APPARATUS AND METHOD FOR BULK COLLECTION OF AMNIOTIC FLUID

CROSS REFERENCE TO RELATED APPLICATIONS

[0001 ] This application claims priority of U.S. Provisional Application No. 62/459,263, filed February 15, 2017, which is hereby incorporated by reference in its entirety for all purposes. FIELD OF THE INVENTION

[0002] This disclosure relates generally to an apparatus and method for the bulk collection of amniotic fluid from a patient undergoing an elective caesarean section birth.

BACKGROUND

[0003] During pregnancy amniotic fluid fills the amniotic sac, sustains fetal growth, and provides mechanical cushioning to the fetus. This complex, dynamic fluid contains nutrients, growth factors, and cells which circulate as the fetus swallows and "inhales" the fluid and then releases it back into the amniotic sac. While in utero, the amniotic fluid helps the developing fetus move within the womb, promotes proper lung development, and regulates the temperature around the fetus, among other functions. [0004] Amniotic fluid has been used for decades for diagnostic purposes, for example, prenatal genetic screening, determining fetal lung maturity, metabolic diseases, fetal infections and intrauterine infections. Removing a sample of fluid through amniocentesis can provide information about the sex, health and development of the fetus. In addition, abnormal amounts of amniotic fluid, too much ("polyhydramnios") or too little ("oligohydramnios") may be a sign of a pregnancy complication. Removal of a sample through amniocentesis is not without risk to the fetus, however. Potential risks include miscarriage, needle injury, introduction of infection, bleeding and other types of adverse events that have led to a very judicious use of this procedure.

[0005] Amniotic fluid changes during pregnancy from a clear yellow solution at three months to a colorless solution in the third trimester. At approximately the 33 ^rd -34 ^rd week on, cloudiness and flocculation occur, at first very slowly, after the 36th-37th week steadily faster. At term, the amniotic fluid is moderately cloudy and contains a moderate number of flakes of vernix. Verpoist, et al., J. Perinatal Med., 4(1 ):12-25 (1976). Amniotic fluid collected at term is referred to as "mature amniotic fluid". This is in contrast to the amniotic fluid collected during amniocentises which has yet to mature. [0006] In addition to diagnostic purposes, amniotic fluid may also be useful in various therapeutic applications. Such therapeutic applications require the collection of greater volumes of amniotic fluid than would be necessary in relation to typical diagnostic tests. The risks of collecting sufficient quantities of amniotic fluid through currently available amniocentesis techniques are generally not acceptable. Moreover, there appears to be no methods for collecting mature amniotic fluid substantially free of cellular debris, blood, vernix, and fetal urine, and corresponding components thereof such as urea.

[0007] Accordingly, there exists a need to collect bulk quantities of amniotic fluid in a safer manner than is currently provided for with typical amniocentesis fluid collection techniques. Ideally, the method of collection would result in the collection of a large volume of high quality amniotic fluid such as mature amniotic fluid substantially free of cellular debris, blood, vernix, and fetal urine, and should be collected while adhering to well-known aseptic technique, while at the same time exposing the mother and fetus to a minimal level of risk.

SUMMARY OF THE INVENTION

[0008] There is accordingly a need in the art to collect bulk quantities of high quality amniotic fluid by providing a novel method and apparatus. The technique of the present invention provides for the aseptic collection of large quantities of amniotic fluid from a patient undergoing an elective caesarean section birth. [0009] In some embodiments, an apparatus is disclosed in which amniotic fluid is drained into a flexible drape funnel. Once the fluid begins to fill the drape funnel, it is then collected via the use of a Poole catheter which is connected to flexible tubing. The tubing is connected to a trap container which is connected to suction provided at the wall or from another source. [0010] In some embodiments, an apparatus is disclosed in which amniotic fluid is collected by the insertion of a cannula into the uterine cavity, wherein the cannula is connected via flexible tubing to a trap or collection canister which is connected to suction.

[0011 ] In some embodiments, the collected amniotic fluid is mature amniotic fluid substantially free of cellular, blood, vernix, and optionally other contaminants. In some embodiments, the amniotic fluid is filtered and desalted. In some embodiments, the amniotic fluid is reduced in nitrogen content. Such compositions, therefore, comprise filtered, desalted, mature amniotic fluid substantially free of cellular, blood and vernix and optionally other contaminants. In some embodiments, the amniotic fluid is filtered after collection. In some embodiments, amniotic fluid is filtered in-line during collection.

[0012] In some embodiments, amniotic fluid of varying levels of turbidity is collected. In some embodiments, the collected fluid is substantially free of blood. In some embodiments, the collected fluid comprises <1 % blood.

[0013] In some embodiments, the collection of the amniotic fluid is carried out using aseptic technique in combination with sterilized instruments.

[0014] In some aspects, this disclosure provides methods for harvesting bulk quantities of amniotic fluid in a safe and efficient manner that constitutes little to no risk to the mother and fetus.

[0015] In some aspects, this disclosure provides an apparatus for harvesting bulk quantities of amniotic fluid in a safe and efficient manner that constitutes little to no risk to the mother and fetus. BRIEF DESCRIPTION OF THE FIGURES

[0016] Fig. 1 is a side view of a fully assembled apparatus for harvesting bulk quantities of amniotic fluid.

DETAILED DESCRIPTION OF THE INVENTION [0017] It is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of this invention will be limited only by the appended claims. [0018] The detailed description of the invention is divided into various sections only for the reader's convenience and disclosure found in any section may be combined with that in another section. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated by reference in their entireties to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0019] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pluripotent stem cell" includes a plurality of pluripotent stem cells.

[0020] For the foregoing embodiments, each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiment. All combinations, sub-combinations, and permutations of the various elements of the methods described herein are envisaged and are within the scope of the invention.

Definitions

As used herein the following terms have the following meanings.

[0021 ] "Comprising" or "comprises" is intended to mean that the compositions, for example media, and methods include the recited elements, but not excluding others. "Consisting essentially of" when used to define methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. "Consisting of" shall mean excluding substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention.

[0022] Optional" or "optionally" means that the subsequently described event or circumstance may, may not, can, or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0023] The term "subject" as used herein is any vertebrate organism including but not limited to mammalian subjects such as humans, farm animals, domesticated pets and the like. The term "patient" may be used interchangeably with "subject." [0024] The term "placental tissue" refers to any and all of the well-known components of the placenta including but not limited to amnion, chorion, Wharton's Jelly, and the like.

[0025] The term "bodily fluid" unless specified otherwise, refers to the entire fluid and non- fluid components present in the amniotic cavity, such as the liquid, solid, semi-solid, or cellular constituents contained therein, whether in suspension or not. Such non-fluid components include cellular debris, blood cells, vernix and the like. "Amniotic fluid" is used in accordance with its meaning in this field. The methods and devices described herein can be used to collect amniotic fluid that is substantially free of undesired components, particularly, blood.

[0026] The term "full term" is used to describe a stage during human pregnancy that is at least 38 gestational weeks.

[0027] The term "post term" is used to describe a stage during human pregnancy that is greater than 41 gestational weeks.

[0028] The term "serosanguinous" in color refers to the color of fluids collected from or leaving the body. The color is yellowish, which may be due to the presence of blood or other components.

[0029] The term "substantially free" refers to a collected fluid that contains little or none of a particular component. If blood or other components are present, it is preferably present in only trace quantities that do not materially alter the quality of the amniotic fluid for its intended purpose. That is, preferably the collected amniotic fluid is substantially free of blood. In embodiments, substantially free refers to less than 10%, less than 5%, less than 1 %, less than 0.5%, 0.4%, 0.3%, 0.2%, 0.1 % or 0.01 % of a particular component.

[0030] The term "passively accumulates" refers to the flow of bodily fluid from the uterus to the collection drape caused by the pressure within the amniotic sac and/or gravity (i.e. without using a pump). [0031 ] The term "passively fill" refers to the flow of bodily fluid from the collection drape to the collection canister caused by the application of negative pressure at the collection canister.

[0032] Where a range of values is provided, it is understood that each intervening value, to the hundredth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of the range and any other stated or intervening value in that stated range, is encompassed. Thus, for example, 400 ml to 1000 ml means that 400.01 , 400.02 ... 400.09; 400.1 , 400.2 ... 400.9; and 401 , 402 ... 999 ml unit amounts are included as embodiments of this invention. The upper and lower limits of these small ranges which may independently be included in the smaller ranges is also encompassed, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included. Amniotic Fluid

[0033] Amniotic fluid originates predominantly from maternal plasma that crosses the amniotic membranes into the amniotic cavity. Amniotic fluid is a dynamic composition that undergoes changes throughout pregnancy and is made up of water and soluble materials such as, for example, electrolytes, carbohydrates, proteins, amino acids, lactate, pyruvate, hormones, growth factors, cytokines, and other bioactive molecules. In humans, fetal urine begins to enter the amniotic fluid at approximately 8-1 1 weeks gestation and becomes the major component of amniotic fluid during the second half of pregnancy. In addition, a heterogeneous population of cells derived from fetal skin, gastrointestinal, respiratory, and urinary tracts and the amniotic membrane all slough off into the amniotic fluid. Stem cells have been isolated from amniotic fluid including, for example, pluripotent amniotic fluid stem cells, placental-derived stem cells, and mesenchymal stem cells. Prior to keratinization of fetal skin there is rapid bi-directional diffusion between the fetus and the amniotic fluid. Thus, the amniotic fluid components can vary drastically depending on the gestational age from which the amniotic fluid sample is collected.

[0034] In humans, amniotic fluid volume vary during gestation. At around 10 weeks gestation the volume of amniotic fluid is approximately 25 mL. At around 20 weeks gestation, the volume is approximately 400 mL. The volume of amniotic fluid plateaus at approximately 800 mL at around 29 weeks gestation and declines to approximately 400 mL at full term.

[0035] An embodiment of the invention may be understood more clearly by the following explanations, which refer to elements shown in Figure 1 .

Methods of Use

[0036] A patient undergoing a planned caesarean section is prepared with an amniotic fluid collection drape 4. The collection drape 4 is positioned at the surgical site. The lower end of the drape 4 (comprising a collection pouch where fluid will collect) is placed below the uterine segment. If a bladder blade is used, the adhesive on the collection drape 4 can be affixed to the inside of the blade. If an Alexis retractor is used, the lower end is placed as low as possible in the operative field.

[0037] Alternatively, a drape and a separate collection pouch may be used in conjunction with one another.

[0038] Once the uterus is visualized, a small 2-3 cm incision is made in the lower uterine segment. [0039] Before the amniotic sac is ruptured, Allis clamps are placed on the superior and inferior cuts of the incision. This should cover as much of the incision as possible in order to minimize blood contaminating the amniotic fluid 6.

[0040] The initial incision is made, with careful attention to hemostasis.

[0041 ] The underlying fetal compartment is exposed. At this point, the amniotic fluid 6 is visualized. Pennington clamps may be used to achieve hemostasis.

[0042] The upper opening of the collection drape 4 is elevated and a small incision is made into the amniotic sac. The amniotic fluid 6 can now be evaluated for clarity. The fluid 6 should flow freely into the collection drape 4, because the drape is below the amniotic sac. If the amniotic fluid 6 streams upward, the upper end of the collection drape 4 can be elevated in order to capture it.

[0043] A Poole catheter 2 attached to a suction device is used to suction the amniotic fluid 6 from the collection drape 4 into a trap or a collection canister 12. The amniotic fluid 6 should be collected until the fluid 6 stops flowing, or the fluid 6 becomes bloody or is no longer clear.

[0044] The collected mature amniotic fluid 6 can be subjected to desalting using conventional osmotic conditions.

[0045] In an alternate method, the bodily fluid is collected directly from the uterine cavity, and not from a collection drape. The abdomen is entered in the standard fashion. Depending on its position, the bladder flap may or may not be created. In a preferred embodiment, the bladder flap is not created, in order to minimize bleeding.

[0046] A small incision is made with a scalpel (approx. 1 .5-2.0 cm long) in the lower uterine segment in a single pass. Pennington clamps are placed on all cut surfaces and the incision is carefully elevated. A second cut is then made with the scalpel deeper in the myometrium, and the clamps are repositioned to include the entire thickness of the muscle that has been incised.

[0047] The clamps are again elevated and continued passes, or cuts, are made with the scalpel with clamp. Repositioning may be done with the clamps until the membranes of the amniotic sac are visible.

[0048] Once the membranes are visible, using the scalpel, a very small superficial incision is made to rupture the membrane. A Yankauer suction catheter, typically plastic, is placed in the uterine cavity, under the superior surface of the uterus. Fluid travels through the catheter tubing and is collected in a collection canister and closely observed. In this embodiment, no drape 4 is utilized. [0049] Once sufficient fluid is collected, or the fluid appears to be any color other than clear, the tubing is quickly clamped or otherwise closed to prevent colored fluid from entering the suction canister. The catheter is removed from the uterus. If a drape is being used, and there is any clear fluid remaining in the drape, it is captured using the suction.

[0050] The suction tubing is removed from the canister. The canister is capped off or otherwise sealed and sent for evaluation.

[0051 ] The above methods provide significant advantages in that they are minimally invasive, passively and aseptically collect mature amniotic fluid 6, and protect the fetus prior to birth.

Apparatuses

[0052] The apparatus may be comprised of clamps to be used for hemostasis at the surgical site, a collection drape 4 where the amniotic fluid 6 pools once the amniotic sac is pierced during the caesarean section delivery, a cannula-type device 2 for inserting into the pooled amniotic fluid 6, a section of flexible tubing 8 to transport the fluid to a collection trap or other container 12 that is connected to a vacuum source 10. In an embodiment, the subject matter described herein is directed to a kit containing a sterile collection drape 6; a cannula 2, flexible tubing 8, a collection canister 12; and instructions for using the kit to collect amniotic fluid 6. The kit is to be used with a vacuum source 10 provided separately from the kit.

[0053] In another embodiment, the apparatus may be comprised of clamps to be used for hemostasis at the surgical site, a cannula-type device for inserting into the uterine cavity in order to collect amniotic fluid, 6, a section of flexible tubing to transport the fluid to a collection trap or other container that is connected to a vacuum source 10. In an embodiment, the subject matter described herein is directed to a kit containing; a cannula 2, flexible tubing 8, a collection canister 12; and instructions for using the kit to collect amniotic fluid 6. The kit is to be used with a vacuum source 10 provided separately from the kit. In a further embodiment, the cannula-type device or cannula is a catheter. In an embodiment, the catheter is a Yankauer catheter. In an embodiment, the Yankauer catheter is a Yankauer suction catheter. In any of these embodiments, the apparatus may not include the drape 4.

[0054] In some embodiments, the apparatus further comprises a filter. In some embodiments, the filter is at either end of, or within, the flexible tubing. In an embodiment, the filter excludes all solid material. In another embodiment, the filter excludes all solid material that does not fit through a 1 μηι pore, 3 2 μπι pore, 3 5 ιπ pore, a 10 μηι pore, a 20 μηι pore, a 30 μηι pore, a 40 μηι pore, a 50 μηι pore, or a 100 μηι pore.

Experimental

[0055] Twelve amniotic fluid samples were collected via a method as described above. The collected samples were evaluated for their volume, color, and turbidity. Certain samples were evaluated for blood content and/or centrifuged, and the resulting pellet and supernatant were examined. An acceptable volume of collected fluid was at least 100 ml. The color was ideally clear, white, or tan, and a light pink tinge could be permissible. Red would be undesirable as this would indicate too much blood was in the sample. Preferable turbidity was clear to slightly turbid with a white/yellow color.

[0056] Table 1 : Details of collected amniotic fluid

[0057] Samples 2, 6, 10, and 12 were collected via a method using a cannula inserted directly in the uterine cavity, and the remaining samples were collected using a method incorporating a collection drape. Sample 12 appeared to have the meconium significantly removed by filtration. Samples 2, 6, and 10 all had desirable colors, indicating sufficiently low blood content.

[0058] Samples 3, 4, and 7 were centrifuged, and found to comprise 20-30% blood. After centrifugation, blood remained in the supernatant. The pellet, if analyzed, was found to contain blood, vernix, and cells.