METHOD FOR PRESERVING URINARY CELLS

Abstract

The present invention relates to a method for preserving urinary cells, the method comprising the step of contacting a urine sample obtained from a patient with a buffer substance suitable to create and/or maintain a pH value in the range of 6 to 8, particularly approx. 7, within said urine sample, and a formaldehyde releasing compound.

Claims

1. A method for analysing urinary cells, the method comprising the steps of: contacting a urine sample obtained from a patient with a. a buffer substance suitable to create and/or maintain a pH value in the range of 6 to 8, particularly approx. pH 7, within said urine sample, and b. a formaldehyde releasing compound yielding a preserved urine sample, analysing urinary cells within said preserved urine sample.

2. The method according to claim 1, wherein said urine sample is contacted with said buffer substance and said formaldehyde releasing compound not later than 6 h, 5 h, 4 h, 3 h, 2 h or 1 h after sampling, particularly immediately after sampling.

3. The method according to claim 1, wherein said formaldehyde releasing compound is selected from imidazolium urea, hexamethylenetetramine chloroallyl chloride, diazolidinyl urea, 1,3-bis(hydroxymethyl)-5,5-dimethylimidazolidine-2,4-dione, 1-(hydroxymethyl)-5,5-dimethyl-2,4-imidazolidindione or hexamethylenetetramine.

4. The method according to claim 1, wherein said buffer substance is selected from 3-(N-morpholino)propanesulfonic acid, phosphate buffered saline sodium hydrogencarbonate, tris(hydroxymethyl)aminomethane and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

5. The method according to claim 1, wherein the buffer substance is provided in a buffer solution.

6. The method according to claim 1, wherein the urine sample is contacted first with the buffer substance and then with the formaldehyde releasing compound.

7. The method according to claim 1, wherein the final concentration of the buffer substance is between 5% (w/v) and 10% (w/v) and/or the final concentration of the formaldehyde releasing compound is between 0.5% (w/v) and 5% (w/v).

8. The method according to claim 1, wherein the preserved sample is stored prior to analysing the urinary cells, particularly at a temperature ≤10° C., more particularly ≤4° C.

9. The method according to claim 1, wherein said analysing comprises a cytometric analysis of said urinary cells with said preserved urine sample.

10. The method according to claim 5, wherein said cytometric analysis comprises a flowcytometric analysis, particularly FACS.

11. A method for diagnosing a medical condition, the method comprising analysing urine cells within a urine sample obtained from a patient with a method according to claim 1.

12. The method according to claim 11, wherein said medical condition is selected from Lupus nephritis, acute kidney injury, rejection after kidney transplantation, ANCA vasculitis, diabetic nephropathy, IgA nephropathy, nephrolithiasis, and bladder cancer.

13. A container suitable for preserving urinary cells in a urine sample comprising an inner volume, wherein a composition is present in said inner volume and wherein the composition consists of a formaldehyde releasing compound selected from imidazolium urea, hexamethylenetetramine chloroallyl chloride, diazolidinyl urea, 1,3-bis(hydroxymethyl)-5,5-dimethylimidazolidine-2,4-dione, 1-(hydroxymethyl)-5,5-dimethyl-2,4-imidazolidindione or hexamethylenetetramine, and a buffer substance able to maintain a pH in the range of pH 6 to pH 8, particularly approx. pH 7, in a urine sample.

14. The container according to claim 13, wherein said buffer substance is selected from 3-(N-morpholino)propanesulfonic acid, phosphate buffered saline, sodium hydrogencarbonate, tris(hydroxymethyl)aminomethane and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

Description

DESCRIPTION OF THE FIGURES

[0107] FIG. 1 shows the relation between pH values and precipitate formation in urine. Especially specimen with low pH values tend to form precipitation. (A) Example of precipitation of patient's urine with added formaldehyde after 22 hours (+/−2 hours). (B) Dipstick results and relation to precipitation of urine with added formaldehyde after 22 hours (+/−2 hours)

[0108] FIG. 2 shows representative dot plots of peripheral blood mononuclear cells (PBMCs) in urine show reduced staining quality after incubation with MOPS buffer and formaldehyde for 22 hours (+/−2 hours). Gating strategy for T Cells: lymphocyte scatter-gate. Selection of CD3+ T Cells. Discrimination CD4+ T Cells vs. CD8+ T Cells. Gating strategy for macrophages: monocyte scatter-gate. Selection of CD14+ CD36+ macrophages. (A) Fresh PBMCs, fully stained. (B) PBMCs incubated in urine with MOPS buffer and 1% formaldehyde for 22 hours (+/−2 hours)

[0109] FIG. 3 shows representative dot plots of urinary leukocytes. Staining quality and percentages of populations of conserved sample after 22 hours and fresh sample are comparable. Gating strategy for T cells: exclusion of debris (low FSC and SSC) and large events (very high FSC and SSC). Selection of CD3+ T cells. Discrimination CD4+ T cells vs. CD8+ T Cells. Gating strategy for macrophages: exclusion of debris (low FSC and SSC) and large events (very high FSC and SSC). Selection of CD14+ CD36+ macrophages. (A) Fresh patient specimen (<6 hours after voiding), unstained. (B) Fresh patient specimen (<6 hours after voiding), stained. (C) Conserved patient specimen after 22 hours (+/−2 hours) at 4° C., stained.

[0110] FIG. 4 shows representative dot plots of urinary epithelial cells. Staining quality and percentages of populations of conserved sample after 22 hours and fresh sample are comparable. Gating strategy for tubular epithelial cells (TECs): exclusion of debris (low FSC and SSC) and very large events (very high FSC and SSC). Selection of epithelial cells (cytokeratin+). Discrimination CD326+ (=EpCAM) TECs and CD10+ or CD13+ TECs. (A) Fresh patient specimen (<6 hours after voiding), fixated and permeabilized for intracellular staining, single stain for cytokeratin, isotype-control for CD326, CD10 and CD13. (B) Fresh patient specimen (<6 hours after voiding), fixated and permeabilized for intracellular staining, fully stained. (C) Conserved patient specimen after 22 hours (+/−2 hours) at 4° C., followed by permeabilization, fully stained.

[0111] FIG. 5 shows the staining quality and percentages of populations of urinary leukocytes from conserved sample remain stable for up to 6 days, whereas leukocyte populations in non-fixated urine specimen disappear after 6 days (see FIG. 4 D). Gating strategy for T cells: Exclusion of debris (low FSC and SSC) and large events (very high FSC and SSC). Selection of CD3+ T Cells. Discrimination CD4+ T Cells vs. CD8+ T cells. Gating strategy for macrophages: Exclusion of debris (low FSC and SSC) and large events (very high FSC and SSC). Selection of CD14+ CD36+ macrophages. Representative FACS plots shown. (A) Conserved patient specimen after 22 hours (+/−2 hours) at 4° C. (B) Conserved patient specimen after 3 days at 4° C. (C) Conserved patient specimen after 6 days at 4° C. (D) Patient specimen without conservation after 6 days at 4° C.

[0112] FIG. 6 shows the staining quality and percentages of populations of urinary epithelial cells from conserved sample remain stable for up to 6 days, whereas cell populations in non-fixated urine specimen almost disappear after 6 days (see FIG. 5 D). Gating strategy for tubular epithelial cells (TECs): Exclusion of debris (low FSC and SSC) and very large events (very high FSC and SSC). Selection of epithelial cells (cytokeratin+). Discrimination CD326+ (=EpCAM) TECs and CD10+ or CD13+ TECs. Representative FACS plots shown. (A) Conserved patient specimen after 22 hours (+/−2 hours) at 4° C. (B) Conserved patient specimen after 3 days at 4° C. (C) Conserved patient specimen after 6 days at 4° C. (D) Patient specimen without conservation after 6 days at 4° C.

[0113] FIG. 7 shows the total cell counts of analyzed urinary leukocyte and epithelial cell populations remain stable for 22 hours, 3 days, 6 days or potentially even longer when conserved and stored at 4° C. Each dot represents the absolute cell number from each patient in both conditions (that is storage times). The diagonal line indicates identical cell amounts (slope=1) in both conditions, therefore dots on or close to this line represents measurements with equal or very similar cell numbers in both conditions. (Column A) Total cell counts from fresh patient urine specimens (<6 hours after voiding) and from equivalent volume of conserved patient urine specimens after 22 hours (+/−2 hours) at 4° C. (Column B) Total cell counts from conserved patient urine specimens after 22 hours (+/−2 hours) at 4° C. and from equivalent volume of conserved patient urine specimens after 3 days at 4° C. (Column C) Total cell counts from conserved patient urine specimens after 22 hours (+/−2 hours) at 4° C. and from equivalent volume of conserved patient urine specimens after 6 days at 4° C.

[0114] FIG. 8 shows the preservation of cells in urine. A. Representative flow cytometry plots of staining on fresh cells and after 20 h preservation. No qualitative deterioration of staining is observed. B. Comparison of cell numbers in urine in the freshly measured sample and after 24 h preservation, each point represents one sample. The gray line shows absolutely identical cell numbers, the dashed line shows the regression analysis of the actually measured values. There is a high degree of agreement, i.e. an almost constant cell count despite preservation.

[0115] FIG. 9 shows the kinetics of the fixation of immune cells using IU. Displayed is the percentage of fixed cells in comparison to cells fixed using a commercial cell fixation kit. The cells were incubated with IU for 5 min, 30 min, 1 h, 5 h and 24 h. Experiments were performed in replicates.

[0116] FIG. 10 shows CD4.sup.+ T cells as biomarkers for active lupus nephritis (LN). A. Correlation of CD4.sup.+ T cell count per 100 ml urine with disease activity (measured as SLEDAI). Each open square represents one SLE patient without renal involvement, filled circles represent one SLE patient with known renal involvement. B. Comparison of the amount of CD4.sup.+ T cells in the urine with a simultaneous kidney biopsy. All patients with inflammatory LN (proliferative LN) show increased levels of T cells in urine. C. ROC curve for the detection of proliferative/inflammatory LN in SLE patients using the amount of CD4.sup.+ T cells in urine (sensitivity of 100% and specificity of 98.0%). D. Patients who normalize their CD4.sup.+ T cell counts under therapy show a significant reduction of their disease activity, patients with persistent or increasing CD4.sup.+ T cell counts in urine show no reduction of their disease activity. E. Patients with normalized CD4.sup.+ T cells in urine have a better creatinine increment after six months of therapy than patients with persistence of T cells in urine. F. The effector/memory (EM) CD4.sup.+ T cell count predicts response to therapy after 6 months of induction therapy.

[0117] FIG. 11 shows different cell populations in urine as markers for acute kidney transplant rejection. A. Representative flow cytometry plots for the detection of T-cells (CD3, CD4, CD8), macrophages (CD45, CD36, CD14), podocalyxin-positive cells (PDX, as surrogate for podocytes) and renal tubule epithelial cells (TEC; cytokeratin, CD10, EPCAM) B. Amount of T cells in relation to TEC and PDX.sup.+ cells in patients with acute cellular rejection (ACR), humoral rejection (HRX), patients with graft degeneration without rejection (No Rx) and patients with stable graft function (Contr.). C. ROC curves show a good diagnostic significance of the combination of T cells and TECs or PDX.sup.+ cells in urine (AUC 0.9). Black line: comparison of patients with rejection with the No RX group; grey line: data including contrast patients with stable graft function. SSC=sideways scatter; FSC=forwards scatter; DAPI=dead cell dye.

EXAMPLES

Example 1: Preserving and Analyzing Urine Cells

[0118] Fixation of cells and tissues with agents such as formaldehyde is considered routine practise in laboratories. The basis of this fixation is cross-linking of proteins at certain amino acids.

[0119] When adding formaldehyde directly to the urine of patients the inventors observed precipitation of a gel-like mass in a significant number of samples after approximately 22 hours, thus making it impossible to analyse the samples on the next day. Dipstick results of patient's urine specimens suggest that especially samples with low pH tend to form precipitation (FIG. 1).

[0120] Following the assumption that low pH triggers precipitation, the inventors added a standard cell culture buffer (MOPS) to urine samples together with formaldehyde. By stabilizing the pH closer to neutral the inventors were able to avoid precipitation. Further neutralizing strategies were tested as well, for example dilution with distilled water or other buffers (PBS), but resulted either in more precipitation or worse quality in FACS analysis when compared to MOPS buffer in corresponding conditions.

[0121] However, incubation of the urine specimen with formaldehyde and MOPS buffer for at least 20 hours resulted in poor staining quality with diminished separation of positive and negative cells (FIG. 2) and thus a decrease in detectable cells.

[0122] In order to achieve a gentler fixation of cells, the inventors next introduced formaldehyde releasers such as, but not limited to, imidazolidinyl urea (IU) instead of direct addition of formaldehyde. The chemical group of formaldehyde releasers is used for example as a preservative in cosmetic products and is characterized by a slow release of formaldehyde-groups from a more complex molecule. When using this chemical agent, staining quality was only marginally impaired in comparison to fresh urine samples (FIGS. 3-6). Hence, the inventors were able to detect similar counts of urinary cells after 22 hours (+/−2 hours) compared to fresh samples (FIG. 7A). Furthermore, detected similar numbers of urinary cells were detected even after 3 and 6 days compared to samples analysed after 1 day (FIGS. 7B and 7C).

[0123] Liquid MOPS buffer and powdery imidazolidinyl urea are added in corresponding concentrations (see 2d) to the urine sample. By inverting the sample several times, the powder (IU) dissolves and all components are mixed sufficiently. Afterwards, the sample can be stored at 4° C. for 22 hours, 3 days, 6 days or potentially even longer for later sample preparation, i.e. antibody-based staining of cells, and flow cytometric analysis.

[0124] The method of the invention allows delayed sample preparation and analysis of human urinary cells through an easy conservation procedure. Both components (IU and MOPS buffer) are simply added to the urine specimen. After inverting the sample several times it can be stored in the fridge (4° C.) for 22 hours, 3 days, 6 days or potentially even longer. This enables centralized and standardized analysis facilitating multicentre studies as well as future diagnostic use in more peripheral clinics and outpatient care. Another important advantage of the method of the invention is increased safety when working with human samples, as cells will be dead after fixation. Furthermore, the conservation procedure leads to fixation of the cells, thus enabling to start directly with permeabilization for intracellular staining after a centrifugation step.

[0125] So far, the inventors have analyzed 15 samples for T Cell counts (which stain positive for CD3 and CD4 or CD3 and CD8) and Macrophages (positive for CD14 and CD36) and 9 samples for counts of kidney-derived tubular epithelial cells (positive for cytokeratin and CD326 (EpCAM) or cytokeratin and CD10 and CD13), respectively, after 22 hours (+/−2 hours). For time points after 3 days and 6 days, the inventors have acquired data from 10 samples for both T cells and tubular epithelial cells, respectively.

[0126] Every urine specimen was split equally allowing to compare cell counts of the conservation method of the invention after a certain time to a reference sample. Conserved samples stored for 22 hours (+/−2 hours) at 4° C. were referenced to a sample freshly prepared and analyzed directly upon receipt. Conserved samples stored for 3 days or 6 days at 4° C. were referred to samples conserved in the same way with the method of the invention and stored for 22 hours (+/−2 hours) at 4° C. before staining and analysis.

[0127] Specimen were acquired from patients with diverse renal pathologies, including: Acute Kidney Injury (AKI), Diabetic Nephropathy, Acute Rejection of a Kidney Transplant, Granulomatosis with Polyangiitis (GPA) with Renal Involvement, Systemic Lupus Erythematosus (SLE) with Renal Involvement, Rapid-progressive Glomerulonephritis (RPGN), IgA Nephropathy.

[0128] Comparison of total cell counts for samples from each time point and corresponding reference samples is presented in FIG. 7.

[0129] It is possible, that the hereby claimed method can be applied in diagnostics and monitoring of various renal diseases. So far, analysis of urinary cells was described for following entities: [0130] Lupus nephritis [0131] acute kidney injury [0132] rejection after kidney transplantation [0133] ANCA Vasculitis [0134] diabetic nephropathy [0135] IgA nephropathy [0136] renal stones (nephrolithiasis) [0137] bladder cancer

[0138] Considering the variety of medical conditions, it is likely that the method according to the invention can be used for any renal or urologic disease or any disease that leads to altered cell composition in the urine.

Methods and Materials

[0139] Concentration imidazolidinyl urea: 20 mg/ml of total volume (Urine+MOPS)

[0140] Concentration MOPS buffer: 1 mol/l

[0141] Composition MOPS buffer (1 mol/l, for 100 ml): [0142] 20.95 g MOPS [0143] 0.82 g anhydrous sodium acetate (NaAc anhydrite) [0144] 1.85 g EDTA [0145] Fill up to 100 ml with Aqua dest. [0146] Adjust pH to 7 with NaOH

[0147] pH MOPS buffer: 7

[0148] Proportion of MOPS buffer of Total Volume: 1:3

[0149] Proportion of Urine of Total Volume: 2:3

[0150] 60 ml urine, 30 ml buffer and 1800 mg imidazolidinyl urea (IU) were mixed. The final concentration of IU was 20 g/l.

Example 2: Establishment of a Sample Preservation System

[0151] Currently, a fresh urine sample is required for the flow cytometric analysis of urine cells (maximum 4-6 h after sample collection), which makes sample logistics difficult. The analysis of fresh samples requires an immediate transport of the sample, and the appropriately trained laboratory personnel and instrument capacities must be available for the analysis. At large clinics with maximum care, an essay based on fresh samples is feasible, but makes the analysis cost-intensive. It is almost impossible to use it in smaller hospitals or outpatient clinics. Currently, there is no established sample logistics for conserving urine cells for later flow cytometric analysis. Therefore, the inventors have developed a preservation method that preserves the cells in a special container for at least 6 days. The difficulty here was not only to preserve the cells, but also not to change the cells in such a way that no meaningful staining for flow cytometry is possible after preservation. In addition, the preservation system must be as simple and uncomplicated as possible in order to be accepted by the users (medical personnel in routine operation). By combining a buffer substance and a formaldehyde releasing compound in a two-step process, the inventors succeeded in preserving the cells for at least six days. All that is required is to add a urine sample to a container containing a buffer and then add a formaldehyde releasing compound (in powder or tablet form). Alternatively, the container is equipped with the buffer compound and formaldehyde releasing compound in a way that enables stable shelf life for both, and the urine sample is added, optionally after addition of water, upon opening of the container from its packaging.

[0152] The sample can then be stored and/or transported. With this preservation, staining and flow cytometry of the cells can be performed without compromising the quality of the analysis. A comparison of the analysis of fresh urine and after one, three or six days of preservation showed almost identical cell counts and a linear correlation of the measured cell counts

[0153] (Pearson correlation for CD3.sup.+CD4.sup.+, CD3.sup.+CD8.sup.+ and proximal and distal TEC, p<0.001 for all, r=0.9530−0.9998, median r 0.9982, FIG. 8).

[0154] The system is user-friendly and does not require a complicated or time-consuming protocol. It is easy to implement within 30 seconds, which is the prerequisite for practical implementation.

Example 3: Comparison with Published Protocols of Cell Preservation

[0155] WO 2014/029791 A1 shows a flow cytometric analysis of cells that have been treated with different variants of a fixation protocol. The following reagents were used to preserve the cells and each solution was A to E was diluted 1:50 with the cell sample:

[0156] A. 25% w/v diazolidinyl urea

[0157] B. 1.5% w/v aurintricarboxylic acid

[0158] C. 0.8% w/v sodium fluoride

[0159] D. 10% w/v EDTA

[0160] E. 4.5% w/v glyceraldehyde

[0161] The mentioned variants for cell preservation were compared with the protocol according to the invention, i.e. the solutions A to E were each diluted 1:50 with urine. Aurintricarboxylic acid rapidly precipitates when added to urine, rendering it unusable for the purpose of the protocol of the present invention. Also EDTA alone (0.2% final concentration) precipitates in urine.

[0162] The three other ingredients (glyceraldehyde, diazolidinyl urea and sodium fluoride) were tried at the concentration given in WO 2014/029791 A1 for the preservation of urine cells in three patient urine samples and compared with the protocol. For all three substances, the quality of the staining in the flow cytometric analysis deteriorated significantly.

Example 4: Kinetics of Fixation of Immune Cells Using IU

[0163] IU fixation was performed with different incubation times: 5 min, 30 min, 1 h, 5 h, 1 day. Subsequentyl, permeabilization was performed using Perm/wash (unfixed cells would have to break). Measurement and cell counting was performed at MACSquant followed by evaluation via SSC/FSC and ZZ.

[0164] Controls: positive: PFA-fixed cells; negative: unfixed cells.

[0165] After 5 h of incubation in all replicated 100% of cells were fixed. The protocol according to the invention even yielded higher amounts of recovered cells in comparison to the commercial kit, therefore values>100% were observed.

Example 5: T cells as Biomarkers for Lupus Nephritis

[0166] Lupus nephritis (LN) is one of the most common and serious complications of systemic lupus erythematosus (SLE). Under standard therapy with cyclophosphamide or mycophenolate mofetil (MMF) there is a risk of serious side effects, such as severe infections under immunosuppression. On the other hand, as repeatedly shown in studies, only about 45% of patients achieve complete remission under therapy, which implies significant morbidity. In addition, uncontrolled disease activity as well as side effects of the therapy repeatedly lead to deaths. Biomarkers could effectively help to improve the prognosis of LN by early diagnosis and individualized therapy based on better prognosis assessment and therapy control.

[0167] In a cohort of 147 SLE patients and 186 urine samples, the inventors were able to demonstrate that the amount of CD4.sup.+ T cells in the urine identified patients with active lupus nephritis with high sensitivity (100%) and specificity (98%) (FIG. 9. area below the ROC curve, AUC 0.99). SLE patients without kidney involvement showed hardly any T-cells in the urine even with increased disease activity, and SLE patients with known kidney involvement but without increased disease activity showed only few T-cells in the urine. In contrast, all patients with proliferative lupus nephritis (the most common form of LN) had increased levels of T cells in their urine. Interestingly, in the few patients with active, non-proliferative LN (such as LN class I or V), hardly any T cells were observed, which could be used to differentiate the individual LN subclasses. In the investigated cohort of 147 SLE patients, the determination of T-cells in urine was clearly superior to routine markers such as creatinine, proteinuria and urine sediment.

[0168] Even more interesting for clinical use is the fact that the amount of T cells in urine in the course of therapy allows a correlation to the response to treatment at an early stage. An early adaptation of the therapy in the sense of a personalized treatment would be conceivable with the aid of biomarkers.

[0169] In a small cohort of SLE patients, the inventors were also able to show that the amount of memory/effector T cells (EM T cells) in urine at the time of diagnosis can predict therapy response 6 months later. All patients with a higher frequency of EM T cells showed no response after induction therapy.

[0170] In summary, lupus nephritis can be i) diagnosed with T-cells in urine, ii) the response to treatment monitored and iii) the prognosis estimated.

Example 6: Combination of T Cells and Tubule Epithelial Cells for Diagnosis of Kidney Transplant Rejection

[0171] Kidney transplantation is probably the best therapy for terminal renal failure. However, acute rejection of the transplant is a constant concern, requiring permanent immunosuppressive use and regular monitoring of graft function. At present, there are no reliable non-invasive markers for acute rejection of the transplant, which is why a kidney biopsy with corresponding risks must be performed if suspected.

[0172] In a cohort of 63 kidney transplant patients, the amount of different immune cells (CD4+ and CD8+ T cells, monocytes/macrophages) as well as renal tubule epithelial cells (proximal and distal TECs) and podocytes (PDX.sup.+ cells) was investigated. A kidney biopsy was performed in 39 of the examined patients due to a deterioration of NTX function and the results were directly compared with the biopsy result as gold standard. In addition, 24 patients with stable NTX function were examined as control groups. By combining the amount of T cells (total amount of T cells or CD4.sup.+ or CD8.sup.+ T cells) and TEC in the urine as T cell/TEC ratio, a good separation of patients with and without rejection was achieved (FIG. 10, area below the ROC curve 0.90). In a cohort of 64 kidney transplant patients in a clinically very relevant scenario, a combination of T cells and tubule epithelial cells in the urine showed good sensitivity and specificity (FIG. 10, area below the ROC curve 0.90).

Example 7: Further Data in Other Diseases

[0173] In patients with acute renal failure (ANV), it was shown that the amount of TECs in the urine correlated with the extent of renal damage (measured as ANV stage). In addition, stem cell-like cells could be detected in urine in patients with ANV, which only occurred in patients recovering from ANV (possible regeneration markers).

[0174] In patients with glomerulonephritis associated with ANCA, similar to LN, it was demonstrated that the amount of T-cells in the urine is a biomarker for active renal inflammation.