Inventors: Macri; James N. (170 Sidney St., Oyster Bay, NY 11771) Notice: The portion of the term of this patent subsequent to May 31, 2011 has been disclaimed. Appl. No.: 051761 Filed: February 3, 1993

Primary Examiner: Rosen; Sam Attorney, Agent or Firm: Kenyon & Kenyon United States Patent 5,324,668 June 28, 1994

Parent Case Text
This application is a continuation of application Ser. No. 07/420,775, filed Oct. 12, 1989, which is a continuation-in-part of application Ser. No. 07/360,603, filed Jun 1, 1989, which is a continuation-in-part of application Ser. No. 07/349,373, filed May 8, 1989, which is a continuation-in-part of application Ser. No. 07/311,808 filed Feb. 17, 1989 which is a continuation-in-part of application Ser. No. 07/297,481, filed Jan. 17, 1989, all now abandoned.

Claims
What is claimed is:

1. A screening method for determining a pregnant woman's risk of carrying a fetus with Down syndrome comprising: measuring said pregnant woman's maternal blood for free beta (human chorionic gonadotropin (HCG)) level, comparing said level of free beta (HCG), and said pregnant woman's gestational age, to reference values at various gestational ages of the level for free beta (HCG) in: (1) pregnant women carrying Down syndrome fetuses and (2) pregnant women carrying normal fetuses, said comparison being indicative of said pregnant woman's risk of carrying a fetus with Down syndrome, wherein a higher level of free beta (HCG) is indicative of a higher probability of carrying a fetus with Down syndrome.

2. The method of claim 1 further comprising: measuring said pregnant woman's maternal blood for intact human chorionic gonadotropin molecule (Intact HCG) level and incorporating said level for Intact HCG and reference values at various gestational ages of the levels Intact HCG in: (1) pregnant women carrying Down syndrome fetuses and (2) pregnant women carrying normal fetuses into said comparison wherein a higher level of Intact HCG is indicative of a higher probability of carrying a fetus with Down syndrome.

3. The method of claim 2 further comprising: measuring said pregnant woman's maternal blood for alpha-fetoprotein (AFP) level and said pregnant woman's maternal blood for unconjugated estriol (UE) level and incorporating said level of AFP, said level of UE, and reference values at various gestational ages of said levels of AFP and UE in: (1) pregnant women carrying Down syndrome fetuses and (2) pregnant women carrying normal fetuses into said comparison wherein lower levels of AFP and UE are indicative of a higher probability of carrying a fetus with Down syndrome.

4. The method of claim 3 further comprising: incorporating a ratio of said measurement of said level of said free beta (HCG) to said measurement of said level of said Intact HCG and reference values at various gestational ages of the ratio of the level of free beta (HCG) to the level of Intact HCG in: (1) pregnant women carrying Down syndrome fetuses and (2) pregnant women carrying normal fetuses, into said comparison wherein a higher ratio is indicative of a higher probability of carrying a fetus with Down syndrome.

5. The method of claim 2, further comprising: measuring said pregnant woman's maternal blood for alpha-fetoprotein (AFP) level and incorporating said measurement of said level of AFP, a ratio of said level of said free beta (HCG) to said level of said intact HCG and reference values at various gestational ages of the ratio of the level of free beta (HCG) to the level of Intact HCG and the level of AFP in (1) pregnant women carrying Down syndrome fetuses and (2) pregnant women carrying normal fetuses into said comparison wherein lower levels of AFP and a higher ratio are indicative of a higher probability of carrying a fetus with Down syndrome.

6. The method of claim 1 wherein the assay comprises a one step free beta-hCG assay.

7. A method for determining the risk that a pregnant woman is carrying a fetus with Down syndrome comprising: measuring said pregnant woman's maternal blood level of a fragment of free beta (human chorionic gonadotropin (HCG)) and comparing the measurement of said level of the fragment of free beta (HCG) to reference data containing reference values at various gestational ages of the level of the fragment of free beta (HCG) in: (1) pregnant women carrying Down syndrome fetuses and (2) pregnant women carrying normal fetuses, said comparison being indicative of the pregnant woman's risk wherein a higher level of the fragment of free beta (HCG) is indicative of a high probability of carrying a fetus with Down syndrome.

8. A method for determining the risk that a pregnant woman is carrying a fetus with Down syndrome comprising: measuring said pregnant woman's maternal blood for a protein portion of free beta (human chorionic gonadotropin (HCG)) level and comparing the measurement of said level for the protein portion of free beta (HCG) to reference data containing reference values at various gestational ages of the level for said protein portion of free beta (HCG) in (1) pregnant women carrying Down syndrome fetuses and (2) pregnant women carrying normal fetuses, said comparison being indicative of the pregnant woman's risk wherein a higher level of the protein portion of free beta (HCG) is indicative of a higher probability of carrying a fetus with Down syndrome.

9. A method for determining the risk that a pregnant woman is carrying a fetus with Down syndrome comprising: measuring said pregnant woman's maternal blood for a carbohydrate portion of free beta (human chorionic gonadotropin (HCG)) level and comparing the measurement of said level for the carbohydrate portion of free beta (HCG) to reference data containing reference values at various gestational ages of the level for said carbohydrate portion of free beta (HCG) in: (1) pregnant women carrying Down syndrome fetuses and (2) pregnant women carrying normal fetuses, said comparison being indicative of the pregnant woman's risk wherein a higher level of the carbohydrate portion of free beta (HCG) is indicative of a higher probability of carrying a fetus with Down syndrome.

10. A method for determining the risk that a pregnant woman is carrying a fetus with Down syndrome comprising: measuring said pregnant woman's maternal blood for a portion of free beta (human chorionic gonadotropin (HCG)) located at about the junction of the carbohydrate and the protein portions of free beta (HCG) and comparing the measurement of said level to reference data containing reference values at various gestational ages of the level of said portion of free beta (HCG) located at about the junction of the carbohydrate and the protein portions of free beta (HCG) in: (1) pregnant women carrying Down syndrome fetuses and (2) pregnant women carrying normal fetuses, said comparison being indicative of the pregnant woman's risk wherein a higher level of the portion of free beta (HCG) is indicative of a higher probability of carrying a fetus with Down syndrome.

11. A method for determining the risk that a pregnant woman is carrying a fetus with Down syndrome comprising: measuring a pregnant woman's maternal blood for free beta (human chorionic gonadotropin (HCG)) level and comparing the measurement for the level of free beta (HCG) to a set of reference data containing reference values of the level for free beta (HCG) in: (1) pregnant women carrying Down syndrome fetuses and (2) pregnant women carrying normal fetuses, said comparison being indicative of the pregnant woman's risk wherein a higher level of free beta (HCG) is indicative of a higher probability of carrying a fetus with Down syndrome.

12. The method of claim 11 further comprising measuring the pregnant woman's maternal blood for alpha-fetoprotein (AFP) level and incorporating said level of AFP and reference values at various ages of the levels AFP in: (1) pregnant women carrying Down syndrome fetuses and (2) pregnant women carrying normal fetuses, into said comparison wherein a lower level of AFP is indicative of a higher probability of risk.

13. A screening method for determining a pregnant woman's risk of carrying a fetus with trisomy 13 comprising:

measuring said pregnant woman's maternal blood for free beta (human chorionic gonadotropin (HCG)) level and comparing said level of free beta (HCG) and said pregnant woman's gestational age to reference values at various gestational ages of the level of free beta (HCG) in: (1) pregnant women carrying trisomy 13 fetuses and (2) pregnant women carrying normal fetuses, said comparison being indicative of said pregnant woman's risk of carrying a fetus with trisomy 13 wherein a higher level of free beta (HCG) is indicative of a higher probability of carrying a fetus with trisomy 13.

14. A method for determining whether a pregnant woman's risk of carrying a fetus with Down syndrome warrants further testing comprising: measuring said pregnant woman's maternal blood level of an analyte using an assay that employs an antibody raised against free beta (HCG) and comparing the level of the analyte to a set of reference data containing reference values of the level of the analyte in: (1) pregnant women carrying Down syndrome fetuses and (2) pregnant women carrying normal fetuses, said comparison being indicative of pregnant woman's risk of carrying a fetus with Down syndrome wherein a higher level of the analyte is indicative of a higher probability of carrying a fetus with Down syndrome.

15. The method of claim 14 wherein the assay comprises an enzyme linked immunosorbent assay for free beta-hCG.

16. The method of claim 14 wherein the assay comprises a one step free beta-hCG assay.

17. A method for screening a pregnant woman to determine if said pregnant woman's risk of carrying a fetus with Down syndrome warrants further testing comprising:

selecting a risk cut-off level that will determine whether said pregnant woman should be offered further testing;

assigning a prior risk level for said pregnant woman's risk of carrying a fetus with Down syndrome;

measuring said pregnant woman's blood for free beta (human chorionic gonadotropin (HCG)) level;

determining said pregnant woman's gestational age; and

comparing said level of free beta (HCG) and said pregnant woman's gestational age to a set of reference data, containing reference values at various gestational ages of the blood level of free beta (HCG) in: (1) pregnant women carrying Down syndrome fetuses and (2) pregnant women carrying normal fetuses said comparison in conjunction with said prior risk being indicative of whether said pregnant woman's risk of carrying a fetus with Down syndrome is greater than the selected risk cut-off level wherein a higher level of free beta (HCG) is indicative of a higher probability of carrying a fetus with Down syndrome.

18. The method of claim 17 further comprising:

measuring said pregnant woman's blood level of alpha-fetoprotein (AFP) and incorporating said levels of AFP and a set of reference data containing reference values at various gestational ages of the blood levels of AFP in: (1) pregnant women carrying Down syndrome fetuses and (2) pregnant women carrying normal fetuses into said comparison in conjunction with said prior risk wherein a higher level of AFP is indicative of a higher probability that the pregnant woman's risk of carrying a fetus with Down syndrome is greater than the selected risk cut-off level.

19. The method of claim 17 further comprising:

measuring said pregnant woman's blood for intact human chorionic gonadotropin (Intact HCG) level and incorporating said level for Intact HCG and a set of reference data containing reference values at various gestational ages of the blood levels of Intact HCG in: (1) pregnant women carrying Down syndrome fetuses and (2) pregnant women carrying normal fetuses into said comparison in conjunction with said prior risk wherein a higher level of Intact HCG is indicative of a higher probability that said pregnant woman's risk of carrying a fetus with Down syndrome is greater than the selected risk cut-off level.

20. The method of claim 18 further comprising:

measuring said pregnant woman's blood for intact Human chorionic gonadotropin level and incorporating said level of Intact HCG and a set of reference data containing reference values at various gestational ages of the blood levels of Intact HCG in: (1) pregnant women carrying Down syndrome fetuses and (2) pregnant women carrying normal fetuses into said comparison in conjunction with said prior risk wherein a higher level of Intact HCG is indicative of a higher probability that said pregnant woman's risk of carrying a fetus with Down syndrome is greater than the selected risk cut-off level.

21. The method of claim 20 further comprising:

measuring said pregnant woman's blood for unconjugated estriol (UE) level and incorporating said level of UE and a set of reference data containing reference values at various gestational ages of the levels of UE in: (1) pregnant women carrying Down syndrome fetuses and (2) pregnant women carrying normal fetuses into said comparison in conjunction with said prior risk wherein a higher level of free beta (HCG) is indicative of a higher probability that said pregnant woman's risk of carrying a fetus with Down syndrome is greater than the selected risk cut-off level.

22. The method of claim 1 further comprising: measuring said pregnant woman's maternal blood level of alpha-fetoprotein (AFP) and incorporating said level of AFP and reference values at various gestational ages of the levels of AFP in: (1) pregnant women carrying Down syndrome fetuses and (2) pregnant women carrying normal fetuses, into said comparison wherein a higher level of AFP is indicative of a higher probability of carrying a fetus with Down syndrome.

23. The method of any one of claims 1, 2, 3, 4, 5 and 17-22 wherein the free beta (HCG) is an aberrant form of free beta (HCG).

24. The method of any one of claims 1, 2, 3, 4, 5 and 22 wherein said pregnant woman's prior risk of carrying a fetus with Down syndrome is a factor incorporated into said comparison.

25. The method of any one of claims 13-21 wherein said pregnant woman's prior risk of carrying a fetus with Down syndrome is based on her maternal age.

Description
BACKGROUND OF THE INVENTION

The present invention relates to a method for detecting fetal Down syndrome (Trisomy 21) during prenatal screening. This method also relates to other more rare but detectable chromosomal trisomies such as Trisomy 13 and Trisomy 18. More particularly the present invention relates to a method for improving detection efficiency in screening for Down syndrome by measuring the amount of the free beta subunit of human chorionic gonadotropin (hCG) in the blood of pregnant women.

Down syndrome, also referred to as Trisomy 21, is the most common congenital cause of severe mental retardation. Generally, fetal Down syndrome can be determined by a diagnostic procedure including amniocentesis and karyotyping. However, this diagnostic procedure is invasive and involves risk to the woman and the fetus. Amniocentesis and karyotyping are not routinely performed during all pregnancies. Instead, one or more screening methods may be utilized to determine when the risk to the pregnancy warrants the risk of undergoing an invasive diagnostic procedure.

The incidence of Down syndrome increases significantly with increasing maternal age. Historically, the prenatal search for Down syndrome has focused on pregnant women at and over the age of 35, at which ages the risks of Down syndrome approach or exceed the risks of diagnostic procedures utilized to detect fetal Down syndrome. Therefore the standard method of prenatal screening has involved selecting women for diagnostic amniocentesis on the basis of maternal age. Age, however, is an inadequate screening criterion in that only about 20% of all Down syndrome pregnancies can be detected by carrying out amniocentesis and karyotyping on the 5% of pregnant women most at risk, that is, those aged 35 years or greater. And, because in actual clinical practice only about half of the women aged 35 years or greater undergo amniocentesis and karyotyping, fewer than 10% of Down syndrome pregnancies are prenatally detected.

In 1984 an association between lowered maternal blood alpha-fetoprotein (AFP) levels and fetal Down syndrome was discovered. For example, see "An association between low maternal serum alpha-fetoprotein and fetal chromosomal abnormalities"; Merkatz, Macri, et al.; Am. J. Obstet. Gynecol. 148:886, 1984; the disclosure of which is hereby incorporated by reference. In this publication it was noted that other chromosomal trisomies, in particular Trisomy 13 and Trisomy 18, were also associated with lowered maternal blood AFP levels. The incidence of these additional chromosomal trisomies (1 in 5000 pregnancies and 1 in 6600 pregnancies, respectively) is significantly lower than the general a priori risk associated with Trisomy 21 (Down syndrome). However, because of the association of these other chromsomal trisomies with lowered MSAFP levels, such abnormalities will also be detected within a screening protocol utilizing maternal blood AFP and the free beta subunit of hCG and possibly additional markers described herein. It is obvious to those skilled in the art that in using the protocol described herein for Trisomy 21, the detection of Trisomy 13 and 18 may also be accomplished. The association between lowered maternal blood AFP levels and fetal Down syndrome presented the opportunity to use a non-invasive blood screening test in the detection of Down syndrome cases in young, apparently unaffected families where approximately 80% of Down syndrome cases occur. It is estimated that the use of a screening test based on low maternal blood AFP (as a screening marker) would lead to the prenatal detection of approximately 20% of all cases of fetal Down syndrome.

Another method for screening involves measuring the level of unconjugated estriol (UE) in maternal blood. For example, see "Maternal blood screening for Down syndrome in early pregnancy"; Wald, et al. British Journal of Obstetrics and Gynocology (BMJ) Volume 95, April 1988, the disclosure of which is hereby incorporated by reference. The measurement of UE however, provides a poor basis for screening.

More recently an association between elevated maternal blood hCG levels, elevated maternal blood level of the alpha subunit of hCG (hCG is composed of two subunits, hereinafter referred to as alpha-hCG and beta-hCG respectively), and fetal Down syndrome was discovered. For example, see "Abnormal Maternal Serum Chorionic Gonadotropin Levels in Pregnancies with Fetal Chromosome Abnormalities"; Bogart, Pandian and Jones; Prenatal Diagnosis, Vol. 7, 623-630 (1987) the disclosure of which is hereby incorporated by reference. In the Bogart article it is estimated that the use of elevated maternal blood hCG levels and elevated maternal blood levels of the alpha subunit of hCG, would detect approximately 68% of the chromosomally abnormal fetuses. However, these results were obtained from a study on pregnancies at 18-25 weeks of gestation and the affected cases appear to be of women previously identified as being at risk for Down syndrome.

Generally, as suggested above, screening by evaluation of maternal blood hCG has involved only the measurement of hCG in general and additionally the measurement of alpha-hCG. Although these screening methods do detect fetal Down syndrome, there is a need and a desire for a method which detects a greater percentage of fetal Down syndrome cases.

I have discovered a previously unknown association between elevated levels of maternal blood free beta-hCG and fetal Down syndrome. I have also discovered a previously unknown association between the maternal blood level of free beta-hCG and the maternal blood level of AFP and fetal Down syndrome, I have further discovered a previously unknown association between the ratio of the maternal blood level of free beta-hCG to the maternal blood level of the intact hCG molecule and fetal Down syndrome. I have still further discovered that using a multivariate discriminant analysis technique improves the detection efficiency of a screening method using the maternal blood level of free beta-hCG, or the maternal blood level of free beta-hCG and the maternal blood level of AFP, or the log of either, or the log of both, especially when gestational age is also incorporated as a variable in the discriminant analysis technique, for a chosen risk cut-off level. Gestational age refers to the age of the pregnant woman's fetus. Detection efficiency refers to the percentage of cases of fetal Down syndrome which are correctly detected for a chosen risk cut off level. The risk cut off level will be more fully explained in a following section. Discriminant analysis is a generally known approach to multivariate analysis involving the separation of a population into two or more groups by a univariate risk assessment. Discriminant analysis is also sometimes described as a way of constructing a linear combination of independent variables, thus reducing the problem of measuring group differences to a univariate problem. Discriminant analysis can also be performed when there is only one variable involved in a problem. A general discussion of discriminant analysis can be found in Marketing Research; Churchill, G.A.; Dryden, 1976; Chapter 15, pages 530-543, the disclosure of which is hereby incorporated by reference. I have discovered that subjecting the maternal blood levels of free beta-hCG, the maternal blood levels of intact hCG, the ratio of the maternal blood level of free beta-hCG to the maternal blood level of the intact hCG molecule, the maternal blood level of AFP, the maternal blood level of UE, and gestational age to multi-variate discriminant analysis detects a greater percentage, with a lower false positive rate, of fetal Down syndrome cases than any other known screening method for the prenatal detection of Down syndrome. I have further discovered that a still greater number of the cases of fetal Down syndrome may be detected by using only the measurements of the maternal blood levels of free-beta hCG and the maternal blood levels of AFP and subjecting the log of each measurement and gestational age to a multivariate discriminant analysis. These and other discoveries will be more fully explained in the Summary of the Invention section and the Detailed Description of the Invention section.

One object of the present invention is to provide a method and process for screening for fetal Down syndrome which detects a greater percentage of fetal Down syndrome cases with a given false positive rate than other known prenatal screening methods.

Another object of the present invention is to provide a method and process for screening for fetal Down syndrome which has a lesser false positive rate for a given detection percentage than other known methods.

A still further object of the present invention is to apply multi-variate discriminant analysis to methods for screening for Down syndrome to detect a greater percentage of fetal Down syndrome cases with a lesser false positive rate.

A further object of the present invention is to provide a method and process for screening for fetal Down syndrome by measuring the level of maternal blood free beta-hCG.

A still further object of the present invention is to provide a method and process for screening for fetal Down syndrome by measuring the maternal blood level of AFP and the maternal blood level of free beta-hCG.

Other objects and advantages of the present invention will become apparent in the following description of the invention.

SUMMARY OF THE INVENTION

To achieve these and other objects, according to the present invention a pregnant woman's (hereinafter the patient) maternal serum levels of free beta-hCG are measured by conventional immunological methods which can include immuno-assay techniques such as those referred to in the papers above, and other techniques known in the art. The level of free beta-hCG is then compared to a set of reference data to determine the patient's risk of carrying a fetus with Down syndrome. To improve detection efficiency, the level of free beta-hCG and the gestational age can be compared to a set of reference data. To further improve detection efficiency, a patient's maternal blood levels of free beta-hCG and AFP (referred to as "markers") are measured by conventional immunological methods, including assay techniques known to the art such as those referred to in the papers above. The levels of each marker are then compared to a set of reference data to determine the patient's risk of carrying a fetus with Down syndrome. A multivariate discriminant analysis technique is used to compare the levels of the markers to a set of reference data. More particularly, a patient specific risk is then calculated using Bayes rule, the patient's a priori risk, and the relative frequencies for unaffected and affected pregnancies which are determined by incorporating the log of the patient's quantitative levels of each marker into the probability density functions for the reference data developed using multivariate discriminant analysis. If the patient's risk of carrying a fetus with Down syndrome is greater than a given risk cut-off level, the patient should be counseled about further diagnostic tests to confirm the presence of Down syndrome. Incorporating gestational age as a marker along with the level of free beta-hCG and the maternal blood level of AFP will further improve detection efficiency. Since the maternal blood level of free beta-hCG and the maternal blood level of AFP for a number of samples tend to be distributed according to a log-gaussian distribution curve, the greatest detection efficiency can be achieved by incorporating the log of the patient's quantitative levels of each marker and gestational age into the probability density functions for the reference data developed using multivariate discriminant analysis.

An advantage of the method and process of the present invention is that it correctly predicts a higher percentage of fetal Down syndrome cases, with a lesser false positive rate than other known methods and processes.