Sysmex® CA-1500 System Sample Processing II Online Training

Welcome to the Sysmex® CA-1500 Sample Processing II Online Training course. Sample processing begins with receiving samples and loading them onto the instrument for processing. It is important to learn how to process samples that require special handling. Samples requiring special handling may include: Samples needing serial dilutions Processing samples in Micro mode Samples that require reflex testing Select Next to continue.   Upon successful completion of this course, you will be able to: Describe how multiple dilution analyses (MDAs) can help distinguish between in-vitro inhibitors and in-vivo factor deficiencies Describe how to order sample specific and group MDAs Identify and evaluate MDA results on the MDA Stored Data Graphic Display screen Describe how to program Micro-Mode, Reflex testing, and Register More options Select Next to continue. Congratulations. You have completed the Sysmex® CA-1500 System Sample Processing II online training course. Listed below are the key points that have been presented. Take time to review the material before you proceed to the final quiz. Describe how multiple dilution analyses (MDAs) can help distinguish between in-vitro inhibitors and in-vivo factor deficiencies Inhibitors can cause falsely prolonged aPTT and PT results as well as falsely low factor assay results. If an inhibitor is present, an MDA will dilute out its inhibitory effects so that you can obtain a valid factor assay test result. MDAs have no effect on test results if a factor deficiency exists. Demonstrate how to order sample specific and group MDAs Use the Work Load List screen to order an MDA for a specific sample or for all samples whenever a particular factor assay is ordered. Identify and evaluate MDA results on the MDA Stored Data Graphic Display screen Like all stored data, you can access MDA results from the Stored Data List screen. The system plots MDA results on a graph that you can view and/or print. Compare the MDA curve to the standard curve, assess dilution ratio linearity, and review the Slope Ratio value when evaluating if the mean MDA result is valid. Describe how to program Micro-Mode, Reflex testing, and Register More options Use the Work Load List screen to program Micro-Mode, Reflex testing, and Register More options. Micro-Mode allows you to conserve low volume samples, Reflex testing saves time by automatically running the next test needed if a sample yields results outside specified limits, and the Register More option allows you to continuously process a batch of samples registered on up to five worklists. Select Next to continue. The presence of circulating inhibitors in a patient's blood can affect hemostasis test results. A common inhibitor, known as a lupus-like anticoagulant (LA), interferes with the phospholipid reagents used to perform aPTT and PT tests yielding falsely prolonged results. LAs also interfere with factor assays, yielding falsely low results for the clotting factors involved in the intrinsic and extrinsic pathways. These abnormal results could also be due to a true clotting factor deficiency. One way to differentiate between an in-vitro inhibitor versus an in-vivo factor deficiency is to program a multiple dilution analysis (MDA) to diminish inhibitory effects by analyzing serial dilutions of the patient sample. If the abnormal factor assay is due to an inhibitor, serial dilutions will dilute out the inhibitor's effect—a valid test result is obtained after two consecutive dilution ratios yield the same activity percent. If the abnormal factor assay result is due to a factor deficiency, serial dilutions will have NO effect on the test result. Hemostasis Terminology Learn about hemostasis. Term Definition Lupus-like Anticoagulant (LA) Autoimmune immunoglobulins triggered by antibiotic use and autoimmune disorders that recognize protein phospholipids and interfere with phospholipid-dependent hemostasis tests, such as aPTT and PT. Activated Partial Thromboplastin Time (aPTT) Hemostasis test that measures the clotting time from factor XII through fibrin formation, which includes factors from the intrinsic pathway and common pathway: XII, HMWK, PK, XI, IX, VIII, X, V, II, and fibrinogen. The APTT is an excellent pre-surgical coagulation-screening test and allows physicians to monitor heparin therapy. Also called PTT test. See also intrinsic pathway, common pathway Prothrombin Time (PT) Hemostasis test that measures the clotting time from factor VII through fibrin formation, which includes factors from the extrinsic pathway and common pathway: II, V, VII, X, and fibrinogen. The PT is one of the most frequently performed hemostasis tests and measures most of the factors depressed by oral anticoagulant drugs. Also called protime. See also extrinsic pathway, common pathway. Intrinsic Pathway Coagulation pathway activated by a foreign surface involving contact factors PK and HMWK, and factors XII, XI, IX, VIII. Most congenital deficiencies involve factors in this pathway. Extrinsic Pathway Normal physiologic coagulation follows this pathway, which is activated by tissue factor and also involves factor VII. Most of the factors depressed by oral anticoagulant drugs are involved in this pathway. Common Pathway Coagulation pathway activated by either the VIIa/tissue factor complex from the extrinsic pathway, or the VIIIa/IXa complex from the intrinsic pathway. Factors involved in the common pathway include factors X, V, II, and fibrinogen. Anticoagulant Chemical that prevents blood coagulation. Anticoagulants can be of natural origin, or administered therapeutically. EDTA, heparin, sodium citrate, and sodium oxalate are examples of anticoagulants used in blood collection tubes to prevent clotting. See also natural anticoagulants, heparin. Heparin Anticoagulant administered to prevent thrombosis and other conditions. Heparin inhibits the activated forms of factors IX, X, XI, and XII, as well as platelet release factor. In-vitro Biological reaction or procedure occurring in a laboratory apparatus or environment, such as a test tube In-vivo Biological reaction occurring in a living organism. Natural Inhibitors Proteins that circulate in the blood to prevent blood clot formation beyond the site of vessel injury. Examples are protein C, protein S, Antithrombin III and tissue factor platelet inhibitor (TFPI). When complete, select the X in the upper-right corner to close the window and continue. Understanding what happens during the intrinsic, extrinsic, and common coagulation pathways will help you understand the theory behind many of the diagnostic tests performed in the Hemostasis lab, such as when to order a multiple dilution analysis. Intrinsic Pathway Learn about the Intrinsic Pathway. Base ImageHotspotsText BlocksImage FileThe change in molecular structure of factor XII, along with Kallikrein and HMWK (High Molecular Weight Kininogen) activates factor XII to factor XIIa.The intrinsic pathway begins when circulating factor XII comes in contact with and is bound to a negatively charged surface, such as collagen.Factor XIIa activates factor XI.Factor XIa plus calcium activates factor IX, which can also be activated by the thromboplastin-factor VII complex via the extrinsic pathway.Factors IXa plus calcium and PF3 from platelet membranes activate factor VIII.The factors IXa-factor VIIIa complex is needed to convert factor X to Xa in the common pathway. Extrinsic Pathway Learn about the Extrinsic Pathway. Base ImageHotspotsText BlocksImage FileThe factor VII-thromboplastin complex rapidly converts factor X to the enzyme Xa in the common pathway.Factor VII forms a complex with tissue thromboplastin and calcium. Common Pathway Learn about the Common Pathway. Base ImageHotspotsText BlocksImage FileVia the extrinsic pathway, the factor VII-thromboplastin complex rapidly converts factor X to the enzyme Xa.Via the intrinsic pathway, the factor IX-factor VIIIa complex slowly converts factor X to the enzyme Xa.Factor Xa, in the presence of factor V, calcium, and PF3, catalyzes the prothrombin to thrombin.Thrombin converts fibrinogen to fibrin to form an unstable clot.The fibrin strands in the unstable clot begin to gel and are acted upon by factor XIIIa to form a stable insoluble clot which the fibrin strands become cross linked to each other in their D-Dimer region. If inhibitors such as lupus-like anticoagulants (LAs) are present in patient samples, PT and APTT results could be falsely prolonged, and factor assay activity percent could be falsely low. Some examples include: LAs are anti-phospholipid antibodies that bind to phospholipids Phospholipid reagents are used to initiate the extrinsic pathway in PT tests (Innovin), and the intrinsic pathway in APTT tests (Actin)—the LAs bind to phospholipid reagents and interfere with the coagulation cascade LAs also interfere with factor complex formations that occur during the coagulation cascade—this affects factor assays involving the extrinsic and intrinsic pathways The APTT test is more sensitive to LA inhibitors than the PT test due to the phospholipid content of the reagent. Note: Many regulatory agencies now require factor assays to be performed on more than one dilution (in addition to the CA-1500 initial 1:10 sample dilution) to detect the presence or determine the absence of inhibitors Prolonged aPTT Learn more about prolonged results. A prolonged aPTT is seen in the following conditions: Heparin Lupus-like anticoagulants Deficiencies of intrinsic factors Liver disease Consumptive states (DIC) Vitamin K deficiency Warfarin therapy The most common cause of a prolonged APTT in an inpatient is heparin therapy. Before running tests to confirm intrinsic factor deficiencies, be sure to exclude heparin as the cause of a prolonged APTT—this may require using the thrombin clotting time to interpret the result correctly. Lupus-like anticoagulants (LAs) are probably the second most common cause of a prolonged APTT—and because LAs manifest in predominately in vitro situations, these patients usually have no clinical bleeding disorder. When complete, select the X in the upper-right corner to close the window and continue.   To process samples with low volumes, analyze the sample in the Micro mode--the sample probe aspirates sample from a primary tube and dispenses it directly into a reaction tube--bypassing the sample plate well. To program a sample to run in Micro mode: On the Main Menu screen, press Work List On the Work Load List screen, press Next to open a new Rack worklist Register samples and order tests Select a sample, and then press Micro The O symbol will appear on the Micro key and the sample will display the M flag Load samples and place the rack in the right input tray Press Start Note: Auto-repeat, re-dilution, and reflex testing are not available for samples being processed in Micro mode. Be sure to manually remove caps when running in Micro mode. Select Next to continue.   If reflex test conditions have been set up, the instrument will automatically run another test method if a test results are outside reflex limits set by the laboratory--for example, if PT >25 sec and APTT > 45 sec, then run Fbg. You can set up to 60 types of reflex tests Reflex testing improves the timeliness of results because the instrument automatically performs the next test needed without having to wait for the physician to see the initial result and ask for the next test To program a sample for reflex testing: On the Main Menu screen, press Work List On the Work Load List screen, press Next to open a new Rack worklist Register samples and order tests Select a sample, and then press Reflex The O symbol will appear on the Reflex key and the sample will display the R flag Load samples and place the rack in the right input tray Press Start Note: Reflex must be selected for each sample. Reflex testing is not available for samples being processed in Micro mode. Select Next to continue. If the host computer is down, or if additional samples arrive that need to be processed after you’ve pressed the Start key, create a new worklist, register the additional samples, and then press the Register More key. The instrument can process up to five registered racks continuously. To register more samples: On the Main Menu screen, press Work List On the Work Load List screen, press Next to open a new Rack worklist Register samples and order tests on the new worklist Load samples and place the rack in the right input tray Press Register More Select Next to continue. To check and correct for the possible presence of inhibitors when running a factor assay, order a Multiple Dilution Analysis (MDA). In an MDA, the instrument removes inhibitory effects by serially diluting a sample—when at least two consecutive dilution ratios yield the same activity percent, you've confirmed that inhibitors have been diluted out and the "true" activity percent has been obtained. The table on the right shows the dilution ratios for three dilution sets: MDA, MDA High, or MDA Low. Note: For abnormal factor assays, which MDA should you order? Keep the following in mind: Order MDA to get started, and then, if needed, order MDA High to run the factor assay using a significantly higher (less concentrated) sample dilution than 1:10. MDA Features Learn about MDA features. MDA features include: Order an MDA for a specific sample, or as a group—every time a particular factor assay is requested. For each factor assay, you can set up multiple dilution ratios by ordering an MDA, MDA High, or MDA Low dilution ratio set. Order MDA to get started, and then, if needed, order MDA High to dilute samples containing high levels of interfering anticoagulants. Order MDA Low to assay low factor levels, or for a low fibrinogen. Define a 3-point dilution series to order three dilutions per assay, or you can define a 2-point dilution series to order two dilutions per assay. On the chart shown here, dilutions displayed in red are NOT performed during 2-point dilution assays. All patient samples are initially diluted 1:10—this means that all MDA dilutions are made starting with a 1:10 sample dilution. For each assay, the instrument plots the analysis results of each dilution on a graph, which you can review to determine if the final result is valid. When complete, select the X in the upper-right corner to close the window and continue. After ordering an MDA for a factor assay, the system obtains analysis results for each dilution ratio and displays the mean analysis result on the Stored Data List screen. But is the result valid? Is an inhibitor present? Could a factor deficiency exist? To determine if the mean result is valid, evaluate the MDA results of each individual MDA dilution ratio. You can review MDA results on the MDA Stored Data Graphic Display screen and/or output results to a graphics printer. To view or print an MDA graph: From the Main Menu screen, press Stored Data On the Stored Data List screen, press Select Display to display only specified stored data On the Select Display window, press MDA to display only MDA results. On the Stored Data List screen, select the sample whose MDA results you want to review, and then do either of the following (make sure that the cursor is on the mean result): -Press Graph to display the MDA graph on the MDA Stored Data Graphic Display screen. Press Output and then press GP Graph -Press Output to output the MDA graph directly to a printer, and then press Current (to print the current sample selected), and then press GP Graph Select Next to continue. If a sample's test request includes a factor assay for which you need to run an MDA, you must first define the number of dilutions—2 or 3—that you want the instrument to perform. Use the MDA Settings screen to define MDA dilution points. To define MDA dilution points: From the Main Menu screen, select Settings Select Analysis Settings Select Replic Select MDA Dilutions On the MDA Settings screen, define the dilution points Select Next to continue. After defining dilution points for an MDA, use the Work Load List screen to specify the sample, and then order an MDA for the factor assay. To order a sample specific MDA: From the Main Menu screen, select Work List Select ID. No. Entry, and the enter the sample ID Order  tests, select Enter, and then select Quit Select a specific sample, and then select Sample Specific Select a dilution set: MDA, MDA High, or MDA Low and then select Return Select Start Note: To order an MDA factor assay for a specific sample, from the Work Load List screen, select the sample and then press Sample Specific to open the Sample Specific screen. Order Sample Specific MDA Learn how to order a sample specific MDA. Instructions:If media does not automatically start, select the play arrow to begin.Flash File:/content/generator/Course_90013574/sim_CA1500_SampleProcessingII_OrderSampleSpecific_scaled/sim_CA1500_SampleProcessingII_OrderSampleSpecific_scaled.htmHTML5 File:/content/generator/Course_90013574/sim_CA1500_SampleProcessingII_OrderSampleSpecific_HTML5_800x600/index.htmlPDF File: After defining dilution points for an MDA, to save time, you can use the Repeat feature on the Work Load List screen to order MDAs for a factor assay every time that test is requested on any worklist. The Sysmex® CA-1500 System holds this programming option in memory until the instrument is turned off. To order a group MDA: Order the test for which you want to program an MDA Order a sample specific MDA Order the selected test with MDA for all samples on all worklists Clear the selected test order on all samples on all worklists—but the MDA program remains Resume processing—if the selected test is ordered, an MDA will automatically be run too Select Next to continue.   In the following example, let's go through the steps to order a group MDA for a Factor VIII assay. To order group MDAs for every Factor VIII test that will be ordered on any worklist, first we need to order ONE Factor VIII test. To order a group MDA: From the Main Menu screen, select Work List On the Work Load List screen, press ID No. Entry, and then enter the sample ID for a sample in position 1, for example Press the VIII parameter key to register the Factor VIII test, select Enter, and then select Quit. Note: Remember to select Other Tests to display additional parameter keys on the Work Load List screen, if necessary Select Next to continue. To order a sample specific MDA: Select the sample in position 1, and then select Sample Specific On the Sample Specific screen, select VIII to order an MDA for the factor VIII test for the sample in position 1 Select a dilution set: MDA, MDA High, or MDA Low, and then press Return to return to the Work Load List screen Select Next to continue. To  repeat this test request so that ALL samples on ALL worklists are registered for a Factor VIII assay with MDA: On the Work Load List screen, select Repeat to open the No. of Repeats Entry screen Enter 99, and then select Enter to program all racks to perform a Factor VIII assay with MDA Select Quit to return to the Work Load List screen Select Next to continue. To cancel the Factor VIII test request for ALL samples on ALL worklists: On the Work Load List screen, select the sample in position 1, and then select the VIII parameter key to cancel the Factor VIII test. Note that the circle changes to a dash in the first position Select Repeat to open the No. of Repeats Entry screen Enter 99, and then select Enter to clear the Factor VIII test order on all racks--but MDA programming will remain Select Quit to return to the Work Load List screen Note: MDA programming for any Factor VIII test that will be ordered in the future will remain unless the instrument is powered off. Select Next to continue. Now sample order information can be registered to process samples...if any samples have Factor VIII test requests, MDAs will automatically be performed. To resume processing, load necessary reagents and samples, and then press Start Select Next to continue. The MDA Stored Data Graphic Display screen displays MDA raw data and calculated results. The system plots MDA raw data on a graph to create the MDA curve. The graph also displays the standard curve for the MDA test that was run, which the system uses to obtain MDA calculated results. Identify MDA Stored Data Learn about identifying MDA stored data. Base ImageHotspotsText BlocksImage FileThe standard curve is created by plotting coagulation time (or dOD) measurements of each calibrator level on the vertical axis against known activity % (or concentration) of each calibrator level on the horizontal axis.  Parameter for which the MDA test that was run.The MDA curve is created using the method of least squares--the system plots coagulation time (or dOD) measurements of each dilution ratio on the vertical axis against dilution ratio % on the horizontal axis, where the 1/1 dilution ratio = 100% , 1/2 dilution ratio = 50%, 1/4 dilution ratio = 25%, etc.This line helps you determine how well the MDA curve correlates to the standard curve. The system plots the clot time (or dOD) of the highest MDA dilution (the 1/4 dilution ratio, in this example) on the MDA curve, and then draws a line parallel to the standard curve line through this point.  MDA dilution set selected for the analysis.  Average coagulation time (or dOD) of each dilution ratio.The system calculates the activity % (or concentration) of each dilution ratio by inserting dilution ratio clot times (or dOD) into the formula used to calculate the standard curve and then multiplying the result by the appropriate dilution factor.  Average activity % for all dilutions run during the MDA.Correlation coefficient of the MDA curve.Abnormal value flags appear if the limit value of MDA SR has been exceeded.  The Slope Ratio (SR) is a ratio of the MDA analysis data to the standard curve data. The SR value is an index used to examine the influence of inhibitor and accelerator.  Correlation coefficient of the standard curve.+s indicate dilution ratio data points used to create the MDA curve.  *s indicate calibrator level data points used to create the standard curve.   Now that you can identify data on the MDA Stored Data Graphic Display screen, review the MDA results shown here for a Factor X assay of normal control plasma. Note the following: The MDA curve is parallel to the standard curve, which indicates that the dilution ratios are linear and the final result is valid Calculated dilution ratio results correlate well—in fact, all three dilution ratios correlate well, which indicates that an inhibitor is NOT present The SR value is close to 1.0, which confirms that the MDA curve is parallel to the standard curve—the MDA dilution ratios are linear and the final result is valid Note: When evaluating MDA results on the MDA Stored Data Graphic Display screen, use the following guidelines: Compare the activity percent values. If they match, then an inhibitor may NOT be present Evaluate if the MDA curve is parallel to the standard curve and if the SR value is close to 1.0 Select Next to Continue.