US3146163A

US3146163A - Apparatus for separating certain components from blood

Info

Publication number: US3146163A
Application number: US168165A
Authority: US
Inventors: John H Brewer
Original assignee: Individual
Current assignee: Individual
Priority date: 1962-01-23
Filing date: 1962-01-23
Publication date: 1964-08-25
Anticipated expiration: 1981-08-25
Also published as: DE1498577A1; CH412405A; FR1361738A; GB969741A; DE1498577B2

Description

Aug. 25, 1964 J. H. BREWER APPARATUS FOR SEPARATING CERTAIN COMPONENTS FROM BLOOD 2 Sheets-Sheet 1 Filed Jan. 23, 1962 k RM 2 W Aug. 25, 1964 J. H. BREWER 3,146,163

APPARATUS FOR SEPARATING CERTAIN COMPONENTS FROM BLOOD 2 Sheets-Sheet 2 Filed Jan. 25, 1962 FIG. 5

FIG. 6

INVENTOR. .rmw xx B/P'h EF United States Patent Office 3,145,163 Patented Aug. 25, 1964 3,146,163 APPARATUS F913 SEPARATHNG CERTAIN CDMPUNENTS FRGM BLOOD John H. Brewer, 4-25 Oak Lane, Towson 4, Mid. Filed Jan. 23, 1962, Ser. No. 168,165 8 Claims. (Cl. 167-74) This invention relates to an improved apparatus and method for separating liquid containing components such as plasma and serum from other components in a small sample of blood.

It is a particular object of the present invention to provide a practical, rapid, simple and inexpensive device and method for use in separating plasma and serum from small samples of blood such as can be obtained from finger punctures.

Most immunological, serological and chemical tests and reactions performed with blood require the use of plasma or serum. Heretofore, this has generally been obtained from the arm vein and the cells are separated from the serum or plasma by centrifuging. With the improvement of micro-chemical techniques, many of these tests can be performed with very small samples of blood obtained from puncturing the finger or ear lobe. This has generally been accomplished by means of a lancet which is used to draw two or three drops of blood from which a usable amount of serum or plasma may be collected. The separation of the serum or plasma from the small samples of blood has generally been accomplished by causing the blood to flow into a capillary tube and then centrifuging the blood in the capillary tube to separate the blood cells from the liquid containing components thereof. In both of these procedures the centrifuging is time consuming and, in addition, requires special centrifuging equipment, electric current and other laboratory facilities.

It is an object of the present invention to overcome the difliculties and disadvantages heretofore encountered and to provide an improved apparatus and method for separating the liquid containing components such as plasma and serum from a small blood sample and which is simple to use, which requires a minimum amount of time and which does not require the use of centrifuging apparatus or laboratory equipment.

A further object is the provision of an improved apparatus and method of the above character in which the apparatus is small and compact and is relatively inexpensive and disposable thereby eliminating the problems of contamination and sterilization.

My invention contemplates the provision of a relatively thin compact member such as a slide having an upper surface formed with a recess having a liquid impervious surface depressed below the surface of the card. The recess has a relatively larger principal recess portion for initially receiving the blood sample and it also has a relatively smaller auxiliary recess portion communicating with the principal recess portion. The principal recess portion contains an agent preferably in the form of a coating which includes a hemagglutinin for causing certain of the components such as the blood cells to separate from the remainder of the blood. The blood sample is placed in the principal recess portion and is stirred around therein to cause the blood to thoroughly mix with the hemagglutinin until the said components including the blood cells separate from the remainder of the blood and precipitate in the principal recess portion. Thereafter, the card is tilted to cause the liquid containing components of the blood to flow into the auxiliary recess portion while the separated components remain in the principal recess portion. The serum or plasma thus collected in the auxiliary recess portion can be used at once or can be dried and thereafter reconstituted to be used for making the desired immunological, serological or chemical tests or reactions.

In the accompanying drawing:

FIG. 1 is a perspective view of apparatus in the form of a slide embodying my invention to be used in separating the liquid containing components from a small sample of blood;

FIG. 2 is a fragmentary cross-sectional view in the direction of the arrows on the line 2 -2 of FIG. 1;

FIG. 3 is a perspective view illustrating my apparatus in horizontal position overlapping the edge of a supporting surface and serving to illustrate the first and second steps of my method;

FIG. 4 is a similiar perspective view showing my ap paratus tilted at an angle on the same supporting surface and serving to illustrate the third step of my method;

FIG. 5 is a top plan view of a kit serving as a commercial package for my apparatus and showing the envelope of the kit in sealed condition; and

FIG. 6 is an exploded view of the kit showing the envelope broken open and revealing the contents thereof including my improved apparatus and other auxiliary equipment which may be conveniently used therewith.

Referring to the drawings, and more particularly to FIGS. 1 and 2 thereof, my improved apparatus comprises a relatively compact, thin, flat member 10, preferably in the form of a slide or card having a relatively flat upper surface 11 with a recess 12 formed therein.

The recess has a liquid impervious surface depressed below the upper surface 11 of the card. The recess is provided with a relatively larger principal recess portion 13 disposed nearer to one end of the card and also with a relatively smaller auxiliary recess portion 14 communicating therewith and disposed nearer to the opposite end of the card.

The slide is made either of a suitable material which is itself inert to the blood components and is impervious to the liquid components of blood or it is made of any other suitable material having applied thereto or at least to the recess 12 a surface or coating which is inert to the blood components and impervious to the liquid components of blood. Thus, the slide may be made of a suitable plastic such as polypropylene, polyethylene, polystyrene or a polyvinyl plastic having a plasticizer which is inert to the blood components. Preferably, however, the slide is made of a suitable fiberboard such as paperboard or newsprint board having a sizing such as a casein sizing. Either the entire upper surface or at least the recess portion thereof has applied thereto a coating inert to the blood components and impervious to the liquid components of blood such as polyethylene, polypropylene, polystyrene or a polyvinyl plastic of the type indicated.

The recess 12 is of sufficient depth to confine the initial blood sample within the principal recess portion 13 when the slide is in horizontal position and to confine the liquid containing components thereof within the auxiliary recess portion when the slide is tilted at an angle as hereinafter described.

The area of the principal recess portion is sufficiently large so that the cohesion of the blood will confine the initial blood sample within the principal portion without flowing into the auxiliary portion when the card is disposed in horizontal position. The area of the auxiliary recess portion is sufficiently large to hold the liquid containing components of the blood when the card or slide is tilted at an angle as hereinafter described.

The present apparatus is intended for use with relatively small blood samples such as are obtained by puncturing a finger or an ear lobe with a lancet. For this purpose, two to five drops of blood generally serve quite satisfactorily. I have found that in using my ap- J paratus in connection with small samples of approximately this size satisfactory results are obtained if the recess is at least approximately 0.1 mm. in depth and if the area of the principal recess portion is at least approximately 300 mm. and if the area of the auxiliary recess portion is at least approximately 75 mm?.

The initial blood sample of two to five drops, preferably approximately three drops, is placed in the principal recess portion 13 and in the principal recess portion certain of the components are caused to separate as by agglutination, coagulation or precipitation. For this purpose, I provide agents within the principal recess portion for causing the separation, agglutination, clotting or precipitation of certain of the components. These agents are preferably applied in the form of a coating to the surface of the principal recess portion 13.

For certain purposes it is desirable to collect serum which consists of the liquid containing components of blood free of the cells and fibrinogen. For other purposes, it is desirable to collect plasma which consists of the liquid containing components free of the cells but including the fibrinogen. In the collection of either serum or plasma the coating should include a suitable homagglutinin such as phytohemagglutinin or that produced by immunological procedures. Where it is desired to collect serum, then the coating should also preferably include a suitable coagulant or clotting agent to accelerate the clotting of the fibrinogen, such as thrombin, or this may be accomplished more slowly by rapid stirring. Where it is desired to collect plasma, then the coating should in clude in addition to the hemagglutinin a suitable anticoagulant or anti-clotting agent such as heparin, dicou marol, sodium citrate, potassium oxalate, ammonium oxalate, lithium oxalate, sodium oxalate or ethylenediaminitetraacetate.

As the hemagglutinin, I prefer to employ a substantially pure phytohemagglutinin, such as those made from the seeds of the Leguminosae. A very satisfactory phytohemagglutinin is the one obtained from kidney beans. A hemagglutinin which causes the precipitation of the globulin fraction should not be used. For a recess of the size indicated for use with a small blood sample of the size indicated, i.e., between two and five drops, I have found that satisfactory results are obtained by using at least approximately 0.3 mg., preferably 0.5 mg., in the coating in the principal recess portion. The coating may be applied by mixing approximately 0.5 mg. of phytohemagglutinin in 0.1 ml. of physiological saline and then spreading it evenly over the surface of the principal recess portion and permitting it to dry.

In the collection of plasma, as the anti-clotting agent or anticoagulant I prefer to employ heparin for use with a blood sample of the size indicated. In a recess of the indicated size I have found that satisfactory results are obtained if I use at least approximately 0.02 mg., preferably approximately 0.1 mg, of heparin in the coating. The heparin may be mixed with the physiological saline along with the phytohemagglutinin and applied to the surface of the principal recess portion.

Where my apparatus is used in collecting serum, I prefer to employ thrombin as the coagulating or clotting agent. I have found that satisfactory results are obtained by using at least 10 units, preferably 12 units, in the coating. The units indicated are N.I.H. units established by the National Institute of Health. The thrombin may be mixed along with the phytohemagglutinin in the physiological saline and applied to the surface of the principal recess portion.

Slides of the type indicated may be packaged in sterile form in sealed containers or envelopes of the type indicated in FIGS. and 6. The envelope is inert and impervious to the coating materials employed in the recess 12 and is also impervious to microorganisms and contaminants. It may be made of any desired inert and impervious sheet materials. Thus, in the illustrated embodiment the envelope 20 is formed of two sheets of metal foil, preferably of aluminum foil, having a thermoplastic lining such as a lining of polyethylene, polypropylene or polyvinylchloride. The card or slide 10 is placed between the two sheets of metal foil with the thermoplastic lining arranged in confronting relationship and the envelope is then heat sealed around the edges thereof as indicated at 21. In order to obtain access to the slide and remove it from the package, it is simply necessary to tear or sever the envelope to expose the contents as indicated in FIG. 6.

The package is preferably prepared in the form of a complete kit and it contains the additional equipment required for using the slide in collecting a small blood sample and separating the serum or plasma therefrom. Thus, as indicated in FIG. 6, the envelope contains a small disposable lancet 22, a stirring rod which may be similar to a toothpick indicated at 23 and a wire loop shown at 24. Instead of a wire loop, a capillary or pipette or other device for removing liquid may be supplied. All of this equipment is packaged within the envelope in sterile form. The lancet may be separately packaged within the envelope in a smaller sealed envelope 25 which may be similar to the envelope 20. It will be appreciated that the inner envelope 25 is torn or severed so as to expose the lancet 22.

It will be seen that the entire kit is of compact form being no more than approximately three inches by six inches in size and with the slide being no larger than approximately two inches by 5 inches. Thus, a number of the kits can be readily transported by a physician or a serologist for the purpose of conducting immunological, serological and chemical blood tests and reactions in the oflice or in the field. No other facilities, such as glassware, laboratory equipment or centrifuges, are necessary in order to collect the small blood sample and separate the serum or plasma therefrom.

The following examples give representative coating procedures and coatings to be used in preparing the slides for the collection of blood and the separation of plasma.

Example 1 Prepare the coating by mixing together the following ingredients:

Substantially pure phytohemagglutinin mg 0.5 Heparin mg 0.1 Physiological saline rnl 0.1

After the materials have been thoroughly mixed to gether, spread the mixture evenly over the surface of the principal recess portion and then permit the coating to dry, leaving a mixture of the phytohemagglutinin, the heparin and the salts uniformly distributed over the principal recess portion. The slide may then be packaged along with a suitable lancet, preferably of the disposable type, a suitable stirring rod and a loop or pipette in a sealed envelope, thereby providing a slide ready for use in collecting a small blood sample and separating plasma therefrom.

Example II Prepare the coating by mixing together the following ingredients:

Hemagglutinin -mg 0.5 Sodium citrate mg 1.0 Norman saline ml 0.1

The materials are mixed together and applied to the surface of the principal recess and thereafter the slide is packaged as in Example I.

Example Ill The quantity of coating materials indicated in Examples I and II are entirely satisfactory for testing blood from human beings and most laboratory animals. However, in the case of bovine or cows blood a much greater quantity is required. The following example is particularly suitable for testing of bovine blood.

Prepare the coating by mixing together the following:

Hemagglutinin mg 3.0 Heparin mg 0.2 Normal saline ml 0.1

After the materials are mixedtogether, the coating material is applied and the slide is packaged as in Example I. However, in using the slide one additional drop of saline is added to the principal recess at the time the blood is added.

The following examples give representative coating procedures and coatings to be applied to the recesses in the slides for use in collecting small blood samples and separating serum therefrom.

Example IV The following materials are mixed together:

Substantially pure phytohemagglutinin 0.5 mg.

Thombin N.I.H.

units.

Physiological saline 0.1 ml.

After the materials are thoroughly mixed together, the mixture is spread evenly over the principal recess portion of the slide and the permitted to dry, leaving a coating of uniformly mixed phytohemagglutinin, alum and salts. The slide thus prepared may be packaged in a sealed envelope along with a lancet, preferably of the disposable type, a stirring rod and a loop or pipette, thereby providing a kit suitable for use in collecting blood and in separating serum therefrom.

Example V The following materials are mixed together:

Hemagglutinin mg 0.5 Normal saline ml 0.1

After the materials are thus mixed together, they are applied to the surface of the principal recess and the slide is packaged as indicated in Example IV. However, in using this slide the blood sample should be stirred more rapidly and for a more prolonged period of time (one or two minutes) to cause the clotting of the fibrinogen since no clotting agent is present in the coating material.

It should be understood that the other hemagglutinins, clotting agents and anticoagulants listed in this specification may be substituted for the specific materials listed in Examples I-V. It should also be understood that where larger blood samples are treated and larger recesses are provided that the quantity of the coating materials should be increased proportionately.

In using my improved apparatus, the envelope is first opened so as to expose the contents thereof. The disposable lancet is then used for the purpose of puncturing a finger or the ear lobe and a small sample of blood from two to five drops, preferably three drops, is collected in the principal recess portion 13 of the slide 10. The slide is then placed on a horizontal surface such as the top 26 of table 27. The stirring rod or toothpick 23 is then used for stirring the blood sarnple around the entire principal portion 13 of the recess causing it to mix thoroughly with the hemagglutinin in the coating. In this connection, care should be taken to avoid stirring the blood into the auxiliary recess portion 14. The stirring continues for about 30 to 50 seconds. During the stirring, the slide should be tilted and rotated to cause the blood to distribute the cells evenly and allow the plasma or serum to begin separation. However, as indicated above, care should be taken to avoid permitting any of the blood to flow into the auxiliary recess portion.

Where the slide is prepared for the collection of plasma, the blood during the stirring operation also mixes with the anticoagulant or anti-clotting agent, thereby preventing the fibrinogen from clotting. On the other hand,

where the slide is prepared for the collection of serum, the blood mixes with the coaguluant or clotting agent, thereby hastening the clotting of the fibrinogen.

During the stirring operation, the hemagglutinin causes the blood cells to agglutinate and to precipitate in the principal recess portion. If the slide were prepared for the collection of serum, then the fibrinogen also clots and precipitates during this period in the principal recess portion. Thereafter, the slide is shifted to the edge of the table or supporting surface so that the tab 17 overlaps the edge with the fold line 16 in registry therewith, as indicated in FIG. 3. The fold down tab 17 is then folded downwardly at right angles to the major portion of the slide, the slide is then shifted backwardly to be fully disposed on the table top as shown in FIG. 4 with the result the it is supported in a tilted position. The liquid containing components of the blood, i.e., the plasma or serum, will then flow downwardly from the principal portion of the recess into the auxiliary portion thereof. When all of the serum or plasma have thus been collected in the auxiliary portion of the recess, any desired amount thereof can be removed, as by means of a pipette or capillary or by means of the loop 24 for purposes of conducting the desired immunological, serological or chemical tests or reactions.

Instead of being used at once, the liquid containing component of the blood can be dried in the auxiliary recess portion and then be reconstituted and tested at a later date.

The specific length of the fold down tab 17 and the angle at which the slide is supported when the tab is folded downwardly may be varied. I have found that in a slide of the type illustrated and described with a recess of the indicated size for collecting a small blood sample satisfactory results are obtained if the slide is supported at an angle of approximately five to twenty degress, preferably ten to fifteen degrees.

It will thus been seen that I have provided an improved apparatus and method for use in separating liquid containing components from other components in a small sample of blood. My method and apparatus have the advantage that it is not necessary that they be used in the laboratory as they can be conveniently transported and used either in an ofiice or in the field. They have the further advantage that serum and plasma may be readily separated from small samples of blood without the use of centrifuges or other laboratory equipment.

It should be understood that modifications may be made in the illustrated and described embodiment of my invention without departing from the invention as set forth in the accompanying claims.

I claim:

1. A device for use in separating liquid containing components from other components in a small blood sample which comprises a relatively thin flat slide presenting an upper surface formed with a recess having a liquid impervious surface depressed below the level of said upper surface and formed with a principal recess portion for initially receiving the blood sample and containing a coating including a hemagglutinin for causing certain of the components including the blood cells to precipitate in said principal recess portion and also formed with an auxiliary recess portion communicating with the principal recess portion for thereafter receiving the unprecipitated and liquid components of the blood.

2. A device for use in separating liquid containing components from other components in a small blood sample as set forth in claim 1 in which the coating in the principal recess portion also includes an anti-clotting agent to prevent the fibrinogen from clotting so that plasma is collected in the auxillary recess portion.

3. A device for use in separating liquid containing components from other components in a small blood sample as set forth in claim 1 in which the coating in the principal recess portion also includes a coagulant to accelerate clotting of the fibrinogen so that serum is collected in the auxiliary recess portion.

4. A device for use in separating liquid containing components from other components in a small blood sample as set forth in claim 1 in which the principal recess portion is sufiiciently large so that the blood sample is retained therein by its cohesion.

5. A device for use in separating liquid containing components from other components in a small blood sample as set forth in claim 1 in which the hemagglutinin is a phytohemagglutinin.

6. A device for use in separating liquid containing components from other components in a small blood sample of approximately 2 to 5 drops which comprises a relatively thin fiat slide presenting an upper surface formed with a recess having a liquid impervious surface depressed below the level of said upper surface at least approximately 0.1 mm. and formed with a principal recess portion for initially receiving the blood sample and large enough so that the blood is retained therein by its own cohesion and containing a coating including a hemagglutinin for causing certain of the components including the blood cells to separate in said principal recess portion and also formed with an auxiliary recess portion communicating with the principal recess portion for thereafter receiving the unseparated and liquid compo- 8 nents ofthe blood, said auxiliary recess portion being at least large enough to accommodate the unseparated and liquid components of the blood sample.

7. A device for use in separating liquid containing components from other components in a small blood sample of approximately 2 to 5 drop as set forth in claim 6 in which the coating also includes an anticlotting agent for the fibrinogen.

8. A device for use in separating liquid containing components from other components in a small blood sample of approximately 2 to 5 drops as set forth in claim 6 in which the principal recess portion has an area of at least approximately 300 mm. and the auxiliary recess portion has an area of at least approximately mm.

References Cited in the file of this patent UNITED STATES PATENTS 2,561,339 Chediak July 24, 1951 FOREIGN PATENTS 989,480 France May 23, 1951 OTHER REFERENCES Eimer and Amend: Chemical and Physical Apparatus, Catalog, pp. 380, New York, 1903, QD/53/E34. (Copy in Group (86).