AgNOR expression in Central Nervous System Tumors - Scientific ...

Volume 4, Issue 1, 2011
AgNOR expression in Central Nervous System Tumours
Dr. Sushil Lakra M.D., Assistant Professor, Department of Pathology, Faculty of Medicine, Misurata University,
Libya. sushil_lakra2003@yahoo.com
Abstract
AgNOR (Argyrophilic nuclear organizers regions) technique is simple, rapid, inexpensive and can be performed
on formalin fixed, paraffin- embedded tissue including archival material. Therefore, unlike most available
‘proliferation’ techniques, it does not require special processing of tissue. When a set of observers and laboratory
become familiar with the technique, counting of AgNOR should be included as one of the techniques in routine
diagnosis along with Haematoxylin and Eosin staining in deciding the distinction between normal brain tissue and
case of definite low grade astrocytoma. Therefore, if AgNOR is used in concert with clinical information and
routine light microscopy, the AgNOR technique may prove adjunct in pathological distinction between normal
brain tissue showing gliosis and low grade astrocytoma and distinction between benign and malignant tumors.
The conclusion drawn from the present study is that the AgNOR technique can be used to distinguish between
normal and benign lesions, benign and malignant lesions, and low versus high grade malignant lesions.
Introduction
Argyrophilic nucleolar organizers regions (AgNOR) which are identified by a silver staining technique have been
reported to reflect cellular activity and proliferative potential (Egan M.J. 1988). Nucleolar organizer regions may
be visualized by number of ways. The simplest and most widely used method is silver colloid impregnation. This
technique has been shown to be specific for carboxyl and sulphydryl-rich proteins associated with NOR (Nuclear
organizer region) (Crocker J. 1990). The technique is rapid and can be performed on formalin fixed paraffin
embedded tissue. The AgNOR method has been used both to grade human tumors and distinguish benign from
malignant lesions (Editorial 1990).
NORs have attracted much attention because of claims that their frequency within nuclei is significantly higher in
malignant cells than in normal, reactive or benign neoplastic cells. Because NORs can now be demonstrated in
routinely processed histological sections, the techniques are obviously of potential value in diagnostic
histopathology (Underwood J.C.E.1988). The principal advantage of the AgNOR technique is the relative
simplicity of the staining method and the ease of its application to archival tissue. Disadvantages include the time
consuming and tedious counting of the little dots, often clustered associated with the usual vagaries of observer
error (Underwood J.C.E.1992).
The AgNOR method can be applied to various materials including cells in smears, semi-thin sections of plastic
embedded cells and sections of paraffin embedded human pathological specimens. Nuclear organizer regions
have been shown to reflect cellular proliferation or malignancy, although they are not directly related to the cell
cycle. NORs are loops of ribosomal DNA present in nucleoli. The DNA poss esses ribosomal RNA genes that are
transcribed to ribosomal RNA by RNA polymerase I. NORs are demonstrated by means of the argyrophilia of
their associated proteins (AgNORs) such as RNA polymerase I, C23 protein and B23 protein. These proteins are
thought to play some role in the transcription of ribosomal RNA (A. Hara 1991). In the human karyotype NORs
are located on the short arms of chromosomes 13, 14, 15, 21, and 22 (Underwood J.C.E.1988).
In Central Nervous System (CNS) tum ors, many times the light microscopic distinction between gliosis and low
grade astrocytoma is difficult with Haematoxylin and Eosin (H&E) stained sections. It can be evaluated with the
use of the AgNOR impregnation (David N. Louis 1992). It has been thought that AgNOR impregnation can be
used as an additional diagnostic index to provide help in differentiating reactive gliosis and benign or malignant
tumors of CNS. It may helpful to assess the prognosis of the lesions (Louis D.N. 1992).
When using cytological imprint preparation in studies, the AgNOR count is found to be much higher for low grade
astrocytoma than in sections of paraffin embedded pathological studies. It also showed a rough correlation
between AgNOR count and astrocytoma grades. For instance the use of cytologic preparation may give higher
count presumably because all AgNORs in nucleus are visible not just those present in 3 micron thick tissue
sections (Plate K.H. et. al 1990).
1

The present study was designed to compare mean AgNOR counts in benign and malignant neoplastic lesions of
the central nervous system and to find out the utility of AgNOR count in diagnosis of central nervous system
neoplasms.
Objectives
1. To compare mean AgNOR count in benign and malignant neoplastic lesions in central nervous system.
2. To find out utility of AgNOR count in diagnosis of central nervous system neoplasms.
3. To compare AgNOR count with histological grading of central nervous system tumors.
4. To compare the present study with other workers’ studies.
Materials and Methods
The present study was conducted on surgical biopsies from 100 cases of CNS which were over a period from
July1996 to July 1998 received in the department of pathology, Gajra Raja Medical College Gwalior, Madhya
Pradesh, India. Surgical biopsies were colleted in 10% buffered formalin from cases undergoing surgery. The
biopsies were subjected to routine paraffin sections. Histopathological diagnosis was first established on these
sections obtained from paraffin blocks using the routine H&E stains. Other paraffin blocks sections were
subjected to the AgNOR staining technique.
Paraffin sections:
The tissue was fixed in 10% formalin.
Pieces of the fixed tissue were subjected to the procedures of the dehydration, cleaning and embedding
in automatic tissue processor.
The dehydrated cleared tissue pieces were further impregnated with paraffin wax by immersion in a
succession of wax bath on the automatic tissue processor.
The treated tissue was embedded in paraffin wax. The blocks were made using a shaped metallic
mould. The embedded tissue blocks were allowed to cool.
The paraffin wax blocks were fixed on a rotary microtome and sections of 4 µm thickness were cut.
The cut sections were transferred to a water bath and thereby picked on albumin filmed glass slides.
The sections were fixed to the slides by keeping them in incubator at 37° C overnight.
The sections were then subjected to the H&E and AgNOR stains.
H&E Stain (Haematoxylin and Eosin stain):
Paraffin sections obtained were incubated at 37° C overnight.
The sections were dewaxed in xylene, hydrated through various grades of alcohol.
Sections were rinsed in running water for one minute and then briefly in distilled water.
Sections were stained in Ehrlich′s Haematoxylin for 8 minutes.
Differentiation was done by dipping the stained sections in 1% acid alcohol (1ml conc. HCl to 99ml of
80% ethyl alcohol).
Sections were rinsed in water.
Sections were blued by placing them in tap water until the sections appeared blue.
Sections were rinsed well in water.
Sections were stained with aqueous eosin Y for 2 minutes.
Sections were rinsed well in water.
Sections were dehydrated through different grades of alcohol.
Dehydrated sections were passed through two baths of xylene.
Sections were dried and mounted with 80 Dibutylphthalate xylene (DPX) mountant.
Preparation of AgNOR staining solution:
A solution was prepared by dissolving 2 gm of gelatin in 1% aqueous formic acid to make 100 ml of
solution at a concentration of 2%.
50%aqueous silver nitrate solution was prepared by dissolving 5 gm of silver nitrate in triple distilled
water to make 10 ml of solution.
The solutions (a) and (b) were mixed in a proportion of 1:2 respectively to obtain the final AgNOR
staining solution.
AgNOR staining technique:
Paraffin sections were incubated at 37° C overnight, further dewaxed in xylene, dehydrated through
various grades of ethanol and washed well with triple distilled water. The sections were dried thoroughly
and subjected to AgNOR stains.
The AgNOR stain prepared as mentioned above, was poured over the tissue sections and left for 60
minutes at room temperature.
The silver colloid was washed off with triple distilled water thoroughly.
Sections were dehydrated through various grades of alcohol and cleared in baths of xylene.
Stained sections were mounted with DPX mountant.

Precautions taken:
Particular attention was paid to the cleanliness of glassware and purity of water in order to avoid background
staining and non specific granular deposits on tissue sections.
Counting procedure:
AgNORs were counted as brown-black dots in the nuclei of cells using a 100X oil immersion objectives.
100 cells were studied in each case; the mean AgNOR per nucleus was calculated.
AgNORs were counted in the normal brain tissue, benign and malignant tumors of CNS cells. The cells
in all above cases were chosen randomly.
The final scores in normal brain tissue, benign and malignant lesions were compared.
The data obtained were subsequently correlated with the histopathological diagnosis.
Results
The present study was conducted on surgical biopsies from 100 cases of CNS tissue received in the department
of pathology, Gajra Raja Medical College Gwalior, Madhya Pradesh.. There were 30 cases of normal brain
tissue, 25 cases of astrocytoma in which there were 15 grades I & II and 10 grade III & IV, 10 cases of
meningiomas, 13 cases of neurofibroma and 22 cases of others in which 4 cases of glioblastoma multiforme, 2
cases of ependymomas, 5 cases of medulloblastomas, 2 cases of Schwanomas, 5 cases of heangioblastoma, 2
cases of craniophangiomas and 2 cases of secondary metastatic tumors. Each of these specimens was
subjected to paraffin. H&E staining was done on one paraffin section and one section was stained by the AgNOR
staining technique.
The sections were examined under oil immersion objective 100X. AgNOR were visible as dark brown intranuclear
dots, against yellowish background. They were counted in 100 cells in each section. Each dot was class ified as
small, medium and large according to its size.
A small dot was defined as just visible but distinct. A dot about three times the size of small was classified as
medium and those about five times or more classified as large dots. Care was taken to count AgNOR in groups
of cells.
Fields were selected at random and 100 cells were taken into count. The mean number of AgNORs per cell is
then calculated. Simultaneous morphology of AgNOR dots was also observed. The AgNOR dots lying in groups
and clusters were counted as one structure.
In benign lesions the AgNORs were small, round and regular while in malignant lesions the AgNORs were often
angulated and irregular. Errors were avoided by counting AgNORs in thinnest part of the sections. Single naked
nuclei present in background were not taken in analysis.
The histopathological diagnosis of each case was based mainly on the H&E stained sections.
Table 01: Comparative mean AgNOR count in normal brain tissue and astrocytoma:
S.NO. Groups Range of AgNOR mean AgNOR count/100 cells
count/100 cells
1 Normal brain tissue 0.68 to 1.28 0.87±0.14
2 Astrocytoma grade I &II 1.00 to 1.80 1.17±0.19
3 Astrocytoma grade III &IV 1.65 to 2.38 2.05±0.17
Table 1 shows the comparative mean AgNOR count in three different grades of brain tissue. In group I or normal
brain tissue the mean AgNOR count is 0.87±0.14. In group II or astrocytoma grade I &II the mean AgNOR count
is 1.17±0.19. In group III or astrocytoma grade III &IV the mean AgNOR count is 2.05±0.17.
Figure 01: Mean AgNOR counts in normal versus astrocytoma tissue.
3

2.5
2
2.05
1.5
1
0.5
1.17
0.87
Astrocytoma grade III &
IV
Atrocytoma grade I &II
Normal brain tissue
0
Figure 01 shows the mean count in a normal versus astrocytoma tissue. X-axis represents the mean AgNOR
count per 100 cells and Y-axis indicates the normal brain tissue, astrocytoma grade I &II and astrocytoma grade III
&IV. Astrocytoma grade III & IV has the highest AgNOR count when compared with astrocytoma grade I &II and
normal brain tissue.
Table 02: Comparative mean AgNOR count in different benign tumors:
S.NO Types of benign tumors Range of AgNOR count/100 Mean AgNOR count /100 cells
cells
1 Schwannoma 1.40 to 1.80 1.60 ± 0.20
2 Neurofibroma 0.78 to 2.10 1.32 ±0.32
3 Meningioma 1.00 to 2.80 1.87 ±0.42
4 Craniopharyngioma 2.0 to 2.20 2.10 ±0.20
Table 02 shows the comparative mean AgNOR count in different benign tumors of CNS. Craniopharyngioma
showed highest mean AgNOR index (2.10 ±0.20) with variation among the individual count (range from 2.00 to
2.20). Other subgroups Schwannoma, neurofibroma and meningioma were found to have mean indices close to
each other 1.60 ± 0.20, 1.32 ±0.32 and 1.87 ±0.42 respectively.
Figure 02: Comparative mean AgNOR counts in different benign tumors:
2.5
2
1.5
1.6
1.32
1.87
2.1 Schwannoma
Neurofibroma
1
Meningioma
0.5
0
Craniopharyngioma
Figure 02 represents the comparative mean AgNOR counts in different benign tumors-including, schwannoma,
neurofibroma, meningioma and craniopharyngioma. The X- axis represents the mean AgNOR count/100cells and
the Y-axis indicates the different types of benign tumors. Craniopharyngiom has the highest AgNOR count and
neurofibroma has the lowest compared to other benign tumors.
Table 03: Comparative mean AgNOR count in different malignant tumors:
S.NO. types of malignant tumors Mean AgNOR count /100
cells
1 Astrocytoma grade I &II 1.17±0.19
2 Astrocytoma grade III &IV 2.05±0.17
3 Glioblastoma multiforme 2.67 ±.64
4 Haemangioblastoma 2.22 ±.44
5 Ependymoma 3.88 ±.43
6 Medulloblastoma 3.52 ±.43
7 Secondary metastatic tumor 3.90 ±.90

Table 03 shows the comparative mean AgNOR count in different malignant tumors. Secondary metastatic tumor,
medulloblastoma and ependymoma had the higher AgNOR count of 3.90 ±.90, 3.52 ±.43 and 3.88 ±.43
respectively. Astrocytoma grade III &VI, glioblastoma multiform and haemangioblastoma had the mean AgNOR
count of 2.05±0.17, 2.67 ±.64 and 2.22 ±.44 respectively. The AgNOR dots were large, regular and easily
identifiable in these cases.
Figure 03: AgNOR counts in malignant CNS tumors :
4
3.5
3.88 3.9
3.52 Astrocytoma grade I &II
3
2.67
Astrocytoma grade III
&IV
2.5
2.22
2.05
Glioblastoma
2
multiforme
Haemangioblastoma
1.5 1.17
1
Ependymoma
0.5
Medulloblastoma
0
Secondary metastatic
tumor
Figure 03 represents the comparative AgNOR count in different malignant tumors of CNS.
Secondary metastatic tumor shows the highest AgNOR count as compared to other malignancies. Ependymoma
the primary malignant tumor showed the highest AgNOR count than other primary tumors.
Discussion
The present series reveal significant qualitative and quantitative difference between normal brain tissue, low
grade astrocytoma and high grade astrocytoma and between benign and malignant lesions of the central nervous
system. Theses reports are discussed in light of other reports on the use of AgNOR count in brain biopsies.
The AgNOR technique has been applied to the study of normal brain tissue in a number of studies. Hussain N.et
al (1997) demonstrated that normal brain specimens showed little variation among the individual counts (range-
0.61 to 1.14) with a mean index 0.94±0.24. The result of present study correlates with their study. The mean
AgNOR count of normal brain tissue is 0.87±0.14.
The AgNOR dots in normal brain tissue (1.40 to 1.80 of AgNOR count/100 cells) were small, discrete and not all
the nuclei showed presence of dots in present study which is similar as N. Hussain described. A astrocytoma
grade I &II (1.00 to 1.80 of AgNOR count/100 cells) showed AgNOR were larger and irregular dots than normal
brain. In astrocytoma grade III &IV (1.65 to 2.38 of AgNOR count/100 cells) AgNOR dots were large, irregular
and more than one in nuclei. Figure 01 shows astrocytoma grade III & IV has the highest AgNOR count when
compared with astrocytoma grade I, II and normal brain tissue.
The AgNOR technique has been applied to study of astrocytoma in number of studies. Most have focused on the
use of AgNOR in astrocytoma grading which were as given below in Table 04:
Table 04: AgNOR count in astrocytoma in various studies:
Studies
Kajiwara et al
1990
Plate et al
1990
Hara et al
1991
Shirashi et
al 1991
Louis et.
Al 1992
H. Hussa
in et al
1997
Present
study
Grade I &II 1.75 3.15 1.80 1.98 2.22 1.75 1.17
Grade III & IV 2.01 4.5 2.87 2.41 2.94 2.2 2.05
Table 04 shows an increase in AgNOR counts with increasing grade of tumors. The present study also indicates
that increase in AgNOR count with increasing astrocytoma grade. On comparing our results with the published
material we found that AgNOR count of astrocytoma grade I &II was lower. Kajiwara K. et. al (1990) showed a
linear relationship between AgNOR count and S phase fraction as determined by in vitro Bromodeoxy-uridine
(BRdU) labeling and found that patients with tumor AgNOR count of less than 1.80 had a better prognosis than
those with tumor AgNOR count of greater than 1.80. Hara A. et. al (1991) showed a correlation between AgNOR
count and astrocytoma grades and a correlation between AgNOR count and proliferative potential as determined
5

y Ki-67 labeling. The AgNOR count was found by Hara A.et. al. in astrocytoma grade I &II was higher than in
our study. Plate et. al (1990) using cytological imprint preparation in three separate studies, obtained much
higher AgNOR count for low grade astrocytoma, but also showed rough correlation between AgNOR count and
astrocytoma grades.
Nicoll and Candy (1991) have demonstrated that AgNOR count, while correlating with mitotic rate, do not relate
to the post operative survival in patients with glioblastoma multiforme. Therefore while AgNOR may provide
additional information in assessing tumor malignancy, the significance of the information remains unclear. In the
present study, as follow up was not possible due to time limited study, it is difficult to comment on survival
prognosis of these grades I &II tumors.
The AgNOR dots in different benign tumors were large, regular and easily identifiable and the AgNOR dots in
different malignant tumors were smaller and more widely scattered throughout the nucleus as compared to those
of benign cases. Craniopharyngioma has the highest AgNOR count, neurofibroma has the lowest compared to
other benign tumors as showed in figure 02 and
Secondary metastatic tumor shows the highest AgNOR count as compared to other malignancies. Ependymoma,
the primary malignant tumor, showed the highest AgNOR counts of the primary tumors as showed in figure 03
which is correlating to the results of other workers.
The present study has shown relatively low AgNOR count in the low grade lesions such as astrocytoma grade
I&II and relatively high count in higher grade lesions such as medulloblastoma, astrocytoma grade III&IV,
metastatic carcinoma and haenagioblastoma.
Silver nuclear organizer regions in neoplastic lesions tended to be larger than those in normal brain tissue. The
increase in AgNOR size may reflect increased nucleolar activity. It is important to note, however that the nucleoli
of low grade astrocytoma, while increasing their size do not become compound and fragmented like the nuclei of
higher malignant cells. Field D.H. (1984) this may be due to rapid proliferation in higher malignant cells, since
AgNOR may not completely aggregated in dividing cells, thereby producing compound and irregular early
lobulated nucleoli.
Furthermore clinical studies have shown close correlation of AgNOR number and proliferation rate and imply that
rapidly proliferating high grade malignant tumor cells have higher AgNOR count and more compound, irregular
AgNOR than less rapidly proliferating low grade malignant tumor cells. These differential features provide a
suitable tool for using AgNOR to distinguish normal brain tissue from neoplastic cells and have been used in the
differential diagnosis of human malignancies (Croker J. 1990).
In the CNS, Manuelidis L. (1984) has reported that there are striking difference in the quantity and quality of
AgNOR between normal and neoplastic cells. It has been also demonstrated that AgNORs are precisely
positioned in the nuclei of normal, differentiated central nervous system cells while AgNOR in neuroectodermal
tumor cells were large, multiple and variable in their nuclear position.
The present study supports the findings of other workers by showing significant quantitative and qualitative
AgNOR difference between normal brain and low grade astrocytoma. Low grade astrocytoma had greater than
1.01 AgNOR/ nucleus and often had large and irregular AgNOR while normal brain tissue had less than 1.20
AgNOR/ nucleus and had small and regular compound AgNOR. The majority of low grade astrocytoma
numbered over 1.00 AgNOR/ nucleus (mean= 1.17), Most cases of astrocytoma were well above the 1.01
AgNOR/ nucleus with large and irregular AgNOR and are likely to be neoplastic.
Variability in AgNOR counting exists between laboratories and probably results from difference in impregnation
and counting techniques. For instance the use of cytologic preparation may give higher count presumably
because all AgNOR in nucleus are visible not just those present in 3 micron thick tissue section. Count may also
be higher if 4 or 5 micron thick sections are used rather than the standard 3 micron. Counts may be lowered and
perhaps even erroneous if one does not count all AgNOR individually i.e. multiple distinct intranucleolar AgNOR
should be counted as multiple AgNOR and not as a single AgNOR.
Malignant lesions of the central nervous system had greater than 1.17 mean AgNOR/ nucleus ( astrocytoma
grade I&II). Most of the malignant lesions had multiple, dispersed and irregular AgNOR while benign lesions had
less than 2.22 mean AgNOR count / nucleus (haemangioblastoma).
There is considerable overlap of AgNOR count while comparing astricytoma grade I & II (>1.01) to normal brain
tissue (1.17) to benign lesions (

me to undertake and complete this work. Special thanks to my Dean, Dr. V.K. Joshi without whose cooperation
this interesting subject could not have been developed.
References
1. Ponti, Massimo Geuna and Giorgio Palestro (1996): P53 expression and proliferative activity predict survival
in non-invasive Thymomad. J. Cancer (pred. Oncol) 69: 180-183
2. Akira Hara M.D., Noboru Saki M.D, Hiromu Yamad, M.D, Hiroshi Hirayama M.D, Takiji Tanaka M.D and
Hideki Mri M.D (1991): Nucleolar Organizer Regions in vascular and neoplastic cells of human Gliomas.
Neurosurg. Vol. 29, No. 2.211- 215.
3. Akira Hara M.D., Noboru Saki M.D, Hiromu Yamad, M.D, M.D, Hiroshi Hirayama M.D, Takiji Tanaka M.D
and Hideki Mri M.D (1991): Assessment of proliferative potential in glomatosis cerebri. J.Neurology 238: 80-
82.
4. Anjali Das Gupta, R.N. Ghosh, Ranu Sarkar, R.N. Laha, T.K. Ghosh, Chanda Mokharjee (1997): Argyrophilic
Nucleolar Organizer Regions (AgNORs) in breast lesions. J. of Indian medical associations. Vol. 95. NO.
492-494.
5. Boom A.P, Sharif H. (1989): Value of AgNOR method in prediting recurrence of meningima. J. Clinical
Pathology.42: 1002-1003.
6. Crocker J. Nucleolar Organizer Regions. Current Top Pathology 1990: 91-149.
7. Crocker J., Boldy DAR, Egan m.j: How should we count AgNOR proposal for a standardized approach. J.
Pathology 1989: 158- 185.
8. Crocker J. and Nar P.: Nucleolar Organizer Regions in lymphomas. J. Pathology 151: 118-128, 1987.
9. Crocker J. Ayres J. & Mc Govern j. : Nucleolar Organizer Regions in small cell carcinoma of bronchus:
Thorax (1987) 42:972.
10. D.A. Miller, J.V.G. Dev, R. Tantravahi and O.J. Miller (1976): Suppression of Human Nuclear Organizer
Activity in Mouse – Human Somatic Hybrid Cells: Experimental cell Research 101: 235-243.
11. David N. Louis M.D., Shane M. Mechan M.D., Robert J. Ferrenite M.S., and E. Tissa Hedly Whyte M.D.
(1992): Use of the Silver Nucleolar Organizer Regions ( AgNOR) Technique in the differential diagnosis of
Centarl Nervous System Neoplasia: Jurnal of Neuropatholgy and Experimental Neurology. Vol. 51, NO. 2:
150- 157.
12. Devid Trere, Fulvia Frabegoli, Alessandra Cancelliere, Claudio Cancelliere, Vineenzo Eusebi and Massimo
Derenzini (1991): AgNOR area in interphase nuclei of human tumors correlated with the proliferative activity
Evaluation by Bromodeoxyuridine Labelling and Ki-67 immunostaining. Journal of pathology Vol. 165: 53-59.
13. D. Ploton, M Menagar, P. Jeannesson, G. Humber, F. Pigeon and J.I. Adnet. (1986): Improvement in the
staining and in the visualization of the argyrophylic proteins of the nucleolar organizer regions at the optical
level. Histochemical Journal 18:5-14
14. D. Pratibha, Sarah Kuruvilla (1995): Value of AgNORs in pre malignant and malignant lesions of the Cervix.
Indian Journal Pathol. Microbiol. 38: 11-16.
15. D.W. Morgan, J.Crocker, A. Watts and P.M. Shenoi. (1988): Salivary Gland tumors studied by means of the
AgNOR technique. J. Histopathology 13: 553-559.
16. Derenzini M., Pession A., Farabegoli F., Tere D., Badiali M.: Relationship between interphasic nucleolar
organizer regions as growth rate in two neuroblastoma cell lines. Am J. Pathol. 1983 134; 925-32.
17. Editorial NORs – a new method for the pathologist. Lancet 1990 1: 1413-14.
18. Egan M.J., F. Raafat, J. Crocker and K. Smith (1987): Nucleolar organizer regions in small cell tumour of
childhood. Juornal of Pathology: Vol. 153:275-280.
19. Egan M J. Raafat F., Crocker J., Smith K. (1988): Nucleolar Organizer Regions in fibrous proliferation of
childhood and infantile fibrosarcoma. J. Clin. Pathol 14: 31-33.
20. Egan M J., Crocker J.: Nucleolar Organizer Regions in pathology. Br. J. Cancer 65: 1-7, 1993.
21. Francisco J. Moreno, Daniel Henandezverdum, Claude Masson and Michel Bouteille (1984): Silver staining
of the Nucleolar Organizer Regions (NORs ) on Lowicryl- Cryo Ultrathin sections. The journal of
Histochemistry and Cytochemistry. Vol. 33- No. 5.
22. Goodpasture G., Bloom S.E. (1975): Visualisation of nucleolar organizer regions in mammalian
chromosomes using silver staining. Chromosoma. 53: 37-50.
23. H.L. Arora, Neelima Arora, Rh. Solanki. (1996): Argyrophilic Nucleolar Organizer Regions in soft tissue
tumors. Indian J. Pathology Microbiol. 39: 40 257-263.
24. Howell W.M., Black A.D. (1980): Controlled silver staining of nucleolar organizer regions with colloidal
developer, a one step method. Experimentia 36: 1014.
25. Hara A., Hirayama H, Sakai N, Yamad H., Tanaka T., Mon H. Correlation between Nucleolar region staining
and Ki- 67 immunostaining in human gliomas. Surg Neurol 1990. 33: 320-324.
26. Hoshino T, Nagashima T., Murovic JA et al: proliferative potential of human Meningiomas of the brain. A
kinectic study with Bromodeoxyuridine. Cancer 1466-1472, 1986.
27. Hoshino T, Wilson CB: (1979) cell kinectic analysis of human malignant brain tumors (gliosis): Cancer: 44:
956-962.
7

28. Hara A., Hirayama H, Sakai N. et al: Nucleolar organizer region score and Ki-67 levelling index in grade
gliomas and metastatic brain tumours. Acta Neurochir (Wien) 109: 37-41. 1991.
29. Hara A., Sakai N, Yamad H.: Nucleolar organizer region in vascular and neoplastic cells of human giomas.
Neurosurgery 29, 2: 211-215. 1991.
30. Iwaki T., Takeshita I., Fukui M. et al: Cell kinetics of malignat evolution in meningothelial memingiomas.
Neuropathol 74: 243-247. 1987.
31. J. Crocker, Carey M.P., Allcock R. (1990): Hemangioblastoma and renal clear cell carcinoma distinguished
by means of the AgNOR method. Am. J. Clin. Pathol. April 93 (4) 555-557
32. J. Crocker and Paramjit Nar (1987): Nucleolar organizer regions in Lymphomas. Journal of Pathology VOL.
151: 111-118.
33. James S. Nelson (1991): athology of the nervous system. Anderson”s Pathology 9 th ediion, C.V. Mosby Co.
2123-2191.
34. K. Kajiwara M.D., Takafuni Nishizaki M.D., Tetsuji Orita M.D, Hisato Nakayama M.D, Hideo Aoki M.D., and
Haruhide Ito M.D. (1990): Silver colloid staining technique for analysis of glioma malignancy. J. Neurosurg.
73; 113-117.
35. Korkolopoulou p., Christodoulou P., Papanikolaou A. Thomas, Tsagli E. (1993): Proliferating cell nuclear
antigen and Nucleolar organizer regions in CNS tumours grade. A comparative study of 82 cases on paraffin
sections. Am.J. Surg. Pathol. Sep. 17(9): 912-919.
36. K. Rajeevan, K.P. Aravindan, Kumari Chandrika B. (1995): Value of AgNOR in fine needle Aspiration
cytology of breast lesions. Indina J. Pathol. Microbiol. 38: 17-24.
37. K.Y. Chiu Loke S.L. and Wong K.K. (1989): Improved silver technique for showing Nucleolar organizer
regions in paraffin wax sections. J.Clin. Pathology 42: 992.
38. Kajiwara K., Nishizaki T., Orita T., Nakayama H., Aoki H.: Silver colloid staining technique for analysis of
glioma malignancies. J. Neurosurg 1990 73: 113-117.
39. Kim T.S, Halliday AL, Hedly, Whyte E.T., Convery K.: Correlation of survival and the Daumas- Duport
grading system for neuroblastomas. J. Neurosurg. 191. 74: 27-37.
40. L.S. Chin M.D. and Devid R. Hiton M.D. (1991): the standerized assessment of argyrophilic Nucleolar
organizer regions in Meningeal tumours. J. Neurosurg. 74: 590-596.
41. L.M.Mc. Nicol, J. Colgan, W. Mc. Meckin and G.M. Teasdale. (1989): Nucleolar organizer regions in pituitary
adenomas. Acta Neuropathologica 77: 547-549.
42. Louis DN., Mechan MS., Ferranta RJ. et. al.: use of AgNOR technique in differential diagnosis of central
nervous system neoplasia: J. Neuropathol. Exp. Neurol. 57:150-157, 1992.
43. M.K. Dude, Anjana Govil. (1995): Evaluation of significance of AgNOR count in differentiating benign from
malignant lesions in the breast. Indian J. Pathol. Microbiol. 38: 5-10.
44. Maier H., Morimura T., Ofner D., Hallbrucker C., Kitz K., Budha H.: Argyrophilic Nucleolar organizer region
proteins (AgNOR) in the human brain tumours. Relation with grade malignancy and proliferation indices.
Acta Neuropathol. (Berl) 1990:80: 156-162.
45. Manuelidis L. Active nucleolus organizers are precisely positioned in adult central nervous system cells but
not in neuroectodermal tumour cella. J. Neuropathol. Exp. Neurol. 1984: 43: 225-41.
46. Nicoll JAR, Candy E. Nucleolar organizer regions and post operative in glioblastoma multiforme.
Neuropahol. Appl. Neurobiol. 1991: 17: 17-20.
47. N. Hussain, Manisha Bagchi , Mazhar Hussain, Bandana Tiwari.(1997): AgNOR expression in CNS
neoplasms. Indian J. Pathol. Mcrobiol: 40 (4) : 503-509.
48. Orita T., Kajiwara k., Nishizaki T. et. al: Nucleolar organizer regions in Meningiomas. Neurosurg. 26: 43-
46.1990.
49. Plate KH. Amdt. D., Hellwig D. Ruschoff J., Mennal HD: Assessment of histogenesis and proliferating
potential in cytologic specimens of human brain tumours. Value of Immunocytochemistry and Nucleolar
organizer regions. Anal Quant Cytol Histol 1990: 12: 157-164.
50. Plate KH. Ruschoff J., Behnke j., Mennal HD: proliferative proteins of human brain tumours as assessed by
Nucleolar organizer regions (AgNOR) and Ki- 67 immunoreactive. Acta Neurochir 1990: 9: 103-104.
51. Plate KH. Ruschoff J., Mennal HD: Cell proliferation in intracranial tumours. Selective silver staining of
Nucleolar organizer regions (AgNOR). Application of surgical and experimental neuro-patholgy. Neuropathol.
Appl. Neurobiol. 1991: 17; 121- 132.
52. Ploton D., Menager M., Jeannesson P. et al: Improvement in staining and visualization of argyrophilic
proteins of Nucleolar organizer regions at the optical level. Histochem j. 18: 5-14 (1986).
53. Ploton D., Menager M., Adnet J.J: Simultaneous high resolution localization of a AgNOR proteins and
nucleoproteins in interphase and mitotic nuclei. Histochem j. 16:1994: 1897-1906.
54. Ross A.D., Mckeever P.E., Sandler H.M, Muraszko K.M. (1993): Myxopapillary ependymoma. Results of
Nucleolar organizer region staining. Cancer: may 15: 71 (10): 3114-3118.
55. Ruschoff T., Plate K. Thomas C. (1989): Nucleolar organizer regions (NORs): Basic concepts and Pathol.
Res. Pract 185: 878-885.
56. Sawada T., Oinuma T. Katada H., Saitoh M., Sakuai I. (1992): Argyrophilic Nucleolar organizer region
proteins (AgNOR) in human central nervous tumours. Rinsho Byori Nov. 40 911) 1179-1184.
57. Shiraishi T., Tabuchi K., Mineta T., Momozaki N., Takagi M.; Nucleolar organizer regions in various human
brain tumours. J. Neuosurg 1991; 74; 979-984.
58. Tetsuji Orita M.D., K. Kajiwara M.D., Takafumi Nishizaki M.D., Norio Ikeda M.D., Toshifumi Kamiryo M.D.
and Hideo Aoki M.D. (1990): Nucleolar organizer regions in meningioma. Neurosurg. Vol. 26, No 1, 43-46.

59. Underwood J.C.E., Giri D.D., (1988): Nucleolar organizer regions as diagnostic discrimination for
malignancy. J.Pathol. 155: 95-96.
60. Wachtler F., Hopman AHN, Wiegnant J., Schwazacher HG. On the position of nucleolus organizer regions
(NORs) in interphase nuclear. Studies with anew non-autoradiographic in situ hybridization method. Exp. cell
Res. 1986; 167: 227-240.
9