CA2231714A1

CA2231714A1 - Additive preparation and method of use thereof

Info

Publication number: CA2231714A1
Application number: CA002231714A
Authority: CA
Inventors: Ajit N. Dastane; Richmond R. Cohen
Original assignee: Becton Dickinson and Co
Current assignee: Becton Dickinson and Co
Priority date: 1997-04-30
Filing date: 1998-03-05
Publication date: 1998-10-30
Also published as: JP3967456B2; EP0875756A3; JPH10323341A; KR100473726B1; AU749445B2; US6024710A; AU6079598A; DE69821670T2; DE69821670D1; BR9801418A; SG83678A1; EP0875756B1; EP0875756A2; US6225123B1; ZA981866B; ES2212166T3; CN1199609A; KR19980081787A

Abstract

The present invention is an additive preparation which comprises an additive, an organic acid and a metal carbonate compound. The preparation effervesces when in contact with a body fluid sample, thereby efficiently dispersing in a body fluid sample.

Description

CA 02231714 1998-03-0~

METHOD C)F USE THEREOF
1~
BACKGROUND~ OF THE INVENTION

1. FIELD C)F THE INVENTION
l'j The present invention is an additive preparation for use in collection devices wherein the additive preparation effervesces when in contact with a body fluid. The additive preparation desirably comprises a formulation comprising an additive such as a clot activator, anticoagulant or urine preservation material, an organic acid and a metal carbonate compound. The effervescence e:Efect of the formula-tion aids in dispersal and delivery of the additive in a blody fluid sample. The formulation is desirably tableted to provide an effective, easily stored and handled preparation. In particular, the formulation of the present invention preferably comprises a clot activator or 2'j anticoagulant, an organic acid and a metal carbonate compound wherein the formulation en:hances clot activation or the anticoagulant effect in a blood speclmen.

2. DESCRIPTION OF RELATED ART

3() Blood collected in evacuated tubes often must be clotted prior to clinical examin~tion because it is desirab~le to form a dense clot as rapidly and completely as possible to facilitate clean separation of the clot from the serumlayer by centri:~ugation. To achiel~e this end, both plastic and glass blood 3'j collection tubes frequently employ a clot activator. Typical clot activators are diatomaceous earth and particles of inorganic silicates, or biochemicals such CA 0223l7l4 l998-03-0~

as ellagic acid, -thrombin, trypsin and thromboplastin.

Typical clot activators used commercially are silica coated on fabric, silica particles in small plastic cups or silicate particles applied to the tubewall with a polyvinylpyrrolidone (PVP) carrier. However, in these type of arrangements, it is necessary for the user to initiate mixing of the sample so that the activator is bioavailabile to the specimen thus providing the desired effect of the aclditive in the sampLe. Therefore, the mixing requirement is critical to obtaining the desired effect of the additives.

Maximum effectiveness is ac:hieved by thorough dispersion of the clot activator throughout the blood sample. Since clot activator materials are generally in powder form or as a wall coating, mixing of the clot activator with the blood sample to achieve dispersion may be a physically awkward operation. Also complete dispersion of the clot activator material in the blood sample tends to be frustrated by the tendency of the clot activator material to agglomerate upon moistening.

In addition, agglomerated clot activator particles tend to settle relatively rapidly, according to Stokes Law, which provides that the settling rate of a particle in a dispensing fluid will be governed by its relative diameter and density as well as the fluid's viscosity and density.

Therefore, there is a need for providing a means for deploying an additive in a body fluid with minimal requirements of the user to initiate mixing of the additive and the body fluid and whereby the additive is able to provide rapid and reliable performance under variable handling conditions.

More particularly, there is a need for a blood collection tube with means for promoting c:lot-acceleration of a blood sample which provides an enhanced rate of blood coagulation (shortened time for blood coagulation) without leaving any substantial amount of soluble or particulate material in the serum layer on centrifugation, thus avoiding potential interference with CA 02231714 1998-03-0~

clinical tests, particularly in blood banking procedures. Whereas there are numerous commercial products available that employ clot activators, these products are unable to satisfactorily provide a shortened time for blood coagulation or provide a sample with minimal soluble or particulate material !, in the serum la'yer.

SUMMARY OF THE INVENTION

The present invention is an additive formulation for use in collection 10 devices wherein the additive preparation effervesces when in contact with a body fluid. The additive preparation desirably comprises a formulation comprising an aLdditive, an organic aLcid and a metal carbonate compound.

Desirably, the additive is a clot activator, anticoagulant, urine 1!~ preservation material or any other body fluid preservative.

In addition, the additive formulation may further comprise a stabilizer and/or a flow improver or a binder.

Desirably, the additive formulation comprises:

(a) from about 40 weight percent to about 90 weight percent of an additive;
(b) from about 5 weight percent to about 30 weight percent of an 2!~ organic acid or mixtures thereof; and (c) from about 5 weight percent to about 30 weight percent of a metal carbonate compound.

The present invention is most preferably an additive formulation for 30 enhancing clot activation of a b]Lood sample. The additive formulation CA 02231714 1998-03-0~

desirably comprises a clot activator, an organic acid and a metal carbonate compound.

Preferably, the clot activator additive formulation comprises:
, (a) from ;about 40 weight percent to' about 70 weight percent of a clot activa,tor;
(b) from about 10 weight percent; to about 26 weight percent of an organic acid or mixtures thereof; and o (c) from about 10 weight percent to about 26 weight percent of a metal carbonate.

The additive formulation ma,y further comprise a binding agent from about 6 weight percent to about 30 ~weight percent of a binder.
1 .~
The clot activator may be diatomaceous earth, particles of inorganic silicates or biochemicals such as ellagic acid, thrombin, trypsin and thromboplastin or combinations thereof.

21~ A significant attribute of t,he additive formulation of the present invention is its use in collection devices wherein the additive preparation effervesces when in contact with a body fluid sample. The effect of the additive preparation therefore aids in the dispersal and delivery of the additive to a body fluid sample.

A significant attribute of the clot activator additive formulation of the present invention is its use in blood collection devices as an effective and efficient means for promoting blood coagulation. Most importantly is that the additive formu],ation and the blood sample does not have to be mixed by the user.

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An import;ant advantage of the clot activator additive formulation of the present invention is its ease of use not requiring lengthy time to promote blood coagulation as compared to conventional techniques.
';
Notably, t,he formulations of t;he present invention rapidly disintegrate and disperse in a body fluid sample, thereby minimi7.ing the requirement that the user assist in mixing the formulation and the body flùid sample.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a typical blood collection tube with a stopper.

~, FIG. 2 is ;a longitudinal sectional view of the tube of FIG. 1 taken along line 2-2, comprising the additive formulation of the present invention.

DETAILED DESCRIPTION

The present invention may be embodied in other specific forms and is not limited to any specific embodim~ents described in detail which are merely exemplary. Various other modifical,ions will be apparent to and readily made by those skilled in the art without departing from the scope and spirit of the invention. The scope of the invention will be measured by the appended claims and their equivalents.

The addit,ive formulation of the present invention comprises:

(a) from about 40 weight percent to about 90 weight percent of a clot aCtiVcltOr;
(b) from about 5 weight percent to about 30 weight percent of an CA 02231714 1998-03-0~

organic acid; and (c) from about 5 weight percent to about 30 weight percent of a metal carbonate.

ci The additive formulation may further comprise a binding agent from about 5 weight percent to about 30 ~eight percent.

Desirably, the pH of the formulation is from about 5 to about 9, preferably from about 6 to about (3 and most preferably from about 6.5 to 10 about 7.5.

A clot activator material is preferably used in the formulation to initiate rapid c lotting of a blood sample that comes in contact with the formulation of the present invention.
1'~
A blood specimen often needs to be clotted to obtain serum. Serum is the specimen of choice for performing blood chemistries.

Desirably, the clot activator :materials, include, but are not limited to, 2l~ diatomaceous earth, inorganic silicates, ellagic acid, thrombin, trypsin and thromboplastin.

The preferred clot activator material for use in the formulation of the present invention is silica. A commercially available silica material is Min-U-Sil (trademark of Pennsylvania Glass Company, PA). Preferably, silica maybe present in the formulation such that the acceleration of the clotting mechanism is substantially achieved while minimi7.ing visual hemolysis or impacting chernistry analytes of the blood sample in an amount from about 40 to about 90 weight percent and most preferably at about 50 weight percent.

An organic acid is also preferably used in the formulation of the present CA 02231714 1998-03-0~

invention to initiate an effervescent reaction in the presence of water.

Desirably, the organic acid includes, but is not limited to, organic compounds containing at least one carboxylic acid functionality such as s tartaric acid, ci-tric acid, malic acid, maleic acid, fumaric acid, succinic acid, ascorbic acid and mixtures thereof.

The preferred organic acid for use in the formulation of the present invention is citric acid. Preferably, citric acid may be present in the o formulation in an amount from about 5 to about 30 weight percent and more preferably at about 25 weight percent.

A metal carbonate compound is also preferably used in the formulation of l;he present invention to initiate the disintegrating reaction in the presence 1!~ of acid and water.

Preferably, the metal carbonate compound is water soluble and includes, but is not limited to an alkali metal salt, sodium bicarbonate, sodium carbonate, sodium sesquicarbonate, potassium carbonate, cadmium carbonate, calcium carbonate, rubidium carbonate, potassium bicarbonate, soclium benzoate, sodium phosphate~ monobasic and sodium glycine carbonate.

Most pref-erably the metal carbonate compound is an alkali metal salt.
The preferred alkali metal salt for use in the formulation of the present invention is sodium bicarbonate. Preferably, sodium bicarbonate may be present in the formulation in an amount from about 5 to about 30 weight percent and more preferably at about 25 weight percent.

Most preferably the additive formulation of the present invention co m priSeS:

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(a) fronn about 50 weight percent of silica;
(b) fronn about 25 weight percent of citric acid; and (c) fronn about 25 weight percent of sodium bicarbonate.
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An alternate embodiment of the present invention includes an additive formulation connprising:

(a) from aLbout 40 weight percent to about 90 weight percent of a clot activator;
(b) from about 5 weight percent to about 30 weight percent of an organic acid;
(c) from aLbout 5 weight percent to about 30 weight percent of a metal carbonate; and 1!~ (d) from about 5 weight percent to about 30 weight percent of a binding agent.

Most preferably the alternate embodiment of the present invention cornprlses:
21~
(a) frorn about 50 weight percent of a clot activator;
(b) frorn about 20 weight percent of an organic acid;
(c) from about 20 weight percent of a metal carbonate; and (d) from about 10 weight percent of a binder.

A binding or bulking agent may be used in the formulation of the present invention to provide binding and lubricating properties to the formulation.

Preferably the binding agent includes but is not limited to polyvinyl-py:rrolidone (PVP) polyvinyl-alcohol (PVA) polyethylene glycol (PEG) CA 02231714 1998-03-0~

carboxymethyl cellulose or corn starch.

The preferred binding agent for use in the formulation of the present invention is polyvinylpyrrolidone (E'VP). Preferably, PVP may be present in s the formulation in an amount from about 5 to about 30 weight percent.

A binding agent enables granulating of the formulation without the forming of a pellet.

o Polyvinylpyrrolidone is soluble in water and in a number of polar and non-polar organic solvents. Polyvinylpyrrolidone therefore serves as a binder and as a lubricant. Therefore, when the pellet of the present invention is dissolved in water, there is substantially no insoluble residue.

~, The polyvinylpyrrolidone used in the present invention is a polymer of vinylpyrrolidone having a molecular weight of about 1~,000 to about 200,000.
PVP is soluble in water and is also soluble in many organic solvents such as aliphatic alcohLols, chlorinated hydrocarbons, esters, nitroparaffins and am.lnes.
21~
Most prel-erably, the components of the formulation are in a physically bound form to maximize the dispersion of the formulation. They may be bound into pellets, pills, capsules, granules, tablets and the like may all be used in the practice of this invention. Most preferably, the formulation of the 2~ present invention is in pellet form. Pellet form is convenient because the formulation will rapidly disperse in the blood sample.

A dry compression techniqu.e may be used to form a pellet of the formulation of the present inventiom CA 0223l7l4 l998-03-0~ ' The dry oompression technique consists of mixing the components of the formulation and then applying sufficient force to form a pellet. The heat of compression binds the dry powder together, to form a pellet. The pellet formulation of l,he present invention is prepared by mixing the ingredients in a standard shaker for about two (2) hours or until a fine blend mixture is obt;ained. Then small portions of the mixed batch of powder are aliquoted into a manual puncher. The manual puncher consists of a piston-cylinder assembly. AlLiquoted powder is placed in the die cavity and force is applied by means of a hydraulic or pneumatic press. Typically, the aliquoting and pressing operal~ions are automatically performed in commercially available tablet presses.

Other ingredients which are conventional or desirable in various pellet formulations m.ay also be added to the formulation as long as they do not adversely affect; the overall properties of the formulation.

The addil,ive preparation of the present invention is preferably located in a collection clevice. The device is most preferably a blood collection deviceand may be eil,her an evacuated blood collection device or a non-evacuated blood collection device. The collection device is desirably made of plastic, such as but not limited to polyethylene terephthalate, or polypropylene or glass.

Referring to the drawings in which like reference characters refer to like parts throughout the several views thereof, FIG. 1 shows a typical blood collection device 10, having an open end 16, a closed end 18, inner wall 12, and a stopper 14 that includes a lower annular portion or skirt 15 which extends into and presses against the inner wall 12 of the tube for maintaining stopper 14 in p]Lace.

FIG. 2 shows device 10 with an additive preparation 20.

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A blood specimen sample of interest can be transferred into device 10, wherein the specimen contacts the additive so that the additive rapidly disperses into t:he specimen and clot activation is initiated.

The following examples are not limited to any specific embodiment of the invention, blut are only exemplary.

PREPARATION OF ADDITIVE FORMULATION

The additive formulation of the present invention was prepared with the following ingredients as listed in Table 1:

Ingredients Weight Percent (%) Grams ~gm) silica 50 1.0 (Min-U-Sil, Pennsylvania Glass Compa:ny) baking soda 48 1.0 (Mixture of sodium bicarbonate and tartaric acid, Mfg: Arm and l~ammer) citric acid 2 0.0 (Sigma Chemicals Catalog No. C1909, Lot No.
~L~H0868) CA 0223l7l4 l998-03-0~

In a mixing vessel, all of the above ingredients were mixed together.
The mixture was then blended u sing a mortar and pestle for about 20 minutes or unt;il a fine blend was obtained. Twenty (20) pellets weighing !~ approximately 1.25 mg each were formed using a puncher. Because of the ma nual force applied on the puncher during the aliquoting process the powdered mate rial formed into a pellet.

1~
EFFECTI~IENESS EVAL,UATlON OF ADDITIVE FORMULATION

The effectiveness of the preparations of Example 1 were assessed by measuring the amount of silica available in the supernatant of added water.
1:5 In other words, the measurement oi-- silica not sedimented at the bottom of the tube whereby the silica in the su pernatant represents silica available for clotting in the blood.

The pellets prepared in Example 1 were added to twenty 16 x 100 mm plaLstic tubes. 'l'hese twenty tubes were separated into groups of ten, Group A
and Group B. Group C consisted of the control tubes~ twenty 13 x 100 mm plastic tubes with a wall coating c:onsisting of a mixture of silica, PVP and surfactant and a gel at the bottom. These tubes are VACUTAINER~' brand PL,US tubes.

The 16x100 mm tubes used in this experiment were marked for 8 ml fil~. There was also a second mark on these tubes, placed near the closed or bottom end of 1,he tube. The bottonn mark acted as an indicator for pipetting wa.ter from the tube, without disturbing the sedimented silica. This ensured that silica that is at the bottom oi- the tube is not aspirated along with the CA 02231714 1998-03-0~

supernatant, thus giving falsely elevated numbers for amount of silica in the solution.

These twenty tubes were further separated into groups of ten tubes each. The reason for the two separate groups was during the experiment one group was to be mixed while other one remained unmixed.

For controls, twenty 13xlO0 mm plastic tubes with wall coating consisting of a mixture of silica, PVP, and surfactant were used (VACUTAINE~'~ brand PLUS tubes, Lot # DG061096tI). These tubes also had gel at the bottom of the tube. These tubes were also separated into groups of 10 each.

De-ionized (DI) water was added to all the prototype and control tubes, one tube at a time. For the unmixed set of tubes, the DI water was immediately pipetted out. Nephelometry (Hach Turbiditimeter), which measures the amount of suspended particles, was done on these collected samples immediately. For the mixed tubes the methodology used was identical, except mixing with five inversions was done before pipetting the DI
water.

Table 2 llsts the amount of silica per mL of water in the supernatant.
The data clearly indicates that l;he pellets in both mixed and unmixed conditions outperformed the wall coated tubes.

SILICA IN SUPERNATANT

s (SAMPL]3 SIZE, N = 10) 'l'ube Type Handling Amt. of Silica Amt. of silica in (wt%) Condition dispensed the Supernatant (#of Inversions) (mg/mL) ~mg/mL) E'rototype 0 0.08 0.07 87.~
Control 0 0.15 0.05 33.33 Prototype ~ 0.08 0.1* 100 Control 6 0.15 0. 09 60 * Over recol)ery was attributed to the turbule~ce caused by m ixing and nephelometry reading the air bubbles irl mixing as silicaparticles.
11) PREPARATION OF ADDITIVE FORMULATION

, The addil,ive formulation of the present invention was prepared with the following ingredients as listed in Table 3:

TAlBLE 3 Imgredients Weight Percent s Llica 50 sodium bicarbonate 25 citric acid 25 CA 02231714 1998-03-0~

EXAMPI,E 4 CLINICAL EFFICACY OF THE PELLETS

r~ To demor-strate the clinical efficacy of the dispersion of the pellets in Example 3, a f:ive donor clinical study of human donors was performed and the clotting performance and select analytes of the samples were evaluated.

The measure of clotting performance was visual evaluation for gelation 10 of blood as well as presence of fibrin mass post centrifugation. The presence of fibrin mass after centrifugation demonstrates an incomplete clotting process even if the blood has completely gelled prior to centrifugation. In this experiment the prototype and control tubes were centrifuged for 10 minutes after waiting for 15 minutes post specimen collection 1~
Table 4 lists the visual observations done for clotting, while Table 5 lisl;s the results from the analyte measurements done on these tubes.

VISUAL OBSERVATIONS (SAMPLE SIZE N=5) Tube Type Number of Tubes not Fibrin Clotted before ~Iass Centrifugation Prototype 1 2 (Formulation per l~xamp]e 3)(13 X 100 mm plastic tube with first coat of surfactant, Pellet wt.=1.5 mg) Control- l 5 5 (Glass, 13xlOO mrn tul)e with wall coated silica) PLUS Control 2 5 (Plastic, 13xl00 mm tube with wall coated silica) CA 0223l7l4 l998-03-0~

TABLE. 5 (Part of Example 4) MEAN OF MEASURED ANALYTES

(SAMPLE SIZE, N=5 FOR EACH TUBE TYPE) Analyte Glass Plastic Prototype Control Control Sodium 139 138.8 ]39.6 (nlmol/l,) Potassium 4.02 4.02 4.06 (mmol/L) Chloride 100.4 100.6 100 (mmol/lJ) C(~2 27.2 27.6 27.8 (~lmol/L) LD 421.6 427.6 425.4 (IlJ/L) pE I 7.62 7.61 7.59 In Table 4, it is important to note that in all cases when fibrin was present in the prototype tubes, the size of the fibrin masses was markedly smaller than those presented by the corresponding control tubes. This was an indication of more complete clotting in the prototype tubes. In Table 5, the results from the analytes demonstrated medically significant differences compared with the control products. This demonstrates that the use of disintegrant material does not have an adverse impact on analyte values.

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PREPARATION OF ADDITIVE FORMULATION

The additive formulation of the present invention was prepared with the following ingredients as listed in Table 6:

Ingredients Weight Percent Ethylene(li~minetetraacetic acid dipotassium salt 50 (K2EDTA) (Anticoagulant) (Mfg.: SIGMA Chemicals, Lot #14H0457) Cil,ric Acid Monohydrate 16.33 (Mfg.: SIGMAC'hemicals, Lot#115H1018) Sodium bicarbonate 33.67 (Mfg.: SIGMA IChemicals, Lot #56H0423) In a mixlng vessel, all of the above ingredients were mixed together.
The mixture was then blended using a mortar and pestle for about 30 minutes or until a fine blend was obtained. Pellets weighing between about 7 to 8 mg each were formed using aL puncher. Because of the manual force applied on the puncher during the aliquoting process the powdered material formed into a pellet.

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CLINICAL EFFICACY OF THE ADDITIVE FORMULATION

c, To demonstrate the clinical efficacy of the additive, the pellets made according to Example 5 were evaluated for anticoagulant performance. Three 13 x 75 mm plastic evacuated tubes of 2ml draw each containing one pellet as made in Example 5 were used to collect blood from 3 human donors. The tubes were then evaluated for anticoagulant performance. The method for 10 evaluating anticoagulant performance was by visual inspection for gelation ofblood, as well as by observing microclots when the blood specimen is filtered through a fine meshed sieve. Any observation of visual clotting and/or the presence of microclots was deemed a failure. Table 7 shows the results from the clinical study.
1, Tube Type Handling Number of Condition Tubes that Passed Prototype no mixing 3 (formulation p~er Example-5 in 13x75 mm plastic evacuated tube, 2mL draw) Control #1 - no mixing (VACUTAINER brand tubes, cat # 367648, 2 mL, draw, K~EDTA sprayed on the tube wall) Control #2 - mixed by 3 (VACUTAINER brand tubes, cat #367648, 2 inverting tube mL draw, K~EDTA sprayed on the tube walV 10 times

Claims

1. An additive preparation comprising an additive, an organic acid and a metal carbonate compound

2. The additive preparation of Claim 1 further comprising a binding agent.

3. The additive preparation of Claim 1 wherein said additive is clot activator, an anticoagulant or urine preservation material.

4. The additive preparation of Claim 1 wherein said organic acid is citric acid, tartaric acid, malic acid, maleic acid, fumaric acid, succinic acid or ascorbic acid, or mixtures thereof.

5. The additive preparation of Claim 1 wherein said metal carbonate compound is an alkali metal salt.

6. The additive preparation of Claim 5 wherein said alkali metal salt is sodium bicarbonate, sodium carbonate, sodium sesquicarbonate, potassium carbonate, cadmium carbonate, calcium carbonate, potassium bicarbonate, sodium benzoate, sodium phosphate monobasic or sodium glycine carbonate.

7. The additive preparation of Claim 6 wherein said alkali metal salt is sodium bicarbonate.

8. The additive preparation of Claim 2 wherein said binding agent is polyvinylpyrolidone, polyvinyl-alcohol, polyethylene glycol, carboxymethyl cellulose or corn starch.

9. An additive preparation comprising:

(a) from about 40 weight percent to about 90 weight percent of additive;
(b) from about 5 weight percent to about 30 weight percent of an organic acid or mixtures thereof; and (c) from about 5 weight percent to about 30 weight percent of a metal carbonate compound.

10. The additive preparation of Claim 9 further comprising about 5 weight percent to about 30 weight percent of a binding agent.

11. The additive preparation of Claim 9 wherein said additive is clot activator, an anticoagulant or urine preservation material.

12. The additive preparation of Claim 9 wherein said organic acid is citric acid, tartaric acid, malic acid, maleic acid, fumaric acid, succinic acid or ascorbic acid or mixtures thereof.

13. The additive preparation of Claim 9 wherein said metal carbonate compound is an alkali metal salt.

14. The additive preparation of Claim 13 wherein said alkali metal salt is sodium bicarbonate, sodium carbonate, sodium sesquicarbonate, potassium carbonate, cadmium carbonate, calcium carbonate, potassium bicarbonate, sodium benzoate, sodium phosphate monobasic or sodium glycine carbonate.

15. The additive preparation of Claim 14 wherein said alkali metal salt is sodium bicarbonate.

16. The additive preparation of Claim 10 wherein said binding agent is polyvinylpyrolidone, polyvinyl-alcohol, polyethylene glycol, carboxymethyl cellulose or corn starch.

17. An additive preparation comprising a clot activator, an organic acid and a metal carbonate compound.

18. The additive preparation of Claim 17, wherein said clot activator is selected from the group consisting of silica, diatomaceous earth, inorganic silicates, thrombin, ellagic acid, trypsin and thromboplastin and mixtures thereof.

19. The additive preparation of Claim 17, wherein said organic acid is selected from the group consisting of citric acid, tartaric acid, succinic acid and mixtures thereof.

20. The additive preparation of Claim 17, wherein said metal carbonate is selected from the group consisting of sodium bicarbonate, ammonium carbonate, sodium carbonate or rubidium carbonate.

21. An additive preparation comprising:

(a) about 50 weight percent to about 90 weight percent of clot activator;
(b) about 25 weight percent to about 30 weight percent of organic acid; and (c) about 25 weight percent to about 30 weight percent of metal carbonate.

22. The additive preparation of Claim 21, wherein said clot activator is silica.

23. The additive preparation of Claim 21, wherein said organic acid is citric acid.

24. The additive preparation of Claim 21, wherein said metal carbonate is sodium bicarbonate.

25. A method for preparing a pellet additive formulation, comprising the steps of:

(a) mixing a clot activator or anticoagulant, citric acid and sodium bicarbonate in a shaker for about 2 hours or until a fine blend mixture is obtained;
(b) adding the mixture to the die cavity of a puncher;
(c) applying hydraulic or pneumatic pressure to the die cavity to form a pellet.

26. An assembly for collecting a body fluid, said assembly comprising:

(a) a container having an open end, a closed end and an inner and outer surface;
(b) an additive preparation contained within the container at the closed inner surface of the container.

27. A method for activating clotting of a blood sample, comprising the steps of:

(a) providing a container having an open end, a closed end, said container further having an additive preparation contained therein at the closed inner surface of the container;

(b) introducing said blood sample in said container with said additive preparation; and (c) mixing said sample in said container with the additive solution whereby the additive solution effervesces and mixes with said blood sample upon contact.

28. An additive preparation comprising:

(a) silica;
(b) sodium bicarbonate;
(c) tartaric acid (d) citric acid

29. An additive preparation comprising:

(a) about 50 weight percent silica;
(b) about 48 weight percent sodium bicarbonate and tartaric acid;
and (c) about 2 weight percent citric acid.

30. An additive preparation comprising an anticoagulant, an organic acid and a metal carbonate compound.

31. The additive preparation of Claim 30, wherein said anticoagulant is ethylenediamineteraacetic acid dipotassium salt.

32. The additive preparation of Claim 30, wherein said metal carbonate is selected from the group consisting of sodium bicarbonate, ammonium carbonate, sodium carbonate or rubidium carbonate.

33. The additive preparation of Claim 30, wherein said organic acid is selected from the group consisting of citric acid, tartaric acid, succinic acid,and mixtures thereof.