Chemistry: analytical and immunological testing – Biological cellular material tested
Reexamination Certificate
1999-09-24
2001-10-30
Warden, Jill (Department: 1743)
Chemistry: analytical and immunological testing
Biological cellular material tested
C436S086000, C436S174000, C436S176000
Reexamination Certificate
active
06309885
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method and a blood-withdrawal vessel for preparing blood samples for detecting homocysteine and/or total folate. In particular, “preparation” is defined here as the stabilization of blood samples for the detection of homocysteine and the lysis of the erythrocytes in order to prepare the detection of total folate. In conjunction with this invention, “total folate” is defined as the sum of erythrocytic and plasma folate.
PRIOR ART
Homocysteine, a sulfur-containing amino acid, occurs in the organism only as an intermediate product in the methionine/cysteine/glutathione metabolic circulation and is not incorporated into proteins. Various hereditary defects of the key enzymes cystathionine-&bgr;-synthetase and methylene-tetrahydrofolate-reductase (MTHFR) or a deficiency of corresponding vitamin cofactors (B
12
, B
6
, folate) cause homocysteine to be insufficiently broken down and therefore it arises in increased concentrations in the plasma (K. Berg et al., Clin. Genet. (1992) 41:315-321; P. Goyette et al., Am. J. Hum. Genet. (1995) 56:1052-1059). Numerous clinical studies over the last few years were able to identify this hyperhomocysteinemia as a risk factor independent of lipid metabolism as regards atherosclerotic disorders and thrombo-embolic consequences thereof. 30-40% of patients who prematurely suffer a coronary (R. Clarke et al., N. Engl. J. Med. (1991) 324: 1149-1154) or a stroke (B. Israelsson et al., Scand. J. Clin. Lab. Invest. (1993) 53: 465:469), angina pectoris or peripheral arterio-occlusive disease (L. Brattström et al., Atherosclerosis (1990) 81: 51-60) exhibit an increased level of homocysteine. The prevalence of the homozygotic MTHFR defect accompanying an increased homocysteine level is given at 59, (Goyette et al., loc. cit.), which lies in the order of magnitude of the prevalence of diabetes mellitus in the entire Caucasian population.
Homocysteine is currently measured with the aid of HPLC (high-pressure liquid chromatography) from EDTA plasma and such a measurement can be performed with reasonable accuracy. It is nevertheless necessary to optimize the pre-analytics of the detection of homocysteine, because the in vitro release of this amino acid from the blood cells, which also continue to produce homocysteine in the withdrawal vessel after withdrawal of the blood and which release this substance from the cells as a result of an active transport process, may considerably distort detection in the plasma. For this purpose, it has so far been necessary to employ centrifugation so as to separate the cells from the plasma immediately after withdrawal of the blood. The homocysteine concentration in the plasma is then stable for at least 48 hours (T. Fiskerstrand et al., Clin. Chem. (1993) 39/2: 263-271). This complicated procedure cannot, however, be routinely performed in standard ward mode or by the General Practitioner (GP).
As a partial aspect, the present invention is therefore based on the problem of avoiding a significant homocysteine increase in the whole blood, an increase which naturally occurs within one to two hours of venous blood withdrawal (FIG.
1
). The hitherto necessary and time-critical separation of the blood cells from the plasma can be avoided by the present invention, and the stabilization of the homocysteine concentration in the resultant lyzed blood can be reached over 48 hours.
It is already known that blood samples should be protected from coagulation by addition of specific reagents (NaF, EDTA, citrate) and stabilized against enzymatic breakdown processes. The use of these reagents in standard blood-withdrawal vessels, however, does not achieve any stabilization of the homocysteine concentration, since these enzyme inhibitors are not absorbed into the blood cells and consequently an intracellular inhibition of the enzyme that produces the homocysteine cannot be achieved (FIG.
1
). Within two hours, the currently valid limit of 15 &mgr;mol/l (K. Rasmussen et al., Clin. Chem. (1996) 42:4 630-636), above which the risk of atherosclerosis increases significantly, is therefore already exceeded or virtually reached by samples exhibiting a very low initial concentration. This is evident from
FIG. 1
which shows the significant increase of different initial homocysteine concentrations in the blood treated with NaF or EDTA at room temperature and at 4° C. This rise can amount to as much as 100% within 6 hours.
As stated above, the homocysteine concentration frequently correlates inversely with the folate concentration, since folate is a cofactor for the enzyme MTHFR that breaks down homocysteine. In consequence, folate is frequently given in order to return a pathologically elevated homocysteine concentration to the normal range. A detection of the basal and actual folate level in addition to the homocysteine level is therefore diagnostically appropriate. Since folate is mainly present and takes effect in the erythrocytes (approx. 98%), the detection of the erthyrocytic folate or total folate is more conclusive than the usual detection of the plasma folate or serum folate. The present invention is therefore based on the further problem of preparing a blood sample such that the concentration of total folate can be immediately detected in addition to or instead of the homocysteine concentration.
DESCRIPTION OF THE INVENTION
In accordance with the invention, the above objects are solved by a method for preparing blood samples for detecting homocysteine and/or total folate, in which method the blood sample is brought into contact, during or immediately after withdrawal of blood, with
a) at least one reagent for lysis of the blood cells and
b) at least one inhibitor of the enzymes which produce and break down homocysteine.
Particular preference is also placed on bringing the blood sample into contact with c) one or more acids.
The invention is also directed at a blood-withdrawal vessel for preparing blood samples, particularly for detecting homocysteine and/or total folate, this blood-withdrawal vessel comprising
a) at least one reagent for lysis of the blood cells and
b) at least one inhibitor of the enzymes which produce and break down homocysteine and preferably
c) one or more acids.
The dependent claims relate to other preferred embodiments.
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“Measurement of Homocysteine Concentrations and Stable Isotope Tracer Enrichments in Human Plasama” MacCoss et al, May 1999.*
Ubbink et al., “Vitamin B-12, vitamin B-6, and folate nutritional status in men with Hyperhomocysteinemia1-3”, Amer. Soc. For Clin. Nutr., 57:47-53, 1993.
Probst et al., “Stabilization of Homocysteine concentration in whole blood,” Clin. Chem., 44:1567-1569, No. 7, 1998.
O'Broin et al., “Erythrocyte, Plasma, and Serum Folate: Speciment Stability before Microbiological Assay,” Clin. Chem., pp. 522-524, 1980.
Israelsson et al., “Homocysteine in frozen plasma samples. A short cut to establish Hyperhomocysteinaemia as a risk factor for arteriosclerosis,” Scand. J. Clin. Lab Invest, 53: 465-469, 1993.
Goyette et al., “Seven Novel Mutations in Methylenetetrahydrofolate Reductase Gene and Genotype/Phenotype Correlations in Severe Methylenetetrahydrofolate Reductase Deficiency,” Am. J. Hum. Genet, 56: 1052-1059, 1995.
Malinow et al., “Population variation and genetics of plasma Homocyst(e)ine level,” Clin. Genet., 41:315-321, 1992.
Clarke et al., Hyperhomocysteinemia: An Independent Risk Factor for Vascular Disease, The New Eng. J. of Med., 324: 1149-1155, Apr. 25, 1991.
Fiskerstrand et al., “Homocysteine and Other Thiols in Plasma
Blümke Matthias
Probst Reiner
Cross LaToya I.
Fitch Even Tabin & Flannery
Reinee Probst
Warden Jill
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