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Oak Ridge Poster Sessions |
Univ. of California Davis Med. Center, 4625 2nd Ave., Rm. 203, Sacramento, CA 95817
a author for
correspondence: fax 916-734-3320, e-mail bgosseli{at}clb.ucdmc.ucdavis.edu
The Sysmex CA-6000 automated coagulation analyzer (Dade International) is a new random access instrument that is capable of performing 20 clot-based and chromogenic assays. The purpose of this study is to determine whether the CA-6000 demonstrates performance characteristics for prothrombin time (PT), derived fibrinogen (DF), activated partial thromboplastin times (aPTT), fibrinogen (FBG), thrombin time (TT), factors VII (FVII) and VIII (FVIII) activity, antithrombin (AT), and plasminogen (PLG) similar to an established coagulation analyzer, the MLA 1000C (Medical Laboratory Automation).
Samples were drawn into 32 g/L sodium citrate tubes (Becton Dickinson) and centrifuged at 1300g for 10 min before analysis or freezing. Fresh and frozen (at -70 °C, then quick-thawed with a 37 °C waterbath before analysis) plasma samples were compared contemporaneously on the CA-6000 and MLA 1000C with the same reagents (Dade, except for TT noted below). Specimens tested within normal limits by the clinical laboratory served as normal samples but were not used to determine reference intervals. Abnormal samples were identified by documented evidence of oral anticoagulant, heparin, or fibrinolytic therapy, and (or) history of liver disease, hemophilia A, or other acquired coagulopathies to include trauma and disseminated intravascular coagulation.
Unless otherwise indicated, the principle and detail of test methods are described by Sirridge (1). Below are the reagents used for each individual assay:
PT: Two separate thromboplastin reagents were used, Thromboplastin C+ (TPC) containing acetone-dehydrated rabbit brain thromboplastin, and Innovin (INN), prepared from recombinant human tissue factor and synthetic phospholipids. These reagents are moderately and highly sensitive as indicated by their International Sensitivity Index (ISI) values of 1.94 and 1.02 respectively.
DF: The DF calibrator was determined with assayed reference material (FACT, George King Biomedical) and run according to each instrument operator's manual for both PT reagent sources. This method of FBG determination is a mathematical extrapolation based on clot formation kinetics analyzed concurrently with every PT assay.
aPTT: Testing was performed with Actin FSL containing ellagic acid and purified soy and rabbit brain phosphatides, and calcium chloride.
FBG: An assay based on the modified Clauss technique (2) involved use of commercially prepared thrombin containing ~100 NIH units/mL. Test plasma was diluted in a buffered saline solution before analysis. A single calibration curve was determined on each respective device with assayed material (George King Biomedical).
TT: The assay was performed with commercially prepared dilute thrombin (Sigma Diagnostics) containing ~3–4 NIH units/mL. For the CA-6000, the thrombin is heated to 37 °C before addition to test plasma. The MLA 1000C method requires addition of room temperature thrombin for this assay.
AT: Exogenous bovine thrombin and heparin are added to test plasma to form a thrombin–heparin–AT complex. The residual thrombin not bound then hydrolyzes the p-nitroalanine substrate to produce a yellow color, which is read at 405 nm. The intensity of color produced is inversely proportional to the AT present. A single calibration curve was performed at the onset and used for the duration of sample testing.
PLG: This method is based on the addition of streptokinase to test plasma, which forms a complex with test plasma PLG to hydrolyze the p-nitroalanine substrate, producing a yellow color that is read at 405 nm. The intensity of color produced is proportional to PLG present. A single calibration curve was performed at the onset and used for the duration of sample testing.
FVII: Testing was performed with immunodepleted FVII-deficient plasma substrate by using the PT with Innovin reagent only. The calibrator, Factor Assay Reference Plasma (Dade), was assayed before each test run with a seven-point curve.
FVIII: Testing was performed with immunodepleted FVIII-deficient plasma substrate by using the aPTT test with Actin FSL. The calibrator, Factor Assay Reference Plasma (Dade), was assayed in duplicate before each test run with a seven-point curve.
All assays requiring calibrator or patient dilutions were automatically prepared by the respective devices.
Imprecision analysis: Designated quality-control material was assayed in quadruplicate, twice daily for 3 consecutive days for all test variables. Three concentrations of controls ranging from normal to high abnormal were used for PT-TPC, DF-TPC, PT-INN, DF-INN, and aPTT assays. All other assays involved two concentrations of controls, normal and abnormal. All samples were tested in duplicate. Replicate values must match within 10% to be accepted into analysis. Any samples that failed replicate precision were repeated on both devices.
Statistical analysis: Calculations were performed by linear regression analysis with an R2 >0.95 predetermined as an acceptable correlation. Imprecision studies are expressed by CVs (for control data) and R2 values for replicate samples.
There was excellent correlation between the CA-6000 and the MLA 1000C
for all test variables except TT (Table 1
). TT showed a significant difference (P <0.05)
between the two devices despite use of the same reagent. TT on the MLA
showed a lower mean prolongation than did the CA-6000 for patients with
low FBG or liver disease or treated with low-dose heparin (not shown)
(3). The imprecision was similar between the two devices.
The replicate testing data (all tests had an R2
0.96) indicate that single-well testing could be used to reduce
reagent and consumable usage.
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The CA-6000 demonstrates operational characteristics similar to another automated coagulation analyzer, the MLA 1000C. However, the reagent carousel on the CA-6000 facilitates the performance of multiple assays concurrently. The CA-6000 may be more sensitive for detection of abnormal TTs than the MLA 1000C.
References
The following articles in journals at HighWire Press have cited this article:
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  M. M. Flanders, R. Crist, S. Safapour, and G. M. Rodgers Evaluation and Performance Characteristics of the STA-R Coagulation Analyzer Clin. Chem., September 1, 2002; 48(9): 1622 - 1624. [Full Text] [PDF]
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