WO2000033853A1

WO2000033853A1 - Anti-coagulation with calcium containing citrate solution

Info

Publication number: WO2000033853A1
Application number: PCT/SE1999/002271
Authority: WO
Inventors: Mats RÅNBY
Original assignee: Global Hemostasis Institute Mgr Ab
Priority date: 1998-12-07
Filing date: 1999-12-06
Publication date: 2000-06-15
Also published as: AU2017400A; SE9804248D0

Abstract

A method of anti-coagulating a blood product and counteracting the reduction of calcium ion activity in the anti-coagulated product, is disclosed. The method comprises addition to the blood product of an anti-coagulant selected from the group consisting of soluble citrate compounds, citric acid, and mixtures thereof in an amount of 13-60 mmole citrate per liter of anti-coagulated blood product, and of a soluble calcium compound in an amount giving a calcium ion activity in the anti-coagulated blood product of 0.2 - 10mM. Further, an anti-coagulated blood or blood plasma product comprising such an anti-coagulant and calcium compound, and its use for the production of coagulation Factor VIII, are described. Additionally, a blood sampling container comprising an aqueous solution of such an anti-coagulant and calcium compound, and its use for transport and storage of the anti-coagulated blood product, are disclosed.

Description

Anti-coagulation with calcium containing citrate solution

The present invention relates to a method of anti-coagulating blood products, and counteracting the reduction of calcium ion activity in the anti-coagulated product. The invention also relates to anti-coagulated blood or blood plasma products comprising an anti- coagulating solution of one or several soluble citrate compounds, citric acid and/or mixtures thereof, and a soluble calcium compound. The anti-coagulated product is useful for the production of coagulation Factor VIII. Further, the invention comprises blood sampling containers comprising an anti-coagulating aqueous solution of such an anti-coagulant and calcium compound, which may be used for transport and storage of the product.

Background.

Biological fluids, in particular blood, lymph, synovial fluid and cerebral liquor and preparations of these may undergo dramatic rheological transformation whereby they become either highly viscose or gelatinous. Such transformation is denoted coagulation or clotting and may serve the physiological function of preventing the biological fluid from leaking out of the confinement in which it belongs. Blood is prevented from leaking out of the vasculature, synovial fluid out of the joint, cerebral fluid out of the spine and brain cavities.

Coagulation often occurs when the biological fluid is removed from its natural environment and collected in some man-made container as is the case when biological fluid is collected for transfusion, laboratory diagnostics or other medical uses. Collection of biological fluid into man-made containers therefore often requires some anti-coagulation procedure. Anti-coagulation is most often accomplished by addition of an anti-coagulating substance, also called anti-coagulant.

One group of commonly used anti-coagulants includes oxalate, EDTA, and citrate. They function, according to prior art theory, by reducing the concentration of free Ca²⁺ in the biological fluid from about 1 mM to non-effective levels. Free Ca²⁺ concentration is also denoted Ca²⁺ activity and non-effective levels are levels below about 0.1 mM.

Anti-coagulation of a biological fluid by reduction of the Ca²⁺ activity provides a technical feature of considerable importance. The anti-coagulation can readily be neutralized by addition of soluble Ca²⁺ salt, such as CaCl₂. The possibility of neutralizing the anti- coagulation makes it possible to use the preparation made from an anti-coagulated biological fluid and containing therapeutic agents for infusion to patients suffering from bleeding disorders, thereby promoting coagulation in the patients. Such agents include thrombocyte concentrates and coagulation Factor NIII. Furthermore, the simple neutralization of the anti- coagulation effect enables in vitro laboratory determination of coagulation analytes. Citrate is the most commonly used anti-coagulation agent for blood and blood plasma products from which therapeutic agents, such as thrombocytes, coagulation Factor VIII and anti-thrombin III, are prepared. In vitro laboratory diagnostic procedures for coagulation analytes are most often performed on blood and blood plasma anti-coagulated with citrate. Coagulation Factor VIII (deficiency in which causes hemophilia A) is prepared from blood plasma anti-coagulated with citrate. In the industrialized part of the world, there are some half a million people in need of periodic infusions of coagulation Factor VIII. Most of the Factor VIII needed is produced by fractionation of citrate anti-coagulated blood plasma. Millions of liters of citrate anti-coagulated blood plasma are processed every year for this purpose. The price of this product for industrial manufacture pivots around the level of

Factor VIII activity that is contained in the plasma, nota bene, not the level in the blood as it is collected but the level that is present in the plasma as it is subjected to fractionation. This in turn is dependent on the stability of this protein in the anti-coagulated blood plasma.

It is well known that Factor VIII is relatively unstable in citrate anti-coagulated blood. In about 24 hours the levels of active Factor VIII is reduced by about 50%. If this decline in activity can be prevented, the efficiency of Factor VIII production can be considerably improved.

An alternative, according to prior art, to improve anti-coagulation is to reduce the levels of citrate in the anti-coagulant. Reduction of citrate has often meant reduction by about 50% and is referred to as anti-coagulation with half citrate. However, this has not been effective in improving the efficacy of Factor VIII production. The reduced amounts of citrate anti-coagulant have rather caused problems by coagulating the anti-coagulated blood plasma. The limited stability of Factor VIII causes problems also in the field of in vitro diagnostics.

In WO 88/08004 is disclosed a method of collecting blood from a donor into a calcium chelating anti-coagulant (e.g. citrate), separating the plasma from the cellular components of the blood, and then adding to the plasma first heparin and then a soluble calcium compound (stabilizing Factor VIII by increasing the calcium ion activity). The rational behind the invention is that heparin acts as a non-chelating anti-coagulant and allows the calcium ion activity to be raised without compromising the anti-coagulation.

EP -A2-0 244 834 is directed to the production of a concentrate of a related coagulation factor, namely Factor V. The Factor V is produced from human blood plasma containing an anti-coagulant. A divalent metal salt is added to plasma obtained from blood anti-coagulated with citrate or other calcium chelating agent anti-coagulant. The amount of added divalent metal salt, e.g. CaCl₂, is lower than the equivalent concentration of the anticoagulant. The highest concentration of citrate mentioned is 11 mmol/L, and highest amount of added soluble calcium compound ions 5 mmol/L. The present invention contrasts the cited prior art through utilization of a surprising discovery disclosed herein. According to this discovery, elevated levels of citrate anti- coagulates blood and blood plasma by some mechanism other than reducing the levels of calcium ion activity. Hence, at elevated levels of citrate, here defined as levels above 13 mol/L, the calcium ion activity can be raised more, without causing coagulation, than what is possible at lower levels of citrate.

A related medical field in which anti-coagulation by reduction of Ca²⁺ causes practical concerns is blood banking. Blood or blood plasma that is to be used in transfusion is commonly anti-coagulated. This is because, with present techniques, native blood and blood plasma can only be handled for a short time period outside the organism without coagulating. Because of low toxicity, rapid metabolization and relatively good anti-coagulant properties, citrate is the commonly used anticoagulant in blood banking. In many cases, the citrate content in transfused blood or blood plasma does not cause adverse effects for the receiving patient. But in some cases, especially in patients with liver dysfunction, citrate anti- coagulated blood and blood plasma are not well tolerated. Due to slow metabolism of citrate in these patients, the citrate causes a notable reduction in Ca²⁺ activity which results in cramps and gastrointestinal discomfort.

It would be desirable to provide anti-coagulated blood and blood plasma wherein the reduction of calcium ion activity due to the addition of the anti-coagulant is counteracted. For practical reasons, it would be desirable to provide blood sampling containers for blood collection which contain an aqueous solution of both an anti-coagulant and a calcium compound, especially for transport and storage of the blood product. Description of the invention.

The present invention provides a method of anti-coagulating a blood product and counteracting the reduction of calcium ion activity in the anti-coagulated product. The method of the invention is for example useful for accomplishing 1) increased stability and reduced denaturation of coagulation analytes in the anti- coagulated product, 2) reduction in the Ca²" lowering effect when citrate anti-coagulated blood or blood plasma is transfused into patients, and 3) increased levels of Factor VIII in anti-coagulated blood plasma after transport and storage.

Thus, one aspect of the invention is directed to a method of anti-coagulating a blood product and counteracting the reduction of calcium ion activity in the anti-coagulated product, comprising addition to the blood product of an anti-coagulant selected from the group consisting of soluble citrate compounds, citric acid, and mixtures thereof in an amount of 13- 60 mmole citrate per liter of anti-coagulated blood product, and of a soluble calcium compound in an amount giving a calcium ion activity in the anti-coagulated blood product of 0.2 - 10 mM. Examples of the amount of citrate per liter of anti-coagulated blood are 13, 20, 30, 40,

50, and 60 mmole, respectively, and any intervals construed there between. It should be noted that even higher amounts are possible, but will not bee used in practice. The range of calcium ion activity may also be extended, but will probably not be used in practice.

In an embodiment of this aspect of the invention the soluble citrate compound is selected from the group consisting of sodium, potassium and lithium salts of citric acid, and the soluble calcium compound is selected from calcium chloride and calcium acetate.

Examples of sodium, potassium and lithium salts of citric acid which may be used in the invention are mono-, di-, and tri- potassium, sodium and lithium citrate compounds and mixtures thereof. In an embodiment of this aspect of the invention the soluble citrate compound is trisodium citrate and the soluble calcium compound is calcium chloride.

In another embodiment the citrate and calcium are dissolved and added in an aqueous solution which is sufficiently acidic to prevent the formation of insoluble compounds of calcium and citrate. Another aspect of the invention is directed to an anti-coagulated blood or blood plasma product comprising an anti-coagulant selected from the group consisting of soluble citrate compounds, citric acid and mixtures thereof citric acid, and mixtures thereof in an amount of 13- 60 mmole citrate per liter of anti-coagulated blood product, or 20- 100 mmole citrate per liter of anti-coagulated blood plasma product, and an added soluble calcium compound in an amount giving a calcium ion activity in the anti-coagulated blood or blood plasma product of 0.2 - 10 mM.

In an embodiment of this aspect of the invention the soluble citrate compound is selected from the group consisting of sodium, potassium and lithium salts of citric acid, and the soluble calcium compound is selected from calcium chloride and calcium acetate. In an a preferred embodiment the soluble citrate compound is trisodium citrate and the soluble calcium compound is calcium chloride.

In another preferred embodiment the anti-coagulated blood product according to the invention comprises 1-20% of added water. In yet another preferred embodiment the anti-coagulated blood plasma product according to the invention comprises 1-35% of added water.

A further aspect of the invention is directed to the use of a blood or blood plasma product according to the invention for the production of coagulation Factor VIII. Still another aspect of the invention is directed to a blood sampling container for collecting nine volumes of a blood product comprising one volume of an aqueous solution containing 130 - 600 mmole per liter of an anti-coagulant selected from the group consisting of soluble citrate compounds, citric acid and mixtures thereof, and a soluble calcium compound in an amount giving a calcium ion activity in an anti-coagulated blood product to be produced of 0.2 - 10 mM.

Examples of blood sampling containers are tubes and bags, especially for blood banking.

In a preferred embodiment the citrate and calcium are dissolved in an aqueous solution which is sufficiently acidic to prevent the formation of insoluble compounds of calcium and citrate.

Yet another aspect of the invention is directed to the use of a blood sampling container according to the invention for transport and storage of an anti-coagulated blood product. Sort description of the drawing Figure 1 shows the coagulation time plotted against the calcium ion activity for four different citrate anti-coagulated plasma samples. Description of experiments Materials and Methods

Blood is obtained from normal individuals, laboratory staff of the Department of Biomedicin and Surgery at the University Hospital of Linkoping, Sweden, who considered themself healthy and who were not on prescribed medication. Trisodium citrate, and calcium chloride, product 32320 are from Riedel-de Haen, Seelze, Germany. In examples 1 and 2, callcium ion activity and pH were determined potentiometrically with the instrument ICA 2, Ionised Calcium Analyser (Radiometer, Copenhagen, Denmark). In Example 3, calcium activity and pH were determined potentiometrically at the routine clinical chemistry laboratory of the Department of Biomedicin and Surgery at the University Hospital of Linkoping in accordance to their routines of November 1998. Coagulation assays are performed with nephelometric clot detection using an ACL 300R (Instrumentation Laboratory, Milan, Italy). Example 1 The effect of citrate on coagulation reactions is demonstrated in a prothrombin (PT) assay of the Owren type, see Owren and Aas. Scand J Clin Invest 1951 ;3:210-8. The assay system is composed of one volume of normal human plasma sample diluted 1 :7 in a 4 mM diethylbarbiturate buffer pH 7.3 containing 132 mM NaCl, one volume of plasma depleted of vitamin K dependent coagulation factors, one half volume of thromboplastin and one half volume of CaCl₂ solution. In the exampled experiment, the concentration of the CaCl₂ solution is varied to give final concentrations of added CaCl₂ in the range 0 to 15 mmol/L. Trisodium citrate is added to diethylbarbiturate buffer to give final concentrations of added citrate of 0, 5, 10 or 20 mmol/L. Coagulation time and calcium ion activity is determined. The latter is determined after the system has coagulated and the coagulated material compacted by centrifugation.

The results, coagulation time and calcium ion activity are plotted in the Figure 1. There are four series of data points. The data points of each series are interconnected to give four curves. The lowest curve (filled circles) connects data points with zero citrate added, the next (open circles) with 5 mM citrate added, the next (filled triangles) with 10 mM citrate added and the highest (open triangles) with 20 mM citrate added. The experiment clearly shows that the coagulation time increases dose dependently with the amount of added citrate and this irrespective of the calcium ion activity. Citrate is thus shown to be inhibitory for coagulative reactions at all coagulation permissive calcium ion activities.

This discovery is important for deeper understanding of the present invention. According to prior art, increased calcium activity levels can be obtained in a citrate anti- coagulated blood product by reducing the amount of added citrate. This, however, gives an anti-coagulated blood product that is more weakly anti-coagulatated than if, according to the present invention, the same calcium ion activity was reach by adding calcium without reducing the citrate level. From the present example, we can understand, that of two samples of citrate anti-coagulated blood product with the same level of calcium ion activitv. one anti- coagulated by addition of citrate (according to prior art) and the other anti-coagulated by addition of citrate and calcium (according to the invention), the one anti-coagulated according to the invention will contain more citrate and enjoy a higher degree of anti-coagulation because of coagulation inhibitory effect of the citrate. The same applies to anti-coagulated blood plasma, synovial fluid and cerebral liquid products. Example 2

Blood collection tubes containing 0.5 mL of anti-coagulant for collection of 4.5 mL of blood are prepared. Six of the tubes contain 0.5 mL water solution with 30, 50, 70, 90, 110 130 and 150 mM of trisodium citrate. Four other tubes contain 0.5 mL water solution with 200, 300, 400 and 500 mM of trisodium citrate and near equimolar amounts of calcium chloride. The calcium ion activity of the anti-coagulant in these latter tubes is adjusted to 1.2 mM. Blood from one healthy person is collected into all 10 tubes. The anti-coagulated blood is stored at room temperature and inspected regularly, over a 48 hours period,for presence of coagulum. After the 48 hours, calcium ion activity and pH is determined. In tubes where coagulation has occured, determination is performed in the blood serum obtained by centrifugation. The results are seen in the Table 1 :

Table 1

Added citrate & calcium, Ca²⁺ activity (mM) Coagulum (C ) or No Coagulation (NC) final concentrations and pH Time intervals (hours)

Citrate Calcium Ca^: 2+ pH 0-1 1-5 5-24 24-48

3 0 0.23 7.50 NC C C C

5 0 0.11 7.46 NC NC NC NC

7 0 0.07 7.44 NC NC NC NC

9 0 0.04 7.44 NC NC NC NC

11 0 0.02 7.44 NC NC NC NC

13 0 0.01 7.43 NC NC NC NC

15 0 O.01 7.43 NC NC NC NC

20 20 0.69 7.43 C C C C

30 30 0.58 7.41 NC NC NC C

40 40 0.57 7.43 NC NC NC NC

50 50 0.48 7.43 NC NC NC NC o

It is to be noted that anti-coagualtion of blood by addition of citrate, according to prior art, requires reduction of calcium ion activity to below 0.1 1 mM. Anti-coagulation of blood by addition of citrate and calcium, according to the present invention, allows anti-coagulation at considerably higher calcium ion activity.

An observation is made during the described experiments. Anti-coagulants containing both citrate and calcium at neutral pH display precipitates after some time period. Since these precipitates most likely are less effective as anti-coagulants than the soluble components, formation of insoluble compounds of citrate and calcium should be avoided. Example 3

To determine the maximum calcium ion activity levels that can be reached in anti- coagulated blood product containing 13 mM of added citrate the following experiments are performed. Anti-coagulant solutions containing 130 mM citrate and 0, 21, 30, 43 or 85 mM calcium are prepared. The 130 mM citrate are obtained by dissolving 65 mmole trisodium citrate and 65 mmole citric acid per liter anti-coagulant which also contains 0, 21, 30, 43 or 85 mmole of dissolved CaCl₂. Blood is anti-coagulated by collecting 4.5 mL into plastic tubes that contain 0.5 mL of each of the five anti-coagulants.

The anti-coagulated blood products are stored at room temperature and inspected often for presence of coagulum during 2 hours. Calcium ion activity and pH are then determined. The results are given in Table 2.

Table 2

Coagulum (C ) or

Added citrate & calcium, Ca²⁺ activity (mM) No Coagulum (NC) final concentrations (mM) and pH Time intervals (hours)

Citrate Calcium Ca^2÷ pH 0-1 1-2

13 0.0 0.07 6.78 NC NC

13 2.1 0.16 6.73 NC NC

13 3.0 0.21 6.78 NC NC

13 4.3 0.40 6.74 NC C

13 8.5 - - C C

Anti-coagulated blood product, according to the invention, can be obtained by addition of 13 mmole of citrate and a maximum of 3 mmole of calcium per liter of the product. This results in a product with calcium ion activity of 0.21 mM. Larger additions of calcium results in greater calcium ion activity levels but also in insufficient anti-coagulation.

No precipitates of citrate-calcium compounds are observed in the acidic anti-coagulant solutions used in the example. This is in contrast to what was observed with pH neutral anti- coagulation solution in Example 2.

The anti-coagulant solution with 130 mM citrate and 30 mM calcium, which is obtained by dissolving 65 mmole trisodium citrate, 65 mmole citric acid and 30 mmole calcium chloride per liter of water, is used in this example to anti-coagulate blood according to the invention. Anti-coagulated blood product is obtained by adding one volume of anticoagulant solution to nine volumes of blood.

In Example 4, below, anti-coagulated blood product is obtained in this way and then centrifuged to sediment the blood cells and allow an anti-coagulated blood plasma product to be harvested as supernatant. In example 4, the stability of coagulation Factor VIII activity in such anti-coagulated plasma product is studied and compared to the stability in anti- coagulated plasma obtained according to prior art.

It should be noted that the concentrations of added citrate and added calcium is considerably higher in the anti-coagulated plasma product than in the anti-coagulated blood product from which the plasma product is obtained. An explanation is that citrate and calcium added to blood only penetrates the blood cells to a limited degree. When the blood cells, which make up some 40% of the blood volume, are removed, the added citrate and calcium are concentrated to the remaining plasma volume. Example 4

Venous blood was collected from one person into tubes containing one of three different anti-coagulant solutions. Each tube contained 0.5 ml of one of the anti-coagulant solutions 1 - 3, and 4.5 ml of blood.

1. Trisodium citrate, 130 mM [13 mmol/L final concentration of citrate in blood] 2. A 50/50 (v/v) mixture of sodium citrate and citric acid, 130 mM [13 mmol/L final concentration of citrate in blood] 3. A 50/50 (v/v) mixture of sodium citrate and citric acid, 130 mM [13 mmol/L final concentration of citrate in blood] + CaCl₂, 30 mM [3 mmol L final concentration of Ca⁺⁺ in blood] The blood was centrifuged for 15 minutes at 2 500 g to obtain platelet poor plasma.

Two samples of each anti-coagulated plasma were frozen to -70 ° C at 0.5 h after blood collection. The rest of the plasma samples were stored at room temperature for 4 h or 6 h before freezing.

The plasma samples were analyzed for Factor VIII content with a spectrophotometric method (Coamatic^® Factor VIII, Chromogenix, Mδlndal, Sweden) on an ACL Futura Plus ( intrumentation Laboratory, Milan, Italy). All plasma samples were thawed just before performing the assay. Duplicate measurements were made from each sample. The results are given in Table 3.

Table 3. Factor VIII content in blood plasma (IU/mL)

Sodium citrate, 1 : 1 Sodium citrate/ citric acid, 1 : 1 Sodium citrate/ citric acid, 13 mmol/L 13 mmol/L 13 mmol/L

+ CaCl₂, 3 mmol/L

0.5 h 1.91 1.97 2.21

1.92 2.04 2.17

2.13 2.10 2.32

2.00 2.17 2.33

4 h 1.78 2.04 2.16

1.83 2.14 2.22

6 h 1.55 1.78 2.22

1.53 1.82 2.17

Statistics:

Values for Factor VIII at 0.5 and 6 h for each different anti-coagulant were compared with a paired t-test:

Sodium citrate, 0.5 v 6 h : p = 0.025

Sodium citrate/citric acid, 0.5 v 6 h : p = 0.046

Sodium citrate/citric acid + CaCl₂ , 0.5 v 6 h : p = 0.50

Factor VIII levels after 6 h (% of mean for 0.5 h) compared with a paired t-test: Sodium citrate v Sodium citrate/citric acid : p = 0.097 Sodium citrate v Sodium citrate/citric acid + CaCl₂ : p = 0.019 Sodium citrate/citric acid v Sodium citrate/citric acid + CaCl₂ : p = 0.13

Claims

192966601/BNCLAIMS

1. Method of anti-coagulating a blood product and counteracting the reduction of calcium ion activity in the anti-coagulated product, comprising addition to the blood product of an anti-coagulant selected from the group consisting of soluble citrate compounds, citric acid, and mixtures thereof in an amount of 13- 60 mmole citrate per liter of anti- coagulated blood product, and of a soluble calcium compound in an amount giving a calcium ion activity in the anti-coagulated blood product of 0.2 - 10 mM.

2. Method according to claim 1, wherein the soluble citrate compound is selected from the group consisting of sodium, potassium and lithium salts of citric acid, and the soluble calcium compound is selected from calcium chloride and calcium acetate.

3. Method according to claim 2, wherein the soluble citrate compound is trisodium citrate and the soluble calcium compound is calcium chloride.

4. Method according to any one of claims 1 - 3, wherein, prior to the additions, the compounds of calcium and citrate are dissolved to form an aqueous solution which is, or is made, sufficiently acidic to prevent the formation of insoluble compounds of calcium and citrate.

5. Anti-coagulated blood or blood plasma product comprising an anti-coagulant selected from the group consisting of soluble citrate compounds, citric acid and mixtures thereof citric acid, and mixtures thereof in an amount of 13- 60 mmole citrate per liter of anti- coagulated blood product, or 20- 100 mmole citrate per liter of anti-coagulated blood plasma product, and an added soluble calcium compound in an amount giving a calcium ion activity in the anti-coagulated blood or blood plasma product of 0.2 - 10 mM.

6. Anti-coagulated blood or blood plasma product according to claim 5, wherein the soluble citrate compound is selected from the group consisting of sodium, potassium and lithium salts of citric acid, and the soluble calcium compound is selected from calcium chloride and calcium acetate.

7. Anti-coagulated blood or blood plasma product according to claim 6, wherein the soluble citrate compound is trisodium citrate and the soluble calcium compound is calcium chloride.

8. Anti-coagulated blood product according to claim any one of claims 5 - 7 comprising 1-20% of added water.

9. Anti-coagulated blood plasma product according to any one of claims 5 - 7 comprising 1-35% of added water.

10. Use of a blood or blood plasma product according to any one of claims 5 - 9 for the production of coagulation Factor VIII.

11. Blood sampling container for collecting nine volumes of a blood product comprising one volume of an aqueous solution containing 130 - 600 mmole per liter of an anti-coagulant selected from the group consisting of soluble citrate compounds, citric acid and mixtures thereof, and a soluble calcium compound in an amount giving a calcium ion activity in an anti-coagulated blood product to be produced of 0.2 - 10 mM.

12. Blood sampling container according to claim 11, wherein the soluble citrate compound is selected from the group consisting of sodium, potassium and lithium salts of citric acid, and the soluble calcium compound is selected from calcium chloride and calcium acetate.

13. Blood sampling container according to claim 11 or 12, wherein the aqueous solution which is sufficiently acidic to prevent the formation of insoluble compounds of calcium and citrate.

14. Use of a blood sampling container according to any one of claims 11 - 13 for transport and storage of an anti-coagulated blood product.