WO2009046447A1 - Fetal open lesion detection system - Google Patents

Fetal open lesion detection system

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Publication number
WO2009046447A1
WO2009046447A1 PCT/US2008/078989 US2008078989W WO2009046447A1 WO 2009046447 A1 WO2009046447 A1 WO 2009046447A1 US 2008078989 W US2008078989 W US 2008078989W WO 2009046447 A1 WO2009046447 A1 WO 2009046447A1
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WO
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Patent type
Prior art keywords
cells
open
fluid
amniotic
specific
Prior art date
Application number
PCT/US2008/078989
Other languages
French (fr)
Inventor
Katalin Polgar
Original Assignee
Katalin Polgar
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date

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Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism

Abstract

The present invention provides a disease-specific, cell-based detection system comprising a safe, simple, and quick test to detect disease-specific diagnostic cells for identifying anyone of a number of specific types of open fetal lesions. The test can be accurately performed in about twenty minutes by a relatively unskilled person having no cyto-morphologic knowledge. The present invention is directed to kits and methods for detecting an open fetal lesion. The test is based on the unexpected observation of a strong correlation between the presence of certain disease-specific cells and specific types of open fetal lesions.

Description

FETAL OPEN LESION DETECTION SYSTEM

RELATED APPLICATION

[001] This application claims the benefit of U.S. Provisional Application No. 60/997,772, filed on October 6, 2007. The entire teachings of the above application are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[002] Presently, alpha- fetoprotein (AFP) screening of a pregnant woman's blood is routinely used to identify an increased risk of birth defects. However the test is not specific for fetal neural tube closure defects (NTD), and fetal abdominal wall defects (AWD), and is not reliable. AFP tests often produce false positive results, non- limiting examples of which include miscalculated gestational age, and twin pregnancy, results that generate a need for additional testing. Ultrasound is also routinely used, but open lesions are often too small to be detected by ultrasound. An alternative to AFP tests and ultrasound is neutral-red staining of pathognomonic cells in amniotic fluid to detect fetal neural tube closure defects (NTD) (Polgar et ah, New Eng. J. Med. 310: 1463-1464 (1984); Polgar, K. et ah, Acta Physiol. Hung. 71: 551-555 (1988); Polgar et al., Am. J. Repro. Immun. Microbiol. 18: 81-86 (1988)). These pathognomonic cells are present in the amniotic fluid only when the open lesion exists, as they originate from the open lesion. Implementation of the test in clinical practice had about 91.5 % sensitivity and about 100 % specificity to detect open fetal NTD. Such a test has not been used to identify other types of fetal open lesions.

The early detection of fetal open lesions remains a serious clinical problem. Thus, an on-going need exists for a reliable test to detect all types of fetal open lesions including fetal neural tube defects and fetal abdominal wall defects.

The clinical adoption of a test based on the analysis of fetal cells present in amniotic fluid would benefit from increased sensitivity and/or rapidity and the development of kits, which comprise the required elements for conducting the test.

SUMMARY OF THE INVENTION [003] The present invention provides a safe, reliable, relatively simple, and quick test for identifying a fetal open lesion, including fetal NTD and fetal AWD, and discriminating between different types of fetal open lesions. The invention inter alia includes the following, alone or in combination. In one embodiment, the invention provides a pre-natal diagnostic tool, such as a diagnostic method, that can be used in place of, or to supplement AFP screening and/or ultrasound tests.

[004] The present invention provides a disease-specific, cell-based detection system comprising a safe, simple, and quick test to detect disease-specific diagnostic cells for identifying any one of a number of specific types of open fetal lesions. The test can be accurately performed in about twenty minutes by a relatively unskilled person having no cyto-morphologic knowledge.

DETAILED DESCRIPTION OF THE INVENTION

[005] A description of preferred embodiments of the invention follows. It will be understood that the particular embodiments of the invention are shown by way of illustration and not as limitations of the invention. At the outset, the invention is described in its broadest overall aspects, with a more detailed description following. The features and other details of the compositions and methods of the invention will be further pointed out in the claims.

[006] The present invention is directed to kits and methods for detecting an open fetal lesion. The test is based on the unexpected observation of a strong correlation between the presence of certain disease-specific cells and specific types of open fetal lesions.

[007] Thus, the test comprises obtaining an amniotic fluid sample from a pregnant woman in need of testing, and testing the amniotic fluid for the presence of the disease-specific cells. The quantity of these cells can then be compared to a standard reference in order to quantify results.

Disease-Specific Cells:

[008] In one embodiment of the invention, the disease-specific cell detected is either a phagocyte or a pinocyte. In another embodiment of the invention, a disease- specific cell detected is both a phagocyte and a pinocyte. In yet another embodiment, two or more types of disease-specific cells are detected, including a pinocyte and a phagocyte. As used herein, the term "phagocyte" refers to a cell that can engulf or "eat" a particle. The terms "phagocyte" and "phagocytic cell" have the same meaning and are cells that exhibit phagocytosis. As used herein, the terms "pinocyte" and "pinocytic cell" have the same meaning and refer to a cell that can consume liquid or "drink" a liquid. A pinocyte exhibits pinocytosis.

[009] If a fetus has an open lesion, even if the lesion is very small and undetectable by ultrasound, a diagnostic fetal open lesion-specific cell is able to move from the fetus into the amniotic fluid only through that open lesion. One embodiment of the invention is a method of detecting diagnostic fetal open lesion-specific cells that are present in the amniotic fluid only if a fetus has an open lesion.

[0010] If the fetus does not have open lesion, no open lesion disease-specific cells are present in the amniotic fluid, because there exists no open lesion from which the disease-specific cells can move from the fetus into the amniotic fluid.

[0011] A fetal open lesion that can be detected according to an embodiment of a method of the invention can be, for example, a neural tube defect, such as spina bifida or anencephaly, or an abdominal wall defect, such as gatrochisis or a ventral wall defect, such as an open omphalocele. These defects are characterized by the presence of disease-specific cells that have pinocytic or phagocityic capacity.

[0012] The method of the invention includes the steps of (1) providing a sample of amniotic fluid from a pregnant subject; and (2) determining whether the amniotic fluid sample includes phagocytic and/or pinocytic cells indicative of a fetal open defect. The pregnant subject can be any mammal, but is preferably a human. In one embodiment, the fetus is at risk for having an open defect, due, for example, to a family history of one or more open defects, or test results, such as results of an AFP test or an ultrasound study, that are inconclusive or suggest the presence of an open defect. Other reasons to suspect an open defect include multifactorial genetical and environmental factors.

[0013] In one embodiment, the amniotic fluid sample is withdrawn for routine chromosome analyses. The recommended minimum maternal age for such testing has been decreasing over the last several years, and testing is now suggested for pregnant women who are slightly more than 30 years old. In this embodiment, a portion of this amniotic fluid is then provided for analysis according to the disclosed method for detecting and identifying a possible fetal open lesion. [0014] The disclosed testing methods can also be utilized in the case of a suspicion of any genetically inherited disease or in a case wherein the routine serum and or amniotic fluid AFP screening results are high. AFP testing results in a significant number of false positive results. Further, serum AFP levels can be elevated for other, unrelated reasons, such as, for example, as underestimated gestational age, or multifetal gestation. Thus, the present method can be used following a positive AFP test, to either verify the diagnosis of an open defect, eliminate a false positive value or suggest an alternate cause of the high AFP level.

[0015] The disclosed testing methods can also be utilized in a case in which the results of ultrasound screening are questionable or inconclusive for the presence of an open defect, or in a case wherein the ultrasound does not have sufficient resolution to detect certain small open lesions.

[0016] In one embodiment, the amniotic fluid is withdrawn from the pregnant subject at between about 11 and about 18 weeks of gestation. However, if through the results of a previous test, the subject's medical history or a physician's clinical judgment, the fetus is believed to be at risk for an open defect, amniocentesis can be performed at a later gestational age. Some forms of fetal open lesions can endanger the life of the mother.

[0017] The amniotic fluid sample can be obtained, for example, using standard methods of withdrawing amniotic fluid from the uterus, such as transabdominal amniocentesis.

[0018] Following withdrawal of the sample of amniotic fluid, at least a portion of the amniotic fluid is preferably treated with an identifiable liquid which includes a detectable property, identifiable small solid particles which include a detectable property, or, preferably, both. The solid particles are in a size range that can be taken up, or phagocytosed, by phagocytes. The term "identifiable", as used herein, refers to a liquid or solid particles characterized by a "detectable property", which is a property of the liquid or solid that allows the identification of cells which have taken up the liquid (by pinocytosis) or the solid particles (by phagocytosis). For example, the liquid or the solid particles can be colored, fluorescent, or tagged with a radioactive marker. Thus, if the identifiable liquid or solid particles are consumed by a disease-specific pinocyte or phagocyte present in the amniotic fluid, the disease-specific cell can be easily identified by its resultant color, fluorescence, or radioactivity. The solid particles present in the cells can also be identified microscopically.

[0019] Suitable liquids for use in the method include dyes and stains, such as the cationic dye neutral red, such as the neutral red stain formulation disclosed in WO 88/07583. Other suitable dyes include acidic dyes, amphoteric dyes, anionic dyes, basic dyes cationic dyes, acridine dyes, azo dyes diagnostic dyes, such as bromsulfthalein (BSP) and phenolsulfonphthalein (PSP)), and vital dyes. Other suitable dyes include colloidal azo dyes, such as pyrrhol blue, Indicator sulfonphthalein dyes: bromphenol blue (BPB), brom- cresol green (BCG), bromeresol purple (BCP); reactive pH indicator_dyes: BCECF, Carboxyfluorescein [5 -(and 6-) carboxyfluorescein, succinimidyl ester], 6-HEX [6-carboxy- 2',4,4',5',7,7'-hexachlorofluorescein, succinimidyl ester], 6-JOE (6-carboxy-4',5'- dichloro-2',7'-dimethoxyfluorescein, succinimidyl ester], Naphthofluorescein [5- (and 6-) carboxynaphthofluorescein, succinimidyl ester (C653)], Oregon Green 488 [Oregon Green 488 carboxylic acid, succinimidyl ester (06147, 06149)], Oregon Green 514 [Oregon Green 514 carboxylic acid, succinimidyl ester (06139)], SNARF-I [SNARF-I carboxylic acid, acetate, succinimidyl ester (S22801)], 5-(and 6-) carboxy SNARF-I [(C1270, Section 20.2)], 6-TET [6-carboxy-2',4,7,7'- tetrachlorofluorescein, succinimidyl ester (C20092, Section 1.5)]. In one embodiment, the dye is not neutral red.

[0020] Suitable solid particles for use in the method include polymer beads, such as Zymosan, Latex, polystyrene beads, microspheres, opsonized Mannozym, and haemolysin sensitized sheep red blood cells (sSRBCs). Following phagocytosis, the particles can be identified visually, for example, under magnification. Optionally, they can be coupled with a reporter moiety, such as a fluorescent moiety or a radioactive moiety.

[0021] The use of a combination or mixture of an identifiable liquid and identifiable solid particles offers the advantage of being able to detect disease-specific cells whether they are pinocytic or phagocytic, thereby increasing the probability that the disease-specific cells will be detected. [0022] The volume of amniotic fluid that is treated with the identifiable liquid and/or identifiable solid particles typically about 5 mL, but is not critical, so long as the volume selected is sufficient to enable a reasonable expectation that it will contain disease-specific cells if present in the amniotic fluid of the subject.

[0023] Following incubation of the amniotic fluid sample with the identifiable liquid and/or identifiable solid particles, the mixture is preferably centrifuged, the supernatant is removed, and the pellet is washed. The cell pellet is then examined, for identification and, optionally, quantification of cells which have taken up the identifiable liquid and/or identifiable solid particles. The method of examination of the cells depends upon the detectable property of the identifiable liquid and/or identifiable solid particles. For example, if the detectable property is color, the cells can be examined under a microscope or other magnification device and quantitated using, for example, a cell counting chamber, such as a hemocytometer. If the detectable property is radioactivity, the cells can be examined by measuring their level of radioactivity using, for example, a scintillation counter. If the detectable property is fluorescence, the cells can be examined using a fluorimeter, or microscopically using fluorescence microscopy.

[0024] According to one embodiment of the invention, both identifiable particles and an identifiable liquid, the identities of the particles or the liquid related to color, fluorescence, radioactivity, or some other marker, is add to the aspirated amniotic fluid. If the fetus has an open lesion disease or condition, non- limiting examples of which include a neural tube defect or an abdominal wall defect, then open lesion- specific cells are present in the amniotic fluid, and only these disease-specific cells can take up the identifiable particle/liquid mixture. In contrast, the normal amniotic fluid cells that originate from the skin of the fetus will remain colorless, non- fluorescent, or non-radioactive, as normal amniotic fluid cells do not have pinocytic or phagocytic activity.

[0025] The number of disease-specific cells identified in the amniotic fluid sample can be compared to the number found in the amniotic fluid of normal control subjects (negative controls). The presence of a number of cells above a threshold number will result in a determination that an open defect is present. This threshold level will depend on how the cells are counted, for example, the type of cytometer used. However, because it is expected that normal controls will have few, if any, disease-specific cells, the threshold number should be less than 2500. For example, in certain embodiments, the threshold number is less than 2500, 2000, 1500, 1200, 1000, 500 or 100 disease-specific cells per mL of amniotic fluid.

[0026] The invention also provides a method for obtaining a quantitative assessment of disease-related, diagnostic cells in amniotic fluid. The number of such cells found in the amniotic fluid of the subject can be compared to the number found in the amniotic fluid of subjects carrying fetuses with confirmed defects (positive controls). The cell numbers found in cases of confirmed fetal defects can be used to establish a quantitative correlation between the cell number and the nature and/or severity of the defect. Thus, the inventive method can be used not only to detect the presence or absence of an open defect, but also the nature of the defect. In comparing the cell number determined using the method of the invention with other cases, such as historic controls, it is preferred that comparisons be made only to cases in which the method utilized the same diagnostic method, including the same volume of amniotic fluid and the same identifiable liquid and/or solid particles.

[0027] The disclosed testing methods can be performed in private medical offices, even in situations in which the actual screening technician or doctor does not have specialized experience. Few countries, other than certain European countries, maintain sophisticated screening centers with trained specialists who perform prenatal testing.

[0028] In yet another aspect, the invention relates to test kits for detecting fetal open lesions. For example, disclosed herein is a one-patient kit system for diagnosing all open fetal lesions, including all types of open NTD, including the smallest spina bifida lesions, and non-NTD-related open fetal lesions, for example, abdominal wall defects, such as gastrochisis.

Fetal Diagnostic Test Kit Example:

[0029] The present invention further provides a diagnostic kit suitable for performing the method disclosed herein. The diagnostic kit comprises: (a) A receptacle containing an appropriate amount of an identifiable liquid, identifiable solid particles or a combination thereof. Preferably the receptacle is a closed tube;

(b) A receptacle marked with at least three lines: a first line, indicating the level to which the amniotic fluid should be added; a second line, indicating the level to which the supernatant should be removed; and a third line, indicating the level to which a washing, buffered solution should be added. This receptacle can be, for example, a tube with a safe, non-spill closure; and

(c) A receptacle, for example, a vial or other container, containing a washing, solution. Suitable washing solutions include aqueous buffer solutions, such as a buffered saline solution.

The kit of the invention can further comprise one or more of the following elements:

(d) A balloon, bulb, eyedropper or pipette to remove the supernatant;

(e) A device, such as a pipette or an eyedropper, capable of transferring the appropriate amount of pellet (e.g. one drop) into a measuring chamber;

(f) A measuring chamber to determine number of colored open defect- related cells. The measuring chamber can be, for example, a disposable cell counting chamber, such as a hemocytometer or a Bϋrker chamber;

(g) A magnifier-slide to aid in seeing the colored open disease-related cells; and

(h) written instructions for using the kit. The written instructions can optionally include a color indicator mini-scale, (e.g. 1 drop/10 microliter: 15 colored cells= red flag). The number of red cells increases as the size of the fetal lesion increases. The number of disease-specific cells is closely related to the size of the lesion. If the lesion is bigger there are more disease-specific cells present in the amniotic fluid; if the lesion is smaller less cells will go through the smaller lesion into the amniotic fluid. [0030] In a preferred embodiment, the measuring chamber is a disposable plastic manual cell counter-chamber with colored lines; and the device for transferring the cell pellet is a small calibrated transfer device. The written instructions can be, for example, in the form of a booklet and can provide directions for using the kit/counting chamber and further includes the threshold number.

[0031] In a particularly preferred embodiment, the measuring chamber is a manual disposable plastic cell counter-chamber which can be used to quantitate the cells, such as colored cells, which are specific to open fetal lesions. The chamber preferably includes a special grid with precisely spaced lines for exact quantification. The grid pattern can be a simple counter grid pattern similar to those found in improved Neubauer, Burker chamber and hemacytometers. With this consistent counting grid design one can employ a standard cell counting procedure as a part of fetal open lesion diagnostic assay. The cell number determined using the following formula on the specially designed chamber: cell concentration = dilution x total cell number/volume can be compared to a pre-determined threshold number to determine the existence of an open defect.

[0032] The small calibrated transfer device can be made, for example, of plastic and is calibrated to enable the exact removal the necessary amount of sample with the cells (e.g. 10-20 microliter) in a standard manner. The necessary amount (e.g. 10-20 microliter) cell-containing sample will be introduced into the counting chamber. After manufacturing the chamber we will include in the kit an instruction booklet exactly providing a cut off level for colored cells for easy determination of presence or absence of fetal open lesion.

[0033] If the measuring chamber utilizes the standard hemacytometer chamber format, two lxlx 0.1 mm areas (divided by grid lines into nine small squares, or counting areas), from the two chambers. The cell suspension is preferably diluted so that each such square has between 20 - 50 cells (2-5 x 10 5 cells/ml). A total of 300 - 400 cells should be counted, since the counting error is approximated by the square root of the total count. Using a microscope with a 1OX ocular (and a 1OX objective), for example, the cells are counted in each of 10 squares (1 mm2 each). [0034] In a preferred embodiment, the hemacytometer consists of two chambers, each of which is divided into nine 1.0 mm2 squares. A cover glass is preferably supported 0.1 mm over these squares so that the total volume over each square is 1.0 mm2 x 0.1 mm or 0.1 mm3, or 10"4 cm3. Since 1 cm3 is approximately equivalent to 1 ml, the cell concentration per ml will be the average count per square x 104.

[0035] The number of cells per 1x1x0.1 mm areas = cells x IxIO"4 cm3 (IxIO"4 ml).

With a 1 :20 dilution the count is: number of cells in two lmm2 squares divided by 10 = cells x 106 / ml.

(2 squares / 2 x 20 x 10,000 / 1,000 = 106 cells / ml) (2 squares x 10 x 10,000 / 1 ,000 = 106 cells / ml)

(2 squares x 100 = 106 cells / ml)

(2 squares / 10 = millions / ml)

[0036] The chamber with grid scale border easily shows the following: the cell count within that area between the grid lines and find more than a predetermined number of cells, for example, more than 200 colored cells the fetus has an open defect. If the number of colored cells in the marked area is less than the predetermined value, then the fetus does not have an open fetal defect.

EQUIVALENTS

[0037] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention.

Claims

CLAIMSWhat is claimed is:
1. A method for detecting the presence of an open defect in a fetus, comprising the steps of: (a) providing a sample of amniotic fluid from a pregnant subject;
(b) mixing the amniotic fluid with an identifiable liquid, thereby forming a mixture;
(c) centrifuging the mixture of step (b) to form a centrifuged mixture; (d) removing supernatant from the centrifuged mixture to yield a cell pellet; (e) determining the number of disease-specific cells in the cell pellet;
wherein if the number of disease-specific cells is greater than a predetermined number, an open defect is determined to be present and wherein the identifiable liquid is not neutral red.
2. A method for detecting the presence of an open defect in a fetus, comprising the steps of: (a) providing a sample of amniotic fluid from a pregnant subject;
(b) mixing the amniotic fluid with identifiable solid particles, thereby forming a mixture;
(c) centrifuging the mixture of step (b) to form a centrifuged mixture; (d) removing supernatant from the centrifuged mixture to yield a cell pellet;
(e) determining the number of disease-specific cells in the cell pellet or a portion thereof;
wherein if the number of disease-specific cells is greater than a predetermined number, an open defect is determined to be present.
3. The method of Claim 2 wherein step (b) further comprises mixing the amniotic fluid with an identifiable liquid.
4. The method of Claim 3 wherein the fetus is at risk for having an open defect due to a family history of one or more open defects, the results of an alpha- fetoprotein test or an ultrasound study.
5. The method of Claim 3 wherein the identifiable liquid and the identifiable solid particles are each characterized by a detectable property independently selected from the group consisting of color, fluorescence and radioactivity.
6. The method of Claim 5 wherein the identifiable liquid is neutral red, a colloidal azo dye; an indicator sulfonphthalein dye; or a reactive pH indicator dye.
7. The method of Claim 5 wherein the identifiable liquid is pyrrhol blue, bromphenol blue, bromcresol green, bromcresol purple; BCECF, carboxyfluorescein [5 -(and 6-) carboxyfluorescein, succinimidyl ester], 6- HEX [β-carboxy-l'^^'^'J^'-hexachlorofluorescein, succinimidyl ester], 6- JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein, succinimidyl ester], naphtho fluorescein [5 -(and 6-) carboxynaphtho fluorescein, succinimidyl ester (C653)], Oregon Green 488 [Oregon Green 488 carboxylic acid, succinimidyl ester (06147, 06149)], Oregon Green 514 [Oregon Green 514 carboxylic acid, succinimidyl ester (06139)], SNARF-I [SNARF-I carboxylic acid, acetate, succinimidyl ester (S22801)], 5-(and 6-) carboxy SNARF-I [(C1270, Section 20.2)], 6-TET [6-carboxy-2',4,7,7'- tetrachlorofluorescein, or succinimidyl ester.
8. The method of Claim 5 wherein the identifiable solid particles are zymosan, latex or polystyrene particles.
9. The method of Claim 3 wherein the volume of the amniotic fluid sample of step (a) is about 5 mL.
10. The method of Claim 3 further comprising the step of washing the cell pellet of step (d).
11. The method of Claim 5 wherein the number of disease-specific cells in the cell pellet is determined using a magnification device and a cell counting chamber; by measuring the level of radioactivity; or by using a fluorimeter or fluorescence microscopy.
12. A diagnostic test kit for conducting a method for detecting the presence of an open defect in a fetus, comprising the steps of: i. providing a sample of amniotic fluid from a pregnant subject; ii. mixing the amniotic fluid with an identifiable liquid, identifiable solid particles or a combination thereof, thereby forming a mixture; iii. centrifuging the mixture of step (ii) to form a centrifuged mixture; iv. removing supernatant from the centrifuged mixture to yield a cell pellet; v. washing the cell pellet; and vi. determining the number of disease-specific cells in the cell pellet;
wherein if the number of disease-specific cells is greater than a predetermined number, an open defect is determined to be present; said kit comprising:
(a) a receptacle containing an identifiable liquid, identifiable solid particles or a combination thereof;
(b) a receptacle marked with a first line indicating the level to which the amniotic fluid should be added; a second line, indicating the level to which the supernatant should be removed; and a third line indicating the level to which a washing solution should be added; and (c) a receptacle containing a washing solution.
13. The diagnostic test kit of Claim 12, further comprising a device to remove the supernatant from the centrifuged mixture.
14. The diagnostic test kit of Claim 12, further comprising a measuring chamber to determine number of disease-specific cells.
15. The diagnostic test kit of Claim 12 wherein the measuring chamber is a hemocytometer or a Bϋrker chamber.
16. The diagnostic test kit of Claim 15 wherein the measuring chamber comprises two chambers having gridlines marked thereon dividing said each of said chambers into nine squares.
17. The diagnostic test kit of Claim 12, further comprising a magnifier-slide.
18. The diagnostic test kit of Claim 12, further comprising written instructions for using the kit.
PCT/US2008/078989 2007-10-06 2008-10-06 Fetal open lesion detection system WO2009046447A1 (en)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5641628A (en) * 1989-11-13 1997-06-24 Children's Medical Center Corporation Non-invasive method for isolation and detection of fetal DNA
US20030210460A1 (en) * 2002-03-27 2003-11-13 Olympus Optical Co., Ltd. Microscope stage and microscope having microscope stage mounted thereon
US20040152125A1 (en) * 1999-12-23 2004-08-05 Zymogenetics, Inc. Novel cytokine ZCYTO18
US20040197930A1 (en) * 2003-03-25 2004-10-07 Ron Rosenfeld Proteomic analysis of biological fluids
US20050214864A1 (en) * 2002-08-26 2005-09-29 The Regents Of The University Of Michigan NPHP nucleic acids and proteins
US20060160105A1 (en) * 2002-05-08 2006-07-20 Ravgen, Inc. Methods for detection of genetic disorders
US20070065339A1 (en) * 2005-09-22 2007-03-22 Blane Huff Urine collection and drug testing cup

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5641628A (en) * 1989-11-13 1997-06-24 Children's Medical Center Corporation Non-invasive method for isolation and detection of fetal DNA
US20040152125A1 (en) * 1999-12-23 2004-08-05 Zymogenetics, Inc. Novel cytokine ZCYTO18
US20030210460A1 (en) * 2002-03-27 2003-11-13 Olympus Optical Co., Ltd. Microscope stage and microscope having microscope stage mounted thereon
US20060160105A1 (en) * 2002-05-08 2006-07-20 Ravgen, Inc. Methods for detection of genetic disorders
US20050214864A1 (en) * 2002-08-26 2005-09-29 The Regents Of The University Of Michigan NPHP nucleic acids and proteins
US20040197930A1 (en) * 2003-03-25 2004-10-07 Ron Rosenfeld Proteomic analysis of biological fluids
US20070065339A1 (en) * 2005-09-22 2007-03-22 Blane Huff Urine collection and drug testing cup

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