DE2008493B2 - Hematological control fluid - Google Patents

Description

The invention relates to a hematological control fluid for use in hematological diagnoses, especially as a reference control means for a number of routine haematological determinations.

Hitherto, in hematology a lack of stable control agents for haematological provisions v> regulations. With the advent of automated devices capable of performing multiple hematological determinations, a need for reference samples for manual and machine analysis has developed. <>"

In US-PS 34 06 121 is a stable suspension of red human blood cells for use as Counting standard described in which a stabilizing medium is used which consists essentially of a Saline solution containing osmium tetraxide consists. ■ Such a liquid cannot, however, be used as a reference control for counting white human Serve blood cells.

In US-PS 3412 037 a method for Preparation of a stabilized standard for calibrating a blood counting apparatus described, wherein the Stabilizer medium is a saline solution of yeast, however, yeast is a non-blood material, while seeking to use reference controls, which correspond as closely as possible to the composition of normal human blood.

The object of the invention was to provide a stabilized hematological control fluid for multiple analyzes to create, with which also white blood cells can be determined, which approximate the composition of normal human blood and contains practically no foreign substances

This problem is solved by a hematological control fluid with the features according to characterizing part of claim 1.

Advantageous further developments of the hematological control fluid according to the invention are shown in Claims 2-8 described.

The invention provides a stable control agent created for multiple analyzes, the reference controls for counting red and white blood cells, for the determination of the hemoglobin content and for the hematocrit determination and combined that a mathematical calculation of the mean volume, the mean hemoglobin concentration and the mean hemoglobin value of the blood cells

The hematological control fluid according to the invention consists of a stable cell suspension which corresponds approximately to the composition of normal human blood. On the other hand, with oxal or citric acid treated human blood only limited storage stability. The red blood cells gradually hemolyze and change in size and shape. Likewise, white blood cells experience degenerative changes. The white blood cells of humans are not suitable for control purposes, especially not in the presence of red blood cells, as they are not persistent and secrete a lysozyme enzyme that attacks red blood cells. Red blood cells birds, on the other hand, are larger than human red blood cells and are roughly the same shape and size of human white blood cells. It was found that red blood cells from poultry such as geese, chickens, ducks and preferably the turkey red blood cells in shape and size to the human white blood cells are most similar and are in favor of the inventive method for stabilization with tannin (tanning process) looks good. These stabilized "simulated" white blood cells are good substitutes for human white blood cells in the control product according to the invention.

It has been found that fresh human red blood cells produced by suspension in human Plasma or serum stabilized with an addition of metabolic preservative, useful red Represent blood cells as a component for the mixture according to the invention. Plasma or serum provide some Electrolytes and proteins necessary for the metabolic stability of red blood cells. Selected purines or pyrimidines can be added as additional preservatives. The red blood cells that have not been stabilized with tannin can before the determination of the number of white ones Blood cells and for the subsequent hemoglobin determination are very easily hemolyzed.

Suspensions of human red blood cells and "simulated" white blood cells are mixed in such a ratio that the final red and white blood cell count, hemoglobin content, and hematocrit result fall within what is considered normal for human blood. _t

The number of red blood cells in human blood is usually in the range of

4,000,000-5,000,000 cells / mm ³ and the number of white blood cells between 5,000 and 10,000 cells / mm ³ . The hemoglobin content is usually 12-16 g / 100 ml. The term "hematocrit" means the ratio of the volume of packed red blood cells to the volume of the whole blood. The normal ratio in humans is around 45%. The mean volume of blood cells is the ratio of the volume of compacted red blood cells in ml / 1 blood to the number of red blood cells in millions per m ³ . The mean hemoglobin concentration of blood cells is an index of the mean or average weight of hemoglobin per 100 ml of densified red blood cells as a percentage. The mean hemoglobin value is the ratio of the hemoglobin content in g / l to the number of red blood cells in millions per mm ³ .

Obviously, a comparative control product must indicate exactly what a comparative product is made of Test blood sample with regard to the above 1 described determinations exists. It is still apparent the importance of the control product being a replica of untreated cells. if For example, a control product contains cells of significantly different sizes, are the test results imprecise, if not completely meaningless. The according to the procedure described here treated cells provide an excellent control and compensation system, which is the case with hematological Provisions is absolutely necessary.

In the following, an example for the production of egg eggs is given to explain the invention Control fluid described.

The term "saline solution" as used below can mean one of the following three solutions: one 0.9% by weight aqueous sodium chloride solution, referred to as "solution A"; a 0.45% aqueous Sodium chloride solution called "solution B" or a buffered saline solution (solution C). In the preparation of of the "simulated" white blood cells as a control substance, it is important that the 0.45% solution (solution B) is used because at this concentration the usually oval shape of the red blood cells of Poultry advantageously in a more rounded shape, like human white blood cells have, is converted.

The first step in the process of stabilizing poultry red blood cells is to have the red blood cells are suspended in the saline solution to form a suspension with a concentration of up to 50% by volume Although a concentration of 50% is useful, a concentration of about

5 to 10% by volume, in particular 8%, are used. This suspension is made with an equal volume of a 1-bis 3% by weight solution of mercury chloride in Saline solution mixed.

The second stage consists of an at least 12 hours, preferably about 19 to 20 hours of heating the resulting mixture at 37 ⁰ C. The solids are recovered by centrifuging and separation, and then again in saline solution of about a 2 to 6%, preferably 4% igen suspension suspended.

In the third stage, the suspension described above is mixed carefully with a equal volume of a 0.0025% (w / v) solution of aqueous tannin in saline united; then the resulting mixture is incubated and the solid mixture is centrifuged ίο and severing won. Containing the tannin Saline is a buffered saline solution with a pH of 7.2 and is that mentioned above "Solution C". It consists of the following components in the specified proportions (per liter):

0.9% saline solution 500 ml

0.15 molar disodium phosphate solution 385 ml
0.15 molar potassium phosphate solution 115 ml

The stabilized cells are washed several times with saline and then in sterile Reconstituted Alsever solution.

The preparation of the suspension of human red blood cells is carried out under sterile conditions Red blood cells of any type of blood can be used for this. However, preferably the Blood group 0 is used because donors can easily be found for this group. That in an anticoagulant group 0 blood collected using acidic citrate dextrose solution is centrifuged; the protruding Fluid is removed and set aside, the white blood cell buffer layer removed and discarded. The compacted red blood cells are washed several times with Alsever solution, in Alsever solution reconstituted and stored at 2 - 8 ° C.

The resuspending medium used for the final human red blood cell suspension is made from a defibrinated plasma that contains suspended human red blood cells is serologically acceptable, d. H. Group A blood cells with group A plasma, group B blood cells with Group B plasma and group 0 blood cells with group 0 plasma. Defibrinated group plasma AB (missing antibodies A and B) is compatible with red blood cells of groups A, B and 0. The

π supernatant plasma fluid is made by treatment defibrinated with bovine thrombin and then diluted in a ratio of 1: 3 with Alsever solution.

To further stabilize the fresh blood cell component of the final cell composition

) (i pension is the suspending medium with a Surine or pyrimidine as a metabolic preservative in such an amount added so that a final concentration of about 34-35 mg per ml of the cell suspension is obtained. A

> ■> Adenine is a particularly effective preservative (6-aminopurine or a pharmaceutically acceptable acid addition salt manufactured by Mann Research Laboratories, Inc., New York, N.Y.).

The following examples serve to further

> Explanation of the invention.

example 1

Group 0 blood collected in an aqueous acidic citrate-dextrose anticoagulant was found under centrifuged under sterile conditions at 200 rpm for 20 minutes. The supernatant became cautious aspirated, taking care that in the border area between the red cells and the

supernatant no red cells or the white buffer layer in the supernatant were withdrawn. The supernatant was diluted with Alsever's solution as described below and use as a resuspending agent

The buffer layer (white blood cells) was carefully removed over the compacted red blood cells, whereby if necessary, a thin layer of the red cells is also peeled off. The red cells were compacted Washed twice with Alsever solution, centrifuged and stabilized at 2-8 ° C until mixed with "Simulated" white blood cells stored

Example II _{| 5}

Red blood cells from poultry, preferably from a turkey, were washed four times with saline (solution A) washed (centrifuging at 3300 rpm for 20 minutes after each wash; and after the final wash to an 8vol.% suspension in saline solution (solution A) resuspended 100 ml of this 8% suspension were mixed with 100 ml of a 1% by weight solution of mercury (II) chloride in Saline solution (solution A) added; the resulting suspension was carefully mixed, then it became Incubated for 19 hours at 37 ° C, four times with distilled Water (although centrifuged at 1200 rpm for 5 minutes after each wash) and in Saline solution (solution A) resuspended to form a 4vol.% Suspension. This 4% suspension was with 3d an equal volume of a 0.0025% (weight per volume) solution of tannin in buffered Salt solution (solution C) was added and the resulting mixture was carefully mixed for 30 minutes at 50 ° C incubated, centrifuged and distilled three times with J5 Water washed.

Example III

The supernatant plasma, which is produced in an aqueous acidic citrate-dextrose anticoagulant collected group 0 blood was diluted 1: 3 with Alsever's solution. That diluted plasma was with the addition of bovine thrombin (manufactured by Parke-Davis% Co, Detroit, Michigan) defibrinated at a concentration of 1 unit / ml and incubated at 37 ° C for 1 hour Lumpy material was shaken vigorously and the lump was broken up and passed through a filter (Whatman No. 50), which was located on a Buchner funnel over a suction bottle, adenine sulfate was filtered in a Concentration of 70 mg / 100 ml added; to speed up the dissolution the mixture was made up Heated to 37 ° C; then the solution was successively through filter bodies with a pore size of 0.45 and 0.22 Millipore (0.45 and 0.22 Millipore filter pads) filtered

Example IV

The procedure of Example III was repeated with the plasma of group 0 replaced by plasma of the Group AB was replaced.

Example V

The "simulated" white blood cells produced according to Example II were reconstituted in sterile Alsever solution to a hematritic value of about 24%. This I solution contains about 750,000-800,000 cells / mm ³ . Two ml of this suspension were further diluted with the addition of 100 ml of the resuspending agent prepared according to Example III or IV.

A volume of the compressed human blood cells of group 0 prepared according to Example 1 (about 90% hematocrit) was with an equal volume of the diluted suspension of the "simulated" white blood cells

The cell suspension obtained as the end product with a content of about 34-35 mg adenine per 100 ml had about 4,000,000-5,000,000 red blood cells per mm ³ , 5000-10,000 white blood cells per mm ³ , 12-16 g hemaglobin per 100 ml and 43-47% hematocrit.

Aliquots (2.5 ml) of the final suspension in glass vials distributed, kept for about 4 weeks when stored at 2-8 ° C.

Claims (8)

Patent claims: 1. Hematological control fluid, characterized in that it consists of a suspension of fresh human red blood cells and of tannin-stabilized red poultry blood cells in a serologically compatible human pias medium containing an anticoagulant, the concentration of poultry blood cells being about 5000 to 10,000 cells per mm ³ and the concentration of human red blood cells is about 4,000,000 to 5,000,000 cells per mm ³ . 2. Hematological control fluid according to claim 1, characterized in that the fresh human red blood cells are group 0 red blood cells. 3. Hematological control fluid according to claim 1, characterized in that the with Tannin-stabilized fowl red blood cells are turkey red blood cells. 4. Hematological control fluid according to claim 1, characterized in that the anticoagulant active agent in the human plasma medium is acidic citrate-dextrose solution 5. Hematological control fluid according to claim 1, characterized in that the plasma medium from defibrinated with Alsever solution in a volume ratio of about 1: 3 human plasma in an anticoagulant consisting of an aqueous acidic citrate-dextrose solution acting means exists. 6. Hematological control fluid according to claim 5, characterized in that the defibrinated human plasma is group 0 plasma. 7. Hematological control fluid according to claim 5, characterized in that the defibrinated human plasma is group AB plasma 8. hematological control fluid according to claim 1, characterized in that it per 100 ml about 34-35 mg 6-aminopurine or one of its pharmaceutically acceptable acid addition salts contains. «