Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/84—Systems specially adapted for particular applications
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/745—Assays involving non-enzymic blood coagulation factors
  • G01N2333/75—Fibrin; Fibrinogen

Definitions

• the invention relates to a method for determining the concentration of fibrinogen in blood plasma using thrombin as a reagent.
• Prior methods of using thrombin to measure fibrinogen concentration include the Clauss method which is based on measuring the time it takes for a plasma-thrombin reaction to occur (clotting time) and the ACL3 fibrinogen assay.
• the Clauss method is described in Manual of Hemostasis and Thrombosis, ed. 3, by Arthur R. Thompson and Laurence A. Harker, Appendix A, p. 179 (1983) and in Gerrinnunqs physioloqische neglected Methode zur Bestimmunq des Fibrinogens by A. Clauss, Acta Haematol, 17:237 (1957).
• the ACL3 method is described in Method for the Determina ⁇ tion of Functional (Clottable) Fibrinogen by the New Family of ACL Coagulometers by E. Rossi, P. Mondonico, A. Lo abar- di, L. Preda, Thrombosis Research 52; 453-469 (1988). These methods rely on the measurement of a relevant parameter such as clotting time or changes in optical transmission and on multiple dilutions of a calibrator plasma to compensate conditions of the instrument and reagent at a given time. Using calibrator plasmas (i.e. plasma having known fibrinogen concentrations) "standard lines" or "calibration curves" must be constructed repeatedly whenever conditions warrant.
• calibrator plasmas i.e. plasma having known fibrinogen concentrations
• the relevant quantity such as clotting time
• concentration is then "read" from the standard curve.
• This process can involve considerable calculation, and is often tedious and time consuming.
• the relevant quantity being measured is often instrument depen ⁇ dent, as well as reaction dependent.
• the instrument used to measure the relevant parameter employs an electro-optical system in which scattered or transmitted light is detected
• the value obtained from the measurement will depend on the signal level measured by the optical sensor, which in turn depends on the amount of light incident on the reaction vessel as well as the electronic gains used in association with the optical sensor.
• the values of these quantities do not remain constant in time, nor do they remain constant from channel to channel or instrument to instrument.
• the present invention provides a method for measuring the concentration of fibrinogen in a blood plasma sample.
• a sample of plasma containing fibrinogen is provided in a container.
• Thrombin is added to the sample and mixed with the sample to form a reaction mixture.
• An initial optical transmit- tance is measured for the reaction mixture.
• the thrombin and fibrinogen are allowed to react with each other in the reaction mixture.
• a final optical transmittance is measured for the reaction mixture.
• the measurements are manipulated in the manner described below and concentration of fibrinogen is determined from a previously established standard curve.
• the standard curve is constructed in such a manner that it remains unchanged by variations in instrument, reagent or sample. Therefore, once established, it is not necessary to repeatedly reconstruct it.
• the present method is preferably used in conjunction with an optical monitoring system such as that disclosed in concurrently filed and copending U.S. Patent Application Serial No. 07/443,952 to Swope et al., entitled “Multichan ⁇ nel Optical Monitoring System", assigned to the assignee of the present application, the disclosure of which is incorporated herein by reference, or in conjunction with commercially available hemostasis instruments such as the assignee's model Coag-A-Mate XC or model Coag-A-Mate XM. Approximate reagent/plasma concentrations that are suitable for the method of the invention are known from the Clauss fibrinogen method noted above.
• the thrombin con ⁇ centration is preferably about 100 NIH units (a strong thrombin concentration) and the plasma sample is preferably diluted in a 1:10 ratio (a weak plasma concentration) with Owren *s Veronal Buffer (sodium barbital) .
• Owren *s Veronal Buffer sodium barbital
• the formation of fibrinogen is photo-optically monitored for total change between the optical transmittance before the onset of the reaction and the optical transmittance at the conclusion of the reac ⁇ tion.
• reagent is added to plasma and, after a time which allows for complete sample-reagent mixing, an initial transmittance signal (Ti) is recorded.
• Ti initial transmittance signal
• Tf final transmittance signal
• the relevant parameter, delta or D is computed the initial and final transmittance measurements by normalizing the difference in the readings to the initial value plus any offset using the following equation:
• D is the normalized digital value of delta
• Ti is the digital value of the transmitted light prior to the onset of the clot
• Tf is the digital value of the transmitted light subsequent to the formation of the clot
• SQ is the digital offset that may have been imposed as part of the instrument design
• K is an arbitrary constant chosen for convenience. It should be noted that in prior methods, D was defined as the difference (Ti ⁇ Tf) only. The denominator in the above expression represents the normalization of D to the initial value of the transmittance.
• the next step in determining the concentration of fibrinogen of an unknown sample is to refer the above determined value of D to the concentration by the use of a standard curve. This is done by first computing the quantity
• D c is the previously determined delta for a calibrator plasma of known fibrinogen concentra- tion.
• Measurements of D c are performed relatively infrequently as changes in test conditions warrant.
• the next step is to use a previously determined correlation equation which describes the relationship between R and fibrinogen concentration to determine the fibrinogen concentration of the sample. It has been discovered that the correlation equation relating R and fibrinogen concentration does not change significantly with different designated reagents and calibrator plasmas. Therefore, it can be permanently stored as part of the computational software and does not require periodic recomputation.
• the correlation equation is preferably derived as follows: Various standard plasmas of known fibrinogen concentration are prepared and a delta value D s is deter- mined for each standard plasma. Next, a value R s is calculated for each standard plasma based on the following equation:
• R s is the R value for a standard plasma
• D s is the measured delta value for the standard plasma
• D c is the previously determined delta for the calibrator plasma.
• the correlation equation is then derived by plotting R s versus log(C s /C c ) for the various standard plasmas where C s is the fibrinogen concentration of a standard plasma and C c is the fibrinogen concentration of the calibrator plasma.

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• Urology & Nephrology (AREA)
• Biotechnology (AREA)
• Cell Biology (AREA)
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• Food Science & Technology (AREA)
• Medicinal Chemistry (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Plasma & Fusion (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Steroid Compounds (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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• 1990
  • 1990-11-23 IE IE424490A patent/IE904244A1/en unknown
  • 1990-11-28 ZA ZA909564A patent/ZA909564B/xx unknown
  • 1990-11-30 EP EP19910900512 patent/EP0502103A4/en not_active Withdrawn
  • 1990-11-30 CA CA002068221A patent/CA2068221A1/en not_active Abandoned
  • 1990-11-30 JP JP3501113A patent/JPH05503008A/ja active Pending
  • 1990-11-30 WO PCT/US1990/006988 patent/WO1991008460A1/en not_active Application Discontinuation
  • 1990-11-30 KR KR1019920701275A patent/KR920704117A/ko active IP Right Grant
  • 1990-11-30 AU AU68983/91A patent/AU641459B2/en not_active Ceased
• 1992
  • 1992-05-21 FI FI922312A patent/FI922312A0/fi not_active Application Discontinuation

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