10
The Haematopoietic and Lymphoreticular Systems
249 For the purposes of this chapter, the haematopoietic and lymphoreticular systems are limited to the lymph nodes, thymus, spleen, bone marrow, and lymphatics. The majority of lymph node groups associated with organs are examined with that particular organ at the time of evisceration and block/organ dissection. More specialised and individual areas of the lym- phoreticular system such as the Kupffer cells in the liver will obviously be examined as part of the organ system within which they lie and usually will be evident only histologically. The following are included:
• Lymph node dissection
• Special techniques applicable to lymph nodes
• Dissection of the spleen
• Special techniques applicable to the spleen
• Bone marrow examination
• Assessment of the thymus
• Dissection of the thoracic duct
• Dissection of a mediastinal mass
• The autopsy in sickle cell disease
The Lymph Nodes
External Appearance
Superficial lymphadenopathy is likely to be noted either clinically or as part of the external examination. If present, nodes can easily be dissected early during the post mortem and fixed for later examination or collected in a sterile container and sent for microbiological examination. It is not usually necessary to weigh individual nodes and normal sizes and weights for lymph nodes are variable depending on their site. It is rarely necessary to apply special methods when removing lymph nodes and indeed all lymph nodes that may need inspection should have been assessed visually during the evisceration stages. The deeper node groups will be removed at the evis-
ceration stage and will accompany their relevant organs or neighbouring structures. Samples can be retained and processed as described for the superficial nodes.
Internal Appearance
If there is evidence of lymphadenopathy the individual enlarged nodes should be sliced through and the cut surface inspected. The normal appear- ance of a lymph node is uniformly light grey/tan. When the node is involved by a pathological process, the appearances may be reasonably distinctive for that process. For example, metastatic tumour tends to present as firm to hard areas of often white discolouration within the node, whereas involve- ment by infection is usually softer and less discrete. Necrosis of course can be present with either. Current tuberculous infection may show as classical zones of caseous necrosis, whereas old tuberculosis tends to calcify.
Histology of Lymph Nodes
When dealing with a lymphoma it is wise to save appropriate tissue (fresh- frozen) for any subsequent molecular studies that may be required (see Chapter 13). With most lymph nodes it is possible to bisect the node, if thought relevant for microscopy, and block one half for histology, but if extremely large a 0.3- to 0.4-cm thick sample should be examined and the remainder retained in case it is required. Special stains may be necessary to characterise nodal processes and these are also discussed in Chapter 13.
Special Techniques for Lymph Nodes
Dissection of Nodal Disease with Perinodal Infiltration
Occasionally the pathological process involving the lymph nodes may be so extensive that it infiltrates local structures or completely replaces the node, making identification difficult. Obviously in these cases the mass of tissue should be removed as completely as possible to assess the extent of the disease process and to sample tissue for subsequent histological character- isation of the pathology.
Lymph Node Imprints
Dab imprints can be made at the time of post mortem if a rapid diagnosis is needed. It is important to be careful not to press too hard and obscure the cytological detail. This is preferably performed in a safety cabinet because of the potential risk of exposure to infected fresh tissue. Imprints are produced by cutting the node in half and dabbing the cut surface onto clean glass slides. Air-dried preparations can be stained with May–
Grunwald–Giemsa and alcohol-fixed slides can be stained with Papanicolou and/or haematoxylin and eosin or kept for any necessary special stains
(Fig. 10.1). These may include special stains for microorganisms or immuno- histochemistry. The latter may be extremely important in lymphoma diagnosis.
Microbiology of Lymph Nodes
When infective lymphadenitis is relevant to the post mortem diagnosis it may be necessary to attempt to identify the causative organism. This then becomes the domain of the microbiologist, but the ability to differentiate confidently between contaminant and pertinent organisms may be very difficult if tissue is sampled in a less than wholly sterile manner. It is im- portant to use new, sterile equipment and to obtain the tissue as early as possible during the examination. A small portion of the node removed in this manner should be sealed in a sterile container and transported to the microbiology department with all of the relevant clinical information included on the request form.
The Spleen
External Appearance
Whichever method is used for evisceration and block dissection, the spleen will ultimately be isolated from the other organs. A careful record of the The Spleen 251
Figure 10.1. Dab imprint of a lymph node removed at post mortem. Pressure should be light to avoid obscuring the cellular detail and at least two slides are usually prepared, one air-dried, the other wet-fixed. (Courtesy of Mr. Ivor Northey.)
splenic weight is made, with a normal range of 155 to 195 g (see Appendix 2). As mentioned previously, the hilum should be inspected for splenunculi before dealing with the spleen proper. Often pathological processes will involve both. Before slicing the spleen it is important to establish whether it is necessary to send tissue for microbiological investigation, as contami- nation is extremely easy in the post mortem room and meaningful micro- biological studies can be obscured by lack of awareness (see later).
Internal Examination
The spleen should then be sliced through completely in a vertical plane using a large sharp knife. The slices should lie no more than 1 cm apart (Fig.
10.2). The slices can be laid out on the dissecting board for closer inspec- tion (Fig. 10.3). With a normal spleen or spleen involved by tumour (par- ticularly lymphoma), amyloid, or portal hypertension this will be easy but in cases of sepsis the spleen will often be soft and liquefied, and slicing may be impossible. In such cases the demonstration of a liquefied “bag” of splenic tissue is indicative of septicaemia and nothing is usually lost in the inability to inspect the cut surface of slices.
Figure 10.2. The spleen is sliced at 1-cm intervals with a large-bladed knife. (Cour- tesy of Mr. Dean Jansen, Whittington Hospital.)
A splenunculus found during an adult post mortem is irrelevant in itself, but if there are many this may be due to previous traumatic rupture and peritoneal seeding. Similar vertical slices should be made to inspect the parenchyma. Samples of the relevant areas need to be taken if there is any macroscopic evidence of splenic pathology. When the spleen appears normal a single block approximately 3 ¥ 2 ¥ 0.3cm should be removed for histology, which should include the capsule on one side.
Special Techniques for the Spleen
Microbiology of the Spleen
This investigation may be required when there is known sepsis or alterna- tively may be indicated following an observation noted during the post mortem examination itself, such as peritonitis. An important clue to septi- caemia is the presence of a large, wrinkled, and extremely soft spleen (dif- The Spleen 253
Figure 10.3. The spleen is sliced at parallel intervals of no more than 1 cm apart to demonstrate the parenchyma.
fluent). Slicing is often difficult and reveals a core of mushy tissue that is liquefied and runs over the capsule as an amorphous mass. If infection is suspected then it is best to obtain splenic tissue or a splenic swab prior to contamination of the spleen during slicing. The capsular surface should be seared with a very hot sterile metal implement such as the flat side of a scalpel blade and a cut made through this area using a sterile scalpel blade.
A small piece of deeper splenic parenchymal tissue should then be removed and placed in a sterile container for transportation to the microbiology department. Alternatively, a sterile swab can be inserted into the original defect and transferred to the microbiological department in a swab tube.
Microbiological sampling of splenic tissue is summarised as follows:
– Sear the outside of the spleen.
– Separate the edges.
– Introduce the swab or remove a cube of tissue.
– Remove the swab and reintroduce into a transport tube or place the tissue into a sterile container.
– Label all specimens and tubes.
– Complete the request form with all relevant information and hazard stickers if necessary.
Fixing the Spleen Whole
Rarely, it may be felt necessary or appropriate to keep the spleen whole for later dissection. In these cases consent must be sought before the spleen is retained. To prevent further autolysis it is possible to inject fixative through the splenic artery at moderate pressure while the splenic vein is clamped.
Bone Marrow
Bone marrow should be examined in cases of hereditary haemoglo- binopathies, haematological malignancies, lymphomas (for staging), dis- seminated cancers, and generalised infections. There are two main sites for examining the bone marrow. The first is to inspect a long bone such as the femur, which gives a good idea of the quantity of any expansion in haemopoeitic tissue. This is fully described in Chapter 11 on p. 261 and is not repeated here. The second area for assessment is within the vertebral bodies. In adults most of the haemopoeitic marrow is concentrated in the axial skeleton and it is very easy to either use the electric saw or even a handsaw to remove the anterior portions of the lumbar vertebral bodies and inspect the exposed marrow. This can be performed most easily by approaching the vertebral column from the anterior aspect after all of the organs have been eviscerated. All of the bulky paravertebral skeletal
muscle (e.g., psoas and paraspinal muscles) and soft tissue should be removed using a PM 40 to allow easier access for sawing. This also prevents the soft tissue from causing dangerous slippage of the electric or hand saw.
Sawing begins along the lateral border of the vertebral bodies bilaterally and the cut is extended around the lower border of the lumbar spine just above the pelvis. A superior cut is then made with a large scalpel (PM40) through the surrounding soft tissue and through an appropriate disc/disc space. This block of tissue can now be lifted off and the undersurface exam- ined. The tissue can be decalcified (following the method discussed later) before sampling for histological examination or a small block cut prior to declacification.
Rarely, it may be necessary to examine bone marrow from the sternum.
For this the sternum is removed in the usual way and a cut is made across the bone with a handsaw to expose the integral marrow tissue. The latter can be scooped out with a curette for fixation and microscopy.
Special Techniques for Bone Marrow
Decalcification of Tissue for Bone Marrow Histology
When histological assessment of the bone marrow is required, it will usually be necessary to decalcify the tissue prior to preparation of the sections. This is also essential for diagnosis of any calcified lesions or bony tumours. It is first important that an adequate sample of the relevant tissue is fully fixed before any of the following methods of decalcification are started. Decal- cifiers are either acids or chelators. The acids may be strong or weak but the latter is preferable, as preservation of staining characteristics is supe- rior. One of the most commonly used techniques is immersion of the fixed tissue in formic acid (5% to 10% solution) which is then left for an appro- priate length of time. With small pieces of tissue, decalcification will require only a matter of 24 to 48 hours, but larger segments of tissue will need longer periods. In the latter situation, it may be necessary to take serial X- ray films of the specimen to assess the amount of calcium still present in the tissue. Once appropriately decalcified, blocks of the usual size can be trimmed from the specimen and sections cut in the routine way. An alter- native is to embed the bone marrow in resin which can be sectioned on a special microtome without the need for decalcification.
If the calcified tissue is not in fact appreciated until the sectioning stage then only brief surface decalcification may be necessary. The block is either bathed in 1% hydrochloric acid for 15 to 60 minutes prior to sectioning or the block is placed face down on a cotton wool pad soaked in 10%
hydrochloric acid before the sections are cut. It should be remembered that with all of these methods there may well be some resultant loss of staining ability in the tissues.
Bone Marrow 255
The Thymus
In the vast majority of adult post mortems the thymus will not be relevant to the cause of death and it is rarely the site of significant pathology. As it naturally atrophies with age it is either glossed over or not appreciated in the course of most examinations. The size of the thymus gland is therefore extremely dependent on age and is given in Appendix 2. Certain patholog- ical processes may involve the thymus in adults, and in these cases the thymus should be removed complete and histology taken. The method for removing a thymic tumour follows the description given later in this chapter for any mediastinal mass. Blocks approximately 3 ¥ 2 ¥ 0.3cm should be removed from a thymic tumour, including samples from the periphery/
normal tissue junction, for histological assessment.
In young children and infants the thymus may be more relevant to the underlying pathological process and is usually more conspicuous. It becomes part of the routine examination. It should be weighed accurately and examined carefully both macroscopically and microscopically with at least one block taken for histology (see Chapter 14).
Other Special Techniques
Dissection of the Thoracic Duct
The thoracic duct is a structure that is conventionally ignored during the post mortem examination. It may be relevant in some settings, however, to dissect out the duct. The most likely situations include necropsies with malignancy, tuberculosis, and situations with a chylous pleural effusion. The thoracic duct is a thin-walled cord-like lymphatic vessel that originates at the cisterna chyli adjacent to the lower end of the abdominal aorta on the right side. It passes superiorly beside the aorta through the diaphragm. In the thorax it passes across the midline, medial to the main azygous vein on the left, at the level of the aortic arch/fifth thoracic vertebral body. It then runs posteriorly above the large arteries and veins passing to or returning from the left shoulder and arm and sweeps down and enters the left bra- chiocephalic or subclavian vein.
It is wise to identify the thoracic duct before the main thoracic eviscera- tion takes place, as this thin-walled structure is rather difficult to assess afterwards. This is best accomplished by lifting the left lung forward and inspecting the posterior mediastinum. The intercostal arteries are tran- sected close to the aorta in the lower part of the mediastinum. The aorta is pulled to the right to expose the retro-aortic adipose tissue and the thoracic duct is identified. This is then held with forceps while the duct is dissected off its neighbouring tissues superiorly and inferiorly, cutting across the remaining intercostal arteries. Dissection can be aided by injecting warm
5% gelatin into the duct in order to aid visualisation and provide a firm structure to grasp, although in practice this a laborious procedure that is very rarely performed. Barium sulphate can also be injected at this time if post mortem lymphangiography is considered necessary. If the thoracic duct is obviously macroscopically abnormal, tissue can be taken for microscopic investigation in as sterile a manner as possible.
Removal of a Mediastinal Mass
The following technique should be followed whenever a large mediastinal mass is present, as it is important to identify the site of origin of the pathol- ogy and this may not be achievable once the individual organs have been removed. This method is also applicable to cases with mediastinitis with or without interstitial emphysema. Any of the general preparation techniques are reasonable but when the sternum is removed this should be done with particular care, as damage to substernal tissue may make further assessment difficult. With large masses it is likely that pleural extension will be present and so it is best to release the parietal pleura from the chest wall to keep it in continuity with the intrathoracic organs (described more fully in Chapter 3). Rarely, it may be necessary to cut the local sternal bone involved by the mediastinal disease with a saw and remove it with the mass in continuity.
The mediastinal structures should now be removed as a unit following either the en bloc or en masse technique as previously described. If the en masse method is used the upper structures will need to be separated from the abdominal organs by dividing all of the soft tissue just above the diaphragm exactly as described for removing the thoracic pluck of Ghon’s evisceration technique. Depending on the suspected pathology and the size of the lesion it should now be possible to separate those organs that are not involved and remove them one by one following standard procedures (i.e., the Ghon or Virchow methods previously described). This will obviously be most appropriate and easiest for small tumours but less suitable for large- scale disease processes.
With larger tumours or inflammatory conditions infiltrating its neigh- bouring structures, there are two possible ways to best demonstrate the pathology macroscopically. The first and probably the easiest is to leave the aggregate of organs intact and make large transverse sections through the whole block of tissue with a large sharp knife, leaving about 1 to 2 cm between slices. This can be performed either on the fresh tissue or after the block of tissue has been fixed complete to be sliced later.
Alternatively, the routine thoracic block dissection method can be fol- lowed. This is probably performed more easily from the posterior aspect for anterior mediastinal tumour and it may be easier to open all of the sur- rounding organs in the mediastinal block and leave the main mass for dis- section after assessing these other structures. It is important to remember that Other Special Techniques 257
mediastinal masses need not necessarily be neoplastic in nature and that some may actually be inflammatory, such as sclerosing mediastinitis or tuberculosis. Obviously with the latter and a risk of infection, this should be handled appropriately.
If consent has been obtained, blocks of the usual size (up to 0.3 to 0.4 cm thick) should be taken for histology in the normal logical way, with areas of tissue sampled from the main mass, the junction between mass and adja- cent tissue, and all other relevant organs.
The Post Mortem in Sickle Cell Disease and Trait
Deaths in patients with sickle cell disease and sickle trait require careful clinicopathological correlation and if the approach described in this text for a routine examination is adhered to, the cause of death should become apparent if it is ascertainable. Experience has shown, however, that pathologists encounter difficulties when assessing sickle-related deaths.
The examination should take place as soon after death as possible to avoid potential problems with interpretation of red cell morphology. Occa- sionally red cells sickle after death and sickled cells unsickle [1]. It is essen- tial to obtain all clinical information, including hospital notes, radiographs, and results of all recent tests, including microbiology. The case should be discussed with the appropriate clinicians prior to the examination to obtain a clear sequence of events leading to death. The clinicians should be invited to attend the examination, or at least attend the demonstration afterwards, to discuss the findings.
If an overdose of painkilling medication is suspected then blood and urine samples should be taken early during the examination and sent for toxicological analysis. Blood and lung tissue samples should be obtained for microbiological assessment. The heart is examined particularly carefully for all causes of sudden cardiac death (in the appropriate clinical setting). His- tology should be taken from all relevant organs. In particular the heart, lungs, kidneys, muscle, and bone (with marrow) should be examined histo- logically. Samples should be fixed in buffered formalin to reduce post mortem intravascular sickling.
The haematopoeitic and lymphoreticular systems examination is sum- marised as follows:
– Lymph nodes are assessed during evisceration or block dissection and sliced if necessary.
– The thymus is examined during evisceration.
– The thoracic duct is identified and examined during evisceration.
– The spleen is sliced after removal.
– Bone marrow is assessed when required.
– Special techniques are employed as necessary.
Reference
1. Royal College of Pathologists. Guidelines on autopsy practice, 2002.
Reference 259
