DE19811739A1 - Determination of efficacy of tumor therapy - Google Patents

Abstract

Determination of apoptotic products in serum samples from tumor patients is used to determine the efficacy of tumor therapy. An Independent claim is also included for determining apoptotic products in samples from tumor patients, where the concentration of apoptotic products in serum samples correlates with the efficacy of tumor therapy and thus serves to monitor the progress of therapy.

Description

The present invention relates to a method for the determination of apoptotic Products in samples taken from tumor patients, the concentration of apoptoti products in serum samples is correlated with the effectiveness of tumor therapy and thus serves to monitor the course of therapy. The serum samples are taken at different times taken and determined. In particular, the subject of the present invention is a ver drive, in which the concentration of nucleosomes in serum samples from tumor patients be is true to determine the effectiveness of tumor therapy. Furthermore is the use Development of a method for the determination of apoptotic products in serum samples from tumor pa The subject of the present invention was used to determine the effectiveness of tumor therapy dung.

A eukaryotic cell can die in two fundamentally different ways. A Necrosis is gone, cell death as a result of damage, e.g. B. due to a insufficient blood supply and thus undersupply with oxygen and nutrients, or as Consequences of poisoning or physical damage (mechanical injury, frost or Heat). The other way, the so-called programmed cell death, is of great importance for the Development and functionality of tissues, organs and organisms as a whole (1991, J. Cohen, Advances in Immunology 50: 55-85). Those who died this way Cells have DNA fragments associated with histones in the cytoplasm as mono- and oligonu kleosome on. Such an appearance is also observed in induced cell death, how he z. B. by ionizing radiation (Yamada et al., Int. J. Radiat. Biol. 53 (1988), 65) or certain monoclonal antibodies such as e.g. B. Anti-Fas (Yonehara et al., J. Exp. Med. 169 (1989), 1747-1756) or anti-APO-1 (Trauth et al., Science 245 (1989), 301-304)  becomes. Cytotoxic T cells and natural killer cells also induce such Cell death with the described appearance (Sanderson, Biol. Rev. 56 (1981), 153-197, p. Curnow et al., Cancer Immunol. Immunother. 36 (1993), 149-155 and Berke, Immunol. Today 12: 396-399 (1991). However, they also cause an increased permeability of the plasma membrane, as observed in necrosis (Krähenbühl et al., Immunol. Today 12 (1991), 399-402). The type of programmed or induced cell death described is called apoptosis (Wyllie et al., Int. Rev. of Cytol. 68 (1980), 251-301). It is characterized by vesicular protuberances of the plasma membrane, condensation of the cytoplasm and Activation of an endogenous endonuclease. In contrast to necrosis, it is the Apoptosis is an active process of the eukaryotic cell. He will therefore not be at killing the cells by heating, freezing and thawing or lysis with a lytic acting antibody (R. Duke et al., Proc. Natl. Acad. Sci. 80 (1983), 6361-6365). The calcium and magnesium dependent endonuclease activated during apoptosis cleaves the DNA double strand in the easily accessible linker regions between the nucleosomes in Mono and oligonucleosomes. The DNA in the nucleosomes, however, is with the core histo Nene H2A, H2B, H3 and H4 closely associated and therefore protected against the cleavage by the En donuclease (Burgoyne et al., Biochem. J. 143 (1974), 67 and Stach et al., J. Neurochem. 33 (1979), 257). Therefore, after extraction of the DNA and separation in the agarose gel, the apop to recognize total cells typical "DNA ladder", a pattern of DNA fragments with a Length of approximately 180 base pairs or a multiple thereof (Wyllie, Nature 284 (1980), 55-556 and J. Cohen, Advances in Immunology 50 (1991), 55-85). The plasma membrane of the cell len remains intact at this early stage of apoptosis. There is an enrichment of the Mono- and oligonucleosomes in the cytoplasm of the dying cell (Duke et al., Lymphokine Research 5: 289-299 (1986).

Apoptotic processes can be observed in patients with malignant and benign tumors become. Increased cell turnover is often observed in malignant tumors. H. the cell proliferation rate - as well as the cell death rate is increased. The latter is an expression of massive apoptoti cell end. Regulated, programmed breakdown of DNA, cell nucleus and organelles, Packaging of the cell contents in "apoptotic bodies", as well as phagocytosis by macrophages and Neighboring cells ensure complete recycling without residues. Is this disposal system  stem overload, cellular components and. a. also in the bloodstream. So in Se rum and plasma from patients with malignant tumors found higher DNA concentrations, the DNA in the circulation mainly bound to histones in the form of mono- and Oligonucleosomes.

The amount of apoptotic products, for example nucleosomes, in the serum can be cor are related to the extent of the malignancy. Advanced diseases white sen z. B. a significantly higher concentration of serum nucleosomes. The is known further that by radiotherapy and by various cytostatic drugs apoptosis is induced.

Surprisingly, it was found that the concentration of apoptotic products in the serum of tumor patients during radio or chemotherapy can be used as a marker for the efficiency of the therapy. And thus is a method for the determination of apoptotic products in samples taken from tumor patients, the concentration of the apoptotic products in serum samples being correlated with the effectiveness of the tumor therapy and thus serving to monitor the progress of the therapy. The serum samples are taken and determined at different times. In particular, the present invention relates to a method in which the concentration of nucleosomes in serum samples from tumor patients is determined in order to determine the effectiveness of the tumor therapy. A method for determining the concentration of nucleosomes in serum samples from tumor patients is particularly preferred in which

• a) the serum of a tumor patient with an anti-histone antibody and an anti-DNA anti body incubated, one of the antibodies before or after this incubation to a solid phase is bound and the other antibody is a labeled antibody,
• b) separates solid and liquid phase and
• c) the marker is determined in one of the two phases in order to determine the concentration of the nucleosomes in serum samples from tumor patients. For example, the commercially available Cell Death Detection ^plus ELISA from Boehringer Mannheim GmbH, Germany, is suitable for determining the concentration of nucleosomes in serum samples from tumor patients.

The invention furthermore relates to the use of a method for determining apoptotic products in serum samples from tumor patients for determining the effectiveness of tumor therapy. It is particularly preferred to use the Cell Death Detection ^plus ELISA kit from Boehringer Mannheim for the determination of apoptotic products in serum samples from tumor patients.

In particular, the nucleosome concentration in the serum was in the course of a radio or chemotherapy treatment of tumor patients and observed with the clinical Course and tumor markers correlated. They occurred very quickly after the start of therapy different strongly increased concentrations of nucleosomes in the serum, which in the therapy ver running were observed and therapy efficiency was to be indicated at an early stage, particularly with Radiato seem to. The nucleosome concentration in the serum was determined, for example, before Therapy, 3 hours, 6 hours, 1 day, 4 days, 7 days and weekly after the start of Therapy. The increase in the concentration of nucleosomes in serum, the maximum value that Delay in the decline and the minimum concentration was determined with the clinical Course correlated.

A correlation was found between the pre- and post-therapeutic concentration the nucleosomes in the serum of tumor patients. In patients with a high spontaneous Apoptosis, high concentration values were also found during therapy (7/16). At Patients with a pretherapeutically low concentration of nucleosomes remained in the serum the value also low (5/16) or showed an increase (4/16). However, it turned out that the Maxi Mal concentration of the nucleosomes in serum has no influence on the clinical course of the Therapy or disease in the tumor patients.

With a decrease in the tumor and a positive clinical course, a decrease could occur the concentration of the nucleosomes in the serum directly after the maximum value on a very low minimum value can be observed (6 of 7). The late sinking and higher minimal  scores (5 out of 5) were observed in patients with poor clinical course or even death.

example 1 Cell Death Detection ^plus ELISA Test principle

The Dell Death Detection ^plus ELISA is based on the quantitative sandwich enzyme immunoas say principle. Two mouse monoclonal antibodies directed against DNA and histones are used. The test can be performed with cytoplasmic lysates, culture supernatants, plasma, serum and other body fluids and allows specific detection of mono- and oligonucleosomes. The description given here applies to measurements in liquid media:

protocol

20 µl each of the material to be tested are coated in a well of a streptavidin Pipetted microtiter plate (MTP). 80 µl of an immunoreagent, consisting of bio tinylated anti-histone antibodies, peroxidase-labeled anti-DNA antibodies and Inku Bationspuffer, added, covered with an adhesive film and for 2 hours on a MTP shaker (500 rpm) incubated. The anti-histone antibody reacts during this incubation period with the histone component of the nucleosomes and fixes the complex using the biotin the streptavidin-coated microtiter plate. The anti-DNA antibody also binds to the DNA component of the nucleosomes. Both ds and ss DNA are recognized. After Remove the still unbound antibodies by washing three times with 300-300 µl each The fixed complexes are incubated with 100 μl ABTS (2,2'-azino-di (3-ethyl benzthiazoline sulfonate)) incubated, the MTP sealed with an adhesive film and at 250 rpm shaken. The substrate reacts with the peroxidase label of the anti-DNA antibodies and causes a photometric proportional to these antibodies at d = 405 nm against the sub strat solution quantified as a blank (reference wavelength d = 492 nm).  

Duplicates of the sample materials are analyzed. In addition, a negative con trolls carried along, for the calculation of the enrichment factor recommended by the manufacturer (see below).

solutions

• - Incubation buffer: PBS with 1% RSA (BM order no. 238 040), 0.5% Tween 20 (BM order no. 1333 465) and 1mM EDTA
• - Anti-histone biotin: biotinylation (Biotin Labeling Kit, BM, order no. 1418 165) one Anti-histone panels (BM, order no. 1492 519); Prepare a 50 mg / ml solution Anti-histone biotin in incubation buffer
• - Anti-DNA-POD: conjugate anti-DNA (BM, order no. 1003 399) with activated POD (BM, order no. 1428 861); Prepare a solution of 5 units / ml anti-DNA-POD in Incubation buffer
• - Immunoreagent: Careful homogenization of 1/20 parts by volume of anti-histone bio tin 1/20 parts by volume of Anit-DNA-POD and 18/20 parts by volume of incubation buffer
• - Substrate buffer: ready-to-use solution from BM (order no. 1204 530); alternatively dissolving 1047.5 mg citric acid, 62.5 mg sodium perborate × 3H ₂ O, 9.77 mg CaCl ₂ × 2H ₂ O, 1335 mg Na ₂ HPO ₄ × 2H ₂ O and establishing a pH of 4.5 a total of 125 ml
• - Substrate: ABTS tablets (BM order no. 1204 521); Dissolve one tablet in 5 ml sub strat buffer
• - Streptavidin-coated microtiter plate: SA-MTP (BM, order no. 1734 776)
• - Adhesive films: every available film

Specificity of the antibodies

The anti-histone antibodies have specific affinity for the histones H1, H2a, H2b, H3 and H4. The anti-DNA antibodies bind to ss- and ds-DNA. Nuclear nucleosomes of eukaryotic cells are detected. In mitochondria, prokaryotic cells, as well as in bacteria, as well as in viruses, DNA is not associated with histones and is therefore not in nucleosome form. RNA is also not bound to histones and therefore cannot be detected ( ^{cell biology book} ).

Sensitivity of the test

The ELISA is able to detect the DNA content of 10 ³ cells.

interpretation

After averaging the measured duplicate values, the manufacturer recommends an interpretation of the nucleosome concentration of the sample materials based on the enrichment factor (AF) according to the following formula:

AF (mU) = mU of the sample / mU of the untreated negative sample.

Example 2 Modification and standardization Enrichment factor

On the basis of the enrichment factor (AF) it is possible to compare the values in given within one test, but not two tests carried out on different days (see below).

Standard curve

A standard curve with highly apoptotic material is therefore used as the basis for interpretation dressed:

The incubation of whole EDTA blood from three healthy blood donors over 24 hours becomes massive Induced apoptosis. After centrifugation, the plasma is lyophilized and stored at 4 ° C. At Resuspension at different times become reproducible maximum values in the upper Measuring range of the photometer achieved with an optical density (OD) of 2500 mE; there is one good long-term stability and a linear dilution behavior in the entire range of 0 up to 2500 mE. Comparability both within a test and between several Other test approaches are guaranteed.  

Measuring time

Pipetting a complete microtiter plate (96 wells) by hand takes approx. 90 seconds with a multipette. In the last pipetting step, this leads to a difference in the Incubation time of the ABTS substrate between the first and last well of 15% (at a Color development time of 10 minutes). Around a reasonable measurement inaccuracy limit of 5% ABTS incubation is therefore not to be exceeded and to keep the total test time low on time set to 30 minutes.

Dilutions

The highest standard value is the 1:24 dilution of the standard material with incubation puff fer (see above), which reaches the required 2500 mE after 30 min. The further dilutions Steps (1:24, 1:32, 1:48, 1:64, 1:96, incubation buffer only) result in a linear dilution on curve that corresponds to a straight line of origin.

Unit of measurement

Nucleosomes may be accessible differently for the binding of antibodies, depending on the form in which they are present: as mono- or oligonucleosomes, the latter with or without a tertiary structure. The amount of bound and POD-labeled anti-DNA antibodies does not necessarily correspond to the exact nucleosome concentration in the sample material.

Therefore, an absolute concentration is not specified and scaling with so-called AUs (arbitrary units) introduced. 1000 AU are the highest standard value (2500 mE after 30 min) equated; scaling is linear, i. H. 500 AU is about 1250 mE, 100 AU about 250 mE etc.

This is the ignorance of the relationship between mono- and oligonucleosomes and there accounted for by conditional, changed behavior of the antibodies, but at the same time comparability between tests increased.  

Example 3 Preanalytical standardization matrix

In the present investigations, serum is used as the matrix. Hemolysis due to improper blood collection (too long stowage) or inadequate storage of the Blood sample or its transport to the laboratory (exposure to heat or shaking, too long ver delay in transport) can lead to false positive test results. The centrifugation therefore takes place within a period of 1-2 hours after removal for 15 min. at 3000 rpm.

stabilization

By adding 10 mM EDTA (pH 8) directly after the serum CA ²⁺ - and MG ² - from dependent endonucleases, z. B. DNase I, and acidic endonucleases, such as DNase II with an optimum activity at pH 4.5 inhibited. This prevents possible splitting off of the anti-DNA antibodies and a decrease in the measured values.

• - EDTA solution: Prepare a solution with 100 mM EDTA in TRIS buffer, adjust one pH of 8.0
• - Serum-EDTA mixture: 1/10 part by volume 100 mM EDTA is mixed with 9/10 parts by volume Mixed serum and homogenized with vortex.

After adding EDTA, the serum can be stored at 4 ° C for several hours the.

stability

200 µl of the serum treated with EDTA are filled into Greiner freezer tubes (2 ml) and frozen at -20 ° C. At this temperature, the serum is stable for up to 6 months. For possible re-measurements are made of 3 tubes of the same serum, for determination of different Pa Freeze 2 untreated serum tubes at -20 ° C.  

Test preparation

After thawing, adjust to room temperature and carefully homogenize the Ensure sample material with no difference between centrifugation and use of a vortex.

The serum samples are diluted 1: 4 with incubation buffer (20 µl serum, 60 µl incubation onspuffer), vortexed and then used in the test. The rest of the serum is discarded fen, as the readings can increase with repeated freezing.

Dilutions with incubation buffer, which are prepared before freezing, lead to Standard material as well as serum for lower measurement results. In comparison with the Standard curve, a parallel line of dilution can be observed when diluting the serum ten.

Patient

In our investigations, sera from 363 subjects were evaluated. There of were 303 sera from patients who were in the period from August 1996 to December 1997 were treated in the Großhadern hospital and 60 healthy people.

Healthy people

60 subjects were examined for their clinical and clinical-chemical health. The ge was done by taking a medical history, a questionnaire and the parameters CRP, creatinine, GGT, transaminases and blood count. The age distribution ranged from 20 to 60 years an average of. . ., 32 women (53%) and 28 men (47%) participated in the study.

Patients with inflammatory diseases

50 of the patients suffered from acute inflammatory diseases, which according to the values of the C-reactive Proteins were divided into five different categories (I: CRP <1 ng / mL, II: 1 ng / mL <CRP <5 ng / mL, III: 5 ng / mL <CRP <10 ng / mL, IV: 10 ng / mL <CRP <20 ng / mL, V: 20 ng / mL <CRP).  

Patients with solid tumors

We examined 253 patients with solid tumors; 69 ovarian tumors, 53 Mammary tumors, 53 colon carcinomas, 41 bronchial carcinomas and 37 other gastrointestinal Carcinomas. Blood samples were taken before surgical primary therapy or before chemotherapy or Radiotherapy taken to increase the spontaneous cell death rate and thus nucleosome release receive.

Follow-up

Of these, 15 were also treated with both primary and secondary radiotherapy (6 with BC, 4 with lymphoma, 4 with ENT-CA, 1 with Colon-CA) and 16 patients under Che motherapy (6 with lymphoma, 6 with colon CA, 2 with pancreas CA, 2 with sarcoma) in the ver run observed.

In the former, blood samples were taken before radiation, 3, 6, 24 and 72 hours and one week after the start of the radiation and also before the start of the first weekly radiation unit. Since most patients' regimen is a daily regimen radiation, it should be taken into account that after the 4th measurement a new therapy unit took place.

Patients on chemotherapy were given therapy one day, three days and one week after the first dose, also weekly or before the next therapy session begins. The Patients were informed and agreed to be included in a study.

Example 5 Methodological results matrix

We tested the serum and plasma of the same subjects in parallel (n = 10), of whom 4 were healthy, 4 were tumor patients and 2 were patients with acute inflammatory diseases. As described by Steinmann, So rensen and Fournie for free DNA ³⁰ , ²⁵ , ²⁶ , we found mostly higher concentrations of nucleosomes in serum than in plasma (n = 9). In a patient with extremely high CRP values above 45 ng / ml, the measurement results in serum and plasma were equally high.

stability

These serum and plasma samples were taken with and without the addition of 10 mM EDTA (pH 8) Store at 25 ° C, 4 ° C, -20 ° C and -80 ° C for a period of 3 weeks. The best stability was observed in serum which was ver. immediately after centrifugation with 10 mM EDTA (pH 8) sets and was stored at a temperature of -20 ° C or -80 ° C.

More matrices

Nucleosomes were also detected in the urine of a healthy subject. With a molecule lar weight of. . . of a mononucleosome and renal mobility up to a molecular size of . . . the appearance of nucleosomes in the urine is quite physiological.

Daily profile

To rule out a circadian rhythm, several were used in a healthy subject Blood samples taken every two hours during a day. During the course no fluctuations (10%) of the measured values beyond the VK.

Preanalytics Influence of hemolysis

Slight hemolysis was demonstrated in a whole blood sample by brief shaking and heat exposure (37 ° C) over 4 hours. After Abseren became a healthy, low apoptosis serum titrated. Even with the addition of 1 µl hemolysate / 250 µl serum there was an increase in To recognize measurement signal that increased steadily with increasing amount of hemolysate.

Influence of the time of centrifugation

Whole blood samples were taken under different storage conditions (37 ° C, 25 ° C, 4 ° C) for 0, 2, 4 and 6 hours before centrifugation. After that they were with EDTA (see above) mixed or frozen untreated. The longer the period, the higher  Values were measured in the serum. Different temperatures, however, do not seem to be an on to have flow.

Influence of the time of EDTA addition

After centrifugation, sera were stored at 37 ° C, 25 ° C and 4 ° C, before 0, 2, 4, 6 or 10mM EDTA (pH 8) was added for 8 hours. After that, they were immediately - Frozen at 20 ° C.

The later EDTA was added to the serum, the lower signals were obtained in the serum While the drop in the first two hours was between 8 and 12% per hour, the values decreased linearly between the 2nd and 8th hour, by about 6% per hour, so that after 8 hours only half of the original measurement signal could be detected. Later flattened the curve. Exceptions were very low or above the measuring range Values that remained constant. The temperature had no significant influence.

Influence of the time of freezing

Sera were obtained after centrifugation and immediate addition of 10 mM EDTA at 37 ° C, 25 ° C and Store at 4 ° C for 0, 2, 4, and 6 hours before freezing at -20 ° C. Neither time temperature also had an influence on the measurement signals obtained.

Long-term stability of the sample material

Long-term stability was lower with three sera frozen at -20 ° C (x = 50 AU), medium-high (x = 300 AU) and high (x = 500 AU) test results after 1, 2, 3, 4 and 6 months checked. With all three sera stable measurement signals were obtained with a CV of 4.2% -9.2%.

Quality criteria of the test Imprecision

The variation coefficients of two sera pools were used to assess the measurement inaccuracy determined. The intra-assay CV moved depending on the values of the optical density (OD)  (n = 10) between 3.0 (x = 327 RU, 882 mE) and 4.1% (x = 235 RU, 473 mE), with higher ODs was accompanied by a lower precision.

Analytical specificity

Can also nucleosomes, free DNA, histones, anti-histone or anti-DNA antibodies alone or together cause disruptive changes in OD? This question was answered by a Titration experiment followed, in which non-biotinylated anti-histone antibodies in un different concentrations of the standard immunoreagent with biotinylated anti-histone anti body was added. The rapidly decreasing signal with increasing concentration, pointed to a competitive displacement competition of those with and without biotin Anti-histone antibodies that asymptomatically approach the LDD to the absence more disturbances. A measurement signal is therefore due to the complex formation of biotinylated anti-histone anti bodies, nucleosomes and peroxidase-labeled DNA antibodies bound.

Lowest detection dose

The lowest concentration to be detected was determined from the mean (n = 20) + 3x Stan The standard deviation of the blank is calculated at 16 RU / 38 mE.

Test modifications Enrichment factor and standard curve

The test was not comparable based on the recommended enrichment factor (AF) possible because the interassay precision at very low ODs is of the order of magnitude As expected, the basic value of the blank used was higher and reached values of up to 46.0%. With a measured OD of 1000 mE, one obtains e.g. B. with a base value A of 20 an AF of 50, with a base value B of 30 an AF of 33, which corresponds to an OD of 660 in test A. talk. Better test comparability and a more direct method of quantification could be achieved by establishing a standard curve as described above. In order to an interassay accuracy of less than 10% was achieved.  

Measuring time

By setting the incubation time with ABTS to 30 min, both the intra-assay VK as well as the Interassay-VK improved. Hung the former above all of the time delays in Pipetting off, the latter is probably due to the compensation of slight material defects, which are less significant after a longer incubation period.

Unit of measurement

The introduction of the new measuring unit AU (arbitrary units) also increased the test comparably speed: While the intraassay precision (n = 10) was constant: 3.3% / 3.0% (x = 882 mE / 327 AU) and 4.0% / 4.1% (x = 480 mE / 181 AU), the inter-assay precision (n = 14) improved 15.3% to 8.6% (x = 1007 ME / 455 AU) and from 17.0% to 13.5% (x = 473 ME / 235 RU).

Example 6 Clinical outcomes Distribution of values Healthy subjects

The distribution of values showed low ODs below 100 RU (approx. 250 mE) in all clinically and clinically and chemically healthy subjects. No differences regarding the age, gender, lifestyle (e.g. smoking, alcohol) of the test persons could be determined. (see ³¹ )

Patients with solid tumors

A wide range of values can be found in patients with various solid tumors, regardless of their location. This corresponds to previous studies, which described different apoptosis rates or found different concentrations of free DNA in plasma and serum in ^{solid tumors 27,31-35} .

After the remaining sera have been measured:

• - Absolute numbers
• - significance

Patients with inflammatory diseases

In patients with acute inflammatory diseases, we all looked for the CRP value divided groups a wide range of values. Nevertheless, there is a tendency towards higher Nucleosome concentrations in the serum can be seen at higher CRP values.

• - Significance is still missing

Follow-up

Patients with inflammatory diseases: In the course is a correlation with the CRP and clinical findings (objectifiable e.g. by sonography).

Example: The patient S.F. developed due to an igniting pigtail cathe cholangitis in cholestasis. After catheter revision and antibiotic treatment In parallel, a decrease in CRP, a decrease in signs of inflammation as well the serum nucleosome concentration was observed.

Patients under chemotherapy: The course under chemotherapy can only be assessed individually and with knowledge of the clinical background. Characteristic for most of the observed patients is a steep increase in the nucleosome concentration in the serum with different latency depending on the drug (24-48 hours) after the start of therapy. In the further course the values often decrease again. Infections or other side effects can cause a temporary increase in the nucleosome concentration and make it difficult to assess the course (see also Fournie ²⁷ ).

Example: In patient H.P., who is treated for metastatic pancreatic cancer the nucleo increased after the start of a cycle with 5-fluorouracil / folinic acid some concentration in the serum rose steeply and then slowly decreased. Because of clinical progre In view of the deterioration, a new therapy regimen with gemcitabine was set up. Here, too, there was initially a peak, but the disease progressed, as on steady rise in the CEA can be seen.  

Patients under radiotherapy: Through massive apoptosis induction occurs at the beginning of the radiothe therapy usually a rapid increase in the serum nucleosome concentration. The latency is shorter than with chemotherapy. In addition, some patients think after 3 or 6 hours intermittent drop in the nucleosome concentration in the serum before it rapidly afterwards increases. Over the course of time, sometimes after persistently high values, a drop is observed in many attention that is associated with proven tumor regression.

Example: In E.K., a patient with a metastatic bronchial carcinoma, came at the beginning of radiation therapy, the first day after a drop, one significant and rapid increase in serum nucleosome concentration. Only after The values decreased in a few weeks, at the same time as a ratiologically verified regression of the tumor. The nucleo increased due to a pericade cast somen concentration slightly on the 40th day, only to drop again. With occurrence of multiple metastases (as secured) and malignant pleural effusion (zyto, hist +?) higher nucleosome concentrations in the serum were again measured. The tumor marker CYFRA 21-1 shows a correlating course.

test

Intra- and inter-assay imprecision were respectively below 10% (3.0% -4.1%) and below 15% (8.6% -13.5%) and met the criteria of a hand assay ^{?? ?} . In order to enable comparability between different test approaches, the recommended enrichment factor was dispensed with, a standard curve with a new measuring unit was introduced and the measuring time was standardized. The analytical specificity could be demonstrated by a titration test.

matrix

Nucleosomes were detectable in different media (plasma, serum, urine). As in other studies for free DNA ^25,26,30 , we also received stronger signals for nucleosomes in serum than in plasma. There are several possible reasons for this:

• 1. The approval process could also a) induce apoptosis in the serum prior to centrifugation (stress situation for all blood cells), b) more DNA could be released ²⁵ .
• 2. The chelators added to the plasma in any case could inhibit the activity of the Ca ²⁺ and Mg ²⁺ -dependent endonuclease, hinder the splitting into oligo- or mononucleotides and the exposure of DNA for the binding of the antibodies.
• 3. Plasma proteins could interact with the nucleosomes and their presence mas kieren.

The very good stability after the addition of 10 spoke in favor of the use of serum as a matrix mM EDTA (pH 8) and the clear measurement signals with good discrimination ability difference between healthy, low-apoptosis and high-apoptosis sera in diseases genesis.

All tumor markers are also measured on a serum basis in everyday clinical practice. in the With regard to a possible, future, routine application and automation of the It seemed appropriate to carry out the tests with serum.

Preanalytics

Treatment of the serum was from blood collection, storage and transportation, centrifuga tion, addition of EDTA, long-term storage, standardized until the test is carried out. Possible Interferences (hemolysis by shaking or heat exposure) were examined. Consequently the foundations for a valid test application were laid.

Claims (5)

1. A method for determining apoptotic products in samples taken from tumor patients, characterized in that the concentration of the apoptotic products in serum samples is correlated with the effectiveness of the tumor therapy and thus serves to monitor the course of the therapy. 2. The method according to claim 1, characterized in that serum samples at different Times can be taken and determined. 3. The method according to claim 1 or 2, characterized in that the concentration of Nucleosomes in serum samples from tumor patients is determined. 4. The method according to claim 3, characterized in that one

• a) the serum of a tumor patient is incubated with an anti-histone antibody and an anti-DNA antibody, one of the antibodies being bound to a solid phase before or after this incubation and the other antibody being a labeled antibody,
• b) separates solid and liquid phase and
• c) the marker is determined in one of the two phases in order to determine the concentration of the nucleosomes in serum samples from tumor patients.

5. Using a method for the determination of apoptotic products in serum samples from Tumor patients to determine the effectiveness of tumor therapy.