IE50322B1 - Serum-separating agent - Google Patents

Description

The present invention relates to a serum-separating agent for separating serum from other components of blood.

Serum-separating agents are known which operate by virtue of the specific gravity difference between the specific component required and other components of the blood to be fractionated, the agent being interposed between these components. For this purpose, the seurmseparating agent has a specific gravity intermediate those of the components, and it should be fluid during centri10 fugal separation, but it should be stable and not flow after the centrifugal separation is completed.

A known conventional serum-separating agent of this kind is a gel-like material consisting of silicone oil, silica and a gelling agent. However, this liquid separating agent gives problems in practice. For example, because its components lack compatibility, they are mechanically mixed to obtain a thixotropic gel by means of a gelling agent which accelerates the formation of hydrogen bonds between the silica particles (specific gravity adjusting agent). Coagulation develops due to this hydrogen bond formation growing as time elapses, resulting in phase separation and poor fluidity during centrifugal separation. To counter this, the addition of a surfactant for avoiding phase separation has been proposed, but this resulted in the further problem of hemolysis caused by the large amount of surfactant.

Serum-separating agents of the above composition have further drawbacks, such as their tendency to change in nature by cross-linking and other chemical changes upon γ-ray sterilisation after encapsulation of a suitable quantity in a blood collecting tube. This reduces the serum-separating capability, and delays blood coagulation and clot deposition by evaporation of low molecular components of the gel-like material, thus rendering the inner surface of the tube hydrophobic. Furthermore, these separating agents are fairly expensive.

Another known serua-separatir.g agent is a gel-like material of polyester base. This material is not always satisfactory in that it renders the inner surface of the tube water-repellent resulting in slower blood coagulation and clot deposition. Furthermore, serum-separating agents of this type smell unpleasant.

We have now devised a serum-separating agent by which many of the problems associated with prior known agents are reduced or overcome.

According to the present invention, there is provided a serum-separating agent having thixotropic characteristics for fractionating a serum component from a coagulated portion of blood by means of centrifugation said agent comprising as the major component by weight, an α-olefin-dialkylmaleate copolymer having a viscosity of 10,000 to 80,000 cp, (25°C) and an agent to control viscosity and specific gravity mixed therewith, for adjusting the specific gravity of the whole composition to 1.035 to 1.055.

The invention also provides a blood serumseparating tube for fractionating a serum component from a coagulated blood portion by means of centrifugation, and thereafter collecting the blood serum portion, which comprises a bottomed glass tube, a rubber plug sealing the glass tube, and a thixotropic blood serum-separsting agent as defined above, said blood serum-separatir.g agent being preliminarily disposed within the tube.

In the present invention, a typical example of the Λ-olefin-dialkylmaleate copolymer has the general f ormula: where R^ is an alkyl group having 2 to 56 carbon atoms which in the copolymer molecule, may either be the same throughout or may differ between repeating units; R- and R^ “re selected from methyl, ethyl, butyl, and 2-ethylhexyl groups; and n is an integer which allows the viscosity of said copolymer to be in the range of 10,000 to 80,000, more preferably 40,000 to 80,000 cp (25°C). When, as is described hereinafter, the agent also includes an α-olefin-dialkylmaleate copolymer wax of the above formula, n is chosen to provide a copolymer whose carbon atom number is in the range of 30 to 60.

Among the preferred copolymers of the above formula are those (a) wherein some R1 is an alkyl group of 4 carbon atoms, and the remainder is an alkyl 2q group of 6 carbon atoms; and (b) wherein some R^ is an alkyl group of 10 carbon atoms and the remainder is an alkyl group of 12 carbon atoms.

The rt-olefin-dialkylmaleate copolymer major 25 component of the agents of the invention is light yellow in colour, transparent, odourless, inert to blood, free of blood absorption, elution end so on, and stable for long periods of time. It allows the inner surface of the blood collecting tube to remain clean since it does 3G net produce any water-repellent material. It does not substantially change its chemical or physical nature upon sterilisation with gamma rays or the like. The specific gravity of this Ck-olefin-dialkylmaleate copolymer is selected to be between 1.00 and 1.038, preferably between 1.027 and 1.035· According to the present invention, the viscosity and specific gravity-adjusting agent may be selected from aliphatic amine derivatives such as aliphatic amine derivatives of smectite clay, and inorganic fine powder materials. The aliphatic amine derivatives of smectite clay may be aliphatic primary amine, aliphatic secondary amine or aliphatic tertiary amine or aliphatic quaternary amine derivatives of smectite clay. These amine derivatives are known. Among these derivatives, aliphatic quaternary amine derivatives of smectite clay are the most desirable, examples of which are aliphatic amine derivatives of smectite clay having Cg - such as Bentone 34, Bentone 38, Bentone 27, and Bentone 128 (quaternary ammonium salts of smectite clay, products of NL Industry Co. (Bentone is a trade mark.)).

The inorganic fine powder which may be used as the viscosity and specific gravity adjusting agent in the present invention may, for example, be selected from calcined silica and precipitated silica, and is added, taking account of the specific gravity and viscosity of the main component, in such an amount that the whole composition is gel-like and has the prescribed specific gravity.

A structure-forming agent may be used in the present invention for making and maintaining the gel state of the liquid separation agent. It can, for example, be used when it is considered difficult to make the whole composition gel-like without the inclusion of such an additive. For example, dimethylpolysiloxane-polyoxyalkylene copolymer (e.g. trade names SH-3771, SH-190, and SH-192 of Toray Silicone Co., Ltd.) or Carbitol (e.g. ethyldiglycol) and the like may be used. (Carbitol is a trade mark. ) The amount of structure-forming agent used may be decided taking into account the kinds of main component and viscosity-and specific gravity-adjusting agent, so that it is sufficient for gelation and is miscible with the other components. Generally, an amount of from 0.04 to 0.6 parts by weight relative to 100 parts by weight of the o<.-olefin-dialkylmaleate copolymer, is used.

In the present invention, in addition to the CColefin-dialkylmaleate copolymer, the viscosity and specific gravity adjusting agent and the structure-forming agent, a nonionic surfactant (e.g. polyoxyethylenehydrogenated castor oil monolaurate, polyoxyethylenehydrogenated castor oil tri-isostearate or the like) may be added as required, for example in an amount of 0.5 to 3.0 parts by weight of the total composition.

In order that the invention may be more fully understood, an embodiment thereof will now be described, by way of example only, with reference to the accompanying drawings, wherein: Fig. 1 is a perspective view illustrating the use of a liquid separating agent of the present invention as a blood serum separator encapsulated in a blood collection tube; and Fig. 2 is a sectional view of the blood collection tube shown in Figure 1 after centrifugal separation.

Compositions of the liquid separating agent of the present invention for serum separation are shown in the following Tables 1 and 2. In these Tables, the rt-olefin-dialkylmaleate copolymer (A) is an n-«-olefindimethylmaleate copolymer having an average molecular weight 3,000 - 4,000; specific gravity 1.027 - 1.035 (25°C); viscosity 40,000 - 70,000 cp (25°C), and consists of a combination of d-olefin components with carbon atom numbers 12 and 14, respectively; the copolymer (B) is an n-O.-olefin-dimethylmaleate copolymer having an average molecular weight 2,000 - 3,000; specific gravity 1.005 (28°C); viscosity 10,000 - 15,000 cp (28°C), and consists of a combination of aL-olefin components with carbon atom numbers 6 and 8, respectively; and the copolymer (C) is a wax made of an n-cf-olefin-dimethylmaleate copolymer of ei-olefin components with an average carbon atom number between 30 and 60. 30322 (examples of serum separation agent compositions) φ rH Λ Γΰ C< u ϋ D •rl < •H MJ w MJ •H • •H 0 □ □ QJ QJ Qj Ch ω • w JJ c JJ c QJ 0 u >. OJ Mi Mi Jj (0 JJ (0 Qj Ui a Qj V Qj <0 *0 10 c 1 © 1 O © a a. ε J «Η rH <-J ε 2 OJ \o *-«. r—J χ-* MJ t—I · • 0 o σ Q 0) Q | 0) N Eh to (*) OJ M J •H U JJ •H to ϋ 10 “ 9 • Q) . Ό o o O o QJ O rH O 0 I « rH U ϋ c Ml rJ ol rH Ol ϋ •H Qj •H J JJ >4 —* JJ IH u w Ml W <0 ω * o O to O QjO >, | | acd Oi (0 rH rH W 0) « τΗ QJ < ϋ σ» (0 (0 z k Z Φ Μ O Φ O JJ o © Φ CM > CM •rl I • > CM to CM - JJ rj m (0 H *H ϋ 2 MJ 2 Φ MJ 0 ε ϋ r- * * ΟΊ O * I o * Λ Ol CD fO * «tt « 4c » * ςΓ Γ. m oj 4c * « 00 OJ f—1 mj mj to 0 M 0 Ml Φ MJ Φ JJ Ό 4J 0 «ϋ ο 5 ϋ 5 3 0 3 to 0 Τ3 QjO Jj Qj 0 0 ι—1 Μ Φ Ml to J4 Φ Qj C Qj to C •Η τ' to •Η MJ ε ε MJ - * 9 rj 10 rH •r| 0) to \ o 9 ϋ cn •η σ» 0 t0 •r| rH E Ml rH O •Η O E JJ •rl KD (0 vo (0 to to JJ □ JJ >1 □ 9 •rl 9 Mi to •H 0 rj 0 to •H A A •ΗΛ c 0 to Λ «J Ml s A Qj 0) i—1 Qj >n 0 >1 JJ •rl 0 jj Mt JJ to to ·Η Ό -H 9 0 Ό > >1 ;> σ Ml >i to 43 to * Q) A Ml a M 4£ Dl c Cn c= r* 250322 Table 2 (examples of servm separation agent compositions) o in o kD ko O vd 0 • . >, m © rd 0 >1 £L Z 1 ·Η o Φ ω X £ 0 o in w to ϋ • z X w rd © Ud ο -H rd £· 0 •rl ιη Ό JJ > C ϋ rd flj CM 3 0 1 , Ό Pa — © 0 rd u Id >0 0, jz o JJ CM Φ ·. ε o Φ •rd © | Jj U co rd to o U *. a 3 r*. rd <0 a rd τι ι © O 0 jj • c μ2 O rd o 0 VO ε » o rd 0 rH © •id υ in rd o >1 φ jj O © Li C ·Η Φ o > JJ a to W •rd u Λ rd σ» ϋ •id to ϋ Ό •rd Φ >Md •Ρ (0 ’id iti Li O * £ 0 Φ * 0) E-< CL « cn to * 0 Ud Jj id 0 43 C Ό JJ Φ >1 JJ -rd σ» rC □ > <0 1 — 3 Φ · Π3 Id cn £ Q 0 Φ c Φ Eh υ ε •rl rd J CL >1 * CM * * 6 M rd rd >1 *—* rd 0 · r* * * 0 Φ +) a * Pa CL o\ O * Ud Φ rd Φ o HOO l © JJ 1 rd 43 >iU r* □ ¢4 CM £ Φ 43 (0 X r* o « Li >0 Ed 0 z cn oi co rd rd JJ 3 >lO 1 £ CM •rl id U jj Φ rd H S Φ 0) (fi (0 0 Φ Φ 0 to W rH 1 0 o Md 3 Φ (fi & >4 * >1 Ll Ll Li Id (fi * □ * O Φ Φ 3 JJ * * 2 * rd kD to to * * * ω * flj CM Next, the preparation method of the liquid separating agent of the present invention will be described.

The a-olefin-dialkylmaleate copolymer is for example, made by low polymerization of ethylene to obtain an η-α-olefin. This is then separated into fractions of carbon atom numbers, for example of 4, of 6, of 8 and 10, of 12 and 14, of 16 and 18, and of 30 to 60 by fraction·ating distillation. In accordance with the specific gravity of the liquid to be fractionated, these fractions may be used either singly or in combination, and a fraction with carbon atom numbers of 12 and 14, or of 6 and 8 is preferable for use in serum separation from the standpoints of viscosity and specific gravity. The selected fraction is subjected to copolymerization with a maleic diester in the conventional manner to obtain the desired product.

Using this α-olefin-dialkylmaleate copolymer with a viscosity between 10,000 and 80,000 cp, preferably between 40,000 and 80,000 cp (25°C) as the base, an aliphatic amine derivative of smectite clay, a viscosity and specific gravity adjusting agent such as fine silica powder, a structure-forming agent and a wax consisting of an a- olefin-dialkylmaleate copolymer (fear exanple, the above η-α-olefin having 30-60 carbon atoms) are added as required.

The mixture is kneaded using either a roll mill, a grinding mill, a planetary mixer or the like.

The liquid separating agent thus prepared should prefer25 ably has a viscosity, for use in serum separation for example, between 250,000 and 800,000 (25°C). Its specific gravity is between 1.035 and 1.055. All of the components in the previous tables are thixotropic, showing flowability upon application of a centrifugal force or the like and staying in the normal condition as a stable uniform gel otherwise. Furthermore, because this liquid separating agent does not contain such a gelling agent as used conventionally, it is not apt to coagulate in time as stated previously, nor does it foster phase separation or deterioration of buoyancy. Its specific gravity is comparatively high being close to that of blood serum, so that even if phase separation does take place the base layer, namely the layer of α-olefin-dialkyImaleate copolymer, does not float suspended in the serum.

Example As shown in Fig. 1, about 1.7 ml of each liquid separating agent composition 1 above (numbers 1 to 12) was put at the bottom of a 10 cc blood collecting tube 2. A polyester unwoven cloth 3 coated with 1 - 5 mg of diatomaceous earth (e.g., Caper Flattery Sand, trade name WG-200, of Kvoritsu Ceramic Materials Co., Ltd.), was then placed at a slant in ''or micro glass powder’ each blood collecting tube. Each tube was then stopped with a butyl rubber plug 4, and the tubes were placed under reduced pressure. Then a blood sample was placed in each blood collecting tube and allowed to stand for 7 to 8 minutes. As a result, the diatomaceous earth dispersed in the blood upon the introduction of the blood, and it accelerated blooc coagulation together with the nonwoven cloth 3. Adequate coagulation was thus attained within this short time. Each blood collecting tube was placed in a centrifuge for 10 minutes at 700 - 1,000 G, and the liquid separating agent compositions were stably distributed between the serum and the clot layers. This stage is shown in Fig. 2. Because the liquid separating agent is thixotropic and has a specific gravity between that of the blood serum 5 and that of the blood clots 6, it stays between the blood serum 5 and blood clot 6, forming a gel that separates these two layers.

Since the diatomaceous earth and the nonwoven cloth 3 have higher specific gravities, they were not included in the layer of blood serum 5. Thus, blood serum 5 obtained was of high purity with no entrainment of fibrin. This blood serum 5 was readily collected from the blood collecting tube by decantation or by suction.

Because the smectite clay particles are not light15 transmitring, their state of dispersion can easily be checked by means of a microscope etc., making quality control easier. Because the aliphatic amine derivatives of smectite clay increase viscosity, the structure-forming agent which was conventionally essential can be eliminated. The use of wax in the above example is especially effective for prevention of phase separation of the liquid separating agent. Because the α-olefin-dialkylmaleate copolymer is used as the main component in the present invention, the manufacturing cost is very low, being about one third the case with liquid silicone.

When being used as a serum separator, the liquid separating agent does not form a water-repellent film in the blood collecting tube by releasing water-repellent substances, and consequently does not cause delay of blood coagulation. Because the blood collecting tube is made of glass, it accelerates coagulation when contacting blood at the surface, so that it is necessary to keep the inner surface clean. Accordingly, compared with conventional liquid separating agents which have the drawback of forming a water-repellent film, the time needed for collecting blood serum is shortened. This is even more effective in combination with the use of the diatomaceous earth and nonwoven cloth. The time saving can amount to as much as 30 minutes.

When encapsulation in a blood collecting tube is performed, sterilization is desirable. In clinical tests, no chemical, or physical changes that cause adverse effects were found after applying gamma-ray sterilization.

In our copending Patent Specification No.5032.3 (divided from the present Application) we have described and claimed a blood-collecting tube containing therein a blood coagulation-promoting medium, which medium comprises a carrier having a specific gravity higher than that of blood cells, and a powdered blood coagulation-promoting agent supported on the carrier, and is held between the inner walls of the bloodcollecting tube in an intermediate portion of the tube.

Reference should be made to our said Patent Specification No. 50333 for further details.

Claims (14)

CLAIMS:
1. A serum-separating agent having thixotropic characteristics, for fractionating a serum component from a coagulated portion of blood by means of centrifugation, said agent comprising, as the major component by weight, an c<-olefin-dialkylnialeate copolymer having a viscosity of 10,000 to 80,000 cp (25°C) and an agent to control viscosity and specific gravity mixed therewith for adjusting the specific gravity of the whole composition to 1.035 to 1.055.
2. 2. An agent according to claim 1, wherein said ti-clefin-dialkylmaleate copolymer has the general formula: H C H R, 0 = C OR. '2 OR where R 1 is an alkyl group having 2 to 58 carbon atoms which, in the copolymer molecule, may either be the same throughout or may differ between repeating units; R^ and R^ are selected from methyl, ethyl, butyl and 2-ethylhexyl groups, and n is an integer.
3. 3. An agent according to claim 1 or 2, wherein said agent to control viscosity and specific gravity is at least one of the following: an aliphatic amine derivative of smectite clay with between 8 and 24 carbon atoms or an inorganic fine powder material.
4. 4. An agent according to claim 3, wherein said aliphatic amine derivative of smectite clay is a quaternary ammonium salt of smectite clay.
5. 5. An agent according to claim 3, wherein said 5 inorganic fine powder material is calcined silica or precipitated silica.
6. 6. An agent according to claim 2, wherein in the copolymer, some R 1 is an alkyl group of 10 carbon atoms and the remainder is an alkyl group of 12 carbon atoms. 10
7. 7. An agent according to claim 2, wherein in the copolymer, some is an alkyl group of k carbon atoms, and the remainder is an alkyl group of 6 carbon atoms.
8. 8. An agent according to any preceding claim, which also contains a structure-forming agent in an amount 15 between 0.04 and 0.6 parts by weight relative to 100 parts by weight of said tX-olefin-dialkylmaleate copolymer.
9. 9. An agent according to any preceding claim, further containing from 1.0 to 3.0% by weight, based on said major component, of a wax consisting of an cC-olefin2o dialkylmaleate copolymer whose o(,-olefin component has between 30 and 60 carbon atoms.
10. 10. An agent according to any preceding claim, further containing 0.5 to 3.0 parts by weight of a surfactant.
11. 11. A serum-separating agent according to claim 1, substantially as hereinbefore described.
12. 12. A blood serum-separating tube for fractionating a serum component from a coagulated blood portion by means of centrifugation , and thereafter collecting the blood serum portion, which comprises a bottomed glass tube, a rubber plug sealing the glass tube, and a thixotropic blood serum-separating agent as defined in any of claims 1 to 11, said blood serum-separating agent being preliminarily disposed within the tube.
13. 13. A tube according to claim 12, wherein a blood coagulation-promoting agent is disposed inside the tube.
14. 14. A blood serum-separating tube according to claim 12 substantially as hereinbefore described with reference to Figures 1 and 2, of the accompanying drawings.