Patent Application: US-71386307-A

Abstract:
a method in which the cleavage profile and size distribution of von willebrand factor multimers is analyzed , includes : providing a sample medium of human body fluids comprising a plurality of vwf multimers of different size ; enrichment or purification of the vwf multimers by cryoprecipitation or chromatography to obtain a separated preparation of the vwf multimers from said sample medium ; exposing the separated preparation of vwf multimers to a light source to produce signals obtained by vibrational spectroscopy ; detecting said signals ; transformation by mathematical alogrithms ; generation of patterns based on computing of data of original resonance spectra and determining the cleavage profile and the size distribution of said separated vwf multimers by chemometrics ; and acquisition of a databank obtained from healthy individuals for identifying subjects at risk of developing at least one of the following diseases : sepsis , coagulopathy , thrombotic disease , infection , and inflammation .

Description:
citrated plasma specimens for measurement of vwf - antigen ( vwf : ag ), vwf multimer analysis and adamts13 activity were stored at − 80 ° c . for later assaying . vwf : ag was measured by an enzyme - linked immuno - sorbent assay , using polyclonal anti - vwf antibody ( dako , hamburg , germany ). adamts13 activity was determined by the collagen binding method using recombinant human vwf as substrate [ 23 , 24 ]. all samples were tested at dilutions 1 : 10 , 1 : 20 and 1 : 40 , and mean values were calculated . intra - and inter - assay coefficients of variation were 12 % on the basis of six replicates and 16 % in six different runs , respectively . one unit / ml ( u / ml ) adamts13 activity is defined as the proteolytic activity of one ml pooled normal human plasma from 45 healthy individuals presenting 100 % activity . the detection limit of the assay was 0 . 05 u / ml . the specificity of the assay was verified by dilution experiments with plasma from ttp patients containing autoantibodies [ 23 ]. by dilution experiments with normal plasma the presence of autoantibodies was excluded in all patient samples . multimer analysis of vwf was carried out by agarose gelelectrophoresis ( 60 v , 16 ° c ., 15 h ) in a multiphor ( amersham pharmacia biotech , freiburg , germany ) using 1 . 2 % lgt - agarose ( sigma - aldrich , seelze , germany ). after blotting on nitrocellulose membranes ( 33v , 2 . 5 ma , 2 h ) luminescent visualisation was performed using hrp - vwf - ab ( dako hamburg , germany ) and ecl - detection kit from biorad [ 25 - 27 ]. plasma samples were normalized to an equal amount vwf : ag ( 0 . 1 - 0 . 05 u / ml ). for comparison , vwf multimers of a normal plasma pool ( npp ; n = 40 ) are shown . illustrations of all gels in the present description are without any use of non - linear adjustment . the amount of high molecular weight multimers in npp was defined as 100 % and compared to those of patients . preanalytical sample preparation : 200 μl citrated plasma from healthy controls as well as from patients with thrombotic microangiopathy of different origin diagnosed by a residual proteolytic activity of adamts13 & lt ; 20 % as determined by the method of gerritsen [ 24 ] was cryoprecipitated by thawing on ice for 45 min after freezing at − 80 ° c . for about 6 h . afterward the samples were centrifugated at 18 , 000 g for 30 min at 4 ° c . the supernatant was discarded and the cryoprecipitated pellet was resolubilized in a total volume of 20 to 30 μl phosphate - buffered saline ( pbs ) solution . for uv - raman analysis , the cryoprecipitated protein mixture was spotted onto fused silica plates and dried in vacuo . the plasma components were purchased by sigma - aldrich ( taufkirchen , germany ). recombinant factor viii was purchased from baxter ( wien ). the substances were diluted in water or pbs in appropriate concentrations and spotted onto fused silica plates and dried in vacuo . for comparison of vwf multimers differing in length and thrombophilic activity , we used two different vwf specimens : ( i ) ultralarge vwf was obtained from recombinant protein synthesis 20 and ( ii ) low - molecular - weight vwf was obtained by limited proteolysis of rhvwf by adamts13 at mild denaturating conditions ( 1 . 5 m urea ) in the presence of ba 2 + over 2 h as described [ 23 ]. spectroscopic instrumentation : the uvrr data were collected on a micro - raman instrument ( hr800 , horiba / jobin yvon ) equipped with a 2400 groove mm − 1 grating and a cryogenically cooled ccd detector . an intracavity frequency doubled argon ion laser ( innova 300 , fred , coherent ) provided the 243 . 993 nm continuous wave laser lines . approximately 1 mw was delivered to the sample . the wavenumber accuracy of the hr800 spectrometer is ± 4 cm − 1 . incident light on the sample and 180 ° backscattered light was collected by a broadband anti - reflection coated uv micro spot objective ( lmu uvb , 40 ×/ 0 . 50 ) with a working distance of 1 mm . photochemical decomposition was limited by rotating the blood plasma samples at 6 rpm on a turning knob , whereby the turning knob has been moved in the xy - direction after each turn . a video camera , which is sensitive in the uv and in the visible spectral range , was used for positioning of the samples under the microscope . the spectrometers entrance hole was set to 300 μm . an accumulation time of 120 s - 240 s was chosen for each spectrum . the uvrr data were collected on a micro - raman instrument ( hr800 , horiba / jobin yvon ) equipped with a 2400 groove mm − 1 grating and a cryogenically cooled ccd detector . an intracavity frequency doubled argon ion laser aq6 ( innova 300 , fred , coherent ) provided the 243 . 993 nm continuous wave laser beam . approximately 1 mw was delivered to the sample . the wavenumber accuracy of the hr800 spectrometer is 4 cm − 1 . incident light on the sample and 180 ° backscattered light was collected by a broadband antireflection - coated uv microspot objective ( lmu uvb , 40 ×/ 0 . 50 ) with a working distance of 1 mm . photochemical decomposition was limited by rotating the blood plasma samples at 6 rpm on a turning knob , whereby the turning knob was moved in the xy - direction after each turn . a video camera , which is sensitive in the uv and the visible spectral range , was used for positioning of the samples under the microscope . the spectrometer &# 39 ; s entrance hole was set to 300 μm . an accumulation time of 120 - 240 s was chosen for each spectrum . an unsupervised classification chemometric method , the hierarchical cluster analysis , was applied to differentiate between cryoprecipitated plasma samples of healthy controls and patients with tma , which was performed by the use of the program opus ident from bruker . in fig1 ( a ) and 1 ( b ) , various uv - resonance raman spectra of plasma samples of healthy donors ( fig1 ( a ) ) and patients with thrombotic microangiopathy ( fig1 ( b ) ) are represented , illustrating considerable variations in the absolute and relative intensities of the bands of 1551 , 1615 , and 1650 cm − 1 between patients with tma and healthy donors . differences of the absolute intensities are due to the inhomogeneous distribution and variations in thickness resulting from surface tension across the plasma spot . variation in the background intensities can be attributed to mild pyrolysis of the sample despite moving the sample during measurement . fig2 illustrates the influence of photochemical degradation of the plasma samples during measurement with an accumulation time of 2 min . when the sample was rotated and moved in the xy - direction after each turn during spectrum recording , the best resolution of the spectrum was demonstrated compared with that obtained when the sample was rotated and not moved in the xy - direction ( fig2 b ) and when the sample was measured by keeping the laser beam position fixed on the sample ( fig2 c ). therefore the samples were rotated and moved during measurement to minimize the photochemical decomposition of the plasma samples . additionally , there is a significant change in the relative intensities of the two principal bands at 1615 and 1650 cm − 1 for healthy donors . most of the samples show an increased intensity at 1615 cm − 1 compared with the signal at 1650 cm − 1 . the signal at 1551 cm − 1 is due to vibrations of tryptophan and the amide ii vibration . the amide ii vibration reflects the n — h bending coupled with the c — n stretching mode . the signal at 1615 cm − 1 can be attributed to in - plane ring stretching vibrations of aromatic amino acids . the band at 1650 cm − 1 can be assigned to the amide i vibration , the c — o stretching , and n — h in - plane bending vibration , and the amino acid phenylalanine . the amide iii mode is located at 1243 cm − 1 and results from the n — h and c — c - vibration . to characterize cryoprecipitated human plasma and to elucidate the differences of the relative intensity of the bands at 1615 and 1650 cm − 1 for healthy controls , different plasma components such as high abundance proteins were investigated . fig3 a represents uv - resonance raman spectra of a plasma sample of a healthy donor ( a ) opposed to clotting factor viii ( b ), ultra large vwf multimers ( c ), proteolyzed vwf ( d ), fibrinogen ( e ), and glucose ( f ). glucose exhibits various bands . the two prominent bands at 1334 and 1124 cm − 1 can contribute slightly to the plasma spectrum . the spectrum of fibrinogen ( e ) reveals nearly the same bands as a plasma sample featuring some differences in the relative intensities of the three bands at 1551 , 1615 , and 1650 cm − 1 . furthermore proteolyzed vwf fragments ( d ) that give an intense band at 1009 cm − 1 and some small signals at 1176 , 1543 , 1580 , 1616 , and 1650 cm − 1 were analyzed . ultra large vwf multimers ( c ) were investigated that are present in a complex with factor viii in patients &# 39 ; plasma samples . this spectrum looks also similar to that of the plasma sample , although the bands are less intense . the spectrum of clotting factor viii ( b ) shows two prominent bands at 874 and 1446 cm − 1 and various weaker peaks at 1145 , 1250 , 1320 , 1366 , 1567 , and 1567 cm − 1 . in fig3 b , uv - resonance raman spectra of blood plasma of a healthy donor ( a ), pbs ( b ), tryptophan ( c ), tyrosine ( d ), and phenylalanine ( e ) are illustrated . the plasma samples were resolubilized in pbs , hence pbs was measured to exclude distortion arising from the buffer . the spectrum of pbs ( b ) shows an intense signal at 960 cm − 1 and some small bands at 860 , 1092 , and 1134 cm − 1 . these bands are not detectable in the human plasma spectrum , excluding an attributable role of pbs in the assay system . since using uv - resonance raman spectroscopy aromatic amino acids are discriminatory enhanced , three important amino acids , tryptophan , tyrosine , and phenylalanine , were analyzed . tryptophan ( c ) exhibits characteristic bands at 758 and 1009 cm − 1 due to symmetric benzene / pyrrole in - phase and out - of - phase breathing modes . the signals at 1340 and 1356 cm − 1 can be attributed to the vibration resulting from the fermi resonance between the n1 - c8 stretching in the pyrrole ring and combination bands of the out - of plane bending . the signal of the c — c stretching vibration of the pyrrole ring is located at 1551 cm − 1 . the c — c stretching mode of all aromatic acids gives a band at 1615 cm − 1 . the symmetric ring stretching mode of tyrosine ( d ) is located with an intense band at 829 cm − 1 connected with a shoulder at 851 cm − 1 . the signal at 1173 cm − 1 can be assigned to the in - plane c — h bending vibration . the band at 1208 cm − 1 can be attributed to the ring c — c - stretching mode of tyrosine and phenylalanine ( e ). the signal of tyrosine at 1615 and of phenylalanine at 1604 cm − 1 is due to the in - plane ring stretching vibration . an additional band of phenylalanine is seen for the ring breathing mode at 1006 cm − 1 . comparing the signals of plasma components with those of cryoprecipitated human plasma , the most common peaks arise from the amino acids tryptophan , tyrosine , and phenylalanine . fig3 a shows uv resonance raman spectra of a human plasma sample of a healthy donor ( a ), recombinant factor viii ( b ), ultralarge vwf multimers ( c ), proteolyzed vwf fragments ( d ), fibrinogen ( e ) and glucose ( f ). fig3 b shows uv resonance raman spectra of blood plasma of a healthy donor ( a ), phosphate buffered saline ( b ), tryptophan ( c ), tyrosine ( d ) and phenylalanine ( e ). some plasma samples differed in intensity of yellowness . this effect could be caused by different endogenous dyes . therefore β - carotene was studied because it is often responsible for pigmentation in biological samples such as human plasma . furthermore whole blood was measured to investigate whether these variations were caused by other components of whole blood , such as cellular components . the dye hemoglobin from erythrocytes and its degradation product bilirubin were also investigated . fig4 a illustrates uv - resonance raman spectra of a human plasma sample of a healthy donor ( a ), whole blood ( b ), β - carotene ( c ), hemoglobin ( d ), and bilirubin ( e ). the spectra of whole blood , hemoglobin , and bilirubin do not reflect an increased band at 1650 cm − 1 as seen in some plasma samples . these components show an intense band at 1615 cm − 1 . hence these variations of the relative intensities between the 1615 and 1650 cm − 1 peaks do not occur because of the availability of some different blood components in the plasma sample . β - carotene shows one dominant broad band at 1640 cm − 1 ( c ). this signal does not occur at the spectrum of human plasma . thus β - carotene does not contribute decisively to the human plasma spectra ; it only contributed to the peak at 1650 cm − 1 with a slight shoulder . in addition to the various dyes , high - density lipoprotein from human plasma as a lead structure for human lipoproteins was investigated to clarify the differences in the relative intensities between the 1615 and 1650 cm − 1 peaks . in fig4 b , uv - resonance raman spectra of blood plasma of a healthy donor showing increased intensity at 1615 cm − 1 compared with the signal at 1650 cm − 1 ( a ), a human plasma sample of a healthy donor showing decreased intensity at 1615 cm − 1 relative to the signal at 1650 cm − 1 ( b ), and lipoprotein ( c ) are represented . lipoproteins exhibit an increased intensity at 1650 cm − 1 compared with the signal at 1615 cm − 1 . therefore the plasma samples with the raised band at 1650 cm − 1 offer a high content of lipoproteins . normally lipids should not be present with high concentrations in the cryoprecipitated plasma . plasma sample spectra with an absence of the increased band at 1650 cm − 1 may serve as a method for quality control of sample preparation . to classify the analyzed plasma samples of healthy donors and patients with thrombotic microangiopathy , an unsupervised method , the hierarchical cluster analysis , was performed . only spectra without an increased peak at 1650 cm − 1 were used for classification . the spectra were pretreated by vector normalization and the spectral range between 600 and 1800 cm − 1 was chosen for classification . the spectral distances between each spectrum were calculated with the standard method . ward &# 39 ; s technique was used to calculate the spectral distances between a newly created cluster and all of the other spectra or identified clusters . fig4 a shows uv resonance raman spectra of a human plasma sample of a healthy donor ( a ), whole blood ( b ), β - carotene ( c ), hemoglobin ( d ) and bilirubin ( e ). fig4 b shows uv resonance raman spectra of a blood plasma of a healthy donor showing an increased intensity at 1615 cm − 1 compared to the signal of 1650 cm − 1 ( a ), of a human plasma samples of a healthy donor showing an decreased intensity at 1615 cm − 1 relative to the signal of 1650 cm − 1 ( b ) and high density lipoprotein from human plasma ( c ). fig5 shows the dendrogram of the resultant classification of the cryoprecipitated plasma samples based on 175 spectra of 8 healthy controls and 10 different patients &# 39 ; samples . the smaller the spectral distances in the dendrogram the more similar are the spectra . the dendrogram shows a clear separation of healthy controls and patients ; however the spectrum of one healthy control was falsely classified to the patients &# 39 ; cluster for unknown reasons . this spectrum is indicated in the figure by an asterisk . the dendogram of fig5 results from hierarchical cluster analysis of plasma sample spectra of the healthy controls and patients with thrombotic microangiopathy based on the spectral range of 600 - 1800 cm − 1 . similar investigations were performed by means of ft - ir spectroscopy . ft - ir spectra were recorded with a ft - ir spectrometer ( ifs66 , bruker ) in the spectral region of 400 and 6000 cm − 1 with a resolution of 4 cm − 1 . as a radiation source a globar was used as well as a dtgs - detector ( deuterated triglycine sulfate ) for detection . in fig6 a and 6b , ft - ir spectra of plasma samples of healthy donors ( fig6 a ) and patients with thrombotic microangiopathy ( fig6 b ) are represented , illustrating an increased band in the region of 2900 cm − 1 and increased ester band in the region of 1740 cm 1 for healthy patients . in order to identify the cause of the increased bands and to characterize the spectra of cryoprecipitate several plasma components such as proteins , glucose and lipids were analyzed . in fig7 a a ft - ir spectra of a human plasma sample of a healthy donor ( a ), cryosupernate ( b ), glucose ( c ), vwf - factor viii complex ( d ) clotting factor viii ( e ), ultralarge vwf ( f ) and fibrinogen ( g ) are depicted . furthermore high density lipoprotein and cholesterol was investigated to detect the cause of the increased bands . fig7 b shows ft - ir spectra of blood plasma of a healthy donor ( a ) ( a ), of a human plasma samples of a healthy donor showing an increased intensity at 1740 cm − 1 and 2900 cm 1 ( b ), cholesterol ( c ) and high density lipoprotein from human plasma ( d ). similar to raman spectroscopy it was possible to assign this effect to lipids showing that also ft - ir spectroscopy is a feasible method for quality control of sample preparation . differences in the spectra of healthy donors and patients with tma are not easily visualizable making a hierarchical cluster analysis necessary for distinguishing between them . only spectra without an increased peak at 1740 and 2900 cm − 1 were used for classification . the spectra were pretreated by baseline correction and vector normalization and the spectral range between 600 and 1800 cm − 1 and 2540 - 3680 cm − 1 was chosen for classification . the spectral distances between each spectrum were calculated with the method scaling to first range . ward &# 39 ; s technique was used to calculate the spectral distances between a newly created cluster and all of the other spectra or identified clusters . fig8 shows the dendrogram of the resulted classification of the cryoprecipitated plasma samples based on 237 spectra of seven healthy controls and of ten different patient &# 39 ; s samples the dendrogram shows a clear separation of healthy controls and patients without any misclassification . the asterisk indicates one wrong classified spectra of a healthy donor as a patient . “ diagnosis ” in the context of the present invention refers to verifying whether an individual has suffered from an inflammatory associated coagulatory disturbance . “ prognosis ” in the context of the present invention refers to the prediction probability ( in %) an individual will suffer from an inflammatory associated coagulatory disturbance . “ therapy stratification ” in the context of the present invention refers to assessing the appropriate therapeutic treatment for the inflammatory associated coagulatory disturbance which may occur or has occurred . “ treatment monitoring ” in the context of the present invention refers to controlling and , optionally , adjusting the therapeutic treatment of an individual . “ therapeutic treatment ” includes any treatment which may alter the pathophysiological state of an individual , and includes , for example , administering of pharmaceutical drugs as well as surgical treatment ( e . g . by application of artificial surfaces like balloon dilatation , stenting ). “ spectroscopical analysis ” in the context of the present invention refers to raman spectroscopy , uv resonance raman spectroscopy , surface enhanced raman spectroscopy as well as fourier transform ( ft ) infrared spectroscopy . the present invention refers to thrombotic diseases such as inflammation associated microangiopathy , pregnancy associated microangiopathy , bone marrow transplatation associated microangiopathy , microangiopathy due to endocrine dysfunction and primary thrombotic microangiopathy such as ttp or hus , which are caused or paralleled by an altered molecular and functional structure of vwf and / or changes in the activity of adamts13 . 1 . bone r c , balk r a , cerra f b , dellinger r p , fein a m , knaus w a , schein r m , sibbald w j ( 1992 ) definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis . the accp / sccm consensus conference committee . american college of chest physicians / society of critical care medicine . chest 101 : 1644 - 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