Chemistry: analytical and immunological testing – Biological cellular material tested
Reexamination Certificate
2001-03-16
2002-06-25
Gitomer, Ralph (Department: 1623)
Chemistry: analytical and immunological testing
Biological cellular material tested
C435S004000, C435S029000
Reexamination Certificate
active
06410334
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to a method for determining the immune defence activity of blood, a test kit for performing the determination and the use of a medical blood sampling system for the determination.
2. Description of the Prior Art
The immune defence activity of blood is determined in order to establish whether blood-conveying organisms, particularly mammals and humans, suffer from a disorder, particularly a weakness of the immune system. In the hitherto known methods blood samples are separated after removal, after which there is a separate treatment and examination of white blood corpuscles. It has been found that as a result of storage and transportation, which can last up to 24 hours and in particular in summer is associated with an undesired thermal effect, a considerable part of the activity of the white blood corpuscles can be lost. Research has revealed that within an hour some cells lose 50% of their activity.
White and red blood cells are separated, because blood red cells are considered responsible for disturbing the activity determination of the white blood cells. Following separation the white blood cells are stored in a buffer medium in the presence of nutrient salts and vitamins. It has been found that an activity change is also possible in such a nutrient solution.
The problem of the invention is therefore to improve the reliability and handling of such activity determinations.
Some blood cells are immediately suitable for testing. However, most cells must be activated in such a way that their metabolism is transposed, i.e. new genes are activated, which generally takes at least 6 to 8 hours. It has now been found that the reliability and reproducibility of activity determination can be significantly improved if an incubation and in particular stimulation of the blood cells is carried out substantially immediately after blood sampling without there being any separation into white and red blood cells. According to the invention the white blood corpuscles are incubated and optionally stimulated for as long as they are in the whole blood. The blood separates itself during incubation without centrifuging being necessary. The supernatant then contains the mesenger substances cytokines) supplied by the white blood cells and which represent a measure for the activity of the cells and which can e.g. be found and determined by ELISA. The expression cytokines, apart from cytokines in the narrower sense, also covers other messenger substances such as e.g. histamine, prostaglandins, leucotrienes, etc.
SUMMARY OF THE INVENTION
Thus, the invention provides a method for determining the immune defence activity of the blood by incubating the blood without prior separation, particularly in conjunction with a stimulation of the white blood cells in the whole blood, separation of the supernatant from the sediment and determination of the activity of the blood cells from the separated supernatant.
In many respects the invention leads to the avoiding of errors.
1. Systematic Errors.
Cells normally react on stimulants, e.g. medicaments, not only in an allergic manner, but also on the combination of the stimulants with proteins (carrier proteins), e.g. albumin or lipoprotein, or in combination with other cells, e.g. antigen-presenting cells (APC). In turn, they have surfaces detectable by receptors. The presence of such costimulating signals is additionally necessary in order to bring about a very precise imaging of the natural activities, also to be expected in vivo, of the cells to be examined. Unlike in the case of cultures with isolated leucocytes, whole blood cultures contain said constituents and signals, so that the reaction of the blood cells, for as long as they are present in the whole blood, corresponds more to the natural conditions than in the isolated state. It has also been found that isolated leucocytes have a much greater reaction on stimulants and medicaments than when contained in whole blood, because in whole blood the stimulants and medicaments are frequently bound to erythrocytes and carrier proteins. As a result of this binding the concentration of the stimulants and medicaments is buffered off, which leads to a more gradual release of the messenger substances delivered by the cells.
2. Elimination of Handling Errors.
As stated above, it has been found that the red blood corpuscles do not disturb the activation of the white blood cells (leucocytes) contrary to what has been standing opinion up to now and instead reflect the conditions such as exist in vivo. However, as a result of the separation of the supernatant following incubation it is possible to ensure in a simple manner that the supernatant is not subsequently influenced by the red blood corpuscles (erythrocytes). This has a favourable effect on the storage or dispatch normally necessary after incubation, in that through the introduction of separation the secretion of cytokines in the supernatant is instantaneously prevented and as a result the cell reactions can be ended at a very clearly defined time.
The invention also makes it possible to establish pharmacological and toxicological effects on the immune system.
In a preferred embodiment, without any prior separation, the whole blood is mixed in extracorporeal manner with a blood clotting agent and preferably at least one stimulant (activating agent) for stimulating blood cells, all the blood is incubated in order to stimulate the blood cells, the supernatant form is separated from the sediment and the immune defence activity is determined from the supernatant. The term blood clotting agent more particularly means an anticoagulant with the aid of which the blood is prevented from clotting or coagulating during and after sampling. Incubation preferably takes place in the presence of a nutrient medium. Preferably the nutrient medium is brought together with the blood clotting agent, particularly anticoagulant and in particular with the at least one stimulant before mixing takes place with the blood. As a result the nutrient medium is mixed with the blood in a volume ratio of 5:1 to 1:5, particularly 1:1 to 1:2. The nutrient medium can be placed beforehand in the vessel in which incubation and/or culturing is performed. To obtain reproducible results it is important that incubation is carried out at a constant temperature, more particularly at approximately 37° C. This can appropriately take place in a heating block, which preferably has several reception openings for incubation vessels. Besides using heating blocks, the invention also covers the use of other thermostatically controllable devices. Thus, incubation can also take place in a water bath or e.g. in a corresponding thermostatically controlled cabinet or the like. The incubation period can be a few minutes to approximately 60 hours and is in particular between 8 and 60 hours. The appropriate incubation period is 24 or 48 hours. This also aids the acquisition of reproducible values. During the incubation period the red and white blood corpuscles normally automatically settle. The separation of the supernatant from the blood corpuscles is consequently advantageously carried out without prior centrifuging. Particularly with short incubation periods, it can also be advantageous to carry out centrifuging for the sedimentation of the solid constituents of the blood. The activity can be determined on the supernatant by determining the messenger substances (cytokines) separated from the blood cells. Determination more particularly takes place in per se known manner by ELISA or other suitable testing methods.
Generally the whole blood is incubated in the presence of at least one stimulant. However, working can also take place without stimulants, e.g. if it is to be established to what extent the cells are already prestimulated in the body.
Generally activity determination takes place at a different location to the incubation, or for other reasons activity determination does not take place directly after the end of incubation. Thus, it is advantage
Chaudhry Mahreen
EDI (Experimentelle & Diagnostische Immunologie) GmbH
Gitomer Ralph
Goldberg Joshua B.
Nath Gary M.
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