CS SERIES Coagulation Curves

Able to Understand Training Objectives Detection Principle Algorithm Types Error Flags

Detection Principle Light from the lamp is split into 340, 405, 575, 660 and 800 nm by 5 different filters. The spectroscopic light is carried to the detector by an optical fibre and then it shines on the mixture of sample and reagent in the cuvette. The light transmitted through the mixture at all wavelengths is detected every 0.1 seconds by a photo-diode and converted into an electronic signal. The clotting time and the dOD/min are derived from this signal by a microprocessor. All detectors can be used for the Clotting assays, Chromogenic assays, Immunoassays by pre-set parameters There are specific detector positions (4 in CS-2x00i or 8 in CS-5100) for Platelet Aggregation assays due to the necessity for mixing during detection.

Evaluation Algorithms The CS series analysers contains 3 evaluation algorithms - the Percentage Detection Method for Coagulation Assays, the Rate Method for Chromogenic Assays and Immunoassays and the VLin Integral Method for Immunoassays. Each evaluation algorithm is specified in the configuration of the test protocol.

Percentage Detection Method After the start of the clotting reaction the transmitted light is monitored and a baseline A/D (Analogue/Digital) value (bH) determined for the reaction. Once this bH is established the reaction will be monitored and the reaction time and Max A/D level (dH) measured until the clotting reaction is completed. The bH is referred to as 0% and dH is referred to as 100%. During the period between 0% and 100%, an optionally set end point is taken and the clotting time determined for this % end point. This percent detection value can be optionally set between 1% and 100%. We use the 50% point as the standard, at which the amount of A/D value per unit time shows the greatest change.

Rate Method After the start of the reaction at a predetermined start and end point the absorbance is measured. The program calculates the rate of absorbance increase per minute between these two time point measurements and the calculated change in absorbance value is expressed as the raw data.

VLin Integral Method The VLin Integral method evaluates the absorbance per minute of an immunologic reaction at the time point when the reaction reaches its steepest increase. Following the lag phase after the start of the reaction the algorithm monitors the reaction and determines when an increase in the absorbance starts. The algorithm monitors the slope of the increasing curve signal to derive a polynomial curve. Linear regression is then used to find the point of maximum velocity from the polynomial curve. The linear range is defined and the integral area between the linear range is used to calculate the reaction in absorbance per minute. VLin integral is a dynamic algorithm based on the strength of the reaction. The VLin Integral evaluation method is used for immunological assays such as D-Dimer and vWF Ag.

Clotting Curve Errors

Normal Coagulation Curve

No Coagulation This message occurs when the analyser is unable to detect a coagulation reaction or when a weak clot is formed. If the dH does not reach the "No Coagulation Threshold" value, the result will be flagged with a "No Coagulation" error. When this condition is detected the result will not be reported. Action Steps 1. Check the sample for possible anticoagulant contamination, haemolysis, etc. 2. Ensure delivery of sample and reagent (this can be confirmed on the basis of QC results). 3. Reanalyse the sample. For fibrinogen, if auto re-dilution is not set, change the dilution ratio and reanalyse. 4. If the No Coagulation flag persists upon reanalysis of the sample, follow the laboratories guidelines for manual result confirmation. Example Low Fibrinogen, over anticoagulated: warfarin or heparin, clotted sample.

Slight Coagulation If the dH does not reach the "Slight Coagulation Threshold“ value, the result will be reported and flagged with a "Slight Coagulation" error. This error is indicative of a low fibrinogen sample or reagent problem. Action Steps 1. Check the sample for possible anticoagulant contamination, haemolysis, etc. 2. Ensure delivery of sample and reagent (this can be confirmed on the basis of QC results). 3. Perform Clauss fibrinogen to confirm fibrinogen result. Example Low Fibrinogen

Analysis Time Over This check determines whether the reaction end point is correct. If the sample reaction end angle is greater than the permitted angle at the Maximum Reading Time, the result will be flagged with an "Analysis Time Over" error. The situation occurs when testing samples with prolonged clotting times Action Steps 1. Check the sample for possible anticoagulant contamination, haemolysis, etc. 2. Set a re-analysis rule in the test protocol such that if analysis time over error then measure for longer using a sub measurement time e.g. 240 seconds. Increasing the sub measurement time will help to confirm whether the coagulation curve becomes a plateau. 3. If re-analysis still gives an “Analysis Time Over” message then the sample may not be able to form a firm clot. Follow the laboratory guidelines for manual result confirmation. Example Over anticoagulated, check for warfarin or heparin

Coagulation Curve Error (CCE) Number of different CCE’s: Stepping Curve Check Sharp Drop Check Terrace Check Fibrinogen Curve Check Jump Up Check Initial Fluctuation Drop Check These messages occurs when there is an unexpected change in the coagulation curve. The most common cause is an air bubble in the reaction cuvette. Air bubbles in the reaction cuvette; along with mechanical problems, will lead to irregular fluctuations in the coagulation curve during the measurement process. This check detects such abnormal fluctuations in the coagulation curve. When this occurs results will be displayed in the joblist however no numerical results will be output. Instead ***.* will be output to host or printed along with the "Coagulation Curve Error" message. Action Steps 1. Check the sample for possible anticoagulant contamination, haemolysis, lipaemia etc. 2. Reanalyse the sample. If upon reanalysis results without an asterisk(*) are obtained the result may be reported. 3. If Coagulation Curve Error occurs upon reanalysis, review the analysis data. If the curves are acceptable and the repeat and initial results are equivalent at the coagulation detection point, then report the final result obtained.

Stepping Curve Check This check prevents a false reaction end point detection in the early stages of the reaction curve. An incorrect result could have been reported if the dH continues to increase after the reaction end point Action Steps Repeat the analysis, if no flag occurs then the result can be accepted. Review the coagulation curve. If it fits within an “reverse S” pattern and the 50% value is defined at the correct position, it is okay to accept the result. 3. If this error occurs on a persistent basis then contact the Sysmex Customer Support Centre.

Sharp Drop Check During the normal clotting process the dH value will always be increasing. If after the start of the clotting assay, if the dH decreases by more than 32 points the result will be flagged with a "Coagulation Curve Error" sharp drop. Action Steps 1. Check sample integrity 2. Re-run sample 3. Change the lamp and re-calibrate. 4. If this error occurs on a persistent basis then contact the Sysmex Customer Support Centre.

Terrace Check This check is used to determine an abnormal flat part in the coagulation curve. The software monitors the clotting time at each 1% interval and if the clotting time is prolonged sharply in any 1% interval the result will be flagged with this error message. Action Steps 1. Check sample integrity. 2. Re-run sample

Fibrinogen Curve Check This check is only for the Clauss Fibrinogen assay. It looks to assess the relationship between the fibrinogen dH threshold and the reaction end point. When the dH value obtained is greater than the threshold value and the reaction end point is greater than 50 seconds then this flag will be generated. When this condition is detected, it is suspected that the Owren's Veronal Buffer is cold or the sample has an inhibitor such as a high concentration of FDP or a high fibrinogen concentration. Action Steps 1. Warm OVB. 2. Re-run sample.

Jump Up Check This check is able to detect an excessively rapid decrease in the reaction curve. If the same clotting time is obtained continuously 10 times (20 times for Innovin) or more within the 2%-80% detection range, the result will be flagged with a "Coagulation Curve Error“ error. Action Steps 1. Check sample integrity. 2. Re-run sample. 3. Change the lamp and re-calibrate. 4. If this error occurs on a persistent basis then contact the Sysmex Customer Support Centre.

Initial Fluctuation Drop Check This check identifies an abnormal fluctuation in the initial part of the coagulation curve based on a check of the dH value at check point T. If the value is greater than the threshold then the error flag will be generated thereby preventing an erroneous result from being released. Action Steps 1. Check sample integrity. 2. Re-run sample. 3. If this error occurs on a persistent basis then contact the Sysmex Customer Support Centre.

Early Reaction Error 128 (ERR128) This message occurs when abnormally short APTT results are produced. ERR128 errors are sub-classified into separate Early Reaction Errors (ERE’s). ERE001 Slow reaction ERE002 Start Angle 1 ERE004 Start Angle 2 ERE008 Drift ERE016 Early % When this occurs no numerical results will be given, instead ***.* will be output along with an early reaction error message. This message occurs when abnormally short APTT results are produced owing to in the majority of cases sample activation. When this occurs no numerical results will be given, instead ***.* will be output along with an early reaction error message.

Summary of ERE Action Steps Check Sample and Reagent Integrity (ERE001; ERE002; ERE008; ERE016) Action Steps 1. Verify sample and reagent integrity. 2. Check the coagulation curve, if the ERE code is 4 (Start Angle 2) go to step 3, if not then proceed to step 4. 3. ERE 004 does not necessarily invalidate the clotting time result. Check the coagulation curve, if the clotting time is defined at the correct position within the clotting curve the result can be accepted. If the result does not fit then go to step 4. 4. The software has detected an unusual early or slow reaction. Repeat the measurement, if no error is generated then the result can be released. If the error persists go to step 5. 5. The software has detected a reproducible early or slow reaction and may be due to a sampling artefact. Repeat the sampling of the patient and repeat the measurement. If the result is free from error then the result can be released. If the error persists even with the repeat sample go to step 6. 6. An unusual or slow reaction that is reproducible has been detected. This may be due to a specific clinical situation in combination with the reagents and technology being used. Follow the laboratories guidelines for result confirmation. DO NOT REPORT ANY RESULTS WITH AN “EARLY REACTION FLAG” WITHOUT REVIEWING THE CURVE . APTT RESULTS <20 SECONDS SHOULD NOT BE REPORTED.

ERE001 – Slow Reaction Check Criteria This method checks the slope of the coagulation curve around the width of the detection point e.g. 44% to 56%. If the reaction time within the % width range (TL1 to TL2) is longer than the slow reaction time limit, the result will be flagged with an Early Reaction Error. Action Steps See flow chart Check Criteria (Time 2 – Time 1) > Slow reaction Time Limit= Err:0008.0128.001 Slow Reaction

ERE002 – Start Angle Check 1 Check Criteria After the start of the reaction, the angle of the coagulation curve is checked using a check criteria. If the angle of the coagulation curve is too steep at the beginning with the dH value less than the defined limit then there is the possibility of an incorrect result being produced and the result will be flagged with Early Reaction Error. Action Steps See flow chart Check Criteria (dH2-dH1)>Start Angle Threshold and dH<Limit dH = Err:0008.0128.002 Start Angle 1

ERE004 – Start Angle Check 2 Check Criteria After the start of the reaction, the angle of the coagulation curve is checked using a check criteria. If the angle of the coagulation curve is too steep at the beginning with the dH value greater than the defined limit then there is the possibility of an incorrect result being produced and the result will be flagged with Early Reaction Error. Action Steps See flow chart ERE 004: Start Angle Check 2 does not necessarily invalidate the clotting time result. Check the coagulation curve, if the clotting time is defined at the correct position within the clotting curve the result can be accepted. Check Criteria (dH2-dH1)>Start Angle Threshold and dH>Limit dH = Err:0008.0128.004 Start Angle 2

ERE008 – Drift Check Check Criteria This check is able to detect a drifting of the reaction curve. If the ratio between dT1 and dT2 is greater than the Drift Check Limit ratio then the result will be flagged with an "Early Reaction Error“. Action Steps See flow chart Check Criteria dT1/dT2>Drift Check Limit ratio = Err:0008.0128.0008 Drift

ERE016 - Early % Check Check Criteria This checks whether the time at a given percentage value (after the start of the reaction) is less than or equal to the threshold. If this is the case the result will be flagged with an "Early Reaction Error". Action Steps See flow chart Check Criteria Checkpoint (sec)<limit = Err:0008.0128.0016 Early %

Trans Light High Check If the A/D value (transmitted light signal) of the test being measured is at or above the A/D threshold value the result will be flagged with a "Trans Light High“ Error. When this condition is detected the result will not be reported. Action Steps 1. Check the sample, reagents and instrument condition. 2. Recalibrate the lamp or replace it with a new lamp and then recalibrate. 3. Reanalyse the sample, if upon reanalysis results are obtained without an asterisk(*) then they may be reported.

Noise Check This check is able to detect whether the reaction curve has been influenced by a noise signal. The analyser monitors the A/D value at the beginning and end plateaus and if the difference between the two checkpoints is less than the noise threshold the error will be reported. Action Steps 1. Check sample, reagents and analyser condition. Make sure there is no other equipment nearby that may be causing mechanical vibrations to the analyser. 2. Check the lamp is seated securely and correctly. 3. Recalibrate the lamp or replace it with a new lamp and then recalibrate. 4. If this is a persistent error then please contact the Sysmex Customer Support Centre

Turbidity Level Over Check The reaction has continued beyond the lowest light limit (gone black) and therefore a dH value can not be calculated. The sample plasma may be turbid or lipaemic. Action Steps 1. Check the sample for turbidity, lipaemia, etc. 2. Reanalyse the sample diluted with appropriate diluent. This step is available only for parameters that are using a diluted sample in the original test protocol such as a Clauss fibrinogen, where Owren’s Veronal Buffer would be used. Check the test protocol for the diluent used. 3. If reanalysis of the sample results in a numerical value without an asterisk(*), the result can be reported. 4. If this is a persistent error then please contact the Sysmex Customer Support Centre

Range Over Check If the clotting time at the 50% detection point is shorter than the minimum reportable time, the result will be flagged with a ‘Range Over’ (short time) error. When this condition is detected the result will not be reported. Action Steps 1. A persistent error message indicates reagent contamination or lack of instrument maintenance. Check the reagents and instrument conditions. 2. If the reagent and instrument conditions are acceptable request a repeat sample. In certain clinical conditions samples may give a PT and APTT "Range Over" (short time) error message may indicate that the PT result was <7 seconds, or the APTT result was <15 seconds. In this scenario follow the laboratories guidelines for manual result confirmation. 3. If this is a persistent error then please contact the Sysmex Customer Support Centre

Flat Curve This check is effective for the PT assay only and looks at the slope of the coagulation curve around the detection point (50%). If the slope of the curve between the width is greater than the slope limit then the result will be flagged with a flat curve error. The result will not be reported. This will occur when the analyser has detected a weak clot which does not have a “true” coagulation end point. Action Steps 1. Check sample integrity 2. Increase the sub measurement time in the test protocol and set a reanalysis rule in the assay setting such that if a “Flat Curve” error is produced then repeat the assay at the sub measurement time. Increasing the sub measurement time will help to confirm whether the reaction is true coagulation or not. 3. If reanalysis of the sample results in a numerical value without an asterisk(*), the result can be reported 4. If reanalysis of the sample still generates a “Flat Curve” error then follow the laboratories guidelines for manual result confirmation.

Chromogenic & Immunoturbidimetric Curve Errors

Trans Light Low If all the light is being absorbed by the sample, very little light is reaching the detector. When this scenario occurs a ‘Trans light low’ error will be generated and the result will not be reported. Action Steps 1. Check the sample and reagent integrity. 2. Reanalyse the sample. 3. Recalibrate the lamp or replace it with a new lamp. 4. If this is a persistent error then please contact the Sysmex Customer Support Centre.

Trans Light High If no light is being absorbed by the sample, too much light is reaching the detector. When this scenario occurs a ‘Trans light High’ error will be generated and the result will not be reported. Action Steps 1. Check the sample and reagent integrity. 2. Reanalyse the sample. 3. Recalibrate the lamp or replace it with a new lamp. 4. If this is a persistent error then please contact the Sysmex Customer Support Centre.

No Linearity If the ‘r’ value that is calculated for the slope is <0.850 then the result will be flagged with a "No Linearity" error. When this condition is detected the result will not be reported. Action Steps 1. Ensure correct reagent labelling and position on board the instrument. 2. Check sample and instrument integrity. 3. Reanalyse the sample, if a result is obtained it may be reported. 4. Recalibrate the lamp or replace it with a new one and then calibrate the new lamp. 5. If this is a persistent error then please contact the Sysmex Customer Support Centre.

Reaction Curve Error If the slope of the reaction curve differs from that defined in the detector setting in the Test Protocol, the result will be flagged with a "Reaction Curve Error" error. When this condition is detected the result will not be reported. Action Steps 1. Check the sample and reagent integrity and re-analyse the sample. 2. Ensure correct reagent labelling and position on board the instrument. 3. If this is a persistent error then please contact the Sysmex Customer Support Centre.

Antigen Excess (Range Over Check) – Immuno Only A dose hook (pro-zone) effect is checked by comparing the dOD in the unstable region (slope A) with the dOD in the linear region (slope B) where the reaction rate is stable and the change in absorbance is constant. When the concentration of antigen is very high, the dOD in the unstable region is significantly raised compared with that in the linear region. The correlation between slope A and slope B is determined. If this value is higher than the threshold value, the result will be flagged with an “Antigen Excess" error and the result will not be reported. Action Steps 1. Check sample, reagent integrity and instrument condition 2. Reanalyse the sample diluted with the appropriate diluent. All immunoassays have an auto re-dilution set for antigen excess ( samples run in micro mode will not trigger a re-dilution). If upon reanalysis results without an asterisk (*) are obtained results may be reported. Please refer to Siemens application guide for the appropriate re-dilutions. 3. If upon re-analysis Antigen Excess is obtained then the sample may need to be diluted manually with the appropriate diluent prior to running the sample on the instrument in order to obtain a result (NOTE: any results obtained from the instrument with samples run in this manner will need to be re-calculated based on the manual dilution applied). 4. Recalibrate the lamp or replace it with a new lamp. A new lamp requires calibration.

No Polynomial Distribution & Range is Non-linear – Immuno Only No polynomial adjustment: The measured data points between the start time and end time cannot be fitted to a polynomial curve due to a lack of agglutination. If this occurs, the result will be flagged with a "No polynomial adjustment" error and the result will not be reported. Range is non-linear: This message occurs if the “r” value of the slope is less than a defined limit or a line could not be approximated. In this instance the result will be flagged with a "Range is non-linear" error and the result will not be reported. Action Steps 1. Check the sample, reagents and instrument condition. 2. Reanalyse the sample. If a result is obtained, the result may be reported. 3. Reanalyse the sample at a different dilution. No Polynomial Distribution : we recommend repeating the sample at a 2:1 dilution in order to increase the antigen level in the sample. Range is Non-Linear : certain assays have automatic repeat rules which will automatically re-dilute the sample if this error flag is generated. If a result is obtained following re-dilution the result can be reported. 4. Recalibrate the lamp or replace it with a new lamp. 5. If this is a persistent error then please contact the Sysmex Customer Support Centre.

Thank you Document ID: SUKBMS-13-179 CD Published Date: 15-10-2013 CD Version: 2.0 Thank you