Immunocytological determination of lymphocytes and monocytes/macrophages in urinary sediments of renal allograft recipients

R. Willi Grunewald, G. Martin Fiedler, Birgit Stock, Julia M. Grunewald and Gerhard A. Müller

Division of Nephrology and Rheumatology, Department of Internal Medicine, University of Göttingen, Germany

Abstract

Background. Urinary studies using Papanicolaou staining following kidney transplantation led to the conjecture that acute allograft rejection might be accompanied by an increased lymphocyturia. However, it is difficult to distinguish lymphoid cells from other urinary cells using conventional stains.

Methods. Staining of urinary lymphocytes using FITC-labelled antibodies is complicated by a high unspecific fluorescence that limits the evaluation. Therefore, we developed a method to stain urinary lymphocytes using enzyme-linked antibodies. The cells were cytocentrifuged onto microscope slides and were fixed.

Results. By means of a combined evaluation of Papanicolaou and immunocytochemical staining, CD3-positive pan T cells, CD4-positive T-helper cells, CD8-positive cytotoxic/suppressor cells, and CD14-positive monocytes/macrophages of urinary sediments were determined in 41 kidney graft recipients following renal transplantation. During periods of normal graft function, neither positive lymphocytes nor positive monocytes/macrophages were found in the urinary sediments. However, in the course of acute allograft rejection a significant increase in positive lymphocytes and positive monocytes/macrophages could be observed. Interestingly, in cases of acute allograft rejection the distribution of urinary lymphocytes and monocytes was comparable to the distribution of infiltrating immunocompetent cells in renal allograft biopsies.

Conclusion. The present study demonstrates that immunocytochemical staining via enzyme-conjugated antibodies is a reliable method to visualize T lymphocytes and monocytes/macrophages in the urinary sediment, and that this technique may be of special diagnostic value in the diagnosis of acute allograft rejection.

Keywords: cyclosporin-A nephrotoxicity; immunocytology; lymphocytes; monocytes/macrophages; renal allograft rejection; urinary sediment

Introduction

The early diagnosis of acute rejection in renal allograft recipients and an immediately induced antirejection therapy are essential to restore and maintain renal graft function. More often the clinical signs are not specific for allograft rejection and have to be differentiated from other causes of declining allograft function, such as infections, cyclosporin A (CsA) nephrotoxicity, or surgical complications. The urinary cytology could be a non-invasive method for monitoring transplant cellular rejections in organ grafts. Several urinary cytology examinations using for example Papanicolaou staining have demonstrated the value of urinary lymphocytes in the diagnosis of allograft rejection [1–9]. However, it is difficult to differentiate the lymphoid cells from other mononuclear cells or even from degenerated or necrotic tubular epithelial cells [7]. The immunotyping of mononuclear cells using monoclonal antibodies has been a widely used method in recent studies of infiltrating cells in renal allograft biopsies. In the present study an immunocytological staining of urinary cells with monoclonal antibodies against CD3 (pan T cells), CD4 (T-helper cells), CD8 (cytotoxic/suppressor cells), and CD14 receptors (monocytes/macrophages) was performed to differentiate T lymphocytes and monocytes/macrophages from other urinary cells. In this way we tried to enhance the diagnostic value of urinary sediments for the early diagnosis of allograft rejection.

Subjects and methods

Patients
Forty-one renal allograft recipients, nine females and 32 males, were followed up to 72 days post-transplant. Thirty-nine patients received their first transplant and two patients received the second transplant.

All patients were treated with a combination of prednisolone and cyclosporin A (CsA). Antirejection therapy commonly consisted of methylprednisolone bolus therapy (500 mg i.v. for 3 consecutive days).

The patients# were divided into four groups:

(i) Stable graft function. In this group serum creatinine, sIL-2R, urine excretion, and ultrasonography were not pathological, and an infection could be excluded. Serum creatinine should not exceed 150 pmol/l. Volume of urine excretion should be >1.8 l/day under constant body weight.
(ii) Acute rejection. Acute rejection was confirmed by allograft biopsy in all cases. The diagnosis of acute rejection was assumed when the serum creatinine level showed an increasing tendency of 20 µmol/l per day in the absence of obstruction (by sonogram) or adverse systemic haemodynamic factors. Plasma sIL-2R concentration also increased [10,11]. In addition, allograft ultrasonography (renal enlargement in all dimensions, prominent hypoechoic pyramids or effacement of renal sinus) and Doppler ultrasonography (resistance index of Pourcelot (RI) >=0.8 with an increment of 0.1–0.2/day) turned pathological [12], and urine excretion declined.
(iii) CsA toxicity. CsA toxicity was diagnosed when the allograft biopsy revealed typical structural lesions consistent with CsA toxicity [13,14]. Characteristically, the impaired renal function showed a rapid improvement in response to cyclosporin dose reduction. For statistical analysis, the measurements of the day with the highest CsA levels and of the following 3 days were chosen.
(iv) Infections. Systemic viral infections were diagnosed by rising of virus-specific IgM titre or a direct virus detection. The diagnosis of CMV was ensured by detection of CMV-pp65-EA or CMV-PCR. Patients with urinary tract infections were excluded.

Samples
Urine samples were obtained two to three times a week. The second voided morning urine were collected and immediately processed.

Creatinine measurement
Creatinine was measured with a Beckman creatinine analyser according to Jaffe's test.

Soluble IL-2 receptor (sIL-2R) assay
The measurements of sIL-2R concentrations in plasma and urine by a commercially available ELISA (Immunotech SA, Marseilles, France) were used in conjunction with serum creatinine as an additional parameter to monitor acute rejection periods.

Urinary cytology
Freshly voided midstream urine samples were collected twice per week. Aliquots of 10 ml of urine were centrifuged at 4°C for 10 min at 1500 r.p.m. and washed twice in PBS with 1% human serum albumin. The resuspended aliquots were cytocentrifuged (Cytospin 2, Shandon, Frankfurt, Germany) onto microscope slides for 5 min at 500 r.p.m. Two cytospin preparations were fixed in palodoine (1 g paladoine in 200 ml ethanol 95% (v/v) and 200 ml ether) and ethanol 95% (v/v). Subsequently they were stained with Papanicolaou for conventional cytology [15]. Ten high-power fields (x500) were scanned to count lymphocytes, monocytes, granulocytes, and renal cells in urine.

Immunocytology of urinary sediments
Seven cytospin preparations were used for immunocytochemical staining. They were fixed in acetone (4°C, 10 min). Immunocytochemical preparation was performed via three-layer alkaline phosphatase/anti-alkaline phosphatase (APAAP) method. From each urine sample seven cytospin preparations were prepared.

i. Cytospin preparations were treated with monoclonal antibodies CD3 (clone T3–4B5, Code. No. M 0756, Dako Diagnostika, Hamburg, Germany, dilution 1 : 10), CD4 (IOT 4a, clone 13B8.2, Cat. No. 0447, Immunotech SA, Marseille, France, dilution 1 : 20), CD8 (clone DK25, Code No. M 0707, Dako Diagnostika, Hamburg, Germany, dilution 1 : 20) and CD14 (IOM 2, clone RM052, Cat. No. 0643, Immunotech SA, Marseille, France, dilution 1 : 20).
ii. One cytospin preparation served as a negative control staining by the Universal–Dako APAAP KitTM (Code No. K 0670, Dako Diagnostika, Hamburg, Germany) utilizing fast red chromogen without antibodies. The negative control serves as a ‘blank’, for any positive staining on this specimen is non-specific and should be ignored when interpreting results.
iii. Another cytospin preparation (positive control) was treated with ‘anti human leukocyte common antigen’ CD45 (clone 2B11 and PD7/26, Code No. M0701, Dako Diagnostika, Hamburg, Germany, dilution 1 : 50). The positive control served as an indicator that specimen processing and staining were carried out correctly.

The air-dried cytospin preparations were fixed in cold acetone (4°C) for 10 min. Thereafter, they were washed in PBS for 5 min and incubated with 50 µl monoclonal antibodies in tested dilution (see above) for 60 min at room temperature. After washing with PBS (three times, each 5 min) the cytospin preparations were incubated with 100 µl alkaline phosphatase-conjugated rabbit-anti mouse immunoglobulin (Dako Diagnostika, Hamburg, Germany, dilution 1 : 10) for 45 min at room temperature. After three further washes in PBS, colour was developed with fast red (20 mg of fast red Tr Salt, 4 mg of naphtol As-Mx phosphate disodium, and 4.8 mg of levamisole (all of Sigma, Munich, Germany) in 20 ml of Tris–MgCl2 buffer pH 8.2) for 30 min. After washing in running tap water the preparations were counterstained with Harris's haematoxylin for 10 s and after further washing in running tap water the preparations were embedded with glycerolgelatin.

Percentages of positive cells were determined by counting up to 100 mononuclear cells (lymphocytes, monocytes/macrophages, and renal cells). By means of Papanicolaou preparations, these percentages were converted into absolute numbers of cells/10 high-power fields.

Statistical analyses
The median (minimum–maximum) values were calculated for each parameter in each group. The significance of differences between groups were calculated using the Mann–Whitney U test for unpaired data and the Wilcoxon test for paired data.

Results

Initially, urinary sediments stained by the Papanicolaou technique were examined. However, urinary lymphocytes and monocytes could not be differentiated exactly. Therefore, both cell types were evaluated together. Urinary tract infections were diagnosed when pyuria was detected by leukocyte esterase dipstick test and microscopic urinalysis (>10 granulocytes/mm3 of unspun urine). Urinary sediments of patients with urinary tract infections were excluded from further immunocytochemical examination.

Table 1Go summarizes the results of urinary examinations by means of Papanicolaou technique and the results of the immunocytological findings. In cases of acute allograft rejection a significantly increased number of granulocytes (P<0.001), lymphocytes/monocytes (P<0.001), and renal cells (P<0.001) could be observed. However, the study groups showed a large dispersion and overlapping between stable graft function and acute allograft rejection. One reason for this finding may be the difficulty of an exact differentiation of these different urinary cells. Therefore, this method lacks sensitivity and specificity for diagnosing acute allograft rejection in practice. In cases of CsA toxicity the number of urinary granulocytes and lymphocytes/monocytes were not significantly influenced compared to stable graft function. However, CsA toxicity induced a remarkable increase in urinary renal cells (P<0.001). The immunocytological investigations showed few or no positive cells in the urine in patients with stable allograft function. In contrast, patients with acute rejecting renal allografts demonstrated characteristic cytological changes in the urine, including significantly increased CD3, CD4, CD8, and CD14 positive cells (P<0.001). Immunocytochemical staining of urinary sediments was found to be equally sensitive and specific with an accuracy over 90% in the diagnosis of acute renal allograft rejection (Table 2Go). CsA toxicity caused no significant changes of the positive cells in urine compared with stable graft function. Figure 1Go shows scatter plots of CD3 and CD4 positive cells. The data emphasize the pronounced increment of the positive cells in the course of acute allograft rejection.


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Table 1. Results of urinary examinations by means of Papanicolaou technique and immunocytology

 

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Table 2. Sensitivity, specificity, and predictive value of positive cells in urine for the diagnosis of acute allograft rejection

 


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Fig. 1. Scatter plots of CD3 and CD4 positive cells in urine. The data emphasize the significant increment of positive cells in urine during acute allograft rejection.

 
Three patients developed a systemic CMV infection, one patient a bacterial venous catheter-related sepsis with acinetobacter. The systemic infections caused no significant elevation of positive lymphocytes or monocytes/macrophages in urine (data not shown).

Discussion

The investigations of Papanicolaou stained urinary sediments confirmed that lymphocytes/monocytes significantly increase in cases of acute allograft rejection. However, the dispersion of the results and the overlapping between acute allograft rejection and stable graft function revealed an insufficient sensitivity and specificity of this method. Thus, a routine use of this method seems not to provide a practical adjunct to the evaluation of renal transplant recipients. The reason for this result might be the inaccurate differentiation of the urinary cells. Therefore, we tried to obtain a better differentiation of urinary T lymphocytes from other urinary cells using immunocytochemical staining. Other authors demonstrated that immunological cells in urine, despite degenerative changes, usually retain their antigenic determinations [7,16–22]. We observed that the technique of immunocytochemical staining enables an excellent identification of positive lymphocytes and monocytes/macrophages. No specimen had to be excluded from examination for reasons of loss of immunocytological staining capacity. Negative and positive controls improved the reliability of the immunocytological identification.

CD4-positive T helper cells and CD8-positive cytotoxic/suppressor cells showed a significant increase without characteristic differences between the two subsets in the course of acute allograft rejection. The increment of the positive cells in the urine usually appeared 1–2 days before the increase in serum creatinine. Under successful antirejection therapy with methylprednisolone, the cell counts fell immediately to values of stable graft function. The number of CD3-positive pan T cells exceed the sum of CD4 and CD8 positive cells because other mononuclear cells such as monocytes/macrophages could partially stain CD3 positive. Other investigators who performed immunocytological staining of urinary sediments also reported that during acute rejection periods, T lymphocytosis can be demonstrated in urinary sediments by incubating cytospin preparations with monoclonal antibodies against T cells [7,16, 19–21]. For example, Kyo et al. [16] found a significant increment of CD25-positive cells as a reliable indicator of rejection. Flow cytometric patterns with fluorescinated monoclonal antibodies were also highly sensitive and specific for the diagnosis of acute allograft rejection. However, the quantity of amorphous or necrotic debris and of mucus interfere with flow cytometry and limit the diagnostic value in the course of allograft rejection [22].

The early presence of lymphocytes in the urine during allograft rejection can be explained by the characteristic histological features of acute cellular rejection that include a pleomorphic interstitial infiltrate of mononuclear cells [13]. The infiltrating mononuclear cells typically include lymphoblasts. Characteristically 30–50% of infiltrating cells are CD3 positive T cells [13,23]. Interestingly, in cases of allograft rejection the distribution of urinary lymphocytes and monocytes seems to be comparable to the distribution of infiltrating immunocompetent cells in renal allograft biopsies.

The results of patients with CsA nephrotoxicity were all in the range of stable graft function. The acute renal dysfunction due to CsA seems to be caused by haemodynamically mediated changes and do not reflect a state of immunological activation [14]. Therefore, the immunocytological determination of urinary lymphocytes, and especially monocytes/macrophages, may contribute to the better differentiation of allograft rejection and CsA nephrotoxicity.

It is clear that immunocytological examination of the urine cannot be performed in patients with absolute anuria. In addition, patients with urinary tract infections have to be excluded. Urinary tract infections, however, can reliable be diagnosed by means of leukocyte esterase dipstick test and microscopic urinalysis. Additionally, a conventional urine culture should be performed for detecting significant bacteriuria. During the early post-transplant period (day 1–6) an enhanced number of positive cells were found in urine even in some cases of normal allograft function, which could lead to false-positive results. Therefore, immunocytological examination of the urine should not be performed during the first week following kidney transplantation.

In conclusion, our results show that immunocytological examination of urinary sediment enables one to obtain a more precise differentiation of T lymphocytes and monocytes/macrophages from other cells that are excreted in urine during rejection episodes. It is an easily feasible and cheap method with minimal patient discomfort, requiring no invasive procedure. This type of investigation can be performed daily without extensive technical equipment. In particular, the sequential determination of urinary CD3 positive pan T cells after the first week following kidney transplantation may contribute to the diagnosis of acute allograft rejection earlier than clinical criteria. No further differentiation of T lymphocytes appears to be necessary for the diagnosis of acute rejection. From the observation that in case of allograft rejection the distribution of urinary lymphocytes and monocytes appears to be comparable to the distribution of infiltrating immunocompetent cells in renal allograft tissue, it can be expected that immunocytological examination of urinary sediment may complement invasive renal biopsy. However, further prospective studies are necessary to confirm this hypothesis.

Notes

Correspondence and offprint requests to: Dr R. W. Grunewald, Division of Nephrology and Rheumatology, Department of Internal Medicine, University of Göttingen, Germany. Back

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Received for publication: 6. 3.99
Revision received 20. 1.00.



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