and approximately 90% relative humidity. 2.5. Statistics Results for individual samples are given as mean and standard deviation (S.D.). Data analyses were performed by Student’s paired t-test, bias analysis, correlation and linear regression with the use of a microcomputer software package (Excel 97, Microsoft Co.). To assess between-method variability of aPTT, individual whole blood aPTT measurements were compared using bias analysis. A P < 0.05 was considered statistically significant.

3. Results

Fig. 1. Frequency response of a PQC sensor with plasma coagulation on electrode surface. A PQC sensor was set in an oscillating circuit and used to monitor the time course of oscillating frequency response to the case of plasma coagulation (real line) and non-coagulation (dashed line). The arrow shows the point of 20 ␮l of plasma sample dropped onto sensor.

sensors with time had been confirmed in our previous study [20] and its related literatures. When 20 ␮l of the samples were separately dripped onto the sensor (arrow mark), the frequency rapidly decreased about 8 kHz. This change could be attributed to the mass loading caused by the effects of the viscosity and density of liquid on the interfacial layer of the quartz crystal. In the case of no activator-containing sample, the frequency kept at a constant value over the observation time (1000 s). Whereas, the frequency would begin to gradually decrease to a relatively low value at approximately 270 s following that the sample mixed with activator reagent was dripped. The descending phenomena lasted for 200 s and then turned to a steady state. From such a frequency response, we could conclude that adding of the aPT reagent and calcium ions induced the coagulation reaction of blood. In fact, the shortened time that the system decreased in frequency response was extremely dependent on the coagulation factors activities present in the sample [16,20,21]. The results confirmed that the system could be used for real-time coagulation monitoring and coagulation time determination.

3.1. Behavior of blood coagulation monitored by PQC sensor

3.2. Determination of PLaPTT and WBaPTT

The behaviors of real-time monitoring in the coagulation reaction of the plasma sample with (continuous line) and without (dashed line) the coagulation activator were shown in Fig. 1. The stability of PQC

Fig. 2 shows the results of typical PQC responses for the coagulation of aPT reagent-added whole blood (WB) samples containing 0 and 0.4 unit/ml of heparin. In a whole blood sample containing no heparin (curve

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statistical ratio was different from our previous report [20] that 90% of 1f was the best regressive method for determining the prothrombin time (PT) of plasma. In this experiment, possible effects of the additives (aPT reagent and heparin solution) on the coagulation mechanism are considered important in evaluating such a different result. The ratio value in 99% of 1f was also used to compare with the corresponding results of PLaPTT obtained by a PQC sensor. 3.3. Comparing the aPTT results among different methods

Fig. 2. Oscillating frequency responses of PQC sensors in whole blood coagulation reaction. Whole blood contained: (a) 0 and (b) 0.4 unit/ml of heparin in the blood sample, respectively. The arrow mark shows the time point as the sample was dropped onto sensor. 1f is a frequency change of PQC sensor from primary to final state that coagulation of blood specimen was accomplished. The aPTT was determined by the ratio (99%) of 1f shown in curve (b).

(a), Fig. 2), the frequency only declined slightly in a period of about 50 s and then went quickly down to a constant value within the next 20 s. However, such a prior slight declination in frequency continued over 120 s and a longer time was required to reach a relatively low steady level for 0.4 unit/ml of heparin (curve (b), Fig. 2). Therefore, the duration of blood coagulation, determined by the time required for the frequency to reach a steady state, would be extremely prolonged owing to the 0.4 unit/ml of heparin presence in blood sample. The computer program was used to determine the end point of WBaPTT by the PQC method in this study as previously described [20]. A scheme for calculating the value of aPTT was obtained from the PQC sensor response curve. The ratios corresponding to 90, 95, 99 and 99.5% of the total frequency change (1f) were statistically calculated from data of nine volunteers with various heparin concentrations and the means of regression (R2 ) were 0.536, 0.871, 0.950 and 0.892, respectively. The WBaPTT determined at 99% of 1f showed the best R2 value in linear regression. The

The aPT reagent-added plasma sample from one of the volunteer (subject 1) was prepared and separately monitored by PQC and optical methods to make a comparison between the results of WBaPTT. Each of the aPTT results based on the three methods was plotted together against the concentrations of heparin to make an individual regression analysis. As shown in Fig. 3, all the regressive results of PLaPTT, WBaPTT and OCaPTT data implied good linear relationships in regions of the adjusted heparin concentration. Based on the above observation, we compared the regression results of the remaining eight volunteers. As summarized in Table 1, all of the regression results showed a good linear relationship with high mean values of R2 (0.9491, 0.9699 and 0.9822 for the data of WBaPTT, PLaPTT and OCaPTT, respectively). In the regression equation, aPTT = aH + b, (Table 1), the slope value of a, represents the sensitivity to heparin for the monitoring methods and the intercept b is the substantial aPTT value of the blood specimen without heparin. From our data, it was found that the mean b value of WBaPTT or PLaPTT is two or three folds larger than that of OCaPTT. All of a mean values for three aPTTs are similar. It is proposed that using WBaPTT and PLaPTT to monitor heparin therapy may offer comparable sensitivity with the other method (OCaPTT) used of optical coagulometer and utilized with plasma. Furthermore, Utilization of WBaPTT that directly makes use of whole blood is more convenient than PLaPTT in processes. 3.4. Regressive analysis of the WBaPTT result The relationship between heparin concentrations and the corresponding aPTT values of the nine volun-

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Fig. 3. Calibration curves of WBaPTT, PLaPTT and OCaPTT to heparin concentrations. The blood specimen was sampled from a normal volunteer (no. 1 in Table 1). Triangle mark curve is WBaPTT; open-circle mark is PLaPTT and fill-up circle mark is OCaPTT.

teer samples are shown in Fig. 4. The regression results of PLaPTT and WBaPTT were carried out and the R2 values were 0.7038 and 0.6789, respectively (shown as Fig. 4a and b). The R2 values are somehow less than the value of 0.80 reported recently, as measured by a portable whole blood coagulation monitor [24]. A relatively wide 95% confidence

interval marked with scattered points of WBaPTT and PLaPTT are shown to judge the divergent results of these three methods. We found that the interval was kept within a narrow margin for the OCaPTT method, even at a high dose of heparin. By comparison, a more significant discrepancy appeared in higher heparin-dosed samples reflected in the PLaPTT and

Table 1 Comparison of heparin responsiveness of whole blood activated partial thromboplastin time (WBaPTT), plasma activated partial thromboplastin time (PLaPTT) with the PQC and plasma activated partial thromboplastin time (OCaPTT) with the OC in specimens from normal subjects spiked in vitro with heparina Normal volunteers

Regression line WBaPTT

PLaPTT

OCaPTT

ab

bb

R2

ab

bb

R2

ab

bb

R2

1 2 3 4 5 6 7 8 9

154.61 182.25 267.50 175.75 147.75 295.43 235.75 324.25 168.25

93.52 95.93 135.15 100.48 85.28 93.80 94.68 88.13 80.33

0.9836 0.9002 0.9833 0.8866 0.9492 0.9099 0.9974 0.9706 0.9613

148.50 270.50 308.50 162.50 141.50 176.00 246.00 306.50 202.00

81.13 73.83 97.13 80.03 76.58 76.80 94.70 75.43 55.90

0.9395 0.9457 0.9126 0.9930 0.9688 0.9932 0.9912 0.9867 0.9984

227.45 255.50 416.05 254.25 179.00 300.10 171.25 241.20 157.35

32.22 36.54 33.89 27.34 31.73 29.15 33.69 34.88 30.60

0.9916 0.9832 0.9972 0.9197 0.9926 0.9779 0.9974 0.9845 0.9956

Mean S.D. C.V.

216.84 65.70 0.30

96.37 15.76 0.16

0.9491 0.0405 0.0427

218.00 66.47 0.30

79.06 12.07 0.15

0.9699 0.0305 0.0314

244.68 79.29 0.32

32.23 2.89 0.09

0.9822 0.0244 0.0248

a

Linear regression and correlation coefficients (R2 ) values are listed. aPTT = aH + b. H: heparin concentration; WBaPTT and PLaPTT were measured with PQC. OCaPTT was measured with a conventional, semiautomated coagulometer in citrated plasma. b

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Fig. 5. Correlation between (a) PLaPTT or (b) WBaPTT obtained with PQC sensors and OCaPTT obtained with an optical coagulometer. Data were collected from nine normal volunteers. Heparin concentrations were adjusted from 0 to 0.4 unit/ml. Fig. 4. Relationship between heparin concentrations and the values of aPTT: (a) PLaPTT; (b) WBaPTT with PQC sensor as well as; (c) OCaPTT with OC. The regression relationships and the corresponding intervals of 95% confidence were shown by solid lines and dotted lines, respectively.

WBaPTT results. A low correlation exists among the nine volunteers measured by the PQC sensor methods. Therefore, a general and reliable calibration curve, which can be used as a diagnostic standard for clinical applications, is hard to be established. 3.5. Evaluation of the usefulness of the PQC method Despotis et al. had developed and employed as a home-care coagulometer to assess the anticoagulant effect of plasma specimen containing heparin from 0 to 0.4 unit/ml in 1995 [24]. According to their results,

a linear relationship (R 2 = 0.79) between WBaPTT and heparin concentration had been demonstrated. In our results, the OCaPTT value possessed a very good linear relationship with heparin concentration (mean of R2 is 0.9822) as shown in Table 1. We compared the results obtained by OCaPTT with the results of a PQC-based measurement. As shown in Fig. 5a and b, both WBaPTT and PLaPTT exhibited a good relationship with OCaPTT (R2 are 0.8429 and 0.7759, respectively). The assay results suggested that a PQC sensor had similar performance as a coagulation test for whole blood or plasma. Furthermore, we individually summarized the statistical analysis of correlation for the two PQC results based on OC according to each subject. From the data, the values of the mean correlation are 0.9441 and 0.9718 (shown in Table 2) for the cases of WBaPTT and PLaPTT, respectively.

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Table 2 Correlations among methods of aPTT determination Subjects

WBaPTT/OCaPTT

PLaPTT/OCaPTT

1 2 3 4 5 6 7 8 9

0.9984 0.9374 0.9902 0.8256 0.9824 0.8267 0.9866 0.9616 0.9883

0.9911 0.9412 0.9682 0.9529 0.9853 0.9810 0.9590 0.9728 0.9947

Mean S.D.

0.9441 0.0694

0.9718 0.0181

This indicated that the two PQC-based methods have a high similarity in performance with traditional coagulometry. 3.6. Evaluation of PQC method used in higher concentrations of heparin Fig. 6a presents calibration curves of heparin in higher concentration range (larger than 1 unit/ml) obtained with three different methods. There are only three conditions (rhombus mark) were carried out for OCaPTT because of aPTT shown to be constant as heparin concentration was over 1 unit/ml. Both PLaPTT and WBaPTT were proportionately correlated with heparin doses within 3 units/ml, but they were significantly distinct as heparin doses in excess of 3 units/ml. The WBaPTT reached saturation, but PLaPTT increased rapidly as the heparin concentration was larger than 3 units/ml (Fig. 6a). This finding could be attributed to erythrocyte accelerated the coagulation time of whole blood to be saturated. Fig. 6b is a zoom-in diagram of Fig. 6a. The means of calibration curves shows that OCaPTT exhibit a poor performance, while PLaPTT and WBaPTT perform adequate (both values of R2 are larger than 0.98) within 3 units/ml of heparin.

4. Discussion Plasma aPTT is the most widely used method for laboratory monitoring of heparin levels in blood. In a clinical setting, the risk of venous thromboembolism

Fig. 6. Calibration for higher concentration of heparin utilizing WBaPTT, PLaPTT and OCaPTT. Diagram (b) is a zoom-in diagram of diagram (a) within 3 units/ml of heparin. Correlation coefficients are 1.0 and 0.9892 for WBaPTT and PLaPTT, respectively. OCaPTT is shown as rhombus, PLaPTT is shown as square and WBaPTT is shown as circular.

can be reduced when heparin is used within aPTT ratio (patient: control) of 1.5–2.5, which is equivalent to 0.2–0.4 unit/ml heparin by protamine titration or 0.35–0.7 unit/ml by anti-Xa assay [1]. However, administration of heparin causes highly variable anticoagulation responses among individuals. For the safety requirement of accurate heparin assay in such a narrow therapy range, a more sensitive and rapid method for aPTT determination is prospected. In this report, we proposed measuring WBaPTT values in whole blood by a portable PQC device that could be considered as a potential method for heparin monitoring in the future. The results in this study indicate that the correlation between WBaPTT (R 2 = 0.6789) or PLaPTT (R 2 = 0.7038) and the heparin concentrations were fairly good (Fig. 4) and similar to other reports [24,25]. Furthermore, individual regressive curves of nine volunteers appeared to be good and responsive

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of whole blood and plasma aPTT with PQC and plasma aPTT with OC by increasing concentrations of heparin (Table 1). The difference of regression intercepts between OCaPTT and PLaPTT was significant (P < 0.001), but insignificant (P = 0.33) in regression slopes. In addition, the regression slope of WBaPTT was insignificantly different compared with both PLaPTT (P = 0.95) and OCaPTT (P = 0.29). Moreover, the difference of regression intercepts of WBaPTT was significant (P < 0.001) compared to OCaPTT. Inconsistency in regression intercepts of the PQC sensor and OC system (Fig. 3) may be resulted from the differences in sensing principles. The PQC sensor possesses as a pseudo-static measurement of coagulation time [20], while the OC system was based on a dynamic reaction condition [21]. Means of individual correlation coefficient between the PQC system and an optical STAGO coagulometer were very adequate (0.94 for WBaPTT and 0.97 for PLaPTT, Table 2). The results had a good agreement with the previous studies that demonstrated linear relationships between whole blood aPTT and in vitro heparin concentration obtained with protamine titration [25] and a Biotrack 512 portable coagulometer [24]. Additionally, the correlation coefficients of PLaPTT and WBaPTT, which relate to OCaPTT for combined data (R 2 = 0.7759 for PLaPTT and R 2 = 0.8429 for WBaPTT, Fig. 5), also supported the utilization of a PQC system in place of popular optical coagulometry systems used in clinical laboratories. This is the first report to conform that the PQC sensor provided another available method (device) for heparin assay in blood in clinical laboratory. Moreover, the PQC method based on viscoelastic sensing has not reported problems [9,10] regarding influence by sunlight and interference from electromagnetic noise in photometry and electromagnetic coagulation analyzers, respectively. The results also implied that the PQC sensor might provide an alternative potential advantage for next generation products. PQC has a wider calibration curve of heparin (0–3 units/ml) according to WBaPTT or PLaPTT than OCaPTT obtained with optical coagulometer (STAGO system). Broader calibration range of heparin by PQC sensors will be a great benefit not only for analytical assessment, but also for clinical applications. This detection range obtained with PQC sensor is sufficient for low heparin levels (0–0.4 unit/ml) administration

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such as post-surgical therapy, critical care procedure as well as hemodialysis, and has a potential to use in moderate heparin levels (0–3 units/ml) administration such as catheter laboratory procedure, electrophysiology laboratory procedure, interventional cardiology and extracorporeal membrane oxygenation. For other commercial coagulation analyzers (e.g. Hemochron, TAS, CoaguCheck, and CL8), the detection range of heparin concentrations obtained with whole blood aPTT is only from 0 to 1.5 units/ml [10]. Results indicated that this developing prototype of coagulation analyzer based on PQC sensors have a better characteristic in detection range of high-doses heparin. The speed of assay (80–600 s), a wide measurable range of heparin concentration (from 0 to 3.0 units/ml) and convenience through utility of whole blood or plasma make PQC sensor particularly attractive as a point-of-care test method. In clinical situations, rapid verification of adequate anticoagulation is critical. Classical laboratory aPTT examined by OC has limitations due to the longer turn-round time required to transport and process the samples. Use of whole blood to identify coagulation factor deficiencies can circumvent some of the drawbacks associated with traditional laboratory plasma prothrombin time (PT) and aPTT determinations as well as reduce operative expenditures. Therefore, a whole blood sample is an essential requirement for development of coagulometry in home-care and monitoring long-term oral anticoagulant therapy. In our recent report, we had utilized whole blood clotting time examined by the PQC sensor to assay heparin level [21]. However, due to the intensely slow coagulation time, it was not satisfactory for clinical or home-care applications. In this report, the WBaPTT determination by adding aPT reagent, requiring less than 300 s in clinically applied doses of heparin (0–0.4 unit/ml). It is indicated that this PQC method may be satisfactory requirements for clinical or home-care applications. Many laboratory clotting tests in the prior art based on the phenomenon of measuring an endpoint which is a change of phase when a test sample changes from a liquid to a coagulated form. The clotting endpoint is physically detected by such turbidity measurements through light scattering and magnetic particle oscillation. These laboratory instruments are relatively large because of the complex technology and require

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large volume of blood samples are also usually required. The smaller blood sample volume will be demanded for the development and improvement of on-site instruments and home-care analyzers in the future [26]. Sample volumes required of marketable analyzer usually are more than 45–300 ␮l [8–10]. It was demonstrated that only a 20 ␮l whole blood sample required for testing by the PQC sensor. It must be a useful coagulometer for long-term or home-care anticoagulant therapy monitoring for patients requiring repetitive testing over short periods of time to minimize blood loss. Additionally, traditional hematological studies and coagulation tests were based on end-point methods for fibrin formation. The dynamic response of physical properties for the samples during the whole coagulation reaction was seldom investigated. Coagulation monitoring based on a PQC sensor can also provide capability for real-time measurements of viscoelastic characteristics of blood during the coagulation process. High price of gold electrodes and inconvenient to handle are the major concernments for PQC as a candidate coagulometer of commercial product. The selling price of a PQC sensor with silver electrodes is somewhere less than US$ 0.5 that is only one thirtieth of cost to gold-electrodes PQC sensor. Moreover, the PQC sensor with silver electrodes showed a feasible element for coagulation monitoring. It was shown no difference compared to gold electrode on PQC sensor based on our experience (data not shown). The inconvenience pertains to handling of fragile PQC sensor has been resolved by engineering technology. Currently, at least one easy-handle PQC kits (p-sensor 2000 chip, ANT Technology Co., Ltd., Taiwan) are available in market. Every kit is constructed of a friendly card-set and a PQC sensor. According to brief surveys of commercial products, the PQC sensor shows a potential to be a candidate of coagulation analyzer for heparin assay in the future.

5. Conclusion A new method has been presented in this study to determine the value of aPTT for heparin dosed whole blood and plasma. The PQC sensor could provide effective measurement for real-time monitoring of heparin effects on blood coagulation time. During

measurements, samples were treated in a pseudo-static condition and the results showed good agreement with traditional optical coagulometry. Thus, values of WBaPTT and PLaPTT obtained from the PQC sensor were confirmed to be accurate and rapid in tracking heparin effects. It might also be a hopeful technique enabling physicians make safe and rapid decisions during anticoagulant therapy. Normal subjects were tested in this study and the results might not fully reflect the patients’ condition of coagulation. This research has made a step towards to develop an effective approach for integrating PQC sensors to be a candidate for commercial development for clinical hematological laboratory. Exploration of the possible applications of PQC in ex vivo test of post-surgery or long-term oral anticoagulant therapy will be the direction of our future studies.

Acknowledgements We gratefully acknowledge funding from the National Science Council of Republic of China (Taiwan) under contract no. NSC 88-2213-E006-017. References [1] M.N. Levine, J. Hirsch, in: V. Fuster, M. Verstraete (Eds.), Thrombosis in Cardiovascular Disorders, WB Saunders, Philadelphia, 1992, p. 515. [2] R.D. Hull, V.V. Kakkar, G.E. Raskob, in: V. Fuster, M. Verstraete (Eds.), Thrombosis in Cardiovascular Disorders, WB Saunders, Philadelphia, 1992, p. 451. [3] J. Hirsh, E.W. Salzman, V.J. Marder, in: R.C. Colman, J. Hirsh, V.J. Marder (Eds.), Hemostasis and Thrombosis: Basic Principles and Clinical Practices, 3rd Edition, Philadelphia, Lippincott, 1994, p. 1346. [4] G.J. Depotis, S.A. Santoro, E. Spitznagel, Anesthesiology 80 (1994) 3338. [5] G.J. Depotis, S.A. Santoro, E. Spitznagel, J. Thorac. Cardiovasc. Surg. 107 (1994) 271. [6] J.M. Hasenkam, L. Knudsen, H. Kimose, H. Gronnesby, J. Attermann, N.T. Andersen, H.K. Pilegaar, Throm. Res. 85 (1997) 77. [7] A.P. Bode, L. Erick, Am. J. Clin. Pathol. 91 (1989) 430. [8] Point-of-Care Analyzers, Clinical Laboratory, in: Health Product Comparison System, ECRI, April 1998, pp. 1–39. [9] Coagulation Analyzer, Automated Semiautomated, in: Health Product Comparison System, ECRI, June 1998, pp. 1–51. [10] Coagulation Analyzers, Whole Blood, in: Health Product Comparison System, ECRI, June 1998, pp. 1–24.

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