General Techniques in Surgical Investigations 10

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Initial Remarks on the Histopathology of the Lymphatic System

For any clinical diagnosis or treatment to be performed it is necessary to have sufficient morpho- logical knowledge about the organization and structures of the lymphatic system. This includes knowing how the network of lymphatic vessels is organized and how this network is connected to regional lymph nodes and recognizing the nature of the synergistic structures and cellular activities of the different cell populations of the nodes, the connections between the nodes, and the significance of junctional node stations.

As already explained in Chapter 7, the lymphatic capillaries begin in the periphery in the in- terstitium of soft tissue parts. This means that they are closed at the end and that there is no stream with peripheral loops, with inflow and out- flow, as there is in the peripheral bloodstream.

The lymph vessels drain the lymphatic fluid into the marginal sinuses of the lymph node via the vasa afferentia (Fig. 1).

The biological processes within the nodes cannot be understood without some basic knowledge.

In the cortex of the nodes we find the B-cell follicles ± these are easily detected by histological procedures ± with their germinal centers, showing high proliferative activity. The periphery of the follicular system is where the dendritic reticulum cells are localized; these are the conductors direct- ing the orchestra made upof the different cell populations. The paracortical T-zone is sited sub- cortically, i.e. inside the follicular B-zone. Here we find the different categories of T-cell populations.

The lymphatic sinus network is located between the follicular structures, beginning with the subcapsular marginal sinuses which drain the lymphatic fluid to the central parts (medullary sinuses) and from there through the channel system and then to the vasa efferentia, where the fluid leaves the node.

The lymphatic sinuses are outlined by and partly filled with so-called sinus histiocytes. These cells show high phagocytic activity and therefore have the functions of macrophages. This makes them the most important cell populations respon- sible for the clearance of the lymphatic stream, which they achieve by ªparenteral digestionº of toxins and proteins and also by digestion of living and necrotic tumor cell material.

Significance of Labeling of the Different Cellular Compartments of the Lymph Nodes for Radiological Diagnosis

At least two features related to components of the lymphatic system must be mentioned in this con- text:

· The B-cell population of the lymph nodes (cor- tical zone) can be labeled by using the antibody CD20. This method is used for radiological staging diagnosis of B-cell lymphomas, but when high sensitivity has developed it may also be possible to use this antibody to demonstrate neoplastic infiltration within the nodes, leading to destruction of parts of the node-specific structures.

· A second approach, which is newer and still experimental, is systemic application of ultrasmall particles of iron oxide (USPIO) colloids (see also Chapters 1, 2, 9).

These particles are taken up by phagocytosis, generally in the reticuloendothelial system of the organs (spleen, liver), and especially also in the peripheral lymphatic tissue (lymph nodes), where the material is strongly phagocytosed and stored by sinus histiocytes.

Both labeling systems have been used by several expert groups in efforts to find areas of cancer infiltration-related defects in lymph nodes that could Chapter 10

General Techniques in Surgical Investigations 10

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be sentinel nodes. However, the preliminary results have not been sufficiently convincing to justify in- troducing such methods into routine clinical investigations.

Critical Points in Blue Dye (Patent Blue) Mapping In use of the blue dye method, whether alone or in combination with tracer methods, various points seem to be important.

· This method is limited to particular tumor types, such as breast cancer, malignant melanoma, and now also colorectal cancer. The topo- graphic overview it allows is optimal in some circumstances, and the lymphatic basin is generally well defined.

· In many other settings, including N-staging of other primaries, it is not so easy to use the usual blue dye methods, because the flow of the dye cannot be followed.

· Depending on the local vascularization, the blue dye can be quickly resorbed into the bloodstream locally and excreted with the urine, which is discolored by it and becomes blue to green; in addition, long-term or even permanent blue staining of the skin can develop.

· Allergic cutaneous reactions and anaphylactic shock are also possible.

Therefore, with due consideration for the side-effects mentioned (points 3 and 4), these possibilities must be discussed with the patients and when informed consent is given special measures must be implemented.

Fig. 1. Normal lymph node structure, showing the vasa afferentia (1) in which the lymphatic fluid is transported into the node and the vas efferens (2) in which the fluid leaves the node. Arborization of the arterial stream (3) passes through the node and small vessels end in the re-

gional fatty tissue (4). After collection of the venous blood the veins concentrate in the apposite direction and the main stream leaves the node (5) [6 marginal sinuses, 7 medullary sinuses, 8 secondary follicles (B-zone), 9 mark strands (T- zone)]

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Timing in Blue Staining Methods

When isotonic aqueous dye solutions are used it is clear that resorption via the lymphatics is very much quicker than with the methods using labeled colloidal particles. Exact timing, accurately based on clinical experience, is therefore very important for successful operation procedures. The following points must be kept in mind:

· When the preoperative injection of the tracer solution is given too early, not only the sentinel lymph node(s) (SLN/s) will be stained, but as a rule all lymph nodes within the basin.

· When the preoperative injection is given too late the bulk of the solution injected will be found around the injection site and too little of the stain will reach the SLN(s).

Because preoperative labeling of SLNs, for instance in colorectal cancers, with ^99mTc-nanocolloids is not yet sufficiently well developed, intraoperative subserous administration of blue dye is used to look for the SLN(s). This preliminary strategy has the advantage that no such precautions as are needed with radioactive material are necessary.

Probe Guidance in Surgical Treatment

In recent years it has become increasingly clear that use of a gamma probe increases the rate of SLN detection. Albertini et al. (1996) demonstrated an increase from 73% to 92%. This successful outcome paved the way for further developments of the devices used in daily routine, to improve handling and sensitivity.

The great advantage of ªprobe-guided surgeryº is that the localization of the SLN/s can be evaluated even preoperatively and the surgical procedure can be guided intraoperatively. In order to determine the exact course of the lymphatics from the primary to the sentinel node, in many clinics a combination of the blue dye method and gamma- guided detection is preferred.

In the circumstances. at the moment it seems we can hope that optimal solutions will be developed. A second important advantage of using the gamma probe is that it is possible to check for complete excision of the SLN intraoperatively by keeping the gamma probe in the wound region

after excision of the sentinel node. If residual activity is found near the site of the sentinel node, re-excision of this region can be performed and the material can be subjected to histological investigation.

Many authors have now come to the conclusion that sentinel node detection is optimal when the blue dye staining method and the gamma probe technique are used in combination, because the two methods work in synergy. The stained lymphatic cord connecting the primary with the sentinel node can be seen with the naked eye, and the gamma probe can detect macroscopically invisible, very small nodes or be used to control for com- pleteness and to guide re-excisions if residual activity is found.

The use of both methods in combination can be seen as a dual-control system aimed at complete locoregional tumor clearance.

Clinical studies must demonstrate how much lower the frequency of locoregional recurrences is than after blind excision on the basis of surgical experience alone. There is no doubt that these new developments are valuable in terms of modern im- proved quality control.

Practical Use and Servicing of the Gamma Probe (see also Chapter 12)

For controlled usage of the gamma probe several points are important to prevent accidental mal- functions with highly unfavorable consequences.

· Battery-supported devices must be checked before use.

Directly operating non-battery-supported devices must also be tested preoperatively for elec- tric functionality.

· Regular sensitivity testing of the gamma probe is necessary to guarantee constant sensitivity and to exclude undetected loss of sensitivity.

Standard sources (iodine-129 or cobalt-57) can be used for reproducible standard measuring.

· When it is the surgeons, and not specialists in nuclear medicine, who are performing the investigations in the patients, the surgeons must be familiar with the handling of the probe.

· When anesthesia has started and the patient is fully prepared for the start of the operation, the gamma probe must be brought into the operating position.

Practical Use and Servicing of the Gamma Probe 91

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The following points must be observed:

± Probe and cable must be covered with sterile tubing

± The probe must be covered with a glove (Fig. 2)

± The probe must be secured during the operation procedure, and accidents must be pre- vented by secure provision of a safe place for the operator to lay down the probe (Fig. 3).

Good Practice in Combined Use of Dye and^99mTc-labeling Procedures

Depending on the localization of the primary, for instance in breast cancer cases (subcutaneous, centrally in the gland, prefascial), patent blue solution is injected subdermally or peritumorally in a volume of 0.2 ml in subdermal or 4 aliquots of 1,5±

2 ml in deeppertumoral injections (quantities vary in the published literature, and different reasons are given for larger and smaller quantities).

The syringe used must be locked against accidental separation from the needle during injection under pressure, because in such an event the dye will flow over the epidermis and label more or less extensive areas of the skin.

In addition, careful handling is necessary at the site of injection (empty syringe at time of withdraw-

al, pressure balance within the injection system).

Gentle massage at the injection site helps to lessen or avoid reflux of the dye or labeling solution within the superficial parts of the injection channel.

Because it is necessary to be prepared for anaphylactic reactions after blue dye injection, corti- sone and catecholamine solutions should be immediately available and ready to hand.

The injection of ^99mTc labeled colloid solution must have been performed 24 hours before the operation.

Determination of the SLN's Location and Procedure for Its Excision

The point on the skin that is as exactly as possible over the SLN located in the deeptissue is marked with the aid of the gamma camera.

Because the hot spot(s) is/are located in deep tissue, a three-dimensional judgment is not possible at this time. These evaluations can be adequately performed with the Neoprobe 1500 or other new probes (see also Chapter 12). One of the devices frequently used in our Department of Nuclear Medicine, the Tec-Probe 2000, is shown in Fig. 4.

When the device reaches the hot spot region, this is indicated by acoustic signals and a high radiation count readable on the device. These counts Fig. 2.Schematic demonstrating how to protect a probe with a sterile glove, which can be drawn off after use. This makes subsequent cleaning and steriliza- tion after the operation easier

Fig. 3.The gamma probe should be put down on a table or box with a rim at the edge so that it can never roll or fall off or be pulled down by a tug on or tension on the cable

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have to be compared with those of the background analysis, which should have been measured earlier in the sternal notch area with the gamma camera held perpendicular at the measuring point.

To find the shortest way to the SLN the ªline of sightº described by Krag et al. (1993, 1998) is rou- tinely used.

The surgeon can find the shortest route quite precisely by changing the angle of the tip of the probe, but it is quite important that the probe is not pointing at the injection site, as this would cause the count to increase and the values indicated would be false.

Blunt preparation with avoidance of bleeding is necessary. When the SLN is reached it must be excised with at least a thin layer of perinodal soft tissue, the purpose of this being to include the vasa afferentia of the nodes or any nodal metastasis in the early phases of a breakthrough of through the lymph node capsule.

When the blue-stained lymphatic draining to the sentinel node becomes visible during the operation it should not be dissected early in the procedure, because there is a danger that the blue dye solution would flow out into the soft tissue in the area surrounding the node, bringing with it lymphatic fluid and also cancer cells.

· It is good practice to compare the counts ob- tained in situ before extirpation of the sentinel node with the counts of the isolated node after extirpation to avoid misinterpretation when the radioactivity measured in situ actually came from the background.

· The second control is to place the probe in the wound bed to make sure that no radioactive nodes have been left.

From the location point of the extirpated SLN the gamma camera has to be angled in all directions in a circle to make sure that no second sentinel node has been left in the vicinity of the excision point. This secondary evaluating procedure is again highly sensitive; it detects even slightly ele- vated activities, which must be regarded as activities in radioactive fluids within lymphatics, which can lead the operator to a SLN that has been left in place.

If there are no areas leading to the suspicion of second or further SLNs, some surgeons conclude the operative lymph node clearance at this point, taking the view that skipmetastases, while possible, are very unlikely.

At present, however, many authors still argue that the function of the SLN concept as an intraoperative ªsignal boxº for limited lymphadenecto- my is still under investigation. The goal of obtaining scientifically based and proven calculations of its value and evaluating the lymph node status in the whole axillary basin is now being approached intensively and it will still be some years before we know more about the real value of sentinel node evaluation.

The most important point in this connection is that we have no statistically evaluated comparative life-tables for the tumor entities discussed, showing that the more limited surgical programs based on the SLN concept ± e.g. avoidance of complete axillary revision ± have long-term survival rates equivalent to or better than those following the more aggressive strategies for locoregional cancer clearance.

Therefore, based on current documented knowledge, after SLN excision full axillary lymphadenec- tomy including all lymph nodes in levels I and II is practiced in many clinics during learning, control and early follow upperiod to be on the safe side.

Experience has shown that there are difficulties when the primaries are located in the lateral quadrants near the outline of the lateral pectoralis mus- cles. In such cases the ªshine-through phenome- nonº has an important role and can disturb the search for the sentinel node quite badly. Most of the false-negative cases in earlier sentinel node investigations were due to this. In Krag's investigations (Krag et al. 1998), in all false-negative cases the primaries were in the lateral part of the breast.

For cases in which the shine-through phenome- non must be assumed, some possible ways of solv- ing this problem can be defined:

Determination of the SLN's Location and Procedure for Its Excision 93

Fig. 4.The Tec-Probe 2000 device connected with the gamma probe

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· The probe can be angled away from the injection site.

· Additional collimation can be implemented to reduce radiation scatter and background activ-

· ity.Radioactivities can be separated by blocking plates (steel, tungsten).

· The more highly loaded primary can be excised before the search for the sentinel node(s) is started.

These possibilities should be adapted to the indi- vidual case, but they must be kept in mind, and such equipment as shields, etc. must be available in the acute situation for when its use seems ap- propriate or necessary.

Pitfalls of SLN Labeling and Detection

The most important pitfalls in SLN detection are based on the following points.

· In totally or almost totally tumor-infiltrated nodes perfusion is generally severely reduced, and sometimes the vasa afferentia are already occluded by cancer cells filling upthe lumens.

In such cases the lymphatic flow bypasses the tumor-infiltrated node and labels a tumor cell- negative node; the ªrealº sentinel node ± which is not detected by the probe ± would be positive, but the ªpseudosentinelº or ªsubstituteº sentinel node is seen as cancer cell negative.

· A further possibility when the sentinel node is unable to take upthe labeled fluid is so-called fatty degeneration of the node in elderly per- sons. This means that 80±90% of the node, especially the central parts, has become substi- tuted by fat cell tissue. This degenerative change corresponds to fat cell proliferation ªe vacuoº from central parts to periphery, filling the space that has resulted from the shrinkage of lymphatic tissue. In this process the lymphatic channels have also disappeared, as they are also involved in the shrinkage process. This means that lymphatic flow is much reduced or no longer present at all.

In practice, such nodes ± primarily sentinel nodes ± cannot be labeled by the methods described.

· A multicentric or multifocal cancer of the breast can simulate a paraglandular SLN when one fo-

cus is localized in an extremely far lateral site in the glandular body.

· Primaries that are located not in the main parts of the glandular structures, but in the axillary tail can:

± Be misinterpreted as metastases of an undetected primary.

± Be accepted as the primary (mostly after surgical excision), but labeling of the sentinel node is then impossible.

· Dystopically located primaries ± in rare cases in the axilla ± can initially be misinterpreted as metastases, with a consequent search for a non- existent primary in the glandular body of the breast.

SLN Investigation by Pathologists in Cooperation with Cytopathologists

In recent years the ªworking formulaº has been:

When pathologists investigate the SLN intensively and do not find any metastatic settlement investigation of the other approximately 12 lymph nodes (average in German breast cancer studies) or even more (usually after revision of the axilla) can be omitted.

This is only partly correct, and mostly emanates from nonpathologists.

It must be stated that investigation of the sentinel node means work-upof a large number of serial sections stained alternately with HE and immu- nohistochemically, using antibodies directed at cytokeratin (Fig. 5).

In view of the heavy responsibility resting on anyone stating that the SLN is really negative, a large number of sections must also be stained im- munohistochemically and evaluated microscopi- cally by qualified pathologists.

In addition, in the case of positive results of the sentinel node investigation, 12 or even more lymph nodes must be paraffin embedded and also evaluated histologically in addition to appraising the status and classifying it according to the TNM- system in view of the exact N-status.

The thesis that serial sectioning of one or two SLNs with HE and cytokeratin stainings takes less time than sectioning of a dozen or some more nodes after axillary revision is not fully correct, because

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· Pathologists bear a much heavier responsibility, especially in breast cancer cases, because false- negative results caused by insufficient investigation of the node(s) can lead to axillary recur- rence with a fatal outcome.

· Before introduction of the sentinel node concept pathology laboratories brought three or more lymph nodes in a single paraffin block for serial sectioning.

Preparation of such a large number of serial sections as dictated by the sentinel node concept was not recommended or even considered. This state of affairs was acceptable insofar as if single tumor cells were not detected in the first node reached by the flow of lymphatic drainage all secondary nodes at levels I and II were already prophylactically removed according to the principles of extended axillary revision.

Altogether, the responsibility for the assurance that sentinel nodes are genuinely tumor-free has become considerably heavier, but the investment required for the investigations is no lower in terms of time or cost.

In total, the new procedure followed since adop- tion of the sentinel node concept demands more manpower than the conventional staging investigations. Therefore, following the introduction of these new and certainly helpful investigative principles, new ground rules on financing the manpower needed and the cost-intensive immunohistochemical staining must be established in every country. Otherwise, these intensive diagnostic efforts cannot correctly be carried out. This is a ser-

ious warning and should be heeded in order to avoid later litigation against clinics and the medi- cal staff working in them.

Handling of Histopathological Procedures

When the SLN(s) is/are taken out, it/they should be immediately transported to the pathology labo- ratory to be opened by cutting with a fresh unused one-way scalpel into two halves, neither of which should be more than 2±3 mm thick. In the case of a large node a middle, third, slice can be taken.

If it is possible, imprint cytology can be performed on material taken from the fresh-cut sur- faces for cytological investigations during the operation. (This method of combined investigation was practiced for a long period in the Department of Pathology at the University Clinic in Gættingen in the 1970s and 1980s, with the aim of obtaining at least a preliminary result by cytology.) In the case of a positive result axillary revision can be performed immediately. This seems to be a very important point, since it can make it possible to avoid a second operation.

Proposals for the use of ultrarapid immunohis- tochemistry, as proposed by Nåhrig et al. (see Chap. 17), are very helpful in other conditions, but should be avoided in sentinel node investigations.

But, imprint cytology, if correctly performed, is not connected with loss of tissue parts. However, when frozen sections of sentinel nodes are taken, a thickness of at least 1 mm of native tissue is lost, and after paraffin embedding and the first sections

Handling of Histopathological Procedures 95

Fig. 5.Segment of a lymph node near to the subcapsular region, showing dissemi- nated single cancer cells stained (red) for cytokeratin. The typical epithelial cell layering is missing. The epithelial cancer cells cannot be detected with certainty in normal HE stainings and cannot be dif- ferentiated from reticulohistiocytic cells

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are discarded unused, which means a second loss of tissue must be calculated in. With this double procedure, it can be that so much tissue is lost that micrometastases are missed.

Benefits of Sentinel Node Evaluation Over Primary Axillary Revision

At the current state of the art there is no doubt that improvements of axillary lymph node staging overall, especially since the fundamental investigations of Krag et al. (1993), have brought advantages with them. This is true insofar as more con- servative surgical treatment is possible and also in that higher degrees of diagnostic safety can now be attained.

The pros and cons of the sentinel node concept are listed in Table 1.

In recent years we have learned from the current literature that the rate of false-negative cases can be reduced to 1±2% when surgical discipline and pathological procedures are optimally dove- tailed. We have also learned that false-negative results and skip metastases cannot be fully ruled out, however. Therefore, optimal monitoring of the axillary node status (palpation, PET, etc.) is necessary in the follow-up period, especially when axillary revision has been omitted in cases with negative sentinel nodes.

Use of Radiodiagnostic Techniques

Basic conditions of usage of a radiopharmaceutical substance are:

· The substance is used as a ªtracer,º which means in very low dosages without pharmaco- logical effects.

· The tracer must have the property of accumulat- ing in target organs (lymph nodes) and being stored there for a sufficiently long period to be recorded intraoperatively by a gamma probe or an imaging device (e.g., gamma camera). so that it can yield sufficient information about its regional distribution (e.g. sentinel node detection).

Choice of the Labeled Contrast Solution

The most frequently used radionuclide in nuclear medicine has long been^99mTc.

Clinical work with this radionuclide is largely unproblematic, because it is constantly available throughout the world at an acceptable price.

Technically there are two options for its production:

· Neutron irradiation of molybdenum-98

· Preparation of a fission product of uranium-235 An important step was the finding of a concept for

99mTc binding to other compounds. As described by Keshtgar et al. (1999), reduction to the more positively charged technetium species III and VI by stannous chloride enhances the binding capaci- ty to other compounds. Another possibility is to use insoluble ^99mTc-sulfur chloride with a TcVIII Table 1. Important advantages, each countered by a disad-

vantage with possibly far-reaching ramifications, of using the sentinel lymph node (SLN) concept without axillary revision

Pros (advantages of the

SLN concept) Cons (danger of postoperative cancer spread within the axillary basin in false- negative cases)

Avoidance of intra- and

postoperative bleeding In false-negative cases [SLN(s) not immunohisto- chemically detected or in- complete investigated, or primary ªskipº metastasis], cancer cells can be left behind, leading to infiltration of secondary nodes

Avoidance of injury to the long thoracic, thoracodor- sal, and intercostobrachial nerves

Consequences: A number of the lymph nodes in the axillary basin can be infiltrated by the cancer after a short time, leading to in- curability!

Avoidance of postoperative axillary infections with de- velopment of abscesses

Conclusion: The status after sentinel node control only is not to 100% calcul- able

Avoidance of lymphedema formation in the arm Excision or radiation of SLNs that would otherwise not be detected in the ªpreaxillaryº (paraglandular) or parasternal regions

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oxidation state. Stabilizing agents such as gelatin or albumin can be used for stabilization.

In consequence of the insolubility and the stabilizing effects mentioned above, labeled colloids were introduced for detection of regional lymph nodes and lymphoscintigraphic analysis. In practical procedures, there are some difficulties in the choice of special colloids for specific purposes:

· National limits on availability

· Difficulties in registration of new radiopharmaceuticals

Therefore, globalization of different companies can help to lower the international transfer problems.

For routine diagnosis and for continuous investigations in clinical studies it is important

· That the preparation of labeled colloids is reproducible with reference to special qualities.

· That stable labeling is maintained.

· That each radiopharmaceutical has a constant well-defined particle size.

The consistency of particle sizes within each batch and continuously throughout different batches used for the same purpose is a very important fac- tor.It is well known that small particles migrate quickly from the injection site to the regional node(s) and then pass through the node within a short period, while larger particles migrate slowly and need late imaging records.

Experience has made it clear that the same particle size cannot be used for lymphoscintigraphy of the nodes of the whole lymphatic basin and for isolated detection of the SLN(s).

Particle sizes are qualified and measured by different modern techniques, such as electron micro- scopy, X-ray fluorescence and gel chromatography.

Besides particle size, which is important for the speed of transport and the rate of phagocytosis by so-called sinus histiocytes of the lymph nodes, there are other factors that have roles in detection of node labeling. These are:

· Number of particles with defined size.

· Charge to the particles with the tracer substance.

· Rate of degradation by enzymatic activity of proteases (higher degradation rates in sulfur than in albumin colloids).

Specialists in nuclear medicine and physicians are familiar with the difficulties encountered in stan- dardizing the charges.

A brief period of heating (in the range of 3 minutes) followed by cooling for a short period makes for greater radiochemical purity and re- duces the quantity of particles that are smaller than 400 lm.

When we select a longer heating time the pro- portion of small particles decreases.

As Keshtgar et al. (1999) emphasize, there is a decisive gap between the experimental studies on injection techniques, maintenance of constant particle size, and visualization of the lymph node chains and experience in clinical practice according to systematic research, and there has so far been no consistent improvement.

It also seems clear that comparative interdiscip- linary studies on national and international bases must be subject to firm controls and that these must also apply to the radiopharmaceuticals used.

Early investigations on evaluation of radiocolloid sizing techniques were performed by Warbick et al. as long ago as 1977.

Some experts are of the opinion that particles in the size range of 50±80 nm should be favored for the detection of the SLN(s). However, evaluations conducted by clinically experienced research groups have demonstrated that imaging depends heavily both on the volume applied and on particle size of the colloids used.

Paganelli et al. published the data compiled in Table 2 as long ago as in 1998, and these have proved valuable for orientation.

Figure 6 illustrates the imaging of a case with a breast cancer located centrally in the upper quadrants, and while it is true that drainage to the axillary nodes only is indicated, at least three nodes of the axillary basin are labeled and must be removed and investigated histologically and immu- nohistochemically as ªsentinel nodesº in serial sections.

Choice of the Labeled Contrast Solution 97

Table 2. Sizing of radiocolloid particles for use in detection of SLN(s): published data for guidance. (Paganelli et al.

1998)

Size of tracer

molecules (nm) Patients positive/

total No. of nodes

imaged

<50 29/30 1±5

<80 26/30 1±4

<200<1000 155/155 1±2

The publications available suggest that preparations with particle sizes between 100 and 200 nm should be aimed for

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Because of existing problems in transporting radioactive materials within the European Union and also in rapid transoceanic exchange, specifically with reference to quality requirements, preference should always be given to national products. Such products, based on national production, are:

Product Manufacturer and comments Nanocoll Manufacturer: Nycomed Amer-

sham Sorin S.r.l., 13040 Sa- luggia, Vercelli Italy, labeled particles 95% < 80 nm

Nanocis Manufacturer: CIS bio Interna- tional, France

This preparation contains colloidal rhenium sulfide (0.48 mg) with average particle size ranging from 3 to 15 nm

Microlite Manufacturer: Du Pont USA This preparation contains 1 mg of human serum albumin with an average particle size of 10 nm

Sulfur colloid Manufacturer CIS ± USA In keeping with the labeling protocol, the labeled particles are between 40 and 1,000 nm Antimony sulfide This preparation is mostly colloid used in Australia

It has a very small particle size of 5±15 nm

Gold-198 This has been withdrawn because of the excessive radiation dose acting on the target tissues

Fig. 6.Imaging in a case of breast cancer located centrally in the upper quadrants, showing drainage exclusively to the axillary nodes. At least three nodes in the axillary basin are labeled and must be removed. They must be regarded as sentinel nodes and sectioned for histological and immunohistochemical investigation. (Illustration kindly donated by Prof. Paganelli and his group(IEO Mi- lan)

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Svensson et al. (1999) relatively recently tested a

99mTc-labeled polyclonal human immunoglobulin, but such approaches are still in the early stages. It could be that, once labeled, FAB fragments of antibodies can be used for particular kinds of labeling, and it is possible that in the future radiopharmaceuticals will be developed that will bypass the lungs and bind to the reticuloendothelial system and, accordingly, also to the analogous cell systems within the lymph nodes.

Some other radiopharmaceuticals, such as

99mTc-dextran (molecular weight 100,000), which has some allergic potential and exhibits capillary transport (Henze et al. 1982), and^99mTc-human serum albumin (Nawaz et al. 1985), were used earlier but only for a time.

Plans for Improvements to the Quality of the Contrast Media

Comparative studies on the colloidal solutions used that include liposomes are in progress. Ac- cording to the investigations of Philips et al.

(1998), in a rabbit model ^99mTc-labeled liposomes with an average size greater than 100 nm migrate more slowly than liposomes with an average size less than 100 nm.#

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