Inventors: Macri; James N. (170 Sidney St., Oyster Bay, NY 11771) Appl. No.: 925683 Filed: August 7, 1992

Primary Examiner: Rosen; Sam Assistant Examiner: Spiegel; Carol A. Attorney, Agent or Firm: Kenyon & Kenyon United States Patent 5,252,489 October 12, 1993

Parent Case Text
This application is a continuation-in-part of application Ser. No. 07/868,160, filed Apr. 14, 1992, which is a continuation-in-part of application Ser. No. 07/420,775, filed Oct. 12, 1989, which is a continuation-in-part of application 07/360,603, filed Jun. 1, 1989, now abandoned, which is a continuation-in-part of application Ser. No. 07/349,373, filed May 8, 1989, now abandoned, which is a continuation-in-part of application serial number 07/311,808 filed Feb. 17, 1989, now abandoned, which is a continuation-in-part of application Ser. No. 07/297,481, filed Jan. 17, 1989, now abandoned.

Claims
I claim:

1. A screening method for determining a pregnant woman's risk of carrying a fetus with Down syndrome comprising:

obtaining a blood sample from the pregnant woman during a time period selected from the group consisting of: the first trimester of pregnancy; the second trimester of pregnancy and the third trimester of pregnancy; on filter paper and allowing the blood sample to dry to form a dried blood spot;

determining said pregnant woman's maternal blood level of free beta (human chorionic gonadotropin (HCG)) from the dried blood spot and comparing said level of free beta (HCG) to reference values of the level for free beta (HCG) during the time period in: (1) pregnant women carrying a fetus with Down syndrome 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 determining the pregnant woman's maternal blood level of alpha-fetoprotein (AFP) from the dried blood spot and incorporating the woman's blood level of AFP and reference values of the levels AFP during the time period in: (1) pregnant women carrying a fetus with Down syndrome and (2) pregnant women carrying normal fetuses, into said comparison wherein a lower level of AFP is indicative of a higher probability of risk.

3. The method of claim 1 wherein the free beta (HCG) is an aberrant form of free beta (HCG).

4. The method of claim 2 wherein the free beta (HCG) is an aberrant form of free beta (HCG).

5. The method of claim 2 wherein the free beta (HCG) is an aberrant form of free beta (HCG).

6. The method of claim 3 wherein the aberrant form of free beta (HCG) is nicked free beta (HCG).

7. A method for determining the risk that a pregnant woman is carrying a fetus with Down syndrome comprising:

obtaining a blood sample from the pregnant woman during a time period selected from the group consisting of: the first trimester of pregnancy; the second trimester of pregnancy and the third trimester of pregnancy; on filter paper and allowing the blood sample to dry to form a dried blood spot;

determining said pregnant woman's maternal blood level of a fragment of free beta (human chorionic gonadotropic (HCG)) from the dried blood spot and comparing the measurement of said level of the fragment of free beta (HCG) to reference data containing reference values of the level of the fragment of free beta (HCG) during the time period in: (1) pregnant women carrying a fetus with Down syndrome 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 higher probability of carrying a fetus with Down syndrome.

8. The method of claim 7:

wherein the fragment of free beta (HCG) is selected from the group consisting of: the protein portion of free beta (HCG); the carbohydrate portion of free beta (HCG); and the portion of free beta (HCG) located at about the junction of the carbohydrate and the protein portions of free beta (HCG).

9. The method of claim 8 further comprising determining the pregnant woman's maternal blood level of alpha-fetoprotein (AFP) from the dried blood spot and incorporating the woman's blood level of AFP and reference values of the levels AFP during the time period in: (1) pregnant women carrying a fetus with Down syndrome and (2) pregnant women carrying normal fetuses, into said comparison wherein a lower level of AFP is indicative of a higher probability of risk.

10. A method for determining whether a pregnant woman's risk of carrying a fetus with Down syndrome warrants further testing comprising:

obtaining a blood sample from the pregnant woman during a time period selected from the group consisting of: the first trimester of pregnancy; the second trimester of pregnancy and the third trimester of pregnancy; on filter paper and allowing the blood sample to dry to form a dried blood spot;

determining said pregnant woman's maternal blood level of an analyte using an assay for free beta (HCG) from the dried blood spot and comparing said level of said analyte to a set of reference values of the level of the analyte during the time period in: (1) pregnant women carrying a fetus with Down syndrome and (2) pregnant women carrying normal fetuses, said comparison being indicative of pregnant woman's risk of carrying a fetus with the chromosomal trisomy wherein a higher level of the analyte is indicative of a higher probability of carrying a fetus with Down syndrome.

11. A screening method for determining a pregnant woman's risk of carrying a fetus with Trisomy 18 comprising:

obtaining a blood sample from the pregnant woman during a time period selected from the group consisting of: the first trimester of pregnancy; the second trimester of pregnancy and the third trimester of pregnancy; on filter paper and allowing the blood sample to dry to form a dried blood spot;

determining said pregnant woman's maternal blood level of free beta (human chorionic gonadotropin (HCG)) from the dried blood spot and comparing said level of free beta (HCG) to reference values of the level for free beta (HCG) during the time period in: (1) pregnant women carrying a fetus with Trisomy 18 and (2) pregnant women carrying normal fetuses, said comparison being indicative of said pregnant woman's risk of carrying a fetus with Trisomy 18, wherein a lower level of free beta (HCG) is indicative of a higher probability of carrying a fetus with Down syndrome.

12. A screening method for determining a pregnant woman's risk of carrying a fetus with a chromosomal anomoly comprising:

obtaining a blood sample from the pregnant woman during a time period selected from the group consisting of: the first trimester of pregnancy; the second trimester of pregnancy and the third trimester of pregnancy; on filter paper and allowing the blood sample to dry to form a dried blood spot;

determining said pregnant woman's maternal blood level of free beta (human chorionic gonadotropic (HCG) from the dried blood spot and comparing said level of free beta (HCG) to reference values of the level for free beta (HCG) druing the time period in: (1) pregnant women carrying a fetus with the chromosomal anomoly and (2) pregnant women carrying normal fetuses, said comparison being indicative of said pregnant woman's risk of carrying a fetus with the chromosomal anomoly.

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

obtaining a urine sample from the pregnant woman during a time period selected from the group consisting of: the first trimester of pregnancy; the second trimester of pregnancy and the third trimester of pregnancy; on filter paper and allowing the urine sample to dry to form a dried urine spot;

determining said pregnant woman's urine level of the beta core fragment of free beta (human chorionic gonadotropin (HCG)) from the dried urine spot and comparing said level of free beta (HCG) to reference values of the level for the beta core fragment of free beta (HCG) during the time period in: (1) pregnant women carrying a fetus with Down syndrome and (2) pregnant women carrying normal fetuses, said comparison being indicative of said pregnant woman's risk of carrying a fetus with Down syndrome.

Description
FIELD OF THE INVENTION

The present invention relates to a method for detecting fetal Down syndrome (Trisomy 21), trisomy 13, trisomy 18, Turners syndrome and other chromosomal anomalies during prenatal screening by analyzing a dried blood sample from a pregnant woman. More particularly the present invention relates to a method for improving detection efficiency in prenatal screening for Down syndrome by measuring the amount of free beta (human chorionic gonadotropin "HCG") and nicked or fragmented or aberrant forms of free beta (HCG) all of which are referenced throughout this application as free beta (HCG) in dried blood samples from pregnant women.

BACKGROUND OF THE INVENTION

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 diagnostic procedures including amniocentesis or chorionic villus sampling and karyotyping. However, these diagnostic procedures are invasive and involve risk to the woman and the fetus. For this and other reasons, amniocentesis or chorionic villus sampling 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 detection of 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. 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, 1 in 800 pregnancies). However, because of the association of these other chromosomal trisomies with lowered MSAFP levels, and elevated or depressed free beta (HCG) levels, such abnormalities will also be detected within a screening protocol utilizing maternal blood AFP and free beta (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, Trisomy 18, Turners syndrome and other chromosomal anomalies 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.

U.S. Pat. No. 4,874,693 to Bogart discloses an association between elevated maternal blood HCG levels and elevated maternal blood levels of the alpha subunit of HCG, during the 18th-25th weeks of pregnancy, and fetal Down syndrome. In the Bogart patent it is estimated that the use of elevated maternal blood HCG levels and elevated maternal blood levels of the alpha subunit of HCG in a screening protocol, would detect a greater percentage of chromosomally abnormal fetuses than the use of AFP or UE alone. In a paper entitled "Human Chorionic Gonadotropin Levels in Pregnancies with Aneuploid Fetuses" (Bogart et al., Prenatal Diagnosis, Vol. 9, 379-384 (1989)) Bogart discloses that a screening method utilizing HCG and the alpha subunit of HCG is not useful at 9-11 weeks gestation (the first trimester of pregnancy) for selecting pregnancies at risk for fetal aneuploidy (including Down syndrome).

In my co-pending U.S. patent applications, Ser. No. 07/868,160, filed Apr. 14, 1992, Ser. No. 07/709,019 filed May 31, 1991 and Ser. No. 07/420,775, filed Oct. 12, 1989, I describe Down syndrome screening methods utilizing free beta (HCG) (and nicked or fragmented or aberrant forms of free beta (HCG)) that detect a greater percentage of chromosomally abnormal fetuses than heretofore utilized screening methods. These applications also describe an apparatus that may be advantageously utilized by laboratories to analyze samples in a screening protocol. The disclosure of each application is hereby incorporated herein by reference.

As described in these applications, a particularly effective Down syndrome screening method includes measuring a pregnant woman's maternal blood level of free beta (HCG), and comparing the measured level of free beta (HCG) to reference data including the levels of free beta (HCG) in (1) pregnant woman carrying normal fetuses and (2) pregnant woman carrying Down syndrome fetuses. I also describe how Down syndrome screening may be further enhanced by utilizing a screening method that includes comparing the pregnant woman's maternal blood level of AFP and the pregnant woman's gestational age to reference data. This comparison is advantageously accomplished using a multivariate discriminant analysis technique. Discriminant analysis is a generally known approach to multivariate analysis involving the separation of a population into two or more groups on the basis of probability. A general discussion of discriminant analysis can be found in Marketing Research; Churchill, G. A.; Dryden, 1976; Chapter 15, pages 530-543. As set forth in my prior applications the use of multivariate discriminant analysis to compare the pregnant woman's levels of free beta (HCG) and AFP, and her gestational age to reference data 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.

As described in the preceding paragraphs, prenatal screening methods generally include an analysis of the pregnant woman's blood. Generally, this blood sample is taken from the pregnant woman at her physician's office, or in a similar clinical setting then transported to a clinical laboratory for analysis. The blood sample may also be stored for analysis or transport at a later time.

In many instances the clinical setting is in a different physical location than the clinical laboratory where the blood is to be analyzed. For example, the clinical setting where the blood sample is drawn from the pregnant woman may be located in a city or population center and the laboratory located in a suburban or rural setting and in some instances in another state.

In generally utilized screening methods, the blood sample taken from the pregnant woman is transported, and/or stored for later analysis, in liquid form in a vial or test tube. Additionally, the test tubes must be sealed to prevent contamination of the sample and also to prevent evaporation of the sample prior to analysis. Transporting and storing blood samples in liquid form has many disadvantages. Special handling techniques must generally be utilized to avoid breaking the test tube or vial including packaging the tubes or vials in shock absorbent packages. It is customary to place the tubes in leak proof bags. Generally, to avoid spills the packages must also be designed so that the test tubes remain upright. The net result can be bulky packages that require large storage or shipment areas.

It is also known that blood borne pathogens may be transmitted by liquid blood samples due to splashes, needle sticks, broken test tubes etc that may occur when the blood sample is taken, stored and/or transported with containers used for handling liquid blood. Additionally, it is generally known that certain constituents of the blood will degrade in vitro if not maintained at cool temperatures.