CN101329353A - Method for detecting trisomy 13 and down syndrome by non-invasive maternal blood screening - Google Patents
Method for detecting trisomy 13 and down syndrome by non-invasive maternal blood screening Download PDFInfo
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- CN101329353A CN101329353A CNA2008100812456A CN200810081245A CN101329353A CN 101329353 A CN101329353 A CN 101329353A CN A2008100812456 A CNA2008100812456 A CN A2008100812456A CN 200810081245 A CN200810081245 A CN 200810081245A CN 101329353 A CN101329353 A CN 101329353A
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Abstract
A method and apparatus for determining if a pregnant woman is at significant risk of carrying a fetus with Down syndrome. The method comprises measuring the pregnant woman's maternal blood levels of the free beta subunit of human chorionic gonadotropin. The level of free beta subunit of human chorionic gonadotropin individually or with other markers may be compared to reference data. A computerized apparatus for making the determination preferably using a probability density function generated from a set of reference data by a linear discriminant analysis procedure is disclosed.
Description
The application be that January 17 nineteen ninety, application number are 90100994.6 the applying date, denomination of invention divides an application for the application for a patent for invention of " with the method and apparatus of the noninvasive method discovery Down's syndrome of checking maternal blood ".
Technical field
The present invention relates to a kind of method of in antenatal exaination, finding fetus Down's syndrome (21 trisome).This method also relates to other rare but trisome that can detectedly dye inclusion as 13 trisomes and 18 trisomes.More particularly, the present invention relates to a kind of method of checking the discovery rate in the Down's syndrome that improves by the content of measuring the free beta subunit of human chorionic gonadotrophin (HCG) in the pregnant woman blood.
Background technology
Down's syndrome also claims 21 trisomes, is the most general congenital reason of serious mental retardation.Down's syndrome adopts a kind of diagnostic method of amniocentesis and karyotype that comprises to determine usually.But this diagnostic method is invasive, can bring danger to pregnant woman and fetus.Amniocentesis and karyotype when using this invasive diagnostic method proof to bring danger to the pregnant woman, can adopt one or more inspection methods to replace this invasive method to diagnose not as the method that all pregnant woman are carried out routine examination.
The incidence of Down's syndrome look like one's mother the age increase and increase rapidly.Antenatal research for Down's syndrome in the past concentrates on the age the pregnant woman more than 35 years old always, at this age bracket, the danger of Down's syndrome near or surpass the danger that diagnostic method brought be used to find Down's syndrome.Therefore, the standard method of antenatal exaination comprises with the pregnant woman age being that benchmark is picked out the pregnant woman who does the amniocentesis diagnosis always.But Nian Ling And checks foundation fully.Because to 5% most probable pregnant woman on the line, be to be 35 years old or bigger pregnant woman carries out amniocentesis and karyotype can only be found about 20% of all Down's syndrome gestation at the age.Maggot is because in actual clinical practice, has only the only about half of age to make amniocentesis and karyotype 35 years old or above pregnant woman, so be lower than 10% in the Down's syndrome gestation of antenatal discovery.
1984, the someone found getting in touch between α-fetus albumen (AFP) content low in the maternal blood and the fetus Down's syndrome.For example referring to " An assooiationbetween low maternal serum alpha-fetoproteinand fetal Chromosomal abnormalities "; (following between the low content of maternal serum α-fetus albumen and the fetal chromosomal abnormalities relation); Merkatz, people such as Macri work; Am.J.Obstet.Gynecol, 148:886,1984; The disclosure content is incorporated among the application by reference.Put down in writing some other trisome in this article, particularly 13 trisomes and 18 trisomes, also with maternal blood in AFP content low relevant.Trisomic incidence (being respectively one of five per milles and 6,006 percentages) in these other the chromosome is far below the general priori morbidity rate of 21 trisomes (Down's syndrome).But because the trisome in these other the chromosome is low relevant with MSAFP content, therefore, the inspection scheme of these chromosomal unusual free beta subunits that also can utilize maternal blood AFP described herein and HCG and possible other index is found.One of skill in the art obviously also can be used to find 13 trisomes and 18 trisomes with the 21 trisomic schemes that are used for described herein.In the maternal blood low relevant this discovery of AFP content with the fetus Down's syndrome make we might those Down's syndrome incidences of disease be about 80% youth, appear in the not ill family blood test of employing Noninvasive and test and find the Down's syndrome case.According to estimates, adopt the check test of hanging down to make the antenatal discovery rate of fetus Down's syndrome reach about 20% of total case based on maternal blood AFP (checking index) as one.
Another kind of inspection method comprises the content of measuring uncombined estriol (UE) in the maternal blood.For example referring to " Maternal blood screening for Downsyndrome in early pregnancy " (finding Down's syndrome when mother's blood test is pregnant in early days); People such as Wald work; British Journal ofObstetrios and Gynocology (BMJ) Volume 95, April 1988, and its disclosure is incorporated among the application by reference.But the measured value to UE only provides a benchmark of poor quality for inspection.
Recently, find that (HCG is made up of two subunits, below divides have another name called α-HCG and β-HCG) and the relevance between the fetus Down's syndrome for the raising of α-subunit's content of raising, HCG of HCG content in the maternal blood.For example referring to " AbnormalMaternal Serum Chorionic Gonadotropin Levelsin Pregnancies With Fetal ChromosomeAbnormalities " (the human chorionic gonadtropin content of maternal serum is unusual in the fetal chromosomal abnormalities gestation); Bogart Pandian and Jones; Prenatal Diagnosis, Vol, 7,623-630 (1987), its disclosed content is incorporated among the application by reference.In the article of Bogart, estimated to utilize the raising of α-subunit's content of HCG and HCG in the maternal blood to find the fetus of about 68% chromosome abnormality.And these conclusions are to obtain by the gestation research to conceived 18-25 week, and these ill cases seemingly those have been identified as the women that may suffer from Down's syndrome in advance.
As mentioned above, in general, only relate to measurement usually, additionally also relate to measurement α-HCG to HCG by the inspection method of measuring the HCG value in the maternal blood.Though these inspection methods really can be found Down's syndrome, also need and wish the method for the fetus Down's syndrome case of the higher number percent of a kind of discovery.
The inventor has found the raising of free content in the maternal blood in prior ignorance road and the relevance between the fetus Down's syndrome.Also found the relevance between the free content and AFP content and fetus Down's syndrome in the maternal blood in prior ignorance road, the inventor has found that also free content is to the ratio of int HCG molecule content in the maternal blood and the relevance between the fetus Down's syndrome in the maternal blood in prior ignorance road, I have also further found to adopt a kind of multivariate discriminant analysis method can improve the discovery rate of following said inspection method under a morbidity rate cutoff of selecting, this method is utilized free content in the maternal blood, or utilize in the maternal blood free β-HCG content and AFG content, or any one logarithm in the two, or both logarithms.Particularly also be taken as a variable in pregnant age and join in the discriminant analysis method, the raising of discovery rate is more obvious.The all numbers that refer to the pregnant youngster of pregnant woman pregnant age, discovery rate are meant the number percent that correctly is found to be the Down's syndrome case under a selected morbidity rate cutoff.The morbidity rate cutoff will be illustrated in the back in more detail.Discriminant analysis method generally speaking is known a kind of multivariable technique, morbidity rate (or level of significance) estimation technique that relates to unitary variant is divided into two or more a plurality of group with research object, discriminant analysis method also is described as a kind of method that constitutes independent variable linear combination sometimes, and the problem reduction that is about to weigh each group's difference becomes a single argument problem.When only comprising a variable in certain problem, also can use discriminant analysis method.Generality discussion visible " Marketing Research " (sales research) about discriminant analysis method; Churchill, G.A.; Dryden, 1976, the 15 chapters P.530～543, the disclosure content is incorporated among the application by reference.I find the content of free in the maternal blood, int HCG content, free are compared the inspection method of ratio, AFP content, UE content and the pregnant age of not impaired HCG molecule handling any known antenatal discovery Down's syndrome of back and other through the multivariate discriminant analysis method, the number percent of finding the fetus Down's syndrome is higher, and false positive rate is lower.I find that also by content and the AFP content of only measuring free in the maternal blood, And handles the two logarithm and pregnant age separately through the multivariate discriminant analysis method, still can find the more fetus Down's syndrome case of more number.The discovery of these and other will be illustrated more fully to overview section of the present invention with in describing in detail partly below.
Summary of the invention
An object of the present invention is to provide and a kind ofly compare, under the situation of given false positive rate, can find the inspection method and the step of the fetus Down's syndrome of higher number percent with other known antenatal exaination method.
Another object of the present invention provides a kind of method and step that has the inspection fetus Down's syndrome of lower false positive rate under given discovery rate condition than other known method.
Of the present invention one further purpose be that the multivariate discriminant analysis method is applied to check that so that the discovery rate of fetus Down's syndrome is higher, false positive rate is lower in the method for Down's syndrome.
Another object of the present invention provides a kind of method and step of checking the fetus Down's syndrome by the content of measuring free in the maternal blood.
The present invention also have one further purpose a kind ofly check the method and the step of fetus Down's syndrome with regard to providing by measuring the content of AFD content and free in the maternal blood.
Other purpose of the present invention and advantage will become apparent in the description of this invention below.
In order to achieve the above object, according to the present invention, the content of free adopts conventional immunological method to measure in pregnant woman's (below pretend illness people) the maternal serum, and this method comprises immunoassay, as other known method in those methods of mentioning in the above-mentioned article and this field.Then, content and the one group of reference data with free compares to determine that this patient nourishes the morbidity rate of suffering from the Down's syndrome fetus.In order to improve discovery rate, with content and pregnant age and one group of reference data comparison of free.Be further to improve discovery rate, adopt conventional immunological method, comprise mention in the above-mentioned paper at assay method well known in the art, measure β-HCG and AFG (to call " index " in the following text) content free in patient's maternal blood.Then every desired value and one group of reference data are compared, nourish the level of significance of suffering from the Down's syndrome fetus to determine this patient.Adopt the multivariate discriminant analysis method that these desired values and one group of reference data are made comparisons.In more detail, adopt pattra leaves phase law exactly, the level of significance of this patient's priori and be updated to by the logarithm with the quantitative value of every index of this patient is determined in the probability density function that utilizes the normal data that the multivariate discriminant analysis method launches and is not illly calculated the concrete level of significance of each patient with the relative incidence of ill gestation.If the level of significance that this patient nourishes the Down's syndrome fetus then should advise this patient to make further diagnostic test greater than given level of significance cutoff, so that whether conclusive evidence suffers from Down's syndrome.In maternal blood, free and the AFP content, add and further to improve discovery rate as an index pregnant age.Because free content and AFP content are tending towards distributing by logarithm one Gaussian distribution curve in the maternal blood of great amount of samples, therefore, can reach high discovery rate in the probability density function by reference data that the logarithm of the every index quantification value of patient and pregnant substitution in age are launched with the multivariate discriminant analysis method.
One of advantage of method of the present invention and step is compared with step with other known method exactly, can predict accurately that the percent of fetus Down's syndrome case is higher, and false positive rate is lower.
Some other advantage of the present invention will become in more detailed description and the example very clear below.
Description of drawings
Fig. 1 is a form of mentioning in the example 2, has represented the effective value of (21 trisome) each index.
Fig. 2 is a form of mentioning in the example 2, has represented the checking efficiency of the Down's syndrome of each index.
Fig. 3 is a form of mentioning in the example 2, has represented the checking efficiency of the Down's syndrome of combined index.
Fig. 4 is a form of mentioning in the example 2, has represented to be higher than in not ill gestation the percentile Down's syndrome case part that the beta subunit of given free HCG distributes.
Fig. 5 is a form of mentioning in the example 2, represents the Down's syndrome rate of each index.
Fig. 6 is a form of mentioning in the example 2, is illustrated in the scope in different pregnant age the checking efficiency of the Down's syndrome with the logarithm of the logarithm of AFP and free β-HCG subunit during as a combined index.
Fig. 7 is a form of mentioning in the example 2, the Down's syndrome recall rate that is drawn by free β-HCG and maternal age in U.S. various places that expression is estimated.
Fig. 8, referred in example 2, represented that in 21 trisome cases free subunit is with respect to the percentage content of ill gestation not.
Fig. 9, referred in example 2, represented the distribution of free subunit content.
Figure 10 is a form of mentioning in the example 3, the recall rate of the Down's syndrome that expression is obtained by the various combinations of some indexs.
Figure 11 represents the employed equipment of Down's syndrome method of checking that is used for carrying out of the present invention.
The computer program process flow diagram be used for determining the basic parameter that level of significance that a patient nourishes ill fetus is relevant is calculated in Figure 12 and 13 expressions.
Figure 14 represents to utilize the basic parameter that calculates in the program shown in Figure 12 and 13 to determine that this patient nourishes the computer program process flow diagram of the level of significance of ill fetus.
Embodiment
In one embodiment of the invention, obtain the parent blood sample from patient, adopt conventional analytical approach then, immunological method is as known in the art measured the content of free in the maternal blood.Then, this value and one group of reference data comparison are nourished in the danger of suffering from the Down's syndrome fetus to determine whether this patient is in.In order to improve discovery rate, the content value of free in pregnant age and the maternal blood and one group of reference data compared to determine whether this patient nourishes suffer from the Down's syndrome fetus.
Although the professional is obvious as this area, the content that uses any known analytical approach to measure free in the parent in the present invention is all right, but the analytical approach that is used for free must be the same with the method that produces the free reference data.If used a kind of new analytical approach to measure free, then must produce one group of new reference data based on the data of this method formation.
Be understood that in the process that forms the special monoclonal body to the beta chain of HCG, some antibody will be special to this protein, and other will be special to the candy related with the antibody position.Though the measured value of the β that in declaratives of the present invention, mentions-HCG content comprised to free HCG use to say protein special, or to free on antigen position or the special antibody of the related candy in other any position.
Professional in this area is understood that, though free α-HCG subunit by an independent gene code, free β-HCG is then encoded by the compound family of seven closely similar genes or pseudogene at least.For example referring to " HumanChorionic gonadotropin beta-subunit is encodedat least eight genes arranged in tandem andinverted pairs " Boorstein, Vamvakopoules ， ﹠amp; Fiddes; Nature Vol300, Dec 2 nineteen eighty-two; Instruction wherein is incorporated among the application by reference.Only there are three in the normal placentation process of free, to be expressed in known seven free genes.For example referring to " Fragmentation of the Beta-Subunit of humanChorionic Gonadotropin produced byChroriocarcinoma "; Nishimura, Ide, Utsunomiya, Kitajima, Yuki, and Mo chizuki; Endocrinology, Vol.123, No.1,1988; Instruction wherein is incorporated among the application by reference.Whether these three same genes as are expressed when suffering from the fetus Down's syndrome and are never had sure answer under morbid state.Therefore, might synthesize on sequence of amino acid or others have the free of the various ways of minute differences.Also might be in the Down's syndrome case, the gene of one or more frees is expressed, and therefore, produces mutation or a plurality of variation kind of a uniqueness of free.According to the present invention; If these variations kind exist, the immunological method of the measurement free that they can be by routine is measured so comes out.If a kind of determination method that the specific free variation relevant with Down's syndrome planted of measuring can be arranged, can further improve discovery rate.
In fact, we have used the monoclonal antibody measuring method to measure the free beta subunit of HCG to distinguish trouble 21 trisomes gestation and not ill gestation effectively.21 trisomic discovery rates are up to 83%.As this area professional knows, utilize antibody quantitatively illustrate specific analytic target can obtain with one have any different but the degree of intersecting activity of similar substance.So the difference between the ill and not ill case may be that these subunits are owing to have the activity of intersecting to a certain degree to be detected with the antibody that is utilized owing to exist due to the free of multi-form HCG.If this anomaly pattern of free subunit is differentiated out that then it will be decided to be a kind of new biochemical.In fact, the data in the scientific literature shows, has identified this anomaly pattern (for example referring to outstanding articles such as Nishimura) of β-HCG
Suffering from 21 trisomic cases can be feature with the anomaly pattern of free subunit also.If 21 trisomes are that the anomaly pattern with free is a feature, then this area professional can be developed and the antibody special to these anomaly patterns, and the discovery rate of Down's syndrome is further improved.
Reference data has reflected to be nourished the content (also claiming " ill ") of suffering from free in the Down's syndrome fetus pregnant woman blood and/or reflects the content (also claiming " not ill ") of nourishing free in the normal fetus pregnant woman body inner blood.For this area professional, be understood that the method that is used to check the fetus Down's syndrome is exactly the process by relatively making decisions.For any decision process, needing with the patient who suffers from this disease or the condition that we were concerned about and/or the patient who does not suffer from this disease or our institute's caring conditions is the basic parameter of benchmark.Among the present invention, reference value is the content of tested index in the maternal blood, as nourishes pregnant woman who suffers from the Down's syndrome fetus and the free of nourishing normal fetus pregnant woman.Set up one group of reference data by the reference value of concentrating great amount of samples.Concerning the professional of this area, obviously this group reference data also can further be improved by the quantity that increases the reference value sample.
Nourish in the danger of suffering from the Down's syndrome fetus in order to determine whether patient is in, must set up a cutoff.Concerning the professional of this area, obviously, setting up a definite patient, whether to nourish the cutoff of suffering from 13 trisomes or 18 trisome fetuses also can be effective in identification 21 trisome cases.This cutoff can be by laboratory, doctor or based on each patient's case and set up.Cutoff also can be based on several discrimination standards, the number that comprises the women that need continue to do further invasive diagnosis test, all women that do further invasive diagnosis test suffer from the average level of significance of Down's syndrome fetus and its specific ill level of significance of any one women just should continue to do that further invasive diagnosis is tested or the decision-making of other discrimination standard that this area professional is known as 1/400 greater than a concrete level of significance value.Cutoff can be set up by certain methods, comprising: percentage, average positive and negative standard deviation, the multiple of intermediate value, the ill level of significance that patient is concrete or other known method of those this fields professional.
Can find in the embodiment of more number fetus Down's syndrome more at of the present invention another, in the patient body, take out the maternal blood sample, then, by the analytic approach of routine, immunoassay is measured int HCG molecule in the maternal blood, free β-HCG, UE and the content of APF (to call " index " in the following text) as known in the art.Although as this area professional is obvious, to measure in the maternal blood content of These parameters all right for any known analytical approach of utilization in this, but the method for the analytical approach that is used for every index and the reference data of this specific indexes of formation must be identical.If a certain index is adopted a kind of new analytical approach, then must produce one group of reference data based on the data of this method generation.
Then, adopt the relative incidence of the not ill and ill gestation of determining in Bayes law, this patient's priori risk rate and the probability density function to calculate the level of significance that patient nourishes trouble Down's syndrome fetus by basic parameter that the quantitative values of every index of patient (int HCG molecule, free, UE and APF) and free are launched with multivariate discriminant the ratio of not impaired HCG molecule content and patient's pregnant substitution in age.
The computer program statistical package " statistical analysis system " that the multivariate discriminant analysis method can have on market (by company of SAS research institute production and selling) is gone up and is calculated, or adopts other multivariate statistical analysis method or known other statistical package of this area professional to calculate.
Probability density function is for relatively providing a method every desired value of patient and basic parameter.Though, as this area professional obviously understands, also can use other probability density function similarly, specifically noted that below a kind of probability density function ， And of pattern can make full use of in the present invention.
The formula that is used for the Down's syndrome level of significance is:
The case that subscript " a " expression is ill
The case that subscript " u " expression is not ill
(X-M) vector of expression, wherein each element is the value that each variable deducts average of variable.
COV-1 is the ill of all variablees in this model and associating covariance matrix contrary of ill case not.
(X-M)
TIt is the transposition of vector (X-M).
EXP represents exponential function.
| COV| represents in this model the determinant as the covariance matrix of all variablees of reference data.
This area the professional obviously know, ill gestation and not ill gestation covariance matrix separately can replace the associating covariance matrix.The formula that is used to suffer from the Down's syndrome level of significance just becomes:
| COV| represents in this model the determinant as the covariance matrix of all variablees of basic parameter.
For the purpose of carrying out discriminant analysis, in the research object of generally not selecting, make the hypothesis that to suffer from the prior probability of Down's syndrome earlier.This prior probability generally is about 1/800.In order to carry out the multivariate discriminant analysis method, also to do a decision-making which type of level of significance cutoff can constitute a positive test result about.For example, if wish that nourishing the probability of suffering from the Down's syndrome fetus for one is equal to or greater than 1/400 pregnant woman and carries out further diagnostic test, then nourish the probability of suffering from the Down's syndrome fetus when being equal to or greater than 1/400 when the table as a result of multivariable analysis with the pregnant woman, this pregnant woman is considered to have a positive test result.If draw the positive test result, should advise patient to make further diagnostic test and whether suffer from Down's syndrome with conclusive evidence.
With reference to Figure 11 to 14, the equipment that is used to calculate reference data and concrete level of significance and the process flow diagram of computer program have been shown among the figure.
As shown in figure 11, for setting up reference data, in case ill and not ill gestation, adopt conventional method to determine GA in pregnant age, the content of AFP content and free.Selected great amount of samples for improving reliability.The measured value that is used to set up reference data is schematically represented with 10.
Calculate basic parameter 22 in case enter the back via suitable input media 15 by processing unit 20, the level of significance 25 concrete as certain patient can calculate according to the measured concrete index value (representing with 30) of this patient.
The flow chart of reference data is determined in Figure 12 and 13 expressions, and Figure 14 represents to calculate the flow chart of concrete level of significance.
Referring now to Figure 12 and 13, in first circulation 100, program is read in identification parameter ID from a reference group, pregnant age GA, whether the quantitative values of AFP and free and a gestation suffer from 21 trisomic codes.See step 102.In process flow diagram, shown in step 104, pregnant age the GA variable X
1Expression, the logarithm X of AFP
2Expression, the logarithm X of free
3Expression, then, at X
1, X
2And X
3Determine on the basis of data or calculate with matrix and and-product matrix, see step 106.Be used for the variable Ncode of ill in the reference group and not ill case counting is increased by 1 then.In case loop ends is as by shown in the flowline 110, by go out mean value by amount I, J, the defined a series of cycle calculations of K shown in reference number 112.In these circulations, utilization circulation 100 definition with matrix and in circulation 100, calculate and-product matrix calculates covariance matrix.After these loop ends, to whether with ill and not the covariance matrix of ill case combine and make one's options.This step 114 that is chosen in is transfused in 116 and 118.If select combination, then covariance matrix just is combined into an associating covariance matrix that is provided by step 120.Shown in step 122, this associating covariance matrix is got contrary, obtains contrary associating covariance matrix IPCM.Should be deposited in file Zhong ， And against the associating covariance matrix is printed in step 124 and 126.If select not make up, then in step 123 and 125, each of two covariance matrixes is got contraryly, and deposit mean value and inverse covariance matrix in Wen spare And and in step 125 and 127, print.These numerical value comprise and are used to calculate the basic parameter that the unique individual nourishes ill FD rate.
Referring to Figure 14, be read in step 130 by carrying out the basic parameter that program is calculated shown in Figure 12 and 13, basic parameter comprises mean value and contrary associating covariance matrix.Read in concrete case notes in step 132, comprise patient identification marking, pregnant age GA, AFP and free.In step 134, calculate more accurately pregnant age, in step 136, calculate pregnant woman's age.In 138, on the basis of pregnant woman age and incidence parameter, determine the priori risk rate.In the example that is discussed below, this result of calculation is 1/800, is a typical numerical value.
In 140, determine this priori risk rate and the product of nourishing the relative frequency of ill fetus (ABT), the molecule of promptly above-mentioned equation (1), (2).In 142, calculate the product (NT) of the relative frequency of nourishing ill fetus and (1-priori risk rate), promptly above-mentioned equation (1), second factor in (2) denominator.In 144, utilize Bayes law to determine concrete level of significance, i.e. ABN=ABT/ (ABT+NT) (equation (1), (2)).In 146, print the result, promptly print specific level of significance ABN of patient and patient identification number.
For this area professional, easily so can also adopt other statistics or the mathematical method that are different from linear discriminent analysis to calculate basic parameter.
An embodiment preferred obtains the maternal blood sample from patient according to the present invention, adopts conventional analytical approach then, comprises that immunological method well known in the art measures the content of free and AFP (following title " index ") in the maternal blood.Although as this area professional is known, all right with the content of These parameters in any known analytical approach measurement maternal blood in the present invention, the analytical approach that is used for every index must be the same with the method that forms each index basic parameter.If a certain index is adopted a kind of new analytical approach, then must on the data basis that forms with this method, produce one group of new basic parameter.
Then, the utilization Bayes law, the level of significance of patient's priori, and the probability density function by reference data that the quantitative values of every index of patient and substitution of pregnant age are together launched with multivariate discriminant in the ill and relative incidence of ill gestation not that obtains calculate the level of significance of nourishing trouble Down's syndrome fetus.Be further to improve discovery rate, in the probability density function with the basic parameter that launches with multivariate discriminant in the logarithm value of patient's free and AFP quantitative values and pregnant age substitution together.The computer program statistical package " statistical analysis system " that the multivariate discriminant analysis method can have on market (by company of SAS research institute production and selling) is gone up computing, or adopts known other multivariate statistical analysis method of this area professional or other statistical package.
For the purpose of carrying out discriminant analysis, in common unselected research object, the prior probability of suffering from Down's syndrome is made a hypothesis earlier, in general, this prior probability is approximately 1/800.In order to carry out the multivariate discriminant analysis method, also to make the decision-making which type of level of significance cutoff constitutes the positive test result.For example, wish to nourish for one and suffer from Down's syndrome fetus probability and be equal to or greater than 1/400 pregnant woman and do further diagnosis examination danger, then nourish the probability of suffering from the Down's syndrome fetus when being equal to or greater than 1/400 when the result of multivariable analysis shows a pregnant woman, this pregnant woman is considered to have a positive test result.If demonstrate the positive test result, should advise this patient to make further diagnostic test and whether suffer from Down's syndrome with conclusive evidence.
For this area professional, obviously, in all embodiments of Tao Luning, change positive level of significance cutoff or in research object, different groups is adopted different priori risk rates in the above, will change the result who each patient is done discriminant analysis.
The present invention not only is confined to above-mentioned embodiment, also comprises all possible embodiment and the combination of disclosed each index in the example below.
Example 1
Relation between AFP content (MSAFP), UE and the not impaired HCG in free content and the maternal serum in employing 400 above case sample study fetus Down's syndromes and the maternal blood.These samples comprise 25 examples from known nourish the sample that obtains in pregnant woman's body of suffering from the Down's syndrome fetus and with the check sample of ill case-control.
To each blood sample, the content value of AFP, not impaired HCG molecule, free and UE (to call " index " in the following text) is measured by following assay method:
Index
Assay method
The immunosorbent assay method (ELISA) of MSAFP enzyme link
The UE radioimmunoassay
Not impaired HCG globule type ELISA
Free ELISA
Every refers to that target value becomes a variable that carries out on the statistical analysis system of the computer software statistical package that has (company of SAS research institute) during juggling and linear discriminent are handled step by step on market, be used to set up one group of reference data.Also being taken as variable the patient's when of free and not impaired HCG molecule pregnant age adds.Juggling solves all variablees and can be added into during linear discriminent handles step by step.Respectively the various combination of each variable and each variable being carried out linear discriminent then handles.The result of these discriminant analysis methods is summarized in the following table.Sensitivity is meant that test result shows the number percent of positive fetus Down's syndrome case, and false positive rate refers to the number percent of the normal fetus that test result is positive.
Variable
Sensitivity
False positive rate
MSAFP 15.4％ 4.2％
UE 15.4％ 2.8％
Not impaired HCG 37% 8.6%
The not impaired HCG of MSAFP, UE 50.0% 7.2%
Free+not impaired HCG 60.0% 8.5%
Compound W/O, ratio 76% 5.3%
Compound W/O UE 76% 5.3%
Compound W/O free 60.0% 5.3%
*Not impaired HCG logarithm+(free
β-HCG logarithm+not impaired HCG) 68% 7.6%
*Not impaired HCG logarithm, MSAFP logarithm+
(free logarithm+not impaired HCG) 88% 7.4%
Compound-the MSAFP+ free+not impaired HCG+UE+ ratio
All comprise pregnant age in every variable.
Level of significance cutoff=1/400, remove (
*) be outside 1/365.
Concerning the professional of this area, obvious, positive level of significance cutoff is determined in change, or will change the result of the discriminant processing that certain patient is done to the different priori risk rate of research object different groups employing.
Example 2
Study the relation between the free content in fetus Down's syndrome and the maternal blood with the above patient's sample of 550 examples.The blood sample that the known pregnant woman that nourish ill syndrome fetus of 29 examples are obtained and be used to contrast the not ill sample of 520 examples of pregnant age (all numbers are identical), maternal age (differing in 3 years) and refrigerated storage time (differing within one month) and analyze at first.All samples all are taken from the white man pregnant woman of the single tire in bosom of not suffering from diabetes.
Droop for fear of estimating to have in the recall rate certain tendency has adopted effective group notion.Effectively group is exactly one group of data that are independent of reference data.Effectively the result who is obtained by patient in the group is not used in and sets up reference data, but is used for comparing to determine recall rate with reference data.This another effectively group comprise 26 additional groups of forming by 159 check samples that are confirmed as 21 trisomic cases (55 cases altogether) and picked at random.Check sample is similarly also taken from the white man pregnant woman of the single tire in bosom of not suffering from diabetes.
All research comprises 4388 judgements to seven of following each desired value in the maternal blood different assay methods:
Index
Assay method
MSAFP enzyme link immunosorbent assay method (ELISA)
Not impaired HCG ELISA
Not impaired HCG+ free ELISA
Free radioimmunoassay (RIA)
Free α-HCG RIA
Two kinds of methods of UE, enzyme immunoassay and RIA
Every desired value all becomes a variable that carries out during juggling and linear discriminent are handled step by step on computer software statistical package available on the market " statistical analysis system " (company of SAS research institute), to set up one group of reference data.Also being taken as variable patient's pregnant age adds.Respectively each independent variable combination execution linear discriminent different with each variable handled then.With 1/365 serves as that the cutoff of suffering from the Down's syndrome level of significance is divided into ill or not ill case with patient.Consider it is false positive rate dividing ill not ill case into.Each patient suffers from level of significance employing Bayes law, the multivariate normal probability density function ill and not ill case of Down's syndrome and calculates as the priori risk rate with 1/800 usually.The associating covariance matrix is used to each probability density function.
The result of relevant preliminary research group is presented in table 1～3 of Fig. 1 to 3.The table 5-7 of Fig. 5 to 7 is the result who obtains according to the classification to patient in effective group.Table 4 shown in Figure 4, Fig. 8 and Fig. 9 draw according to preliminary research group and all ill cases.
Analyzed by the result that assay method obtains, to determine whether there is evident difference between every desired value between the ill and not ill case.All indexs between table 1 (Fig. 1) ill case of expression and the not ill case except that UE all show tangible difference.In addition, determine the false positive rate and the discovery rate of every index, seen Table 2 (Fig. 2).Obtain the highest discovery rate by the HCG determination method of measuring not impaired HCG and free subunit.
Can be combined into composite index by the index that each is independent and further improve discovery rate.Wherein a kind of compound HCG determination method that comprises not impaired HCG molecule of measurement and free subunit, it produces the highest discovery rate in compound shown in the table 3 (Fig. 3).
Separately the assessment of the β of HCG and alpha subunit is shown that α-subunit does not have notable difference (P=0.23) between ill and not ill case and beta subunit is significantly increased (P=0.001) in ill case.The professional is in common knowledge as this area, and probability P is by showing that a result who is observed is the occurrent power of weighing evidence in the scientific research with the possibility of so at least maximum value.The P value is more little, illustrates that observing is not that the accidental evidence that occurs is strong more.
Fig. 8 represents the curve of 10%, 50% and 90% free with pregnant age.Discovery has continuous downward trend along with increasing pregnant age in not ill gestation.Free analysis on Content in the fetus Down's syndrome case has been disclosed 86% to be dropped on ill patient's not the mean value.
The content of free is all followed logarithm Gaussian distribution (P=0.78 and 0.86) in the ill and not ill case.Fig. 9 this distribution of having drawn, table 5 (Fig. 5) has provided the discovery rate data of independent dependence free subunit and free α-HCG subunit.Shown in the table 5 that free has high discovery rate.
Analyze with the combination of AFP and free and can obtain higher discovery rate.By the logarithm of free and the logarithm of MSAFP are attached in the linear discriminent processing of above-mentioned software package " statistical analysis system " (company of SAS research institute), can obtain the more superior discovery rate shown in table 6 (Fig. 6).
Opposite with its logarithm, also can obtain high discovery rate with free content and AFP content.
To data further the analysis showed that AFP and free all with age irrelevant (P=0.8394 and P=0.5214, AFP and free to four age groups are done Kruskal-Wallis test (age=30 respectively, 31～35,36～40 and 40)).In addition, the related coefficient (r) of free beta subunit content and AFP content and zero does not have marked difference (not ill and ill case is respectively r=0.04, P=0.39; R=-0.06, P-0.81).
In our data, the basic result of discovery has been affirmed such fact, and promptly You Li beta subunit makes the discovery rate of Down's syndrome reach the highest helpful.In fact, adopt discovery rate that determination method produced and the false positive rate of only measuring free to be respectively 65.4% and 5.2％。These ratios and other people utilize the report of the combination of three kinds of determination methods to be more or less the same.Therefore, that crosses as noted is such, and reducing the mensuration number is an advantage of the present invention.
We about the discovery of free effect according to as follows: (a) see separately to the contribution of Down's syndrome discovery rate maximum be the free determination method; (b) mensuration of not decreasing the HCG molecule is obtained lower discovery rate; (c) measure not impaired HCG molecule and obtain the discovery rate that a ratio is only measured not impaired HCG molecular assay Fa Genggao with the determination method that free makes up.
Verified, the morbidity rate of fetus Down's syndrome increased with the parent age.Therefore, as preceding selected,, will different priori risk rate be updated in the multivariate discriminant analysis processing with the parent age in order in clinical practice, to obtain the concrete ill level of significance of patient with the present invention.Because the content of AFP and free is all irrelevant with the parent age, therefore, we have done analysis to our data, have positive result to find out how many ill with not ill women under a given priori risk rate situation of the age different to each.Above-mentioned information is distributed as the basis, estimates to carry out false positive rate and sensitivity in the national widely generaI investigation in the U.S. according to the U.S. childbirth baby's pregnant woman age of living.Shown in table 7 (Fig. 7), this estimation shows might reach 80% discovery rate and 5% false positive rate.
Example 2 described samples are made the further discovery rate of analyzing with other combination of finding each index.More particularly, with the level of significance of same level of significance cutoff and priori the linear discriminent that the various combination of each index carries out (multiple of index intermediate value and the logarithm of each index are adding or do not adding under pregnant age) example 2 is handled.Utilize reference data and valid data to carry out linear discriminent and handle, the result is summarized in the table 8 of Figure 10.
Example 3
The one step determination method of following example explanation free and preliminary work and their application in the methods of the invention of two step determination methods.
The preliminary work of one step of free determination method
1. has covering one deck " catch " antibody special on the microtitre plate of 96 wells to the free of human chorionic gonadotrophin (HCG) molecule.Antibody can be monoclonal or polyclonal antibody.The antibody concentration that is used for covering plate is each well 0.8 microgram, but as need, also can be different.Plate was cultivated 16 hours down at 4 ℃ at least.
2. the phosphate buffer with the PH 7.2 that contains 0.05% polysorbas20 washes plate, also available other suitable cleaning buffer solution.Use the phosphate buffer of the PH7.2 that contains 3% hydrolyzed animal protein and 0.05% polysorbas20 with plate " obstruct " then.Also other solution that can use this area professional to be familiar with, the bovine serum albumen solution as 1%.The moon of 300 microlitres is added in each well every solution, allows plate at room temperature cultivate 1 hour.Also available other blocking method is as " being coated with the glaze method ".
3. clean plate then as previously mentioned, 100 microlitres are contained the inferior single special biotinylated antibody test damping fluid of free is added in each well.Used assay buffer is the phosphate buffer that contains 3% hydrolyzed animal protein and contain the PH 7.2 of 0.05% polysorbas20, but the known more any suitable solution of also available this area professional.Antibody according to the operator to like can be monoclonal, also can be polyclonal, can be the antibody that combines with a kind of material except that the biotin as horseradish peroxidase or alkaline phosphatase.The concentration of antibody is adjusted to the optimal absorption rate that reaches in the assay buffer.
4. the sample copy of getting 20 microlitres adds in each well, sample can be that assay buffer is as the blank that confirms test performance, a kind of free solution that is used for standardization unknown sample value, or the serum sample that takes out in trimestral pregnant woman's body to mid-term by pregnancy.With 30 seconds of plate vortex, be placed on ρ then and be on 200 rev/mins the spinner, at room temperature cultivated 30 minutes.
5. clean plate then as previously mentioned, the 100 microlitre assay buffer that will contain the strepavidin that combines horseradish peroxidase add in each well.If used second kind of antibody combines with a kind of material except that biotin, then do not need this step.The concentration of the strepavidin-peroxidase in the assay buffer is every microlitre 2.0 micrograms.It is last 5 minute of spinner of 200 rev/mins that plate at room temperature is placed on ρ.
6. clean plate as previously mentioned.An a kind of diamino-benzene matrix of neighbour solution of 100 microlitres is added in each well.In the also available this area of this matrix solution in the more known suitable stains of professional any replaces ， And to depend on what the material with second kind of antibodies is.Then plate is placed on ρ and is on 200 rev/mins the spinner, at room temperature place the dark place to cultivate 8 minutes.
7. then, the dilute sulfuric acid (1.0N) with 100 microlitres adds in each well to end the reaction of this matrix.
8. under 492nm, determine the absorptivity of each well by spectrophotometry.
The preliminary work of the two step determination methods of β-HCG
1. has covering one deck " catch " antibody special on the microtitre plate of 96 wells to the free beta subunit of human chorionic gonadotrophin (HCG) molecule.Antibody both can be monoclonal, also can be polyclonal.The antibody density that is used to cover plate is every well 0.8 microgram, but as need, also can be different.Plate was cultivated under 4 ℃ 16 hours at least.
2. the phosphate buffer with the PH7.2 that contains 0.05% polysorbas20 cleans plate, also available other suitable cleaning buffer solution.Then, with the phosphate buffer of PH7.2 that contains 3% hydrolyzed animal protein and 0.05% polysorbas20 with plate " obstruct ", other solution that also can use those skilled in the art to be familiar with, bovine serum albumen solution as 1%.The obstruct solution of 300 microlitres is added in each well, allows plate at room temperature cultivate 1 hour, also available other blocking method is as " being coated with the glaze method ".
3. then, clean plate as previously mentioned, a kind of assay buffer of 100 microlitres is added in each well.Used assay buffer is the phosphate buffer that contains the PH7.2 of 3% hydrolyzed animal protein and 0.05% polysorbas20, any in known some appropriate solution of also available this area professional.
4. then 20 microlitre samples are added in each well.Sample can be: as the assay buffer of the blank that confirms test performance; A kind of free solution that is used for standardization unknown sample value; Or the serum sample that from the pregnancy to the mid-term, takes out in the trimestral human female.With 30 seconds of plate vortex, be placed on ρ then and be on 200 rev/mins the spinner and at room temperature cultivated 30 minutes.
5. then, clean plate as previously mentioned, the assay buffer that 100 microlitres are contained the special biotinylated antibody of free subunit adds in each well.According to liking of operator, antibody can be monoclonal or polyclonal, also can be to combine with material except that the biotin as horseradish peroxidase or alkaline phosphatase
Regulate the concentration of antibody, to reach the optimal absorption rate.With 30 seconds of plate vortex, be placed on ρ then and be on 200 rev/mins the spinner and at room temperature cultivated 30 minutes.
6. clean plate then as previously mentioned.The assay buffer that 100 microlitres is contained the strepavidin that combines horseradish peroxidase joins in each well.If used second kind of antibody combines with a kind of material except that biotin, then do not need this step.The concentration of assay buffer streptococcus intermedius avidin one peroxidase is every microlitre 2.0 micrograms.It is last 5 minute of spinner of 200 rev/mins that plate is at room temperature placed ρ.
7. clean plate then as previously mentioned.An a kind of adjacent diamino-benzene matrix solution of 100 microlitres is added in each well.This matrix solution also can depend on the material that combines with second kind of antibody with any replacement ， And in the more known suitable stains of those skilled in the art.Then plate is placed on ρ and is on 200 rev/mins the spinner.At room temperature place the dark place to cultivate 8 minutes.
8. then, the dilute sulfuric acid (1.0N) with 100 microlitres adds in each well to end the reaction of this matrix.
9. under 492nm, determine the absorptivity of each well by spectrophotometry de termination.
These two kinds of determination methods are used for carrying out method of the present invention.With 178 routine patient's sample study the relation between the free content in fetus Down's syndrome and the maternal blood.To from known nourish 26 samples obtaining in pregnant woman's body of suffering from the Down's syndrome fetus and 152 examples known, not ill sample analyzes.All sample standard deviations are taken from the white man pregnant woman who does not suffer from diabetes of the single tire in bosom.
Then, we analyze patient's sample with the ELISA determination method, and to obtain the quantitative values of MSAFP, And draws the content of free individually with a step determination method and two step determination methods.MSAFP value that is obtained by every kind of determination method and free value become the variable of the linear discriminent that carries out in handling to set up one group of reference data on computer software statistical package available on the market " statistical analysis system ".Also be added in the discriminant processing as a variable patient's pregnant age.Discriminant analysis result between 14 to 16 weeks is summarized as follows to all pregnant ages and pregnant age:
All pregnant ages
The contrast of false positive rate discovery rate is ill
Log(β-1) 6.6％ 69.2％ 152 26
Log(β-1)+Log(AFP) 5.9％ 72.0％ 152 25
Log(β-2) 8.2％ 33.3％ 138 18
Log(β-2)+Log(AFP) 10.1％ 64.7％ 138 17
Log(β-2)
* 9.6％3 3.3％ 136 18
Log(β-2)+Log(AFP)
* 10.3％ 52.9％ 136 17
14 to 16 weeks
The contrast of false positive rate discovery rate is ill
Log(β-1) 5.8％ 68.4％ 104 19
Log(β-1)+Log(AFP) 4.8％ 73.7％ 104 19
Log(β-2) 7.1％ 45.4％ 98 11
Log(β-2)+Log(AFP) 9.2％ 63.6％ 98 11
Log(β-2)
* 10.4％ 54.6％ 96 11
Log(β-2)+Log(AFP)
* 8.3％ 63.6％ 96 11
Annotate: all analyses include pregnant age.
*Be with in the back
*Analysis in, other two groups of sample values are that 260 and 316 result has been removed.
Suffix-1 expression single stage method is handled, and-2 expression two step methods are handled.
Discovery rateThe percent that refers to the fetus Down's syndrome of the positive test result of demonstration.
False positive rateThe number percent that refers to the normal fetus of the positive test result of demonstration.
ContrastThe number that refers to analyzed not ill sample.
IllThe number that refers to analyzed ill sample.
Utilize the step determination method of free β and the combination of AFP can obtain the highest discovery rate and minimum false positive rate to all conceived all numbers and conceived 14 to 16 all pregnant woman.
Our result has supported further that in pregnancy serum inspection scheme this good performance comprises: (a) noninvasive method with feasible and effective method carries out the good performance that Down's syndrome is checked; (b) low false positive rate and high discovery rate; (c) use essentially independent each other index; (d) to blood sample do not have harsh restriction (a day when, feed situation, personal habits etc.) and (e) with the compatibility of other antenatal exaination method.
Our result shows, though utilization might obtain higher discovery rate than other method biochemical analysis still less in the inspection of Down's syndrome.In Down's syndrome is checked, only use the most effective index just can provide non-intrusive inspection information in the gestation in early days to the family that the overwhelming majority is suffered from the common reason of serious mental retardation.
For those skilled in the art, obviously, change the level of significance cutoff of judging positive findings, or the different groups of research object are adopted different priori risk rates, patient is carried out the result that discriminant is handled changing.
Therefore, should be clear and definite, present invention includes all and drop on improvement within the following claims scope.
Especially, the present invention relates to following embodiment:
1. whether a definite pregnant woman is in and nourishes the method for suffering from the obvious danger of Down's syndrome fetus, comprising:
Measure the content of the free of human chorionic gonadotrophin in the pregnant woman body blood, in probability density function of pregnant substitution in age with the measured value of said free in the said human chorionic gonadotrophin and pregnant woman, with said free content and pregnant age of said pregnant woman and one group of reference data comparison of said pregnant woman, nourish the level of significance of suffering from the Down's syndrome fetus thus to determine said pregnant woman.
2. whether a definite pregnant woman is in and nourishes the method for suffering from the obvious danger of Down's syndrome fetus, comprising: measure the free of human chorionic gonadotrophin in the pregnant woman body blood, measurement result is the sign that fetus Down's syndrome level of significance increases.
3. as enforcement scheme 2 described methods, wherein also comprise: measure the α-fetus albumen in the pregnant woman blood, the measurement result of free and α-fetus albumen is the sign that fetus Down's syndrome level of significance increases in the human chorionic gonadotrophin.
4. whether a definite pregnant woman is in and nourishes the method for suffering from the obvious danger of Down's syndrome fetus, comprise: the content of free in the human chorionic gonadotrophin in the measurement pregnant woman body blood, And nourishes the level of significance of suffering from the Down's syndrome fetus with this measured value and one group of normal data comparison to determine the pregnant woman.
5. whether a definite pregnant woman is in and nourishes the method for suffering from the obvious danger of Down's syndrome fetus, comprise: measure a kind of content of analyte in the pregnant woman body blood with the determination method of measuring free in the human chorionic gonadotrophin, And will nourish the level of significance of suffering from the Down's syndrome fetus to determine the pregnant woman to measurement result and one group of reference data comparison of this analyte.
6. as enforcement scheme 4 or 5 described methods, wherein also comprise: measure the content of α-fetus albumen in the pregnant woman body blood, And compares pregnant age and one group of reference data of this measurement result and pregnant woman.
7. as enforcement scheme 4,5 or 6 described methods, wherein comparison step comprises measurement result is updated in the probability density function that is produced by one group of reference data of being handled by linear discriminent.
8. as enforcement scheme 4,5,6 or 7 described methods, wherein comparison step comprises that logarithm and one group of reference data with measurement result compare.
9. one is used for carrying out embodiment 1 to 8 any is used for determining whether the pregnant woman is in the assay method of nourishing the method for suffering from the Down's syndrome FD, comprising:
Be used for testing the means of free in the pregnant woman blood human chorionic gonadotrophin.
10. free Determination on content method of measuring human chorionic gonadotrophin in the blood of human body.
11. an equipment comprises:
Be applicable to the device that receives the test result of the free content of human chorionic gonadotrophin in the pregnant woman body blood and be used for test result and one group of normal data are compared computer installation with definite fetal chromosomal abnormalities.
12. as enforcement scheme 11 described equipment, fetal chromosomal abnormalities wherein is meant Down's syndrome.
13. be used for determining whether the pregnant woman is in the equipment of nourishing in the obvious danger of trouble Down's syndrome fetus, comprising:
Be applicable to the device that receives the test result of the free content of human chorionic gonadotrophin in the pregnant woman body blood, and computer installation, be used for calculating one group of reference data, with be used for the measurement result of the free of said human chorionic gonadotrophin and probability density function of pregnant substitution in age of said pregnant woman, thus said pregnant woman's free beta subunit value and pregnant age and one group of reference data comparison of said pregnant woman are nourished the level of significance of suffering from the Down's syndrome fetus to determine said pregnant woman.
14. be used for determining whether the pregnant woman is in the equipment of nourishing the obvious danger of trouble Down's syndrome fetus, comprising:
Be applicable to the device that receives the measured value of a certain analyte in the pregnant woman body blood that the free determination method with a kind of human chorionic gonadotrophin obtains, reach
Computer installation, be used for calculating one group of reference data and the probability density function of pregnant substitution in age that is used for said analyte Determination on content value and said pregnant woman, with pregnant age of the content of said pregnant woman's free and said pregnant woman and one group of reference data relatively, nourish the level of significance of suffering from the Down's syndrome fetus thus to determine the pregnant woman.
15., wherein also comprise as enforcement scheme 13 or 14 described equipment:
Be applicable to the device that receives the measured value of α-fetus protein content in the pregnant woman body blood, and
Computer installation wherein also in this probability density function of test result substitution with α-fetus albumen, is compared α-fetus albumen value of this pregnant woman thus with one group of normal data.
16. as enforcement scheme 13,14 or 15 said equipment, wherein this group reference data is that logarithm by each content measurement value calculates, and
The logarithm of these measurement results is used for the content of each index of pregnant woman and this group reference data are compared.
Claims (8)
1. a use is to determine that whether the pregnant woman should accept the method for measuring of diagnostic test, comprises the steps:
Determination method at the free of human chorionic gonadotrophin is provided specifically;
Obtain maternal blood from the pregnant woman; With
Measure described maternal blood with described determination method, to measure the free content of the human chorionic gonadotrophin in the described maternal blood.
2. the method for claim 1 further comprises free content and one group of reference data step relatively with said human chorionic gonadotrophin.
3. claim 1 or 2 method further comprise pregnant age and one group of reference data step relatively with said pregnant woman.
4. claim 1,2 or 3 method further comprise α-fetus protein content of measuring in the said maternal blood and with the step of said α-fetus protein content with one group of reference data comparison.
5. claim 1,2,3 or 4 method, wherein said comparison step comprise measurement result are updated in the probability density function that is produced by this group reference data by the linear discriminant analysis method.
6. claim 1,2,3,4 or 5 described methods, wherein said comparison step comprise with the logarithm of measurement result and this group reference data relatively.
7. claim 1,2,3,4,5 or 6 described methods, further comprise the steps: by nourish the known fetus that suffers from Down's syndrome known pregnant age the pregnant woman maternal blood in human chorionic gonadotrophin free content and nourish the known fetus that does not suffer from Down's syndrome known pregnant age the pregnant woman maternal blood in the free cubage of human chorionic gonadotrophin obtain one group of reference data.
8. claim 1,2,3,4,5,6 or 7 described methods, further comprise the steps: by nourish the known fetus that suffers from Down's syndrome known pregnant age the pregnant woman maternal blood in α-fetus protein content and nourish the known fetus that does not suffer from Down's syndrome known pregnant age the pregnant woman maternal blood in α-fetus protein content calculate one group of reference data.
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| US29748189A | 1989-01-17 | 1989-01-17 | |
| US297,481 | 1989-01-17 | ||
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| CN90100994A Division CN1047390A (en) | 1989-01-17 | 1990-01-17 | Find the method and apparatus of Down's syndrome with the noninvasive method of checking maternal blood |
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| CNA2008100812460A Pending CN101329354A (en) | 1989-01-17 | 1990-01-17 | Method for detecting trisomy 13 and down syndrome by non-invasive maternal blood screening |
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