METHOD AND ENZYME SOLUTION FOR FLOW-CYTOMETRIC DETECTION OF LIGHT CHAIN RESTRICTION

Abstract

The invention relates to an enzyme solution, which is used preferably for pre-treating mammalian cells for flow-cytometric detection of light chain restriction of the cells, the enzyme solution comprising at least two proteases, which are proteolytic and also collagenolytic, in a buffer. Preferably, the enzyme solution comprises trypsin, collagenase 4, dispase and astacin. The invention also relates to uses of the enzyme solution and methods for flow-cytometric detection of light chain restriction of cells.

Claims

1. An enzyme solution, for pre-treating mammalian cells for flow-cytometric detection of light chain restriction of the cells, the enzyme solution comprising at least two proteases, which are proteolytic and also collagenolytic, in a buffer.

2. The enzyme solution of claim 1 comprising at least three enzymes selected from trypsin, collagenase 4, dispase and astacin.

3. The enzyme solution of claim 2 comprising trypsin, collagenase 4, dispase and astacin.

4. The enzyme solution according to claim 1, wherein the cells are from a cell sample selected from the group consisting of blood plasma, bone marrow, lymph, liquor, endolymph, vitreous humour, synovial fluid, pleural fluid, pericardial fluid (liquor pericardii), peritoneal fluid and a combination thereof.

5. The enzyme solution according to claim 1, wherein the buffer is phosphate buffered salt (PBS).

6. The enzyme solution according to claim 1, wherein trypsin is present in a concentration of 0.25-25 mg/ml, preferably 1.25-2.5 mg/ml.

7. The enzyme solution according to claim 1, wherein collagenase 4 is present in a concentration of 0.05-2 mg/ml, preferably 0.05-0.2 mg/ml.

8. The enzyme solution according to claim 1, wherein dispase is present in a concentration of 0.05-2 mg/ml, preferably 0.05-0.2 mg/ml.

9. The enzyme solution according to claim 1, wherein astacin is present in a concentration of 0.05-25 mg/ml, preferably 0.05-0.2 mg/ml.

10. The enzyme solution according to claim 1, comprising: 0.25-25 mg/ml trypsin, 0.05-2 mg/ml collagenase 4, 0.05-2 mg/ml dispase and 0.05-25 mg/ml astacin.

11. A flow-cytometric detection method for pre-treating cells comprising the enzyme solution of claim 1.

12. The method of claim 11, wherein the flow-cytometric detection method determines a light chain restriction of plasma cells and/or B lymphocytes.

13. The method of claim 12, wherein the plasma cells and/or B lymphocytes are from bone marrow, liquor or peripheral blood.

14. An in vitro method for determining a light chain restriction of plasma cells and/or B lymphocytes by a flow-cytometry detection method, comprising: (a) providing plasma cells and/or B lymphocytes obtained from bone marrow, liquor or peripheral blood, (b) contacting the plasma cells and/or B lymphocytes with an enzyme solution of claim 1, (c) subjecting the cells to the flow-cytometric detection method, and (d) determining a light chain restriction of the cells.

15. A method for flow-cytometric detection of light chain restriction comprising the steps of: a. removing peripheral blood or bone marrow from an EDTA tube and transferring to a falcon tube, b. erythrocytolysis by means of distilled water and balancing by means of ten-times-concentrated phosphate buffer solution (PBS), and centrifuging to pellet cells, c. absorbing the cells in PBS, transferring the absorbed cells to Eppendorf vessels and pelleting the cells again, d. resuspending the pelleted cells in the enzyme solution according to claim 1, e. stopping the enzyme reaction after an incubation time by rinsing with PBS, absorbing the cells in PBS and staining the blood/bone marrow aspirate with monoclonal antibodies for flow cytometric analysis.

16. The enzyme solution according to claim 1, wherein trypsin is present in a concentration of 1.25-2.5 mg/ml.

17. The enzyme solution according to claim 1, wherein collagenase 4 is present in a concentration of 0.05-0.2 mg/ml.

18. The enzyme solution according to claim 1, wherein dispase is present in a concentration of 0.05-0.2 mg/ml.

19. The enzyme solution according to claim 1, wherein astacin is present in a concentration of 0.05-0.2 mg/ml.

20. The enzyme solution according to claim 1, comprising: 1.25-2.5 mg/ml trypsin, 0.05-0.2 mg/ml collagenase 4, 0.05-0.2 mg/ml dispase and 0.05-0.2 mg/ml astacin.

Description

[0018] The object of this invention is achieved by the enzyme solutions, uses and methods in the claims. Advantageous embodiments are to be found in the description.

[0019] Subject of the invention is an enzyme solution, which is used preferably for pre-treating mammalian cells for flow-cytometric detection of light chain restriction of the cells, the enzyme solution comprising at least two proteases, which are proteolytic and also collagenolytic, in a buffer.

[0020] Preferably, the enzyme solution comprises at least three enzymes selected from trypsin, collagenase 4, dispase and astacin. Preferably, the enzyme solution comprises trypsin, collagenase 4, dispase and astacin. As used herein, the term astacin refers to an enzyme from the astacin family of metalloproteases. Thus, the enzyme solution comprising astacin should comprise at least one enzyme from the astacin family of metalloproteases.

[0021] Antigens, in this case specific light chains of the lymphocytes, can be exposed and coloured with the inventive enzyme solution. This was only limitedly possible until now, even though it is highly important as the proof for malignancy can only be provided with evidence of light chain restriction.

[0022] Preferably, the cells are from a cell sample selected from the group comprising blood plasma, bone marrow, lymph, liquor, endolymph, vitreous humour, synovial fluid, pleural fluid, pericardial fluid (liquor pericardii), peritoneal fluid or a combination thereof.

[0023] Preferably, the buffer is phosphate buffered salt (PBS). Preferably, the PBS buffer does not comprise calcium and/or magnesium. Preferably, the enzyme solution comprises a cell culture medium, such as RPMI1640. The cell culture medium may comprise fetal calf serum (FCS). The enzyme solution may also comprise other common components for pre-treating cells, such as EDTA.

[0024] Preferably, the proteases comprise a mixture of preferably trypsin, collagenase 4, dispase and astacin in a buffer, preferably phosphate buffered salt solution (PBS).

[0025] Preferably, the enzyme solution is present in the sample tube before or after the cell sample that can be introduced.

[0026] Preferably, trypsin is present in a concentration of 0.25-25 mg/ml, preferably 1.25-2.5 mg/ml, and/or

[0027] collagenase 4 is present in a concentration of 0.05-2 mg/ml, preferably 0.05-0.2 mg/ml, and/or

[0028] dispase is present in a concentration of 0.05-2 mg/ml, preferably 0.05-0.2 mg/ml, and/or

[0029] astacin is present in a concentration of 0.05-25 mg/ml, preferably 0.05-0.2 mg/ml. Preferably, astacin is present in a concentration of 0.3-times 0.5-times.

[0030] Preferably, the enzyme solution comprises

[0031] 0.25-25 mg/ml, preferably 1.25-2.5 mg/ml trypsin,

[0032] 0.05-2 mg/ml, preferably 0.05-0.2 mg/ml collagenase 4,

[0033] 0.05-2 mg/ml, preferably 0.05-0.2 mg/ml dispase and

[0034] 0.05-25 mg/ml, preferably 0.05-0.2 mg/ml astacin.

[0035] In a specific embodiment, the enzyme solution does not comprise other proteases or other enzymes.

[0036] Another subject of the invention is the use of the enzyme solution for pre-treating cells in a flow-cytometric detection method. Preferably, the flow-cytometric detection method is for determining a light chain restriction of plasma cells and/or B lymphocytes. Preferably, the plasma cells and/or B lymphocytes are from bone marrow, liquor or peripheral blood.

[0037] Preferably, the method is for detecting malignant cells. Preferably, the method is a diagnostic method, preferably for diagnosing multiple myeloma or B-cell non-Hodgkin lymphoma.

[0038] Another subject of the invention is an in vitro method for determining a light chain restriction of plasma cells and/or B lymphocytes by a flow-cytometry detection method, comprising: [0039] (a) providing plasma cells and/or B lymphocytes, which preferably have been obtained from the mammalian body, preferably from bone marrow, liquor or peripheral blood, [0040] (b) contacting the plasma cells and/or B lymphocytes with the enzyme solution, [0041] (c) subjecting the cells to the flow-cytometric detection method, and [0042] (d)determining a light chain restriction of the cells.

[0043] In step (a), the cells are preferably isolated from the mammalian body, such that a cell pellet is obtained. In step (b), the cells are incubated with the enzyme solution for a sufficient time such that the proteases can act as desired on the cell. For example, the cells are incubated with the enzyme solution for 15 to 90 min, preferably at a temperature between 15 and 60 C., preferably between 20 and 40 C. Typically, step (c) comprises staining of the cells with monoclonal antibodies.

[0044] Another subject of the invention is a method for flow-cytometric detection of light chain restriction comprising the steps of:

[0045] remove peripheral blood or bone marrow from an EDTA tube and transfer to a falcon tube, erythrocytolysis by means of distilled water and balancing by means of ten-times-concentrated phosphate buffer solution (PBS), centrifuge of the cells, absorb the cells in PBS, transfer to Eppendorf vessels and pellet again, resuspend the pelleted cells in the inventive enzyme solution, stop the enzyme reaction after the incubation time by a rinsing step with PBS, absorb the cells in PBS and staining of the blood/bone marrow aspirate with the monoclonal antibodies for flow cytometric analysis.

[0046] The preparation of the cells to be investigated is made in a manner, which is obvious for the person skilled in the art and known from the prior art, and which is illustrated here by way of example:

[0047] Up to 2 ml of peripheral blood or bone marrow are removed from an EDTA tube (ethylenediaminetetraacetic acid preincubated tube for the anticoagulation of whole blood) and transferred to a 50 ml falcon tube. Without or after double hypotonic erythrocytolysis by means of distilled water for 25 seconds in each case and corresponding balancing by means of ten-times-concentrated phosphate buffer solution (PBS), the cells are centrifuged down (pelleted). Subsequently, these cells are absorbed in 1.2 to 1.6 ml PBS, transferred to 400 l portions in 1.5 ml Eppendorf vessels in each case and pelleted again. These cell pellets are resuspended in 300 l total volume in the enzyme solution according to the invention. After the respective incubation time (15 to 90 min at room temperature or 37 C.), the enzyme reaction is stopped by a rinsing step with PBS. The cells are initially absorbed in 100 l PBS and subsequently the staining of the blood/bone marrow aspirate with the monoclonal antibodies for flow cytometric analysis is undertaken.

[0048] The enzyme solution according to the invention consists by way of example of:

[0049] Trypsin (0.25-25 mg/ml PBS) without calcium and magnesium (w/o Ca&Mg), collagenase 4 (0.05-2 mg/ml) cell culture medium, such as for example RPMI1640 incl. 10% fetal calf serum (FCS), dispase (0.05-2 mg/ml) RPMI incl. 10% FCS and astacin (such as for example accutase 0.3-times-5-times in PBS w/o Ca&Mg+0.5 mM EDTA)