Original article The influence of vascular diathesis on the localization of inflammatory foci in renal allografts with a specific antigranulocyte antibody Rainer W. Lipp 1, Gerhard H. Wirnsberger 2, Manfred Ratschek 3, Vinzenz Stepan 1, Herwig Holzer 2, Georg Leb 1 1 Division of Nuclear Medicine, Department of Internal Medicine, KarI-Franzens-University, Auenbruggerplatz 15, A-8036 Graz, Austria 2 Division of Nephrology, Department of Internal Medicine, KarI-Franzens-University, A-8036 Graz, Austria 3 Institute of Pathology, KarI-Franzens-University, A-8036 Graz, Austria Received 7 August and in revised form 18 November 1995
Abstract. Immunoscintigraphy with technetium-99m labelled BW 250/183, a murine monoclonal antibody specific for granulocytes, yielded a false-positive result in a patient suspected of having an abscess in his renal graft. To substantiate the presumption that diathesis and unspecific accumulation of the antibody may have caused this result, ten selected patients were investigated who presented with chronic vascular graft rejection but without signs of bacterial infection. Scintiscans were recorded 4 and 24h after administration of 99mTc-labelled BW 250/183. Graft-background ratios (GBRs) were calculated for each transplant. These were compared with the mean of physiological kidney-background ratios (KBRs) and with bone marrow-background ratios (BMBRs). After removal, the grafts were examined with pathological and immunohistological methods. Seven transplants demonstrated 4-h GBRs (mean: 3.9_+1.1, P <0.001) significantly outside the range of normal KBRs while three were within the normal range (mean: 1.8_+0.4). The relation between 4-h and 24-h GBRs varied. After 24 h five GBRs still remained increased (mean: 3.2_+1.4, P <0.05). By contrast the BMBRs decreased uniformly by 18%_+5%. After graft removal, histopathology demonstrated no dominant granulocyte accumulations but various degrees of chronic vascular and tubulo-interstitial rejection. Immunohistochemical studies did not indicate cross-reactivity of BW 250/183. Increased GBRs of longstanding renal allografts indicate the passage of the antibody through injured vascular walls rather than the presence of granulocyte accumulations. Therefore, variability of GBRs with time reflects changes in transitory concentrations of 99mTc-labelled BW 250/183 in the tissues. Key words: Immunoscintigraphy - Renal allograft - An-
tigranulocyte antibody Eur J Nucl Med (1996) 23:395-400
Introduction
The murine IgG1 monoclonal antibody (MAB) BW 250/183, labelled with technetium-99m, has been reported to detect specifically accumulations of granulocytes [1, 2] as well as displacements of these cells, e.g. by metastases in bone marrow [3]. This MAB is also recommended for whole-body scans in the search for pyogenic foci of unknown localization [4]. Most of the injected BW 250/183 is bound to the epitopes in bone marrow and spleen and to circulating granulocytes but a fraction of approximately 19% is known to circulate as free immunoglobulin [5, 6]. This raises the question of whether the unbound BW 250/183 may accumulate without specific binding in areas of increased vascular permeability even in the absence of granulocyte accumulations, i.e. in a way similar to human unspecific polyclonal immunoglobulin G, thereby mimicking the presence of pyogenic foci [7]. A patient who presented with renal dysfunction and nondescript inflammatory symptoms led us to undertake the study. Immunoscintigraphy with 99mTc-labelled BW 250/183 was positive, apparently indicating the patient's long-standing renal allograft to be the site of infection. However, while histopathology after graft extirpation showed a few interstitial granulocytes and alterations in accordance with chronic vascular rejection, there was no pyogenic infiltration. Since relevant references are missing, this misleading result called for further investigation to substantiate the hypothesis that the specific antibody may also accumulate non-specifically after passage through altered vascular walls. Therefore, additional patients were investigated by scintigraphy immediately prior to transplant removal. The results of scintigraphy were subsequently compared with those of pathological and immunohistological examinations to determine the prevalence of unspecific MAB accumulations in the absence of pyogenic foci.
Correspondence to: R.W. Lipp
European Journal of Nuclear Medicine Vol. 23, No. 4, April 1996- © Springer-Verlag 1996
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Materials and methods Patients. The initial patient was a 43-year-old male who had had a renal allograft for 13 years and for 11 months had required chronic haemodialysis for about 4 h, 3 times a week. Subsequently, ten afebrile patients (mean age: 40_+14 years) without clinical signs of bacterial infection but presenting transplanted kidneys (mean graft survival: 43_+27 months) affected by chronic tubular and glomerular impairment were included in the study. At the time of investigation, immunosuppressive therapy was administered on a constant schedule based on cyclosporine (dose 2-3 mg/kg per day) along with an adjunctive steroid therapy (methylprednisolone 2.5-5 rag/day). In all cases intermittent chronic haemodialysis thrice weekly was necessary (mean duration: 4_+0.5 h).
bedded material. Specific immunoreactivity was demonstrated using the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique with Fast Red TR salt (Fluka, Germany) as chromogen [9]. In brief, BW 250/183 was applied to 5 to 6-gin-thick sections for 60 rain. Sections were then washed in PBS and incubated with a secondary antibody to mouse IgG (1:30) in human plasma/PBS (Dakopatts, Denmark) for 30 min. Following a PBS wash, the sections were incubated with the mouse APAAP complex (1:80) in PBS for 30 min. After a tap-water rinse, the sections were counterstained with haematoxylin and coverslipped with water-based Gel/Mount (Biomeda, USA). Concomitant negative control studies used irrelevant antibodies instead of primary antisera and omitted essential steps of the procedure.
Statistical analysis. All data are expressed as mean+SD. StatistiImmunoscintigraphy. The patients received 0.5-1 mg BW 250/183 (Behringwerke, Germany) labelled with 400-800 MBq 99mTc according to the method of Steinstr~iger [8]. Planar images (anterior and posterior views, 106 cts, matrix: 256x256) were recorded 4 h and, without intervening haemodialysis, 24 h after the intravenous injection of BW 250/183 using a single-head Elscint gamma camera fitted with a low-energy all-purpose collimator system. Singlephoton emission tomographic (SPET) images (recording: 180°; matrix: 64x64) were recorded after 4 or 24 h in cases of unclear planar scintigraphy (n=3). The results were evaluated by comparing individual graft-background ratios (GBRs) with the mean of ten kidney-background ratios (KBRs). KBRs were obtained from patients presenting with normal renal function (creatinine less than 1.2 rag/100 ml) and without a history of renal infections, renal stones or pyelogenic cysts. These patients were subjected to immunoscintigraphy for the detection of extrarenal abscesses. The KBRs were multiplied by a factor of 1.35 [calculated using the formula (a/b) 2] in order to compensate for the difference in distance between recording collimator and kidney in situ (mean distance: 50 cm) or transplant (mean distance: 43 cm), respectively. Both GBRs and KBRs were computed as follows: the gamma count within a circular region of interest (ROI) inside the graft or kidney and encompassing the respective centre of mass was divided by the lowest gamma count within a neighbouring background area of equal size. Bone marrow-background ratios (BMBRs) served as a standard for the stability of granulocyte-BW 250/183 binding and were computed as follows: the gamma count within a circular ROI inside the bone marrow of lumbar vertebra 3 or 4 was divided by the lowest gamma count within a neighbouring background area of equal size.
cal analysis of the data was performed using the Student's unpaired t test. A P value of <0.05 was considered as significant in all analyses.
Results The initial patient (see Materials and methods for details) was referred for i m m u n o s c i n t i g r a p h y for abscess detection because he presented elevated parameters as follows: leucocytes 12.2x109/1, C-reactive protein 129 mg/1, f i b r i n o g e n 8130 mg/1 and temperature u n d u l a t i n g b e t w e e n 36.8 ° and 39.4 ° C. The scintiscan showed the renal allograft only faintly after 4 h, but distinctly outlined it after 24 h (Fig. 1). This result was interpreted as indicative of the presence of a p u r u l e n t lesion, s e e m i n g l y
Pathological examination. Patients underwent transplant removal within 1 week after immunoscintigraphy. Representative specimens of well-defined areas of the upper/lower pole, of the midportion and of the renal hilum were routinely fixed with formaldehyde and embedded in paraffin. Serial sections were stained with haematoxylin-eosin and periodic acid-Schiff. For grading, different lesions were semiquantitatively defined as predominant (+++), focal (++) or scattered (+).
Immunohistochemistry. First, different dilutions of BW 250/183 (l:l 0-1:500 in 1% bovine serum albumin/PBS) and different fixation protocols including aldehydes, alcohols and heavy metal fixatives were tested to establish a proper procedure. Proteolytic predigestion was not necessary for antigen detection. For testing purposes a variety of fresh tissue samples with granulocytic infiltrates including stomach, breast, tonsil, spleen and lung were obtained from surgery and immediately prepared. A dilution of 1:50 of BW 250/183 proved to be optimal for routinely fixed and paraffin-em-
Fig. 1. Twenty-four-hour planar scintiscan of the initial patient. The renal allograft (arrow) is distinctly outlined below the liver and above the right iliac fossa. The haemopoietic bone marrow is also imaged, but the kidneys are not. The spleen has been extirpated
European Journal of Nuclear Medicine Vol. 23, No. 4, April 1996
397
Fig. 2. Initial patient; immunohistochemical demonstration of BW 250/183 specificity. Only a few granulocytes (arrows) are visible in an area of diffuse tubular atrophy with dilatation of tubular lumina and interstitial oedema. Only the granulocytic cytoplasm is stained dark due to MAB binding. No cross-reactivity is visible. Cell nuclei are counterstained with haematoxylin. Original magnification x400 Table
1.
Clinical data
Patient no.
Sex
Age (yrs)
Graft survival (months)
Creat. clearance (ml/min/1.73 m 2 BSA)
Leucocytes (xl091)
CRP (mg/1)
FBG (mg/1)
1 2 3 4 5 6 7 8 9 10
f m m m f m f m f f
60 49 28 60 29 36 20 31 50 36
27 20 60 15 31 44 48 46 32 109
10 20 . 5 4 0 0 0 5 0
11 5.6 . 7 4 3.5 5.4 9.9 5.4 -
33 35
6860 3500
21 25 5 59 12 17 -
4810 4440 4420 2850 6080
.
.
-
Creat. clearance, Creatinine clearance (normal range: 85-160 ml/min/l.73 m 2 BSA); BSA, body surface area; CRR C-reactive protein (normal range: 0-5 rag/l); FBG, fibrinogen (normal range: 1700-4000 mg/1); f, female; m, male; -, not recorded e x p l a i n i n g the clinical state o f the patient. However, the p a t h o l o g i c a l e x a m i n a t i o n after graft r e m o v a l p r o v e d differently: it s h o w e d no p y o g e n i c infection but solely features o f chronic v a s c u l a r rejection such as signs o f necrosis and b l e e d i n g as well as interstitial o e d e m a . This result was c o n f i r m e d b y m i c r o s c o p y , w h i c h d e m o n s t r a t ed in tissue sections o n l y scattered g r a n u l o c y t e s too few in n u m b e r to e x p l a i n the distinct scintigraphic v i s u a l i z a tion o f the renal transplant. T h e M A B r e a c t e d specifically o n l y with g r a n u l o c y t e s , as shown b y Fig. 2, d e m o n strating the specificity o f B W 250/183. T h e r e was no ind i c a t i o n o f c r o s s - i m m u n o r e a c t i v i t y o f the M A B with other tissue antigens. F o r clarification, ten additional patients w e r e investigated. Table 1 s u m m a r i z e s their clinical data, and Tables 2 and 3 show the scintigraphic and p a t h o l o g i c a l results. A l t h o u g h the f i b r i n o g e n values o f s o m e patients w e r e increased, the values for C - r e a c t i v e p r o t e i n and the h u m -
European Journal of Nuclear Medicine Vol. 23, No. 4, April 1996
Table 2. Scintigraphic results Patient no. 1 2 3 4 5 6 7 8 9 10
MAB (mg)
0.5
1 1 1 1 1 0.5 0.5 1 1
BMBR
GBR 4h
24 h
4h
24h
5.0"
2.1"* 5.4**
3.1 2.2 3.0 2.9 3.7 3.4 3.0
2.3 1.9 2.6 2.5 2.9 2.9 2.5
-
2.7
-
1.4
3.3
2.8
1
3.7
2.7
4.8* 4.8* 4.3* 2.9* 2.7* 2.5* 1.9 1.5 1
1.9
3.7** 2.2** 1.6
2.6**
Mean 4-h KBR=1.8+0.4; mean 24-h KBR=I.7+0.4 -, Not recorded * P <0.001, ** P <0.05
398 Table 3. Histomorphological data
Patient no.
Glomerulohyalinosis
Tubular atrophy
Tubular inflammation
Necrosis
Interstitial infiltrates
Haemorrhage
Vascular lesions Arteries
Arterioles ++
1
++
++
+
+
++
+
++
2 3 4
+++ ++ +
++ +/++ ++
(+) + -
+ (+) +
++
++
+++
+
+
+
++
+++
+++a
+
+
++
5 6
+++ +++
++ ++/+++
+ (+)
(+) ++b
+
(+)
++
+++
+/++
+
+++
+/++
7
++
+++
+
++
+
++
++
++
8
++
++
(+)
+
++
+
+++
++
9
+++ +++
++ +++
-
++
+
++
+++
++
-
+
+
+
+++
++
10
+++, Predominant diffuse; ++, marked focal; +, scattered through the tissue; (+), minimal histological changes found in some specimens
a Focal polyclonal lymphocytic infiltrations, which lead to the diagnosis of a reactive lymphoprotiferative disorder b Focal calcifications
Fig. 3. Four-hour planar scintiscan of patient 1. The graft (GBR:
Fig. 4. Twenty-four-hour coronal SPET images of patient 10. Se-
5) is distinctly outlined
quence of four images through the pelvic area at distances of 1.8, 1.8 and 2.4 cm, respectively. The binding of BW 250/183 to granulocytic cells demonstrates the bone marrow within the spine, the pelvic bones and, on images 2 and 3, also the femora. The renal transplant is not visible due to a GBR of 1
b e r o f l e u c o c y t e s d i d not p o i n t to b a c t e r i a l infectious disease. Nevertheless, seven patients d e m o n s t r a t e d 4-h G B R s distinctly e x c e e d i n g the n o r m a l r a n g e o f K B R s (P <0.001). The scintiscans o f o n l y three patients were negative, with G B R s w e l l inside the n o r m a l range. Twentyfour-hour G B R s were o b t a i n e d f r o m nine patients. T h e r e was no constant relation b e t w e e n 4-h and 24-h G B R s , the latter b e i n g s i m i l a r in six and distinctly l o w e r in three cases. Hence, after 24 h four G B R s fell within the n o r m a l range o f K B R s , but five transplants still r e v e a l e d f a l s e - p o s i t i v e results (P <0:05). The transplants o f patients 1, 3 and 6 d e m o n s t r a t e d distinct w a s h o u t effects o f l a b e l l e d M A B , their G B R s d e c r e a s i n g b y 58%, 60% and
41%, respectively. In contrast, the BMBRs revealed a rather constant decrease of 18%+_5% between 4- and 24h recordings. Comparison of Figs. 3 and 4 demonstrates the obvious difference in BW 250/183 uptake by renal transplants during chronic vascular rejection. The kidney allograft is easily visible in Fig. 3 due to its high GBR. However, the transplant in Fig. 4 is indiscernible, even on a sequence of SPET images.
European Journal of Nuclear Medicine Vol. 23, No. 4, April 1996
399 Gross pathological examination of transplants after removal showed the texture of the shrunken organs to be tough and fibrous. Small areas of infarction with haemorrhagic borders were scattered throughout the cortex in most cases, and in some cases extended to the medulla. In these grafts careful investigation often documented arterial thrombosis within the hilum. Microscopic examination never revealed significant granulocytic infiltrations. However, the tissue samples showed various degrees of vascular alteration, glomerular hyalinosis, tubular atrophy, interstitial fibrosis and in some cases also lymphocytic infiltration, in accordance with chronic vascular rejection processes (Table 3). The quantitative differences in the pathological appearance of the specimens did not yield conclusive evidence explaining the physiological MAB uptake by patients 8-10.
Discussion The labelling of isolated granulocytes with indium-111 oxine, first described by Thakur et al., is still regarded as the gold standard for detecting infectious disease [10]. However, the method is cumbersome and necessitates in vitro handling of blood. In addition, virally infected blood may be a danger to patients and technicians [11]. Alternatively, lllIn-labelled platelet scintigraphy has been used to determine the origin of febrile syndrome in patients with non-functioning renal grafts [12]. However, this method suffers from drawbacks similar to those of in vitro leucocyte labelling [13]. The development of 99mTclabelled antibodies that bind specifically to neutrophils, either directly in the bone marrow or after their release into the circulation, has simplified leucocyte scintigraphy. The method is quite straightforward and can be performed readily in any nuclear medicine laboratory. BW 250/183 is an intact murine monoclonal antibody that reacts with an ep~tope of NCA 95 (non-specific cross-reacting antigen) on the surface of granulocytes [14]. This binding to the membrane, though very strong, does not cause antibody- or complement-dependent lysis of the cells [15]. The high stability of 99mTc-labelled MAB binding and the high affinity of BW 250/183 for the epitope are thought to prevent the diffusion of free activity into the tissue [5, 8]. According to the literature, most of the injected MAB is bound to the epitopes in bone marrow and spleen but 10%-20% of radiolabelled injected BW 250/183 is carried by circulating granulocytes and approximately 19% circulates as free immunoglobulin [5, 6]. The MAB-labelled circulating granulocytes remain viable and functionally normal and may still target infectious areas due to their undisturbed chemotactic behaviour. A secondary labelling of still-unlabelled granulocytes may occur in these foci because the increased permeability of capillaries at inflammatory sites facilitates the passage of freely circulating 99mTc-labelled MAB. These characteristics may explain the result demonstrated by the initial patient, which became distinctly positive only after 24 h.
European Journal of Nuclear Medicine Vol. 23, No. 4, April 1996
Histopathology proved the scintigraphic result in the initial patient to be misleading. Therefore, immunocompromised patients presenting transplants with chronic vascular rejection processes but without signs of bacterial infection were investigated in order to determine the prevalence of unspecific accumulations of 99mTc-labelled BW 250/183. The GBRs of the ten transplants varied considerably. In the 4-h recordings, seven (70%) GBRs were significantly higher than the mean of the KBRs, which served as the standard for comparison (P <0.001). In the 24-h recordings five GBRs (56%) were still elevated (P <0.05). Within this period of 20 h, three transplants showed a striking decline of up to 60%, two demonstrated a modest decline of up to 24%, and two transplants demonstrated a slight increase of up to 13%. During the same period the mean of the KBRs remained nearly stable and thus appeared suitable as a standard for comparison. The variability of the GBRs contradicts specific binding of BW 250/183 to epitopes, pointing rather to freely mobile extravascular accumulations. In contrast, the mean of the BMBRs decreased quite uniformly by 18%_+5% over 20 h. It has been demonstrated that the binding of BW 250/183 to granulocyte epitopes is stable at least for the same period [8]. Thus, the decrease seems to reflect the emigration of matured granulocytes into the circulation during the period of 20 h [6]. Plasma extravasation might occur through vascular endothelia damaged by chronic vascular rejection, this extravasation being enhanced by cyclosporine therapy [16]. Such a hypothesis is supported by the fact that cyclosporine may cause a significant albumin loss due to capillary leakage, as shown in an animal model: radiolabelled albumin disappeared significantly faster from the circulation of rats injected with cyclosporine than in control animals [17]. Pathological examination failed to demonstrate extensive granulocytic infiltrations of the transplants. Therefore, the variability of GBRs again points to the presence of fluctuating concentrations of unbound 99mTc-labelled MAB within interstitial fluid. The immunohistological examinations supported this view strongly. They demonstrated only the specific binding of BW 250/183 to granulocytes without any indication of cross-immunoreactivity with other tissue elements, even though in vitro experiments cannot disprove this possibility completely. All facts considered, the increased GBRs of renal transplants compromised by chronic vascular rejection and immunosuppressive therapy seem to result from transitory accumulations of BW 250/183 in the interstitial tissue due to pathological changes in the permeability of local blood vessels. The extent and duration of the GBR increase reflect dynamic differences in the passage of protein through altered vascular walls rather than the number of granulocytes accumulated in the tissue. In this respect BW 250/183 distribution seems to resemble the imaging characteristics of human polyclonal immunoglobulin G [18]. In conclusion, this study has shown that BW 250/183
400 i m m u n o s c i n t i g r a p h y o f renal transplants with chronic vascular rejection m a y y i e l d a m b i g u o u s results due to variations in the p a s s a g e o f u n b o u n d M A B t h r o u g h d a m aged v a s c u l a r walls. Consequently, distinct stable increases in lesion to b a c k g r o u n d ratios during a p e r i o d o f 24 h r e l i a b l y reflect the p r e s e n c e o f g r a n u l o c y t i c accum u l a t i o n s only in the a b s e n c e o f c o m p a r a b l e disturbances o f h o m e o s t a s i s .
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8. Steinstr~iBer A, Berberich R, Kuhlmann L, Zabori S, Schwarz A. Binding of the monoclonal antibody BW 250/183 to human granulocytes. Nucl Med 1992; 31 : 57-63. 9. Wirnsberger GH, Becker H, Ziervogel K, H6fler H. Diagnostic immunohistochemistry of neuroblastic tumors. Am J Surg Pathol 1992; 16: 49-57. 10. Thakur ML, Segal AW, Louis L, Welch MJ, Hopkins J, Peters TJ. Indium-l 1l-labeled cellular blood components: mechanism of labeling and intracellular location in human neutrophils. JNucl Med 1977; 18: 1022-1025. 11. Rojas-Burke J. Health officials reactions to infection mishaps. JNucl Med 1992; 33: 13N-27N. 12. Torregrosa JV, Bassa R Lomena FJ, et al. The usefulness of ill-In-labeled platelet scintigraphy in the diagnosis of patients with febrile syndrome and a nonfunctioning renal graft. Transplantation 1994; 57: 1732-1735. 13. Khanuja PS, Taylor D, Toledo-Prereyra LH, Mittal VK. Diagnostic value of indium-111 leucocyte scans for localizing infective foci in renal or combined renal and pancreatic transplant patients. Dialysis Transplant 1993; 22: 90-100. 14. Bosslet K, Lfiben G, Schwarz A, et al. Immunohistochemical localization and molecular characteristics of the three monoclonal antibody-defined epitopes detectable on the carcinoembryonic antigen (CEA). Int J Cancer 1985; 36: 75-84. 15. Bosslet K, Schorlemmer HU, Steinstraeger A, Schwarz A, Sedlacek HH. Molecular and functional properties of the granulocyte specific Mab 250/183 suited for the immunoscintigraphic localization of inflammatory processes. In: H6fer R, ed. Radioactive isotopes in clinical medicine and research. New York: Schattauer; 1988: 15-20. 16. Mason J. Renal side-effects of cyclosporine. Transplant Proc 1990; 22: 1280-1283. 17. Moore LC, Mason J, Feld L, van Liew JB, Kaskel FJ. Effect of cyclosporine on endothelial albumin leakage in rats. J Am Soc Nephrol 1992; 3: 51-57. 18. Corstens FHM, Claessens RAMJ. Imaging inflammation with human polyclonal immunoglobulin: not looked for but discovered. Eur J Nucl Med 1992; 19: 155-158.
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