Patent Application: US-201013515323-A

Abstract:
a method for the early prediction of risk of hypertensive disorders in pregnant women , including for example eclampsia , mild pre - eclampsia , chronic hypertension , eph gestosis , gestational hypertension , superimposed pre - eclampsia , hellp syndrome , or nephropathy .

Description:
the invention is generally based on the finding that the levels of certain metabolites are associated with pre - eclampsia , and that these metabolites may therefore function as diagnostic and / or prognostic variables of pre - eclampsia , typically at an early gestation stage . the data presented herein shows that the abundance of these biomarkers change at an early stage in the development of the syndrome , earlier than the normal symptoms of pre - eclampsia appear , and the biomarkers can therefore function as prognostic variables of pre - eclampsia risk enabling early detection of the syndrome . this is especially important as the morbidity and mortality associated with pre - eclampsia increases the later the syndrome is detected . the present invention therefore provides a means for early detection of pre - eclampsia allowing earlier clinical intervention and therefore improved outcome . embodiments of the invention also provide for systems ( and computer readable media for causing computer systems ) to perform a method for detecting and / or identifying that a pregnant woman is at risk of developing pre - eclampsia . embodiments of the invention can be described through functional modules , which are defined by computer executable instructions recorded on computer readable media and which cause a computer to perform method steps when executed . the modules are segregated by function for the sake of clarity . however , it should be understood that the modules / systems need not correspond to discreet blocks of code and the described functions can be carried out by the execution of various code portions stored on various media and executed at various times . furthermore , it should be appreciated that the modules may perform other functions , thus the modules are not limited to having any particular functions or set of functions . referring generally to fig8 , the computer readable storage media # 30 can be any available tangible media that can be accessed by a computer . computer readable storage media includes volatile and nonvolatile , removable and non - removable tangible media implemented in any method or technology for storage of information such as computer readable instructions , data structures , program modules or other data . computer readable storage media includes , but is not limited to , ram ( random access memory ), rom ( read only memory ), eprom ( erasable programmable read only memory ), eeprom ( electrically erasable programmable read only memory ), flash memory or other memory technology , cd - rom ( compact disc read only memory ), dvds ( digital versatile disks ) or other optical storage media , magnetic cassettes , magnetic tape , magnetic disk storage or other magnetic storage media , other types of volatile and non - volatile memory , and any other tangible medium which can be used to store the desired information and which can accessed by a computer including and any suitable combination of the foregoing . computer - readable data embodied on one or more computer - readable storage media may define instructions , for example , as part of one or more programs , that , as a result of being executed by a computer , instruct the computer to perform one or more of the functions described herein , and / or various embodiments , variations and combinations thereof . such instructions may be written in any of a plurality of programming languages , for example , java , j #, visual basic , c , c #, c ++, fortran , pascal , eiffel , basic , cobol assembly language , and the like , or any of a variety of combinations thereof . the computer - readable storage media on which such instructions are embodied may reside on one or more of the components of either of a system , or a computer readable storage medium described herein , may be distributed across one or more of such components . the computer - readable storage media may be transportable such that the instructions stored thereon can be loaded onto any computer resource to implement the aspects of the present invention discussed herein . in addition , it should be appreciated that the instructions stored on the computer - readable medium , described above , are not limited to instructions embodied as part of an application program running on a host computer . rather , the instructions may be embodied as any type of computer code ( e . g ., software or microcode ) that can be employed to program a computer to implement aspects of the present invention . the computer executable instructions may be written in a suitable computer language or combination of several languages . basic computational biology methods are known to those of ordinary skill in the art and are described in , for example , setubal and meidanis et al ., introduction to computational biology methods ( pws publishing company , boston , 1997 ); salzberg , searles , kasif , ( ed . ), computational methods in molecular biology , ( elsevier , amsterdam , 1998 ); rashidi and buehler , bioinformatics basics : application in biological science and medicine ( crc press , london , 2000 ) and ouelette and bzevanis bioinformatics : a practical guide for analysis of gene and proteins ( wiley & amp ; sons , inc ., 2nd ed ., 2001 ). the functional modules of certain embodiments of the invention include at minimum a determination system # 40 , optionally , a storage device # 30 , a comparison module # 80 , and a display module # 110 . the functional modules can be executed on one , or multiple , computers , or by using one , or multiple , computer networks . the determination system has computer executable instructions to provide e . g ., sequence information in computer readable form . the determination system # 40 , can comprise any system for detecting at least one of the biomarkers from table 1 . such systems will typically determine the relative abundance of the metabolite in the biological sample . standard procedures such as uplc - ms can be used . additionally one can determine other factors such as blood pressure , bmi , age , height , weight , circumference of waist , arms , legs . these factors can be used in conjunction with the biomarkers in assessing risk of pre - eclampsia . the information determined in the determination system can be read by the storage device # 30 . as used herein the “ storage device ” is intended to include any suitable computing or processing apparatus or other device configured or adapted for storing data or information . examples of an electronic apparatus suitable for use with the present invention include a stand - alone computing apparatus , data telecommunications networks , including local area networks ( lan ), wide area networks ( wan ), internet , intranet , and extranet , and local and distributed computer processing systems . storage devices also include , but are not limited to : magnetic storage media , such as floppy discs , hard disc storage media , magnetic tape , optical storage media such as cd - rom , dvd , electronic storage media such as ram , rom , eprom , eeprom and the like , general hard disks and hybrids of these categories such as magnetic / optical storage media . the storage device is adapted or configured for having recorded thereon metabolite abundance information . such information may be provided in digital form that can be transmitted and read electronically , e . g ., via the internet , on diskette , via usb ( universal serial bus ) or via any other suitable mode of communication . as used herein , “ stored ” refers to a process for encoding information on the storage device . those skilled in the art can readily adopt any of the presently known methods for recording information on known media to generate manufactures comprising information relating to these metabolites and other pregnancy factors . in one embodiment the reference data stored in the storage device to be read by the comparison module is compared , e . g ., level of metabolite in sample with a normal or pre - eclampsia control . the “ comparison module ” # 80 can use a variety of available software programs and formats for the comparison operative to compare sequence information data determined in the determination system to reference samples and / or stored reference data . in one embodiment , the comparison module is configured to use pattern recognition techniques to compare information from one or more entries to one or more reference data patterns . the comparison module may be configured using existing commercially - available or freely - available software for comparing patterns , and may be optimized for particular data comparisons that are conducted . the comparison module provides computer readable information related to the pre - eclampsia related metabolites . the comparison module , or any other module of the invention , may include an operating system ( e . g ., unix ) on which runs a relational database management system , a world wide web application , and a world wide web server . world wide web application includes the executable code necessary for generation of database language statements ( e . g ., structured query language ( sql ) statements ). generally , the executables will include embedded sql statements . in addition , the world wide web application may include a configuration file which contains pointers and addresses to the various software entities that comprise the server as well as the various external and internal databases which must be accessed to service user requests . the configuration file also directs requests for server resources to the appropriate hardware — as may be necessary should the server be distributed over two or more separate computers . in one embodiment , the world wide web server supports a tcp / ip protocol . local networks such as this are sometimes referred to as “ intranets .” an advantage of such intranets is that they allow easy communication with public domain databases residing on the world wide web ( e . g ., the genbank or swiss pro world wide web site ). thus , in a particular preferred embodiment of the present invention , users can directly access data ( via hypertext links for example ) residing on internet databases using a html interface provided by web browsers and web servers . the comparison module provides a computer readable comparison result that can be processed in computer readable form by predefined criteria , or criteria defined by a user , to provide a content based in part on the comparison result that may be stored and output as requested by a user using a display module # 110 . the content based on the comparison result , may be from normal pregnant women . alternatively , the content based on the comparison result may be women with pre - eclampsia . in one embodiment of the invention , the content based on the comparison result is displayed on a computer monitor # 120 . in one embodiment of the invention , the content based on the comparison result is displayed through printable media # 130 , # 140 . the display module can be any suitable device configured to receive from a computer and display computer readable information to a user . non - limiting examples include , for example , general - purpose computers such as those based on intel pentium - type processor , motorola powerpc , sun ultrasparc , hewlett - packard pa - risc processors , any of a variety of processors available from advanced micro devices ( amd ) of sunnyvale , calif ., or any other type of processor , visual display devices such as flat panel displays , cathode ray tubes and the like , as well as computer printers of various types . in one embodiment , a world wide web browser is used for providing a user interface for display of the content based on the comparison result . it should be understood that other modules of the invention can be adapted to have a web browser interface . through the web browser , a user may construct requests for retrieving data from the comparison module . thus , the user will typically point and click to user interface elements such as buttons , pull down menus , scroll bars and the like conventionally employed in graphical user interfaces . the methods described herein therefore provide for systems ( and computer readable media for causing computer systems ) to perform methods for detecting and / or identifying risk of a pregnant woman developing pre - eclampsia . systems and computer readable media described herein are merely illustrative embodiments of the invention for performing methods of diagnosis in an individual , and are not intended to limit the scope of the invention . variations of the systems and computer readable media described herein are possible and are intended to fall within the scope of the invention . the modules of the machine , or those used in the computer readable medium , may assume numerous configurations . for example , function may be provided on a single machine or distributed over multiple machines . the invention also provides a computer program which when executed on a computer causes the computer to perform a process for early detection of risk of a pregnant woman developing pre - eclampsia , the process comprising : inputting an abundance of at least one metabolite biomarker of table 1 obtained by assaying a test biological sample obtained from the woman at 11 to 17 weeks gestation ; comparing the abundance of the at least one metabolite biomarker with a reference abundance for the at least one metabolite biomarker ; and determining a quantitative estimate of pre - eclampsia risk based on the comparison step . typically , the abundance of at least one metabolite biomarker selected from the group of 5 - hydroxytryptophan , monosaccharide , decanoylcarnitine , methylglutaric acid and / or adipic acid , oleic acid , docosahexaenoic acid and / or docosatriynoic acid , γ - butyrolactone and / or oxolan - 3 - one , 2 - oxovaleric acid and / or oxo - methylbutanoic acid , acetoacetic acid , hexadecenoyl - eicosatetraenoyl - sn - glycerol , sphingosine - 1 - phosphate , sphinganine - 1 - phosphate , and vitamin d3 derivatives , is inputted . ideally , the abundance of a plurality of metabolite biomarkers including 5 - hydroxytryptophan , monosaccharide , decanoylcarnitine , methylglutaric acid and / or adipic acid , oleic acid , docosahexaenoic acid and / or docosatriynoic acid , γ - butyrolactone and / or oxolan - 3 - one , 2 - oxovaleric acid and / or oxo - methylbutanoic acid , acetoacetic acid , hexadecenoyl - eicosatetraenoyl - sn - glycerol , sphingosine - 1 - phosphate , sphinganine - 1 - phosphate , and vitamin d3 derivatives , is inputted . preferably , step of determining the quantitative estimate of pre - eclampsia risk comprises determining the likelihood of pre - eclampsia using a multivariate analysis which typically comprises using the abundance of the or each metabolite biomarker and distribution parameters derived from a set of reference abundance values . ideally , the multivariate analysis employs a multivariate discriminant pls - da model or a multivariate discriminant pc - cva model . the invention also provides a computer program recording medium storing a computer program according to the invention . the scope study is a prospective , multi - centre cohort study with the main aim of developing accurate screening methods for later pregnancy complications , including preeclampsia ( actrn12607000551493 ), small for gestational age ( sga ) infants and spontaneous preterm birth . full ethical approval has been obtained from local ethics committees ( new zealand akx / 02 / 00 / 364 and australia rec 1712 / 5 / 2008 ) and all patients gave written informed consent . healthy nulliparous women with a singleton pregnancy were recruited between 14 and 16 weeks and tracked throughout pregnancy . women attending hospital antenatal clinics , obstetricians , general practitioners or community midwives prior to 15 weeks &# 39 ; gestation were invited to participate in the scope study . exclusion criteria included 1 ) considered at high risk of preeclampsia , sga or spontaneous preterm birth due to underlying medical conditions ( chronic hypertension , diabetes , renal disease , systemic lupus erythematosus , anti - phospholipid syndrome , sickle cell disease , human immunodeficiency virus ), gynaecological history , ≧ 3 previous terminations or ≧ 3 miscarriages ; 2 ) had a known major fetal anomaly or abnormal karyotype or 3 ) received intervention that may modify pregnancy outcome ( e . g . aspirin therapy ). participants were interviewed and examined by a research midwife at 15 + 1 and 20 + 1 weeks of gestation and underwent an ultrasound scan at 20 + 1 weeks . at the time of interview , data were entered into an internet accessed , central database with a complete audit trail . data collected at 15 weeks included detailed demographic , obstetric and medical and family information . current pregnancy data included an early pregnancy scan to accurately calculate the estimated date of delivery . if the woman had a certain last menstrual period ( lmp ) date , the estimated date of delivery was only adjusted if either 1 ) a scan performed at & lt ; 16 weeks &# 39 ; gestation found a difference of ≧ 7 days between the scan gestation and that calculated by the lmp or 2 ) on 20 - week scan a difference of ≧ 10 days was found between the scan gestation and that calculated from the lmp . if her lmp date was uncertain , then scan dates were used to calculate the estimated date of delivery . information was collected on current pregnancy complications such as vaginal bleeding and dietary information pre - conception and during pregnancy was obtained using food frequency questions . use of folate and multivitamin , cigarettes , alcohol and recreational drugs was recorded for preconception , 1st trimester and at 15 weeks . a lifestyle questionnaire was completed by participants asking about work , exercise and sedentary activities , snoring , domestic violence and social supports . validated psychological scales measuring perceived stress ( perceived stress scale ), depression ( edinburgh postnatal depression scale ), anxiety ( short form of the state trait anxiety index measuring anxiety ) and behavioural responses to pregnancy ( adapted from the behavioural responses to illness questionnaire ) were completed . maternal physical measurements included two blood pressure recordings with mercury or aneroid sphygmomanometers , height , weight and the circumference of her waist , hip , arm and head . proteinuria in a midstream urine specimen was measured by dipstick or a protein creatinine ratio . at 20 ± 1 weeks &# 39 ; gestation , the information collected included any pregnancy complications since the 15 week interview , maternal physical measurements and the participant completed the lifestyle questionnaire . ultrasound examination at 20 ± 1 weeks included measurements of the fetus ( biparietal diameter , head circumference , abdominal circumference and femur length ) and doppler studies of the umbilical and uterine arteries . participants were followed prospectively , with pregnancy outcome data and baby measurements collected by research midwives . data monitoring included 1 ) individually checking all data for each participant , including for any data entry errors of the lifestyle questionnaire , and 2 ) using customised software to detect any systematic data entry errors . the primary outcome was preeclampsia , defined as gestational hypertension ( systolic blood pressure ≧ 140 mmhg and / or diastolic blood pressure ≧ 90 mmhg on at least two occasions four hours apart after 20 weeks of gestation , but before the onset of labour , or postpartum systolic blood pressure ≧ 140 mmhg and / or diastolic blood pressure ≧ 90 mmhg on at least two occasions four hours apart ) with proteinuria ( 24 hour urinary protein ≧ 300 mg or spot urine protein : creatinine ratio ≧ 30 mg / mmol creatinine or urine dipstick protein ≧ 2 +) or any multi - system complication of preeclampsia . multi - system complications included any of the following 1 ) acute renal insufficiency defined as a new increase in serum creatinine ≧ 100 umol / l antepartum or & gt ; 130 umol / l postpartum ; 2 ) liver involvement defined as raised aspartate transaminase and / or alanine transaminase & gt ; 45 iu / l and / or severe right upper quadrant or epigastric pain or liver rupture ; 3 ) neurological included eclampsia , imminent eclampsia ( severe headache with hyper - reflexia and persistent visual disturbance ) or cerebral haemorrhage and 4 ) haematological included thrombocytopenia ( platelets & lt ; 100 × 109 / l ), disseminated intravascular coagulation or haemolysis . an uncomplicated pregnancy was defined as a pregnancy not complicated by preeclampsia , sga , spontaneous pre - term birth or any other pregnancy complication such as gestational hypertension . in the discovery phase of the investigation , a nested case control study was performed within the initial 1628 recruits in auckland , new zealand , of whom pregnancy outcome was known in 1608 ( 98 . 8 %). sixty seven ( 4 . 2 %) women developed preeclampsia and 1021 ( 63 . 5 %) had uncomplicated pregnancies . the remainder had other pregnancy complications . sixty women who developed preeclampsia were matched for age , ethnicity and bmi to 60 controls who had uncomplicated pregnancies . the study was limited to 120 samples to guarantee optimal signal stability from the uplc - ms systems . 1 in the validation - phase of the investigation , a nested case control study was performed within the initial 596 recruits in adelaide , australia , of whom pregnancy outcome was known in 595 ( 99 . 8 %). forty six ( 7 . 7 %) women developed preeclampsia and 267 ( 44 . 9 %) had uncomplicated pregnancies . the remainder had other pregnancy complications . thirty - nine women who developed preeclampsia were matched for age , ethnicity and bmi to 40 controls who had uncomplicated pregnancies . preeclampsia was defined as a blood pressure ≧ 140 / 90 mmhg after 20 weeks gestation ( but before the onset of labor ) or in the postnatal period , with either proteinuria ( 24 h urinary protein ≧ 300 mg , spot urine protein : creatinine ratio ≧ 30 mg / mmol or urine dipstick ≧++) and / or evidence of multi - organ complications 2 . venipuncture was performed at 15 ± 1 weeks &# 39 ; gestation , and plasma samples were collected into bd edta - vacutainer ® tubes , placed on ice and centrifuged at 2400 × g at 4 ° c . according to a standardized protocol . plasma was stored in aliquots at − 80 ° c . the collection and storage conditions were identical for cases and controls , with the time between collection and storage being 2 . 07 ( sd 0 . 90 ) and 2 . 02 ( sd 0 . 96 ) hours , respectively p = 0 . 78 . all chemicals and reagents used were of analytical reagent or hplc grade and purchased from sigma - aldrich ( poole , uk ) or thermofisher scientific ( loughborough , uk ). all samples were prepared using a protein removal procedure followed by analysis using an ultra performance liquid chromatography - mass spectrometer ( waters acquity uplc system coupled to a thermofisher scientific ltq - orbitrap mass spectrometer ). raw profile data was deconvolved into a peak table using xcms software . 3 data was then subjected to strict quality assurance procedures so that statistical analysis was only performed on reproducible data . full details of all methods pertaining to sample preparation and uplc - ms analysis are described in the attached supplementary methodology file . samples were prepared by reconstitution in 70 μl hplc grade water followed by vortex mixing ( 15 seconds ), centrifugation ( 11 337 g , 15 minutes ) and transfer to vials . samples were analysed by an acquity uplc ( waters corp . milford , usa ) coupled to a ltq - orbitrap mass spectrometry system ( thermo fisher scientific , bremen , germany ) operating in electrospray ionisation mode . samples were analysed consecutively in positive ion mode followed and then consecutively in negative ion mode . chromatographic separations were performed employing an acquity uplc beh 1 . 7 μm - c 18 column ( 2 . 1 × 100 mm , waters corp . milford , usa ). solvent a and solvent b were 0 . 1 % formic acid in water and 0 . 1 % formic acid in methanol , respectively . in positive ion mode a flow rate of 0 . 40 ml min − 1 was applied with a gradient elution profile ( 100 % a for 1 minute and subsequently ramped to 100 % b ( curve 5 ) over 15 minutes , followed by a 4 minute hold at 100 % b before a rapid return to 100 % a and a hold for 2 minutes ). in negative ion mode a flow rate of 0 . 36 ml min − 1 was applied with a gradient elution program ( 100 % a for 2 minutes and subsequently ramped to 100 % b ( curve 4 ) over 15 minutes , followed by a 5 minute hold at 100 % b before a rapid return to 100 % a and a hold for 2 minutes ). the column and samples were maintained at temperatures of 50 ° c . and 4 ° c ., respectively . a 10 μl sample volume was introduced onto the column and 50 % of the column effluent was transferred to the mass spectrometer . centroid ms scans were acquired in the mass range of 50 - 1000th using the orbitrap mass analyser operating with a target mass resolution of 30 000 ( fwhm as defined at m / z 400 ) and a scan time of 0 . 4 s . mass calibration was performed before each analytical batch using an instrument manufacturer defined calibration mixture ( thermofisher scientific , bremen , germany ). comparisons of clinical data between cases and controls were performed using the student &# 39 ; s t - test , mann - whitney test , chi square test or fisher &# 39 ; s exact test , as appropriate ( sas ® system 9 . 1 ). for each metabolite peak reproducibly detected in the discovery phase study , the null hypothesis that the means of the case and control sample populations were equal was tested using either the mann - whitney test or student &# 39 ; s t - test , depending on data normality . the critical p - value for significance was set to 0 . 05 . no correction for multiple comparisons was performed at this point as the aim was to reduce the many thousands of detected features down to a subset of potentially ‘ information rich ’ peaks while keeping the number of probable false negatives ( type ii error ) to a minimum . false positive candidate biomarkers are identified in the validation phase of this investigation . in order to uncover multivariate latent structure in the data , which in turn helps assess the combinatorial predictive ability of the candidate biomarkers , the significant peaks were combined into a single multivariate discriminant model using principal components ( pc ) analysis followed by canonical variate analysis ( cva ) known as pc - cva . 4 the pc stage is necessary to orthogonalize the data and reduce it down to a set of lower - dimensional latent variables . these variables ( principal components — pcs ) contain the fundamental variance structures within the data . the optimal number of pcs ( and hence the most robust multivariate model ) was found by employing 0 . 632 + bootstrap substitution 5 , using 100 bootstrap samples to produce an unbiased estimate of prediction error all peak data were pareto scaled before analysis . 6 7 further analysis to assess the combinatorial predictive ability of the candidate biomarkers was done by combining the significant peaks into a single multivariate discriminate model using partial leas - squares discriminant analysis ( pls - da ). the optimal number of latent factors used in the pls - da model was selected using stratified 5 - fold cross - validation and model quality assessed during the standard r 2 and q 2 measures , where r 2 , the squared correlation coefficient between the dependant variable and the pls - da prediction , measures “ goodness of fit ” ( a value between 0 and 1 , where 1 is a perfect correlation ) using all of the available data to build a given pls - da model . q 2 provides a measure of “ goodness of prediction ” and is the averaged correlation coefficient between the dependent variable and the pls - da predictions for the 5 houdout data sets generated during cross - validation process . further validation was performed to check the robustness of the final pls - da model by comparing the r 2 value to a reference distribution of all of the possible modles uwing permutation testing ( n = 1000 ) following the standard protocol for metabolomic studies . here a reference r 2 distribution is obtained by calculating all of the possible pls - da models under random reassignment of the case / control labels for each measured metabolic profile . if the correctly labeled model &# 39 ; s r 2 value is close to the centre of the reference distribution , then the model performs no better than a randomly assigned model and is , therefore , invalid . for all of the pls - da models described here , the associated reference distribution plots are provided , from which an estimate of the probability of the candidate modle randomly occurring can be estimated . in addition , for each pls - da model , a receiver operator characteristic ( roc ) curve was determined so that an accurate assessment of discriminatory ability could be made . as a preprocessing step to remove any structured nose in the data set , direct orthogonal signal correction was performed using a single correction factor and a tolerance setting of 1 × 10 − 3 . all of the peak data were scaled to unit variance before multivariate analysis . for identification of uplc - ms related peaks , the accurate mass for each peak were searched against the manchester metabolomics database 8 constructed with information from both the human metabolome database ( hmdb ) ( http :// www . hmdb . ca /) and lipidmaps http :// www . lipidmaps . org /). a metabolite name ( s ) was reported when a match with a mass difference between observed and theoretical mass was less than 5 ppm . using uplc - ms , metabolites are often detected multiple times due to chemical adduction , dimerization , multiple - charging , isotope peaks and fragmentation . after removal of duplicate identifications , a list of unique metabolites was compiled . definitive identifications were reported only for metabolites with retention time errors & lt ; 10 seconds and an accurate mass match & lt ; 5 ppm . once identified , the metabolites were grouped into metabolite classes using the hmdb ‘ class ’ hierarchy ( http :// www . hmdb . ca /). for each named metabolite , a receiver - operator characteristic ( roc ) curve was determined to assess each metabolite &# 39 ; s effectiveness as a univariate discriminatory biomarker . in addition , for each metabolite , the optimal unbiased discriminatory decision boundary was estimated using the optimal youden &# 39 ; s index method and then the associated discriminatory odds ratios with 95 % confidence intervals ( or 95 % ci ) calculated . 9 - 10 the identified metabolites found to be significant in the discovery phase study were matched to the metabolite peaks detected in the validation study . if a match was found , then the metabolite was univariately assessed as a potential biomarker using the same protocol as for the discovery stage . in addition , a pc - cva and / or pls - da model was constructed to assess the multivariate discriminatory ability of the validation peaks . finally , an ‘ optimal ’ multivariate discriminatory model drawn from the named metabolites observed in both the discovery and validation studies was searched for . a genetic algorithm - based search program was used to obtain the subset of metabolites which produced an effective predictive rule for the onset of pe . this search method has been shown to be very successful in previous studies . 11 , 12 , 13 , 14 , 15 , 16 . in this algorithm a set of candidate solutions evolve over time toward an optimal state . the evolution is pushed by computational techniques inspired by evolutionary biology . in the algorithm devised by the inventors , each candidate solution ( subset of metabolites ) is assessed by building two independent linear discriminant analysis models , one modeling the discovery data , and the other modeling the validation data . a candidate &# 39 ; s fitness is proportional to the sum of the root mean square error of prediction ( rmsep ) of these two models . once the optimal subset of metabolites was found , its predictive ability was assessed using canonical variate analysis / partial least - squares discriminant analysis and hotelling &# 39 ; s t - square test . 4 assessment was performed independently for the discovery and validation data . all statistical analyses were carried out using the matlab ® scripting language ( http :// www . mathworks . com /). all univariate algorithms were implemented such that any missing values are ignored . all multivariate algorithms were implemented such that missing values were imputed using the nearest - neighbor method . 17 the genetic algorithm search program was written in - house . 11 scripts are available upon request . maternal characteristics and pregnancy outcome in the women with preeclampsia and controls are shown in table 2 . after quality assurance , and univariate screening , the uplc - ms analysis revealed 457 information - rich metabolite peaks . a multivariate discriminant model ( pc - cva ) using these 457 peaks proved to discriminate well , producing an area under the roc curve of 0 . 96 with an odds ratio 68 ( 95 % ci 21 - 216 ). this was far better than any individual metabolite peak . model selection was performed using the 0 . 632 + bootstrap resampling method . fig1 shows the pc - cva scores plot , where a third of the samples were randomly removed from the modeling process and used as a test set . the distribution of the test set model predications was similar to the training set , and only 10 % of the test set data was outside the respective 95 % confidence region ; this suggests there was robust multivariate discriminatory information within these data . further analysis using the multivariate discriminant model pls - da resulted in a model having an r 2 of 0 . 76 , q 2 of 0 . 68 , and area under the roc curve ( auc ) of 0 . 99 . model selection was performed using 5 - fold cross - validation , and the final model was further validated using permutation testing . the final model used a single latent factor and the probability of this model randomly occurring was & lt ; 0 . 001 . fig3 shows the pls - da scores plot and the permutation testing . of the 457 metabolite peaks detected by the uplc - ms , 70 were successfully identified chemically as known metabolites , of which 45 were ‘ unique ’ in the sense of being defined molecular entities ( table 1 ). when grouped into metabolite classes ( based on the human metabolome database ( hmdb )) eleven clear classes emerged . these were : amino acids ( aa ), carbohydrates ( cb ), carnitines ( cam ), eicosanoids ( eic ), fatty acids ( fa ), keto or hydroxy acids ( kha ), lipids ( lip ), phospholipids ( pl ), porphyrins ( por ), phosphatidylserine ( ps ) and steroids ( st ). a pc - cva model was built using only the 45 named metabolites . this produced an area under the roc curve of 0 . 90 and the odds ratio 23 . 5 ( 95 % ci 21 - 216 ). similarly for a pls - da model built using the 45 named metabolites ( 1 latent factor ), a predictive model with r 2 of 0 . 58 , q 2 of 0 . 57 and auc of 0 . 96 ( fig4 ). these results proved to be only a slight reduction of diagnostic performance when compared to the full 457 - peak model . the maternal characteristics and pregnancy outcome in the women with preeclampsia and controls are shown in table 2 — characteristics and pregnancy outcome of women who later developed preeclampsia and controls . of the 45 significant metabolites named in the discovery study , 34 were also detected in the validation study . all these metabolites showed similar changes in peak response ( 29 were raised in patients who went on to develop pe ; 5 were lowered ). the multivariate pc - cva model using the 34 metabolites proved to be reasonably predictive with an auroc of 0 . 86 and odds ratio of 15 ( 95 % ci 4 . 4 - 50 . 6 ). a pls - da model using the 34 metabolites ( 1 latent factor ) proved to be predictive , with r 2 of 0 . 57 , q 2 of 0 . 53 and auc of 0 . 95 ( fig5 ). finally , the data from both studies were mined using a genetic algorithm - based search program to find the subset of named metabolites which produced the most robust predictive general model . the genetic algorithm chose 13 metabolites ( preferred metabolites ). fig2 shows the cva model predictions using these metabolites for both the discovery study and the validation study . the auroc for both models was 0 . 90 and the odds ratios were 16 . 4 ( 95 % ci 6 . 6 − 40 . 6 ) and 39 . 5 ( 95 % ci 8 . 2 − 189 . 4 ) for discovery and validation data respectively . in addition , fig6 shows the pls - da model predictions had an r 2 of 0 . 54 , q 2 of 0 . 52 and auc of 0 . 94 , and an optimal odds ratio of 36 ( 95 % ci : 12 to 108 ) for the discovery study , and an r 2 of 0 . 43 , q 2 of 0 . 39 and auc of 0 . 92 , and an optimal odds ratio of 23 ( 95 % ci : 7 to 73 ) for the validation study . permutation testing showed that the probability of both of the pls - da models randomly occurring was & lt ; 0 . 001 ( fig7 ). the combined effect of the 13 metabolites was also tested using the hotelling &# 39 ; s t - square statistic . for the discovery study data , this produced a p - value of 2 × 10 − 6 , and for the validation study data , a p - value of 0 . 006 . the p - values were obviously affected by the differing sample sizes ( discovery n = 120 ; validation n = 79 ). the embodiments in the invention described with reference to the drawings comprise a computer apparatus and / or processes performed in a computer apparatus . however , the invention also extends to computer programs , particularly computer programs stored on or in a carrier adapted to bring the invention into practice . the program may be in the form of source code , object code , or a code intermediate source and object code , such as in partially compiled form or in any other form suitable for use in the implementation of the method according to the invention . the carrier may comprise a storage medium such as rom , e . g . cd rom , or magnetic recording medium , e . g . a floppy disk or hard disk . the carrier may be an electrical or optical signal which may be transmitted via an electrical or an optical cable or by radio or other means . in the specification the terms “ comprise , comprises , comprised and comprising ” or any variation thereof and the terms include , includes , included and including ” or any variation thereof are considered to be totally interchangeable and they should all be afforded the widest possible interpretation and vice versa . the invention is not limited to the embodiments hereinbefore described which may be varied in construction and detail without departing from the spirit of the invention . 1 zelena , e . et al . development of a robust and repeatable uplc - ms method for the long - term metabolomic study of human serum . anal chem 81 , 1357 - 1364 ( 2009 ). 2 brown , m . a . et al . the detection , investigation and management of hypertension in pregnancy : full consensus statement . aust n z j obstet gynaecol 40 , 139 - 155 ( 2000 ). 3 smith , c . a ., want , e . j ., o &# 39 ; maille , g ., abagyan , r . & amp ; siuzdak , g . xcms : processing mass spectrometry data for metabolite profiling using nonlinear peak alignment , matching , and identification . anal chem 78 , 779 - 787 ( 2006 ). 4 krzanowski , w . j . principles of multivariate analysis : a user &# 39 ; s perspective . ( oxford university press , 1988 ). 5 efron , b . & amp ; tibshirani , r . j . improvements on cross - validation : the 0 . 632 + bootstrap method . journal of the american statistical association 92 , 548 - 560 ( 1997 ). 6 eriksson , l ., johansson , e ., kettaneh - wold , n . & amp ; wold , s . introduction to multi - and megavariate data analysis using projection methods ( pca & amp ; pls ). 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