The significance of C4d staining

with minimal

histologic abnormalitiesMark Haas

Department of Pathology & Laboratory Medicine,Cedars-Sinai Medical Center, Los Angeles, California,USA

Correspondence to Mark Haas, MD, PhD, Departmentof Pathology & Laboratory Medicine, Cedars-SinaiMedical Center, 8700 Beverly Blvd., Los Angeles, CA90048, USATel: +1 310 248 6695; fax: +1 310 423 5881;e-mail: mark.haas@cshs.org

Current Opinion in Organ Transplantation 2010,15:21–27

Purpose of review

C4d deposition within peritubular capillaries (PTCs) in renal allograft biopsies is a

specific marker for interaction of antibody with the graft, and is an extremely useful tool

for the diagnosis of antibody-mediated rejection (AMR). Whereas PTC C4d itself is not

diagnostic of AMR, it is usually accompanied by histologic features of acute and/or

chronic AMR. However, some biopsies, mainly from ABO-incompatible renal allografts,

show C4d staining without histologic findings of AMR or cell-mediated rejection. The

significance of such C4d staining remains a topic of debate, and this finding may have

different implications in ABO-incompatible versus conventional renal allografts.

Recent findings

In biopsies of ABO-incompatible renal allografts (particularly protocol biopsies), C4d

deposition in the absence of histologic evidence of rejection is a common finding, is not

associated with an increased risk of graft scarring, and may even indicate a decreased

risk of scarring, at least in the short term. By contrast, in positive cross-match and

conventional renal allografts such C4d deposition is uncommon, and may indicate

potentially reversible graft injury. However, a state of C4d staining without associated

graft injury may be inducible in positive cross-match grafts by complement inhibition.

Summary

C4d staining without associated histologic findings of rejection may represent a marker

for stable graft accommodation, at least in ABO-incompatible renal allografts. However,

further studies are clearly needed to determine what the long-term implications of such

C4d deposition are.

Keywords

ABO-incompatible, accommodation, antibody-mediated rejection, complement, renal

transplantation

Curr Opin Organ Transplant 15:21–27� 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins1087-2418

IntroductionDuring the past two decades, the role of alloantibodies

in both acute and chronic rejection of renal allografts

has become increasingly documented [1–4,5�,6�]. Still,

whereas certain histologic features of acute rejection in

the presence of donor-specific antibody (DSA), such as

margination of neutrophils in peritubular capillaries, have

long been recognized [1,2], such findings are not specific

for antibody-mediated rejection (AMR), nor are these

seen in all cases of AMR [7��,8].

For these reasons, immunostaining of renal allograft

biopsies for C4d has represented such an important

development in the field of renal allograft pathology.

C4d is a degradation product of complement factor C4,

a component of the classical complement pathway that is

typically initiated by antibody bound to antigen. After

cleavage of remaining C4 domains, C4d remains cova-

1087-2418 � 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins

lently bound at sites of C4 activation, and is thus a

relatively long-lived marker of a humoral response

[9,10]. Furthermore, C4d is easily detected in biopsy

specimens by indirect immunofluorescence on frozen

sections using a monoclonal antibody [8,10] or by immu-

nohistochemistry (IHC) on paraffin sections of formalin-

fixed tissue using a polyclonal antiserum [11], both of

which are now commercially available.

Feucht et al. [10], in a study of 93 renal allografts biopsied

for early dysfunction (mean 11 days post-transplantation),

first demonstrated an association between C4d staining

in peritubular capillaries (PTCs) by indirect immuno-

fluorescence and reduced graft survival. Feucht et al. [10]

also found an association between PTC C4d staining and

both re-transplants and elevated panel-reactive anti-

bodies (PRAs), suggesting an association between such

staining and humoral immunity, and indeed several

groups [8,11–13] later documented a strong correlation

DOI:10.1097/MOT.0b013e3283342ebd

22 Mechanisms of rejection

between C4d staining of renal allograft biopsies and the

presence of DSA in serum, as well as histologic findings

previously shown to be present in acute rejection epi-

sodes associated with the presence of DSA [2]. Additional

studies [13–15] established an association between C4d

staining and morphologic findings of chronic rejection,

including transplant glomerulopathy and PTC basement

membrane multilayering, leading to further work clearly

documenting the association between these findings and

DSA [13,14,16,17,18��].

The strong association between histologic changes

including margination of leukocytes (neutrophils and/or

monocytes/macrophages) in PTC and/or glomerular

capillaries and (less commonly) arterial fibrinoid necrosis,

PTC C4d staining, and the presence of anti-HLA DSA

has led to the Banff consensus process formulating

criteria for the diagnosis of active AMR based on the

presence of this triad of histologic, immunohistologic, and

serologic findings [19]. In ABO-incompatible renal allo-

grafts, however, PTC C4d staining and antibodies to

donor blood group antigens are commonly seen in the

absence of histologic findings of acute or chronic rejection

[20,21��,22��], and cases of C4d staining without histo-

logic evidence of rejection have also been reported in

Figure 1 PTC C4d staining in biopsies of ABO-incompatible versus

For ABO-incompatible grafts, there were 55 protocol biopsies, 17 biopsiesperformed for other causes). For positive cross-match grafts, there were 103total biopsies (including 12 performed for other causes). Intensity of stainingPTC C4d staining (diffuse, focal, or absent) was significantly different betwamong protocol biopsies, biopsies performed for graft dysfunction, and when[20]; reproduced with permission from Wiley-Blackwell.

conventional (ABO-compatible but HLA-mismatched)

grafts [20,23,24], albeit much less commonly. This review

will focus on the significance of PTC C4d staining with

minimal associated histologic alterations, whether these

findings indicate graft injury warranting consideration of

appropriate treatment, and if the significance of such

findings is different in ABO-incompatible versus conven-

tional renal allografts.

ABO-incompatible renal allograftsThe presence of preformed anti-blood group antibodies

was a cause of hyperacute rejection of renal allografts in the

early years of solid organ transplantation [25]. However,

the use of pretransplant desensitization protocols to

remove such antibodies and improved immunosuppres-

sive regimens to prevent their recurrence postoperatively

has allowed an increasing number of renal transplants to be

performed across this immunologic barrier, first in Japan

and subsequently in North America and Europe, although

low levels of anti-blood group antibodies typically persist

in recipients of ABO-incompatible grafts [26�,27,28,29�].

A number of centers, initially in Japan and later in the

USA, reported cases of C4d-positive acute AMR in ABO-

incompatible renal allograft recipients associated with

positive cross-match (HLA-incompatible) renal allografts

performed for graft dysfunction, and 75 total biopsies (including threeprotocol biopsies, 129 biopsies performed for graft dysfunction, and 244was graded on a scale of 0–4þ. By chi-squared analysis of 2�3 tables,een biopsies of ABO-incompatible versus positive cross-match grafts,all biopsies were considered (P<0.0001 in each case). FromHaas et al.

Significance of C4d staining with minimal histologic abnormalities Haas 23

elevated levels of anti-blood group antibodies [30–33].

However, with the growing utilization of routine C4d

staining in the diagnostic work-up of renal allograft bio-

psies, particularly those from recipients felt to be at

increased risk of developing AMR, it became apparent

that PTC C4d staining in biopsies of ABO-incompatible

grafts was far more commonly seen than in biopsies

of ABO-compatible grafts, including those in recipients

who were desensitized prior to transplantation to remove

preformed anti-HLA antibodies (positive cross-match

transplants). In a study of 32 recipients of ABO-incompa-

tible renal allografts from the Mayo Clinic, Fidler et al. [33]

found that 13 (41%) developed AMR, nine with associated

graft dysfunction and four subclinical, diagnosed on a

protocol biopsy. Notably, eight of the nine patients diag-

nosed with clinical AMR had a protocol biopsy done 1–

7 days prior to the biopsy showing AMR, and five of these

protocol biopsies were C4d-negative, indicating that

the C4d associated with AMR was recently deposited.

In addition to the patients with clinical or subclinical

AMR, Fiedler et al. [33] also noted four patients who

had diffuse PTC C4d staining on a protocol biopsy done

approximately 1 year post-transplantation but no histologic

evidence of acute AMR or transplant glomerulopathy.

Figure 2 Correlation between PTC C4d staining and histologic findi

positive cross-match renal allografts

Among biopsies of ABO-incompatible grafts, 34 showed diffuse, >1þ PTCnegative). Among biopsies of positive cross-match grafts, 83 showed diffuse,(C4d-negative). All 20 positive cross-match biopsies and one of two ABO-incshowed PTC neutrophil margination. The kappa statistic was used to assesbetween categories of PTC C4d staining (diffuse >1þ, diffuse <1þ or focalnone). For biopsies of ABO-incompatible grafts, kappa was –0.01 with PP<0.0001. From Haas et al. [20]; reproduced with permission from Wiley

Figure 1 shows C4d staining results in 55 protocol biopsies

done during the first year post-transplantation in 24 reci-

pients of ABO-incompatible renal allografts at Johns Hop-

kins Hospital. We found PTC C4d staining in 80% of these

biopsies, with strong (>1þ on a 0–4þ scale) and diffuse

staining in 51% [20]. By contrast, 76/103 protocol biopsies

from recipients of ABO-compatible, positive cross-match

grafts were C4d-negative (Fig. 1). Furthermore, unlike the

case in positive cross-match grafts in which the vast

majority of C4d-positive biopsies are associated with

histologic features of AMR, including neutrophil and/or

mononuclear leukocyte margination in peritubular and/or

glomerular capillaries or less commonly changes of throm-

botic microangiopathy, most C4d-positive biopsies of

ABO-incompatible grafts show no histologic evidence of

AMR (Fig. 2) or cell-mediated rejection [20]. Subsequent

studies of protocol biopsies of ABO-incompatible renal

allografts from Japan [21��] and Switzerland [34�] have

confirmed that most such biopsies show PTC C4d staining,

diffuse in the majority of cases, without histologic features

of AMR.

We investigated whether diffuse PTC C4d deposi-

tion in the absence of histologic features of AMR or

ngs suggestive of AMR in biopsies of ABO-incompatible versus

C4d, 22 diffuse, �1þ or focal PTC C4d, and 19 no PTC C4d (C4d->1þPTCC4d, 29 diffuse,�1þ or focal PTCC4d, and 132 no PTCC4dompatible biopsies that showed thrombotic microangiopathy (TMA) alsos the degree and significance of correlation (probability of agreement), negative) and those of neutrophil margination (>1þ and/or TMA, trace,¼0.55; for biopsies of positive cross-match grafts kappa was 0.65,-Blackwell.

24 Mechanisms of rejection

Figure 3 Comparison of mean chronic sum scores

(cgR ciR ctR cv) on 6-month and 12-month protocol biopsies

(upper panel), and mean increases in this score from the initial

biopsy to the 6 and 12-month biopsies (lower panel) in group A

and group B patients

7

6

5

4

3

2

1

0

5

4

3

2

1

06 12

Group AGroup B

P = 0.22

P = 0.032

P = 0.030P = 0.044

Months after transplantation

Meanincrease in(cg + ci +ct + cv)

Mean(cg + ci +ct + cv)

Error bars indicate 1 standard deviation (SD). P values shown are all bythe Wilcoxon rank-sum test. From Haas et al. [22��]; reproduced withpermission from the American Society of Nephrology.

Table 1 12-Month cg scores in patients having one or more

diffusely C4d-positive protocol biopsies at 1, 3, or 6 months,

without clinical AMR

cg 0 cg �1

Positive cross-match (n¼17)No histologic evidence of AMR 0 1Subclinical AMR 6 10

ABO-incompatible (n¼25)No histologic evidence of AMR 22 0Subclinical AMR 1 2

All patients had donor-specific anti-HLA (positive cross-match) or anti-blood group (ABO-incompatible) antibodies at the time of biopsy.Biopsies diagnosed as showing subclinical AMR all had marginationof neutrophils and/or mononuclear leukocytes in cortical PTC, meetingcriteria for a ptc score of >1 [7��], whereas biopsies with no histologicevidence of AMR had a PTC score of 0, with no more than mildglomerulitis (Banff g score <2) and no evidence of TMA or arteritis.

cell-mediated rejection is associated with development of

graft dysfunction or scarring in ABO-incompatible renal

allografts during the first year post-transplantation [22��].

Findings in 21 patients (group A) having diffuse C4d

staining without evidence of rejection on a protocol

biopsy taken 1 or 3 months post-transplantation were

compared with those in 12 patients (group B) whose

protocol biopsies showed negative or weak and focal

C4d staining, also in the absence of histologic findings

of AMR or cell-mediated rejection. None of the patients

had donor-specific anti-HLA antibodies. At most recent

follow-up (mean 2.3 years post-transplantation in group A

and 2.4 years in group B), mean serum creatinine levels in

the two groups were identical (1.3 mg/dl), and there have

been no graft losses due to AMR in either group [22��].

However, as shown in Fig. 3, the mean chronic sum score

(sum of Banff indices cgþ ciþ ctþ cv) on 12-month

protocol biopsies was significantly lower in group A than

in group B, and the mean increase in (cgþ ciþ ctþ cv)

from the initial (1 or 3 months) protocol biopsy to both 6

and 12-month protocol biopsies was also lower in group A.

Oettl et al. [34�] likewise observed mild chronic changes

(ci and ct) in only one of six 12-month protocol biopsies of

ABO-incompatible grafts, despite the presence of diffuse

C4d staining on prior protocol biopsies. Furthermore, we

found that none of 22 recipients of ABO-incompatible

grafts having one or more diffusely C4d-positive protocol

biopsies with no histologic evidence of AMR during the

first 6 months post-transplantation had transplant glomer-

ulopathy (cg score >0) at 12 months (Table 1); develop-

ment of transplant glomerulopathy is closely correlated

with donor-specific antibody and prior acute AMR

(clinical or subclinical), and is felt in most cases to

represent chronic AMR [13,16,17,18��]. Among 25 reci-

pients of ABO-incompatible renal allografts having one or

more diffusely C4d-positive protocol biopsies during the

first 6 months post-transplantation, only two had trans-

plant glomerulopathy at 1 year, and both of these patients

had histologic findings of acute AMR on at least one of

their protocol biopsies (Table 1). Consistent with this,

Toki et al. [35��] found transplant glomerulopathy at

1 year in 12/19 ABO-incompatible grafts with prior clinical

AMR, but in only 1/38 such grafts without prior AMR,

despite the fact that most of the latter grafts had PTC C4d

staining on one or more protocol biopsies. Gloor et al. [36]

found transplant glomerulopathy in 3/24 (13%) ABO-

incompatible grafts at 1 year. Whereas this percentage

was higher than that in conventional renal allografts at the

same center (8%), it is notable that 11 (46%) of the 24 ABO-

incompatible grafts had acute AMR (clinical or subclinical)

during the first year post-transplantation, compared with

only 2% of the conventional grafts [36].

Taken together, these findings provide strong evidence,

for the specific case of ABO-incompatible renal allografts,

that C4d deposition within PTC in the absence of histo-

logic findings of AMR or cell-mediated rejection is not

injurious to the graft, at least during the first year post-

transplantation. Whether this situation remains true long

term will clearly require further studies, and only such

Significance of C4d staining with minimal histologic abnormalities Haas 25

longer-term studies will enable us to assess whether

ABO-incompatible grafts showing C4d in PTC without

margination of leukocytes within these capillaries or

other histologic evidence of rejection represents a state

of stable graft accommodation. It is notable, however,

that the great majority of our ABO-incompatible grafts

with these findings showed persistent, diffuse PTC C4d

on each protocol biopsy taken during the first year post-

transplantation together with persistent low levels of

circulating anti-blood group antibodies [22��]. This find-

ing clearly contrasts with findings of Fidler et al. [33] in

ABO-incompatible grafts developing acute AMR; as noted

above the majority of these grafts were C4d-negative

during the week prior to the diagnosis of AMR. Likewise,

in conventional grafts, C4d associated with late AMR and

development of transplant glomerulopathy appears to

cycle on and off the graft, with persistent C4d positivity

on serial biopsies of the same graft being the exception

rather than rule [15].

Conventional and positive cross-match renalallograftsIn our experience, the overwhelming majority of C4d-

positive biopsies of ABO-compatible, HLA-mismatched

renal allografts, irrespective of whether there was a pre-

transplant positive cross-match requiring desensitization,

exhibit margination of leukocytes in PTC and/or other

histologic findings of AMR [20]. This situation is true for

both for-cause and protocol biopsies, although our experi-

ence with the latter has been limited to positive cross-

match and ABO-incompatible grafts (Figs. 1 and 2). We

feel that the combination of PTC C4d staining, leukocyte

margination in PTC and/or glomerular capillaries on a

protocol biopsy of a normally functioning graft, and the

presence of DSA represents subclinical AMR [37], and

indeed have found that for positive cross-match grafts this

triad is frequently associated with subsequent develop-

ment of chronic changes within the graft, including

transplant glomerulopathy (Table 1), just as has been

found for clinical AMR [14–17,18��].

In a study of 551 protocol biopsies of conventional renal

allografts from three European centers, Mengel et al. [23]

found PTC C4d staining (by immunohistochemistry on

paraffin sections, which is somewhat less sensitive than

immunofluorescence on frozen sections [38]) in 4.4%,

with diffuse staining in 2.0%. Notably, 86% of biopsies

showing diffuse C4d staining had PTC leukocyte mar-

gination, although the latter was seen in only 38% of

biopsies showing focal C4d staining. Thus, it appears

that, as with positive cross-match grafts, diffuse C4d

staining in protocol biopsies of conventional grafts

appears to reflect subclinical AMR in the great majority

of cases. However, in a small fraction of conventional

grafts there is C4d staining, usually focal, in the absence

of histologic features of AMR. Dickenmann et al. [24]

studied clinical outcomes in 22 patients with convention-

al renal allografts who had C4d-positive (by immuno-

fluoresence) biopsies lacking histologic features of AMR

or cell-mediated rejection; all biopsies were done for graft

dysfunction and in 21/22 the C4d staining was focal. Five

of the 22 patients received additional immunosuppres-

sive therapy following the biopsy, whereas the remaining

17 continued to receive just maintenance immunosup-

pression. Whereas mean serum creatinine (SCr) levels at

the time of biopsy were virtually identical in these two

patient groups, at 4 weeks post-biopsy the mean SCr was

significantly lower in the patients receiving additional

immunosuppression. Furthermore, graft survival at 3 years

post-biopsy was 100% in the patients receiving additional

immunosuppression compared with 69% in the remaining

patients, although this difference did not reach statistical

significance [24]. Nonetheless, these findings suggest that,

unlike the case in ABO-incompatible grafts, C4d staining

without histologic findings of rejection in conventional

renal allografts is associated with the potential for short-

term and longer-term graft injury, and should be treated

as such.

Therapeutic effects and implicationsPrevention of complement-mediated injury to the graft

can potentially be accomplished by two means: pharma-

cologic inhibition of the complement cascade or up-

regulation of endogenous complement regulatory

proteins, such as CD59 (protectin), which inhibits the

formation of the membrane attack complex (MAC), and

CD55 (decay-accelerating factor), which accelerates the

decay of C3 and C5 convertases. There is presently

evidence from both human renal allografts and rodent

xenografts that complement inhibition via these different

pathways has the potential to induce a state of C4d

positivity without clinical or histologic evidence of rejec-

tion, and possibly stable accommodation of these grafts.

In a preliminary study of 50 patients with positive cross-

match renal allografts from the Mayo Clinic, treatment

with the anti-C5 antibody eculizumab did not reduce the

fraction of C4d-positive biopsies (10/17 in eculizumab-

treated patients versus 18/45 in controls); however, none

of the 10 C4d-positive biopsies in the eculizumab group

showed histologic features of AMR, compared with 11

(61%) of the C4d-positive biopsies in controls [39�].

Furthermore, 22/45 biopsies in the control group (includ-

ing 15 of the 18 C4d-positive biopsies) but none of the

biopsies in the eculizumab group showed reactive

changes of the glomerular endothelium by electron

microscopy during the first year post-transplantation

[39�]; such ultrastructural changes may first become

apparent during the initial 1–3 months post-transplan-

tation and are associated with subsequent development

of transplant glomerulopathy [13]. In a rat-to-mouse

26 Mechanisms of rejection

model of heart xenograft accommodation [40�], the

latter process was found to be associated with strong

up-regulation of CD55 and CD59 on the graft endo-

thelium with prevention of endothelial C5 deposition

and histologic changes of rejection despite the binding of

antidonor IgG [40�].

ConclusionDiffuse PTC C4d deposition with circulating anti-blood

group antibodies in the absence of clinical or histologic

evidence of rejection is a common finding in ABO-

incompatible renal allografts. Furthermore, in these

grafts such C4d staining is not associated with an

increased risk of graft scarring, and may even indicate

a somewhat decreased risk of scarring, at least in the short

term. However, it will be important to determine if this

persists over the long term; if true such findings would

strongly suggest a state of stable graft accommodation.

Unlike the case in ABO-incompatible grafts, in positive

cross-match and conventional renal allografts C4d depo-

sition in the absence of histologic evidence of AMR

appears to be uncommon, and may indicate potentially

reversible graft injury. However, a state of C4d staining

without associated graft injury may be inducible in such

grafts by inhibition of the complement cascade at one or

more points subsequent to C4 cleavage but prior to C5

cleavage and formation of the MAC. Further develop-

ment of such approaches should increase our understand-

ing of humoral immunity in solid organ transplantation,

and may well improve long-term outcomes for transplant

recipients, especially those who are highly sensitized and

most at risk for AMR.

AcknowledgementsOriginal work of the author presented in this article was supported inpart by a grant from the Kidney & Urology Foundation of America, Inc.The author has no commercial associations or financial relationshipsthat might pose a conflict of interest in connection with any of thematerial presented in this article.

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27 Gloor JM, Lager DJ, Moore SB, et al. ABO-incompatible kidney transplantationusing both A2 and non-A2 living donors. Transplantation 2003; 75:971–977.

28 Tyden G, Donauer J, Wadstrom J, et al. Implementation of a protocol for ABO-incompatible kidney transplantation: a three center experience with 60 con-secutive transplantations. Transplantation 2007; 83:1153–1155.

29

�Montgomery RA, Locke JE, King KE, et al. ABO incompatible renal trans-plantation: a paradigm ready for broad implementation. Transplantation 2009;87:1246–1255.

This article summarizes the Johns Hopkins Hospital experience with ABO-incom-patible renal transplantation over a 9-year interval comprising two separate eraswith respect to desensitization and immunosuppression. The article presents keypathologic as well as clinical data.

30 Onitsuka S, Yamaguchi Y, Tanabe K, et al. Peritubular capillary deposition ofC4d complement fragment in ABO-incompatible renal transplantation withhumoral rejection. Clin Transplant 1999; 13 (Suppl 1):33–37.

31 Kato M, Morozumi K, Takeuchi O, et al.Complement fragment C4d depositionin peritubular capillaries in acute humoral rejection after ABO blood group-incompatible human kidney transplantation. Transplantation 2003; 75:663–665.

32 Kanetsuna Y, Yamaguchi Y, Horita S, et al. C4d and/or immunoglobulinsdeposition in peritubular capillaries in perioperative graft biopsies in ABO-incompatible renal transplantation. Clin Transplant 2004; 18 (Suppl 11):13–17.

33 Fidler ME, Gloor JM, Lager DJ, et al. Histologic findings of antibody-mediatedrejection in ABO blood-group-incompatible living-donor kidney transplanta-tion. Am J Transplant 2004; 4:101–107.

34

�Oettl T, Halter J, Bachmann A, et al. ABO blood group-incompatibleliving donor kidney transplantation: a prospective, single-centre analysisincluding serial protocol biopsies. Nephrol Dial Transplant 2009; 24:298–303.

This paper describes a prospective clinical and pathologic study of 10 recipients ofABO-incompatible renal allografts in Basel, Switzerland from 2005 to 2007.Patients underwent protocol biopsies at the time of transplantation and at 7 days,and 3, 6, 12, and 18 months post-transplantation.

35

��Toki D, Ishida H, Setoguchi K, et al. Acute antibody-mediated rejection in livingABO-incompatible kidney transplantation: long-term impact and risk factors.Am J Transplant 2009; 9:567–577.

This study analyzes clinical and serologic risk factors for development of acuteAMR in 57 recipients of ABO-incompatible renal allografts, and examines theimpact of AMR on long-term survival of these grafts.

36 Gloor JM, Cosio FG, Rea DJ, et al. Histologic findings one year after positivecrossmatch or ABO blood group incompatible living donor kidney transplan-tation. Am J Transplant 2006; 6:1841–1847.

37 Haas M, Montgomery RA, Segev DL, et al. Subclinical acute antibody-mediated rejection in positive crossmatch renal allografts. Am J Transplant2007; 7:576–585.

38 Seemayer CA, Gaspert A, Nickeleit V, et al. C4d staining of renal allograftbiopsies: a comparative analysis of different staining techniques. Nephrol DialTransplant 2007; 22:568–576.

39

�Cornell LD, Burns JM, Stegall MD. Prevention of endothelial activation with C5inhibition in positive-crossmatch kidney transplants. Am J Transplant 2009; 9(Suppl 2):304.

This is an abstract of a study from the Mayo Clinic presented at the 2009 AmericanTransplant Congress meeting. Biopsies from 4 recipients of positive cross-matchrenal allografts receiving the C5 inhibitor eculizumab were compared with biopsiesfrom 46 recipients of positive cross-match grafts not receiving this drug; electronmicroscopy was performed on all biopsies to assess endothelial injury and earlychanges of transplant glomerulopathy.

40

�Ding JW, Zhou T, Ma L, et al. Expression of complement regulatory proteins inaccommodated xenografts induced by anti-a-Gal IgG1 in a rat-to-mousemodel. Am J Transplant 2008; 8:32–40.

This study examines the expression of the complement regulatory proteins CD55,CD59, and Crry in the endothelium of accommodated versus rejecting heart graftsin a xenograft model in which baby Lewis rat hearts are transplanted into Rag/GT-deficient mice, and accommodation is induced by repeated injections of low-doseanti-a-Gal IgG1.