A composition of platelet rich fibrin and platelet rich plasma and a method thereof

Abstract

The present invention provides a novel composition of platelet rich fibrin and platelet rich plasma for sealing amniotic fluid leak in premature rupture of amniotic membranes (PROM) in pregnant women. The present invention further provides a method to prepare said composition.

Description

[0001] A COMPOSITION OF PLATELET RICH FIBRIN AND PLATELET RICH PLASMA AND A METHOD THEREOF

[0002] FIELD OF INVENTION

[0003] The present invention provides a composition of platelet rich fibrin and platelet rich plasma for use to seal amniotic fluid leak in premature rupture of amniotic membranes (PROM) in pregnant women. More specifically, the present invention provides a composition for sealing ruptured amniotic fluid membrane and actively manage pre-viable PROM (PV-PROM) at an early stage of pregnancy, before 24 weeks 0 days of gestational period.

[0004] BACKGROUND OF INVENTION

[0005] Premature rupture of membranes (PROM) is the rupture of gestational membranes prior to the onset of labor. Currently there is no curative treatment for the condition where amniotic membrane ruptures before the foetus becomes mature.

[0006] The incidence of PROM ranges from about 5% to 10% of all deliveries, and leak prior to 37 weeks of gestational age which is called as pre-term PROM (PPROM), occurs in approximately 3% of all pregnancies. PROM is one of the major causes of neonatal sepsis, fetal distress, and birth asphyxia. When it happens before 24 weeks, it is called as Pre-Viable PROM (PV-PROM), the prognosis is very poor for the baby with significant psychological and financial burden for the parents as these very premature babies require extensive neonatal care and may have long term physical or cognitive disabilities. Worldwide, around 0.7 million pregnancies are lost every year due to PV-PROM.

[0007] The management of a PV-PROM is quite a challenge as there are no definitive curative options yet. In contrast to PPROM, where there are guidelines supporting expectant management, PV-PROM lacks consensus on recommended lines of management. If left to the natural course of events, up to 80% of women diagnosed with preterm PROM deliver within one week of the rupture of membranes and in subsequent weeks, the most of women deliver or need termination of pregnancy in the maternal interest.

[0008] According to a study conducted in India, 33% of perinatal morbidity and 15% perinatal mortality have occurred due to premature rupture of membranes. Also, looking after a premature infant puts immense burden on the family, and on the economy and healthcare resources of the country.

[0009] The surviving children have higher risks of physical and developmental disabilities, including chronic respiratory disease, cardiovascular diseases and neurodevelopmental and behavioural issues. The most seen maternal complications are chorioamnionitis (24%) and placental abruption (8%) apart from significant psychological trauma.

[0010] Spontaneous sealing of the membranes does occur occasionally (< 10% of all cases), mostly after PPROM that has occurred subsequent to amniocentesis; however, this is the exception rather than the rule.

[0011] As there are no official clinical guidelines, in day-to-day practice, in instances of sPV-PROM, generally three options are offered: An expectant management with antibiotics and tocolysis to prolong the pregnancy closer to viability or a radical management where termination of pregnancy is considered due to poor prognosis.

[0012] An alternative to these two lines of management is the experimental or active, aggressive management where interventions done focus on stopping the leak by adding autologous or synthetic sealants or leaking fluid volume is replaced by amnioinfiision.

[0013] Many different treatments in the form of injecting sealants like a combination of platelet rich plasma (PRP) and cryoprecipitate into the amniotic cavity (Amniopatch), gel foam into the cervical canal, or giving oral immunomodulators are used in an attempt to seal the perforation in the amniotic membrane. In the Amniopatch procedure, the volume of PRP used in platelet rich plasma (PRP) and cryoprecipitate (CP) varies from 30 - 200 ml and that of CP varies from 20-150 ml. Further, preparation of cryoprecipitate requires a large volume of blood around 450 ml. Further treatments include Cervical adapter, Oral immunological membrane sealant, Transcervical Gelatin Sponge, Collagen plugs and Fibrin Sealants, etc which have not shown any significant improvement in clinical outcomes.

[0014] Another study noted that serial Amnioinfusion might improve neonatal outcomes in PPROM by preventing umbilical cord compression, postural deformities, pulmonary hypoplasia, and intrauterine infection. However, these benefits are more pronounced in third trimester PROM.

[0015] However, most of these treatments have not been effective enough to recommend their use in routine clinical practice. Also, a Cochrane review investigating amnioinfusion in PPROM occurring before 26 weeks of gestation found no eligible trials. So, at present, there has been no approved treatment for pre-viable PV-PROM which makes it crucial to have more research in this field and to find a solution to this widely prevalent problem.

[0016] Taking the drawbacks of the prior art, the present invention provides a composition for use in sealing amniotic fluid leak in premature rupture of amniotic membranes (PV-PROM) in pregnant women.

[0017] OBJECT(S) OF THE INVENTION

[0018] The main object of the invention is to provide a composition of autologous Platelet Rich Fibrin (PRF) and autologous platelet rich plasma (PRP) as a sealant for amniotic fluid leak for management of PROM, more specifically, PV-PROM.

[0019] Another object of the invention is a method for preparing said composition of autologous PRF and autologous PRP as a sealant for amniotic fluid leak.

[0020] Yet another object of the invention is to provide a minimal amount of whole blood, 30 - 60 ml, for preparation of amniotic fluid leak sealant which does not affect mother’s health. SUMMARY OF THE INVENTION

[0021] In carrying out the above objects of the present invention, in the main embodiment, the present invention provides a composition for sealing amniotic fluid leak for management of PROM, more specifically, PV-PROM. Said composition comprises of autologous platelet rich fibrin (PRF) and autologous platelet rich plasma (PRP), more specifically comprising of 8 - 14 ml of autologous PRF and 4.5-14 ml of autologous PRP for application derived from the pregnant women to be treated. The quantity of autologous PRF and PRP required for sealing amniotic fluid leak in a pregnant woman depends on the gestational age and intrauterine volume. Generally, when the gestational age is larger, the intrauterine volume is automatically larger. Therefore, to seal a amniotic fluid leak at later gestational age, a higher quantity of autologous PRF and PRP are required.

[0022] In another embodiment, the present invention provides a method for preparation of a composition for sealing amniotic fluid leakage for management of PROM comprising of autologous PRF and autologous PRP comprising steps of: a) obtaining 30-60 ml of whole blood from pregnant woman suffering from PROM; b) preparing PRF by taking 20-30 ml of the whole blood into a polystyrene tube without any additives or anticoagulants, centrifuging at 800-2200 rpm for 8-12 minutes at 22-26°C, collecting the entire upper supernatant containing the platelet-poor plasma (PPP) and the middle buffy layer together forming 8 - 17 ml of PRF; c) preparing PRP by taking 10-30 ml of whole blood, adding ImL ml of sodium heparin per every 10ml of whole blood, centrifuging at 2200 rpm for 12 minutes, collecting the entire supernatant including the buffy layer containing platelets amounting to 4.5-14 ml of PRP; and d) Mixing PRF and PRP as a sealant.

[0023] Said PRF is obtained as liquid PRF unlike other commonly used protocols which yield a solid fibrin plug. Further, the time interval between drawing of the blood to beginning of centrifuge was kept at less than 10 minutes and that between preparing the PRF and PRP solution to injecting into the Intraamniotic cavity is kept at less than 15 minutes.

[0024] Said sealant has potential to seal amniotic fluid leak in premature rupture of amniotic membranes (PROM) in pregnant women. The composition comprises of autologous PRF and autologous PRP, therefore there is no immune reactivity or risk of transmission of viral infections as in methods using allogenic blood.

[0025] The prior existing PRF and PRP preparation protocols focus on getting a small volume or membrane of highly concentrated platelets and fibrin which can be either applied on tissue surfaces or injected into solid tissues like skin or small cavities like joints. However, injecting a concentrate of fibrin and platelets into a large cavity filled with amniotic fluid has a different pharmacodynamics and needs a different kind of preparation which so far has not been optimized in any studies. Said invention provides a detailed method to obtain PRF and PRP which is suitable for injecting into sac of amniotic fluid.

[0026] Also, use of PRF would give a concentrate of platelets and white blood cells in a mesh of fibrin, releasing growth factors over a much longer period. Use of Mouse Embryo Assay (MEA) tested polystyrene tubes to centrifuge the uncoagulated blood aids in slowing the blood clotting cascade.

[0027] Platelet activators are not used in said method, as exposed collagen at the site of membrane rupture, inflammation and infection would also work as powerful activators of platelets. In fact, clotting of platelets and release of their granules at the site of membrane injury is the fundamental process that the present composition achieves when injected into site of leakage of amniotic fluid in the pregnant women.

[0028] In yet another embodiment, the invention provides a composition for sealing amniotic fluid leak for management of PROM at advanced gestations beyond 24 weeks, wherein around 60 ml-450 ml of whole blood could be used as the quantity of PRF required may be around 50 - 100 ml as third trimester pregnancies will have significantly higher intrauterine volume.

[0029] DETAILED DESCRIPTION OF INVENTION

[0030] The present invention will now be described more fully hereinafter with reference to the examples in which a preferred embodiment of the invention is shown. This invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiment set forth herein. Rather, the embodiment is provided so that this disclosure will be thorough, and will fully convey the scope of the invention to those skilled in the art.

[0031] Definitions

[0032] The term “amniotic fluid” means a clear, slightly yellowish liquid that surrounds the unborn baby (foetus) during pregnancy.

[0033] The term “amniotic fluid leakage” usually indicates that the amniotic sac has ruptured, which can happen before or during labour.

[0034] The term “autologous” means obtained from the same individual.

[0035] The term “autologous blood” or “autologous whole blood” means blood obtained from the same individual on whom it is going to be used. In the context of the present invention, the blood of the pregnant woman who needs to be treated for pre- viable premature rupture of amniotic membranes (PV-PROM) or premature rupture of amniotic membranes (PPROM).

[0036] The term “gestational age” means a measurement of how far along a pregnancy is, usually in weeks. It's calculated from the first day of the last menstrual period to the current date.

[0037] The term “PROM” means premature rupture of amniotic membranes. The term “PV-PROM” means pre-viable PROM which occurs before 24 weeks and 0 days of gestational age.

[0038] The term “PPROM” means pre-term PROM which occurs between 24 weeks 0 days of gestational age to 33 weeks 6 days of gestational age.

[0039] The term “whole blood” means human whole blood that flows through a human’s body, made up of red blood cells, white blood cells, platelets, and plasma.

[0040] The term “PRP” means platelet rich plasma derived from whole blood.

[0041] The term “autologous PRP” means PRP derived from the blood of the same individual on whom it needs to be used.

[0042] The term “PRF” means platelet rich fibrin derived from whole blood.

[0043] The term “autologous PRF” means PRF derived from the blood of the same individual on whom it needs to be used.

[0044] The term “rpm” means rotations per minute.

[0045] EXAMPLE 1

[0046] Use of PRF and PRP sealant for stopping leakage of amniotic fluid in 14 weeks pregnant women

[0047] A 26-year-old woman with 14 weeks of pregnancy (who earlier had a miscarriage due to PV-PROM at 18 weeks gestation period), who showed a recurrent PV-PROM was treated with combination of autologous PRF and autologous PRP derived from her whole blood. 30 mL of whole blood from the patient was drawn in three conical Falcon MEA tested 15 mL tubes. lO mL of whole blood with 1.5ml of sodium citrate was taken to prepare PRP, and 20 mL of whole blood without any additives were taken to prepare PRF. They were centrifuged at 2200 rpm for 12 min at 26°C in a centrifuge. After one spin, all the upper and buffy layer was taken out into a separate tube which amounted to 9 mL of PRF. The supernatant and buffy layer from PRP tube was mixed well in another tube which amounted to 4.5 mL of PRP. Under ultrasound guidance PRF followed by PRP was injected into the amniotic cavity.

[0048] Notably, for 36 hours immediately after the procedure, there was no leak. Later she had two more episodes of small leaks following which it stopped completely. She was closely monitored for early signs of infection along with documentation of serial CRP, ESR, TC, and High Vaginal Swab (HVS) once in 3 weeks. Foetal wellbeing and amniotic fluid volume and cervical length were monitored with serial ultrasound scans. Concurrently, the infection was controlled with local asepsis and antibiotics. At 35 weeks and 4 days, an alive, healthy baby weighing 3.4 Kg was delivered and mother and baby did well postnatally.

[0049] Only 30 mL of whole blood to prepare PRP and PRF in contrast to many studies on Amniopatch where a large volume of fluid, up to 350 mL, is used which includes a platelet concentrate, cryoprecipitate and normal saline.

[0050] The current invention highlights the potential of a simple technique of combining PRP and PRF in sealing off the amniotic leak.

[0051] The protocol for fixing the leak is very simple, easy to use and can be done in resource poor settings which is a big advantage as women in developing countries are at more risk of PV-PROM.

[0052] EXAMPLE 2

[0053] Use of PRF and PRP sealant for stopping leakage of amniotic fluid in 18 weeks pregnant woman

[0054] A 32-year-old woman with previous two early pregnancy losses had amniotic leak at 18 weeks in her third pregnancy and was also diagnosed to have a large cervical polyp. She underwent Cervical polyp excision with cervical encirclage procedure.

[0055] Following which she was treated with the composition of the present invention comprising of 8 ml of autologous PRF and 9 ml of autologous PRP derived from 40 mL of maternal blood which led to complete stoppage of leak. The method of preparing the composition comprised the steps of: a) obtaining 40 ml of whole blood from the pregnant woman suffering from PROM; b) preparing PRF by taking 20ml of the whole blood into a polystyrene tube without any additives or anticoagulants, centrifuging at 2200 rpm for 12 minutes at 22°C, and collecting the entire supernatant and the middle buffy layer forming 9 ml of PRF; c) preparing PRP by taking 20 ml of whole blood, adding 1ml of sodium heparin, centrifuging at 2200 rpm for 12 mins at 22°C, and collecting the entire supernatant with buffy layer forming 6 ml of PRP; and d) mixing 8 mb of PRF and 9 mb of PRP as a sealant.

[0056] The pregnant woman continued the pregnancy to full term, 37 weeks, and delivered a healthy baby of 2.6 kg.

[0057] EXAMPLE 3

[0058] Use of PRF and PRP sealant for stopping leakage of amniotic fluid in 17 weeks pregnant woman

[0059] A 38-year-old second gravida with 16 years of infertility with previous early pregnancy loss conceived dichorionic diamniotic twins by IVF treatment. She underwent cervical encirclage for short cervix at 17 weeks. After the procedure she started to have amniotic fluid leak. Without much delay, she was treated with the composition of the present invention comprising of 8ml of PRP and 8 ml of PRF, which resulted in immediate cessation of leak.

[0060] The method preparing the composition comprised the steps of: a) obtaining 40 ml of whole blood from the pregnant woman suffering from PROM; b) preparing PRF by taking 20ml of the whole blood into a polystyrene tube without any additives or anticoagulants, centrifuging at 800 rpm for 8 minutes at 24°C, and collecting the entire supernatant and the middle huffy layer forming 8 ml of PRF; c) preparing PRP by taking 20 ml of whole blood, adding 1 ml of sodium heparin, centrifuging at 2200 rpm for 12 mins at 22°C, and collecting the entire supernatant with buffy layer forming 6 ml of PRP; and d) mixing 8 mb of PRF and 8 mb of PRP as a sealant.

[0061] The rpm and duration of the centrifuge to prepare PRF was reduced in view of the observation that in two other women who underwent the procedure couldn’t get sufficient volume of PRF to inject as it clotted very early.

[0062] The 16 ml sealant was injected into the intraamniotic cavity in the pregnant woman who delivered a healthy boy and a girl at term with birth weights of 2.4 kg and 2.5 kg each.

[0063] EXAMPLE 4

[0064] Use of PRF and PRP sealant for stopping leakage of amniotic fluid in 14 weeks 6 days pregnant woman

[0065] A 33 -year-old, 4th gravida with one live child with two mid trimester losses due to PV-PROM underwent elective cervical encirclage at 14 weeks 6 days. She developed amniotic fluid leak 12 hours after the procedure. She was treated with the composition of the present invention comprising of 14 ml of PRP and 9 ml of PRF 12 hours after leak. The leak stopped immediately and at present she is at 24 weeks of gestation.

[0066] The method preparing the composition comprised the steps of: a) obtaining 50 ml of whole blood from the pregnant woman suffering from PROM; b) preparing PRF by taking 30ml of the whole blood into a polystyrene tube without any additives or anticoagulants, centrifuging at 800 rpm for 8 minutes at 24°C, and collecting the entire supernatant and the middle huffy layer forming 14 ml of PRF; c) preparing PRP by taking 20 ml of whole blood, adding 1 ml of sodium heparin, centrifuging at 2200 rpm for 12 mins at 22°C, and collecting the entire supernatant with buffy layer forming 9 ml of PRP; and d) mixing 14 mb of PRF and 9 mb of PRP as a sealant.

[0067] The 23 ml sealant was injected into the intraamniotic cavity in the pregnant woman under ultrasound guidance. The woman is continuing her pregnancy and is currently at 23 weeks of gestation showing no complications so far.

[0068] EXAMPLE 5

[0069] The cases of successful stoppage of amniotic fluid leakage

[0070] A total of 7 pregnant women showing PV-PROM were treated with the present composition of autologous PRP and autologous PRP to stop amniotic fluid leakage, of which pregnancies of 4 women were saved. Three women successfully delivered healthy babies of which one women also delivered twins. One of the four women whose pregnancy was saved is in her 24 weeks pregnancy and awaits to deliver a healthy baby.

[0071] Table 1 provides details of the composition of PRP and PRF used in the four women whose pregnancies were saved successfully after treating amniotic fluid leakage.

[0072] Table 1

[0073] Table 2 provides details of the composition of PRP and PRF used in the other women whose pregnancies could not be saved successfully after treatment with PRF and PRP. Table 2

Claims

CLAIMS im,1. A composition for sealing amniotic fluid leak to treat and manage premature rupture of amniotic membranes (PROM), wherein, the composition consists of: autologous platelet rich fibrin (PRF), and autologous platelet rich plasma (PRP).

2. The composition as claimed in claim 1, wherein, the quantity of autologous PRF is 8 - 14 ml.

3. The composition as claimed in claim 1, wherein, the quantity of autologous PRP is 4.5-14 ml.

4. The composition as claimed in claim 1, wherein, the quantity of whole required to obtain autologous PRF and autologous PRP is 30-50 ml.

5. The composition as claimed in claim 1, wherein, the composition is injected in the intraamniotic cavity of pregnant women suffering from PV-PROM The composition as claimed in claim 1, wherein, the composition is used for treating previable premature rupture of amniotic membranes (PV-PROM) in pregnant women.

6. The composition as claimed in claim 1, wherein, the composition is used for treating pre-term premature rupture of amniotic membranes (PPROM) and term premature rupture of amniotic membranes (TPROM) in pregnant women.

7. A method to prepare a composition for sealing amniotic fluid leak to treat and manage premature rupture of amniotic membranes (PROM), the method comprising the steps of: a) obtaining 30-60 ml of whole blood from pregnant women suffering from PROM; b) preparing autologous PRF by taking 20-40 ml of the whole blood into a polystyrene tube without any additives or anticoagulants, centrifuging at 800 - 2200 rpm for 8 -12 minutes at 20-26°C, andcollecting the entire supernatant and buffy layer into another tube forming 8 - 14 ml of PRF; c) preparing autologous PRP by taking 10-20 ml of whole blood, adding 1.5ml Sodium Citrate per lOmL of blood or ImL of Sodium Heaprin per 9-10 mb of whole blood, centrifuging at 800-2200 rpm for 8-12 minutes, and collecting the entire supernatant and buffy layer forming 4.5-14 ml of PRP in a separate tube; and d) mixing PRF and PRP as a sealant.

8. The method as claimed in claim 8, wherein, the time interval between drawing of the whole blood to beginning of centrifuge is kept less than 5- 10 minutes.

9. The method as claimed in claim 8, wherein, the method provides a composition of autologous PRF and PRP for treating PV-PROM, PPROM, and TPROM in pregnant women.

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