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The Detection of Prion Protein in CJD

Unique to Creutzfeldt-Jakob disease (CJD) is the accumulation of an abnormal protein in the central nervous system (CNS) known as prion protein (PrPSC). Immunocytochemistry is essential for the demonstration of PrPSC along with rigorous pretreatments to ensure that normal PrPC is destroyed and only PrPSC is demonstrated. The emergence of variant CJD (vCJD) has resulted in a wider spectrum of PrPSC distribution, including lymphoid tissue involvement (e.g. tonsil) where small quantities of PrPSC have been detected. The labeling of endogenous biotin can be a problem, especially in the peripheral tissue when amplification techniques using streptavidin-biotin were used.

Using 5 sporadic CJD cases (sCJD) and 3 vCJD cases with consent for research, immunocytochemistry was performed using the NovolinkTM Polymer Detection Kit to ensure the level of PrPSC detection was acceptable in both the CNS and peripheral tissue, and to ensure the problem of endogenous biotin was removed. We also decided to expand our trial to look at the other neurological markers with a view to including this kit in our repertoire.

The results showed that the Novolink System is sensitive enough to detect small quantities of PrPSC in both CNS and peripheral tissue, removed the biotin problem and is equally successful with other routine neuropathological antibody markers.

Introduction

Creutzfeldt-Jakob disease (CJD) belongs to a group of fatal neurological disorders known as the transmissible spongiform encephalopathies (TSE). These diseases were brought to the forefront of media attention due to the emergence of bovine spongiform encephalopathy (BSE).

Neuropathological examination of the brain is essential for the diagnosis of these disorders, which are characterized by spongiform change, neuronal loss, astrocytosis and amyloid plaque formation in the grey matter.1 The severity and distribution of these lesions are markedly variable in different cases of this disease.

Unique to all human TSEs is the accumulation of an abnormal protein which is protease resistant and known as prion protein (PrPSC). Normal PrP (PrPC) is a cell membrane associated glycoprotein that is encoded on the human chromosome 20 and is expressed in normal neurones, glia and many cells outside the central nervous system (CNS).2 In TSEs, this protein changes shape from an alpha-helix to a beta-pleated structure and this abnormal isomer is particularly resistant to denaturation. Immunocytochemistry is essential for the demonstration of this abnormal isoform in sections of the brain for the diagnosis of CJD. The immunocytochemistry for PrP has revealed a variety of deposition patterns which may reflect host genetic differences and protein strain types.3

In 1996 a new variant form of CJD was identified, which represents the effect of the BSE agent in humans, presumably acquired through the food chain.4,5 This variant CJD (vCJD) showed new pathological features for a human TSE, including peripheral tissue involvement. PrPSC has been identified in the lymphoid tissue of tonsil, appendix, spleen, and lymph node.6 This involvement of lymphoid tissue has led to speculation concerning the role of the lymphoid system in carrying infectivity, for example from the gut to the lymphoid tissues, if one assumes an oral route of infection. The deposition of PrPSC in peripheral tissue is relatively low compared to that found in the CNS7, so it is important that the immunocytochemical technique selected is capable of detecting very small deposits of PrPSC.

Since the currently available antibodies recognize both isoforms of the protein rigorous pretreatments are needed to ensure that the normal protein is denatured and hence not demonstrated. In order to maximise the sensitivity and specificity of staining a number of pretreatment stages have been developed and tested on infected tissue. These pretreatments involve the use of antigenic retrieval with hydrated autoclaving, enhancement with formic acid, and digestion with Proteinase K followed by an immunocytochemical technique. Several different immunocytochemical methods can be employed to detect PrPSC and the majority of methods used today are based on an avidin or streptavidin-biotin enzyme complex reacting with a biotinylated secondary antibody (ABC).7,8 The introduction of the catalysed signal amplification (CSA) in which the streptavidin-biotin interactions are greatly amplified has played an important role in the detection of small quantities of PrPSC and resulted in a method, which is sensitive enough to detect antigenic sites which may have gone undetected previously. However non specific staining due to endogenous biotin binding is a problem that occurs with the streptavidin-biotin detection systems and unfortunately in the peripheral tissue unwanted biotinylated sites are occasionally being detected with CSA, even though an avidin-biotin block is incorporated. The introduction of the Novolink Polymer Detection System, which is based on a polymeric HRP-linked antibody conjugate is a system which should theoretically remove this problem. We decided to try the Novolink Polymer Detection System on both CNS and peripheral lymphoid tissue using a panel of PrP antibodies to ensure that it was capable of detecting the various patterns of PrPSC deposition that can occur in the CNS and also the small amounts of PrPSC that may occur in the peripheral tissues. We also decided to expand the trial to include our routine neuropathological markers such as beta-amyloid, Glial Fibrillary Acidic Protein (GFAP), Ubiquitin (Dakocytomation, UK), alpha-synuclein (Invitrogen, UK), betaAPP (Chemicon) and Tau (Autogen Bioclear, UK) with a view to incorporating this kit in our laboratory repertoire if successful. These antibodies did not require the same rigorous pretreatments that were applied to the PrP antibody detection.

Material and Methods

Five sporadic (sCJD) and three vCJD cases with full consent for research use were selected. Formalin fixed paraffin wax blocks were taken from frontal cortex, temporal cortex, occipital cortex, basal ganglia, thalamus and the cerebellum. Peripheral lymphoid tissue including tonsil, spleen, lymph node and appendix were also selected. Prior to processing all the blocks were inactivated by immersion in 96% formic acid for 1 hour then washed thoroughly in water. The paraffin embedded CNS blocks were cut at 5 µm and the peripheral blocks cut at 4 m and then dried overnight in a 60 °C incubator.

Prior to immunocytochemistry, the sections were pretreated with hydrated autoclaving at 121 °C for 10 minutes, followed by 96% formic acid for 5 minutes and finally immersion with proteinase K (10 g/ml) for 5 minutes at room temperature. After these pretreatments, immunocytochemistry was performed using the Novolink Polymer Detection System from Leica Microsystems. The mouse monoclonal PrP antibodies used were KG9 (IAH, TSE Resource Centre, UK), 3F4 (Dakocytomation, UK), 6H4 (Prionics, Switzerland) and 12F10 (IDS, UK). Diaminobenzidine (DAB) was used for visualization and the sections were lightly counterstained with haematoxylin.

Two Alzheimer cases with consent for research use were selected to test our routine neuropathological markers. Formalin fixed paraffin wax blocks were taken from frontal cortex, temporal cortex and occipital cortex. Prior to processing all the blocks were inactivated by immersion in 96% formic acid for 1 hour then washed thoroughly in water. The paraffin embedded CNS blocks were cut at 5 µm and then dried overnight in a 60 °C incubator. Immunocytochemistry was performed using the Novolink Polymer Detection System from Leica Microsystems. The antibodies used were beta-amyloid, Glial Fibrillary Acidic Protein (GFAP), Ubiquitin (Dakocytomation, UK), alpha-synuclein (Invitrogen, UK), APP (Chemicon) and Tau (Autogen Bioclear, UK) and all these did not require the rigorous pretreatments used for PrPSC detection.

Immunocytochemical Results

Sporadic CJD

The immunocytochemistry for PrP revealed a variety of deposition patterns in the sporadic CJD cases.

When severe spongiform change was seen in the grey matter and the vacuoles had coalesced to form confluent spongiform change the chromogen was seen as dense granular deposits within and around the vacuoles. This is referred to as Perivacuolar staining (Figure 1). Sometimes the amount of chromogen deposited was quite small, even though dense staining was being produced in the same area. When the vacuoles present were smaller and scattered evenly throughout the cortex i.e. microcystic, there was no evidence of PrP positivity.

Some of the sporadic cases showed the PrP positivity in the form of plaques. These characteristic unicentric kuru type plaques are composed of a homogenous centre surrounded by a densely stained, slightly stellate ridge, which is enclosed by a paler periphery. The overall effect is that of a halo surrounding a PrP positive centre (Figure 2).

In some cases a generalized staining pattern in the neuropil was noted. On closer inspection this was revealed as very small granular depositions of the chromogen along the cell processes in the neuropil and this pattern of staining is referred to as diffuse or synaptic staining. In some cases the synaptic labeling can appear as a ribbon of positivity running through the cortex, along with prominent perineuronal deposits surrounding the neuronal somata and processes (Figure 3). Variations within the same case also occurred, e.g. vacuolation in the grey matter plus plaques in the cerebellum.

The Novolink Polymer Detection System demonstrated these different patterns. All of the PrP antibodies were able to detect the coarser and plaque type deposits, but the 3F4 did demonstrate the synaptic staining more intensely. The KG9, 12F10 and 6H4 antibodies gave good, consistent results along with a clean background.

The Novolink kit also successfully demonstrated the deposition of PrPSC with little or no endogenous biotin staining and there was no cross reaction with the blood vessels. The lymphoid tissues in the sCJD cases all gave negative results.

vCJD

Unlike the sCJD cases, where focal PrPSC deposition occurred, there was extensive deposition of PrPSC in the CNS of the vCJD cases, along with different patterns of PrPSC deposition. One of the pathological patterns observed was the extensive “florid” plaque formation, which was found throughout all areas of the grey matter of the cerebral (Figure 4) and cerebellar cortex. Numerous smaller plaques were also seen, arranged in irregular clusters. These all stained strongly with the Novolink Polymer Detection System. Other features unique to vCJD, are pericellular depositions of PrPSC around small neurones and astrocytes. In the basal ganglia and thalamus a linear and perineuronal pattern is identified. These were also clearly demonstrated by the Novolink System. Like the sporadic cases, the Novolink System also successfully demonstrated the deposition of PrPSC with no evidence of endogenous biotin-linked staining and there was no cross reaction with the blood vessels.

Positive staining of PrPSC in lymphoid tissue was found in the follicular dendritic cells situated within the germinal centres (Figure 5) of the tonsils from the vCJD cases but not in the sCJD cases. The sections did not have endogenous biotin-linked staining which is a common problem with biotin based amplification kits. PrPSC was also detected in small amounts in the appendix, spleen and lymph node. The sections had a clean background, and again no endogenous biotin-linked staining was present.

Routine Neuropathological Markers

All the antibodies labelled very strongly with a clean background. The antigenic sites ie Alzheimer plaques and tangles were all detected well. Figure 6 demonstrates Beta-Amyloid, a routine marker for Alzheimer disease.

Discussion

There was a need in our laboratory to find an immunocytochemical kit, which was capable of detecting the different patterns of PrPSC deposition in the CNS, and small deposits in the peripheral tissue, but which would also prevent the endogenous biotin-linked staining that can occur resulting in interpretation problems. It was important that there was no loss of sensitivity and specificity in the technique and it would also be advantageous to employ a technique which was not time consuming and so allow a greater flexibility during the working day for staff members. Unfortunately the existing amplification techniques did not fulfill this role. When testing the Novolink System we found it was able to fulfil these needs. The Novolink System provided all the components necessary to undertake this immunocytochemical procedure and the comprehensive step by step instructions were easy to follow. By incorporating the Novolink antibody diluent we were able to remove the protein block stage, and with only two incubation steps following incubation in the primary antibody, the method was completed by mid afternoon despite the long pretreatments that are essential for PrPSC demonstration.

The only disadvantage we found using the kit was during the microscopic visualization step. DAB precipitate caused difficulty in identifying small amounts or fine staining of PrPSC deposition in the sections. We experimented with several different methods in an attempt to alleviate this problem. Filtering the working DAB solution resulted in labelling that was less intense, so this was not an option. It was found that with the addition of several wash steps after the DAB, the problem seems to be resolved.

The kit also has the advantage of being a universal kit for both monoclonal and polyclonal antibodies which reduces the possibility of the operator using an inappropriate kit for the antibody. When tested with the other routine neuropathological markers we were able to reduce the working concentrations of the antibodies, an important impact on laboratory costings. In addition the kit is available in different size formats, and there is also the option to buy the components separately, thus giving individual laboratories a greater buying flexibility.

Conclusion

The results show that the Novolink System is sensitive enough to detect small quantities of PrPSC in both CNS and peripheral tissue. The use of polymer technology means that the problem of endogenous biotin labelling has been removed and so there are no longer difficulties in the interpretation of small deposits of PrPSC. In this current climate of staffing shortages, it is important to employ a method which is time effective without compromising the specificity of the technique. The use of the Novolink System has been a welcome addition to our laboratory repertoire with its successful demonstration of PrPSC in brain and lymphoid tissue without compromising sensitivity and specificity and is equally successful with other routine neuropathological antibody markers.

Figures

Figure 1. Demonstrates the PrPSC deposition around perivacuolar staining in the temporal cortex from a sCJD case (KG9, x20).
Immunocytochemical staining demonstrating PrPSC deposition around perivacuolar staining in the temporal cortex from a sCJD case (KG9, x20).
Figure 2. The “classic kuru plaque” located in the granular layer of the cerebellum which is a characteristic of sCJD (6H4, x 20).
Immunocytochemical staining demonstrating “classic kuru plaque” located in the granular layer of the cerebellum which is a characteristic of sCJD (6H4, x 20).
Figure 3. Fine synaptic staining (red arrows) along with the perineuronal staining on the somata of the neurones (black arrows) in a frontal cortex from a sCJD case (3F4, x20).
Immunocytochemical staining demonstrating fine synaptic staining along with the perineuronal staining on the somata of the neurones in a frontal cortex from a sCJD case (3F4, x20).
Figure 4. Heavy deposition of PrPSC in a vCJD case showing the characteristic “florid plaque” (black arrows) along with pericellular staining (red arrows) in the temporal cortex (12F10, x 20).
Immunocytochemical staining demonstrating heavy deposition of PrPSC in a vCJD case showing the characteristic “florid plaque” along with pericellular staining in the temporal cortex (12F10, x 20).
Figure 5. PrPSC positivity in the germinal centre of a tonsil from a vCJD case (12F10, x 20) showing a clean biotin free background.
Immunocytochemical staining demonstrating PrPSC positivity in the germinal centre of a tonsil from a vCJD case (12F10, x 20).
Figure 6. Demonstrates classic beta-amyloid plaques (black arrows) and diffuse plaques (red arrows) in an occipital cortex from a case of Alzheimers disease (beta-amyloid, x 20).
Immunocytochemical staining demonstrating classic beta-amyloid plaques and diffuse plaques in an occipital cortex from a case of Alzheimers disease (beta-amyloid, x 20).