CA2445204A1

CA2445204A1 - Method and device for collecting and preserving cells for analysis

Info

Publication number: CA2445204A1
Application number: CA002445204A
Authority: CA
Inventors: Wayne L. Ryan
Original assignee: Streck Laboratories, Inc.; Wayne L. Ryan; Streck, Inc.
Current assignee: Streck Laboratories Inc
Priority date: 2002-10-16
Filing date: 2003-10-16
Publication date: 2004-04-16
Anticipated expiration: 2023-10-16
Also published as: ES2784011T3; US11647743B2; DK1816461T3; AU2003254755B2; AU2003254755A1; CA2445204C; US10966421B2; US20160143268A1; US20100317107A1; US20210267191A1; US20040137417A1; EP1413874A1

Abstract

The claimed subject matter comprises a device to collect and preserve cells comprising of: (1) a collection container comprised of a tube having an open end and a closed end, a closure in the open end of the tube, a vacuum drawn to a predetermined level inside the container, and (2) compounds including an anticoagulant agent and a fixative agent, wherein the compounds are in a sufficient amount to preserve said cells' original morphology and antigenic sites without significant dilution of said cells, and thereby allowing said cells to be directly analyzed by a flow cytometer without further treatment. The claimed subject matter further comprises of a method of making a collection device for cells comprising of : (1) providing a tube having an open end and a closed end; (2) preloading compounds including: an anticoagulant agent, and a fixative agent into the tube, wherein the compounds are in a sufficient amount to preserve the cells' original morphology and antigenic sites without significant dilution of the cells, and thereby allowing the cells to be directly analyzed by a flow cytometer without further treatment; (3) inserting a closure into the open end of the tube; and (4) drawing a vacuum inside the tube to a predetermined level to form the collection device.

Description

~zs4z.ooss.N~susuo NIE'T~I~h A1~11~ IDIaJ~ICE FAR Ct~LLIt;CTIN'Iy A,l~tl) PS~~IhTG CEIL~,S Ft7I~
AI~T 1'SIS
This is a non-provisional of 1J.S, provisional application Iqo. 60/4I8,97$, filed October 16, 2002 the contents of which are incorporated herein by reference.
13ACKGIt~UN13 In biological and biochemical analysis, and related arts, it is open necessary to collect and preserve biological tissues (i.e,, celIs and cellular coynponents}, for useful periods of tune.
The collected and preserved calls are o$~e:n utilized in a wide variety of applications, including but not limited to instructional aids and the diagnosis and treatur~ent of diseases. For example, such cells are often utili2ed in histological, cytological, immunological, and proteinaceous studies and the like, ~larious methods are hnowri in the art for analyzing histological, cytological, immunological, and proteinaceous materials. For example, surface marker analysis has developed as a Laboratory tool, which is particularly useful for clinical diagnosis through the investigation of immutaodeficiency states, differentiation of cell types and development stages, and other cell processes. 'The expansion of uses for surface marker analysis has resulted in the use of flow cytometry and antibody probes to evaluate cellular properties.
'~lhile other means of assaying for surface marker analysis exist, flow cytometry provides rapid, objective and quantitative assessment ~f surface markers. FurChermore, even though the microscope is still the conventional means for examining preserved and stained biological materials, biological materials rnay also be examined with a flow cytometer. The flow cytorrleter is an important method for examining a plurality of cells in a brief tune.
Flaw cytometry and flow cytonteters are generally described in l~eran's tent, la'low cytometry in Clinical Diagnosis (1989). Flow eytometers operate in principle by tnultiparameter analysis of heterogeneous cell populations (or cellular compotaents) on a cell-by-cell basis. Flow cytometry allows biological and physical properties of cells and cellular components to be determined. In flow cytometry, cells in suspension are forced single file, via hydrodynamic focusing, through the path of a laser beam. lttteraction of the cells with the laser beam scatters some of the light and causes excitation and emission from fluorescent molecules present on the surface or interior c~f tlae cell. A series of lenses collect the scattered or emitted light and pass it _1_ Ft: i49093(fiR L.O11.DOC) 12G42.0065.NPtJS00 t~ a photomultiplier. Each photomultiplier trieasures a separate parameter.
Parameters measured iraclttde_ forward light scatter, r~thich measures relative particle size;
sade light scatter, which measures relative granularity or other internal structure; and fluorescence emission. The optical signals axe converted by a computex to a data display for analysis and interpretation. Cells collected and preserved using conventional methods and instruments generally require further dilution andlor tresxment before they can be analyzed by flow cytometry. Thus, it is desirable in fihe art to obtain a method and a collection device that allow the cells to be directly analyzed by flow cytornetry without further dilution and/or treatment. (There is need for a method to collect and transport human blood specimens for flow cytomctric analysis. Current methods are inadequate in that the samples have to be analyzed soon after collection.) The primary objective of tissue preservation is to provide as much structural detail of celDs and components thereof as possible. To do this, it is necessary to maintain the cells in their original unaltered morphology so that maximum cellulax detail may be observed.
With the clinical application of immtmostaining, there is alsa the requirement that antigens are not altered by the method of preservation. Thus, it is desirable in the art to obtain a method and a collection device that maintain the cells in their original unaltered morphology and preserve their antigenic sites.
The usual formulations for preservation of cells contain one or more agents, which react vigorously with the proteins of the cells to denature and insolubilize the components of tfe cell.
Typical of this type: of agent is picric acid, mercuric ions, formaldehyde and glutaraldehyde. In addition, some less toxic compounds can also be utilized which denature and stabilize the proteins such as acetic and formic acid. Unfortunately, the toxicity associated with such compounds renders their use less than satisfactory. For example, a 37%
solution of formaldehyde, the most common of these fixatives, is a noxious gas which is also toxic, flammable, and carcinogenic. Although effoxts are made when this chemical is used to protect workers and avoid contamination of the drainage system when disposed, these efforts are usually both expensive and inconvenient, and fixatives such as formaldehyde still present a danger to laboratory workers and health care professionals. Thus, it is highly desirable to develop a method and a collection device, which can preserve the ceDls in a low toxicity and non-flammable environment so that it can be used safely, effectively and conveniently in histologieal and other 9ltlC1li;S.
_zJ
H: w9093(B0. Go~LOpCj 12fr32.0065.AdPdISDD
For even easier handlixag, it is also desirable to develop a method and a collection device that allow transportatiozt (e.g., from the collection situ to the amal~rsis site) of the ceps in ambient temperature.
Sl<llVVl«MARY
The claimed subject matter addresses many of the challenges encountered when using conventional methods and instruments to collect and preserve cells by providing a method and a collection device that are capable of maintaining the calls in their original unaltered morphology;
preserving the cell antigenic sites; and allowing the cells to be transported at ambient temperature, to be handled in a low toxicity and non-flammable erwironment, and to be directly analyzed by :~o~r cytometry without further dilution andior treatment. The claimed subject matter more specifically relates to a method and a device that allow cells (e.g., whole blood, epithelial cells, spinal fluid, and the like.) to be collected and preserved for analysis and addresses many of the challenges encountered when using conventional methods and instruments. Specifically, the claimed subject matter describes a method and a collection device that (1} use a less toxic and non-flammable reagent for fixing and stabBlizing cells; (2) allow the cells to stay in their original unaltered morphology; (3) allow the cell antigenic sites to be preserved for a useful period of time; (4) allow the cells to be transported at ambient temperature; and/or (S) allow the cells to be directly analyzed by flow cytometry without further dilution andlor treatment.
The claimed subject matter includes a device to collect and preserve cells cornp~'ising of:
(1) a collection container comprised of a tuba having an opeet end and a closed end, a closure in the open end of the tube, a vacuum drawn to a predetermined level inside the container; and (2) compounds including an anticoagulant agent and a fixative agent selected from the group consisting of: diazolidinyl urea, imidazolidinyl urea, dimethoylol-5,5 dimethylhydantoin, dirnethylol urea, 2-bromo-2.-nitropropane-1,3-dial, oxaLOlidimes, sodiurri hydroxyrnethyl glyeinate, 5-hydroxymethoxymetlnyl-1-laze-3, 7-dioxabicyclo [3.3.0]octane, 5-hydroxymethyl-1-la~a-3,7~dioxabicyclo [3.3.0]octane, 5-hydroxypoly[methyleneaxy]methyl-1-laze.-3,T-dioxabicycto [3.3.0]octane, quaternary adamantine and combinations thereof, The claimed subject matter may optionally include polyaerylic acid or a suitable acid having a pH ranging from about one to about seven inside the tube. The compounds of the device must be in a e: savoo3~aH~~ov.ua~

t 2642.0065.NPUS00 sufficient amount to preserve the collected cells' original morphology and antigenic sites without significant dilution of the cells (i.e., in a volume that is not clir~acally significant), and thereby allowing the cells, stored with the compounds, to be directly analyzed by a flow cytometer.
The claimed subject matter also includes a method comprised of (1) providing a tube vith an open end and a closed end, (2) preloadimg the tube with compounds including: an anticoagulant agent, a fixative agent selected from the group consisting of diazolidi~ayl urea, imidazolidinyl urea, dimethoyloh5,5-dimethylhydantoin, dimethylol urea, ~-bromo-2-nitropropane-1,3-diol, oxazolidines, sodium hydroxymethyl glycinate, 5-hydroxymethoxymefihyl-1-laza-3,?-dioxabicyclo[3.3.0]octane, 5-hydroxymethyl-1-laza-3,7-dioxabicyclo[3.3.0]octane, S-hydroxypoly[methyleneoxy]methyl-1-1 aza-3,7-dioxabicyclo[3.3.0]octane, and quaternary adamantine, and optionally a polyacrylic acid or a suitable acid having a pIFI ranging from about one to about seven, wherein the compounds are in a s>rtfficient amount to preserve the collected cells' original morphology ttnd antigenic sites without significant dilution of the cells, and thereby allowing the cells, stored with the compounds, to be directly analyzed by a flown cytometer; inserting a closure into the open end of the tube; and drawing a vacuum to a predeternuned level inside the tube.
The anethod and device of the claimed subject anatter may also optionally include other art-disclosed components conventionally used in cell collection and analysis such as gauze, glove, tourniquet, lancet, needle, test strip (e.g., immunoassay ), alcohol swab, tube holder, additional cell collection tubes (with or witl'tout conventional cell analysis additives inside these tubes), adhesivE strip, syringe, glass or plastic strip, packaging means to store the desired components and the device, and packaging means to transport at least the collected and preserved cells stored in the device< °I he method of the claimed subject matter may also optionally include additional art disclosed methods and instruments used for cell analysis such as a ~lovv cytometer, a hematology analyzer, and other hematology instruments, etc.
BRIEF DESCRIPTION OF TIE DRAWINGS
FIG. 1. A cross-sectional illustration of an exemplary embodiment of the collection device of the claimed subject matter; and FIG. 2 A flow diagram illustrating a metlhod for making the collection device illustrated in the FIG. 1.
~q_ F1: 549099(DR LOl!.DOC) m64~.oass.~rusoo DESC~tIPTI~hI ~F °1C1F~E I~ItEFE ID E1VIIEObIt~l~lV~' The claimed subject matter can be satisfied by embodiments in many different forms, the drawings at2d the description herein describe in detail a preferred embodiment of the claimed subject matter. It is understood that the present disclosure is to be considered exemplary of the principles of the claimed subject mafiter attd is not intended to limit the claimed subject matter to the embadiment illustrated. The scope of the claimed subject matter is measured by the appended claims and their equivalents.
Turning now to the drawings, FIG. 1 shows a cross-sectional ilIustratior~ of a device 100 that incorporates a preferred embodiment of this claimed subject matter and can be used to Collect and preserve biological tissues such as Cells and cellular components for analysis. The device 100 H5 particularly useful it1 the collection of whole blood, but can be use to collect other types of bodily fluids and/or biological tissues (e.g., epithelial cells, bone marrov~r, spinal fluid and the like) including, without limitation, abnormal tissue samples such as leukernias, cancex tissue cancez~, and the like as long as the tissue saanples can be transformed into a cellular suspension.
The device 100 includes an evacuated collection container 10 comprised of (1) a tube 12 having an open end 1d and a Closed end 16; a closure (e.g., stopper) I8 in the open end of the tube I 2, and a predetermined level of vacuum (not shown) inside the container 10. It is preferred that the tube 1215 made of a transparent material such as glass or :plastic for bettee visibility.12 is also preferred that the tube 12 has an interior surface that is sterile said resists adherence to the cells 20 (not shown) during collection, storage, and analysis- The closure 18 is preferably puncturable by a needle and resealable allowing easy transfer of the cells 20 (e.g., the cells 20 from its host to the container 10 and f~'om the container 10 to another substrate if desired). It is also preferred that the closure 18 and the tube 12 together form a seal capable of maintaining a pressure differential between atmospheric pressure and a press~.ue less than atmospheric pressure ~.vithin the tube 18.
The 5lZe Of the COntalner 10 is not narrowly critical and is depealdent upon the cell sample volume that is desired to be collected arid preserved. For example, a typical size for the Container may hare an internal volume of between 100 gel to 10 ml. The cantainer 10 can be constructed using art-disclosed methods and is commercially available (e.g., Vacutainer Plus H: Sd9093(BR LOIf,DOC) t 2442.0065.1~1F'llSOll Plastic Tubes with Hemogard Closure available from Becton T~ickinson and Company located in Franklin Lakes, New Jersey; the evacuated sample collection tube described ia~
iJ'.~. Patent No.
5,860,937, which is incorporated by reference). ~f course, it should be understood that a wide range of changes and modifications can be made to the preferred embodiment described above for the container 10.
For preservation (e.g., fixation, stabilization and the like) of the cells 20, the device 100 further includes compounds 22 including an anticoagulatat agent ~4, a fixative agent 26 selected from the group cotlsisting of: diaaalidinyl ua'ea, imidazol:idinyl urea, dimethoylol-5,5-dimethylhydantoin, dimethylol urea, 2-bromo-2-nitropropane-1,3-diol, oxaizolidines, sodium hydroxymethyl glycinate, 5-hydroxymethaxymethyl-1-1 aza-3,7-dioxabicyclo [3.3.~)octane, 5-hydroxymethyl-1-laze-3,7-dioxabicyclo X3.3.0]octane, 5-l~ydroxypoly[methyleneoxy]methyl-1-la2a-3,7-dioxabicyclo [3.3.0]octane, surd quaternary adamantine, and optionally a polyacrylic acid 28 or any suitable acid having a plri ranging from about one to about seven, wherein the compounds are in a sufficient amount to preserve the collected cells' original morphology and antigenic sites without significant dilution of the cells 20, and thereby allowing the cells 2D, stored with the compounds 22, to be directly analyzed by a flow cytometer. It is preferred that the compounds 22 have been sterilized (e.g., by sterilizing hltratior~).
A suitable amount of any art-disclosed anticoagulant agent sash as ethylene diamine tetra acetic acid (E13T A) a.nd its salts, ethylene glycol tetra acetic acid (EC,TA) and its salts, hirudin, heparin, citric acid, salts of citric acid, oxalic acid, salts of oxalic;
acid, or mixtures thereof may be used. A preferred anticoagulant agent 24 is K3E1~'fA. The suitable amount of the anticoagulant agent 24 for the claimed subject matter is that e'ffecdve to prevent coagulation of the cells 20 (e.g., whole blood) without causing significant dilution of the cells 2~ (i.e., not clinically signif cant), and thereby allowizlg the cells 20, stored with the compounds 22, to be directly analyzed by a flow cytometer). For example, in a preferred embodiment, K,3EI33TA is the anticoagulant agent 24 and its concentration weight/volume is preferably less than about 0.3 glrrnl, more preferably less than about n.2 glrnl, and most preferably about less than about 0.15 g/ml.
Tlae preferred faxative agent 26 is a heterocyclic urea {e.f;., iazolidinyl urea {known as l~l.l), imidazolidinyl urea {known as IL~U) or a mixture thereof). The anost preferred fixative agent. is diazolidinyd urea. The suitable amount of tlm fixative t~~;era~e 26 vor the claimed subjGCv -6_ c~: sasogxnR c.on.ooct 12$A2.OD65.NP8JS00 matter is that effecti.'ve to fix or stabilize the cells 20 without causing significant dilution of the cells 20 (i.e., not clinically significant), and thereby allowing the cells 20, stored with the compounds 22, to be directly analyzed by a flow cytometer. For example, in a preferred embodiment, di~olidinyl urea is the fixative agent 26 and its concentration weighdvolume is preferably about less than about 1 glml, more preferably less than about 0, 75 g/anl, and most preferably less than about 0.5 g/m(concentration af' solution of ~7f1 before blv~c~ sample is odder) it is known that the acid 2$ stay rise signal to noise ratio during flow cytometry; and therefore, the acid 28 may be optionally added as one of tYae compounds 22 in the device 100.
'fhe preferred acid 28 is a polycarboxylic acid, and more preferably a polyacrylic acid with a molecular weight of 5,000. The suitable amount of 'tire acid 28 for the claimed subject matter is that effective to rise signal to noise ratio during flow cytornetry but without causing signiFcant dilution to the cells 20 (i.e., not clinically significant) so that the cells 20, stored with the compounds 22, can be directly analy;~ed by a flow cytometry. Fox e~cartlple, in a preferred en~bodixnent, polyacrylic acid with a molecular weight of 5,000 is included in the container 10.
Additional compounds may optionally be added as one of the compounds 22 in the de~rice 10t?. Such additional and optional compounds may inclLade: cell permeabilizang agents for substantially gaining access to intracellular analytes/epitopes and/o1r for lysing red blood cells; proteins that substantially protect the cells during processing andJor substantially reduce non-specific binding of probes; serwn/lipoproteins that substantially protect cells during processing and/or substantially reduce non-sp~cifla binding of probes; RNAse inhibitors which substantially inhibit digestion of RTdA andlor substantially maintain RhIA
integrity; nucleic acid stabilizers which substantially inhibit the degradation of r~uo3eic acids and nucleic acid containing compounds; amino acids f polypeptides which substantially enhance binding of probeslantibodies to epitopes and/or substantially increases the observable signal; fixatives which substantially preserve cell integrity especially for permieabilization agents, and rnay preserve some epitopes; anticoagulants which substantially decreases clotting of red blood cells, chelates calcium and / or may help maintain V~~C integrity/viability; protease inhibitors which substantially decreases degradation of protein epitopes; antioxidants/
reducing agents which substantially prevent hemolysis of red blood cells andl or substantially prevent oxidation of pCi7Cid>rS, rlClCil ~r 5albstagliitally nl~erttt~a~, Cpit~rp~rx; n~acl~iu ~fc:icf 6iyC~ tlWE ~cracs~sily servo ~o H:549093(RR L8fI.OQC) 12642.OOb5.NPUS00 label/identify nucleic acid; carbohydrates which substantially maintain cellular ar~tegrity andlor osmolarity; and, polyacrylic acids vahich substantially ettltaztce the binding of probes and/or antibodies to epitopes; and. /or substantially inCt°eases signal. Gne of skill in the art should be able to determine the usefulness attd quantities of such optional compounds by routine fiesting and knowledge of the art. within multiple specific embodiments the above additional and optional compounds may be moxe specifically include; Cell permeabilizing agents such as:
DhISO {Dimethyl Sulfoxide), Ethylene glycol, Polyethylene glycol, Glycerin, CellosolvEs {ethylene glycol dimethyl ether) (phenoxyeihanol), Triton ~ :100, Triton ~ 705 (non-ionic detergents), 1-methyl-2-pyrrolidinone, Tr~een 2~, Tween 40 (r~orc-ionic), Brij 35 (detergent), Polyoxyethylene ether {Polyox) , Sodium cholate, )Ethylene oxide polymers, MonensizR, Monactin, Pentachiorophenol, 2,4 dinitrophenol, saponRZt, SI3S (sodium dodecyl sulfate);
Proteins such as: biotin, Albumins {egg, bovine), Gelatin, and siraailar such compounds as should be known to one of skill in the art; RI~TAse inhibitors such as: human placenta derived RNAse inhibitor, and similar such compounds should be known to one of skill in the art; Nucleic acid stabilizexs such as: Guanidinium hydrochloride, Polycation.s such as Polyethylenitnine}, and similar such compounds as should be knowct to one of skill in the art; Amino acids/polypeptides such as: Glutamic acid, Glycine, Aspartic acid, and similar such compounds as shoc.bld be known to one of skill in the art; Fixatives such as: Aldehydes (form,aldehyde and glutaraldehyde), Aleohols (ethanol, methanol), and similar such compounds as should be known to one of skill in the art; Anticoagulants such as: EDTA {Ethylene Diamine Tetra acetic acid.), and similar such compounds as should be known to one of skill in the art; ACTS (Acid Citrate Dextrose), I-3feparin, and similar such compounds as should be known to one of skill in the art;
Protease Inhibitors such as: EDTA, PMSF (phenyl methyl sulfonyl fluoride), AEBSF (2~~iminoethyl benzene sulfonyl fluoride), and similar such compounds as should be known to one of skill in the art;
Antioxidants) Reducing agents such as: Trolox, a-tocopherol, B-rnercaptoethanol, and similar such compounds as should be known to one of skill it1 the art; Nucleic Acid Dyes such as: L~API
(Diamidino 2-phenylindole), I'ropidium Iodide, ~°luorescein diacetate, and similar such compounds as should be known to one of skill in the aat; Carbohydrates such as: Sugars (sucrose), cellulose, and similar such compounds as should be known to one of skill in the art. Tt should be appreciated that the above specific listings of compounds cvay contain a measure of _g_ r~: saoo93feR~~we.ooC7 12542.OObi.IVPUS00 overlap, rnhich recognizes the sometil~es-overlapping function of certain specific compounds.
One of skill in the art should understand and appreciate this aspect of the disclosure.
'The claimed subject matter allows the final composition 30 to be transported in ambient temperature. Thereafter, it is preferred that the fuial composition 30 be stored at temperature less than about ~0°C. The cells 20 stored in the final composition 30 have more than about 3 days, preferably more than about 5 days, more preferably more than about 7 days stability. The claimed subject matter allows the cells 20 stored in the final composition 30 to be directly analyzed by a flow cytometer without further dilution andlor treatment because the compounds 22 can preserve the cells 20 without significantly diluting the cells 20. Any significant dilution of the cells 20 is likely to cause error in flow eytometry measurements (e.g., lowering the lymphocytes' count). To avoid significant dilution, the compounds ZZ
(comprising of the anticoagulant agent 24, the fixative agent 2d, and optionally, the acid 28) are in concentrated forms, preferably in a ratio with the final cora~position 30 that is less than about 2:100, more preferably less than about 1.5:100, and most preferably less than about 1:100.
'The device 100 may be included in a kit of the claimed subject matter containing components 32 {not shown) conventionally used to collect and analyze the cells 30 such as alcohol swab, gauze, tube holder, tourniquet, glove, other cell collection tube (with or without conventional cell analysis additives inside such tube), needle (with hub, part of a syringe assembly including barrel and plunger, or with wings connected via a hub and tubing to another needle far delivery to the device 100 or other collection tubes), lancet, adhesirre strip, syringe, test strip (allowing the cells 20 to flow directly onto a glass or plastic strip containing reagents for cell analysis), glass or plastic strip containing reagents for cell analysis (e.g., immunoassay), packaging means (e.g., plastic bag, compatttl~entalized plastic enclosure, and the like) to store the desired components 32 and the device 10a, and packaging means go taansport the cells 20 stored in the device 100 after collection. It is preferred that the packaging means and any other components 32 that may become in physical contact with the Cells 20 be sterilized and the packaging means is constructed to maintain this sterile environane~nt.
Llr~like the t~rpical histological fixing agents, the fixative agent 26 of the cIaamed subject matter has extremely low toxicity. For example, toxicity studies comparing diazolidinyl urea with formaldehyde of the prior arC show the following:
FP:549097(8R L011.DOC) 264Z.Q065.~1PUS00 - lalhalatron Taercnal Toxicity ~'oxicity LD50 Formaldehyde 500 mg/kg 270 mg/kg g00 mglkg Diazolidinyl urea None 2000 mg/kg 270 mglkg This reduced toxicity makes disposal and handlitag less of a problem. In addition, since there is no inhalation toxicity, there are no badge detection devices required as there are fox formaldehyde.
Another advantage offered by the fixative agent 26 is the fact that it is not flammable and therefore does not present a fire hazard as do many of the prior art fixative agents.
The mechanism by which the fixative agent 2b provides the desired tissue and cell membrane stabilizatioc~ is not known for certaia~. It is believed that the fixative agent binds in some fashion to the cell membrane or tissue. This hypothesis is drawn because malty members of the active agent are Dcnown disinfectants, which kill bacteria by binding to ceh strlacture. This is not a full explanation of the mechanism responsible for the results of the clairraed subject matter since many other disinfectants such as rCATHON and OMr~DIfN~ fail to provide tissue and cell stabilizing effects.
The ability of the fixative agent 2h to preser~re antigenic sites is also not understood but it is probably due to a difference in the reaction between protei~rs and the fixative agent 26 compared to prior art preservatives such as formaldehyde, Formaldehyde cross-links with itself and proteins to obscure the a~ttigen. To determine if this is trim, diazolidinyl urea was added to the protein, albumin. Afier ineu6ation of the diazolidinyl urea and protein mlixture for 24 hours, disc-gel electrophoresis indicated no change in the rate of migration of the protein. ~lVhen this experiment is conducted with formaldehyde, a large number of insoluble proteins result and the electrophoretic migration is altered..
Referring to FIG. 2, a method of making the device 100 of the claimed subject matter is comprised of providing the tube 12 having the open end 14 and the closed end 16 (202). It is preferred that the tube 12 is sterile< The method is further comprised of preloading (i.e., introducing) the compounds 22 comprising of the anticoagulant agent 24, the fixative agent Z6, and Qptionally the acid 28 into the tube 12 using art~disclosed methods (204).
The types and amounts of the anticoagulant agent 2~+, the fixative agent ~o, as~d optionally, tt-ee aced 2a -1~-6i: SA9097(!iR 1.O11.DQG) 12642.~065.NPtJSQO
including the ratio between the compounds 22 and the final corraposition 30 are the same as described above for the device 100 of the claimed subject matter. It is preferred the compounds have been sterilized (e.g., by sterile filtration}. The method of the preloading step 204 may optionally include freeze drying the corllpounds in the tube 12. The: method 200 further includes inserting the closure 18 into the open end 14 of the tube 12 (2416). 'fhe method 200 further includes drawing a vacuum inside the tube 12 to a predetermined level (208 using art-disclosed methods. The amount of vacuum to be drawn is dependent upon the nature and volume of the cells desired to be collected and preserved. For example, for ~vhoie blood collection, the vacuum should be drawn to a level that allows khe pressure of the whole blood to cause it to flour into and fill the tube 12 to the desired level. The method 200 may optionally include providing the components 32 conventionally used to collect and analyze the cells 20. The components 32 are the same as for the device 100 of the claimed subject matter as described above.
The method may also optionally include collecting the cells 20 using art-disclosed methods {e.g., venipuncture, use of a lancet, etc.). It rnay optionally include screening the cells 20 using art-disclosed iz~stnnnents such as flow cytometers {cg., FACScan, FACSCaIibur by 1~D
and EPICS by 1'3eckman Coulter?, other hematology instruments (e.g., Ii3 by layer Corporation, the l3eckynan Coulter STKS or Gen-S Systems, the Abbott Cell-D;yn 4000 Hematology System, gayer ADVIA 12Q System, the Sysmex XE2I00 Systerr~, and the like. 'The screening of the cells may be for any purpose including, without littlitation, for I-II~i, I-1PV, hepatitis, leukemia, cancer, and the like; other art-disclosed screening sucl~a as imrnur~oassay, AIi~S panel, and the likes and sereenang by methods disclosed in commonly owned Ifnited States Patent I~o.
4,788,139 (Ryan titled "Platelet aggregation reagent, reagent coattainer and method oI
determining platelefi aggregation in EDTA-antieoagulated blood", which I5 hereby incorporated by reference. Cells 20 collected and preserved using the claimed subject a»atter may undergo histological study in a»y known conventional manner, such as through the use of paraffin sectioning equipment, staining, mounting on slides, or other common steps utilized prior to microscopic or other e~tamir~ation. 'The claimed subject matter thus provides a safe, convenient, and effective solution to collect and preserve cells for analysis.
It should be noted that the method and device a~f the claimed subject rrlatter may be used by those skilled in the art to preserve antigenic sites on or within cells (or components thereof) deraved from any source includlt~g nonmal t~looai, bane rrmc~rflvv, lynapl~, or solid tissu~s9 or aYaay -I I-E1:5A9097(DR L0ll.DOC) 12bd2.0065.NP1JS00 be derived from abnormal tissr~es such as leukertiias or solid tissue cancer's. Tlie claimed subject ar<atter may also be utilized with any cellular component or biological material that has at least one antigerlle site.
It should be noted that in preferred embodiments o;~ tlxe claimed subject matter cell clumping is prevented, light scattering properties axe preserved, antigenic sites are preserved, and nucleic acids may be analyzed.
The foregoing detailed description has discussed only a few of the many forms that the claimed subject matter can take. For this reason, this detailed desct~ptic~n is intended only by way of ihttstration. It is only the following claims, including all equivalents that are itltended to define the scope of the claimed subject matter.

H:549093(DR LOILDDC)

Claims

1. A method of making a collection device for cells comprising of:
providing a tube having an open end and a closed end;
preloading compounds including:
an anticoagulant agent, and a fixative agent into said tube, said fixative agent selected from the group consisting of:
diazolidinyl urea, imidazolidinyl urea, dimethoylol-5,5 dimethylhydantoin, dimethylol urea,

2-bromo-2-nitropropane-1,3-diol, oxazolidines, sodium hydroxymethyl glycinate, hydroxymethoxymethyl-1-laza-3, 7-dioxabicyclo [3.3.0]octane, 5-hydroxymethyl-1-laza-

3,7-dioxabicyclo [3.3.0]octane, 5-hydroxypoly[methyleneoxy]methyl-1-laza-3,7-dioxabicyclo [3.3.0]octane, quaternary adamantine and combinations thereof;
wherein said compounds are in a sufficient amount to preserve said cells' original morphology and antigenic sites without significant dilution of said cells, and thereby allowing said cells to be directly analyzed by a flow cytometer without further treatment;
inserting a closure into said open end of said tube; and drawing a vacuum inside said tube to a predetermined level to form said collection device.

2. The method of Claim 1, wherein said anticoagulant agent is selected from the group consisting of ethylene diamine tetra acetic acid (EDTA), salts of EDTA, ethylene glycol tetra acetic acid (EGTA), salts of EGTA, hirudin, heparin, citric acid, salts of citric acid, oxalic acid, salts of oxalic acid, and a combination thereof.

3. The method of Claim 1, wherein concentration of said fixative agent is less than about 1 g/ml.

4. The method of Claim 1, wherein concentration of said anticoagulant agent is less than about 0.3 g/ml.

5. The method of Claim 1, wherein said preloading step includes preloading a polyacylic acid into said tube.

6. The method of Claim 1, wherein ratio of said compounds and a final composition comprising said cells and said compounds is less than about 2.100.

7. The method of Claim 1, wherein said cells are selected from the group consisting of epithelial cells, bone marrow, spinal fluid, abnormal tissue sample in a cellular suspension, and a combination thereof.

8. The method of Claim 1, further comprising of sterilizing said compounds before said compounds are preloaded into said tube.

9. The method of Claim 1, further comprising of sterilizing at least all surface areas of said tube and said closure that can come into physical contact with said collected and preserved cells before said compounds are preloaded into said tube.

10. The method of Claim 1, further comprising of providing at least one component selected from the group consisting of an alcohol swab, a gauze, a tube holder, a tourniguet, a glove, other cell collection tube, a needle, a lancet, adhesive strip, syringe, a test strip, a strip containing reagents for cell analysis, a packaging means for storing said at least one component and said collection device to form a kit, and a packaging means for transporting said collection device.

11. The method of Claim 1, wherein said preloading step further comprises of freeze drying said compounds inside said tube.

12. The method of Claim 1, further comprising of screening said collected and preserved cells using an instrument selected from the group consisting of: a flow cytometer, a hematology analyzer, H3 by Bayer Corporation, the Beckman Coulter STKS System, the Beckman Coulter Gen-S System, the Abbott Cell-Dyn 4000 Hematology System, Bayer ADVIA 120 System, the Sysmex XE2100 System, and other analyzers and a combination thereof.

13. The method of Claim 1, further comprising of screening said collected and preserved cells for a purpose selected from the group consisting of HIV, HPV, hepatitis, leukemia, cancer, and a combination thereof.

14. A collection device for cells comprising of:
a collection container comprising of a tube having an open end and a closed end, a closure in said open end of said tube, a vacuum drawn to a predetermined level inside said container; and compounds including an anticoagulant agent and a fixative agent selected from the group consisting of: diazolidinyl urea, imidazolidinyl urea, dimethoylol-5,5 dimethylhydantoin, dimethylol urea, 2-bromo-2-nitropropane-1,3-diol, oxazolidines, sodium hydroxymethyl glycinate 5-hydroxymethoxymethyl-1-laza-3, 7-dioxabicyclo [3.3.0]octane, 5-hydroxymethyl-1-laza-3,7-dioxabicyclo [3.3.0]octane, 5-hydroxypoly[methyleneoxy]methyl-1-laza-3,7-dioxabicyclo [3.3.0]octane, quaternary adamantine and combinations thereof, inside said tube, wherein said compounds are in a sufficient amount to preserve said cells' original morphology and antigenic sites without significant dilution of said cells, and thereby allowing said cells to be directly analyzed by a flow cytometer without further treatment.

15. The device of Claim 14, wherein said anticoagulant agent is selected from the group consisting of ethylene diamine tetra acetic acid (EDTA), salts of EDTA, ethylene glycol tetra acetic acid (EGTA), salts of EGTA, hirudin, heparin, citric acid, salts of citric acid, oxalic acid, salts of oxalic acid, and a combination thereof.

16. The device of Claim 14, wherein concentration of said fixative agent is less than about 1 g/ml.

17. The device of Claim 14, wherein concentration of said anticoagulant agent is less than about 0.3 g/ml.

18. The device of Claim 14, wherein compounds further includes a polyacrylic acid.

19. The device of Claim 14, wherein ratio of said compounds and a final composition comprising said cells and said compounds is less than about 2:100.

20. The device of Claim 14, wherein said cells are selected from the group consisting of epithelial cells, bone marrow, spinal fluid, abnormal tissue sample in a cellular suspension, and a combination thereof.

21. The device of Claim 14, wherein said compounds are sterile.

22. The device of Claim 14, wherein at least all surface areas of said tube and said closure that can come into physical contact with said cells are sterile.

23. A kit comprising the device of Claim 14 and further comprising of at least one component selected from the group consisting of an alcohol swab, a gauze, a tube holder, a tourniquet, a glove, other cell collection tube, a needle, a lancet, adhesive strip, syringe, a test strip, a strip containing reagents for cell analysis, a packaging means for storing said at least one component and said collection device to form a kit, and a packaging means for transporting said collection device.

24. The device of Claim 14 wherein compounds contained in said tube are freeze dried.

25. The device of Claim 14 wherein said device is used along with an instrument selected from the group consisting of: a flow cytometer, a hematology analyzer, H3 by Bayer Corporation, the Beckman Coulter STKS System, the Beckman Coulter Gen-S
System, the Abbott Cell-Dyn 4000 Hematology System, Bayer ADVIA 120 System, the Sysmex System, and a combination thereof to provide screening of said cells.

26. The device of claim 14 wherein said device is used in screening said cells for a purpose selected from the group consisting of: HIV, HPV, hepatitis, leukemia, cancer, and a combination thereof.

27. A method for transporting cells for analysis, said method comprising:
providing a collection container under vacuum; and containing compounds including an anticoagulant agent and a fixative agent selected from the group consisting of:
diazolidinyl urea, imidazolidinyl urea, dimethoylol-5,5 dimethylhydantoin, dimethylol urea, 2-bromo-2.-nitropropane-1,3-diol, oxazolidines, sodium hydroxymethyl glycinate, 5-hydroxymethoxymethyl-1-laza-3, 7-dioxabicyclo [3.3.0]octane, 5-hydroxymethyl-laza-3,7-dioxahicyclo [3.3.0]octane, 5-hydroxypoly[methyleneoxy]methyl-1-laza-3,7-dioxabicyclo [33.0]octane, quaternary adamantine and combinations thereof inside said tube, wherein said compounds are in a sufficient amount to preserve said cells' original morphology and antigenic sites without significant dilution of said cells, collecting said cells in said collection container and;
transporting said cells for analysis.