Antibody accumulation in small tissue samples: Assessment by quantitative autoradiography

Nucl. Med. Biol. Vol. 17, No. 6, pp. Inr. J. Radiar. Appl. Inswum. Parr B

585-596, 1990

0883-2897/9053.00+ 0.00 Copyright 0 1990Pergamon Press plc

Printed in Great Britain All rights reserved

Antibody Accumulation in Small Tissue Samples: Assessment by Quantitative Autoradiography UDO SCHMID,’ HEINER

BIHL and SIEGFRIED

MATZKU’*

‘Institute of Radiology and Pathophysiology, German Cancer Research Center, P.B. 101949, D-6900 Heidelberg, F.R.G. and 2Division of Nuclear Medicine, Kopfklinik, University of Heidelberg, Im Neuenheimer Feld 400, D-6900 Heidelberg, F.R.G. (Received 21 February 1990) Uptake of radiolabeled ‘25I monoclonal antibodies in small metastases can only be characterized by autoradiographic techniques. To obtain quantitative data out of autoradiographic images, a transformation of the essentially two-dimensional signal into the Bq per unit volume information is needed. Part of the calibration problem could be solved by using tissue-equivalent standard preparations. However, when aiming at a quantification of radioactivity in small areas (d 2 mm diameter), special criteria had to be expanded upon for the reconstruction of the area in the dose matrix and for the correct integration of the radioactivity content.

Introduction Macroautoradiography producing image of radioactivity distribution and in experimental animals

an x-ray film in tissue samples

is superior to scintigraphy and to y counting of dissected tissues in at least two respects: the method offers high spatial resolution down to clusters of cells and at the same time an overview of the pertinent tissues. Moreover, autoradiography allows the analysis of radioactivity levels in cases where the actual site of accumulation is not known beforehand, the most notable example being spontaneous (micro-)metastases. However, the method is not a priori suited for quantification because the signal, i.e. the density of the x-ray film, is essentially two-dimensional and because it relates in a complex manner to tissular radioactivity concentration. The choice of the nuclide is determined by the fact that high spatial resolution is needed when aiming at an analysis of material transport and retention in tumors, since this will focus on tissue boundaries, endothelial cell layers and tumour cell layers in the vicinity of these structures, as well as on minimal residual disease processes and micrometastases. Other premises are high specific activity, and, out of practical considerations, a reasonably long half life. All of them can be met by “‘1 as the tracer radionuclide. However it has to be accepted that “‘1 emits not only short range electrons *All correspondence should be addressed to: Dr S. Matzku, P.B. 101949, D-6900 Heidelberg, F.R.G.

but also photons with an inherently longer range (Table 1). The techniques of the whole body (macro)autoradiography (Ullberg, 1954) and quantitative autoradiography (Landau et al., 1955) have been developed independently in the early Fifties. Progress achieved since this time has been traced in several reviews (Rogers, 1979; Blasberg et al., 198 1; Larsson and Ullberg, 1981; Yonekura et al., 1983; Som et al., 1983; Kuhar and Unnerstall, 1985; Davenport et al., 1989). With respect to the present investigation, the introduction of tissue paste standards (Reivich et al., 1969) and subsequent refinements of the method (Blasberg et al., 1981; Geary et al., 1985; Kuhar and Unnerstall, 1985; Baskin er al., 1986; Clark and Hall, 1986; Davenport and Hall, 1988; Davenport et al., 1989) deserve considerable credit, since this important detail helped us to overcome problems associated with intra-sectional absorption of low energy radiation. Previous work dealing with the assessment of radioactivity distribution after application of 12’1labeled antibodies was restricted to digital densitometry and to bona fide transformation of density into radioactivity per pixel (Fand et al., 1986; Nelp et al., 1987; Matzku et al., 1989). Griffith et al. (1988) have used similar methods for quantitatively evaluating the intra-tumoral distribution of i3’I-labeled MoAbs. Yet, problems associated with differential absorption of izsI radiation and with quantification of radioactivity in small objects have not been brought to a generally applicable solution until now. 585

Uno Scnhfm et al.

586

Table 1. ADDroximate disintegration of “‘1 [according to Rogers, 1979) Intensity* 7 19 11 3

3.5 3 30 34

139 106-139 O-33 24

27.2-27.5 31.0-31.7 3.1-4.6

22 155

21.9-31.8 2.34.9

Unconverted gammas Internal conversion electrons

Fluorescence K fluorescence total K roentgen rays L roentgen rays total

Energyt

Auger electrons From K vacancies From L vacancies

Range of electronsf ca 0.4 ca 30 ca 35

ca 25 co 0.6

*Intensity in number of emissions per 100 disintegrating nuclei tEnergy or energy limits in keV. SRange of electrons in H,O in flrn (Harder, 1974).

Our efforts to establish QAR for the evaluation of MoAb accumulation in small spontaneous metastases were mainly concerned with control and correction procedures, the goal being to achieve concordance within a range of k 10% standard deviation of QAR readings and conventional biodistribution studies in dissected animals. The murine lymphoma ESB-Mp was chosen as an experimental system because this lymphoma variant yields a reproducibly high number of spontaneous liver metastases (Benke et al., 1988) and because a selectively binding MoAb 12-15A (Pfhiger et al., 1988) was available. The ultimate questions to be .answered by QAR in this system are how small metastases would compare to macroscopic tumor nodules (Matzku et al., 1988) with regard to the absolute amount and the intra-lesional distribution of accumulated radioactivity.

Materials and Methods Tumor cell lines The characteristics of mouse lymphoma variant Esb-Mp have been described previously (Benke et al., 1988; Matzku et al., 1989). In short: tissue cultured ESb-Mp cells (2 x lo5 per animal) were injected intradermally into the flank of DBA/2 mice. Three weeks later, the local tumor was excised. After another 10 days, animals had a varying number of liver metastases ranging from 0.14 mm diameter. Human B-lymphoma line OCI.Ly (kindly provided by B. Darken, Heidelberg) was inoculated subcutaneously into the flank of nude mice (10’ tissue cultured cells per animal), giving rise to nodules of ca 20 mm diameter after 3-5 weeks. Monoclonai antibodies MoAb 12-15A (rat IgGl; Pfliiger et al., 1988) binding to the murine ESb-Mp tumor was kindly donated by V. Schirrmacher (Heidelberg). MoAb HD37 (murine IgGl; Pezzuto et al., 1987; donated by B. Diirken and G. Moldenhauer, Heidelberg) binds to the OCI-Ly tumor. MoAbs were purified from

ascitic fluid by chromatography on Protein A and Mono Q columns (Pharmacia, Freiburg, F.R.G.) and labeled with lz51according to the Iodo-Gen method (Fraker and Speck, 1980). Immunoreactivity was checked in Lineweaver Burk experiments (Matzku et al., 1985). Quantitative autoradiography Mice received 1.1 MBq ‘2sI-labeled MoAb via the tail vein. 48-72 h later, animals were sacrificed by extensive ether anesthesia. Whole animals or dissected livers (one experiment) were frozen in isopentane/dry ice and were embedded in precooled methylcellulose (2.5% w/v). Blocks kept at -20°C were cut with a whole body cryotome (Jung, N&loch, F.R.G.) or with the successor cryotome (Cry0 Polycut, Reichert-Jung, N&loch, F.R.G.), sections with a nominal thickness of 20pm being collected on adhesive tape. After lyophilization adhesive parts of the mounted sections were dusted with talcum powder. Sections were then placed on x-ray film (Kodac X-OMAT AR), which was exposed at -20°C for 2-4 weeks in the absence of intensifier screens. Development was carried out in a Agfa-Gevaert Curix 2428 apparatus adjusted to a temperature of 26°C (2 min cycle, developer G138, fixer G334). The hardware configuration used for digital densitometry and transformation of density matrices into dose matrices was essentially similar to that described by Gochee et al. (1980). Film densities were determined by a scanning densitometer (Joyce Loebl, Scandig Colour Scanner Model 2605) with an aperture size of 50pm. Information was transferred to a MicroVAXII computer and further processed by the DENSTODOSE software establishing dose matrices out of density information (G. Sroka and G. Hartmann), which was feeding an optical evaluation set-up (LOCODI designed by R. Kubesch), both developed at our Institute. Calibration with respect to radioactivity per unit area Calibration was achieved by exposing step-wedge ‘*‘I standards together with the tissue sections. In

587

Quantification of autoradiography initial experiments, commercially available ‘25I standards composed of polymer stripes (20 pm thickness, The Radiochemical Centre, Amersham, U.K.) were compared to lyophilized tissue-equivalent standards. The latter were prepared as described by Clark and Hall (1986), i.e. graded doses of ‘Z51-labeled protein were admixed to batches of brain paste or liver paste, which were homogenized and centrifuged (20,000 rpm, IO min). Supernatant liquid was discarded, the remaining paste was vortexed and again centrifuged (2000 rpm, 5 min). Radioactive pastes contained in the centrifuge tubes were freeze-embedded in methylcellulose and lyophilized sections were prepared therefrom. In subsequent experiments, lyophilized tissue-equivalent standards of both liver and brain origin were used for calibration exclusively. Calibration curves were calculated from the area and the count rate (determined in the scintillation counter) of standard samples as well as the grey-level integrals. These were collected in a region of interest (ROI) placed in the center of standard spots, curve fitting being achieved by a 5th order polynome. Due to the contribution of intermediate range photons emitted by “‘1 (Table l), the x-ray film is blackened at sites with no underlying radioactivity. To determine radioactivity concentrations out of dose matrix data, the dose distribution outside the boundaries of small radioactive sources had to be considered (see below). Hence, the result of transforming a density matrix by virtue of the density-toradioactivity calibration curve will be called “dose matrix”, while the result of ROI integration of a given area within the dose matrix will be referred to as “radioactivity per unit area”. The precision of the calibration procedure was controlled by measuring the total radioactivity content of 160 segments (ca 7 x 7 mm size) produced from 60 sections of OCI.Lyl xenografts, having accumulated ‘2SI-labeled MoAb HD37, in a scintillation counter (Compugamma, LKB, Fteibutg). Thereafter, the total radioactivity content of individual segments was determined by QAR. The counting efficiency of the y counter as determined by measuring 0.1 mL aliquots of a reference “‘1 preparation (British Calibration Service, Amersham) was 0.796. Calibration with respect to unit tissue oolume or weight The correct assignment of the volume represented by a unit area element of QAR (i.e. a pixel of 50 x 50 pm) depends on the congruence between the nominal and the actual thickness of sections produced by the cryotome and on the reproducibility of section thickness. This was analyzed by taking a defined number of sections off a Teflon rod and measuring the thickness of serial slices as well as the total shortening of the rod with a digital micrometer. It was found that sections being produced with a 20 pm setting on the Jung cryotome had an actual

thickness of 22.3 &-2 pm, while actual and nominal thickness of slices obtained with the Cryo Polycut cryotome were in a range of f 5% (standard error). Factors attributable to volume changes as well as to photon absorption differences in lyophilized vs wet tissue were assessed altogether by comparing scintillation count rates of aliquot samples from wet range liver paste (radioactivity radioactive 22-190 Bq/mg) with QAR readings from lyophilized sections of these radioactive liver pastes. The resulting correction factor k was then included into the equation used for scaling the x-axis of calibration curves: X=

cps r*A*T*s*k

where [X] = Bq/mg tissue; cps, counts per second of standard; r, counting efficiency of scintillation counter, i.e. 0.796; A, area of standard section; T, thickness of section; s. specific gravity of tissue; k. correction factor. To control the validity of the equation, normal mice were injected with ‘251-labeled liposomes which are known to accumulate uniformly in the RES, especially in the liver. Labeled liposomes were kindly provided by H. Sinn and H. Krempel (Heidelberg). Two hours after i.v. application of l-4 MBq into 6 mice, livers were excised and 4 small biopsies (SO-200 mg) were taken from different parts of each liver and counted in the scintillation counter. The remaining liver tissue was frozen and subjected to QAR evaluation. RadioactitGty determination in small objects The contribution of low energy photons to the autoradiographic image of a “‘1 source results in halo formation. This was studied systematically by analysis of gtey-level and dose matrices of small circular phantoms, which were produced by filling Teflon tubing (i.d. 0.35-2 mm) with liver paste containing 250 Bq ‘251/mgwet weight. The tubing was embedded and then cut in a plane perpendicular to the long axis. The images of these sources were subjected to various procedures of analysis in order to (1) reconstruct the known geometry of the source and (2) determine the known radioactivity content of the source out of autoradiographic information. Results Problem of quenching Due to the low energy of particles emitted upon radioactivity decay of 12’1,penetration in water does not exceed 20-35pm (Harder, 1974; Table 1). It could be anticipated that signals obtained with a given amount of “‘1 per unit area would differ depending on the consistence of the matrix/tissue. In fact, commercially available “‘1 standards contained in polymer strips of 20 pm thickness yielded calibration curves that related to lyophilized tissue-equivalent

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standards in a non-linear manner (Fig. l(A)). On the other hand, liver-paste and brain-paste standards gave virtually identical calibration curves, This prompted us to use lyophilized tissue-paste standards irrespective of tissue type. The first experiment designed to verify the accuracy of calibration per unit area was performed with sections derived from OCI.Lyl lymphoma xenografts after injection of ‘2SI-labeled MoAb HD37. This model was chosen because tumor accumulation was found to be non-uniform (S. Matzku, unpublished finding), thus requiring integration over areas with great density variation. 160 tumor section segments were scintillation counted and subsequently subjected to the QAR procedure, the ROIS being placed outside of individual autoradiographic images. Excellent correlation of measurements was ob-

2

160

4 6 RadioactIvIty

8 (Balmm?

y-=’

if/.Pf----

10

12

1

tained throughout the range of radioactivity covered (Fig. 2(A)), although the experimental error was clearly greater than what had to be expected from mere counting statistics. Calibration with respect to volume and weight

Differences in the absorption of low energy photons in wet tissue vs lyophilized sections have to be considered when comparing scintillation counting to QAR. In addition, tissue paste standards may undergo artifactual changes upon freezing and sectioning, e.g. volume changes and formation of wrinkles, the latter resulting in an incorrect ROI averaging due to the nonlinear density-to-radioactivity relationship. These factors were jointly evaluated in the second control experiment (Fig. 2(B)), which consisted of a comparison of radioactivity readings obtained by scintillation counting of small aliquots of wet radioactive pastes, after centrifugation but prior to freezing, and by QAR determinations from lyophilized sections of these radioactive liver pastes. Multiple replicates with different radioactivity levels were tested. QAR readings were found to overestimate the radioactivity concentration by a factor of 1.07, which was then included as a correction factor into the equation used for scaling the x-axis of calibration curves (Fig. l(B)). The validity of this equation was tested in the third control experiment (Fig. 2(C)). When using an organ which is accumulating a radioactive substance fairly homogeneously, i.e. liver tissue of normal mice after application of graded doses of ‘2s1-labeled liposomes, there is good reason to assume that samples picked out at random from the liver and cryotome sections obtained from the same tissue do contain the same amount of radioactivity per unit volume. In Fig. 2(C) it is demonstrated that indeed both measurements coincided fairly well, the ratio being 1.02 i 0.1 (SD) over a range of 37-230 Bq/mg. QAR of small radioactive sources

When proceeding from large organs with uniform radioactivity uptake towards small tissue elements, a new dimension of complexity was encountered. As soon as the radioactive source was no longer much larger then the range of photons emitted, marked 400 600 discrepancies between the actual radioactivity distriR~dlo~ctlrlty f8almp tiawe) bution in the sample and density distribution on the Fig. 1. Calibration of x-ray film grey-levels with ‘251 x-ray film, and, hence, dose distribution in dose standard preparations. Three types of standard preparations matrices were noted. Halos became apparent around were exposed for 14 days. After scanning densitometry, the average grey-level per pixel was determined by ROI integrahot spots on the x-ray film as well as in grey-level tion within the autoradiographic images of individual matrices, especially at high radioactivity levels (see standard samples. The actual radioactivity content of Figs 3(A), (B); S(D)). In dose matrices halos were standard samples was either indicated by the manufacturer found to increase with increasing radioactivity conor determined in a scintillation counter. The area of centration of the source (compare Fig. 3(C) to Fig. standard samples was determined with a micrometer microscope. (A) Calibration per unit area. Comparison of 3(D)). The effect has two practical consequences: (1) commercially available polymer standards to lyophilized the outline of a radioactive source could not be standards prepared from mouse brain paste and mouse liver directly deduced from its image in the dose matrix; (2) paste. (B) Calibration per unit weight of tissue (paste). The the signal level over a radioactive source depended on x-axis was scaled according to the equation specified in “Material and Methods”. the size of the source because of a “build-up effect”,

Quantification of autoradiography

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Fig. 2. Validation of QAR measurements by comparison to scintillation counter measurements. (A) Measurement of individual tumor sections by the two methods. QCLLy tumors showing a non-uniform accumulation of ‘Wabeled MoAb were sectioned and lyophilized sections were cut in segments of co 7 x 7 mm size. These were measured in the counter and thereafter subjected to QAR in order to determine the total radioactivity content. The correlation achieved and the ratios of count rates are shown for every single sample. The mean ratio (i.e. 0.97) and the standard deviation (i.e. kO.06) are indicated as full and dotted lines, respectively. (B) Comparative measurement of lyophilized tissue paste sections and wet tissue paste samples. Prior to embedding, freezing and sectioning of tissue pastes (4 radioactivity concentrations in the range of 22-190 Bq/mg), 5 aliquots were withdrawn and measured in the scintillation counter to get the Bq/mg wet weight information. Mean scintillation counter readings were compared to QAR readings (Bq/mg) of multiple sections. Ratios were independent of radioactivity concentrations of individual paste preparations with an overall mean value of k = 1.07 (+O.l I). Factor k was included as a corrective term in the equation used for scaling the x-axis of calibration curves. (C) Validity of QAR calibration as assessed by comparative measurement of liver samples. After injection of ‘2sI-liposomes in normal mice and removal of livers, small samples (4 replicates, ca SO-200 mg) were excised and the remainder was sectioned. The graph shows the ratio of QAR measurements (multiple replicates per liver) and mean scintillation counter measurements of wet tissue aliquots as obtained from 6 livers with radioactivity levels in the range of 37-230 Bq/mg. The mean value of all ratios was 1.02 (*O. 10) as indicated in the graph. reached its maximum only in macroscopic sources such as the tissue-paste standards. Both phenoma are schematically illustrated in Fig. 4(A) showing the typical dose distribution derived from a small cylindrical source, i.e. a section through a plastic tubing of defined diameter filled with radioactive paste. With such sections it could be shown that the boundary of the radiation source corresponded to the steepest part of dose profiles placed through the center of the hot spot in the dose matrix, since the number of pixels between the steepest parts of profiles corresponded fairly well to the inner diameter of tubings. In 35 independent measurements (5 tubings, 7 sections each) the diameter could be determined in dose matrices with a maximal error of

which

+ 1 pixel. The procedure of calculating the cross-sectional area of a source was carried out as follows: two profiles were placed at 90” through the center of the spot, the highest steps in the profiles were identified and the mean bottom level of those was calculated. To this level, an increment of 2% relative dose was added in order to be sure that a thus defined isodose line (isodose level 2 in Fig. 4(A)) was placed around the inner rather than the outer pixels of the source. A ROI procedure then yielded the area contained within the isodose line. However, the radioactivity content of the source could not be determined by integration within the area defined by this isodose line because a considerable part of the dose is deposited in the halo region

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(see Fig. 4(A)). Phantom measurements designed to find out how to identify the exact radioactivity content of small radioactivity sources are shown in Fig. 4(B). Two tubes of different diameter which were filled with identical radioactive material (250 Bq/mg), and sections were exposed for different periods of time to produce increasing halos. When ROI integration was carried out within the putative area of the objects as defined by the above isodose line, cumulative radioactivity (open bars) was clearly underestimated. Bm when integration was performed in an area defined by a 1.5% isodose line (1% above averaged background; isodose level 1 in Fig. 4(A)), thus including all the film density/dose contribution by the halo region, fairly correct values were obtained irrespective of object size and film density level. Hence, radioactivity accumulation in small objects was evaluated by a two-step procedure applied to every single hot spot, which consisted of a determination of the putative contour and an integration of external and internal dose elements as outlined above. The process is illustrated and summarized in Fig. 5, which shows the sequence of events comprised in the QAR procedure: a section through an animal with a liver metastasis (Fig. 5(A); an exceptionally large metastasis was chosen for the sake of demonstration), the x-ray film image (Fig. S(B)), the digital density matrix (Fig. 5(C)), which by virtue of the calibration curve obtained from co-exposed lyophilized tissuepaste standards (Fig. 5(D)) was transformed into a dose matrix (Fig. 5(E)). The hot spot produced by radioactivity accumulation in the metastasis was then analyzed to identify the putative outline according to the steepest slope criteria (the corresponding isodose line is visible as a black track in Fig. 5(F)) and by integration of radioactivity within the 1.5% isodose line, which largely followed the outer boundary of the light blue zone in Fig. 5(F). It is important to realize that this procedure essentially measures radioactivity contained in hot spot areas, but not necessarily in the corresponding metastasis, since isodose lines set at level 2 would spare segments of low radioactivity accumulation. This becomes immediately evident by comparing Fig. 5(A) to Fig. 5(F). A small area in the upper part of the lesion showed low accumulation and was thus excluded by the steepest slope-defined

isodose line (Fig. 5(F)).

Discussion of

There are two major limitations to an analysis radioactivity distribution by straightforward

autoradiography and visual inspection of x-ray film images: (a) lack of quantitative information, and (b) lack of resolution of areas with high film density. Depending on the exposure time, areas of low radioactivity concentration may either vanish or become structured while areas of high radioactivity concentration may either be resolved into discrete structures or merge to form uniformly black spots. This sort of an experimental bias can be eliminated to some extent by densitometry, but especially by transformation of digital film density into radioactivity per unit area/ volume. The problems encountered when establishing a QAR procedure for the evaluation of antibody accumulation in rodent tissue were several-fold. (1) In autoradiography, the signal is produced mainly by the interaction of low energy particles with the film emulsion, while the reference method of scintillation counting of (macroscopic) tissue samples is based on y ray detection. (2) The objects producing the autoradiographic signal are essentially two-dimensional. When aiming at data relating to tissue volume or weight, volume changes occurring during the process of freezing and sectioning have to be considered. (3) The geometry of the object/tissue segment to which QAR is referring is not directly accessible but has to be reconstructed from the dose matrix. (4) The simultaneous contribution by radiation products of different range, i.e. electrons and photons, results in dependence of the autoradiographic image on the size of the radiating object. Problems (1) and (2) were found to be linked. They have been settled by the use of tissue-paste standard preparations, since absorption of low energy electrons within the section as well as changes in thickness and density due to the process of lyophilization would be essentially the same in standard and tissue samples. A control of the actual thickness of sections was necessary with the 1st generation cryotome but proved to be obsolete with the subsequent cryotome model. All other corrections were covered by the experimentally determined correction factor k = 1.07 which was elaborated by comparing QAR readings of lyophilized liver paste sections to scintillation counter measurements of wet liver paste samples. The validity of this correction was proven by QAR measurements of mouse liver preparations obtained after objection of a RES-seeking radiopharrnaceutical. Thus, it was indeed possible to achieve

(Fig. 3 opposite)

Fig. 3. Density and dose distribution around small sources of lz51 Tubing with an i.d. of 0.35-0.45 mm was filled with liver paste containing 250 Bq ‘251/mg.Sections were prepared and exposed for either 1 or 14 days to simulate different radioactivity levels. Halos produced by photons were particularly pronounced at the long exposure time (B, D), but they also became apparent in the dose matrix obtained after the short exposure time (C). Using the evaluation criteria illustrated in Fig. 4(A), the contours of the sources (black isodose lines in Figs 3(C) and (D)) could be reliably identified irrespective of the exposure time.

IB)

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ram/!&

1 day

KmJnter)

7 days

14 days

Fig. 4. Identification of contours and determination of total radioactivity content in small sources of “‘I. (A) Schematic presentation of criteria used for reconstruction of contours and integration of radioactivity content of small sources of 12JI.The idealized source is represented by the area under the bold line. In the dose matrix, the source is reflected by a bell-shaped step profile (shaded area). At the boundary of the source, the steepness of the profile and, hence, the step height (open arrows), is highest. An isodose line set at 2% above the level of the bottom of the step (isodose level 2) detines the cross-sectional area of the source. For determination of the total radioactivity content of the source, ROI integration follows an isodose line set at 1.5% (relative dose; isodose level 1). Note the point that dose components in the halo region have to be added to the central region in order to regain the correct radioactivity integral. (B) Criteria for the integration of radioactivity content. Tubings with different diameters were filled with liver paste (250 Bq/mg) and sections were exposed for increasing periods of time. Integration of dose was then performed within the ROIs defined in Fig. 4(A). Open columns, integration within the putative area of the source (&dose level 2, Fig. 4(A)); hatched columns, integration within the 1.5% isodose tine (isodose level 1, Fig. 4(A)).

592

Fig. S(E) and (F) (legend opposite) 594

Quantification of autoradiography concordance between QAR readings and scintillation counter measurements of radioactivity concentration in macroscopic tissue. Our work profited from extensive methodological efforts by other authors which were mainly dealing with receptor mapping. As it has been previously reported by Davenport and Hall (1988), commercial polymer standards of lzsI were found to give erroneous calibration because of a different density and a different thickness of polymer material as opposed to lyophilized tissue sections. Control measurements with homogeneously accumulating tissue as proposed by Yonekura et al. (1983) were found to be of great value for testing the validity of the overall calibration procedure. However, calibration with macroscopic tissue paste standards was found to be applicable only to radioactive sources of similar size. The reason for this restriction relates to problems (3) and (4). This became obvious when autoradiographic images of small phantoms were produced with increasing exposure times (equivalent to a variation of the radioactivity content). In these images it was not easy to identify the contours of the object in the dose-matrix. In situations where halo formation was low (see e.g. Fig. 3(C)), the flanks of dose profiles were very steep, thus indicating that resolution was fairly high. Resolution was ultimately determined by the 50 pm aperture, which was deliberately chosen for scanning densitometry; a higher level of resolution, e.g. 25 pm, would have produced an unmanageable amount of data. Sources with high radioactivity content were surrounded by pronounced halos, thus precluding a direct determination of the outlines. Moreover, peak film densities were found to decrease with decreasing diameter despite identical specific radioactivity. Hence, a novel procedure had to be established to cope with the problem of quantification of small radioactive sources. The only published method to correctly relate autoradiographic patterns to distinct areas of (pathological) tissue is based on grid overlay matching of the autoradiographic image and adjacent section stained with conventional methods (Blasberg et al., 1981). This approach was not feasible with lyophilized sections mounted on adhesive tape because they could not be stained in a way to correctly identify small lesions. Our approach to a solution of the problem was elaborated and tested with sections

595

from plastic tubings of various diameters filled with radioactive pastes. It consists of two integrations of isodose line-defined areas to yield the putative area of the source and the total radioactivity contained within the area. The approach relies on two premises, i.e. background radioactivity has to be low and radioactivity distribution within the source has to be homogeneous. The first premise was found to be reasonably well fulfilled in most instances of tumor targeting with ‘2SI-labeled MoAbs, because high resolution autoradiography almost invariably yielded varying patterns of hot spots above a very low background (U. Schmid. to be published). In cases of low inherent resolution, e.g. when using ‘3’I-labeled MoAbs, some coalescence of spots,.and, hence, a general increase in background film density was noted. By the same token, the second premise was not reliably fulfilled, since non-homogeneous radioactivity distribution was encountered even in metastatic processes with diameters as small as 0.8-l mm. Consequently area determinations in dose matrices could only be based on hot spots, and not on otherwise defined tissue elements. Thus, a measurement of MoAb uptake by the QAR method presented here will indicate percent injected dose per mg accumutating tissue but not per mg metastasis. A further drawback inherent to the QAR analysis of tissue sections stems from the fact that the upper and lower surrounding of a given area in the section is not known, i.e. a small hot spot could equally well represent the center of a small or the pole of a large metastasis. The problem must be solved by compiling stringently oriented serial sections with the aid of an appropriate computer program. Despite these limitations, the method offers considerable progress in analyzing MoAb accumulation in small lesions, a problem which is intimately linked with the goal of therapeutic targeting of minimal residual disease. Acknowledgements-This

work was supported by the Tumorzentrum Heidelberg-Mannheim. The help of H. Haas, G. Hartmann, D. Meenenga, and G. Sroka with computer software is gratefully acknowledged.

References Baskin, D. G.; Davidson, D. A.; Corp, E. S.; Lewellen, T. K.; Graham, M. An inexpensive microcomputer digital imaging system for densitometry: quantitative autoradiography of insulin receptors with 1251and LKB Ultrofilm. J. Neurosci. Meth. 16: 119-129; 1986.

(Fig. 5 on p. 593 and opposite)

Fig. 5. Sequence of images illustrating the process of QAR applied to a liver with one prominent metastasis. (A) Photograph of the mouse abdominal area immediately before preparation of the 20 pm section. The metastasis is clearly visible in the lower left part of the liver. (B) Autoradiograph of the section. The metastasis showed high accumulation of ~2sI-labeled MoAb 12-15A. The other radioactive spots are due to residual radioactivity in vessels. (C) Grey-level matrix. (D) Autoradiograph of tissue paste standards used to establish the density-to-radioactivity (Bq/mg) calibration curve. (E) Dose matrix. (F) Zoomed image of the metastasis as used for the determination of the putative area of the MoAb accumulating segment of the lesion, i.e. area circum&rib-ed by the black isodose line. As can be seen, non-uniformity of radioactivity accumulation in the upper part of the lesion led to the exclusion of some pixels.

UW

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