hiClot - hiLab
Contents
1. Preheat the sample for at least 2 min or the time indi cated on the reagent box Mm BB WN 6 Move the cup in the reading well the system automati cally senses its presence and asks the operator to insert I The selected method is the one between parentheses on the second row of the display Select it by means of the Q and hiClot user manual 2 2 PT Prothrombin time the reagent 7 Press a three second countdown starts When it 11 is over dispense 200 uL of preheated reagent into the mensa sample cup and the timer starts 8 Once the clot is formed the timer stops and the results pops are shown aue 2 2 2 Calibration A four point calibration is needed in order to calculate the PT Activity the calibrators are obtained from the normal control plasma diluting it with a salt solution as shown by the following schema Activity Dilution Preheat the cups with the calibrators for no less than 2 min and then Preparation plasma solution 1 Measure calibrator A 100 with the procedure de scribed in the last paragraph When the assay is over press the key CAL is displayed lower right to indi cate that the first calibration point has been accepted 2 Measure the B calibrator 50 and press to enter the second point 3 Measure the C calibrator 25 and press to enter the third point 4 Measure the D calibrator 12 5 and press to en ter the fourth p2. FDP heparin or by changes in the structure of the Fibrinogen protein and in these cases it is underestimated 2 5 1 Assay execution WARNING For this assay the samples must be diluted 1 10 into IBS tampon 1 Select the FIB assay hiClot user manual 2 5 FIB Fibrinogen Clauss method 17 Preheat the FIB reagent in a reagent well Insert a void cup into a sample well Pipet 200 uL of diluted sample plasma into it Preheat the sample for at least 5 min or the time printed on the reagent box An BW WN 6 Move the sample cup into the reading well the Sys sma tem automatically senses its presence and prompts the Ee teagen operator to insert the reagent 7 Press starts a three seconds countdown at the end of which pipet 100 uL of preheated reagent into the a sample cup The stopwatch gets started ELEM Gea Wad l 8 When the clot is formed the timer stops and shows the results FIER IS 2 5 2 Calibration This assay needs a three point calibration with calibrators that are produced from a normal control plasma which is to be diluted with the following schema Dilution Preparation plasma IBS 100 4L 400 pE 50 uL 450 uL 25 uL 475 uL Preheat the calibrators for 2 min in the sample wells and operate as follows 1 Measure the A calibrator with the procedure described ponm a in the previous paragraph When the assay is finished al A T EAL press bottom right appears CAL to indicate th
3. E 73 23 CEE It conforms moreover to the product requisites as stated by the following rules CEI EN 50082 1 CEI EN 50081 1 CEI EN 60335 1 hiLab S r l I amministratore unico ing Francesco Iovine Aruana Lou UA Roma li 5 5 2004 hiClot user manual Index 1 General information 3 1 1 Layout of the device 3 1 2 Structure of the instrument 4 1 2 1 Thermostatic block 4 b22 Control pan hosana eea 5 1 3 Working principle ooa aaa aaa 5 2 Assays 8 Zev MUSIC awe em a ae Ge es os a rn ln SR RE 8 2 2 PT Prothrombin time 10 22k ASSAY EXECULION coaie peas 10 2 2 2 CahDratiot soc svs m n ed Sm Be aes 11 2 2 3 Insertionofthe IST 12 2 2 4 Delivered results 13 2 2 5 Reset ofthe PT calibration 14 2 3 PTT Partial Thromboplastin Time 14 2 3 1 Assay execution 14 2 4 TT Thrombintime 15 2 4 1 Assayexecution 15 2 5 FIB Fibrinogen Clauss method 16 2 5 1 Assayexecution 16 2 5 2 Calibration 17 hiClot user manual 26 2 5 3 Insertion of the concentration 2 5 4 Delivered results 2 5 5 Reset of the FIB calibration 2 6 General advice Security and installation rules Sok Security TUNES leg Grae hod eee 3 2 Package content and installation rules 3 3 Declaration of conformity INDEX hiClot user manual
4. and safe usage of the system 11 Place the system as near as possible to the power plug 12 Do not lay anything on the cables 13 Do not surcharge the power plug using multiple plugs 14 Do not insert objects into the system 15 The system has been tested for a typical room temper ature of 25 C in case the room temperature is much higher than this air conditioning is strongly recom mended 16 Do not open the system any repair should be carried out by trained personnel 17 Disconnect the system from the power net and call the technical assistance in case of danger 18 Use only original spare parts for reparation 19 The system must be used only by trained personnel 20 Do not use flammable matter near the instrument 3 2 Package content and installation rules The hiClot is forwarded inside a carton box that contains a shockproof soft and elastic plastic frame that itself contains the instrument The box contains The hiClot The power supply hiClot user manual 3 2 Package content and installation rules 23 A flyer containing the basic instructions 30 sample cups 6 reagent cups hiClot user manual 24 Security and installation rules 3 3 Declaration of conformity The builder company hiLab S r l www hilab it sited in via Piero Aloisi 36 00158 ROMA ITALY hereby declares that the instrument named hiClot conforms to the following EC directives 89 336 CEE 92 31 CEE 93 68 CEE 93 97 CE
5. at the first point of the calibration curve has been ac cepted hiClot user manual 18 Assays 60 40 30 20 10 7 5 4 3 2 20 30 40 277 60 100 200 300 500 1 001 Fil E H aE 2 Measure the B calibrator and press to accept the second value AL 3 Measure the C calibrator and press to accept the third The calibration curve is the regression straight line ap proximating the calibration points placed on a bi logarithmic plane as indicated in figure where the logarithm of the con centration runs along the x axis mg dl and the clotting time sec is on the y axis Verification of calibration data oemteeergemeng The calibration data can be shown and edited pressing fok TE ERIEI when the selected assay is FIB The clotting time T of the A ea calibrator Pressing on and on the T clotting time of the ee a B calibrator appears and then the 73 clotting time for the C TT A calibrator hiClot user manual 2 5 FIB Fibrinogen Clauss method 19 2 5 3 Insertion of the concentration Pressing after the last calibration point has been shown the concentration in mg dl of the control plasma is displayed Insert the new values entering each digit as seen for the PT assay 2 5 4 Delivered results When the hiClot is calibrated for FIB with the procedure described in the previous paragraph the following results are delivered an nu e un E e un n E En 8 u mu f I I m
6. ation The typical absorbance graph after the reagent addition is shown in the following plot hiClot user manual 1 3 Working principle 7 For about ten seconds nothing happens then after 12 13 seconds the sample starts clotting and the absorbance raises as it is clearly seen in the figure As the Fibrin is being synthesized the absorbance raises until the upper limit that corresponds to the total clot formation hiClot follows completely the absorbance curve and builds the straight segments that control the stopwatch This results in a good repeatability and improves hiClot insensibility to external disturbances Please note that the vertical absorbance scale is arbitrary hiClot user manual ch oh EEE EEE HEE Chapter 2 Assays 2 1 Basics Whet switched on hiClot shows its name and the software version for a while then automatically starts heating the mea surement block up to 37 C During the heating phase lasting s for about 5 8 minutes depending on the external tempera ture it displays an arrow that moves slowly rightwards as the temperature rises When the heating is over the message Insert sample is shown on the upper row and on the second the method list begins with PT PTT and continues rightwards as implied by the arrow Please note that PT is surrounded by square parentheses to show that it is the current method i e the one the instru ment is ready to execute You can change
7. he central well incorporates the optical reader and is where to insert the reaction cup to hiClot user manual 1 3 Working principle 5 be read Reagent wells Sample wells 1 2 2 Control panel The control panel is composed by an alphanumeric LCD dis play and a three button keyboard It is seldom used during the normal work as it serves only to select the assay type and start the assay The two upper keys Q and select the items of a list while the lower one accepts the current selection 1 3 Working principle All the clot assay determine chain reactions that end by trans forming the Fibrinogen into Fibrin using Thrombin as a cat alyst The Fibrin forms a high turbidity jelly clot that can be monitored by measuring the absorbance augmentation hiClot user manual 6 General information Actually hiClot is fundamentally a mono channel blue light colorimeter that can be schematized as follows Thermostatic plate Optic sensor The semitransparent cup is crossed by the light beam generated by the emitter whose intensity variation is mea sured by the optical sensor on the opposite side The assay sequence begins by inserting a cup into the measuring well containing the needed volume of sample The cup insertion is revealed by the photometer When the key is pressed a three second count down begins at the end of which the operator adds the reagent from now on hiClot starts monitoring the clot form
8. hiClot User Manual version March 19 2010 Chapter 1 General information 1 1 Layout of the device hiClot is a single channel low cost clot meter very simple to use and capable of performing the most common clot assays PT Prothrombin time PTT Partial thromboplastin time TT Thrombin time FIB Fibrinogen Clauss A piece of software is available for connecting the hiClot to an external computer in order to manage an archive of the assay results and to print reports It is a small box that has an LCD back lit display a key board and 12 positions to preheat the samples 2 positions for the reagents I reading well The most recent version of this package can be downloaded from www hilab it hiClot user manual hiClot right side hiClot left side 4 General information The device can be controlled by means of a three button keyboard and the display on top of it The power switch and the plug for the external supply on the right side the connec tor of the PC interface is on the left side 1 2 Structure of the instrument The hiClot upper side has the control panel and the thermo static block 1 2 1 Thermostatic block The thermostatic block is made of aluminum and has some wells to preheat the samples and the reagents as well as the reading station It is covered with a plastic surface to lower its dissipation thus assuring temperature uniformity which is between 36 5 C and 37 5 C T
9. int the figure 9 ap pears pressing once more it turns into 8 Continue seems pressing until 0 is selected accept it pressing IPT 1 4 Press five times until besides 0 a 5 appears accept ET ieee the figure with oe o 5 Now the selected number is correct enter it pressing another time moak Suossa i 25 S ARE H ORELO asan a ies ice e iy Cka eae Actually the procedure consists in inserting a decimal number selecting each single digit selects the next digit the previous one The decimal point lies after 9 and be fore 0 and the system inhibits to insert more than one 2 2 4 Delivered results When the instrument is well calibrated the assay results are four values organized as follows hiClot user manual 14 Assays top left PT clotting time in seconds 15 0 in the exam ple top right Ratio ratio between the PT of the sample and that of the A calibrator 1 23 in the shown ex ample bottom left INR Ratio 1 20 in the example bottom right Activity expressed as a percentage and based on the described calibration procedure 78 4 in the example 2 2 5 Reset of the PT calibration The aforementioned procedure is aimed at inserting automat ically the calibration points from the calibrator assay results In order to recalibrate the instrument it is mandatory to clear the current calibration switch the instrument off and switch it on agai
10. l Pipet 200 uL of sample plasma Preheat the sample for at least 3 min or the time you can find on the reagent box hiClot user manual Er le gen UTE SRE ate PH send inn g r 5 ii Per en aee E a n a a When the clot is developed the stopwatch stops and pee fens 16 Assays 6 Move the sample cup into the reading well the system senses automatically its presence and asks the operator to insert the reagent 7 Press a three seconds countdown starts and when it ends pipet 100 uL of preheated reagent into the sam ple cup The stopwatch gets started 8 When the clot is formed the timer is stopped and the results are shown The first result 1s the Thrombin time in seconds the sec ond is the ratio between this value and the normal TT 2 5 FIB Fibrinogen Clauss method The Fibrinogen or factor I is a dimer of three polymeric chains a P and y with a residual carboxylic terminal whose nor mal concentration in plasma is 200 400 mg dL The Fib rinogen is converted into Fibrin by means of a polymeriza tion catalyzed by Thrombin or other similar enzymes hiClot uses the Clauss method to determine it that is an indirect as say based on a modified Thrombin Time In presence of high Thrombin concentration the polymerization time of Fibrin of a diluted citrated plasma is correlated to the concentration of that protein This reaction is influenced by additives having antithrom binic activity
11. n keeping pressed the button until the software version is disappears 2 3 PTT Partial Thromboplastin Time It is the clotting time of a platelet deficient plasma after the insertion of phospholipids in place of platelets and of cal cium ions It verifies the extrinsic coagulation pathway 2 3 1 Assay execution 1 Select the PTT assay 2 Preheat the Ca Clg in a reagent well 3 Insert a void cup in a sample well hiClot user manual 2 4 TT Thrombin time 15 Dispense 100 uL of sample plasma and incubate for 2min Dispense 100 uL of PTT reagent ad incubate for an other 3 5 min Move the sample cup to the reaction well the system automatically senses its presence and asks the operator to insert the reagent Press a three second countdown starts and when it is over dispense 100 uL of preheated C a Cla into the sample cup The stopwatch starts counting shows the results The first result is the PTT in seconds the second is the ratio between this time and the normal PTT value 2 4 TT Thrombin time It is the clotting time of a citrated platelet deficient plasma to which a known volume of Thrombin is dispensed This assay investigates the final pathway of the coagulation reaction the transformation of Fibrinogen into Fibrin and its polymeriza tion 2 4 1 Assay execution na BOM Select the TT assay Preheat the TT reagent in a reaction well Insert a void cup into a sample wel
12. oint which is supposed to have 100 activity hiClot user manual 12 Assays 100 50 25 Taa After such procedure the system is calibrated and can cal culate the Ratio and the Activity The calibration curve is the regression straight line based on the calibration points as shown in the figure with the inverse Activity 1 A on the x axis and the clot time in seconds on the y axis Verification of the calibration data i gt The calibration data can be shown and changed pressing fi 12 3 when the current test is PT The coagulation time To of the o i n calibrator A appears Pressing con and on the coagula FIT tion time T of the B calibrator is shown then T gt of the C Fa calibrator and 73 of the last calibrator 2 2 3 Insertion of the ISI If you press once more after the last calibration point the current value of the ISI is displayed which as known is used in the calculation of the INR This parameter is nor 3International Sensitivity Index 4International Normalized Ratio hiClot user manual 2 2 PT Prothrombin time 13 mally found on the reagent box and generally depends on the production lot rey Let us suppose that the current ISI value is 1 00 and it HE should be 1 08 Proceed as follows 1 Press the previous value disappears and a ap pears accept it pressing 2 Press a decimal point is shown accept it pressing T81 3 Press besides the decimal po
13. second row of the display shows the time elapsed to the right the measured absorbance in an arbitrary scale to the left 7 Each hiClot is identified by a serial code written on a sticker under the instrument composed by HC four digits a dash and four more digits This code is needed to download the software package to interface the hiClot to a PC hiClot user manual Chapter 3 Security and installation rules In order to use the hiClot at its best please follow carefully the security rules and installation advices contained in the following paragraph 3 1 Security rules 1 Read carefully and preserve this manual 2 Before cleaning disconnect all the power plugs 3 For cleaning the instrument do not use cleaning liq uids aerosols or any other detergent that can penetrate into the it use only a damp cloth 4 Do not modify in any way the instrument without an explicit authorization of the builder 5 Do not store or use the system in a wet or excessively damp environment 6 Do not place the system on an unstable plane hiClot user manual 22 Security and installation rules 7 The back and bottom of the unit has holes aimed at cooling the instrument keep them clear 8 Connect the system only to a power supply conforming the rules and able to deliver the needed power 9 An uninterruptible power supply UPS is strongly rec ommended 10 A ground connection of the power lines is required for a correct
14. the current method by means of the and keys Pressing the selection goes to the next method in the middle of the row now the PTT method is surrounded by the hiClot user manual 2 1 Basics 9 parentheses Pressing once more the selection goes to the a subsequent method now TT is among parentheses Please note that the arrow to the right disappears so we are at the end of the list while an arrow is now to the left since the head of the list is no more shown Pressing Q goes back and the previous method is selected The methods are ordered as follows PT PTT TT e FIB In order to start an assay a cup must be inserted into the measurement well the photometer reveals its presence and starts waiting for the reagent dispensation Now the display shows the selected method and Reagent If the instrument is not calibrated for the chosen method an asterisk is shown in such case no other result but the clot formation time will be delivered Press and the displays shows a three second count down Insert the reagent when the countdown ends the stop p watch starts an it will stopped by the clot formation After that the instrument shows the time and if calibrated the cal F culated results depending on the chosen method as indicated by the following table display P Prothrombin time P Ratio PT PT oot INR Ratio Activity PTT Partial Prothrombin time sec XXX X Ratio PT PLT yee TAXE TT Thrombin
15. time Sec XXX X Ratio TT TT hoot XX XX FIB Derived Fibrinogen sec XXX X Concentration mg dl XXX The last column to the right contains the significant fig ures for instance XXX X means that the result will be shown hiClot user manual ETT pod Erg pes Saree a td 10 Assays by means of a number having one decimal figure and up to three figures before the decimal point example 12 3 If you take out the cup from the reading well during the clot formation the measurement is interrupted and hiClot sets for a new reaction The maximum time for the clot for mation is 300 sec after which the test is anyway interrupted For the PT PTT and TT assays the ratio computations needs the PIpool PLT oot and T I poo Coefficients that should be determined on a pool of sera that represents well the healthy population of the laboratory 2 2 PT Prothrombin time The Prothrombin or factor II is a glycoprotein synthesized by the liver whose concentration in plasma is 10 16 mg dl The PT test detects the activity of the coagulation fac tors of the extrinsic pathway specifically the III V VII ad X some of which are influenced by the oral anticoagulant therapy For this reason the PT assay is used for monitoring patients under such therapy 2 2 1 Assay execution Select the PT method Preheat the PT reagent in a reagent well Insert a hollow cup in a preheating well Put 100 uL of sample in it plasma
16. top left Clotting time in seconds 16 3 in the example top right Ratio between the sample time and that of the A calibrator 1 21 in the example bottom Fibrinogen concentration in mg dl 228 in the example 2 5 5 Reset of the FIB calibration The calibration procedure described permits to insert auto matically the calibration values measuring the calibrators In order to recalibrate the instrument it is mandatory to reset the current calibration data This can be done switching the instrument off and switching it on again pressing the key until the software version disappears hiClot user manual Thiet ans 20 Assays 2 6 General advice 1 Cold reagents stored in the refrigerator should stay at least 5 minutes in the heated reagent cups before usage 2 The insertion of reagents or starters at the end of the countdown should be fast If the insertion is too slow poor mixing of the components turns into wrong assay results 3 Please check properly the pipettes Non repeatable re sults normally are due to poor repeatability of liquid dispensation 4 Preparation of the calibration samples by means of di lutions is critical Errors made in this phase cause wrong measurements 5 This is a manual instrument so good results depend on proper manipulation of reagents and samples and on good liquid dispensation as well The operator should be trained in using both the reagents and the pipettes 6 During the assay the
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