1. Glomerular CD45 Immunostaining in post perfusion renal transplant biopsies predicting rejection
Anand Yuvaraj
International Transplant Fellow
University Hospitals Coventry and Warwickshire NHS Trust
Coventry, United Kingdom
01/05/2017
Supervisors : Dr. Kishore Gopalakrishnan , Consultant Pathologist
&
Prof. Nithya Krishnan , Consultant Transplant Nephrologist, UHCW, Coventry

2. Disclosures
No financial disclosures

3. Introduction
Recipients of incompatible renal allografts remain at risk of harbouring persistent DSA and developing AMR after transplantation, with increased risk of graft loss when compared with compatible transplants
(Bagnasco SM. Time Course of Pathologic Changes in Kidney Allografts of Positive Crossmatch HLA-Incompatible Transplant Recipients. Transplantation 2014:97(4); )
Glomerulitis is a significant finding in renal transplant biopsies and is an important feature in diagnosing AMR. CD45 or leucocyte common antigen is a useful immunohistochemical stain that can identify leucocytes and has the advantage over CD3, CD8 or CD68 in being able to stain a wider range of leucocytes
(Nithya Krishnan. BANFF Poster CD45 counts in post perfusion biospy predicts AMR)

4. Introduction
Glomerular margination of leucocytes occur early after transplantation and was associated with DSA level and early graft dysfunction
(Higgins R, Zehnder D, Chen K, Lowe D, McKinnell J, Lam FT, Kashi H, Tan LC, Imray C, Fletcher S, Krishnan N, Hamer R, Briggs D. The histological development of acute antibody-mediated rejection in HLA antibody-incompatible renal transplantation. Nephrol Dial Transplant Apr;25(4): )
Analysis of renal allograft biopsies showed similar disturbances in the selected microarray sets in AMR and T cell-mediated rejection (TCMR), suggestive of significant T cell involvement in AMR.
(Mueller TF, Einecke G, Reeve J et al. Micoarray analysis of rejection in human kidney transplants using pathogenesis-based transcript sets. Am J Transplant 2007; 7: 2712–2722)

5. Aim
To determine the significance of increased number of glomerular leucocytes (which are CD45 positive cells) in HLA and ABO incompatible transplants, in predicting rejection, type of rejection and deciding subsequent graft function.

6. Materials and Methods
Retrospective study: 45 Sensitized patients: 43 to HLA antigens and 2 to both HLA and ABO antigens to 2013
Immunosuppression:
MMF 1000 mg BD started 10 days before transplant
Tacrolimus was started 4 days before transplant: 0.15 mg/kg/day, target trough level of 8-12 μg/L in the first month.
Prednisolone 20mg OD, and methylprednisolone 500 mg.
Two doses of basiliximab 20 mg were given at days 0 and 4
Graft biopsies were performed approximately 30 minutes after perfusion in the operation theatre.

7. Materials and Methods
CD45 Immunostaining on de-waxed sections were performed using the antibody, DAKO CD45, Cat. No. M0701 or Vision Biosystems CD45 X16/99, Cat. No. PA0042
CD45 cells were counted manually under high resolution microscope:
Total number in one glomeruli
Average CD45 count

8. Materials and Methods
Banff 2007 criteria for glomerulitis, tubulitis, interstitial inflammation, peritubular capillaritis and intimal arteritis. Similarly, subsequent biopsies were scored and were looked in for rejection, AMR or TCMR.
Crossmatch: CDC/Flow/Bead
MFI- Median Flouroscence intensity: Pre treatment Class I,II, Highest, Cumulative
Statistical analysis of comparison between groups was performed using Student’s t-test on SPSS for Windows, version 22.0

9. Results 45 Patients, 18 M, 27 F Mean: 41.31 ± 11 years, 23-68 years
Rejection: 23, No Rejection: 22
Type of Renal Transplant Rejection in
<3 Months
3 Months- 1 Year
1-3 Years
AMR*- 11
AMR- 4
AMR- 2
TCMR**- 8
TCMR- 5
*AMR: Antibody Medicated rejection
**TCMR: T cell mediated rejection

10. Results Glomerulitis Tubulitis Interstitial Inflammation
Tubulitis
Interstitial
Inflammation
Peritubular Capillaritis
Intimal Arteritis
Inflammation in subsequent Graft biopsies according to BANFF 2007
(number of patients)
g0- 34
g1-10
g2- 0
g3- 1
T0- 37
T1- 4
T2- 4
T3- 0
ti0- 32
ti1- 6
ti2- 5
ti3- 2
ptc0- 32
ptc1- 11
ptc2- 1
ptc3- 1
v0- 39
v1- 5
v2- 1
v3- 0

11. CD45 cell count <5 or >5 and Rejection

12. CD45 cell count <5 or >5 and Rejection <3months

13. CD45 cells and Rejection(ALL)
Average CD45 cells
Rejection(ALL)
Number
Mean
Standard Deviation No 22
2.97
1.63
Yes 23
6.63
6.25
p=0.0118
CD45 cell count and Rejection <3Months
Rejection- Type
Average CD45 Cell count
Mean
Standard Deviation
p value
No Rejection (0)
2.95
1.63
AMR (1)
7.11
6.54
(1 Vs 0)
TCMR (2)
7.82
7.08
(2 Vs 0)

14. CD45 cell count and tubulitis
Tubulitis score
BANFF’ 07
Average CD45 Cell count
Mean
Standard Deviation
p value
t0 (0)
4.30
4.23
t1 (1)
2.55
2.13
(2 Vs 1)
t2 (2)
12.15
7.34
(2 Vs 0)

15. CD45 cell count and Intimal arteritis
Intimal Ateritis
Score BANFF’ 07
Average CD45 Cell count
Mean
Standard Deviation
p value
v0 (0)
4.82
1.90
v1 (1)
2.82
1.13
(2 Vs 1)
v2 (2)
15.6
(2 Vs 0)

16. Conclusions
An average of five or more CD45 positive cells in the post perfusion renal transplant biopsy was significantly associated with rejection, both AMR and TCMR
Higher the average CD45 cell count and the total count in one glomeruli, greater is the chance of developing rejection, AMR and TCMR, in the immediate post transplant period
Initial highest CD45 count in a single glomeruli and a greater average CD45 count is associated with a greater risk of developing tubulitis and intimal arteritis

17. “All AIT transplants should have a post perfusion biopsy and an higher average/total number of CD45 cell count patients should be closely followed up for rejection-AMR and TCMR”