American Journal of Transplantation 2010; 10: 24422452

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2010 The Authors
Wiley Periodicals Inc. Journal compilation
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2010 The American Society of
Transplantation and the American Society of Transplant Surgeons

doi: 10.1111/j.1600-6143.2010.03261.x

A Critical Appraisal of Methods to Grade
Transplant Glomerulitis in Renal Allograft Biopsies


Ibrahim Batal
a,
*, John G. Lunz III
a,

,
Nidhi Aggarwal
a
, Adriana Zeevi
a
,
Eizaburo Sasatomi
a
, Antik Basu
b
,
Henkie Tan
b
, Ron Shapiro
b
and Parmjeet
Randhawa
a


a
Department of Pathology,
b
Department of Surgery,
University of Pittsburgh Medical Center, Pittsburgh, PA
*Corresponding authors: Ibrahim Batal,
ibatal@partners.org; corresponding for
histocompatibility: J. G. Lunz, lunzjg@upmc.edu

Transplant glomerulitis is an increasingly recognized
lesion in renal transplant biopsies. To develop a re-
fined grading system, we defined glomerulitis by the
presence of 5 leukocytes/glomerulus and evaluated
111 biopsies using three different grading systems:
(i) percentage of glomerular involvement, (ii) peak in-
flammation in the most severely affected glomerulus
and (iii) presence/absence of endocapillary occlusion by
inflammatory cells. Endocapillary occlusion had no
impact on graft survival, but was associated with in-
creased serum creatinine, proteinuria and subsequent
transplant glomerulopathy. Grading based on either
percent or peak glomerular involvement correlated with
graft failure and peritubular capillaritis. However, the
percent glomerular involvement method had the
additional advantage of displaying associations with:
concurrent proteinuria, focal or diffuse immunoperox-
idase peritubular capillary C4d staining, 1-year post-
biopsy serum creatinine, subsequent detection of
donor-specific antibody and development of trans-plant
glomerulopathy. Patients with >75% glomeru-lar
involvement also revealed persistent high-grade
glomerulitis on follow-up biopsies despite antirejec-tion
treatment. In conclusion, grading of glomerulitis is a
meaningful exercise, and a quantification system based
on percentage of glomerular involvement shows the
most robust associations with clinical parameters and
prognosis.

Key words: Acute allograft rejection, glomerular dis-
ease, transplant glomerulitis, transplant glomerulo-
pathy

Abbreviations: AMR, antibody-mediated rejection;
DSA, circulating donor-specific antibody; ELISA,
enzyme-linked immunoabsorbent assay; FSGS, focal
segmental glomerulosclerosis; PRA, panel reactive
an-tibody; PTC-C4d, peritubular capillary C4d
staining; TCMR, T-cell-mediated rejection; TGP,
chronic trans-plant glomerulopathy.


Received 20 April 2010, revised 26 June 2010 and
accepted for publication 14 July 2010


Introduction

Transplant glomerulitis is defined as intracapillary glomeru-
lar leukocytic inflammation in kidney allografts. It was first
described by Richardson et al. in 1981 (1) and has been
increasingly recognized as a form of allograft injury likely
linked to rejection reaction (2,3). While glomerulitis is not
currently integrated into the Banff grading schema for the
diagnosis of T cell-mediated rejection (TCMR) (4), it is rec-
ognized as a component of antibody-mediated rejection
(AMR) (3). The work by Magil and coworkers (57) suggests
that intraglomerular capillary macrophages accumulate in
AMR while T cells predominate in TCMR.

Methodological aspects of histological grading of trans-plant
glomerulitis are the subject of ongoing debate. No clearly
enunciated cut-off value exists to define the num-ber of
glomerular leukocytes needed to render a diagno-sis of
glomerulitis. In addition to glomerular inflammation, some
authors stress the concurrent enlargement of en-dothelial
cells (4), while others suggest a need to demon-strate
endocapillary occlusive lesions (8,9). Several studies have
attempted to investigate the significance of transplant
glomerulitis (Table 1) (2,58,1013). Many of these stud-ies
lacked a precise definition of this lesion. Furthermore,
glomerulitis was usually not graded but rather expressed as
a binary variable (presence/absence) (57,11,13). In two
studies where glomerulitis was graded, g2 and g3 lesions
were lumped together to define high-grade glomerulitis
while g1 lesions were excluded from analysis (2,12). An-
other caveat in the currently available literature is that data
on peritubular capillary C4d staining (PTC-C4d), circulat-ing
donor-specific antibodies (DSA), and association with
chronic transplant glomerulopathy (TGP) and proteinuria
have not been consistently presented. A need to refine the
currently used scoring criteria for glomerulitis was ac-
knowledged at the 2009 Banff meeting on allograft pathol-
ogy (14). In this study, we have sought correlations be-tween
clinical parameters and glomerulitis grade assessed by three
different histologic methods. It is shown that grad-ing based
on% of glomerular involvement is preferable to scoring
methods which rely on the peak glomerular inflam-mation, or
the presence of glomerular capillary occlusion by
inflammatory cells.

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Table 1: Brief review of the transplant glomerulitis in the literature
Association with

Intimal TCMR
Authors (year) Country Selection Time post-tx No. of g Frequency Grading Proteinuria Graft failure DSA C4d arteritis grade TGP

Axelsen et al. 1985 Australia All bxs 1st 6 months 47 Up to 28.8% Yes, Complex
1
NA NS (66%) NA NA No No yes
Tuazon et al. 1987 USA TCMR 1st year 12 7.3% No yes
2
NS

(58%) NA NA yes NA NA
Olsen et al.
3
1995 Canada All bxs 1st 90 days 60 33% Banff yes
2
NS (23%)
2
NA NA NA yes
2
NA
Colvin at al. 1997 USA All bxs 4700 days 33 19% No NA NA NA NA yes NA NA
Messias at al. 2001 USA TCMR 1st 3 years 25 NA Banff NA NS (28%) Yes PRA NA Yes NA NA
Magil et al. 2003 Canada TCMR 1st 6 months 16 NA No NA NA NA yes NA NA NA
Magil. 2005 Canada TCMR & g 1st 6 months 42 NA No NA NA NA NA NA NA NA
Hara et al. 2005 Japan CAN NA 40 29% No NS NS (27%) Yes (81%) Yes (59%) NA NA yes
Tinckam et al. 2005 Canada TCMR 1st year 31 32% Banff NA yes (35%)
3
NA NA NA NA NA
Current study USA TCMR NA 56 56% Banff No yes Yes Yes No No yes
Abbreviations: g = glomerulitis; bxs = biopsies; TCMR = T cell-mediated rejection (including borderline changes); TGP = transplant glomerulopathy; CAN = chronic allograft
nephropathy; PRA = panel reactive antibody; NS = not significant; NA = not available.
1
A composite score for individual glomeruli was calculated based on the extent of hypercellularity (03) and the number of infiltrating cells in the most severely affected capillary
loop (03) in each glomerulus. Glomerulitis score was calculated by averaging the score of all glomeruli. One-year graft failure was numerically but not statistically higher in
samples with glomerulitis (66% vs. 45%).
2
Descriptive association but no comparison with patients with no-glomerulitis and no correction for the presence of confounding factors.
3
Incidence of
glomerulitis is calculated for all glomerulitis (g13). However, the study focused only on high-grade glomerulitis (g2 and g3).

7/12 (glomerulitis) vs. 5/19 (No glomerulitis), p = 0.12. However, the increased number of intraglomerular T cells was significantly associated with 1-year graft loss even in
patients with no-glomerulitis.
2
The authors mentioned that when biopsies were stratified according to TCMR grade, the difference disappeared. However, 40% of biopsies with glomerulitis were not
associated with overt TCMR (borderline for rejection or less).
3
Associated with poor prognosis (GFR<30 at 1 year) on univariable but not multivariable analysis. On multivariable analysis 1 monocyte/ glomerulus was associated with poor
prognosis.
Higher incidence of glomerulitis was observed in the current study. This is attributed to use the WHO criteria to define glomerulitis (5 inflammatory cell/ glomerulus) in order to
recognize the mildest form of glomerulitis.





























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Batal et al.

Material and Methods

Clinical information and surgical biopsy slides were retrospectively
reviewed from patients who (i) underwent kidney transplantation between
January 2005 and March 2006 and (ii) had at least one biopsy showing
tubulointersti-tial inflammation (Banff grade: borderline and above). The
first biopsy with tubulointerstitial inflammation was regarded as the index
biopsy. Most other investigators followed a similar approach by only
including samples with tubulointerstitial inflammation (57,12,13). The
initial data set con-tained only four biopsies with Banff grade 3 glomerulitis
(g3). In order to increase this number, 11 additional g3 cases were
subsequently retrieved by searching the years 2003 through 2008 in an
Electronic Database main-tained by the Thomas E. Starzl Transplantation
Institute using the same inclusion criteria.

The majority of the patients received pretransplantation alemtuzumab
(Cam-path 1-H, Berlex, Seattle, WA) depletion therapy followed by
posttransplan-tation steroid-free tacrolimus monotherapy. Graft function
was followed up to December 2009. All the biopsies were for cause
biopsies performed for rising serum creatinine.

Clinical and laboratory assessment
Enzyme-linked immunosorbent assay (ELISA) was used to screen the pa-
tients sera for IgG-anti-HLA antibodies. Panel reactive antibody (PRA)
val-ues <10% were regarded as unsensitized. DSA detection was
performed by ELISA (One Lambda, Canoga Park, CA, USA) or by single
and multiplex bead arrays (Luminex, One Lambda, Inc., Canoga Park,
CA, USA and Tep-nel Life Codes Corporation, Stamford, CT, USA)
usually when the PRA was 10% as previously described (15). Antibody
testing was performed at dif-ferent time points. For the purpose of the
study, the results were divided into:

(i) DSA at biopsy (n = 86): Testing was performed on the day of biopsy
(n = 83) or within 10 days of biopsy (n = 3).
(ii) DSA prebiopsy (n = 111): from transplantation to 30 days before biopsy.
(iii) DSA postbiopsy (n = 111): >30 days after biopsy.

Whenever available within 14 days of biopsy, BK virus genomic load in
the urine and plasma was quantitatively assessed (copies/mL) as
previously described (16) and immune cell function values (ng ATP/mL
whole blood) using the Cylex ImmuKnow assay were documented as
described else-where (17).

Histologic evaluation of index biopsies
Standard H&E, periodic acid -Schiff (PAS), methenamine silver and
trichrome-stained sections were evaluated.

Transplant glomerulitis: The Banff schema does not explicitly state the
minimum number of intraglomerular inflammatory cells needed to render a
diagnosis of transplant glomerulitis. The WHO defines glomerular leuko-
cytic infiltration in native kidneys by the presence of 5 leukocytes per
glomerulus (18). We applied this criterion on PAS-stained renal allograft
biopsies. Only unequivocal inflammatory cells (mononuclear cells and
neu-trophils) in the glomerular capillary loops were counted. Neutrophils
were included since the number of neutrophils correlates with the
diagnosis (19) and outcome (20) of AMR. The severity of glomerulitis was
graded using three scoring systems:

i. Method A, based on the percentage of the involved glomeruli as
recom-mended by Banff 1997. Grades g0, g1, g2 and g3 refer to 0%,
<25%, 2575% and >75% of glomeruli being affected, respectively.
ii. Method B, based on the peak number of leukocytes in the most
severely affected glomerulus. For the purposes of this study, we de-


fined grades g0, g1, g2 and g3 to refer to biopsies with 04, 59, 10
15 and >15 leukocytes/glomerulus, respectively.
iii. Method C based on the presence/absence of endocapillary occlusive
lesions as defined by Nickeleit (9). This lesion requires the presence of

3 endocapillary cells within a glomerular capillary loop, typically with
>75% luminal occlusion.

Semiquantitative scores (03) for interstitial inflammation, tubulitis, inti-mal
arteritis, double contour of glomerular basement membrane, inter-stitial fibrosis,
tubular atrophy, arteriolar hyaline thickening and arterial fi-brous intimal
thickening, were recorded according to Banff 97 criteria (4). Peritubular
capillaritis was semiquantitatively assessed (03) according to Banff 2007
recommendations (21). The presence/absence of protein re-absorption droplets
and tubular vacuolization was documented. BK virus replication in biopsies was
assessed using in situ hybridization for viral DNA (Enzo Diagnostics, New York,
NY, USA) as previously described (22). Polyclonal C4d (ALPCO Diagnostics,
Windham, NH, USA) was performed using published methodology (23). PTC-
C4d was graded according to Banff 2007 recommendations (21). Since
immunoperoxidase C4d was utilized on formalin-fixed paraffin-embedded
tissue, both diffuse and focal PTC-C4d were regarded as a positive stain as
recently recommended (15,21,23).

Response to treatment
Serum creatinine values obtained within 3 days, 30-day before and 30-day after
the index biopsy were recorded as biopsy, baseline and follow-up serum
creatinine, respectively. The difference between baseline and follow-up serum
creatinine (_Cr) was calculated by subtracting baseline from follow-up serum
creatinine. In addition, the serum creatinine response to treatment in individual
cases was categorically assessed based on the reversal in the rise of serum
creatinine from the baseline to biopsy as pre-viously published (24,25): (i)
complete response >70% reversal (ii) partial response 3070% reversal (iii)
unchanged serum creatinine <30% re-versal and (iv) progressive graft
dysfunction a rise in serum creatinine >30%. Follow-up biopsies performed
<100 days postindex biopsies were assessed. Improvement, maintenance or
deterioration of Banff 97 grade for TCMR was considered the basis of histologic
response to treatment.

Follow-up information
The incidence and the date of postbiopsy graft failure were recorded. Six-
month and 1-year postbiopsy serum creatinine was documented. De-
velopment of postbiopsy DSA was recorded as previously explained. As-
sessment of glomerulitis and TGP grades was performed for all follow-up
biopsies.

Research protocol and statistics
The study was approved by the University of Pittsburgh Institutional
Review Board (IRB protocol# 9030095). Statistical analysis was
performed using Sigma Stat 2.0.3 software (SPSS Inc, Chicago, IL, USA).
Except for serum creatinine values, which were presented as median and
interquartile range (IQR: 25th75th percentile), data were presented as
mean standard devi-ation. Continuous values were compared using
MannWhitney Rank Sum Test. Categorical values were compared using
Fishers exact test. Correla-tion coefficient was performed using
Spearman rank correlation. P values <0.05 with a two-sided hypothesis
testing were considered statistically significant.

Results

Demographic information
A total of 111 index renal allograft biopsies were evaluated.
Follow-up biopsies were available for 82 of 111 patients,

2444 American Journal of Transplantation 2010; 10: 24422452


Table 2: Histologic grading of glomerulitis using three different
methods
Method B vs. A Method C vs. A

Method A (peak (endocapillary

(% glomeruli) inflammation) occlusive lesion)


g1 (n = 29) g1 (24/29, 83%) Presence EOL (2/29, 7%)

g2 (n = 23)
g2 (5/29, 17%) Absence EOL (27/29, 93%)

g1 (8/23, 35%) Presence EOL (10/23, 43%)

g2 (10/23, 43%) Absence EOL (13/23, 57%)

g3 (n = 15)
g3 (5/23, 22%)
Presence EOL (8/15, 53%)

g2 (9/15, 60%)

g3 (6/15, 40%) Absence EOL (7/15, 47%)

Endocapillary occlusive lesions: p = 0.03 (g1 vs. g2) and p <

0.001 (g1 vs. g3) (Fishers exact).

and ranged in number from 1 to 10. There were 62 (56%)
males and 49 (44%) females aged 54 13 years [range:
1981 years]. This cohort included Caucasians (n = 88,
79%), African Americans (n = 22, 20%) and Indian (n = 1,
1%). The biopsy samples were obtained 504 370 days
posttransplantation. At the time of biopsy, whole blood
tacrolimus levels were 7.5 +/- 5.6 ng/mL. Median serum
creatinine values were 2.4 (IQR: 1.83.2 mg/dL).

All analyzed biopsies contained 7 glomeruli (12.1 4.6)
and all except one biopsy had arteries [no arteries (n =
1), 1 artery (n = 7), 2 arteries (n = 29) and >2 arteries (n
= 74)]. The single biopsy devoid of arteries had low-
grade glomerulitis (g1). Eighty-six patients with episodes
of graft dysfunction received antirejection treat-ment
while the remaining 25 patients [borderline (n = 23), IA (n
= 1) and IIA (n = 1)] did not receive any therapy. The
latter two patients had concurrent positive urine cul-ture
(Staphylococcus aureus) and renal vein thrombosis,
respectively.

Grading transplant glomerulitis using three
different scoring systems
In general, a higher grade of glomerulitis using one scor-
ing system was associated with higher grades assigned
by the other two scoring methods (Table 2). The correla-
tion between glomerulitis grade (g1-g3) assessed by ei-
ther method A or B was good (r = 0.6, p < 0.001). The
discrepancy in glomerulitis grade using A and B schemas
was never >1. The best agreement (83%) was observed
in g1 while the worst (40%) was observed in g3 sam-ples
(Table 2). The presence of endocapillary occlusive lesion
was a relatively infrequent finding (20/67, 30% of
samples with glomerulitis), which significantly increased
in frequency from g1 (7%) to g2 (43%) and g3 (53%)
(Table 2).

Clinicopathologic correlates of glomerulitis graded by the
three scoring systems are summarized in Table 3. Grad-ing
based on the presence/absence of endocapillary oc-clusive
lesion had no impact on graft survival (Table 3 and Figure
1C). However, the presence of endocapillary occlu-sion was
associated with concurrent increase in serum cre-atinine and
proteinuria and with subsequent development
Histologic Grading of Transplant Glomerulitis

of TGP on follow-up biopsies (Table 3). Glomerulitis grad-ing
using the peak of glomerular inflammation (method B)
showed significant association with peritubular capillaritis
and graft failure (Table 3 and Figure 1B). The development
of TGP on follow-up biopsies was more prominent in g2
lesions. Glomerulitis grading based on the percentage of
glomerular involvement (method A) also correlated with
peritubular capillaritis, development of TGP on follow-up and
graft failure (Table 3, Figure 1A). In addition, it revealed
significant associations with: concurrent proteinuria, posi-tive
PTC-C4d, 1-year postbiopsy serum creatinine and sub-
sequent development of DSA (Table 3). For this reason, all
subsequent clinicopathologic analyses of the severity of
glomerulitis were carried out using the latter grading system
(method A) (Tables 47).

Distribution of Banff schema lesions in biopsies
stratified by the severity of glomerulitis
The sensitivity and specificity of glomerulitis (g > 0) for
concurrent diagnostic AMR were 7/10 (70%) and 31/74
(42%), respectively (Table 4). Glomerulitis could be
detected in diseases other than AMR: 23/42 (55%)
TCMR biopsies with no concurrent DSA and 22/36 (61%)
biopsies with borderline changes and no con-current DSA
showed glomerulitis. Fifteen of 32 (47%) DSA(-) C4d(-)
TCMR and 16/26 (62%) DSA(-) C4d(-) borderline
samples had g>0; most often in the form of g1 [9/15
(64%) and 8/16 (50%), respectively]. Three of the latter
patients (2 TCMR, 1 borderline changes) subsequently
developed DSA. Glomerulitis was also detected in two
cases (both g2) with recur-rent glomerular disease
[membranoproliferative glomeru-lonephritis (n = 1) and
membranous nephropathy (n = 1)]. Both cases were
negative for PTC-C4d, peritubular capil-laritis and intimal
arteritis. The former subsequently devel-oped DSA while
the latter did not.

Biopsies with g3 glomerulitis had a higher peritubular cap-
illaritis score compared to g0 and g1 (p 0.047) (Table 4)
and a higher proportion of positive PTC-C4d (50%) and con-
current TGP lesions (20%) compared to g0 biopsies (21%
and 2%, respectively, both p = 0.047). These biopsies were
more frequently associated with the presence of protein
reabsorption droplets when samples showing concurrent
TGP, focal segmental glomerulosclerosis (FSGS) and mem-
branous nephropathy were excluded (g3 vs.g1, p = 0.02).
Similarly, g2 samples had more frequent concurrent TGP [g2
vs. g0 (p = 0.016)] and a higher proportion of biopsies
showing protein reabsorption droplets [g2 vs. g0 and g1 (p
0.04)], as well as score for interstitial fibrosis [g2 vs. g0 and
g3 (p 0.02)].
Biopsies with different grades of glomerulitis did not dif-
fer with respect to Banff grades for TCMR, intimal arteri-
tis, interstitial inflammation, tubulitis, tubular atrophy, ar-
teriolar hyalinosis or arterial fibrointimal thickening. The
absolute number but not the percentages of glomerular
neutrophils increased from g0 to g3 biopsies reflecting a

American Journal of Transplantation 2010; 10: 24422452 2445
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Table 3: Clinical correlates of glomerulitis graded by three different methods
Method B: peak of inflammatory cells in the Method C: presence/absence of

Method A: percentages of affected glomeruli most severely affected glomerulus EOL


g0 (n = 44)





g-1 (n = 29) g2 (n = 23) g3 (n = 15) g = 1 (n = 32) g2 (n = 24) g3 (n = 11) No EOL (n = 47) EOL (n = 20)
History of prior renal 4/44 (10%) 3/29 (10%) 2/23 (9%) 6/15 (40%) 3/32 (9%) 6/24 (25%) 2/11 (18%) 7/47 (15%) 4/20 (20%)

transplantation
1


Graft failure at 2 yrs 7/31 (23%) 5/22 (23%) 8/14 (57%) 10/13 (77%) 8/25 (32%) 8/15 (53%) 7/9 (78%) 16/36 (44%) 7/13 (54%)

postbiopsy
2

Development of TGP 1/32 (3%) 2/17 (12%) 2/13 (15%) 6/11 (55%) 3/20 (15%) 5/14 (36%) 2/7 (29%) 6/31 (19%) 4/10 (40%)

in follow-up

biopsies
3


Serum creatinine at 2.2 (1.73.2) 2.4 (1.73.3) 2.8 (1.93.6) 2.2 (1.73.2) 2.6 (1.83.6) 2.2 (1.83.2) 2.8 (1.83.2) 2.4 (1.73.2) 2.9 (2.23.9)
biopsy (mg/dL)
1


Serum creatinine 1.8 (1.52.5) 1.5 (1.33.1) 2.2 (1.52.9) 4.0 (1.77.6) 1.6 (1.33.1) 2.1 (1.54.1) 2.3 (1.98.5) 1.6 (1.43.6) 2.3 (1.64.3)
1-year post-

biopsy (mg/dL)
2

16 31 13 24 77 148 160 184 45 110 46 89 145 208 19 35 200 204

Adjusted urine

protein (mg/dL)
3


DSA postbiopsy
1
5/44 (11%) 6/29 (21%) 7/23 (30%) 6/15 (40%) 8/32 (25%) 7/24 (29%) 4/11 (36%) 13/47 (28%) 6/20 (30%)

Positive PTC-C4d
2
9/43 (21%) 9/28 (32%) 7/23 (30%) 7/14 (50%) 11/31 (36%) 7/23 (30%) 5/11 (45%) 16/46 (34%) 7/19 (37%)

PTCitis
3
0.5 0.8 0.8 0.9 1.0 1.0 1.5 0.9 1.0 1.0 0.8 0.9 1.6 0.8 1.0 1.0 1.2 1.0

Abbreviations: g = transplant glomerulitis; EOL = endocapillary occlusive lesions; TGP = transplant glomerulopathy; DSA = circulating donor-specific antibody; PTC = peritubular
capillary; PTCitis = peritubular capillaritis.

Development of TGP in follow-up biopsies was calculated for samples revealing cg0 on index biopsies.

Adjusted urine protein: urine protein values after excluding samples with concurrent TGP, focal segmental glomerulosclerosis and membranous
nephropathy. Bold font corresponds to values showing significant differences as summarized below:
Method A
1
History of prior renal transplantation: p 0.04 (g3 vs. g0 or g1 or g2) (Fishers exact).

2
Graft failure at 2 years: p 0.003 (g3 vs. g0 or g1), p = 0.04 (g2 vs. g0) (Fishers exact).
3
Development of
TGP on follow-up: p 0.03 (g3 vs. g0 or g1) (Fishers exact).


2
Serum creatinine 1-year postbiopsy: p 0.02 (g3 vs. g0 or g1) (MannWhitney).
3
Adjusted urine protein: p 0.003 (g3 vs. g0 or g1) (MannWhitney).
1
DSA postbiopsy: p = 0.02 (g3 vs. g0) (Fishers exact).

2
Positive PTC-C4d: p = 0.047(g3 vs. g0) (Fishers exact).

3
PTCitis: p 0.047 (g3 vs. g0 or g1) (MannWhitney). Method B


2
Graft failure 2 years: p 0.025 (g3 vs. g0 or g1), p = 0.049 (g2 vs. g0) (Fishers Exact).

3
Development of TGP on follow-up: p = 0.007 (g2 vs. g0 or g1) (Fishers exact test).

3
PTCitis: p 0.02 (g3 vs. g0 or g2) (MannWhitney).


Method C
3
Development of TGP on follow-up: p = 0.03 (g with EOL vs. g0) (Fishers exact test).
1
Serum
creatinine at biopsy: p = 0.02 (g with EOL vs. g0) (MannWhitney).
3
Adjusted urine protein: p 0.002 (g with EOL vs. g0 or g without EOL) (MannWhitney).
B
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A

Kaplan-Meier Survival Curves, Percentage of Glomerular Involvement
1.00

0.75

0.50

0.25

0.00
0 500 1000 1500 2000
Post-biopsy time (days)

g0 g1


g2

g3



p <0.01: g2 vs. g0 or g1 and p <0.003 g3 vs. g0 or g1 (log-rank test)
B
Kaplan-Meier Survival Curves, Peak of Inflammation
1.00

0.75

0.50

0.25

0.00
0 500 1000 1500 2000
Post-biopsy period (days)

g0 g1


g2

g3



p =0.01 g2 vs. g0 and p <0.001 g3 vs. g0 (log-rank test)
C
Kaplan-Meier survival curves, Presence of Endocapillary Occlusion
1.00

0.75

0.50

0.25

0.00
0 500 1000 1500 2000
Post-biopsy period (days)

g0 g without EOL


g with EOL



Abbreviation: EOL, endocapillary occlusive lesion
p =0.008 g with EOL vs. g0 and p =0.01 g without EOL vs. g0 (log-rank test)

Figure 1: KaplanMeier survival curves for postbiopsy
cumu-lative renal allograft survival in patients stratified by
the sever-ity of glomerulitis using grading methods based
on the per-centage of glomerular involvement (A),
inflammation in the most severely affected glomerulus (B)
or the presence of en-docapillary occlusive lesions (C).
Histologic Grading of Transplant Glomerulitis

general increase in inflammatory cells rather than a
selec-tive increase in neutrophils (Table 4).

Correlation of glomerulitis grade with clinical
and laboratory parameters
G3 patients had more frequent history of prior renal trans-
plantation (Table 5). Immune cell function levels were higher
in g3 compared to g1 (p = 0.007) and tended to be higher
than in g0 (p = 0.12) suggesting higher level of immune
system activation. Concurrent higher urine pro-tein values
were observed in g3 compared to g0 and g1 (p 0.001)
patients. These differences persisted when samples showing
concurrent TGP, FSGS and membranous nephropathy were
excluded from the analysis (p 0.007). G2 patients tended
to be somewhat more frequently sen-sitized to class-II HLA
antigens compared to g1 (p = 0.07). Although they had
concurrent higher urine protein values compared to g0 and
g1 (p 0.03) patients, this difference was no longer
significant after adjustment for the presence of TGP, FSGS
and membranous nephropathy.

Response to antirejection therapy in
patients stratified by glomerulitis severity
No difference in the type or dose of administered antire-
jection treatment was observed in patients with different
grades of glomerulitis (Table 6, row 1). Following antirejec-
tion therapy, delta serum creatinine (_Cr) indicated greater
worsening of graft function in g3 compared to g0 (p = 0.01)
and g2 (p = 0.013) patients (Table 6). In addition, pro-
gressive serum creatinine deterioration in individual cases
tended to be higher in g3 compared to g1 (p = 0.1). The
inflammatory infiltrate within glomerular capillaries tended to
persist despite treatment for rejection. Thus, 3/11 (27%) of
g1 patients showed persistence of g1 lesion while 2/11
(18%) patients showed deterioration in glomerulitis grade
(from g1 to g2). Similarly, 4/11 (36%) of g2 patients showed
persistence of g2 lesion while 1/11 (9%) of g2 patients
showed deterioration in glomerulitis grade (from g2 to g3).
G3 glomerulitis persisted in 6/10 (60%) of follow-up biopsies.


Follow-up data in samples stratified by severity
of glomerulitis
G3 patients had significant elevation of 6-month and 1-
year postbiopsy serum creatinine values (Table 7). These
patients also had more frequent detection of high-grade
glomerulitis (g2 or g3) on follow-up biopsies (g3 vs. g0,
g1 or g2, p 0.01). When TGP was absent in index
biopsies, a significant number of g3 patients
subsequently developed TGP compared to g0 and g1 (p
0.03) patients. TGP was documented 311 216 days
postindex biopsy where PTC-C4d was often negative
(4/6, 66%). Postbiopsy detection of DSA was also more
frequent in g3 compared to g0 (p = 0.02) (Table 7).

Similarly, when compared to g0, g2 patients tended to
have more frequent high-grade glomerulitis on follow-up

American Journal of Transplantation 2010; 10: 24422452 2447
Batal et al.

Table 4: Pathology findings
g0 (n = 44) g-1 (n = 29) g2 (n = 23) g3 (n = 15)

No. of Glomeruli 12.2 4.9 11 4.5 13.7 4.8 10.7 3.7

No. of GS 1.1 2.8 1.1 1.5 1.1 1.3 0.9 1.2

No. of arteries 3.5 1.5 2.9 1.5 3.2 1.4 2.7 1.1

TCMR

Borderline 19/44 (43%) 13/29 (45%) 8/23 (35%) 4/15 (27%)

1A 14/44 (32%) 8/29 (28%) 7/23 (31%) 5/15 (33%)

1B 6/44 (13%) 7/29 (24%) 4/23 (17%) 4/15 (27%)

2A 2/44 (5%) 1/29 (3%) 2/23 (9%) 2/15 (13%)

2B 2/44 (5%) 0/29 (0%) 1/23 (4%) 0/15 (0%)

BKVN 1/44 (2%) 0/29 (0%) 1/23 (4%) 0/15 (0%)

Concurrent AMR
1


Diagnostic 3/34 (9%) 3/26 (12%) 3/17 (18%) 1/7 (14%)

Suspicious 7/34 (21%) 6/26 (23%) 3/17 (18%) 3/7 (43%)

Negative 24/34 (70%) 17/26 (65%) 11/17 (64%) 3/7 (43%)

PTC-C4d

Diffuse
2
3/43 (7%) 5/28 (18%) 4/23 (17%) 3/14 (21%)

Focal
2
6/43 (14%) 4/28 (14%) 3/23 (13%) 4/14 (29%)

Minimal 15/43 (35%) 8/28 (29%) 8/23 (35%) 4/14 (29%)

Negative 19/43 (44%) 11/28 (39%) 8/23 (35%) 3/14 (21%)

PTCitis
3
0.5 0.8 0.8 0.9 1.0 1.0 1.5 0.9

No. of neutrophils per glomerulus
1
0.3 0.3 0.5 0.5 0.8 0.8 1.9 4.4

% of neutrophils within glomeruli 16 19 12 13 12 19 14 22

Protein reabsorption droplets
2
28/44 (64%) 14/29 (48%) 21/23 (91%) 11/15 (73%)

Adjusted protein reabsorption droplets
3
26/41 (63%) 10/25 (40%) 13/14 (93%) 10/12 (83%)
Interstitial inflammation (i) 1.8 0.7 1.7 0.7 1.9 0.7 2.1 0.7

Tubulitis (t) 1.8 0.8 1.9 0.8 2.0 0.9 1.9 0.9

t3 tubulitis 10/44 (23%) 7/29 (24%) 9/23 (39%) 4/15 (27%)

Intimal arteritis (v) 0.14 0.47 0.04 0.2 0.2 0.5 0.13 0.4

TGP score (cg) 1

0.02 0.02 0.07 0.3 0.4 0.9 0.3 0.6

Presence of TGP 1/44 (2%) 2/29 (7%) 5/23 (22%) 3/15 (20%)

Tubular atrophy (ct) 2 1.1 0.5 1.1 0.6 1.3 0.6 1.0 0.7

Interstitial fibrosis (ci) 1.0 0.5 1.0 0.7 1.4 0.5 0.7 0.7

Mesangial matrix increase (mm) 0.2 0.5 0.3 0.5 0.7 1.0 0.3 0.6

Arteriolar hyalinosis (ah) 0.8 0.6 0.8 0.6 1.0 0.9 0.7 0.7

Arterial fibrointimal thickening (cv) 0.9 0.6 1.0 0.6 1.2 0.8 0.7 0.6

Tubular vacuolization 19/44 (43%) 7/29 (24%) 10/23 (43%) 5/15 (33%)


Abbreviations: GS = globally sclerosed glomeruli; TCMR = T cell-mediated rejection; AMR = antibody-mediated rejection; TGP =
transplant glomerulopathy; PTC-C4d = peritubular capillary C4d staining; PTCitis = peritubular capillaritis.
1
Assessment for AMR was performed when concurrent information on PTC-C4d staining and ELISA PRA/DSA was
available. Sensitivity of g > 0 for concurrent diagnostic of AMR: 3 + 3 + 1/ (3 + 3 + 1 + 3) = 7/10 (70%).
Specificity of g > 0 for concurrent diagnostic of AMR: 24 + 7/ (24 + 7 + 17 + 6 + 11 + 3 + 3 + 3) = 31/74 (42%).
3
Adjusted protein reabsorption droplets: after excluding samples with concurrent TGP, focal segmental glomerulosclerosis and mem-
branous nephropathy.
Bold font corresponds to values showing significant differences as summarized below:
2
Positive PTC-C4d: p = 0.047 (g3 vs. g0) (Fishers exact).
3
PTCitis: p 0.047 (g3 vs. g0 or g1) (MannWhitney).


1
No. of neutrophils per glomerulus: p 0.006 (g3 vs. g0 or g1) and p 0.02 (g2 vs. g0 or g1) (MannWhitney).
2
Protein
reabsorption droplets: p = 0.001 (g2 vs. g1) (Fishers exact).
3
Adjusted protein reabsorption droplets: p = 0.02 (g3 vs. g1) and p 0.04 (g2 vs. g0 or g1) (Fishers exact).
1
Presence of TGP: p = 0.047 (g3 vs. g0) and p = 0.016 (g2 vs. g0) (Fishers exact).
2
Interstitial fibrosis: p 0.02 (g2 vs. g0 or g3) (MannWhitney).


biopsies (p = 0.07) and subsequent detection of DSA (p =
0.09). Only two g2 patients developed TGP on follow-up
(152 and 207 days later) and both had neg-ative PTC-
C4d in their index biopsies. G1 patients had numerically,
but not statistically, higher percentages of subsequent
high-grade glomerulitis, TGP and DSA (Table 7).

Analysis of graft failure
G3 patients had the worst postbiopsy graft survival while
g2 patients had survival curve running between g3 and
g1 and had worse 2-year graft survival than g0 patients
(p = 0.04) (Figure 1A and Table 7). Similar results were
obtained by utilizing posttransplantation graft survival
data (not presented).

2448 American Journal of Transplantation 2010; 10: 24422452
Histologic Grading of Transplant Glomerulitis

Table 5: Clinical and laboratory information
g0 (n = 44) g1 (n = 29) g2 (n = 23) g3 (n = 15)
Age (years)
1
55 12 53 13 56 16 47 13
Female sex 19/44 (43%) 12/29 (41%) 14/23 (61%) 4/15 (27%)
Black race 9/44 (20%) 3/29 (10%) 7/23 (30%) 3/15 (20%)
Etiology of ESRD
Diabetes 10/44 (23%) 10/29 (34%) 6/23 (26%) 4/15 (27%)
Hypertension 13/44 (30%) 5/29 (17%) 5/23 (22%) 4/15 (27%)
Cystic kidney diseases 5/44 (11%) 4/29 (14%) 4/23 (17%) 2/15 (13%)
Glomerulonephritis 5/44 (11%) 3/29 (10%) 5/23 (22%) 1/15 (7%)
Toxicity 6/44 (14%) 4/29 (14%) 0/23 (0%) 0/15 (0%)
Reflux 1/44 (2%) 1/29 (4%) 1/23 (4%) 1/15 (7%)
Others 4/44 (9%) 2/29 (7%) 2/23 (9%) 3/15 (19%)
History of prior renal transplantation
2
4/44 (10%) 3/29 (10%) 2/23 (9%) 6/15 (40%)
Deceased donor 35/44 (80%) 21/29 (72%) 16/23 (70%) 13/15 (87%)
Donor age
3
50 12 41 18 51 10 35 14
Posttransplant date (days)
1
438 361 463 296 663 373 529 470
Whole blood tacrolimus level (ng/mL) 7.1 5.3 8.4 7.5 6.7 3.8 7.6 4.2
Serum creatinine at biopsy (mg/dL) 2.2 (1.73.2) 2.4 (1.73.3) 2.8 (1.93.6) 2.2 (1.73.2)
Urine protein (mg/dL)
2
16 31 25 63 91 138 229 230
Adjusted urine protein (mg/dL)
3
16 31 13 24 77 148 160 184
BK viruria 4/37 (11%) 5/26 (19%) 4/17 (24%) 2/10 (20%)
BK viremia 1/36 (3%) 0/25 (0%) 0/18 (0%) 0/10 (0%)
ELISA PRA > 10% class I 6/38 (16%) 4/27 (15%) 4/19 (21%) 3/8 (38%)
ELISA PRA > 10% class II 9/38 (24%) 3/27 (11%) 7/19 (37%) 3/8 (29%)
DSA at biopsy 4/35 (11%) 3/27 (11%) 3/17 (18%) 1/7 (14%)
History of DSA 8/44 (18%) 2/29 (7%) 2/23 (9%) 1/15 (7%)
Immune cell function (ng ATP/mL)
1
210 117 131 65 228 137 277 111
Abbreviations: ESRD = end-stage renal disease; TGP = transplant glomerulopathy; DSA = circulating donor-specific antibody.
Adjusted urine protein: urine protein values after excluding samples with concurrent TGP, focal segmental glomerulosclerosis and
membranous nephropathy.
Bold font corresponds to values showing significant differences as summarized below:
1
Age: p = 0.03 (g3 vs. g0), (MannWhitney).

2
History of prior renal transplantation: p 0.04 (g3 vs. g0, g1, or g2) (Fishers exact).

3
Donor age: p 0.03 (g3 vs. g0, g1, or g2) and p = 0.04 (g0 vs. g1) (MannWhitney).
1
Posttransplant
date: p = 0.02 (g2 vs. g0) (MannWhitney).

2
Urine protein: p 0.001 (g3 vs. g0 or g1) and p 0.03 (g2 vs. g0 or g1) (MannWhitney).
3
Adjusted
urine protein: p 0.007 (g3 vs. g0 or g1) (MannWhitney).

1
Immune cell function p = 0.007 (g3 vs. g1) and p = 0.02 (g0 vs. g1) (MannWhitney).


Information on urine protein values was available for 39 g0, 24 g1, 18 g2 and 11 g3 patients.
Information on adjusted urine protein values was available for 37 g0, 20 g1, 11 g2 and 8 g3 patients.
Information on concurrent immune cell function was available for 25 g0, 14 g1, 12 g2 and 6 g3 patients.

None of the g0 patients underwent nephrectomy while
eight patients with glomerulitis on index biopsy did [g1(2),
g2(1) and g3(5)]. Of the latter, 7/8 (88%) had moderate-
to-severe interstitial fibrosis/tubular atrophy (IF/TA), while
graft failure was attributed to primary dysfunction in the
remaining specimen (1/8, 12%). TCMR was detected in
all nephrectomies and positive PTC-C4d was detected in
the majority (7/8, 88%). The latter suggested a
contribution of AMR but DSA was not tested at the time
of nephrectomy. However, 5/8 (63%) of these patients
had developed DSA at some earlier time-point. Of note,
6/8 (75%) of these patients had previous episodes of
TCMR and positive PTC-C4d.

12/15 (80%) had moderate-to-severe IF/TA and 3/15 (20%)
had recurrent/de novo glomerulonephritis. These causes of
graft loss were comparable to those observed in pa-tients
without glomerulitis (n = 7) [5/7 (72%) moderate-to-severe
IF/TA, 1/7 (14%) recurrent FSGS and 1/7 (14%) primary
dysfunction]. The frequency of previous episodes of TCMR
were also comparable [12/15 (80%) glomerulitis vs. 5/7
(72%) g0]. However, previous episodes of PTC-C4d were
more frequent in glomerulitis group [12/15 (80%) glomerulitis
vs. 1/7 (14%) g0, p = 0.007)] suggesting more prominent
contribution of AMR in the former.

In patients who did not undergo nephrectomy, renal biop-
sies were used to analyze the cause of graft failure. In
patients with glomerulitis (n = 15) [g1(3), g2(7) and g3(5)],
Discussion

Transplant glomerulitis was first described in 1981 (1). Since
then, there has been some uncertainty regarding its

American Journal of Transplantation 2010; 10: 24422452 2449
Batal et al.

Table 6: Summary of therapeutic interventions
Groups g0 (n = 32) g1 (n = 23) g2 (n = 19) g3 (n = 12)

Antirejection therapy

Solumedrol (mg) 25 (78%) 20 (87%) 14 (74%) 9(75%)

(830 390) (810 355) (1035 365) (944 300)

Campath 30 mg 0 (0%)
(210 95)
2 (9%) 3 (16%) 1/12(8%)

Thymoglobulin (mg) 2 (6%) 1 (4%) (200) 0 (0%) 0 (0%)

IVIG (g) 2 (6%) (40 25) 0 (0%) 1(5%) (76) 2(17%) (93 25)

Increase tacrolimus 3 (10%) 0 (0%) 0 (0%) 0 (0%)

Mycophenolate 0 (0%) 0 (0%) 1(5%) 0 (0%)

Serum creatinine response

Complete response 13/31 (42%) 8/23 (35%) 7/17 (41%) 4/12 (33%)

Partial response 7/31 (23%) 4/23 (17%) 2/17 (12%) 1/12 (8%)

Unchanged serum creatinine 6/31 (19%) 8/23 (35%) 5/17 (29%) 2/12 (17%)

Progressive deterioration 5/31 (16%) 3/23 (13%) 3/17 (18%) 5/12 (42%)

_Cr 0.4 1.2 0.7 3.2 0.5 0.7 0.2 0.9

Histologic response

Better 13/23 (57%) 5/11 (45%) 8/11 (73%) 6/10 (60%)

No change 7/23 (30%) 6/11 (55%) 2/11 (18%) 4/10 (40%)

Deterioration 3/23 (13%) 0/11 (0%) 1/11 (9%) 0/10 (0%)

Glomerulitis response

Better NA 6/11 (55%) 6/11 (55%) 4/10 (40%)

No change 16/23 (70%) 3/11 (27%) 4/11 (36%) 6/10 (60%)

Deterioration 7/23 (30%) 2/11 (18%) 1/11 (9%) NA


Abbreviations: _Cr = difference in serum creatinine between baseline and follow-up 4 weeks posttreatment; NA = not applicable.
Bold font corresponds to values showing significant differences as summarized below:
_Cr: p = 0.01 (g3 vs. g0) and p = 0.013 (g3 vs. g2) (MannWhitney).
Histologic response is assessed by Banff grade for acute T-cell-mediated rejection.

Serum creatinine response is not available in 1 (g0) episode and 2 (g2) episodes treated with increased immunosuppression.

Follow-up biopsies were available for 23, 11, 11 and 10 episodes that were classified as g0, g1, g2 and g3 and treated with
increased immunosuppression.

histologic assessment and clinical significance. No clear cut-
off value has existed to define the minimum number of
inflammatory cells needed to render the diagnosis of
glomerulitis. Several researchers have studied glomeruli-tis
(Table 1), but most did not assess the significance of
glomerulitis grade, evolution of glomerulitis to TGP and the
correlation with DSA, PTC-C4d or response to antirejection

treatment. In our study, we sought to perform a detailed
clinicopathologic study addressing all the aforementioned
issues. To define glomerulitis, we used the cut-off value
recommended by the WHO to define increased glomeru-lar
inflammation (5 leukocytes/glomerulus) (18). While the
presence of endothelial enlargement has been re-quired by
some authors (4), we had difficulty distinguishing

Table 7: Follow-up information
g0 (n = 44) g1 (n = 29) g2 (n = 23) All g3 (n = 15)
Graft failure at 1 yr postbiopsy
1
4/37 (11%) 3/27 (11%) 6/19 (32%) 8/14 (57%)
Graft failure at 2 yrs postbiopsy
2
7/31 (23%) 5/22 (23%) 8/14 (57%) 10/13 (77%)
Presence of HG-g on follow-up biopsies
3
4/33 (12%) 5/19 (26%) 6/17 (35%) 11/13 (85%)

Development of TGP in follow-up biopsies

1
1/32 (3%) 2/17 (12%) 2/13 (15%) 6/11 (55%)
Serum creatinine 6 months postbiopsy (mg/dL)
2
1.9 (1.52.6) 1.7 (1.43.1) 2.4 (1.42.8) 2.7 (1.75.3)
Serum creatinine 1-year postbiopsy (mg/dL)
3
1.8 (1.52.5) 1.5 (1.33.1) 2.2 (1.52.9) 4.0 (1.77.6)
DSA postbiopsy
1
5/44 (11%) 6/29 (21%) 7/23 (30%) 6/15 (40%)
Abbreviations: TGP = transplant glomerulopathy; DSA = circulating donor-specific antibody, HG-g = high-grade transplant glomerulitis
(g2 & g3).

Development of TGP in follow-up biopsies was calculated for samples revealing cg0 on index
biopsies. Bold font corresponds to values showing significant differences as summarized below:
1
Graft failure 1 year: p 0.003 (g3 vs. g0 or g1) (Fishers exact).

2
Graft failure 2 years: p 0.003 (g3 vs. g0 or g1) and p = 0.04 (g2 vs. g0) (Fishers exact).
3
Presence
of HG-g on follow-up biopsies: p 0.01 (g3 vs. g0, g1 or g2) (Fishers exact).

1
Development of TGP in follow-up biopsies: p 0.03 (g3 vs. g0 or g1) (Fishers exact).
2
Serum
creatinine 6 months postbiopsy: p = 0.04 (g3 vs. g1), (MannWhitney).
3
Serum creatinine 1-year postbiopsy: p 0.02 (g3 vs. g0 or g1) (Mann
Whitney).
1
DSA postbiopsy: p = 0.02 (g3 vs. g0) (Fishers exact).

2450 American Journal of Transplantation 2010; 10: 24422452


swollen endothelial cells from macrophages adherent to
the glomerular capillary wall using standard light
microscopy. It is worth mentioning that the recognition of
other rejection-related endothelial lesions such as inti-
mal arteritis, arterial fibrinoid necrosis and peritubular
cap-illaritis do not require the presence of endothelial
enlarge-ment (4,26). Two prior studies support our
contention that it may not be necessary to insist on
endothelial swelling as a criterion for recognition of
transplant glomerulitis, Tuazon et al. showed that the
number of intraglomeru-lar T cells correlated with 1-year
graft failure irrespec-tive of endothelial enlargement (13).
Tinckam et al. (7) demonstrated that an average of one
or more CD68+ monocytes/glomerulus is similarly
associated with worse outcome.

To grade the severity of glomerulitis, we first compared
three different scoring systems: (i) percentage of affected
glomeruli, (ii) peak number of leukocytes in the most
severely affected glomerulus, and (iii) presence/absence
of endocapillary occlusive lesions. Methods B and C
were explored since they are conceptually easier to apply
with-out the need of evaluating all glomeruli individually.
How-ever, our analysis indicates that the assessment of
the per-centage of glomerular involvement (method A)
provides the most clinically meaningful grading system.
Parenthet-ically, while both methods A and B correlate
with graft survival and peritubular capillaritis, method A is
superior in that it correlates better with proteinuria, PTC-
C4d, in-creased serum creatinine on follow-up and
subsequent DSA.

Positive PTC-C4d was observed in 50% of g3 samples
compared to 20% g0 samples (p = 0.047). This frequency of
C4d deposition is in the same range as that reported by
others. Thus, Magil showed that half of glomerulitis sam-ples
were associated with positive (focal or diffuse) PTC-C4d (5).
Hara et al., using an immunofluorescence tech-nique,
detected diffuse PTC-C4d in 54% of late transplant biopsies
showing both glomerulitis and TGP (11). In addi-tion to the
association with PTC-C4d, we showed that g3, and to a
lesser extent g2, was associated with higher per-itubular
capillaritis score and subsequent development of DSA and
TGP. Sis et al. made this same observation using silhouette
plot correlations and unsupervised hierarchical clustering
(27). Even though glomerulitis showed the afore-mentioned
associations with lesions used to define AMR, glomerulitis
(often grade g1) could be detected in 47% and 62% of DSA-
,C4d- TCMR and DSA- C4d- borderline sam-ples,
respectively. Magil noted that the glomerulitis associ-ated
with TCMR or borderline changes is characterized by the
predominance of intraglomerular lymphocytes rather than
monocyte infiltrates which is more typical for AMR (5,6). In
our study, glomerulitis grade did not correlate with interstitial
inflammation, tubulitis, intimal arteritis or Banff 97 grade of
TCMR. An association between glomerulitis and intimal
arteritis has been described in many (2,8,12,13) but not all
(10) earlier studies.
Histologic Grading of Transplant Glomerulitis

Even when samples showing concurrent TGP, FSGS and
membranous nephropathy were excluded from the analy-
sis, severe glomerulitis (g3) samples were associated
with significant proteinuria and more frequent detection of
pro-tein reabsorption droplets. Two previous studies
described an association between glomerulitis and
proteinuria (2,13). However, the authors did not perform a
quantitative com-parison and did not correct for the
presence of other chronic glomerular lesions that can
cause protein leak-age. The mechanism of proteinuria in
glomerulitis is uncer-tain, but may reflect immunologic
injury to endothelium (28) mediated by DSA, or injury to
the glomerular base-ment membrane possibly mediated
by antibodies directed against basement membrane
components (29). The pos-sibility of a direct injury to the
podocytes is also raised by studies showing fusion of
podocyte foot processes in glomerulitis (13,28,30).

To the best of our knowledge, a clinicopathologic assess-
ment of posttreatment biopsies has not been previously
performed in patients in whom the severity of glomeruli-
tis was carefully graded. Following antirejection therapy,
delta serum creatinine (_Cr) indicated worsening of graft
function in g3 patients. No improvement and persistence
of high-grade glomerulitis on follow-up biopsies was more
frequently observed in g3 samples. Olsen et al. also
noted the persistence of glomerulitis on follow-up
biopsies (2). Whether more potent medications are
needed to clear such inflammation, and if this turns out to
be the case, whether clearing glomerulitis inflammation
can improve prognosis remains to be determined.

Finally, the most severe grade of glomerulitis (g3) had the
worst follow-up serum creatinine and graft survival. Pa-
tients with grade g2 glomerulitis had a graft survival curve
located at an intermediate position between patients with
g3 and g1 lesions (Figure 1A), and had worse 2-year
graft survival compared to g0 patients. In most previous
stud-ies, the incidence of graft failure was higher in
glomerulitis patients and reached statistical significance
in the hands of some (7) but not all investigators (1012).
This can be partially attributed to the fact that glomerulitis
was not graded but rather categorically assessed as
present or ab-sent (11,13). In one study, the small
number of g3 biopsies led the authors to group g2 and g3
lesions together into one category for analysis (12).

In conclusion, our study based on for cause biopsy sam-
ples has shown that grading glomerulitis based on the
percentage of affected glomeruli is superior to grading based
on the most inflamed glomerulus, or the presence of capillary
loop occlusion by inflammation. We also demon-strate that
grading glomerulitis is a clinically meaningful exercise in that
higher grades of this lesion are more of-ten associated with
proteinuria, peritubular capillaritis, per-itubular capillary C4d
staining, donor-specific antibodies, suboptimal response to
antirejection therapy, persistence of glomerulitis lesions in
follow-up biopsies, subsequent

American Journal of Transplantation 2010; 10: 24422452 2451
Batal et al.

development of transplant glomerulopathy and worse
graft survival.


Acknowledgments

This study was supported by Research Fellowship Grant from the College
of American Pathologists Foundation, Chicago, IL (IB).

Disclosure

The authors of this manuscript have no conflicts of inter-
est to disclose as described by the American Journal of
Transplantation.

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