automated calculation coagulation activity and ...printer parallel adapter disk controler irma...

Journal of Automatic Chemistry of Clinical Laboratory Automation, Vol. 9, No. 4 (October-December 1987), pp. 189-193

Automated calculation of coagulationactivity and International Normalized Ratioin oral anticoagulant control

R. H. M. Peters, A. C. T. H. Gehring and F. M. F. G.Olthuis*Department ofClinical Chemistry, Hospital ’De Stadsmaten’Arinsplein 1, 7511JX Enschede, The Netherlands

Thromboplastin reagents are used to perform the prothrombin timetest in the laboratory control of oral anticoagulant therapy.Previously the measured clotting time was transformed to activityeither by reading the valuefrom a correlation curve orfrom a tablesupplied by the reagent’s manufacturer. An interface whichconnects the coagulometer to a personal computer, and software tohandle the clerical work, has been developed by the authors. Up to12 channels can be analysed simultaneously with this system. Theresults are calculated on-line and can be reported as activity, as wellas by the WHO’s International Normalized Ratio. Because ofthereduction in clerical work, a time-saving of35% was achieved inthe authors" laboratory; additionally, the chance of makingmistakes is reduced.

Introduction

In the laboratory control of oral anticoagulant therapythe prothrombin time (PT) test is the first test of choice.Most methods for measuring PT are based on themeasurement ofthe clotting time, in seconds, ofa sample.Instruments have been developed for automatic measure-ment ofPT, based on electrical or photo-optical detectionof clotting. For clinical use different methods of express-ing the PT result, for example seconds, ratio, index oractivity, are used. Moreover, thromboplastin reagentsdiffer greatly in response to the degree of anticoagulationor clotting factor deficiency. Because ofthe normalizationof the PT in oral anticoagulant control the World HealthOrganization (WHO) developed a system of thrombo-plastin calibration, and introduced the InternationalNormalized Ratio (INR) as a common scale for reportingthe PT result 1]. Whether expressing the PT result as aratio, index, activity or INR, the primary result inseconds has to be transformed to the unit used, bycalculation or by reading the value from a correlationcurve or table. This transformation can be easily carriedout by a computer connected to the coagulometer,thereby reducing the time of analysis and the chance ofmaking mistakes.

Four years ago the authors developed an interface toconnect a coagulometer to an Apple IIe microcomputer(Apple Computer, Inc., Cupertino, California, USA).Software was written in Applesoft Basic to handle theclerical work [2]. This system has been in operation forthree years without any technical failure. Later it was

* Correspondence to Dr Olthuis.

decided to use the IBM PC to connect laboratoryanalysers to the hospital’s mainframe computer. Becausethe interface for the coagulometer had been designedspecifically for an Apple II computer, an interface wasdeveloped according to the commonly used RS232protocol. The computer program was rewritten in Pascal.In this paper the way of calculating the coagulationactivity and the INR, as well as some features of thecomputer program, are described.

Materials ana methods

ApparatusThe LC-6 coagulometer (Lode Instruments, Groningen,The Netherlands) is a six-channel coagulometer, whichdetects clot formation photo-optically. The coagulometeris equipped with a pipette and the start signal is remotelycontrolled. An IBM PC-XT microcomputer (operatingsystem DOS 3.1), equipped with 640 kB random accessmemory, two disk-drives (360 kB), an asynchronouscommunication adapter, and a parallel adapter (Interna-tional Business Machines Corporation, Boca Raton,Florida, USA) (see figure 1). An IRMA 3278/79 Termi-nal Emulator Card (Digital Communications AssociatesInc., Alpharetta, Georgia, USA) was used to link theIBM PC-XT on-line to the hospital’s mainframe (anIBM 4361), which contains the patient data-base. Forprinting Epson FX85 printer (Seiko Epson Corporation,Nagano, Japan) was used.

An interface (RS 232) was developed to connect the LC-6to a microcomputer (see Appendix). This interface wasbuilt in collaboration with Lode Instruments and wasbuilt into the LC-6. The interface was designed to allow asecond LC-6 to be connected to the interface. Thecomputer program was written in Pascal (IBM Pascalcompiler, version 2.0).

ReagentsThrombotest TM thromboplastin reagent was purchasedfrom Nycomed & Co. (Oslo, Norway).

Calculations

Expression of the PT result as activity

Loeliger et al. [3] observed for the Thrombotest reagent alinear relationship between the clotting time ratio(patients’ time/normal time) and 100/c, in which c is themean concentration of the clotting factors II and X inpercentage of normal. As is shown in figure 2, a linear

189

R. H. M. Peters et al. Automatic calculation of coagulation activity

IBM PC-XT monitor

asynchronous

communicationadapter

printer

parallel

adapter

disk controlerIRMA 3278/79

terminal

emulator card

interface

coagulometerEpson printer

Figure 1. Schematic diagram of the computerized coagulometer.

4O

20

0 O0 200 300

Clotting time (s)

Figure 2. Relation between % coagulation activity and clottingtime for the thrombotest reagent batch No. 916.

190

2 diskdrivesIBM 4361

mainframecomputer

relationship exists between the clotting time and 100/%coagulation activity, where the coagulation activity isread from the correlation table supplied by the manufac-turer. This principle is used to calculate the coagulationactivity: for every new batch of thrombotest reagent thelinear regression line of the clotting times and 100/%coagulation activity is calculated. With the calculatedslope and intercept the coagulation activity of the patientsamples is calculated from the measured clotting time bythe equation: Coagulation activity(%)

100/(clotting time slope + intercept).

Expression of the PT result as INR

INR is calculated with the following equation:INR (PTpatient/PYnormal) ISI in which PTpatien is themeasured prothrombin time in s, PTnormal the meannormal prothrombin time in s and ISI the InternationalSensitivity Index [4]. The ISI is supplied by themanufacturer and is specific for one batch of thrombo-plastin reagent. PTnormal has to be measured by thelaboratory itself and is specific for a batch of thrombo-plastin and the method used.

Results

To investigate the method of calculating the percentagecoagulation activity, the calculated coagulation activitywas compared with that read from the correlation cable in


initialization

changing

batch data

preparing

analysisstatistics

building up

worklistanalysis

reporting

results

Figure 3. Schematic diagram ofthe main modules ofthe program.

the package insert for 13 different batches of thrombotestreagent. The regression lines of the clotting times and100/% coagulation activity were calculated for fourdifferent sets of data per batch:

(1) All of the 30 data-points supplied by the manufac-turer.

(2) The data-points of 100, 60, 30, and 15% activity.



With the calculated slope and intercept for each data-setthe percentage coagulation activity was calculated fromthe clotting time values of the 30 data-points read fromthe correlation table. The coagulation activities calcu-lated with data-set (1) and (3) did not differ significantly(Wilcoxons’ Signed Rank Test) from the coagulationactivities read from the correlation table for all the 13batches tested. However, when the activities were calcu-lated with data-set (2) and (4), three (p < 0"001)respectively, two (p < 0"01 batches differed significantly.Because the results with the four-point regression line ofdata-set (3) were as good as that with the 30-pointregression line ofdata-set (1) data-set (3) was used in theprogram to calculate the coagulation activity.

Briefdescription of the computer programThe main modules of the program are shown in figure 3and will be described below.

Initialization: the devices (printer, asynchronous com-munication adapter and IRMA card) will be initializedand the batch data are loaded from disk into memory.

Changing batch data: when a new batch of thrombotestreagent is used, the data concerning that batch have to beentered into the microcomputer through the keyboard asfollows"

ch

Intel

yes

yes

V

I aoelon

ohannel

to patient

,lput result

wo’klist

(b)

Figure 4(a). Flowchart of the subprogram ’analysis’.

Figure 4(b). Flowchart of the subroutine ’check interface’.

(1) Batch number.

(2) The clotting times corresponding to 80, 40, 20 and10% coagulation activity, respectively. From thisdata the regression parameters slope and interceptare calculated automatically.

(3) The ISI and the mean normal PT, to compute theINR. These data are stored on disk. In this part oftheprogram one can define whether the samples shouldbe analysed singly or in duplicate, and whether theresult should be reported in percentage activity, INRor both.

Preparing analysis: a previously compiled work-list isloaded from disk into memory.

Building up the work-list: to create the work-list suchpatient information as sample identification number,name and date of birth can be entered in the computerthrough the keyboard or can be downloaded from themainframe by the PC- mainframe link. Depending onthe lay-out of the work-list, the information and testresults of at least 750 patients can be held in memory atany one time.

191


Figure 5. Diagram ofthe electronic part ofthe interface.

Analysis: the samples can be either randomly analysed oranalysed in ascending order of sample identificationnumber. The operator selects a sample for analysis andstarts the determination by pipetting the citrate blood inthe tube with preheated thrombotest reagent. Theprogram checks which channel of the coagulometer isstarted and the next sample can be selected (see figure4[a]). The timer of a channel stops when a clot is formedand the result is sent to the microcomputer (figure 4[b]).The resolution of the clotting time is 0" s. If the clottingtime is less then 14"0 s, the result is rejected, because thetimer has stopped by other reasons than clot formation.The sample has to be reanalysed. From the acceptedresults, the percentage coagulation activity and/or theINR is calculated from the clotting time. Every 15samples the results are stored on disk. Samples can beanalysed in duplicate; when the difference betweenduplicate clotting times is less than by the user predefinedpercentage, the mean is calculated.

Reporting results: the results, expressed as activity, INR orboth, can either be sent to the mainframe, or can beprinted out selected by ward or by patient identificationnumber.

Statistics: for quality control up to five different controlsamples can be analysed; the results are stored on disk.

192

Means, standard deviations and coefficient of variationscan be calculated and printed on request.

Discussion

In most laboratories the percentage coagulation activityis determined from the measured clotting time by readingthe value from a correlation curve or table. With thepresent method, the coagulation activity (as a percentage)can be calculated automatically. The computed coagula-tion activities are comparable with that read from thecorrelation table supplied by the manufacturer in thewhole range of activities from 3 to 100%, when theregression line was calculated from the clotting timescorresponding with 80, 40, 20, and 10% activity and100/% coagulation activity. We also found a linearrelationship between 100/% coagulation activity and theclotting time for the PT reagent Normotest (Nycomed,Oslo, Norway), which is used to evaluate haemostaticfunction, for example in preoperative screening or inpatients with liver disease.

Several studies have shown that the instruments used forautomated measurement of the prothrombin time have a

significant effect on the result. Some of the bias due todifferent instruments is eliminated by the use ofratios [5].The INR is calculated from the ratio of the patients’


prothrombin time to the mean normal prothrombin timeand the ISI. Until now, the INR for each patient has hadto be calculated, or a correlation table has had to beprepared to avoid the need to use a calculator.

With the described system of a coagulometer connectedto a microcomputer by the interface and the computerprogram, the clotting time is transformed automaticallyto percentage coagulation activity and/or INR. Themicrocomputer controls which of the six channels (or 12when two coagulometers are used) is in use for aparticular patient sample, and the result is calculatedon-line and stored on disk. Because of the reduction inclerical work, the chance of making mistakes, as well asthe time ofanalysis, is reduced. In our laboratory about a35% time-saving was achieved.

The present system of interface and software is alsoapplicable to other coagulometers.

With the developed computer program it is easy to reportthe Thrombotest result as activity, as well as INR. Thisdouble reporting system, as recommended by Loeliger etal. [6], will hold to introduce the INR calibration systemto clinicians and general practitioners.

References

1. WHO Expert Committee on Biological Standardization,Technical Report Series 687 (WHO, Geneva 1983), p. 81.

2. PETERS, R. H. M. and OLTHUIS, F. M. F. G., Abstract in theproceedings of the 3rd symposium on Automatisering inMedische Laboratoria Groningen, 1985).

3. LOELIGER, E. A., MEUWISSE-BRAUN, J. B., Muis, H.,BUITENDIJK, F. J. J., VELTKAMP, J. J. and HEMKER, H. C.,Thrombosis et Diathesis Haemorrhagica, 23 (1970), 569.

4. International Committee for Standardization in Haema-tology/International Committee on Thrombosis and Hae-mostasis, Thrombosis and Haemostasis, 53 (1985), 155.

5. VAN DEN BESSELAAR, A. M. H. P., Haemostasis, 15 (1985),271.

6. LOELIGER, E. A., POLLER, L., SAMAMA, M., THOMSON, J. M.,VAN DEN BESSELAAR, A. M. H. P., VERMYLEN, J. andVERSTRAETE, M., Thrombosis and Haemostasis, 54 (1985), 515.

Appendix: Brief description of the interface

The electronic components of the interface are shown infigure 5. The interface’s microprocessor checks the statusof the channels of the coagulometer 10 times a second:when the timer of a channel is running the status is 1,otherwise it is 0. Using the change in status themicroprocessor calculates the clotting time. The personalcomputer checks the status of each channel by sending acode to the interface. The interface responds by sendingthe status to the PC. When a timer of the coagulometer isstopped, the status is changed and the PC sends a codespecific for that channel to the interface. The interfaceresponds by sending the clotting time of that channel tothe PC.

SOME 39th PITTSBURGH SYMPOSIA22-26 February 1988, New Orleans, USAAlternatives in atomic spectrometry arranged byG. M. Hieftje, Indiana UniversityAnalysis ofhigh Tc superconducting oxides arrangedby W. A. Byers, Westinghouse Research and DevelopmentThirty-year anniversary of capillary (Gas) chro-matography arranged by L. S. Ettre, Perkin-ElmerFlow injection analysis in process control andbiotechnology arranged by J. F. Coetzee, University ofPittsburgh and J. Ruzicka, University of WashingtonFuture potential of microcolumn separation tech-niques arranged by M. L. Lee, Brigham Young UniversitySeeing atoms: building, buying and using scanningtunneling microscopes arranged by B. J. Bulkin andD. A. Chernoff, Standard Oil Research and DevelopmentSpectroscopic investigation of antiques arranged byG. L. Vassilaros, Crucible Materials CorporationField-flow fractionation arranged by J. C. Giddings,University of UtahNear, infrared, spectroscopy arranged by E. W. Starck,KES AnalysisCapillary zone electrophoresis arranged by A. G.Ewing, Pennsylvania State UniversityMS/MS in analytical chemistry: trends and fore-casts arranged by K. L. Busch, Indiana University

Mass spectrometry of large molecules arranged byF. W. McLafferty, Cornell University and A. Benning-hoven, University ofMinsterOn-site environmental analysis arranged by S. L.Shockey, Center for Hazardous Materials Research, andK. R. Martin, PENN-RUN CorporationEnergy dispersive X-ray fluorescence spectroscopyarranged by F. W. Kunz, Ford Motor CompanyAdvances in Raman spectroscopy arranged by S. A.Asher, University ofPittsburghASTM E-42: probing chemistry at surfaces arrangedby R. W. Linton, University ofNorth CarolinaDrug testing in the workplace arranged by H. S. Hertz,National Bureau ofStandardsArtificial intelligence applications in analyticalchemistry arranged by S. H. Peterson, WestinghouseResearch & Development CenterMicroelectronic chemical sensors arranged by R. E.Dessy, Virginia Polytechnic Institute and State UniversityOrganizing and managing the analytical laboratoryarranged by J. G. Grasselli, Standard Oil Research &DevelopmentUser-manufacturer information exchange sympo-sia (UMIX) arranged by Robin L. Garrell, University ofPittsburghDetails from Pittsburgh Conference, Dept. UPD, 12Federal Drive, Suite 322, Pittsburgh, PA 15235, USA.

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