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United States Patent (19) 11 Patent Number: 5,358,853 Butler Et Al

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United States Patent (19) 11 Patent Number: 5,358,853 Butler Et Al US005358853A United States Patent (19) 11 Patent Number: 5,358,853 Butler et al. 45 Date of Patent: Oct. 25, 1994 LIQUID THROMBOPLASTIN REAGENT (54) OTHER PUBLICATIONS (75) Inventors: James R. Butler, Chapel Hill; Juan L. Day et al, NCCLS Document H28-T, vol. 12 No. 22, Torres, Durham; Rajesh Sharma, pp. 1-14 (1992). (NCCLS is National Committee for Cary, all of N.C. Clinical Lab Standards). Z. Boda et al., “Thromboplastin reagent for prothrom 73 Assignee: Akzo AV, Arnhem, Netherlands bin time determination' Chemical Abstracts, vol. 112, 21 Appl. No.: 924,211 No. 11, Mar. 12, 1990, Abstract No. 95033f, USA. S. Kitchen et al., “A method for the determination of 22 Filed: Aug. 3, 1992 activated factor VII using bovine and rabbit brain thromboplastins: demonstration of increased levels on 51 Int. Cl. ......................... C12Q 1/56; C12Q 1/34; disseminated intravascular coagulation' Chemical Ab G01N 33/48; G01N 1/00 stracts, vol. 109, No. 1, Jul. 4, 1988, Abstract No. 2785c, (52 U.S. Cl. ........................................ 435/13; 435/18; USA. 435/63; 435/68.1; 435/810; 435/212; 435/226; G. Palareti et al. “Use of a new rabbit brain thrombo 424/2; 536/18.7; 436/63; 436/69 plastin reagent (Thromboplastin FS) in the coagulation 58 Field of Search ..................... 435/13, 18, 810, 63, laboratory' Chemical Abstracts, vol. 104, No. 15, Apr. 435/69; 514/21; 436/69, 63; 536/18.7; 424/2 14, 1986, Abstract No. 12602x, USA. 56) References Cited Primary Examiner-Jeffrey E. Russel Assistant Examiner-Louise N. Leary U.S. PATENT DOCUMENTS Attorney, Agent, or Firm-Mary E. Gormley; William 2,516,216 7/1950 Kazal .................................... 435/13 M. Blackstone 2,842,480 7/1958 Singher et al. ........................ 436/69 2,847,347 8/1958 Singher et al......................... 436/69 57 ABSTRACT 2,847,350 8/1958 Singher et al. ........................ 436/69 2,921,000 1/1960 Singher et al......................... 436/69 The invention is a stable liquid thromboplastin reagent 3,522, 148 7/1970 Adam, Jr. et al. .................... 435/13 used for measuring the performance of the coagulation 4,416,812 11/1983 Becker ............... ... 435/3 system, with a shelf life of greater than 16 months, and 4,458,015 7/1984 Jering et al. ... ... 435/13 a method of producing this reagent. 4,755,461 7/1988 Lawson et al. ... 435/13 5,091,304 2/1992 LaDuca et al. ....................... 435/13 7 Claims, 9 Drawing Sheets O.8 O7 O6 E So5O Het < C5 O4 s3 O.3 O C O2 OO O.2 O4 O6 O.8 O 1.2 TIME (MINUTES) U.S. Patent Oct. 25, 1994 Sheet 1 of 9 5,358,853 s C g 3 S O is to a s r. to g O O O O Cs CS UU9Ot? IW 30NWSOSSW U.S. Patent Oct. 25, 1994 Sheet 2 of 9 5,358,853 O o O O3 O?) CN Z P CD - dV. LL Q O O C to C to O to C to 5d no no ci ai - - d . MLAOW U.S. Patent Oct. 25, 1994 Sheet 3 of 9 5,358,853 O O (f) H O $3 2Z No r Z O S d LL N O O o OO N. co o so O O O O O O MALOV/ U.S. Patent Oct. 25, 1994 Sheet 5 of 9 5,358,853 8 O O s LO O 2 . 8g CD- Ll O N O LO Q NO CN vP O SLIN NSW OSWOH U.S. Patent Oct. 25, 1994 Sheet 6 of 9 5,358,853 O no N?) O cN NO Sa O CN Slso O 6 CD - N 1 O NS O O LO O o C to C2S CN CN O O SLIN? 'Ndl Ul U.S. Patent Oct. 25, 1994 Sheet 7 of 9 5,358,853 3. O N CD o - O CN l O O o to V. Q. C. O. C. V. O - - - - O O C. SLIN?h'Nd U.S. Patent Oct. 25, 1994 Sheet 8 of 9 5,358,853 O S OO 3Er CD E - l O O CN O OO N- O O so U.S. Patent Oct. 25, 1994 Sheet 9 of 9 5,358,853 r3 C : 3 O O O 8 3 CN CN ww. O SONOOSld 5,358,853 1 2 with sodium chloride and sodium tartrate (U.S. Pat. No. LIQUID THROMBOPLASTIN REAGENT 3,522,148). This extract can be further processed. For example, calcium lactate, glycine, carboxymethylcellu BACKGROUND OF THE INVENTION lose and imidazole can be added to the thromboplastin This invention relates to a stable liquid thromboplas 5 extract. Each additive has an effect on the sensitivity of tin reagent with a long shelf-life and a method of pro the reagent. In general, an acceptable thromboplastin ducing it. reagent must produce a PT of 9-15 seconds with a The operation of the coagulation and fibrinolysis normal blood sample. pathways of the blood system can be tested at many There are also a number of other processes used to stages for abnormalities. One of the most commonly 10 manufacture more sensitive rabbit brain thromboplastin. used tests is the prothrombin time test (PT). A sample of Hawkins et al., in WO 90/05740, published on May 31, blood or plasma is added to thromboplastin in the pres 1990, disclose a method of extracting thromboplastin ence of calcium, and the time needed to form a clot is from tissue using barium sulfate, chaotropic agents and measured. Factor VII is activated by the thromboplas nonionic detergents. However, the process produces tin, which through factors V and X, causes the forma 15 only a lyophilized thromboplastin reagent and not a tion of thrombin from prothrombin (Factor II). The liquid one. thrombin formed cleaves fibrinogen to insoluble fibrin. The time measured from the mixing of thromboplastin Another patent application, DE 3150594A1, discloses and calcium with a blood sample to the formation of a a similar process. Rabbit brain powder is mixed with clot is a measure of the concentration or activity of the cellulose powder and washed with sodium acetate coagulation factors involved. This test is used to moni buffer at Ph 6.5-8 to remove contaminants. It is then tor oral anticoagulant therapy in order to insure that the extracted with surfactants in the presence of calcium proper amount of anticoagulant is given the patient. It is ions. Again, the thromboplastin produced is stable only also used to test the performance of the coagulation in lyophilized form, with a short shelf life once it has system. 25 been reconstituted. Thromboplastin is the primary reagent for the above A liquid thromboplastin is currently available from tests. Currently, it is obtained from mammalian tissue, Pacific Hemostasis, Inc. Although it would appear that usually rabbit brains. Other thromboplastin-rich tissue, this reagent has overcome some problems associated such as human brain, human placenta and bovine brain with lyophilized reagents, it is not available as a single can be used, but is has been found that for cost, perfor 30 vial reagent. Two solutions, in separate vials must be mance and availability, rabbit brain tissue is a suitable combined to yield a reagent with only a one month source of thromboplastin. stability. The sensitivity of a thromboplastin reagent rests on a Therefore, in terms of convenience, stability and number of factors, such as the final reagent composi reliability, a liquid thromboplastin reagent would be of tion, which may include buffers, salts and stabilizers; the 35 value to the clinical and research laboratories. method of extracting the thromboplastin from tissue; and the original source of the tissue. Most of the pre BRIEF SUMMARY OF THE INVENTION pared thromboplastin reagents on the market today are The present invention is a liquid thromboplastin rea only available as lyophilized materials, primarily for gent composed of thromboplastin tissue extract, cal reasons of reagent stability. Reconstituted lyophilized cium ions, stabilizers and antimicrobials. This reagent thromboplastin reagent has a shelf life of approximately has a shelf life, in the unopened final container, of at four days. s least 16 months and once opened, of at least 10 days. Stability of this reagent is important to the clinician, Also claimed is a method for producing this liquid or user, as it is expensive and the longer the shelf life, thromboplastin reagent, the steps being: both of the opened and unopened reagent container, the 45 a) cleaning the thromboplastin-rich tissue, such as less reagent that must be discarded due to expired shelf life time. rabbit brain acetone powder (RBAP) to remove There are inherent problems associated with a lyophi extraneous, primarily non-thromboplastin contain lized product that are either reduced or eliminated in a ing matter; liquid product. These include (1) variability in the fill 50 b) extracting the thromboplastin from the cleaned ing of the vials before lyophilization; (2) shelf-to-shelf, RBAP by mixing with extraction solution at tem and shelf positional differences in the lyophilization peratures that are conducive to extraction of cycle (freezing and heating); (3) pipette errors associ thromboplastin; ated with reconstitution and/or wrong volume addi c) diluting the thromboplastin-containing supernatant tions when reconstituting the powder; and (4) water 55 with albumin and calcium ions under conditions used to reconstitute may not be pure and/or may be favorable to the formation of stable vesicles or contaminated with microorganisms. micelies; A lyophilized reagent is inherently more turbid than d) stabilizing the solution of step c) further by adjust a liquid reagent.
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