WO1996012956A1 - Dosage immunologique conjugue a une fluorescence directe et s'appliquant a l'activation plaquettaire - Google Patents

Dosage immunologique conjugue a une fluorescence directe et s'appliquant a l'activation plaquettaire Download PDF

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Publication number
WO1996012956A1
WO1996012956A1 PCT/US1995/014041 US9514041W WO9612956A1 WO 1996012956 A1 WO1996012956 A1 WO 1996012956A1 US 9514041 W US9514041 W US 9514041W WO 9612956 A1 WO9612956 A1 WO 9612956A1
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Prior art keywords
sample
platelets
samples
selectin
platelet
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PCT/US1995/014041
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English (en)
Inventor
Bruce A. Kottke
Original Assignee
Watson Clinic Foundation
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Publication date
Application filed by Watson Clinic Foundation filed Critical Watson Clinic Foundation
Priority to JP8514149A priority Critical patent/JPH10512951A/ja
Priority to BR9507579A priority patent/BR9507579A/pt
Priority to AU40168/95A priority patent/AU689346B2/en
Publication of WO1996012956A1 publication Critical patent/WO1996012956A1/fr
Priority to NO963780A priority patent/NO963780L/no
Priority to MXPA/A/1996/004205A priority patent/MXPA96004205A/xx
Priority to FI963848A priority patent/FI963848A0/fi

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Classifications

    • 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
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • 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
    • G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
    • 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
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/7056—Selectin superfamily, e.g. LAM-1, GlyCAM, ELAM-1, PADGEM
    • G01N2333/70564—Selectins, e.g. CD62

Definitions

  • P-selectin also known as granule membrane protein-140 (GMP-140), or PADGEM protein
  • GMP-140 granule membrane protein-140
  • PADGEM protein PADGEM protein
  • the other known selectins are ELAM- 1 , a cytokine-inducible endothelial cell receptor for neutrophils, and a leukocyte surface structure which plays a role in directing the homing of lymphocytes to high endothelial venules of peripheral lymph nodes. It has recently been shown by J.-G. Geng et al., Blood.24, 65a (1989), that human neutrophils bind in a Ca 2+ -dependent manner to purified P-selectin immobilized on plastic. Furthermore, adhesion of neutrophils to endothelium stimulated with rapid activators such as histamine is mediated at least in part by P-selectin.
  • P-selectin is also involved in binding of activated platelets to monocytes and neutrophils. See, S.A. Hamburger et al., Blood.25, 550 (1990) and E. Larsen, £sll, 5_9_, 305 (1989).
  • PF4 platelet factor 4
  • ⁇ -thromboglobulin ⁇ -TG
  • metabolites of thromboxane A 2 Several changes in surface membrane glycoprotein expression can be detected during platelet activation with specific murine monoclonal antibodies. For example, as reported by S.J. Shattil, in B fifid, 2Q, 307 (1987), and C.S. Abrams et al..
  • the present invention provides a method to determine the extent of mammalian platelet activation.
  • an assay for platelet activation was disclosed which involved pre-isolation of platelets in vitro , i.e., in platelet-rich plasma.
  • this method greatly increased the accuracy and rapidity of the assay for activated platelets
  • the method of the present invention constitutes an improvement over this method in that it may be practiced on whole blood without a pre-isolation step, thus further reducing the processing time. Furthermore, cell loss that may result due to pre-washing and centrifugation is eliminated.
  • a sample of whole blood is obtained from a patient whose level of platelet activation is to be determined and divided into two portions.
  • One control sample is treated with an activation agonist to maximally activate the platelets contained therein, employing a suitable agonist such as ADP, while the other sample is not treated with exogenous .activation agonists.
  • both samples are treated with a prefixing solution, such as paraformaldehyde, and allowed to incubate for a period of time sufficient to partially fix the platelets.
  • the term "partially fixed” indicates a state in which the platelets contained in said samples will not be further activated or damaged by vortexing in the subsequent step, while the red blood cells in the sample will maintain their ability to react with the lytic agent subsequently employed. While undergoing vortexing, an erythrocytic lytic agent is added to the samples. After allowing a sufficient time for the lysis of the erythrocytes to occur, a leukocyte stabilizer is added to stop the lysis reaction. Subsequently, an amount of a cell membrane fixative is added to fully fix and to stabilize the samples.
  • the term "stabilize” indicates a state in which the samples can be stored for up to at least about 72 hours before completing the analysis.
  • Anti-P-selectin antibody is then added to each sample in an amount effective to bind to the activated platelets in each sample.
  • the antibody-activated platelet complexes in each sample are determined fluorometrically, by means of a fluorescent label that is attached to the anti-P-selectin antibody, or by addition to the complexes of a fluorescent label which binds to a binding site on the bound antibody.
  • a ratio of the fluorescence of the complexed activated platelets in the sample not exogenously activated to the fluorescence of the maximally activated platelets provides a measure of the extent of platelet activation in the mammalian donor of the platelets.
  • the present invention provides a fluorescence-conjugated immunobinding assay ⁇ FCIBA), for measuring endogenous platelet activation comprising:
  • step (d) is performed by placing the samples in a Q- Prep machine (Coulter® Corporation, Hialeah, FL) and performing a 30 second cycle.
  • the Q-Prep instrument consists of a matched, three reagent system that provides a gentle, no-wash erythrocyte lysing and fixing system which maintains leukocyte morphology and cell surface integrity. Previous to the development of this assay, the Q-prep machine had been used primarily to prepare white blood cells for flow cytometry.
  • the intra-assay coefficient of variation (CV) was 6.97%
  • the time-based inter-assay CV was 6.17%.
  • the measurement of the available sites for fibrinogen binding on platelets may be used to determine the extent of mammalian platelet activation. This analysis can be carried out on platelets isolated from a tissue or physiological fluid such as human blood, e.g., the first and second samples may comprise whole blood or platelet-rich plasma.
  • the present assay can be used to evaluate, monitor and stage platelet activation-related events associated with acute coronary syndromes, and in restenosis following percutaneous transfemoral coronary angioplasty (PTC A).
  • PTC A percutaneous transfemoral coronary angioplasty
  • the role of circulating activated platelets in these states see, for example, 1. Weinberger et al., Am. J. Card..2Q, 981 (1992); E. Minar et al., £ajj _J_ ⁇ , 767 (1989); R.S. Schwartz et al., J. Am. Col. Card.. _S_, 267 (1992) and D. Tshoepe et al., Cji ⁇ ,, £&, 37 (1993). 6/12956 PCI7US95/14041
  • Figure 1 is a schematic depiction of one embodiment of the present assay.
  • FIG. 4 Fluorescence intensity was log-transformed and the coefficient r was computed using linear regression.
  • PE phycoerythrin
  • Closed circles represent the fluorescence intensities detected using PE-conjugated CD62, while triangles represent the values detected using unconjugated CD62, and the open circles represent the levels of fluorescence intensity detected by using unconjugated CD62 and PE-conjugated CD62.
  • a dose of 2.5 ⁇ M of ADP was used in the determination of aggregation by aggregometry and by the present assay in platelets in plasma sampled from three normal donors. Values are the means ⁇ standard deviation (SD) of three samples in triplicate.
  • Coefficient r was computed using linear regression.
  • ADP-stimulated platelets in plasma from three normal donors were sampled and assayed by the present assay. Values are the means ⁇ SD of three samples in triplicate.
  • Monoclonal antibodies and polyclonal antibody preparations comprising fluorescent labels or binding sites for ligands comprising fluorescent labels are commercially available, available to the art or preparable by art-recognized procedures.
  • Representative murine anti-P-selectin antibodies are listed in Table I, below.
  • CD62 Phyoerythrin or no label Becton-Dickinson
  • Unlabelled antibodies can be conjugated to fluorescent labels such as fluorescein isocyanate (FTTC) by standard techniques. See, for example, S.J. Shattil et al., Blood.2 ⁇ , 307 (1987) and J.W. Goding et al., Monoclonal Antibodies: Principles and Practice - Production and Application of Monoclonal Antibodies in Cell Biology. Biochemistry and Immunology. Academic Press, London (1986) at pages 255-280.
  • the antibodies can be prepared as biotinylated conjugates and reacted with phycoerythrin-streptavidin as taught by Goding, _ ' ___., McEver et al., it ⁇ d., and S.J.
  • ADP is a preferred platelet activation-aggregation agonists
  • other useful agents for platelet activation include thrombin, serotonin, collagen and throboxane, as well as bioactive subunit polypeptides thereof.
  • the erythrocytic lytic agent of step d(i) of the present invention is preferably formic acid.
  • the lytic agent may also contain a stabilizer that serves to increase the shelf life of the agent.
  • the leukocyte stabilizer is a combination of sodium carbonate, sodium sulfate and sodium chloride.
  • the three components are in aqueous solution.
  • the leukocyte stabilizer may further comprise a stabilizing agent which serves to extend the shelf life of the solution.
  • the cell membrane fixative employed in step d(iii) is paraformaldehyde. Although paraformaldehyde is the preferred fixative, several other sell membrane fixatives are known to those of skill in the art.
  • the cell membrane fixative may further comprise a buffer solution.
  • step (d) of the assay is carried out using automated instrumentation which vortexes the samples and rapidly adds the recited reagents.
  • automated instrumentation shortens the preparation time of samples and thus allows a more rapid response in urgent clinical situations.
  • One example of such an instrument is the Q-Prep machine manufactured by Coulter® Corporation, Hialeah, FL.
  • the Q-Prep instrument consists of a matched, three reagent system that provides a gentle, no-wash erythrocyte lysing and fixing system which maintains leukocyte morphology and cell surface integrity.
  • platelets prepared in this manner offers an alternative to the use of PRP, since the results obtained using whole blood that has been treated in the Q-prep machine correlate to those obtained using PRP. See Examples 7 and 8.
  • the invention will be further described by reference to the following detailed examples, wherein adenosine diphosphate (ADP, Catalog No. 885-3), paraformaldehyde (Catalog No. 62H0174) and other chemicals were obtained from Sigma (Sigma Chemical Co., St. Louis, MO). Phycoerythrin (PE)-conjugated (Catalog No. 348107) and pure (Catalog No.
  • murine monoclonal anti-human platelet P-selectin antibodies (CD62) and PE-conjugated isotype specific mouse IgGl (Catalog No. 340013) were purchased from Becton-Dickinson (Mountain View, CA).
  • FITC -conjugated murine monoclonal anti-human platelet GPHb-IIIa antibody (Catalog No. 0649) was obtained from AMAC (Westbrook, ME).
  • FTTC- conjugated sheep anti-human fibrinogen antibody (Catalog No. K90056F) was obtained from BIO-DESIGNE (Kennebunkport, ME).
  • Antibodies were diluted using 1% fetal calf serum phosphate-buffered saline (PBS) solution.
  • PBS fetal calf serum phosphate-buffered saline
  • Platelet-poor plasma was prepared by further centrifugation of the remaining blood at 1500 xg for 10 minutes. Platelet counts were performed on the Coulter Counter (Coulter Electronics, Inc.) and PRP was adjusted with PPP to a constant count of 3.0 x 10 e-8/ml.
  • Platelet aggregation studies were performed at 37°C on a dual channel aggregometer (Dayton Dual Channel Aggregation Module), at a stirring speed of 900 rpm.
  • Optical density for PRP and PPP was set at 10% and 90%, respectively.
  • Adenosine diphosphate (ADP) (0.05 ml) was added to 0.45 ml of stirred suspension of PRP up to the final concentration as shown. The maximal or steepest slope of the aggregation tracing curve was measured.
  • FCA Fluorescence Concentration Analyzer
  • the total cell/antibody-bound fluorescence is determined by front-surface fluorimetry.
  • the instrument has a fluorimeter capable of exciting and reading at several wavelengths (400/450 nm-590/620 nm).
  • a 20 ⁇ l aliquot of diluted samples was placed in 96 well plates.
  • a 20 ⁇ l aliquot of 1:20 diluted CD62, or 1:40 diluted CD41a, or 1:50 diluted antifibrinogen Ab was added and the samples were incubated in the dark at room temperature for 30 minutes.
  • the cells in the Fluoricon assay plates were concentrated and washed three times in washing buffer (1% Tween PBS) by applying a vacuum membrane from below the plates of 25 mm Hg in the Pandex FCA machine. This step removed any unbound antibody and free fluorescence marker from the complexes.
  • Antibody- bound fluorescence was determined by reading plates at a gain of 1 for CD41a and antifibrinogen Ab and of 10 for CD62 on the FCA instrument at the appropriate wavelength. Data was recorded as relative fluorescence units (FU), after subtracting the blank.
  • Platelet concentrates were obtained from the Mayo Clinic blood bank within 24 hours of collection of blood from volunteer donors.
  • Platelet concentra t e (PC) was prepared by collecting blood (450 ⁇ 45 ml) from random donors in 63 ml of CPD in a pyrogen-free (Fenwal Laboratories, Morton Grove, TL) quad blood collection pack with an attached satellite bag containing 100 ml of ADSOL solution. After blood collection, whole blood was centrifuged for 5.2 minutes at 1400 g's at 20-24°C. The platelet-rich plasma was pressed into an empty satellite bag, leaving approximately 50 ml of PC. The PC was left undisturbed for 1 hour, was resuspended on a platelet rotator, and stored on a horizontal flatbed shaker. Twenty individual PC units were sampled after 24 hours of storage.
  • Samples were prepared for analysis by fixing 100 ⁇ l of platelets with 1 ml cold 1% paraformaldehyde for 1 hour at 4°C.
  • the platelets were washed (x2) with phosphate-buffered saline EDTA (PBS/EDTA), the pellet was resuspended in 1 ml PBS/EDTA .and stored at 4 * C in the dark.
  • the following day (within 24 hours) 50 ⁇ l of the resuspended platelets were labeled with 10 ⁇ l of monoclonal antibody CD41 (AMAC, Inc., Westbrook, ME).
  • Reactivity of platelets in plasma to ADP as measured by aggregation or by expression of P-selectin from three healthy donors were measured both by an aggregometer and by the procedure of Example 1, at time intervals ranging from 1.5 to 10 hours after blood was drawn. Platelets in plasma aggregated in response to increasing stimulating doses of ADP in parallel with the expression of P-selectin at time intervals ( Figure 9). The reactivity of platelets to ADP was increased at time intervals, and reached similar peak levels at the seventh hour ( Figure 9).
  • Detection of P-selectin translocation to the platelet surface as determined by the present method is a sensitive and specific measure of platelet activation, which correlates well with more complex traditional measures of this phenomenon.
  • simpler cells could be employed for fluorometry, the methodology using microtiter plates with filters and the measurement of front-surface fluorimetry to measure this translocation permits measurements to be made in 96 wells as a single semiautomated procedure.
  • a fluorescence analyzer with the ability to read 10 plates (960 wells) as an even more automated procedure (screen machines, IDEXX, Portland, ME) is also available.
  • a 6 ml sample of whole blood was isolated, divided into twenty 100 ⁇ l samples, and added to round-bottomed polystyrene tubes. Thirty ⁇ l of phosphate buffered saline (PBS, 0.01 M, pH 7.4, as baseline) or ADP (2.5 ⁇ M) were added to each sample and all were allowed to incubate at room temperature for five minutes. One hundred ⁇ l of 1% of paraformaldehyde PBS solution was added to each tube, and the samples were incubated at room temperature for fifteen minutes. Following this incubation, each sample was placed in a Q-Prep machine (Coulter® Corporation, Hialeah, FL) for a 30-second cycle.
  • PBS phosphate buffered saline
  • ADP 2.5 ⁇ M
  • the sample was vortexed while a series of three reagents was added.
  • the first reagent added was a solution of formic acid at a concentration of 1.2 mlJL.
  • a solution of sodium carbonate (6.0 g/L), sodium chloride (14.5 g/L) and sodium sulfate (31.3 g/L) was added.
  • a solution of paraformaldehyde was added (10.0 g/L).
  • the fixed samples were then washed twice by centrifugation in a microcentrifuge using PBS solution.
  • the washed platelets were diluted to 8.5 x 10 e-7/ml in PBS solution. All samples were analyzed within 72 hours of fixing.
  • ADP-activated PBS Control ADP Activated PBS Control
  • a 6 ml sample of whole blood was isolated, divided into twelve 100 ⁇ l samples, and added to round-bottomed polystyrene tubes. Thirty ⁇ l of fibrinogen, labeled with FTTC (1:100 dilution) were added to each sample. Subsequently, 30 ⁇ l of either ADP (2.5 ⁇ M) or phosphate buffer saline (PBS, 0.01 M, pH 7.4, as baseline) was added to each tube and the samples were allowed to incubate at room temperature for five minutes. One hundred ⁇ l of 1% of paraformaldehyde PBS solution was added to each tube, and the samples were incubated at room temperature for fifteen minutes.
  • ADP 2.5 ⁇ M
  • PBS phosphate buffer saline
  • each sample was placed in a Q-Prep machine (Coulter® Corporation, Hialeah, FL) for a 30-second cycle. During this cycle, the sample was vortexed while a series of three reagents was added. The first reagent added was a solution of formic acid at a concentration of 1.2 mlJL. Next, a solution of sodium carbonate (6.0 g/L), sodium chloride (14.5 g/L) and sodium sulfate (31.3 g/L) was added. Finally, a solution of paraformaldehyde was added (10.0 g/L). The fixed samples were then washed twice by centrifugation in a microcentrifuge using PBS solution. The washed platelets were diluted to 8.5 x 10 e- 7 /ml in PBS solution. All samples were analyzed within 72 hours of fixing.
  • Q-Prep machine Coulter® Corporation, Hialeah, FL

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Abstract

L'invention se rapporte à un procédé de mesure de l'étendue de l'activation plaquettaire qui consiste à déterminer par fluorimétrie in vitro l'étendue de l'expression de la sélectine P dans un échantillon de sang entier au moyen d'un échantillon de plaquettes activées au maximum, utilisé comme standard de référence.
PCT/US1995/014041 1994-10-25 1995-10-24 Dosage immunologique conjugue a une fluorescence directe et s'appliquant a l'activation plaquettaire WO1996012956A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
JP8514149A JPH10512951A (ja) 1994-10-25 1995-10-24 血小板活性化の直接蛍光−結合免疫測定法
BR9507579A BR9507579A (pt) 1994-10-25 1995-10-24 Processo fluorométrico para medir ativaçao de plaqueta endógena em amostra de plaquetas
AU40168/95A AU689346B2 (en) 1994-10-25 1995-10-24 Direct fluorescence-conjugated immunoassay for platelet activation
NO963780A NO963780L (no) 1994-10-25 1996-09-10 Direkte fluorescenskonjugert immunoassey for blodplateaktivering
MXPA/A/1996/004205A MXPA96004205A (en) 1994-10-25 1996-09-20 Direct immunovation by fluorescence conjugated for the activation of plaque
FI963848A FI963848A0 (fi) 1994-10-25 1996-09-26 Suora fluoresenssi-konjugoitu immunotesti verihiutaleiden aktivoimiseksi

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US32847594A 1994-10-25 1994-10-25
US08/328,475 1994-10-25
US47098095A 1995-06-06 1995-06-06
US08/470,980 1995-06-06

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JP (1) JPH10512951A (fr)
AU (1) AU689346B2 (fr)
BR (1) BR9507579A (fr)
CA (1) CA2184826A1 (fr)
FI (1) FI963848A0 (fr)
NO (1) NO963780L (fr)
WO (1) WO1996012956A1 (fr)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998021591A1 (fr) * 1996-11-13 1998-05-22 Centocor, Inc. Dosages de p-selectine et procedes d'utilisation correspondants
EP0967269A1 (fr) * 1998-06-13 1999-12-29 AEA Technology plc Lyse de cellules microbiologiques
JP2001131078A (ja) * 1999-11-09 2001-05-15 Iatron Lab Inc 新規活性固定化血小板及びその製造方法
EP1194173A1 (fr) * 1999-04-29 2002-04-10 Vanderbilt University Administration de medicament guidee par rayon x
US6913932B2 (en) * 2002-08-23 2005-07-05 Beckman Coulter, Inc. Formaldehyde-ammonium salt complexes for the stabilization of blood cells
US7049140B1 (en) 1999-04-29 2006-05-23 Vanderbilt University X-ray guided drug delivery
US8012945B2 (en) 2001-11-09 2011-09-06 Vanderbilt University Phage antibodies to radiation-inducible neoantigens
US9340581B2 (en) 2001-10-03 2016-05-17 Washington University Ligands to radiation-induced molecules
US10266488B2 (en) 2013-10-10 2019-04-23 Eastern Virginia Medical School 4-((2-hydroxy-3-methoxybenzyl)amino)benzenesulfonamide derivatives as potent and selective inhibitors of 12-lipoxygenase
US10449261B2 (en) 2014-07-24 2019-10-22 Washington University Compositions targeting radiation-induced molecules and methods of use thereof
WO2021224236A1 (fr) * 2020-05-05 2021-11-11 Etablissement Français Du Sang Methode de detection de l'activation plaquettaire liee a l'inflammation

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110250588A1 (en) * 2008-12-18 2011-10-13 Sysmex Corporation Method for detecting cancer cells in blood sample

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4783330A (en) * 1984-11-15 1988-11-08 New England Medical Center Hospitals, Inc. Monoclonal antibodies to activated platelets
US5030554A (en) * 1987-12-04 1991-07-09 Coulter Corporation Conservative whole blood sample preparation technique

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4783330A (en) * 1984-11-15 1988-11-08 New England Medical Center Hospitals, Inc. Monoclonal antibodies to activated platelets
US5030554A (en) * 1987-12-04 1991-07-09 Coulter Corporation Conservative whole blood sample preparation technique

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998021591A1 (fr) * 1996-11-13 1998-05-22 Centocor, Inc. Dosages de p-selectine et procedes d'utilisation correspondants
US6699706B1 (en) 1998-06-13 2004-03-02 Accentus Plc Cell lysis method using a vortex mixer
EP0967269A1 (fr) * 1998-06-13 1999-12-29 AEA Technology plc Lyse de cellules microbiologiques
US7875454B2 (en) 1999-04-29 2011-01-25 Vanderbilt University X-ray guided drug delivery
EP1194173A4 (fr) * 1999-04-29 2003-05-14 Univ Vanderbilt Administration de medicament guidee par rayon x
EP1194173A1 (fr) * 1999-04-29 2002-04-10 Vanderbilt University Administration de medicament guidee par rayon x
US7049140B1 (en) 1999-04-29 2006-05-23 Vanderbilt University X-ray guided drug delivery
JP2001131078A (ja) * 1999-11-09 2001-05-15 Iatron Lab Inc 新規活性固定化血小板及びその製造方法
US10086073B2 (en) 2001-10-03 2018-10-02 Washington University Ligands to radiation-induced molecules
US9340581B2 (en) 2001-10-03 2016-05-17 Washington University Ligands to radiation-induced molecules
US8927288B2 (en) 2001-11-09 2015-01-06 Vanderbilt University Phage antibodies to radiation-inducible neoantigens
US8012945B2 (en) 2001-11-09 2011-09-06 Vanderbilt University Phage antibodies to radiation-inducible neoantigens
US6913932B2 (en) * 2002-08-23 2005-07-05 Beckman Coulter, Inc. Formaldehyde-ammonium salt complexes for the stabilization of blood cells
US10266488B2 (en) 2013-10-10 2019-04-23 Eastern Virginia Medical School 4-((2-hydroxy-3-methoxybenzyl)amino)benzenesulfonamide derivatives as potent and selective inhibitors of 12-lipoxygenase
US10752581B2 (en) 2013-10-10 2020-08-25 Eastern Virginia Medical School 4-((2-hydroxy-3-methoxybenzyl)amino)benzenesulfonamide derivatives as potent and selective inhibitors of 12-lipoxygenase
US11274077B2 (en) 2013-10-10 2022-03-15 Eastern Virginia Medical School 4-((2-hydroxy-3-methoxybenzyl)amino)benzenesulfonamide derivatives as potent and selective inhibitors of 12-lipoxygenase
US10449261B2 (en) 2014-07-24 2019-10-22 Washington University Compositions targeting radiation-induced molecules and methods of use thereof
WO2021224236A1 (fr) * 2020-05-05 2021-11-11 Etablissement Français Du Sang Methode de detection de l'activation plaquettaire liee a l'inflammation
FR3109999A1 (fr) * 2020-05-05 2021-11-12 Etablissement Français Du Sang Methode de detection de l’activation plaquettaire liee a l’inflammation

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AU4016895A (en) 1996-05-15
JPH10512951A (ja) 1998-12-08
AU689346B2 (en) 1998-03-26
NO963780D0 (no) 1996-09-10
NO963780L (no) 1997-08-25
FI963848A (fi) 1996-09-26
BR9507579A (pt) 1997-09-09
MX9604205A (es) 1998-05-31
FI963848A0 (fi) 1996-09-26
CA2184826A1 (fr) 1996-05-02

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