for many years , cosmetic plastic surgeons have recognized the value of low - pressure 
 lipoaspiration for successful transplantation of adipose tissue for structural augmentation . in the 
 introductory years ( 19801990 ) of liposuction techniques , autologous fat grafting ( afg ) was 
 considered unpredictable . once bioengineers discovered the actual mechanisms by which lipoaspiration 
 worked , the closed syringe system for gentle harvesting and transplantation was developed and 
 patented . early belief that effective lipoaspiration was directly related to force of vacuum was 
 replaced by understanding , that , introduction of fluid into the fat layers permitted the adipocyte 
 cells and stroma elements to enter into suspension . this suspension was easily extracted through use 
 of closed syringes , and provided adipose tissues with reduced damage and improved grafting 
 results.1 in the mid-1980s , the importance of tumescent fluid distribution was first appreciated , and more 
 value was placed on extensive pre - tunneling ( moving cannula without applying vacuum ) . this technique 
 better distributes local solution and enhances the ability to mobilize the adipose tissues into a 
 suspension state , which yields more successful and predictable autologous fat grafts ( afg).1 in the early 2000s , appreciation of the potential of adipose tissue and its related stromal 
 elements led to examination of the adipose - derived adult mesenchymal stem cell content 
 ( ad - msc).2,3 evidence has clearly shown the key importance of the progenitor cells 
 ( svf ) and extracellular matrix ( ecm ) as integral contributors to the tissue maintenance and healing 
 processes.4 studies of adipocyte replenishment 
 ( following normal senescence and cellular death ) showed that these attached progenitor cells were 
 activated to form adipocytes and thereby maintain adipose tissue integrity over time.5,6 
 since there is easy accessibility and greater availability of multipotent progenitor cells for all 
 mesogenic lines within adipose tissues , utilization of ad - msc / svf has become a central focus in 
 optimizing effectiveness of autologous fat acquisition and grafting in cosmetic plastic surgery and 
 clinical regenerative medicine.710 preclinical and clinical applications have been reported in many scientific studies in the 
 biological , bioengineering , and clinical medical literature.11 cosmetic plastic surgeons initially focused on understanding the 
 mechanisms to achieve safe and effective afg . it was believed that intact cellular ( mature 
 adipocytes ) transplantation was the most important goal.12 however , it has become crystal clear that mature adult adipocytes transplanted may be the 
 least important feature producing long - term success , even in structural fat augmentation graft 
 applications . current beliefs are that success in long - term afg is actually due to activation of 
 adherent progenitor cells ( attached to mature adipocytes , ecm and svf ) , and proliferation of those 
 progenitor cells to differentiate into the target cell for replacement.4,1315 as an example , placement of lipoaspirants into existing adipose tissue favors proliferation and 
 differentiation into adipose cell phenotypes . as understanding of the maintenance ( homeostatic ) and 
 replenishment of adipose cell cycles in vivo increases , extensive research has been devoted to the 
 study of microenvironment ( niche ) , cell - to - cell / cell - to - matrix factors , and autocrine / paracrine 
 signaling system functions . since ad - msc / svf are capable of differentiation for all mesogenic lines , 
 including : 1 ) chondrogenic ; 2 ) fibro - muscular ( including tendon , ligament , and skeletal and cardiac 
 muscle ) ; 3 ) osteogenic ; and 4 ) adipogenic cell lines , uses in clinical applications have 
 increased.1622 rapidly accumulating clinical data on the safety and efficacy of ad - msc / svf in vivo provides 
 clear evidence that adipose tissue grafts possess extensive potentials in wound healing , well beyond 
 the structural augmentation in cosmetic plastic surgical uses . understanding these mechanisms results in important application potentials for aesthetic , 
 reconstructive , and regenerative medicine . reporting of preclinical , early clinical , and controlled 
 studies in animal and human models shows worldwide recognition of the potential uses of these cells 
 in diverse areas of medicine and surgery.2325 research and clinical applications have led to appreciation of the existence of a large 
 heterogeneous , undifferentiated nucleated cell populations and extensive native bioactive 
 scaffolding that is an integral component of the total svf components within the adipose 
 tissues.4 most reports examining the nature and components of the stromal vascular fraction ( svf ) have come 
 from decades of study on bone marrow - derived stem cells ( bm - scs).26 it is now recognized that ad - mscs have essentially the same 
 capabilities while offering significant advantages over bm - mscs utilization . these include : 1 ) very 
 similar progenitor differentiation capabilities ; 2 ) important cellular subsets ( such as mesenchymal 
 and perivascular cells ) are found in much higher concentrations compared to bone marrow aspirants 
 ( > 1500x ) ; 3 ) less expensive to harvest ; 4 ) less invasive ( safer ) ; 5 ) readily available tissue ; 
 and 6 ) less technically demanding than bone marrow penetration and cellular harvest . these features 
 have led to increased uses in many areas of medicine and surgery.2733 with identification of the near - terminally differentiated cells ( pre - adipocytes ) attached 
 directly to the cytoplastic surface of all mature adipocytes by granneman et al , homeostatic 
 mechanisms with adipose metabolism have been better understood.34 with advanced understanding of the processes of homeostatic adipose 
 replacement , many reasons why carefully harvested autologous fat grafts for structural augmentation 
 is effective have become much more predictable and understood than in years past.3537 technology is now available to effectively isolate / concentrate these progenitor cells , but such 
 manipulation involving chemical digestion is not permitted for clinical use within the us . 
 therefore , utilization of non - manipulated afg / prp concentrates remains the only option at this time . 
 with recognition that lipoaspiration ( versus en bloc excised fat ) acquires a bit over 50% of 
 the native multipotent , nucleated cell populations , such isolation and concentration protocols have 
 gained favor in efforts to help restore the multipotent cell numbers to near native levels.38 at this time , such digestion and isolation procedures 
 are prohibited by the regulatory agencies within the us , as they are considered more - than - minimally 
 manipulated tissues and subject to such rules . laboratory findings suggest that use of closed - syringe harvesting techniques produces higher 
 nucleated cellular counts as compared with low - pressure wall suction or detuned lipoaspiration 
 mechanical pumps ( alexander rw , mandle r ; harvard biosciences laboratory , unpublished data , 2012 ) 
 ( see figure 1 ) . the patented tulip closed - syringe system ( tulip medical systems , san diego , ca , usa ) , 
 with its array of microcannulas ( small volumes-,100 cc ) and standard volume reduction cannulas ( for 
 > 100 cc liporeduction and contouring ) , is a recognized and proven lipoaspiration system . explanation and detailed discussion of a repeatable , effective , and safe protocol for adipose tissue 
 complex harvest for cosmetic plastic surgeons will be provided in this paper . research and clinical applications have led to appreciation of the existence of a large 
 heterogeneous , undifferentiated nucleated cell populations and extensive native bioactive 
 scaffolding that is an integral component of the total svf components within the adipose 
 tissues.4 most reports examining the nature and components of the stromal vascular fraction ( svf ) have come 
 from decades of study on bone marrow - derived stem cells ( bm - scs).26 it is now recognized that ad - mscs have essentially the same 
 capabilities while offering significant advantages over bm - mscs utilization . these include : 1 ) very 
 similar progenitor differentiation capabilities ; 2 ) important cellular subsets ( such as mesenchymal 
 and perivascular cells ) are found in much higher concentrations compared to bone marrow aspirants 
 ( > 1500x ) ; 3 ) less expensive to harvest ; 4 ) less invasive ( safer ) ; 5 ) readily available tissue ; 
 and 6 ) less technically demanding than bone marrow penetration and cellular harvest . these features 
 have led to increased uses in many areas of medicine and surgery.2733 with identification of the near - terminally differentiated cells ( pre - adipocytes ) attached 
 directly to the cytoplastic surface of all mature adipocytes by granneman et al , homeostatic 
 mechanisms with adipose metabolism have been better understood.34 with advanced understanding of the processes of homeostatic adipose 
 replacement , many reasons why carefully harvested autologous fat grafts for structural augmentation 
 is effective have become much more predictable and understood than in years past.3537 technology is now available to effectively isolate / concentrate these progenitor cells , but such 
 manipulation involving chemical digestion is not permitted for clinical use within the us . therefore , utilization of non - manipulated afg / prp concentrates remains the only option at this time . 
 with recognition that lipoaspiration ( versus en bloc excised fat ) acquires a bit over 50% of 
 the native multipotent , nucleated cell populations , such isolation and concentration protocols have 
 gained favor in efforts to help restore the multipotent cell numbers to near native levels.38 at this time , such digestion and isolation procedures 
 are prohibited by the regulatory agencies within the us , as they are considered more - than - minimally 
 manipulated tissues and subject to such rules . laboratory findings suggest that use of closed - syringe harvesting techniques produces higher 
 nucleated cellular counts as compared with low - pressure wall suction or detuned lipoaspiration 
 mechanical pumps ( alexander rw , mandle r ; harvard biosciences laboratory , unpublished data , 2012 ) 
 ( see figure 1 ) . the patented tulip closed - syringe system ( tulip medical systems , san diego , ca , usa ) , 
 with its array of microcannulas ( small volumes-,100 cc ) and standard volume reduction cannulas ( for 
 > 100 cc liporeduction and contouring ) , is a recognized and proven lipoaspiration system . explanation and detailed discussion of a repeatable , effective , and safe protocol for adipose tissue 
 complex harvest for cosmetic plastic surgeons will be provided in this paper . the lower abdomen and flank areas of both males and females are considered ideal adipose donor 
 sites due to distribution of human adipose tissues and relatively large deposits . choice of 
 aspiration sites in the medial and lateral thigh / buttocks areas is sometimes favored for 
 lipoaspiration and adipose graft harvesting in female patients due to genetic distribution within 
 the gynoid body type . in patients with very low percentage body fat needing autologous grafts , use 
 of a high - definition ultrasound probe is helpful to determine the thickness and depth of adipose 
 deposits that can be acquired . the patients may be placed in either supine or lateral decubitus position to facilitate the 
 preparation and complete sterile isolation of the proposed donor area(s ) . it is considered important 
 to follow a standard sterile protocol for both the harvesting and placement sites . routine operative 
 site asepsis should be maintained in all cases , with patients marked in an upright or standing 
 position to effectively mark the area of available or unwanted adipose tissue deposits . the patented tulip closed - syringe system for lipoaspiration features very smooth 
 cannulas and a  super  luer - lock connection for use with standard luer - lock syringes 
 ( figure 2 ) . this hub connection is a very important 
 component of the closed - syringe and microcannula system , in that it provides an excellent seal for 
 maintaining even vacuum forces desirable during lipoaspiration ( figure 3 ) . since the super - luer lock connection seals at both the 
 internal luer connection and at the outer ring of the standard luer connection , it thereby provides 
 a very stable , rigid base . internal female luer - type connectors on some microcannula systems on the market become less 
 efficient when cannulas are redirected , placing a torque on the junction of cannula - syringe barrel , 
 and allowing air leakage into the closed system ( particularly in the longer cannula selections ) . this does not usually prevent aspiration capabilities , but it does decrease the efficiency and may 
 introduce cavitation to the harvest tissues . two standard options for microcannula selection offered within the tulip system are : cell - friendly microcannula option ( autoclavable ) ( figure 
 4 ) . these cannulas are internally polished by microabrasive extrusion process to maximize 
 internal smoothness and reduce adipose tissue damage to the adipocytes , precursor cells , and their 
 accompanying matrix . external cannula anodizing processes provide a smoother surface for ease of 
 passage within the subdermal adipose plane . this is a popular design utilized by cosmetic plastic 
 surgeons for liporeduction as well as harvesting of afg for structural augmentation procedures . references the closed syringe system instruments are also available for larger volume liporeduction 
 ( liposuction ) cases and in large volume structural graft cases . in cannulas of less than 3.0 mm , it 
 is important to thoroughly flush with water - prep soap mix , followed by ultrasonic cleaning , and 
 thorough re - flushing with water prior to steam or gas sterilization . use of disposable microcannulas in small diameters of less than 3.0 mm ( range 
 0.92.4 mm outside diameter [ od ] ) presents a significant challenge to ensure 
 proper and effective cleaning  sterilization cycles . this makes a disposable option 
 attractive , particularly in the smaller diameter cannula group . these are packaged and labeled in a 
 sterile wrap , and can be opened directly onto the sterile field or back table . featuring the super 
 luer - lock base , these stainless steel cannulas are totally coated internally and externally with a 
 hydrophilic material that provides an extremely smooth coating , which passes through adipose tissues 
 with minimal resistance and trauma . initially , hydrogel coatings were applied on both internal and 
 external surfaces , but now have been replaced by more efficient and effective coating materials , 
 featuring more than 20 times more lubriciousness than previous coating materials . it is believed 
 that the least cellular and tissue trauma created , the better the quality of the adipose grafts . with increased recognition of difficulties in effectively cleaning the nondisposable 
 microcannulas ( < 3.0 mm ) , most surgeons and operating facilities are choosing use of completely 
 disposable infiltration , harvesting , and injection cannulas . for small volume applications ( < 100 cc ) , it is recommended to use a small , multiport 
 infiltrator cannula for even and thorough distribution of local anesthesia throughout the adipose 
 donor layer . openings near the tip are multiple and oriented for 360-degree distribution of the 
 tumescent local solution while moving through the subdermal fat layers . it is common for 
 practitioners to use this infiltration cannula in diameters of 2.12.4 mm od and a length of 
 1020 cm ( figure 6 
 [ top ] ) . harvesting cannulas are designed to actually acquire the adipose tissue grafts from the subdermal 
 fat plane , following the same pattern and location of local anesthesia distribution ( figure 6 [ middle ] ) . the openings on the harvesting cannulas are usually provided as inline or offset openings 
 ( meaning in a nonlinear pattern near the tip of the cannula ) . these vary in diameter from 1.67 mm to 
 2.4 mm ( od ) , with a length of 1020 cm . selection of a slightly shorter harvesting cannula 
 compared to length of infiltrator makes it somewhat easier to remain within the local anesthesia 
 distribution areas in awake patients . the external locks are specifically designed for use on bectin 
 dickinson co. ( franklin lakes , nj , usa ) or monoject ( covidien - kendall , mansfield , ma , usa ) 10/12 and 
 20 cc luer - lock syringes and 60 cc toomey tip syringes to hold the syringe plunger in a fully drawn 
 position during the application of vacuum . before application of vacuum by pulling the syringe 
 plunger to the desired level , it is essential to draw sterile saline fluid into the cannula to 
 completely displace all air within the system . when pulled and twisted into the locked position , the 
 edge of the lock engages the side of the plunger permitting the physician to apply even and gentle 
 vacuum pressure while moving the cannula through the tumesced adipose layer . the internal type lock , 
 called a snap lock , universally fits a range of different syringe sizes from various manufacturers . anaerobic transfers ( luer - to - luer ) are utilized to facilitate loading of treatment syringes prior 
 to grafting procedures , and for optional use of additives to the grafts ( such as combining platelet 
 concentrates [ hd prp ] and adipose grafts into one syringe [ figure 5 ( bottom ) ] ) . they are also useful for 
 transferring the treatment mix into syringe sizes of physician preference for injection and avoiding 
 undesirable exposure of the harvested graft to air ( figure 
 8) . it is considered important to avoid excessive air exposure to grafts due to the potential 
 for contamination by airborne particles or pathogens . techniques described as helpful in free lipid 
 removal ( such as telfa  rolling  ; johnson & johnson , new brunswick , nj , 
 usa ) are vulnerable to such contamination . a controlled aliquot injector gun is useful for placement of controlled 0.5 cc aliquots of graft 
 into the prepared tunnels and locations . when additives are added to the adipose tissue graft , the 
 density of the injection material may be increased ( eg , use of hd prp plus thrombin to provide rapid 
 release of platelet - derived growth factors and signal proteins ) . this may result in the physician 
 requiring more force to inject into the tissue site , or , to encounter sudden and uneven distribution 
 of desired small aliquots of graft with prepared tunnels associated with adipose matrix density 
 within the graft itself . single - trigger pull provides exact volumes of solution to be placed with 
 less pressure required by the provider ( figure 
 9 ) . it is recommended that the area of donor and recipient sites be outlined with the patient in 
 upright position and using a skin - marking pencil . this area will then be prepped , draped , and 
 isolated to expose the thickest deposit of palpable fat tissues , and serve as a distribution pattern 
 for local anesthetic infiltration ( see figure 10 
 for an example ) . after marking , preparation , and sterile isolation of the donor area , an 1820 g beveled 
 needle is held such that the side edge is held vertical to the skin . this side edge is utilized to 
 create a small , vertical ( slit - like ) opening , extending through the epidermis and dermis into the 
 subdermal fat plane of the donor site . it is very important to avoid too large an opening , as 
 closed - syringe - system vacuum depends on maintaining a tight side wall opening to ensure even vacuum 
 application . use of stab incisions with scalpel blades of # 15 or # 11 sizes tends to 
 create larger than necessary or desired openings . the opening is made larger by selectively cutting 
 the dermal layer ( under the skin surface ) with the edge of the needle bevel . in large cannula sizes , 
 use of a tapered stainless sharp trocar ( 3 mm ) is utilized to permit a snug fit of the aspiration 
 cannulas into the desired space for cannulas of > 3 mm od . this allows the introduction of the 
 multiport infiltration cannula through the skin and the subdermal fascia ( and in the anterior 
 abdominal wall example , should perforate and remain beneath scarpa s fascia ) ( figure 11 ) . following entry , the multiport infiltrator cannula should be passed in a horizontal fashion 
 within deeper aspects of the subcutaneous donor fat deposit , above the muscular layer , in a 
  spokes - of - a - wheel  pattern . pinching the skin - fat tissues may help in passing the 
 cannula . during movement of the infiltrating cannula , very slow injection of tumescent local 
 anesthesia fluid , during both entry and withdrawal strokes placement in 
 layers , beginning in the deeper aspects , and moving to more superficial levels is most effective . the importance of avoiding  pooling  of the tumescent local anesthesia is that 
 evenly distributing liquids improves the efficiency of harvest by providing a suspensory fluid 
 carrier for the adipose graft tissues , as well as excellent patient comfort . if tumescent fluids are 
 rapidly injected or injected only in a few locations within the fat deposits , pooling is often the 
 result . this pooling is recognized by the surgeon by the presence of larger amounts of infranatant 
 fluids seen after gravity decantation or centrifugation . in typical small volume grafting cases , use of tumescent local anesthesia in a range of 
 2030 cc is common . as a general guideline , it is important to place at least 1 cc of 
 tumescent local per 1 cc of anticipated graft to be harvested . for example , if the plan is to 
 aspirate 50 cc of adipose tissues , then use of 50 cc or greater fluid volume is distributed with the 
 adipose layer to provide the fluid carrier for extraction of the grafts . a common example of the 
 component of tumescent solution is to add the contents of a 50 cc multidose vial of local anesthetic 
 ( eg , 0.5% to 1.0% xylocaine with or without epinephrine [ 1:100,000 ] ) 
 to 1 l of sterile saline or balanced salt solution to provide sufficient tumescent fluid for 
 lipoaspiration . upon completion of even distribution of tumescent fluid within the proposed donor area , it is 
 recommended that repetitive passage of the infiltrating cannula throughout the tumesced donor area 
 ( termed  pre - tunneling  ) multiple times is important and very helpful to attain an 
 even and high quality graft . this more thoroughly distributes local anesthetic fluid for patient 
 comfort , and it also provides the needed  carrier fluid  to effectively suspend the 
 adipose tissues prior to harvesting with low pressure and minimal bleeding . this is a very important 
 step that will improve comfort during harvest , plus make extraction more efficient and result in 
 markedly less volume of the unwanted infranatant fluid layer . in small - volume transfers , most practitioners select a 20 cc luer syringe attached to the 
 harvesting microcannula with a mounted locking device . a very small volume ( 12 cc ) of 
 sterile 0.9% saline is drawn into the cannula to displace air from the system prior to 
 insertion into the harvest ( donor site ) . this is termed  charging  the syringe 
 device , and is necessary to eliminate all air from within the cannula and syringe , thereby avoiding 
 cavitation produced when using mechanical pump suction devices ( wall suction , detuned lipoaspiration 
 machines , etc ) . once the harvesting cannula is inserted into the locally tumesced and pre - tunneled adipose layer , 
 the syringe plunger is drawn to partial or full extension depending on desired vacuum pressure , and 
 twisted to provide a lock ,  if using an external type , or a ledge , which snaps to 
 hold the plunger in one of three positions . after application of vacuum , the physician is free to 
 move the harvesting cannula in a forward and back series of passages . it is important that these 
 passages are within the same plane and of the same pattern used during the placement of tumescent 
 solution . adipose return , at first , will be somewhat slower , as the graft tissue must be in 
 suspension to be able to be easily extracted . continuing these movements with vacuum applied will 
 yield adipose tissues with minimal bleeding in most patients . during the displacement of air , it is recommended that 4 inch by 4 inch sterile gauze should be 
 held over the harvesting tip openings to avoid spraying contents . occasionally , in cases where there 
 is a larger volume of infranatant fluid ( the layer immediately below the fat tissues ) , simply 
 express the liquid portion , reinsert the harvesting cannula into the harvest site , lock the plunger , 
 and gather more graft tissue . in the event of vacuum pressure loss during the harvesting process , it 
 is sometimes necessary to completely remove the harvesting cannula from the donor site , carefully 
 expressing all air from within the cannula . when this is completed , reinsertion of the cannula is 
 performed , and vacuum is re - established by withdrawing ( pulling ) the plunger and re - locking it . the yellow adipose grafts will quickly gravity separate from the underlying ( infranatant ) fluid , 
 resulting in the graft floating on top of the small fluid volume within the syringe system . test 
 tube / syringe stands or decanting stands are available to facilitate this initial gravity separation 
 ( figure 12a and b ) . remember that a common cause of increased infranatant volume in the decanted syringe is 
 inadequate distribution of local fluid , creating a pooling effect , which reduces efficiency of 
 adipose harvest . it is for this reason that extensive pre - tunneling is highly recommended prior to 
 application of any vacuum to the tissues . upon completion of aspiration of the desired graft volume , 
 the harvester cannula is removed , and the syringe end - capped and placed in a vertical position in a 
 standard test tube rack or directly mounted on a luer base of a decanting stand to allow gravity to 
 separate three distinct layers within the harvesting syringe . this usually requires decantation for 
 approximately 23 minutes or centrifugation ( discussed below ) at 1000 g for 
 34 minutes . after layer separation , practitioners expel the unwanted liquid layer 
 ( infranatant liquid ) on which the fat graft floats , which is expressed into sterile containers for 
 disposal . following removal of the infranatant fluid layer , if additional graft is needed , it is possible 
 to reinsert the harvester into the donor site to acquire more graft tissue ( adipocytes and 
 adipose - derived tissue stromal vascular fraction ) , additional decanting / centrifugation , followed by 
 anaerobic transfer loading of the graft into the treatment syringes of choice . when the desired volume is attained , lipoaspiration is completed , and the small opening is 
 covered with a triple antibiotic ointment and a coverlet ( bsn medical , charlotte , nc , usa ) bandage 
 ( adhesive on all sides ) . in the event of loss of vacuum ( evidenced by a hissing sound ) , the cannula is simply removed from 
 the adipose tissues , fluid advanced into the harvesting cannula to eliminate air from the system , 
 cannula reinserted , and plunger pulled open and locked to restart the aspiration process . ideally , the more thorough removal of unwanted fluids from the graft yields a more dense cellular 
 graft . it is important to effectively avoid the top layer ( free lipid layer ) from being included in 
 the final afg transplantation . in addition , the free lipid layer ( clear yellow liquid above the 
 harvested graft ) should be avoided , as it is an irritant and prolongs the healing of the graft 
 tissues in that it must be removed during the process by macrophages , etc , over time . the desire to produce the best compact grafts with minimal remaining fluid excess has led many to 
 include use of optimal centrifugation ( 1000 g for 34 minutes ) to 
 effectively compress the graft materials , help to more precisely separate fluid from harvested 
 graft , and facilitate removal of the unwanted free lipid component ( figure 13)39 
  several providers of tissue processing syringes offer the ability to more easily remove the 
 lipid layer from the graft tissues desired ( see figure 
 14 ) . whether by disk or weighted plunger , the free lipids are allowed to pass out of the 
 upper graft tissues and remain in the harvesting syringe itself . the ability to thoroughly remove 
 unnecessary fluid from the graft provides the surgeon an opportunity to avoid the mandatory 
  overcorrection  needed due to the amount of remaining carrier fluids within the 
 graft . further , use of a disposable separator disk , within the centrifuge processing syringe , 
 ensures more complete separation of the free lipid free lipid layer . its removal prevents the 
 resulting irritation and inflammatory response when removing the unwanted additional free lipid from 
 within a recipient site ( figure 14 ) . some practitioners perform one to two rinses with sterile saline to help reduce any residual 
 local anesthetic solution and red blood cells.40 after decantation and/or centrifugation steps , the graft preparation is ready for placement into 
 treatment syringes of physician s choice . it is important to use clear anaerobic transfers 
 ( luer - to - luer connectors ) to load the individual application syringes from the prepared , compressed 
 graft ( figure 15 ) . they are advantageous in that 
 they prevent external air exposure and potential for contamination and permit use of additives , such 
 as high - density platelet - rich plasma ( hd prp ) concentrates . use of hd prp concentrates are believed 
 to further enhance the healing , graft acceptance , and provide high concentrations of helpful growth 
 factors and signal proteins . treatment - standard luer syringes are then mounted with the desired injection cannulas , using 
 coated , single - port cannulas or the surgeons , preferred injector needles . use of blunt - tip , coated 
 cannulas is recommended , particularly within the facial recipient areas to lower the risk of 
 embolism caused by inadvertent injection of the adipose graft intravascularly . the injection 
 cannulas are available in a variety of lengths and diameters ( ranging from 0.9 mm to 1.47 mm od ) , to 
 accommodate the surgeon s preference and specific areas to be grafted . some elect to inject 
 with sharp needles ranging in size from 18 g to 25 g ( figure 16 ) . the typical graft recipient bed is prepared and developed by pre - tunneling to create a 
  potential  space , which is subsequently filled in small aliquots and in layers as 
 the injection cannula is being withdrawn . use of small , sterile 
 absorbing gauze dressing placed over the skin opening created to place the tumescent fluids into the 
 subdermal tissues . further , use of compression and closed cell , medical grade foam 
 ( tenderfoam ; t&n industries , san diego , ca , usa ) will eliminate or 
 minimize post - harvest bruising of the donor area . it is believed that use of foam and compression 
 reduces the lymphatic dispersion of small amounts of red blood cells or breakdown pigments remaining 
 within the surgical harvest site in the form of bruising or discoloration . firm compression of the 
 gauze and tenderfoam for 2448 hours is typically effective ( figure 17a and b ) . the lower abdomen and flank areas of both males and females are considered ideal adipose donor 
 sites due to distribution of human adipose tissues and relatively large deposits . choice of 
 aspiration sites in the medial and lateral thigh / buttocks areas is sometimes favored for 
 lipoaspiration and adipose graft harvesting in female patients due to genetic distribution within 
 the gynoid body type . in patients with very low percentage body fat needing autologous grafts , use 
 of a high - definition ultrasound probe is helpful to determine the thickness and depth of adipose 
 deposits that can be acquired . the patients may be placed in either supine or lateral decubitus position to facilitate the 
 preparation and complete sterile isolation of the proposed donor area(s ) . it is considered important 
 to follow a standard sterile protocol for both the harvesting and placement sites . routine operative 
 site asepsis should be maintained in all cases , with patients marked in an upright or standing 
 position to effectively mark the area of available or unwanted adipose tissue deposits . the patented tulip closed - syringe system for lipoaspiration features very smooth 
 cannulas and a  super  luer - lock connection for use with standard luer - lock syringes 
 ( figure 2 ) . this hub connection is a very important 
 component of the closed - syringe and microcannula system , in that it provides an excellent seal for 
 maintaining even vacuum forces desirable during lipoaspiration ( figure 3 ) . since the super - luer lock connection seals at both the 
 internal luer connection and at the outer ring of the standard luer connection , it thereby provides 
 a very stable , rigid base . using microcannula sized instruments are very flexible compared to 
 standard cannula types . internal female luer - type connectors on some microcannula systems on the market become less 
 efficient when cannulas are redirected , placing a torque on the junction of cannula - syringe barrel , 
 and allowing air leakage into the closed system ( particularly in the longer cannula selections ) . this does not usually prevent aspiration capabilities , but it does decrease the efficiency and may 
 introduce cavitation to the harvest tissues . two standard options for microcannula selection offered within the tulip system are : cell - friendly microcannula option ( autoclavable ) ( figure 
 4 ) . these cannulas are internally polished by microabrasive extrusion process to maximize 
 internal smoothness and reduce adipose tissue damage to the adipocytes , precursor cells , and their 
 accompanying matrix . external cannula anodizing processes provide a smoother surface for ease of 
 passage within the subdermal adipose plane . this is a popular design utilized by cosmetic plastic 
 surgeons for liporeduction as well as harvesting of afg for structural augmentation procedures . references the closed syringe system instruments are also available for larger volume liporeduction 
 ( liposuction ) cases and in large volume structural graft cases . in cannulas of less than 3.0 mm , it 
 is important to thoroughly flush with water - prep soap mix , followed by ultrasonic cleaning , and 
 thorough re - flushing with water prior to steam or gas sterilization . use of disposable microcannulas in small diameters of less than 3.0 mm ( range 
 0.92.4 mm outside diameter [ od ] ) presents a significant challenge to ensure 
 proper and effective cleaning  sterilization cycles . this makes a disposable option 
 attractive , particularly in the smaller diameter cannula group . these are packaged and labeled in a 
 sterile wrap , and can be opened directly onto the sterile field or back table . featuring the super 
 luer - lock base , these stainless steel cannulas are totally coated internally and externally with a 
 hydrophilic material that provides an extremely smooth coating , which passes through adipose tissues 
 with minimal resistance and trauma . initially , hydrogel coatings were applied on both internal and 
 external surfaces , but now have been replaced by more efficient and effective coating materials , 
 featuring more than 20 times more lubriciousness than previous coating materials . it is believed 
 that the least cellular and tissue trauma created , the better the quality of the adipose grafts . with increased recognition of difficulties in effectively cleaning the nondisposable 
 microcannulas ( < 3.0 mm ) , most surgeons and operating facilities are choosing use of completely 
 disposable infiltration , harvesting , and injection cannulas . for small volume applications ( < 100 cc ) , it is recommended to use a small , multiport 
 infiltrator cannula for even and thorough distribution of local anesthesia throughout the adipose 
 donor layer . openings near the tip are multiple and oriented for 360-degree distribution of the 
 tumescent local solution while moving through the subdermal fat layers . it is common for 
 practitioners to use this infiltration cannula in diameters of 2.12.4 mm od and a length of 
 1020 cm ( figure 6 
 [ top ] ) . harvesting cannulas are designed to actually acquire the adipose tissue grafts from the subdermal 
 fat plane , following the same pattern and location of local anesthesia distribution ( figure 6 [ middle ] ) . the openings on the harvesting cannulas are usually provided as inline or offset openings 
 ( meaning in a nonlinear pattern near the tip of the cannula ) . these vary in diameter from 1.67 mm to 
 2.4 mm ( od ) , with a length of 1020 cm . selection of a slightly shorter harvesting cannula 
 compared to length of infiltrator makes it somewhat easier to remain within the local anesthesia 
 distribution areas in awake patients . the external locks are specifically designed for use on bectin 
 dickinson co. ( franklin lakes , nj , usa ) or monoject ( covidien - kendall , mansfield , ma , usa ) 10/12 and 
 20 cc luer - lock syringes and 60 cc toomey tip syringes to hold the syringe plunger in a fully drawn 
 position during the application of vacuum . before application of vacuum by pulling the syringe 
 plunger to the desired level , it is essential to draw sterile saline fluid into the cannula to 
 completely displace all air within the system . when pulled and twisted into the locked position , the 
 edge of the lock engages the side of the plunger permitting the physician to apply even and gentle 
 vacuum pressure while moving the cannula through the tumesced adipose layer . the internal type lock , 
 called a snap lock , universally fits a range of different syringe sizes from various manufacturers . anaerobic transfers ( luer - to - luer ) are utilized to facilitate loading of treatment syringes prior 
 to grafting procedures , and for optional use of additives to the grafts ( such as combining platelet 
 concentrates [ hd prp ] and adipose grafts into one syringe [ figure 5 ( bottom ) ] ) . they are also useful for 
 transferring the treatment mix into syringe sizes of physician preference for injection and avoiding 
 undesirable exposure of the harvested graft to air ( figure 
 8) . it is considered important to avoid excessive air exposure to grafts due to the potential 
 for contamination by airborne particles or pathogens . techniques described as helpful in free lipid 
 removal ( such as telfa  rolling  ; johnson & johnson , new brunswick , nj , 
 usa ) are vulnerable to such contamination . a controlled aliquot injector gun is useful for placement of controlled 0.5 cc aliquots of graft 
 into the prepared tunnels and locations . when additives are added to the adipose tissue graft , the 
 density of the injection material may be increased ( eg , use of hd prp plus thrombin to provide rapid 
 release of platelet - derived growth factors and signal proteins ) . this may result in the physician 
 requiring more force to inject into the tissue site , or , to encounter sudden and uneven distribution 
 of desired small aliquots of graft with prepared tunnels associated with adipose matrix density 
 within the graft itself . single - trigger pull provides exact volumes of solution to be placed with 
 less pressure required by the provider ( figure 
 9 ) . it is recommended that the area of donor and recipient sites be outlined with the patient in 
 upright position and using a skin - marking pencil . this area will then be prepped , draped , and 
 isolated to expose the thickest deposit of palpable fat tissues , and serve as a distribution pattern 
 for local anesthetic infiltration ( see figure 10 
 for an example ) . after marking , preparation , and sterile isolation of the donor area , an 1820 g beveled 
 needle is held such that the side edge is held vertical to the skin . this side edge is utilized to 
 create a small , vertical ( slit - like ) opening , extending through the epidermis and dermis into the 
 subdermal fat plane of the donor site . it is very important to avoid too large an opening , as 
 closed - syringe - system vacuum depends on maintaining a tight side wall opening to ensure even vacuum 
 application . use of stab incisions with scalpel blades of # 15 or # 11 sizes tends to 
 create larger than necessary or desired openings . the opening is made larger by selectively cutting 
 the dermal layer ( under the skin surface ) with the edge of the needle bevel . in large cannula sizes , use of a tapered stainless sharp trocar ( 3 mm ) is utilized to permit a snug fit of the aspiration 
 cannulas into the desired space for cannulas of > 3 mm od . this allows the introduction of the 
 multiport infiltration cannula through the skin and the subdermal fascia ( and in the anterior 
 abdominal wall example , should perforate and remain beneath scarpa s fascia ) ( figure 11 ) . following entry , the multiport infiltrator cannula should be passed in a horizontal fashion 
 within deeper aspects of the subcutaneous donor fat deposit , above the muscular layer , in a 
  spokes - of - a - wheel  pattern . pinching the skin - fat tissues may help in passing the 
 cannula . during movement of the infiltrating cannula , very slow injection of tumescent local 
 anesthesia fluid , during both entry and withdrawal strokes placement in 
 layers , beginning in the deeper aspects , and moving to more superficial levels is most effective . the importance of avoiding  pooling  of the tumescent local anesthesia is that 
 evenly distributing liquids improves the efficiency of harvest by providing a suspensory fluid 
 carrier for the adipose graft tissues , as well as excellent patient comfort . if tumescent fluids are 
 rapidly injected or injected only in a few locations within the fat deposits , pooling is often the 
 result . this pooling is recognized by the surgeon by the presence of larger amounts of infranatant 
 fluids seen after gravity decantation or centrifugation . in typical small volume grafting cases , use of tumescent local anesthesia in a range of 
 2030 cc is common . as a general guideline , it is important to place at least 1 cc of 
 tumescent local per 1 cc of anticipated graft to be harvested . for example , if the plan is to 
 aspirate 50 cc of adipose tissues , then use of 50 cc or greater fluid volume is distributed with the 
 adipose layer to provide the fluid carrier for extraction of the grafts . a common example of the 
 component of tumescent solution is to add the contents of a 50 cc multidose vial of local anesthetic 
 ( eg , 0.5% to 1.0% xylocaine with or without epinephrine [ 1:100,000 ] ) 
 to 1 l of sterile saline or balanced salt solution to provide sufficient tumescent fluid for 
 lipoaspiration . upon completion of even distribution of tumescent fluid within the proposed donor area , it is 
 recommended that repetitive passage of the infiltrating cannula throughout the tumesced donor area 
 ( termed  pre - tunneling  ) multiple times is important and very helpful to attain an 
 even and high quality graft . this more thoroughly distributes local anesthetic fluid for patient 
 comfort , and it also provides the needed  carrier fluid  to effectively suspend the 
 adipose tissues prior to harvesting with low pressure and minimal bleeding . this is a very important 
 step that will improve comfort during harvest , plus make extraction more efficient and result in 
 markedly less volume of the unwanted infranatant fluid layer . in small - volume transfers , most practitioners select a 20 cc luer syringe attached to the 
 harvesting microcannula with a mounted locking device . a very small volume ( 12 cc ) of 
 sterile 0.9% saline is drawn into the cannula to displace air from the system prior to 
 insertion into the harvest ( donor site ) . this is termed  charging  the syringe 
 device , and is necessary to eliminate all air from within the cannula and syringe , thereby avoiding 
 cavitation produced when using mechanical pump suction devices ( wall suction , detuned lipoaspiration 
 machines , etc ) . once the harvesting cannula is inserted into the locally tumesced and pre - tunneled adipose layer , 
 the syringe plunger is drawn to partial or full extension depending on desired vacuum pressure , and 
 twisted to provide a lock ,  if using an external type , or a ledge , which snaps to 
 hold the plunger in one of three positions . after application of vacuum , the physician is free to 
 move the harvesting cannula in a forward and back series of passages . it is important that these 
 passages are within the same plane and of the same pattern used during the placement of tumescent 
 solution . adipose return , at first , will be somewhat slower , as the graft tissue must be in 
 suspension to be able to be easily extracted . continuing these movements with vacuum applied will 
 yield adipose tissues with minimal bleeding in most patients . during the displacement of air , it is recommended that 4 inch by 4 inch sterile gauze should be 
 held over the harvesting tip openings to avoid spraying contents . occasionally , in cases where there 
 is a larger volume of infranatant fluid ( the layer immediately below the fat tissues ) , simply 
 express the liquid portion , reinsert the harvesting cannula into the harvest site , lock the plunger , 
 and gather more graft tissue . in the event of vacuum pressure loss during the harvesting process , it 
 is sometimes necessary to completely remove the harvesting cannula from the donor site , carefully 
 expressing all air from within the cannula . when this is completed , reinsertion of the cannula is 
 performed , and vacuum is re - established by withdrawing ( pulling ) the plunger and re - locking it . the yellow adipose grafts will quickly gravity separate from the underlying ( infranatant ) fluid , 
 resulting in the graft floating on top of the small fluid volume within the syringe system . test 
 tube / syringe stands or decanting stands are available to facilitate this initial gravity separation 
 ( figure 12a and b ) . remember that a common cause of increased infranatant volume in the decanted syringe is 
 inadequate distribution of local fluid , creating a pooling effect , which reduces efficiency of 
 adipose harvest . it is for this reason that extensive pre - tunneling is highly recommended prior to 
 application of any vacuum to the tissues . upon completion of aspiration of the desired graft volume , 
 the harvester cannula is removed , and the syringe end - capped and placed in a vertical position in a 
 standard test tube rack or directly mounted on a luer base of a decanting stand to allow gravity to 
 separate three distinct layers within the harvesting syringe . this usually requires decantation for 
 approximately 23 minutes or centrifugation ( discussed below ) at 1000 g for 
 34 minutes . after layer separation , practitioners expel the unwanted liquid layer 
 ( infranatant liquid ) on which the fat graft floats , which is expressed into sterile containers for 
 disposal . following removal of the infranatant fluid layer , if additional graft is needed , it is possible 
 to reinsert the harvester into the donor site to acquire more graft tissue ( adipocytes and 
 adipose - derived tissue stromal vascular fraction ) , additional decanting / centrifugation , followed by 
 anaerobic transfer loading of the graft into the treatment syringes of choice . when the desired volume is attained , lipoaspiration is completed , and the small opening is 
 covered with a triple antibiotic ointment and a coverlet ( bsn medical , charlotte , nc , usa ) bandage 
 ( adhesive on all sides ) . in the event of loss of vacuum ( evidenced by a hissing sound ) , the cannula is simply removed from 
 the adipose tissues , fluid advanced into the harvesting cannula to eliminate air from the system , 
 cannula reinserted , and plunger pulled open and locked to restart the aspiration process . ideally , the more thorough removal of unwanted fluids from the graft yields a more dense cellular 
 graft . it is important to effectively avoid the top layer ( free lipid layer ) from being included in 
 the final afg transplantation . in addition , the free lipid layer ( clear yellow liquid above the 
 harvested graft ) should be avoided , as it is an irritant and prolongs the healing of the graft 
 tissues in that it must be removed during the process by macrophages , etc , over time . the desire to produce the best compact grafts with minimal remaining fluid excess has led many to 
 include use of optimal centrifugation ( 1000 g for 34 minutes ) to 
 effectively compress the graft materials , help to more precisely separate fluid from harvested 
 graft , and facilitate removal of the unwanted free lipid component ( figure 13)39 
  several providers of tissue processing syringes offer the ability to more easily remove the 
 lipid layer from the graft tissues desired ( see figure 
 14 ) . whether by disk or weighted plunger , the free lipids are allowed to pass out of the 
 upper graft tissues and remain in the harvesting syringe itself . the ability to thoroughly remove 
 unnecessary fluid from the graft provides the surgeon an opportunity to avoid the mandatory 
  overcorrection  needed due to the amount of remaining carrier fluids within the 
 graft . further , use of a disposable separator disk , within the centrifuge processing syringe , 
 ensures more complete separation of the free lipid free lipid layer . its removal prevents the 
 resulting irritation and inflammatory response when removing the unwanted additional free lipid from 
 within a recipient site ( figure 14 ) . some practitioners perform one to two rinses with sterile saline to help reduce any residual 
 local anesthetic solution and red blood cells.40 after decantation and/or centrifugation steps , the graft preparation is ready for placement into 
 treatment syringes of physician s choice . it is important to use clear anaerobic transfers 
 ( luer - to - luer connectors ) to load the individual application syringes from the prepared , compressed 
 graft ( figure 15 ) . they are advantageous in that 
 they prevent external air exposure and potential for contamination and permit use of additives , such 
 as high - density platelet - rich plasma ( hd prp ) concentrates . use of hd prp concentrates are believed 
 to further enhance the healing , graft acceptance , and provide high concentrations of helpful growth 
 factors and signal proteins . treatment - standard luer syringes are then mounted with the desired injection cannulas , using 
 coated , single - port cannulas or the surgeons , preferred injector needles . use of blunt - tip , coated 
 cannulas is recommended , particularly within the facial recipient areas to lower the risk of 
 embolism caused by inadvertent injection of the adipose graft intravascularly . the injection 
 cannulas are available in a variety of lengths and diameters ( ranging from 0.9 mm to 1.47 mm od ) , to 
 accommodate the surgeon s preference and specific areas to be grafted . some elect to inject 
 with sharp needles ranging in size from 18 g to 25 g ( figure 16 ) . the typical graft recipient bed is prepared and developed by pre - tunneling to create a 
  potential  space , which is subsequently filled in small aliquots and in layers as 
 the injection cannula is being withdrawn . use of small , sterile 
 absorbing gauze dressing placed over the skin opening created to place the tumescent fluids into the 
 subdermal tissues . further , use of compression and closed cell , medical grade foam 
 ( tenderfoam ; t&n industries , san diego , ca , usa ) will eliminate or 
 minimize post - harvest bruising of the donor area . it is believed that use of foam and compression 
 reduces the lymphatic dispersion of small amounts of red blood cells or breakdown pigments remaining 
 within the surgical harvest site in the form of bruising or discoloration . firm compression of the 
 gauze and tenderfoam for 2448 hours is typically effective ( figure 17a and b ) . late 
 1980s , the ability to provide small- and large - volume liposuction via the closed - syringe system was 
 proven safe and more predictable and effective . by use of low vacuum pressure and avoiding air 
 within the system , the closed syringe system improved the results of liporeduction surgery and 
 autologous fat grafting success . this included removal of significantly larger volumes in a single 
 session and reduction of deposits within the superficial plane . the ability to safely and 
 effectively reduce unwanted fat deposits , coupled with improved skin redraping resulted in improved 
 surgical outcomes . besides volume implications , closed - syringe lipoaspiration launched the beginning 
 of more consistent and predictable autologous fat grafting procedures , with improved safety , 
 efficacy , and predictability within aesthetic surgical applications . structural fat grafting , 
 utilizing the exact techniques described herein , has been completed many thousands of times by many 
 cosmetic plastic surgeons ( figure 
 18a  j ) . scientific research has now provided important information to help explain the homeostatic and 
 transplant acceptance mechanisms accomplished by autologous fat . as appreciation of the biocellular 
 nature of the adipose tissue complex increases , the importance and value of the svf has gained major 
 attention.4 the authors believe that high - quality , dense cellular grafts are very important to the 
 effectiveness of structural fat grafting procedures . to that end , ensurance of complete layer 
 separation of the harvested grafts provided by optimized centrifugation is considered advantageous . in addition to use of such centrifugation , the use of a disposable processing syringe ( with 
 separator disk ) further ensures that free lipids are effectively removed during graft preparation . the adiprep system ( harvest - terumo , plymouth , ma , usa ) provides such a system using the 
 same centrifuge as used for producing hd prp concentrates ( smartprep ii ; harvest - terumo , 
 plymouth , ma , usa ) . use of closed systems for the harvesting and preparation of high - quality grafts 
 is considered of great importance . it is becoming mainstream knowledge that the actual transplanted mature adipocytes are gradually 
 lost , while serving an important role in their own replacement from attached near - terminally 
 differentiated cells ( pre - adipocytes ) . for several years , leading practitioners sought to achieve 
 pure , adipocyte grafts , without regard to the svf components and the effects of the local 
 microenvironment available with the recipient fat tissues . the importance of signaling and growth factor secretion associated with certain paracrine 
 effects , have changed the treatment paradigm of small volume structural grafting . while it is now 
 clear that the loss of most of the mature adipocytes is inevitable , it is appreciated that they 
 still play an important integral role , as they participate in stimulation of the microenvironment , 
 within the associated stromal vascular fraction . the near - terminally differentiated pre - adipocytes 
 are activated to initiate their final differentiation into adipocytes and contribution to lipid 
 metabolism and volume storage . further , it is now known that the cellular and extracellular matrix 
 ( ecm ) associated within the stromal elements serves an equally important role , serving as a living 
 bio - scaffold ( attachment to adipocytes and svf ) that is critical in encouraging available 
 multipotent cells to effectively activate and proliferate . both the afg and additive effects are 
 further enhanced in the surgically damaged recipient tissues through complex 
  signaling  mechanisms of autocrine and paracrine pathways in vivo.4,36 this paper presents a simple and effective method of lipoaspiration with which to harvest 
 adipocytes and their accompanying progenitor and stromal elements using a disposable , closed syringe 
 and microcannula system . effective for lipoaspiration of small and large volumes , the use of a 
 closed - syringe system and its accessories offers a full range of options by which to fulfill all afg 
 requirements . the safety and efficacy of using closed - syringe systems has evolved to the use of a 
 coated , disposable microcannula system specifically designed for use in structural autologous fat 
 harvest and transfer , which currently serves as the most complete and effective gold standard for 
 all closed - syringe systems .
objectivesprovide background for use of acquiring autologous adipose tissue as a tissue graft and source of 
 adult progenitor cells for use in cosmetic plastic surgery . 
 discuss the background and mechanisms of 
 action of closed syringe vacuum lipoaspiration , with emphasis on accessing adipose - derived 
 mesenchymal / stromal cells and the stromal vascular fraction ( svf ) for use in aesthetic , structural 
 reconstruction and regenerative applications . 
 explain a proven protocol for acquiring high - quality 
 autologous fat grafts ( afg ) with use of disposable , microcannula systems.designexplain the components and advantage of use of the patented super luer - lock and microcannulas 
 system for use with the closed - syringe system . 
 a sequential explanation of equipment selection for 
 minimally traumatic lipoaspiration in small volumes is presented , including use of blunt injection 
 cannulas to reduce risk of embolism.resultsthousands of afg have proven safe and efficacious for lipoaspiration techniques for large and 
 small structural fat grafting procedures . 
 the importance and advantages of gentle harvesting of the 
 adipose tissue complex has become very clear in the past 5 years . 
 the closed - syringe system offers a 
 minimally invasive , gentle system with which to mobilize subdermal fat tissues in a suspension form . 
 resulting total nuclear counting of undifferentiated cells of the adipose - derived -svf suggests that 
 the yield achieved is better than use of always - on , constant mechanical pump applied vacuum 
 systems.conclusionuse of a closed - syringe lipoaspiration system featuring disposable microcannulas offers a safe 
 and effective means of harvesting small volumes of nonmanipulated adipose tissues and its 
 accompanying progenitor cells within the svf . 
 closed syringes and microcannulas are available as 
 safe , sterile , disposable , compact systems for acquiring high - quality afg . 
 presented is a detailed , 
 step - by - step , proven protocol for performing quality autologous structural adipose 
 transplantation .