Diagnostic analysis of lupus anticoagulant using clot waveform analysis in activated partial thromboplastin time prolonged cases: A retrospective analysis

Abstract Background and Aims Hemophilia was diagnosed in precedence research of clot waveform analysis (CWA) using the activated partial thromboplastin time (APTT). In patients with antiphospholipid syndrome (APS), lupus anticoagulant (LA) causes an increase in APTT, suggesting that the waveform would probably be distorted. Therefore, we evaluated using clinical samples. CWA may be useful low cost for clinical detection of LA. We assessed the clinical value of CWA for detection of LA and coagulation using clinical blood samples collected from patients with a prolonged APTT. Methods We used patient samples inspected between April 2011 and March 2013 in Yamagata University Hospital. CWA was performed using the ACL TOP coagulation analyzer, and the associated software program was used to calculate APTT clotting endpoints. An atypical peak was defined as a derivative plot that did not conform to the normal S‐shaped clot reaction curve. Results In total, 162 patients, including 66 men and 96 women, with an average age of 46 years (range: 24‐89 years) were included. We also collected control samples from unmatched healthy donors. All 162 patients were divided according to medication history or condition into the following five groups: heparin (n = 20), warfarin (n = 23), hepatic dysfunction (n = 13), normal (n = 20), and LA‐positive antiphospholipid syndrome (APS; n = 86). Twenty healthy individuals were included as controls. Eighty patients had an atypical peak. Among all, 78 patients (90.6%) were LA‐positive, and 2 patients (2.5%) were treated with warfarin. The remaining two patients had prothrombin time international normalized ratios (PT‐INR) >4.0 while taking warfarin. Those who were APS LA positive with thrombosis and without thrombosis had split the reaction of clotting time, deceleration/acceleration time (D/A) ratio of 2.36 (1.99,3.24) vs 2.34 (2.04,2.86), respectively. Conclusion The significant atypical peak and D/A ratio extension may be explained by the clotting waveforms observed specifically in patients with LA‐positive APS.

IgG or IgM antibodies, or anti-β2-glycoprotein I antibodies. 2 LA is measured using the dilute Russell's viper venom time (dRVVT) test or the STACLOT LA assay, which is based on the activated partial thromboplastin time (APTT). Both tests require expensive equipment and reagents. In contrast, a less expensive APTT method is used commonly at many hospitals. 2,3 Recently, the development of a fully automated coagulationmeasuring device, the ACL TOP (Instrumentation Laboratory: IL, Lexington, KY, USA) and the associated software program enabled clinicians to conduct a waveform analysis of patients with coagulation disorders. Waveform analysis is generally low cost, and a lot of information can be obtained by from APTT.
In this analysis, the optical solidification reaction waveform curve (ie, clotting curve) can be visualized using absorbance measurements. [4][5][6][7][8][9] Complex waveform analyses, which can be performed using ACL TOP software, have been used to evaluate the prognoses of patients with disseminated intravascular coagulation (DIC) and hemophilia. [10][11][12][13][14][15][16][17] In patients with APS, LA induces abnormal extension of the APTT, which suggests that the waveform would probably be distorted. Therefore, clot waveform analysis (CWA) may be a useful low-cost alternative for the clinical detection of LA. In this study, we assessed the clinical value of the CWA for detecting LA in blood samples from patients with various blood coagulation disorders and those treated with warfarin and heparin at a tertiary care university hospital in Japan.

| Samples
Yamagata University Hospital is a tertiary acute care hospital with departments that provide specific medical care to patients with autoimmune diseases such as systemic lupus erythematosus and APS, hematologic diseases such as coagulation disorders, and obstetric and gynecological diseases. According to the distribution of the cases from the Yamagata University Hospital, 3 years of collection were required.

| Methods of measurement
One hundred forty-eight patients whose blood samples indicated prolonged PT and APTT were divided into the following six groups. The first group comprised 86 LA-positive patients, we numbered the patients with APS consecutively, and two samples were collected per person, those patients whose samples were included had a clinically confirmed diagnosis by the doctors." The second group comprised 20 patients, who received unfractionated porcine heparin therapy (1000-2000 units/d) and whose APTT values were within our therapeutic range (47-77 seconds) ( Table 1). These patients had been admitted to the cardiac, renal, and vascular wards and were receiving treatment for conditions such as acute coronary syndrome, myocardial infarction, and deep vein thrombosis/pulmonary embolism (DVT/PE). The third group comprised 23 patients who had DVT/PE and cerebrovascular diseases who were treated with warfarin (10-20 mg). 18 The fourth group included 13 patients who had liver disease and met the diagnostic criteria of impaired liver function. The patients were diagnosed according to the most recent guidelines established by the American Association for the Study of Liver Diseases (AASLD). 19 From all patients, a 1.8-mL blood sample was collected into a tube containing 0.109 mol/L trisodium citrate (Venoject II, Terumo Company, Tokyo, Japan). Blood samples were centrifuged at 2000 g and approximately 23 C for 15 minutes. Plasma was separated from each sample and transferred to a fresh collection tube, followed by centrifugation at 2800 g and 23 C for 15 minutes. This doublecentrifugation process renders the plasma platelet-poor (final platelet count <1 × 10 9 /L), an essential characteristic for LA detection. All samples were freshly obtained on the day of assessment. The samples were assessed for PT and APTT as described previously, 20  STACLOT LA was also analyzed using STart 4 (Young Instruments, Stago, France) automated coagulation analyzer according to the manufacturer's instructions. We performed measurements according to LA guidelines 2,3 on evaluation of the effectiveness of the CWA.

| Clot waveform analysis (CWA)
We conducted the CWA using the APTT method, as described by  Reference range 0.9-1. plot. The second derivative plots associated with this shoulder-like peak are termed the atypical peak. 9 An example of this pattern is illustrated in (Figure 1). We used EZR software on R commander (version 2.13.0) to conduct the statistical analysis. 21     In conclusion, our data suggest that the CWA observed conventional tests for LA. As shown in Table 2, however, the atypical peak appeared highly LA-positive, and the CWA of data obtained in a previous study was different from that in the present study. 9 Although a previous study also involved CWA using an ACL TOP analyzer, the authors reported that the atypical peak was associated not only with LA but also with FVIII and FIX deficiency. 11 Moreover, the variation in the appearance and features of atypical peaks varied according to APTT reagents and the presence of LA, FVIII deficiency, or FIX deficiency, when measured using the analyzer. Finally, previous findings regarding the atypical peak are limited to CWA using ACL TOP analyzers, as in the present study. Further studies should explore whether an atypical peak also appears when using other analyzers.