Urinary Cytodiagnosis of Acute Renal Allograft Rejection Using the Cytocentrifuge G. BERRY SCHUMANN, M.D., RICHARD L BURLESON, M.D., JOHN BERNARD HENRY, M.D., AND DAVID B. JONES, M.D.
WHEN A RENAL TRANSPLANT between two histoincompatible individuals is performed, a complex immunologic process is usually initiated which, if not suppressed, leads to the destruction of the allograft. The clinical representation of immunologic intolerance is the appearance of a rejection crisis, characterized by an acute deterioration of renal allograft function. Renal allograft rejections manifest a continuous spectrum involving both cellular and humoral immune reactions, and may be referred to as hyperacute, acute (early and delayed), or chronic. Acute allograft rejection is the most persistent and difficult, and different immunosuppressant protocols have been used to prevent it. The ability to detect or confirm early signs of threatened or impending acute renal rejection is of paramount importance because therapy can be instituted to control this reversible reaction. Received February 25, 1976: accepted for publication April 7, 1976. Address reprint requests to Dr. Schumann: Department of Pathology, Division of Clinical Pathology, State University of New York. Upstate Medical Center. 750 East Adams Street, Syracuse, New York 13210.
From the Division of Clinical Pathology, Departments of Anatomical Pathology and Surgery, State University of New York, Syracuse, New York
Several investigators1-1589 have undertaken serial cytologic examinations of the urinary sediment from renal allograft recipients on the assumption that renal parenchymal disease, and thus acute rejection, might be reflected in the exfoliated cells present in the urine. Urinary sediment examinations in all the reported studies were either qualitative (direct smears or membrane filter technics) or semiquantitative (counting cell chambers combined with differential staining technics). The purpose of our study was to evaluate the effectiveness of the Cytocentrifuge* as a means of preparing cytologic urinary specimens from renal allograft recipients. We have found this technic to be far superior, and we have studied the characteristic urinary sediments from patients with acute renal allograft rejection using it. Methods and Materials Cytologic examinations were performed on 224 urine specimens obtained from 15 renal allograft recipients at various intervals: in the immediate postoperative period, during clinically recognizable rejection episodes, and in periods of normal renal function. When possible, early morning urine (volumes ranging from 10 to 30 ml) was collected, and the container placed in an ice bath and immediately carried to the cytopathology laboratory with an accompanying requisition noting history of transplantation, radiation and chemotherapy. A 10-ml sample of urine was immediately spun in a standard centrifuge at 1,500 rpm for 10 minutes. The supernatant was discarded * Shandon Southern Instruments, Sewickley. Pennsylvania.
134
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Schumann, G. Berry, Burleson, Richard L., Henry, John Bernard, and Jones, David B.: Urinary cytodiagnosis of acute renal allograft rejection using the cytocentrifuge. Am. J Clin Pathol 67: 134-140, 1977. The clinical diagnosis of acute renal allograft rejection in immunosuppressed recipients can often be predicted or confirmed on the basis (if characteristic urinary cytologic findings. Use of cytocentrifiigation permits a simple, rapid, reproducible and semiquantitative means of preparing cytologic urinary specimens of diagnostic quality from small quantities of urine. The cytodiagnosis of acute renal rejection was established before or on the same day a clinical diagnosis of rejection was made in the majority of renal transplant eases studied over a 12-month period. Renal tubular cells were found to be the exfoliated cells of greatest value in predicting an acute rejection episode, and their persistence has prognostic importance. (Key words: Cytocentrifuge; Cytodiagnosis; Renal allograft rejection; Renal tubular cells.)
135
CYTODIAGNOSIS OF ACUTE RENAL REJECTION
Vol. 67 . No. 2
Table 1. Summary of Cases of 15 Renal Allograft Recipients Number of Clinical Acute Rejections
Cadaver (C)or Live Donor (L) Kidney
Number of Urinary Cytologies
Patient 1
C
6
+4
Patient 2
C
4
+3
+3
Mild
Patient 3
L
15
-1
+1
Mildmoderate
Patient 4
C
6
-2
14
0
Marked
0
Mildmoderate
Patient 5
C
Patient 6
Cytologic Diagnosis vs. Clinical Diagnosis*
Cytologic Diagnosis vs. Chemical Diagnosist
Estimated Severity of Rejection Episodet
Nephrectomy
Acute Tubular Necrosis
Other Cytologic Findings
Mild Viral inclusions
Mild
L
12
+1
+1
Mild
Patient 8
C
29
-1
-1
Marked
Patient 9
C
17
0
+1
Mild
+2
Moderatemarked
Patient 10
C
26
+2
Patient 11
C
24
+2
+3
Mild
Acute cystitis
Patient 12
C
26
+4
+3
Mild
Viral inclusions
Patient 13
C
9
-1
-1
Mildmoderate
Patient 14
L
20
0
0
Patient 15
L
12
TOTAL
224
Marked Recurrent disease
15
* Days between cytologic diagnosis of rejection and clinical diagnosis ( - = days cytologic diagnosis was made before clinical diagnosis; lifter clinical diagnosis. t Days between cytologic diagnosis of rejection and chemical diagnosis (>0.3 mg/dl increase in serum creatinine from previous level). t Predicted from urinary sediment.
by hand-pipetting to 1 ml and the sediment resuspended. Using a Cytocentrifuge, four slides were prepared using four drops of resuspended specimen per chamber and spinning at 900 rpm for 3 minutes. After the filter was discarded, one to two drops of Parlodiont were applied to the cellular area of a horizontally held slide. The slide was then fixed for 15 minutes in acetic acid-alcohol$ and stained by the Papanicolaou technic.§ All four slides were screened, noting background pattern, cellularity, erythrocytes, viral inclusions, and abnormal cells. Ten high-power fields were counted on the most cellular slide for t 200 ml 95% EtOH, 200 ml anhydrous ether, 1 g Parlodion (Mallinckrodt). t One part glacial acetic acid to nine parts 95% ethanol. § Aqueous alum hematoxylin, OG-6, EA-36.
= days cytologic diagnosis was made
neutrophils, lymphocytes, renal tubular cells, and casts. All specimens were counted separately by at least two cytotechnologists. Results A preliminary summary of our interpretations of cytologic findings and the clinical courses of 15 renal transplant recipients using the Cytocentrifuge is presented in Table 1. Urine specimens from 15 renal allograft patients examined as long as three months after transplantation revealed 15 documented, clinical acute rejection episodes. In more than 50% (8/14) of the episodes, the cytologic diagnosis was established before or on the day of clinically recognizable rejection. Clinical criteria for rejection were localized
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Patient 7
A.J.CI'. • February 1977
SCHUMANN ET AL.
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CYTODIAGNOSIS OF ACUTE RENAL REJECTION FIG. I (upper, left). Common urinary sediment findings during an acute renal rejection episode. Note marked cellularity. "dirty" background, and granular cast formation. Papanicolaou stain. xI50.
FIG. 2 (upper, right). Urinary cytologic findings from a different patient during an acute renal rejection episode. A renal tubular cast (arrow) is present. This type of cast is strong cytologic evidence for renal parenchymal disease. Papanicolaou stain. x430. FIG. 3 (lower left). Urine cytology from another transplant recipient (Patient 12) with one of several multinucleated cells with several eosinophilic intranuclear inclusions (arrow) which were consistent with a recurrent Herpes simplex infection. Papanicolaou stain. x430. FIG. 4. Urine cytology consistent with an acute renal rejection episode. Note the granular cast and numerous renal tubular cells (arrows). The majority of the renal tubular cells (RTC's) appeared as cuboidal cells measuring approximately 10-14 ^.m in diameter with distinct cell borders, light staining cytoplasm and central or slightly eccentric nuclei. RTC's represent the significant cell to follow during acute renal rejection. (Papanicolaou stain—x430)
simplex), with diagnostic viral inclusion cells. In both patients, the intranuclear inclusion cells were present for several days at a time when the clinical picture was consistent with a viral disease (Fig. 3). In the case of the one patient who had acute cystitis, the cytologic diagnosis of acute rejection could not be made until three days after the clinical diagnosis was established, due to the obscuring inflammatory process.
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abdominal pain over the transplant site, fever, and increased serum creatinine (increase of 0.3 mg/dl from previous level). Two patients (Patients 1 and 5) required hemodialysis for posttransplant acute tubular necrosis, so serum creatinine could not be used directly as a diagnostic indicator. Baseline levels of renal tubular cells, lymphocytes, and neutrophils were established during the first postoperative week. Several patients were followed serially after the seventh postoperative day. Increased numbers of lymphocytes, neutrophils, and histiocytes were commonly present during acute rejection periods; however, correlation with these cells was less accurate than with renal tubular cells. More than 15 renal tubular cells per ten high-power fields (HPF) was found to be indicative of rejection. If a second consecutive specimen showed this increase, a cytodiagnosis of acute rejection was made. In several cases, the concentration of renal tubular cells was greater than 100/10 HPF during a rejection episode. During post-rejection therapy, renal tubular cells decreased to fewer than 15/10 HPF. Failure to decrease the number of renal tubular cells represented a poor prognostic sign. In five of seven patients who exfoliated significant numbers of renal tubular cells for more than three days during a rejection episode or failed to decrease the number of renal tubular cells after antirejection therapy, nephrectomies were eventually necessary due to infarction of the kidneys. Two patients (Patients 5 and 12) had severe acute tubular necrosis postoperatively, so renal tubular cells could not be used in diagnosis. In these cases, lymphocytes (>50/10 HPF) were apparently more predictive evidence of rejection. Other common cytologic findings during acute rejection included granular or amorphous background, increased cellularity, and cast formation (Fig. 1). The finding of a renal tubular cast is strong cytologic evidence for renal parenchymal disease (Fig. 2). Urine samples from two patients showed cytologic evidence of viral disease (cytomegalovirus and herpes
A.J.C.P. • February 1977
SCHUMANN ETAL.
138
Daily postoperative serum creatinine determinations were compared with urinary renal tubular cells and lymphocytes in the cases of two renal transplant recipients. After one mild acute renal rejection episode on December 20, 1974, Patient 9 responded to therapy, and is currently asymptomatic (Fig. 6). The diagnosis of acute renal rejection was made both clinically and cytologically on the same day. Patient 14 had a moderate to severe rejection episode on January 27, 1975, which was diagnosed both clinically and cytologically on the same day (Fig. 7). In contrast, this patient continued to exfoliate increased numbers of renal tubular cells after anti-rejection therapy. A nephrectomy was performed on February 19, 1975, due to eventual infarction of the kidney.
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16
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14
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FIG. 6. Transplant recipient (A.S.) was followed with serial urinary cytologic examinations. Daily postoperative serum creatinine determinations were compared with urinary renal tubular cells (RTC's) and lymphocytes. After one acute rejection episode (12/20/74), the patient responded to therapy and is currently asymptomatic. Diagnosis of acute renal rejection was made both clinically and cytologically on the same day. Radiotherapy (. . .) was initiated after the clinical diagnosis was established. The top of the diagram shows the amount of immunosuppression used.
The majority of the renal tubular cells present appeared as cuboidal epithelial cells approximately 1014 jitm in diameter with distinct cell borders, lightstaining granular cytoplasm, and central or slightly eccentric nuclei (Fig. 4). Renal tubular cells were also present in casts and as tissue fragments. Radiation changes occurring in tubular epithelial cells, with occasional binucleate forms, and increased mitotic activity were frequently seen during post-rejection therapy (Fig. 5).
Previous investigators have followed the urinary sediment during acute renal allograft rejection. The following cytologic findings have been reported to occur during acute rejection: hematuria, cast formation (hyaline, granular, erythrocyte, renal tubular cell, lymphocytes, and mixed epithelial cell), increased mononuclear cells, epithelial cells, lymphocytes, and renal tubular cells. Initial cytologic studies were based on the hypothesis that since lymphocytes are the cells involved in the immune response and are seen as a diffuse interstitial infiltrate of renal parenchyma during rejection, they represent the diagnostic cell to follow in the urinary sediment. In 1974, Kaufman and associates4 looked for lymphocytes in the stained sediments of urines from renal-transplanted patients and observed lymphocytes in 8/11 rejection episodes of 7/9 patients. Kline and Craighead, in 1967,5 found that two kinds of epithelial cells were present in the urines of recipients of transplanted kidneys. One cell type, 7-9 /urn in diameter, slightly larger than lymphocytes, clustered in groups of five to 20 cells with scant cytoplasm and hyperchromatic nuclei, was found in urine immediately following transplantation, one to two weeks prior to death, and during episodes of threatened rejections. The second cell type was 10-40 /im in diameter, scattered individually, often had irregular cell borders, enlarged nuclei, clumped nuclear chromatin, and was predominant at times other than during rejection. Transplanted kidneys examined histologically following acute renal rejection showed scattered tubules in the majority of the kidneys that were lined by or contained within their lumen atypical cells with enlarged hyperchromatic nuclei, apparently corresponding to Kline and Craighead's second type. Spencer and Peterson followed 11 patients during 14 allograft rejection episodes using a quantitative
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CYTODIAGNOSIS OF ACUTE RENAL REJECTION 2 0
Imuron(mg)
° 0
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Prednisone (mg)
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Solumedrol(mg) 8 0 0 p
Radiotherapy
le
450
"
16
80
400
14
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60
3 0 0 •= o
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1 1 20
100
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FIG. 7. Transplant recipient (T.A.) was also followed postoperatively with serial urinary cytologic examinations and serum creatinine determinations. Acute renal rejection was diagnosed both clinically and cytologically on the same day. This patient had a moderate to severe rejection episode and continued to exfoliate increased numbers of RTC*s after anti-rejection therapy. A nephrectomy was performed on 2/19/75 due to renal infarction.
the appearance of renal tubular cells in large numbers in the early stage of acute rejection. Histologically, early acute rejection seems to consist largely of a mild ischemic process, an inflammatory component, and a prominent interstitial lymphocytic infiltrate.7 It appears that the renal tubular cells present in the urinary sediment are the result of desquamation of tubular epithelial cells secondary to this ischemic process. A suitable explanation for the lymphocytes that appear in urinary sediment is not apparent. In several of our cases, a peak increase in the urinary lympho-
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cell-chamber count. They found the most reliable indication of threatening renal allograft rejection was a urine containing more than 25,000 lymphocytes/hour. In 2/14 episodes, lymphocyturia appeared before clearcut clinical evidence of rejection, making early diagnosis possible in these cases. In the other 12 rejection episodes, increased lymphocyte excretion could first be demonstrated on the day when the signs of rejection appeared. The number of lymphocytes in the urine tended to decrease with initiation or increase in therapy. One patient had increased lymphocytes without apparent rejection. In 1970, Bossen and co-workers,1 using a membranefilter technic, followed 17 acute rejection episodes in 16/33 patients. By following a cytologic profile that includes several modalities—background debris, lymphocytes, renal tubular cells, casts, etc.—they predicted rejection before clinical evidence was apparent. After rejection therapy, there was a dramatic decrease in exfoliated cells. They considered no one profile modality to be pathognomonic for rejection. Also in 1970, Papamiditrou and colleagues,6 using a semiquantitative technic (counting chamber and differential stains), showed increased excretion of lymphocytes/hour during rejection. Hrushesky and associates2 serially followed pyroninophilic lymphocytes during 14 acute rejection episodes; frequently the lymphocytes were present before clinical evidence of rejection. Although there are several modalities to follow in urinary sediment (background debris, cells, and casts), results of our study confirm that the appearance of renal tubular cells more reliably documents an acute renal rejection episode than do lymphocytes. Using Papanicolaou-stained sediments, renal tubular cells were readily distinguishable from transitional cells, histiocytes, and lymphocytes. The diagnostic renal tubular cells were seen as single cells (10-14 fjon in diameter), as cell clusters (3 to 8 cells) and in cast formation (Fig. 4). Unpublished evidence using touch preparations of normal and rejected kidneys confirmed these cells as renal tubular cells. Large atypical renal tubular cells (Kline and Craighead's second type5) were observed, but these cells did not correlate with rejection episodes. Currently we are investigating cytochemical staining to improve accuracy in counting these small diagnostic renal tubular cells. Although Hrushesky and associates2 employed pyroninophilic staining technics in following lymphocytes during rejection episodes, methyl pyronin-green staining has not been useful in our laboratory, because regenerating renal tubular cells, lymphocytes, and plasma cells all stain positively. It is appropriate to speculate regarding the cause of
139
tubular
Vol. 67 • No. 2
140
A.J.C.I'. . Febniiin 1977
SCHUMANN ET AL.
sions, fungal organisms, crystals, and inflammation were clearly demonstrated. References 1. Bossen EH, Johnston WW, Amatulli J, et al: Exfoliative cytopathologic studies in organ transplantation. III. The cytologic profile of urine during acute renal allograft rejection. ActaCytol 14:176-181. 1970 2. Hrushesky W, Sampson D; Murphy GP: Lymphocytouria in Human renal allograft rejection. Arch Surg 105:424-426, 1972 3. Hume DM, Magee JH, Kaufman HM. et al: Renal homotransplantation in man in modified recipients. Ann Surg 158: 608-644, 1963 4. Kaufman HM, Clark RF, Magee JH, et al: Lymphocytes in urine as an aid in the early detection of renal tiomograft rejection. Surg Gynecol Obstet 119:25-36, 1964 5. Klihe TS, Craighead JE: Renal homotransplantation. The cytology of the urine sediment. Am J Clin Patriol 47:802806, 1967 6. Papadimitriou GP, ChisHolm AE, Kulatilake AE. et al: Clinical evaluation of the urinary sediment after renal allotransplantation. J Clin Pathol 23:99-103, 1970 7. Porter, KA: Pathological changes in transplanted kidneys. Experience in Renal Transplantation. Edited by Starzl TE. Philadelphia, W. B. Saunders, 1964 8. Spencer ES, Petersen VV: The urinary sediment after renal transplantation. Quantitative, changes as an index of the activity of the renal allograft reaction. Acta Med Scand 182:73-82, 1967 9. Taft PD, Flax MH: Urinary cytology in renal transplantation. Transplantation 4:194-204, 1966
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cytes followed a peak increase in the renal tubular cells, suggesting that after desquamation of renal tubular cells there is disruption of tubular basement membrane, and a leakage of proteinaceous material and mononuclear cells (lymphocytes). Use of cytocentrifugatiori permitted a simple, rapid, reproducible and semiquantitative means of preparing cytologic urinary specimens of diagnostic quality. A major advantage over the membrane technic is the ability to process smail specimen volumes (5-20 ml) and concentrate the sediment on a small area of the slide (a circle 6 mm in diameter). Use of Papanicolaou-stained sediments allowed for serial examination of urinary sediments, and documentation of progressive renal disease (Fig: 7). Also, the slide could be reviewed at a later date. Currently, in our institution, urinary cytologic results are routinely included in the management of patients; a positive urinary cytology is considered a clinical indication for increased immunosuppressive therapy. More important, urinary cytology can be an excellent technic to rule out the possibility of renal rejection when unexplained fever occurs in a renaltransplanted patient. Using the cytocentrifuge technic and the Papanicolaou stain, bacteria, viral inclu-
WHEN A RENAL TRANSPLANT between two histoincompatible individuals is performed, a complex immunologic process is usually initiated which, if not suppressed, leads to the destruction of the allograft. The clinical representation of immunologic intolerance is the appearance of a rejection crisis, characterized by an acute deterioration of renal allograft function. Renal allograft rejections manifest a continuous spectrum involving both cellular and humoral immune reactions, and may be referred to as hyperacute, acute (early and delayed), or chronic. Acute allograft rejection is the most persistent and difficult, and different immunosuppressant protocols have been used to prevent it. The ability to detect or confirm early signs of threatened or impending acute renal rejection is of paramount importance because therapy can be instituted to control this reversible reaction. Received February 25, 1976: accepted for publication April 7, 1976. Address reprint requests to Dr. Schumann: Department of Pathology, Division of Clinical Pathology, State University of New York. Upstate Medical Center. 750 East Adams Street, Syracuse, New York 13210.
From the Division of Clinical Pathology, Departments of Anatomical Pathology and Surgery, State University of New York, Syracuse, New York
Several investigators1-1589 have undertaken serial cytologic examinations of the urinary sediment from renal allograft recipients on the assumption that renal parenchymal disease, and thus acute rejection, might be reflected in the exfoliated cells present in the urine. Urinary sediment examinations in all the reported studies were either qualitative (direct smears or membrane filter technics) or semiquantitative (counting cell chambers combined with differential staining technics). The purpose of our study was to evaluate the effectiveness of the Cytocentrifuge* as a means of preparing cytologic urinary specimens from renal allograft recipients. We have found this technic to be far superior, and we have studied the characteristic urinary sediments from patients with acute renal allograft rejection using it. Methods and Materials Cytologic examinations were performed on 224 urine specimens obtained from 15 renal allograft recipients at various intervals: in the immediate postoperative period, during clinically recognizable rejection episodes, and in periods of normal renal function. When possible, early morning urine (volumes ranging from 10 to 30 ml) was collected, and the container placed in an ice bath and immediately carried to the cytopathology laboratory with an accompanying requisition noting history of transplantation, radiation and chemotherapy. A 10-ml sample of urine was immediately spun in a standard centrifuge at 1,500 rpm for 10 minutes. The supernatant was discarded * Shandon Southern Instruments, Sewickley. Pennsylvania.
134
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Schumann, G. Berry, Burleson, Richard L., Henry, John Bernard, and Jones, David B.: Urinary cytodiagnosis of acute renal allograft rejection using the cytocentrifuge. Am. J Clin Pathol 67: 134-140, 1977. The clinical diagnosis of acute renal allograft rejection in immunosuppressed recipients can often be predicted or confirmed on the basis (if characteristic urinary cytologic findings. Use of cytocentrifiigation permits a simple, rapid, reproducible and semiquantitative means of preparing cytologic urinary specimens of diagnostic quality from small quantities of urine. The cytodiagnosis of acute renal rejection was established before or on the same day a clinical diagnosis of rejection was made in the majority of renal transplant eases studied over a 12-month period. Renal tubular cells were found to be the exfoliated cells of greatest value in predicting an acute rejection episode, and their persistence has prognostic importance. (Key words: Cytocentrifuge; Cytodiagnosis; Renal allograft rejection; Renal tubular cells.)
135
CYTODIAGNOSIS OF ACUTE RENAL REJECTION
Vol. 67 . No. 2
Table 1. Summary of Cases of 15 Renal Allograft Recipients Number of Clinical Acute Rejections
Cadaver (C)or Live Donor (L) Kidney
Number of Urinary Cytologies
Patient 1
C
6
+4
Patient 2
C
4
+3
+3
Mild
Patient 3
L
15
-1
+1
Mildmoderate
Patient 4
C
6
-2
14
0
Marked
0
Mildmoderate
Patient 5
C
Patient 6
Cytologic Diagnosis vs. Clinical Diagnosis*
Cytologic Diagnosis vs. Chemical Diagnosist
Estimated Severity of Rejection Episodet
Nephrectomy
Acute Tubular Necrosis
Other Cytologic Findings
Mild Viral inclusions
Mild
L
12
+1
+1
Mild
Patient 8
C
29
-1
-1
Marked
Patient 9
C
17
0
+1
Mild
+2
Moderatemarked
Patient 10
C
26
+2
Patient 11
C
24
+2
+3
Mild
Acute cystitis
Patient 12
C
26
+4
+3
Mild
Viral inclusions
Patient 13
C
9
-1
-1
Mildmoderate
Patient 14
L
20
0
0
Patient 15
L
12
TOTAL
224
Marked Recurrent disease
15
* Days between cytologic diagnosis of rejection and clinical diagnosis ( - = days cytologic diagnosis was made before clinical diagnosis; lifter clinical diagnosis. t Days between cytologic diagnosis of rejection and chemical diagnosis (>0.3 mg/dl increase in serum creatinine from previous level). t Predicted from urinary sediment.
by hand-pipetting to 1 ml and the sediment resuspended. Using a Cytocentrifuge, four slides were prepared using four drops of resuspended specimen per chamber and spinning at 900 rpm for 3 minutes. After the filter was discarded, one to two drops of Parlodiont were applied to the cellular area of a horizontally held slide. The slide was then fixed for 15 minutes in acetic acid-alcohol$ and stained by the Papanicolaou technic.§ All four slides were screened, noting background pattern, cellularity, erythrocytes, viral inclusions, and abnormal cells. Ten high-power fields were counted on the most cellular slide for t 200 ml 95% EtOH, 200 ml anhydrous ether, 1 g Parlodion (Mallinckrodt). t One part glacial acetic acid to nine parts 95% ethanol. § Aqueous alum hematoxylin, OG-6, EA-36.
= days cytologic diagnosis was made
neutrophils, lymphocytes, renal tubular cells, and casts. All specimens were counted separately by at least two cytotechnologists. Results A preliminary summary of our interpretations of cytologic findings and the clinical courses of 15 renal transplant recipients using the Cytocentrifuge is presented in Table 1. Urine specimens from 15 renal allograft patients examined as long as three months after transplantation revealed 15 documented, clinical acute rejection episodes. In more than 50% (8/14) of the episodes, the cytologic diagnosis was established before or on the day of clinically recognizable rejection. Clinical criteria for rejection were localized
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
Patient 7
A.J.CI'. • February 1977
SCHUMANN ET AL.
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137
CYTODIAGNOSIS OF ACUTE RENAL REJECTION FIG. I (upper, left). Common urinary sediment findings during an acute renal rejection episode. Note marked cellularity. "dirty" background, and granular cast formation. Papanicolaou stain. xI50.
FIG. 2 (upper, right). Urinary cytologic findings from a different patient during an acute renal rejection episode. A renal tubular cast (arrow) is present. This type of cast is strong cytologic evidence for renal parenchymal disease. Papanicolaou stain. x430. FIG. 3 (lower left). Urine cytology from another transplant recipient (Patient 12) with one of several multinucleated cells with several eosinophilic intranuclear inclusions (arrow) which were consistent with a recurrent Herpes simplex infection. Papanicolaou stain. x430. FIG. 4. Urine cytology consistent with an acute renal rejection episode. Note the granular cast and numerous renal tubular cells (arrows). The majority of the renal tubular cells (RTC's) appeared as cuboidal cells measuring approximately 10-14 ^.m in diameter with distinct cell borders, light staining cytoplasm and central or slightly eccentric nuclei. RTC's represent the significant cell to follow during acute renal rejection. (Papanicolaou stain—x430)
simplex), with diagnostic viral inclusion cells. In both patients, the intranuclear inclusion cells were present for several days at a time when the clinical picture was consistent with a viral disease (Fig. 3). In the case of the one patient who had acute cystitis, the cytologic diagnosis of acute rejection could not be made until three days after the clinical diagnosis was established, due to the obscuring inflammatory process.
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Ffc * FIG. 5. Urine cytologic preparation from a transplant recipient following radiation therapy. Note the large atypical epithelial cell cluster with enlarged nuclei and foamy cytoplasm. Papanicolaou stain. x410.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
abdominal pain over the transplant site, fever, and increased serum creatinine (increase of 0.3 mg/dl from previous level). Two patients (Patients 1 and 5) required hemodialysis for posttransplant acute tubular necrosis, so serum creatinine could not be used directly as a diagnostic indicator. Baseline levels of renal tubular cells, lymphocytes, and neutrophils were established during the first postoperative week. Several patients were followed serially after the seventh postoperative day. Increased numbers of lymphocytes, neutrophils, and histiocytes were commonly present during acute rejection periods; however, correlation with these cells was less accurate than with renal tubular cells. More than 15 renal tubular cells per ten high-power fields (HPF) was found to be indicative of rejection. If a second consecutive specimen showed this increase, a cytodiagnosis of acute rejection was made. In several cases, the concentration of renal tubular cells was greater than 100/10 HPF during a rejection episode. During post-rejection therapy, renal tubular cells decreased to fewer than 15/10 HPF. Failure to decrease the number of renal tubular cells represented a poor prognostic sign. In five of seven patients who exfoliated significant numbers of renal tubular cells for more than three days during a rejection episode or failed to decrease the number of renal tubular cells after antirejection therapy, nephrectomies were eventually necessary due to infarction of the kidneys. Two patients (Patients 5 and 12) had severe acute tubular necrosis postoperatively, so renal tubular cells could not be used in diagnosis. In these cases, lymphocytes (>50/10 HPF) were apparently more predictive evidence of rejection. Other common cytologic findings during acute rejection included granular or amorphous background, increased cellularity, and cast formation (Fig. 1). The finding of a renal tubular cast is strong cytologic evidence for renal parenchymal disease (Fig. 2). Urine samples from two patients showed cytologic evidence of viral disease (cytomegalovirus and herpes
A.J.C.P. • February 1977
SCHUMANN ETAL.
138
Daily postoperative serum creatinine determinations were compared with urinary renal tubular cells and lymphocytes in the cases of two renal transplant recipients. After one mild acute renal rejection episode on December 20, 1974, Patient 9 responded to therapy, and is currently asymptomatic (Fig. 6). The diagnosis of acute renal rejection was made both clinically and cytologically on the same day. Patient 14 had a moderate to severe rejection episode on January 27, 1975, which was diagnosed both clinically and cytologically on the same day (Fig. 7). In contrast, this patient continued to exfoliate increased numbers of renal tubular cells after anti-rejection therapy. A nephrectomy was performed on February 19, 1975, due to eventual infarction of the kidney.
200 'lmuran(mg) I Q O 0 80 40
Prednisone (mg)
Solumedrol (mg) 6 0 0 [
Radiotherapy 90
450
16
80
400
14
70
350
12 -
2 60
3 0 0 a. o
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FIG. 6. Transplant recipient (A.S.) was followed with serial urinary cytologic examinations. Daily postoperative serum creatinine determinations were compared with urinary renal tubular cells (RTC's) and lymphocytes. After one acute rejection episode (12/20/74), the patient responded to therapy and is currently asymptomatic. Diagnosis of acute renal rejection was made both clinically and cytologically on the same day. Radiotherapy (. . .) was initiated after the clinical diagnosis was established. The top of the diagram shows the amount of immunosuppression used.
The majority of the renal tubular cells present appeared as cuboidal epithelial cells approximately 1014 jitm in diameter with distinct cell borders, lightstaining granular cytoplasm, and central or slightly eccentric nuclei (Fig. 4). Renal tubular cells were also present in casts and as tissue fragments. Radiation changes occurring in tubular epithelial cells, with occasional binucleate forms, and increased mitotic activity were frequently seen during post-rejection therapy (Fig. 5).
Previous investigators have followed the urinary sediment during acute renal allograft rejection. The following cytologic findings have been reported to occur during acute rejection: hematuria, cast formation (hyaline, granular, erythrocyte, renal tubular cell, lymphocytes, and mixed epithelial cell), increased mononuclear cells, epithelial cells, lymphocytes, and renal tubular cells. Initial cytologic studies were based on the hypothesis that since lymphocytes are the cells involved in the immune response and are seen as a diffuse interstitial infiltrate of renal parenchyma during rejection, they represent the diagnostic cell to follow in the urinary sediment. In 1974, Kaufman and associates4 looked for lymphocytes in the stained sediments of urines from renal-transplanted patients and observed lymphocytes in 8/11 rejection episodes of 7/9 patients. Kline and Craighead, in 1967,5 found that two kinds of epithelial cells were present in the urines of recipients of transplanted kidneys. One cell type, 7-9 /urn in diameter, slightly larger than lymphocytes, clustered in groups of five to 20 cells with scant cytoplasm and hyperchromatic nuclei, was found in urine immediately following transplantation, one to two weeks prior to death, and during episodes of threatened rejections. The second cell type was 10-40 /im in diameter, scattered individually, often had irregular cell borders, enlarged nuclei, clumped nuclear chromatin, and was predominant at times other than during rejection. Transplanted kidneys examined histologically following acute renal rejection showed scattered tubules in the majority of the kidneys that were lined by or contained within their lumen atypical cells with enlarged hyperchromatic nuclei, apparently corresponding to Kline and Craighead's second type. Spencer and Peterson followed 11 patients during 14 allograft rejection episodes using a quantitative
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FIG. 7. Transplant recipient (T.A.) was also followed postoperatively with serial urinary cytologic examinations and serum creatinine determinations. Acute renal rejection was diagnosed both clinically and cytologically on the same day. This patient had a moderate to severe rejection episode and continued to exfoliate increased numbers of RTC*s after anti-rejection therapy. A nephrectomy was performed on 2/19/75 due to renal infarction.
the appearance of renal tubular cells in large numbers in the early stage of acute rejection. Histologically, early acute rejection seems to consist largely of a mild ischemic process, an inflammatory component, and a prominent interstitial lymphocytic infiltrate.7 It appears that the renal tubular cells present in the urinary sediment are the result of desquamation of tubular epithelial cells secondary to this ischemic process. A suitable explanation for the lymphocytes that appear in urinary sediment is not apparent. In several of our cases, a peak increase in the urinary lympho-
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cell-chamber count. They found the most reliable indication of threatening renal allograft rejection was a urine containing more than 25,000 lymphocytes/hour. In 2/14 episodes, lymphocyturia appeared before clearcut clinical evidence of rejection, making early diagnosis possible in these cases. In the other 12 rejection episodes, increased lymphocyte excretion could first be demonstrated on the day when the signs of rejection appeared. The number of lymphocytes in the urine tended to decrease with initiation or increase in therapy. One patient had increased lymphocytes without apparent rejection. In 1970, Bossen and co-workers,1 using a membranefilter technic, followed 17 acute rejection episodes in 16/33 patients. By following a cytologic profile that includes several modalities—background debris, lymphocytes, renal tubular cells, casts, etc.—they predicted rejection before clinical evidence was apparent. After rejection therapy, there was a dramatic decrease in exfoliated cells. They considered no one profile modality to be pathognomonic for rejection. Also in 1970, Papamiditrou and colleagues,6 using a semiquantitative technic (counting chamber and differential stains), showed increased excretion of lymphocytes/hour during rejection. Hrushesky and associates2 serially followed pyroninophilic lymphocytes during 14 acute rejection episodes; frequently the lymphocytes were present before clinical evidence of rejection. Although there are several modalities to follow in urinary sediment (background debris, cells, and casts), results of our study confirm that the appearance of renal tubular cells more reliably documents an acute renal rejection episode than do lymphocytes. Using Papanicolaou-stained sediments, renal tubular cells were readily distinguishable from transitional cells, histiocytes, and lymphocytes. The diagnostic renal tubular cells were seen as single cells (10-14 fjon in diameter), as cell clusters (3 to 8 cells) and in cast formation (Fig. 4). Unpublished evidence using touch preparations of normal and rejected kidneys confirmed these cells as renal tubular cells. Large atypical renal tubular cells (Kline and Craighead's second type5) were observed, but these cells did not correlate with rejection episodes. Currently we are investigating cytochemical staining to improve accuracy in counting these small diagnostic renal tubular cells. Although Hrushesky and associates2 employed pyroninophilic staining technics in following lymphocytes during rejection episodes, methyl pyronin-green staining has not been useful in our laboratory, because regenerating renal tubular cells, lymphocytes, and plasma cells all stain positively. It is appropriate to speculate regarding the cause of
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sions, fungal organisms, crystals, and inflammation were clearly demonstrated. References 1. Bossen EH, Johnston WW, Amatulli J, et al: Exfoliative cytopathologic studies in organ transplantation. III. The cytologic profile of urine during acute renal allograft rejection. ActaCytol 14:176-181. 1970 2. Hrushesky W, Sampson D; Murphy GP: Lymphocytouria in Human renal allograft rejection. Arch Surg 105:424-426, 1972 3. Hume DM, Magee JH, Kaufman HM. et al: Renal homotransplantation in man in modified recipients. Ann Surg 158: 608-644, 1963 4. Kaufman HM, Clark RF, Magee JH, et al: Lymphocytes in urine as an aid in the early detection of renal tiomograft rejection. Surg Gynecol Obstet 119:25-36, 1964 5. Klihe TS, Craighead JE: Renal homotransplantation. The cytology of the urine sediment. Am J Clin Patriol 47:802806, 1967 6. Papadimitriou GP, ChisHolm AE, Kulatilake AE. et al: Clinical evaluation of the urinary sediment after renal allotransplantation. J Clin Pathol 23:99-103, 1970 7. Porter, KA: Pathological changes in transplanted kidneys. Experience in Renal Transplantation. Edited by Starzl TE. Philadelphia, W. B. Saunders, 1964 8. Spencer ES, Petersen VV: The urinary sediment after renal transplantation. Quantitative, changes as an index of the activity of the renal allograft reaction. Acta Med Scand 182:73-82, 1967 9. Taft PD, Flax MH: Urinary cytology in renal transplantation. Transplantation 4:194-204, 1966
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cytes followed a peak increase in the renal tubular cells, suggesting that after desquamation of renal tubular cells there is disruption of tubular basement membrane, and a leakage of proteinaceous material and mononuclear cells (lymphocytes). Use of cytocentrifugatiori permitted a simple, rapid, reproducible and semiquantitative means of preparing cytologic urinary specimens of diagnostic quality. A major advantage over the membrane technic is the ability to process smail specimen volumes (5-20 ml) and concentrate the sediment on a small area of the slide (a circle 6 mm in diameter). Use of Papanicolaou-stained sediments allowed for serial examination of urinary sediments, and documentation of progressive renal disease (Fig: 7). Also, the slide could be reviewed at a later date. Currently, in our institution, urinary cytologic results are routinely included in the management of patients; a positive urinary cytology is considered a clinical indication for increased immunosuppressive therapy. More important, urinary cytology can be an excellent technic to rule out the possibility of renal rejection when unexplained fever occurs in a renaltransplanted patient. Using the cytocentrifuge technic and the Papanicolaou stain, bacteria, viral inclu-
