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Saudi Journal of Kidney Diseases and Transplantation
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ORIGINAL ARTICLE Table of Contents   
Year : 1995  |  Volume : 6  |  Issue : 1  |  Page : 15-21
The Diagnosis of Acute Renal Allograft Rejection Using T-lymphocyte Subsets in the Peripheral Blood: A Better Test Now?


Jeddah Kidney Center, King Fahd Hospital, Jeddah, Saudi Arabia

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   Abstract 

Measuring the T-lymphocyte subsets CD4/CD8 ratio is known to be useful in the early diagnosis of acute rejection. A ratio of 1.3 or more is said to indicate the presence of acute rejection, but the sensitivity and specificity rates of the test are too low to render it of diagnostic value. We revisited this test on twenty-three renal allograft recipients who developed graft dysfunction within the first three months following transplantation. All cases were investigated thoroughly to identify the cause of graft dysfunction. Two groups of patients were identified. Group 1: patients confirmed to have acute rejection as the cause of graft dysfunction (13 patients); and group 2: patients found to have other causes of graft dysfunction (10 patients). Results of subsets with cluster of differentiation ratio CD4/CD8 were correlated with incidence of acute rejection. Of the 13 patients with acute rejection 10 revealed a CD4/CD8 ratio more than 1.7, while in the other three CD4/CD8 ratio was less than 1.7. In the second group of 10 patients in whom the cause of graft dysfunction was not acute rejection, the CD4/CD8 ratio was less than 1.7 in all cases. Our study indicates that T-lymphocyte subset ratio is useful in diagnosing acute rejection with a sensitivity and specificity of 81% and 100% respectively when the cutoff point is taken as 1.7. Studies with larger series of patients are needed to confirm this observation.

Keywords: Renal transplantation, Acute rejection, T-lymphocyte subsets.

How to cite this article:
Sheikh IA, Al-Menawy L, Shaheen FA, Al-Koussi M, Shehab AB. The Diagnosis of Acute Renal Allograft Rejection Using T-lymphocyte Subsets in the Peripheral Blood: A Better Test Now?. Saudi J Kidney Dis Transpl 1995;6:15-21

How to cite this URL:
Sheikh IA, Al-Menawy L, Shaheen FA, Al-Koussi M, Shehab AB. The Diagnosis of Acute Renal Allograft Rejection Using T-lymphocyte Subsets in the Peripheral Blood: A Better Test Now?. Saudi J Kidney Dis Transpl [serial online] 1995 [cited 2020 Apr 3];6:15-21. Available from: http://www.sjkdt.org/text.asp?1995/6/1/15/40893

   Introduction Top


Acute rejection episodes still constitute a major obstacle in the path of successful renal transplantation and carry the largest impact over renal allograft survival [1] . Varying degrees of progress has been made in order to overcome this problem. On the one hand new immunosuppressive agents have been developed and on the other, many techniques are being tried to predict or diagnose rejection episodes. One major problem is the difficulty in the differentiation between the five major diagnostic dilemmas of early graft dysfunction, namely: acute rejection, acute tubular necrosis, drug toxicity, infection and obstruction. The last two are perhaps easy to diagnose with the available facilities. However, differentiation between acute rejection, acute tubular necrosis and drug toxicity is still difficult and during most episodes of rejection a diagnosis is made only after substantial damage has occurred to the graft. Workers at different centers are trying to develop a diagnostic tool more sensitive and specific for early prediction of rejection and easy differentiation between the other causes of graft dysfunction. Biopsy of the graft by Tru-cut needle or fine needle aspiration is the gold standard but is not easily accepted by all patients. Transplant immunology seems to be promising and many tests for the diagnosis of acute rejection are available. An ideal test is one which would predict rejection before any damage occurs to the graft and at the same time indicate when acute rejection is over. It would thus enable the physician to give just enough anti-rejection treatment. While we are still away from such a test, immunology has many markers to help.

Activation of the T-lymphocytes is an early event in the process of acute rejection. Since the early 80's, a growing interest has been shown in adopting the determination of CD4/ CD8 ratio for early diagnosis and monitoring of acute rejection episodes during the first three months following kidney transplantation. Cosimi and colleagues in their initial work reported that patients having early graft dysfunction associated with high peripheral blood T-helper and T-suppressor lymphocyte (CD4/CD8) ratio are most likely to have rejection episodes [2] . They suggested that this ratio might be of considerable value in predicting the HLA non-identical kidney recipients who will have a rejection episode in the first three months post transplantation. This was confirmed by many reports particularly among renal allograft recipients on azathioprine and prednisolone [3],[4],[5],[6],[7] . Many centers considered a CD4/CD8 ratio higher than normal (>1.3) as a predictor of rejection [2],[8],[9] . However, the sensitivity and specificity of the ratio at this level were low.

In the present study, we re-evaluated the validity of T-lymphocyte subset characterization and its utility in the diagnosis of acute rejection episodes.


   Materials and Methods Top


Twenty-three renal transplant recipients who developed graft dysfunction during the first three months of follow-up following transplantation constituted the material of this study. All these patients had undergone renal transplantation with living related or cadaveric donors at the Jeddah Kidney Center, Jeddah, Saudi Arabia. There were 12 male and 11 female patients, their age ranging from 12 to 65 years with a mean of 37 years. All recipients were on triple drug immunosuppression including prednisolone, azathioprine and cyclosporine-A (CyA). Clinical suspicion of acute rejection episode was based on the presence of at least two of the following criteria; fever, hypertension, graft tenderness, decrease in urine output and a rise in serum creatinine level.

A complete work-up was performed in order to establish the diagnosis of graft dysfunction in each patient. Blood samples were collected for culture, renal and liver functions tests, and complete blood picture including total and differential white cell count as well as for whole blood cyclosporine trough level. Urine samples were microscopically and bacteriologically studied. Abdominal ultrasound and Tc 99m DTPA renogram were performed on all patients. Renal graft Tru-cut needle biopsies were carried out on seven patients.

At the very early indication of graft dysfunction, blood samples were taken for T-cell subsets study. T-helper (CD4) and T­suppressor (CD8) lymphocytes in the peripheral blood sample were counted using a flowcytometer (Becton-Dickinson Immunocy­tometry Systems, California, USA). Whole blood lyzed samples were studied using the direct immunofluorescence technique. The samples containing T-lymphocytes were incubated with 2 fluorochromes conjugated monoclonal antibodies. T-helper cells monoclonal antibody (anti CD4) was conjugated with fluorescein isothiocyanate giving green color, while that for T-Suppressor cells, (anti CD 8) was conjugated with plycoerythrin giving red color. A subsequent two color fluorescence activated sorter cell scan analysis was employed for cell count and ratio (CD4/CD8) as described by Colvin [10] . The patients who were found to fulfill the criteria for the diagnosis of acute rejection and who did not have a clear evidence of any other cause of graft dysfunction were given anti­rejection treatment empirically, in the form of steroid pulse and when necessary, anti- thymocyte globulin (ATG). The patients with a diagnosis for graft dysfunction due to a cause other than rejection were treated accordingly.


   Results Top


Twenty-three renal transplant recipients who had graft dysfunction were investigated as detailed before. Accordingly, two groups of patients were identified. The first (Group 1) consisted of 13 patients who were diagnosed to have acute rejection as the cause of graft dysfunction based on criteria described earlier, and were subjected to anti­rejection treatment. The second group, (Group 2) consisted of 10 patients who had a definite cause of graft dysfunction other than rejection.

[Table - 1] shows the detailed characteristics of the 13 patients who were diagnosed to have acute rejection (Group 1). Graft ultrasound was reported to be normal in 10 of these 13 cases, while two cases showed cortical hyperechogenecity and pyramidal dilation was noted in a third patient. Cyclosporin-A whole blood trough level was within the therapeutic window in these patients. Only four patients in this group accepted to undergo graft biopsy. All of them had histologic evidence of acute cellular rejection. All patients in this group received anti­rejection treatment. The T-cell subsets ratio was found to be more than 1.7 in 10 of the 13 patients in this group and all of them responded well to pulse steroid therapy. Three patients in this group had a CD4/ CD8 ratio less than 1.7. Positive response to anti­rejection treatment could be obtained in two of them with ATG administration and in the third, with a second dose of steroid pulse therapy.

[Table - 2] describes the characteristics of the second group of patients (Group 2) who did not have rejection. Two patients were diagnosed to have obstruction by ultrasonography. One patient, who had sudden onset of oliguria, hematuria and tender swollen graft was diagnosed to have renal vein thrombosis. Two patients had urinary tract infection and septicemia as confirmed by urine and blood cultures. One patient had acute tubular necrosis which was confirmed histologically. Four patients were diagnosed to have cyclosporine toxicity of whom two had high whole blood trough levels while in the remaining two, the diagnosis was made after graft biopsy. All the patients in group 2 recovered graft function upon correcting the underlying cause except the patient with renal vein thrombosis who lost his graft. The ratio of T-cell subsets was less than 1.7 in all the patients in this group.

Our results yield specificity and sensitivity rates of 100% and 81% respectively for T-cell subsets ratio in the diagnosis of acute rejection.


   Discussion Top


Renal graft survival is greatly dependent on the incidence and frequency of rejection episodes [1] . Histological evidence of rejection is considered the gold standard for diagnosis. However, this invasive technique is not without hazards, and many of our renal transplant patients refuse to undergo graft biopsy.

T-cell subset ratio which was suggested as a diagnostic tool for acute rejection episodes carried low specificity and sensitivity rates with 1.3 as the cutoff point and as such the test did not gain much popularity [2],[8],[9] . However, after analysis of our data, it would seem reasonable to take a CD4/CD8 ratio of 1.7 or more as indicative of rejection. At this level, 10 of our 13 study patients with acute rejection were correctly diagnosed using this test. Also, renal transplant patients with low CD4/CD8 ratio have been reported to have poorer response to anti-rejection treatment mostly due to associated glomerulopathy [9] . We had three such cases that had similar low ratio two of whom showed positive response to ATG after being resistant to steroid therapy and the third responded to a second dose of steroid pulse therapy.

None of the 10 patients in the second group had T-cell subset ratio more than 1.7. All these patients had CD4/CD8 ratio varying from 0.5 up to 1.6. A definite cause for graft dysfunction was found in each case. This group of patients was considered as an internal control in this study. Urinary tract infection was diagnosed on clinical and bacteriologic grounds in two patients and the T-subset ratio was 1.6 and 1.2 respectively. The presence of infection is reported to decrease CD4/CD8 ratio but this seems particularly true for severe infection with the Epstein-Barr virus complicating transplantation which results in an increase CD8 population [11],[12] . Cyclosporine nephrotoxicity was proved to be responsible for graft dysfunction in four cases. More interestingly, two out of these four cases had CyA level within the normal therapeutic window. Their CD4/CD8 ratio was 0.7 and 1.3 respectively; toxicity was diagnosed on histological grounds. Such toxicity might take place in spite of therapeutic blood levels of CyA [13] . Consequently, toxicity has to be considered in recipients with graft dysfunction particularly when CD4/CD8 ratio is less than 1.7.

Chatenoud et al [14] reported a slightly high CD4/CD8 ratio in patients treated by maintenance hemodialysis. None of our patients were on hemodialysis treatment after transplantation. However, this finding is important and has to be considered while interpreting the results of T-cell subsets study.

In conclusion, CD4/CD8 determination is a very good technique to help in the diagnosis of acute cellular rejection during the first 3 months following kidney transplantation. Moreover, it compliments other investigatory techniques to exclude causes other than acute rejection as a cause of graft dysfunction if the ratio is less than 1.7. This technique is particularly valuable among patients who refuse graft biopsies and/or whenever there is a contraindication for such procedures. Studies with larger series of patients are needed to confirm this observation.

 
   References Top

1.Tilney NL. The early course of a patient with, a kidney transplant. In: Morris PJ (ed). Kidney transplantation principles and practice. W.B. Saunders, Philadelphia, U.S.A. 1988;263-83.  Back to cited text no. 1    
2.Cosimi AB, Colvin RB, Burton RC, et al. Use of monoclonal antibodies to T-cell subsets for immunologic monitoring and treatment in recipients of renal allografts. N Engl J Med 1981;305:308-14.  Back to cited text no. 2    
3.Smith WJ, Burdick JF, Williams GM. Immunologic monitoring of renal transplant recipients. Transplant Proc­1983;15:1182-3.  Back to cited text no. 3    
4.Binkley WF, Valenzuela R, Braun WE, Deodhar SD, Novick AC, Steinmuller DR. Flow cytometry quantitation of peripheral blood T-cell subsets in human renal allograft recipients. Transplant Proc 1983;15:1162-5.  Back to cited text no. 4    
5.Carter NP, Cullon PR, Thompson JF. Monitoring lymphocyte subpopulations in renal allograft recipients. Transplant Proc 1983;15:1157-9.  Back to cited text no. 5    
6.Chatenoud L, Chkoff N, Kreis H, Bach JF. Correlation between immunoregulatory T-cell imbalance and renal allograft outcome. Transplant Proc 1983;15:1184-5.  Back to cited text no. 6    
7.Guttmann RD, Poulsen R. Fluorescence activated cell sorter analysis of lymphocyte subsets after renal transplantation. Transplant Proc 1983;15:1160-2.  Back to cited text no. 7    
8.Colvin RB, Cosimi AB, Burton RC, et al. Circulating T-cell subsets in 72 human renal allograft recipients. The OKT4 + /OKT+ cell ratio correlates with reversibility of graft injury and glomerulopathy. Transplant Proc 1983;15:1166-9.  Back to cited text no. 8    
9.Ellis TM, Lee HM, Mohanakumar T. Alterations in human regulatory T­lymphocyte subpopulations after renal allografting. J Immunol 1982; 127:2199-203.  Back to cited text no. 9    
10.Colvin RB, Preffer FI. New technologies in cellanalysis by flow cytometry. Arch Pathol Lab Med1987;lll:628-32.  Back to cited text no. 10    
11.Nagington J, Gray J. Cyclosporin A immunosup-pression, Epstein-Barr antibody, and lymphoma. Lancet 1980;l:536-7.  Back to cited text no. 11    
12.Sweny P, Tidman N. The effect of cyclosporin A on peripheral blood T cell subpopulations in renal allografts. Clin Exp Immunol 1982;47:445-8.  Back to cited text no. 12    
13.Meyers BD. Cyclosporine nephrotoxicity. KidneyInt 1986;30:964-8.  Back to cited text no. 13    
14.Chatenoud L, Chkoff N, Kreis H, Bach JF.Interest in and limitations of monoclonal anti-T-cell antibodies for the follow-up of renal transplant patients. Transplantation 1983;36:45-50.  Back to cited text no. 14    

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Correspondence Address:
Faissal A.M Shaheen
Consultant Nephrologist and Director, Jeddah Kidney Center, King Fahd Hospital, Jeddah
Saudi Arabia
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PMID: 18583838

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