HRP940754A2 - Medium for the storage and suspension of cells, particularly erythrocites - Google Patents
Medium for the storage and suspension of cells, particularly erythrocites Download PDFInfo
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- HRP940754A2 HRP940754A2 HRP-2228/90A HRP940754A HRP940754A2 HR P940754 A2 HRP940754 A2 HR P940754A2 HR P940754 A HRP940754 A HR P940754A HR P940754 A2 HRP940754 A2 HR P940754A2
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- cells
- agent according
- erythrocytes
- agent
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- 239000000725 suspension Substances 0.000 title claims description 11
- 238000003860 storage Methods 0.000 title description 4
- 210000003743 erythrocyte Anatomy 0.000 claims description 35
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 27
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- 239000008103 glucose Substances 0.000 claims description 15
- 210000004027 cell Anatomy 0.000 claims description 14
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 13
- 239000011780 sodium chloride Substances 0.000 claims description 13
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 12
- 239000003795 chemical substances by application Substances 0.000 claims description 12
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 10
- 239000002738 chelating agent Substances 0.000 claims description 9
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical group OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 8
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- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 7
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- ICFJFFQQTFMIBG-UHFFFAOYSA-N phenformin Chemical compound NC(=N)NC(=N)NCCC1=CC=CC=C1 ICFJFFQQTFMIBG-UHFFFAOYSA-N 0.000 description 1
- DTBNBXWJWCWCIK-UHFFFAOYSA-N phosphoenolpyruvic acid Chemical class OC(=O)C(=C)OP(O)(O)=O DTBNBXWJWCWCIK-UHFFFAOYSA-N 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229960003975 potassium Drugs 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000001508 potassium citrate Substances 0.000 description 1
- 229960002635 potassium citrate Drugs 0.000 description 1
- QEEAPRPFLLJWCF-UHFFFAOYSA-K potassium citrate (anhydrous) Chemical compound [K+].[K+].[K+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O QEEAPRPFLLJWCF-UHFFFAOYSA-K 0.000 description 1
- 235000011082 potassium citrates Nutrition 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 150000003212 purines Chemical class 0.000 description 1
- 229960001285 quercetin Drugs 0.000 description 1
- 235000005875 quercetin Nutrition 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 235000011083 sodium citrates Nutrition 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 229960004793 sucrose Drugs 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
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- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
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Description
Izum se odnosi na sredstvo za konzerviranje, odlaganje i suspendiranje stanica, naročito eritrocita, koje je pored ostalih stručnjacima poznatih tvari, kao npr. elektrolita i šećera, sadrži sredstvo za tvorbu helata za visevalentne metalne ione. The invention relates to an agent for preserving, depositing and suspending cells, especially erythrocytes, which, in addition to other substances known to experts, such as electrolytes and sugar, contains a chelating agent for multivalent metal ions.
Za proizvodnju reagenasa za određivanje određenih parametara (npr. komplementno određivanje) potrebni su stabilizirani eritrociti izolirani iz nezgrušane čiste krvi. Stabilized erythrocytes isolated from non-coagulated pure blood are required for the production of reagents for the determination of certain parameters (e.g. complement determination).
U okvirima terapije krvnih sastojaka iz čiste krvi (konzerve krvi) proizvode se odvajanjem plazme koncentrati eritrocita. Ti koncentrati danas se mnogo primjenjuju u terapiji, npr. kod postojeće anemije, npr. kad postoji indikacija za dovođenje eritrocita. Da bi se namjestilo koncentrat eritrocita na fiziološki prihvatljivu viskoznost, treba dodati otopinu za razređenje s kojom se mogu unijeti tvari potrebe također i za povišenje sposobnosti skladištenja. Within the framework of blood component therapy, erythrocyte concentrates are produced by separating plasma from pure blood (canned blood). Today, these concentrates are widely used in therapy, for example, in existing anemia, for example, when there is an indication for bringing erythrocytes. In order to adjust the erythrocyte concentrate to a physiologically acceptable viscosity, a diluting solution should be added with which necessary substances can also be introduced to increase the storage capacity.
Za suspendiranje i odlaganje stanica, naročito eritrocita, prikladna su različita sredstva. Tako se npr. u WO 81/02239 opisuje otopinu koja sadrži adenin, manitol, glukozu ili fruktozu. U WO 86/00809 opisuje se otopinu koja sadrži adenin, manitol, glukozu, kalijev citrat i amonijev klorid, odnosno amonijev acetat. U WO 87/04072 opisana je otopina koja može sadržavati L-amino kiseline ili njihove derivate, masne kiseline ili njihove derivate, intermedijarne proizvode glikolize kao fosfenol piruvat i nlegove analoge, nukleozide kao adenozin ili gvanozin, adenozin monofosfat i iz njega izvedene proizvode, glikogen, acetil-CoA i iz njega izvedene proizvode, alantoin, 4-etiloksaloacetat, feniletil bigvanidid i analoge, kvercetin, sulfat kobalta, sulfat nikla, magnezijev klorid, te razne inhibitore di-fosfo-glicerat-fosfataze kao dodatke standardnim komponentama antikoagulanta, šećerni alkohol, glukozu i natrijev klorid. U WO 89/02274 opisana je otopina koja sadrži glukozu, natrijev klorid, kalijev klorid, kalcijev klorid, magnezijev sulfat, natrijev fosfat, natrijev citrat i natrijev bikarbonat. U US-PS 4 267 269 opisuje se otopinu koja pored adenina sadrži šećerni alkohol manit, glukozu ili fruktozu i natrijev klorid. US-PS 4 356 172 opisuje otopinu adenina ili inozina, saharozu ili laktozu, te limunsku kiselinu ili jednu njenu sol. US-PS 4 704 352 opisuje otopinu koja za stabilizaciju krvnih stanica ima pH ispod 7,0, te sadrži adenin manit, dekstrozu, natrijev klorid i L-askorbat-2-fosfat. US-PS 4 769 318 opisuje stabilizaciju i aktivaciju krvi dobivene transfuzijom pomoću derivata fesfoenol piruvata kao i adenin, saharid (saharoza, galaktoza ili manitol) limunsku kiselinu, odnosno njenu natrijevu sol (takoder i EP 0 275 198). Different means are suitable for suspending and depositing cells, especially erythrocytes. Thus, for example, WO 81/02239 describes a solution containing adenine, mannitol, glucose or fructose. WO 86/00809 describes a solution containing adenine, mannitol, glucose, potassium citrate and ammonium chloride, i.e. ammonium acetate. WO 87/04072 describes a solution that can contain L-amino acids or their derivatives, fatty acids or their derivatives, intermediate products of glycolysis such as phosphophenol pyruvate and nleg analogues, nucleosides such as adenosine or guanosine, adenosine monophosphate and products derived from it, glycogen , acetyl-CoA and products derived from it, allantoin, 4-ethyloxaloacetate, phenylethyl biguanide and analogs, quercetin, cobalt sulfate, nickel sulfate, magnesium chloride, and various di-phospho-glycerate-phosphatase inhibitors as additives to standard components of anticoagulants, sugar alcohol , glucose and sodium chloride. WO 89/02274 describes a solution containing glucose, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, sodium phosphate, sodium citrate and sodium bicarbonate. US-PS 4,267,269 describes a solution that, in addition to adenine, contains the sugar alcohol mannitol, glucose or fructose, and sodium chloride. US-PS 4 356 172 describes a solution of adenine or inosine, sucrose or lactose, and citric acid or one of its salts. US-PS 4 704 352 describes a solution which for stabilizing blood cells has a pH below 7.0 and contains adenine mannitol, dextrose, sodium chloride and L-ascorbate-2-phosphate. US-PS 4 769 318 describes the stabilization and activation of blood obtained by transfusion using derivatives of phosphoenol pyruvate as well as adenine, saccharide (sucrose, galactose or mannitol) citric acid, or its sodium salt (also EP 0 275 198).
EP 0 099 315 opisuje fosfatno puferiranu otopinu koja sadrži adenin, glukozu, saharozu i natrijev klorid. EP 0 099 315 describes a phosphate buffered solution containing adenine, glucose, sucrose and sodium chloride.
EP 0 100 419 opisuje fosfatno puferiranu otopinu koja sadrži adenin i/ili gvanozin, šećerni alkohol (sorbit ili ksilit), glukozu ili fruktozu, natrijev klorid kao i koloide. EP 0 301 250 opisuje fosfatno puferiranu otopinu koja sadrži adenin i/ili gvanozin, šećerni alkohol (sorbit, manit ili ksilit), glukozu, ili fruktozu, natrijev klorid kao i koloide (vidi također DE-A37 22 984). DE 3 220 232 opisuje postupak i otopine za stabiliziranje krvnih pločica pomoću jod-acetamida, imuno-octene kiseline i bakteriostatičkog sredstva. DE-A-3 225 408 opisuje otopinu koja sadrži adenin ili gvanizin, šećerni alkohol (sorbit ili ksilit), glukozu ili fruktozu. Nadalje, Heaton et al. (u British Jouraal of Haematology 57, 467-478, 1984) opisuju otopinu (ADSOL(R)) za stabiliziranje eritrocita, koja -se uglavnom sastoji od adenina, manitola, dekstroze i natrijevog klorida. Također je poznato stabilizacijsko djelovanje humano toksičkog omekšivača dietilheksilftalata (Horowitz B. et al. Vox Sang. 48, 150-155, 1985). EP 0 100 419 describes a phosphate-buffered solution containing adenine and/or guanosine, a sugar alcohol (sorbitol or xylitol), glucose or fructose, sodium chloride as well as colloids. EP 0 301 250 describes a phosphate-buffered solution containing adenine and/or guanosine, a sugar alcohol (sorbitol, mannitol or xylitol), glucose, or fructose, sodium chloride as well as colloids (see also DE-A37 22 984). DE 3 220 232 describes a process and solutions for stabilizing blood platelets using iodine-acetamide, immuno-acetic acid and a bacteriostatic agent. DE-A-3 225 408 describes a solution containing adenine or guanisine, a sugar alcohol (sorbitol or xylitol), glucose or fructose. Furthermore, Heaton et al. (in British Journal of Haematology 57, 467-478, 1984) describe a solution (ADSOL(R)) for stabilizing erythrocytes, which consists mainly of adenine, mannitol, dextrose and sodium chloride. The stabilizing action of the human toxic softener diethylhexyl phthalate is also known (Horowitz B. et al. Vox Sang. 48, 150-155, 1985).
Dodatkom takovih suspenzijskih otopina koncentratima eritrocita poboljšava se doduše omjer preživljavanja i time sposobnost uskladištenja eritrocita, ali ta su poboljšanja bila neznatna, da bi bila dovoljna za visoke zahtjeve npr. za minimalna vremena trajanja jednog dijagnostika. Time se također nije mogao proizvesti reagens gotov za upotrebu, budući da se namještena koncentracija eritrocita kao i svojstva eritrocita mijenjaju tijekom skladištenja. The addition of such suspension solutions to erythrocyte concentrates does indeed improve the survival ratio and thus the ability to store erythrocytes, but these improvements were insignificant, to be sufficient for high requirements, for example, for the minimum duration of one diagnostic. This also could not produce a ready-to-use reagent, since the set concentration of erythrocytes as well as the properties of erythrocytes change during storage.
Izum se stoga temelji na zadatku da se poboljša omjer preživljavanja i time sposobnost skladištenja stanica, odnosno omjer hemolize eritrocita. The invention is therefore based on the task of improving the survival ratio and thus the storage capacity of the cells, i.e. the hemolysis ratio of erythrocytes.
Predloženi izum odnosi se na sredstvo za suspendiranje, konzervaciju i skladištenje biološki aktivnih stanica ili dijelova stanica, naročito eritrocita, pri čemu sredstvo, pored ostalih stručnjacima poznatih tvari, kao npr. antikoagulanata, elektrolita, šećera i šećernih alkohola, sadrži sredstvo za tvorbu helata za viševalentne metalne ione u puferiranoj, vodenoj otopini. Na iznenadujući način je pronađeno da stanice, naročito eritrociti, pokazuju značajno povišen omjer preživljavanja i jako smanjenje omjera hemolize kad se skladište u takovoj otopini. The proposed invention relates to an agent for suspending, preserving and storing biologically active cells or parts of cells, especially erythrocytes, wherein the agent, in addition to other substances known to experts, such as anticoagulants, electrolytes, sugar and sugar alcohols, contains a chelating agent for multivalent metal ions in a buffered, aqueous solution. Surprisingly, it was found that cells, especially erythrocytes, show a significantly increased survival ratio and a strong reduction in hemolysis ratio when stored in such a solution.
Sredstva za tvorbu helata za viševalentne ione su stručnjacima poznati spojevi. Upotrebljavaju se ponajprije sredstva za tvorbu helata za dvovalentne metalne ione, a posebnu prednost se daje upotrebi etilendiamin-tetraacetata (EDTA), etilenglikol-bis-aminoetileter-tetraacetata (EGTA) ili oksalnoj kiselini, a posve posebnu prednost ima EGTA. Chelating agents for multivalent ions are compounds known to those skilled in the art. Chelating agents for divalent metal ions are primarily used, and special preference is given to the use of ethylenediamine-tetraacetate (EDTA), ethylene glycol-bis-aminoethylether-tetraacetate (EGTA) or oxalic acid, and EGTA is especially preferred.
Sredstva za tvorbu helata stavljaju se koncentracijom od 0 do 100 mmola/1, ponajprije od 0,1 do 50 mmolova/1 i s posebnom prednošću od 2 do 10 mmolova/1. Kao elektroliti naročito dolaze u obzir natrijeve, kalijeve i magnezijeve soli u obliku klorida, sulfata ili citrata. Chelating agents are added at a concentration of 0 to 100 mmol/1, preferably from 0.1 to 50 mmol/1 and with a particular advantage from 2 to 10 mmol/1. As electrolytes, sodium, potassium and magnesium salts in the form of chloride, sulfate or citrate are particularly suitable.
Kao šećeri ponajprije se upotrebljavaju saharoza i fruktoza, a kao šećerni alkohol manit, glukoza i sorbit. Sucrose and fructose are primarily used as sugars, and mannitol, glucose and sorbitol are used as sugar alcohols.
Kao daljnji dodaci u obzir dolaze koloidi, pri čemu prednost imaju želatina i poliželin. As further additives, colloids come into consideration, with gelatin and polygelin being preferred.
Kao antikoagulanti prednost imaju heparin i -citrat. Postojanost se nadalje može poboljsati dodatkom antibiotika neškodljivog za stanice kao npr. Kathona DP® (Rohm & Haas) ili kromamfenikola. The preferred anticoagulants are heparin and -citrate. The stability can be further improved by the addition of a cell-friendly antibiotic such as Kathona DP® (Rohm & Haas) or chromamphenicol.
Sredstvo prema izumu može nadalje sadržavati stručnjacima poznate tvari iz razreda nukleozida (npr. adenozin ili gvanozin), iz razreda purinskih baza (npr. adenin) ili intermedijarne proizvode ili supstrate glikolize. The agent according to the invention may further contain substances known to experts from the class of nucleosides (eg adenosine or guanosine), from the class of purine bases (eg adenine) or intermediate products or substrates of glycolysis.
Ostali dodaci, koji se upotrebljavaju u poznatim sredstvima, mogu se također kombinirati sa sredstvom prema izumu. Other additives, which are used in known agents, can also be combined with the agent according to the invention.
Najpovolinija svojstva otopine te vrste mogu se još poboljšati ako se doda adenozin trifosfat (ATP) i/ili kao druga puferska tvar doda se natrijeva sol fosforne kiseline. The most favorable properties of this type of solution can be further improved if adenosine triphosphate (ATP) is added and/or the sodium salt of phosphoric acid is added as a second buffer substance.
Puferske tvari su sve fiziološki podnošljive puferske tvari, a ponajprije se upotrebljava HEPES ili fosfat topiv u vodi. Posve naročitu prednost imaju sredstva u skladu s primjerima 2 i 3. Buffer substances are all physiologically tolerable buffer substances, and HEPES or water-soluble phosphate is preferred. Means in accordance with examples 2 and 3 are particularly preferred.
Tipična otopina gotova za upotrebu priprema se primjerice kako slijedi. Za litru otopine u destiliranoj vodi otopi se 0 do 100 mmolova glukoze, 20 do 200 mmolova natrijevog klorida, 0 do 50 mmolova kalijevog klorida, 0 do 10 mmolova magnezijevog klorida, 2 do 100 rnmolova HEPES-a, 0 do 100 mmolova natrijevog fosfata, 0,1 do 50 mmolova etilendiamin-tetraacetata (EDTA), etilenglikol-bis-amino-etileter-tetraacetata (EGTA) ili oksalne kiseline, ponajprije EGTA, 0 do 20 000 jedinica heparima, 0 do 5 g želatine, 0 do 50 mmolova ATP-a i 0 do 1 g Kathona ili kloramfenikola, pH 5,0 do 9,0, ponajprije 6,5 do 7,5, a posebno korisno 6,8 kod upotrebe Kathona i 7,4 kod upotrebe kloramfenikola. Posebno je korisno pripremiti otopinu tako da je otprilike izotonična. A typical ready-to-use solution is prepared, for example, as follows. For one-liter solutions, dissolve 0 to 100 mmol of glucose, 20 to 200 mmol of sodium chloride, 0 to 50 mmol of potassium chloride, 0 to 10 mmol of magnesium chloride, 2 to 100 rnmol of HEPES, 0 to 100 mmol of sodium phosphate, 0.1 to 50 mmol ethylenediamine tetraacetate (EDTA), ethylene glycol bis-aminoethylether tetraacetate (EGTA) or oxalic acid, preferably EGTA, 0 to 20,000 units of heparim, 0 to 5 g gelatin, 0 to 50 mmol ATP -a and 0 to 1 g of Kathon or chloramphenicol, pH 5.0 to 9.0, preferably 6.5 to 7.5, and especially useful 6.8 when using Kathon and 7.4 when using chloramphenicol. It is particularly useful to prepare the solution so that it is approximately isotonic.
Za primjenu te otopine postupa se obično tako da se priprenti čistu krv humanog ili animalnog podrijetla stabiliziranu sa citratom ili heparinom. Čista krv se centrifugira i plazma, uključiv granični sloj do eritrocita, se odbaci. Na kraju se eritrociti isperu dva puta u puferiranoj otopini bez ATP-a. Isprani eritrociti se konačno dovedu na koncentraciju s potpunim puferom i odlažu se pri 2 do 8°C ili se odmah upotrebljavaju. For the application of this solution, it is usually done by adding pure blood of human or animal origin stabilized with citrate or heparin. Clean blood is centrifuged and the plasma, including the boundary layer to the erythrocytes, is discarded. Finally, the erythrocytes are washed twice in a buffered solution without ATP. Washed erythrocytes are finally concentrated with complete buffer and stored at 2 to 8°C or used immediately.
Slijedeći primjeri služe za objašnjenje izuma i ni na koji način ga ne ograničavaju. The following examples serve to explain the invention and do not limit it in any way.
Primjer 1 Example 1
Priprava suspenzije eritrocita za dijagnostičke svrhe Preparation of erythrocyte suspension for diagnostic purposes
Pod sterilnim uvjetima uzme se 300 ml krvi iz ovce u citratni predložak kao antikoagulant. Za odvajanje plazme krv se centrifugira 15 minuta kod 1000 x g. Plazma se odsisa zajedno s graničnim slojem staničnog taloga i eritrociti se preuzmu u jednaki volumen (u odnosu na volumen taloga eritrocita) pufera za ispiranje (sredstvo za stabilizaciju prema primjeru 3 bez EGTA, ATP i heparina) i pomiješaju. Zatim se stanice odvoje centrifugiranjem kao gore i ispiranje se ponovi još jednom. Za namještanje pravilne koncentracije eritrocita oni se ponovno suspendiraju u istom volumenu sredstva za stabiliziranje prema primjeru 3 i za razredenje od 1:100 u puferu za stabiliziranje odredi se ekstinkciju kod 578 nm. Iz te vrijednosti izračuna se ciljano razređenje i eritrociti se razrijede na odgovarajuću vrijednost. Konačno se suspendirani eritrociti pune u obrocima. Utvrđeno je da se na taj način pripremljena suspenzija može skladištiti 2 godine pri 2-8°C, bez pojave relevantne hemolize ili bilo kakovog gubitka s obzirom na dijagnostičku upotrebu (tablica 1). Under sterile conditions, 300 ml of blood from a sheep is taken into a citrate template as an anticoagulant. To separate the plasma, the blood is centrifuged for 15 minutes at 1000 x g. The plasma is aspirated together with the boundary layer of the cell sediment and the erythrocytes are collected in an equal volume (relative to the volume of the erythrocyte sediment) of the washing buffer (stabilizer according to example 3 without EGTA, ATP and heparin) and mix. The cells are then separated by centrifugation as above and the wash is repeated once more. To adjust the correct concentration of erythrocytes, they are resuspended in the same volume of the stabilizing agent according to example 3 and for a dilution of 1:100 in the stabilizing buffer, the extinction at 578 nm is determined. From this value, the target dilution is calculated and the erythrocytes are diluted to the appropriate value. Finally, the suspended erythrocytes are filled in portions. It was found that the suspension prepared in this way can be stored for 2 years at 2-8°C, without the appearance of relevant hemolysis or any loss with regard to diagnostic use (table 1).
[image] [image]
Primjer 2 Example 2
Priprava suspenzijske otopine Preparation of suspension solution
Odvaže se 80 mmolova glukoze, 150 mmolova natrijevog klorida, 10 rnmolova kalijevog klorida, 0,5 mmolova magnezijevog klorida, 10 mmolova HEPES-a, 10 mmolova EDTA, 1 g želatine i 2 mmola ATP-a i otopi u 900 ml destilirane vode. Zatim se doda 10 000 jedinica heparina kao i 0,05% Kathona. Kad se pH vrijednost namjesti na 6,8 nadolije se s destiliranom vodom na 1 litru. Na kraju se otoplnu sterilno filtrira pod pritiskom kroz membranski filter širine pora od 0,2 μm. Weigh out 80 mmol of glucose, 150 mmol of sodium chloride, 10 rnmol of potassium chloride, 0.5 mmol of magnesium chloride, 10 mmol of HEPES, 10 mmol of EDTA, 1 g of gelatin and 2 mmol of ATP and dissolve in 900 ml of distilled water. Then 10,000 units of heparin as well as 0.05% Kathon are added. When the pH value is adjusted to 6.8, it is topped up with distilled water to 1 liter. Finally, the solvent is sterile filtered under pressure through a membrane filter with a pore width of 0.2 μm.
Primjer 3 Example 3
Priprava modificirane suspenzijske otopine Preparation of modified suspension solution
Odvaže se 80 mmolova glukoze, 150 mmolova natrijevog klorida, 10 mmolova kalijevog klorida, 0,5 mmolova magnezijevog klorida, 10 mmolova HEPES-a, 40 mmolova NaH2PO4, 2 mmola EDTA, 1 g želatine i 2 mmola ATP-a i otopi u 900 ml destilirane vode. Zatim se doda 10 000 jedinica heparina kao i 0,5 g Kathona. Kad se pH vrijednost namjesti na 6,8 nadolije se B destiliranom vodom na 1 litru. Na kraju se otopinu sterilno filtrira pod pritiskom kroz membranski filter širine pora od 0,2 μm. . 80 mmol of glucose, 150 mmol of sodium chloride, 10 mmol of potassium chloride, 0.5 mmol of magnesium chloride, 10 mmol of HEPES, 40 mmol of NaH2PO4, 2 mmol of EDTA, 1 g of gelatin and 2 mmol of ATP are weighed and dissolved in 900 ml of distilled water. Then 10,000 units of heparin as well as 0.5 g of Kathon are added. When the pH value is adjusted to 6.8, B is poured with distilled water to 1 liter. Finally, the solution is sterile filtered under pressure through a membrane filter with a pore width of 0.2 μm. .
Slika 3 prikazuje vremenski tijek hemolize tijekom 2 godine pri 4°C u usporedbi s otopinom prema stanju tehnike (80 mmolova/1 glukoze, 150 mmolova/1 NaCl, 10 mmolova/1 HEPES-a, 10 mmolova/1 KCl, 0,5 ntmola/1 adenina, 0,1% želatine) (x-x); prema stanju tehnike i sa helatorom EDTA (+-+); s otopinom iz primjera 2 (*-*), te s otopinom iz primjera 3 (□-□). Figure 3 shows the time course of hemolysis over 2 years at 4°C compared to a prior art solution (80 mmol/l glucose, 150 mmol/l NaCl, 10 mmol/l HEPES, 10 mmol/l KCl, 0.5 ntmol/1 adenine, 0.1% gelatin) (x-x); according to the state of the art and with the chelator EDTA (+-+); with the solution from example 2 (*-*), and with the solution from example 3 (□-□).
Primjer 4 Example 4
Provedba određivanja aktivnosti C4 pomoću suspenzije eritrocita Implementation of determination of C4 activity using erythrocyte suspension
Priprava reagensa: Preparation of the reagent:
0,5 ml suspenzije eritrocita pomiješa se s 5 ml otopine koja sadrži antitijela prema eritrocitima i zagrije se na 37°C. Time je reagens gotov za upotrebu. Provedba određivanja: 0.5 ml of the erythrocyte suspension is mixed with 5 ml of a solution containing antibodies against erythrocytes and heated to 37°C. The reagent is now ready for use. Implementation of the determination:
10 μl uzorka pomiješa se sa 100 μl C4 deficitne plazme. K tome se s pipetom doda 1 ml reagensa i odredi se vrijeme koje prođe, do pada ekstinkcije kod 578 nm za 0,1 E zbog lize eritrocita. Usporedba dobivenog vremena s vrijednostima iz prethodno analogno utvrđenih baždarnih krivulja daje sadržaj funkcijski aktivnih C4. 10 μl of the sample is mixed with 100 μl of C4 deficient plasma. To this, 1 ml of reagent is added with a pipette and the time that elapses is determined until the extinction at 578 nm drops by 0.1 E due to the lysis of erythrocytes. Comparison of the obtained time with the values from the previously analogically determined bázdar curves gives the content of functionally active C4.
Slika 4 prikazuje ovisnost vremena lize stabiliziranih eritrocita o aktivnosti C4 u uzorku. Figure 4 shows the dependence of the lysis time of stabilized erythrocytes on the activity of C4 in the sample.
Primjer 5 Example 5
Provedba određivanja aktivnosti cjelokupnog komplementa pomoću suspenzije eritrocita Implementation of the determination of the activity of the entire complement using a suspension of erythrocytes
Priprava reagensa: Preparation of the reagent:
0,5 ml suspenzije eritrocita pomiješa se s 5 ml otopine koja sadrži antitijela prema eritrocitima i zagrije se na 37°C. Time je reagens gotov za upotrebu. Provedba određivanja: 0.5 ml of the erythrocyte suspension is mixed with 5 ml of a solution containing antibodies against erythrocytes and heated to 37°C. The reagent is now ready for use. Implementation of the determination:
K 100 μl uzorka s pipetom se doda 1 ml reagensa i mjeri se vrijeme koje prođe do pada ekstinkcije kod 578 nm za 0,1 E zbog lize eritrocita. Usporedba dobivenog vremena s vrijednostima iz prethodno analogno utvrđenih baždarnih krivulja, iz razređenja tekuće plazme s izotoničkom otopinom natrijevog klorida, daje komplementnu aktivnost uzorka. 1 ml of reagent is added to 100 μl of the sample with a pipette and the time that elapses until the extinction at 578 nm falls by 0.1 E due to the lysis of erythrocytes is measured. Comparison of the obtained time with the values from the previously determined analogue curves, from the dilution of the liquid plasma with an isotonic sodium chloride solution, gives the complementary activity of the sample.
Slika 1 prikazuje ovisnost vremena lize stabiliziranih eritrocita o cjelokupnoj komplementnoj aktivnosti uzorka. Figure 1 shows the dependence of the lysis time of stabilized erythrocytes on the overall complement activity of the sample.
Claims (8)
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE3938907A DE3938907C2 (en) | 1989-11-24 | 1989-11-24 | Means for storing and suspending cells, in particular erythrocytes |
| YU222890A YU47648B (en) | 1989-11-24 | 1990-11-22 | THE MEANS OF SUSPENSION, PRESERVATION AND STORAGE OF CELLS, IN THE FIRST LINE OF ERITROCITES AND ITS APPLICATION |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| HRP940754A2 true HRP940754A2 (en) | 1997-06-30 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| HRP-2228/90A HRP940754A2 (en) | 1989-11-24 | 1994-10-25 | Medium for the storage and suspension of cells, particularly erythrocites |
Country Status (1)
| Country | Link |
|---|---|
| HR (1) | HRP940754A2 (en) |
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1994
- 1994-10-25 HR HRP-2228/90A patent/HRP940754A2/en not_active Application Discontinuation
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