Birsa Agricultural University, Ranchi

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Studies on Pancreatic Pathology and Its Correlation with Different Wasting Disease Conditions of Poultry
(Birsa Agricultural University, Ranchi, 2019) KUMAR, BRAJESH; Gupta, M. K.
3796) birds examined showed definite pancreatic pathology. Maximum incidence of pancreatic pathology was observed in fungal (83.33%) and metabolic (82.86%) diseases whereas least pancreatic pathology was registered in viral diseases (16.41%). The overall incidence of pancreatic pathology in viral disease other than RD has been observed to be 66.95%. Disease wise incidence of pancreatic involvement is significantly higher in cases of IBD (94.74%) followed by IB (93.33%) and pasteurellosis (90.48%). Contrary to rest of the viral diseases the pancreatic pathology was extremely low (13.61%) due to RD though mortality of birds registered was highest, followed by pneumonia of bacterial origin ( 16.58% ). Incidence of pancreatic pathology in different poultry varieties showed that broiler birds were most susceptible (75.34%) to develop pancreatic pathology whereas Vanraja (14.29) variety showed significantly low susceptibility. Age group wise maximum pancreatic pathology was registered in grower birds (33.69%) followed by chicks (20.06%) and adult birds (19.54%). A significant variation was also observed in susceptibility to increased pancreatic pathology under different disease conditions between the three age groups. It was observed that highest percentage of pancreatic pathology was observed in monsoon season (28.84%) followed by summer (22.57%) and least was observed in winter season (12.92%). Though the incidence of pancreatic pathology was less in summer season the percentage of pancreatic involvement was significantly higher for most of the disease conditions.
Open Access
AMELIORATING EFFECT OF Curcuma longa ON OCHRATOXICOSIS IN BROILER BIRDS WITH SPECIAL REFERENCE TO NEPHROTOXICITY
(Birsa Agricultural University, Kanke, Ranchi, Jharkhand, 2011) KIRAN, DIPTI; GUPTA, M. K.
Open Access
LOCALIZATION OF ZINC BINDING SITES IN NUCLEI TO BE USED AS PROLIFERATION MARKER FOR NATURALLY OCCURRING EPITHELIAL TUMOURS
(Birsa Agricultural University, Kanke, Ranchi, Jharkhand, 2012) Verma, Ashok; Singh, K.K.
Therapeutic decisions are determined by proliferative behaviour of a tumour and this is measured by determining the mean growth fractions and mean cell cycle time. AgNORs (Argyrophilic Nucleolar Organising Regions) are only parameter available on formalin fixed tissues to measure proliferation fraction of tumours. AgNORs have the advantage that their numbers are increased only in actively and fastly dividing cells. Most of the sites of the NOR proteins reactive with silver are their Zinc binding motifs. These motifs remain bound with zinc particularly in their active states. The chemical reaction and mechanism involved in impregnation of silver at these sites of NORs are such that they can be greatly influenced by presence or absence of zinc in these zinc motifs. The process of cell division is initiated by replication of DNA along with Abstract……… Page 2 increased rate of transcription. Both these processes are initiated and mediated by a number of Zinc binding replication and transcription proteins. These zinc binding proteins are highly dynamic and transportable to different nuclear compartments engaged in transcription or replication of genes. Thus we hypothesised that presence of zinc bound proteins at different levels and their distribution in different compartments of nuclei as well as nucleoli in different metabolic and proliferative states of the cells have definite impact on AgNOR number and clusters which need to be explored. This warranted localization of zinc binding sites in nuclei and their further co-localization with silver by the method of AgNOR in order to throw more insight on mechanism involved in formation of AgNORs and variation in their number, size, shape and clusters in varying metabolic and proliferating states of the cell as well as in different phases of cell cycle. Keeping these facts in mind the present study aimed at development and standardization of some new protocol for co-localization of zinc and silver binding sites in nuclei and to devise a system of morphologic pattern of these granules to identify and count the cells in different phases of cell cycle, which can evolve as a more perfect marker of speed of proliferation. For localization of zinc, three methods were tested which were zinc impregnation, zinc precipitation and zinc-cysteine complex formation methods. Attempt to localize zinc by impregnation and precipitation methods caused nonspecific deposits in nuclei as well as in cytoplasm. This made intranuclear specific deposition of zinc obscured. The problem of nonspecific deposition of zinc was removed by treating the sections with solution of ionic Zinc, Na2SO3, acetic acid which caused deposition of fine amorphous to crystalline, well discernible and discrete granules or dots mostly restricted in nuclei. These granules were taken as specific since this staining reaction aimed at formation of insoluble complex of Cysteine and Cysteine bound zinc. Abstract……… Page 3 Moreover, these granules or dots were further histochemically confirmed for presence of zinc with Dithizone method. This reaction was based on previously known reaction for Cu during which there is formation of insoluble Cu-Cysteine-Acetic Acid complex when solution of Cu and Cysteine were treated with solution of Na2SO3 and acetic acid. Keeping this reaction in mind it was hypothesised that cysteine bound with zinc may lead to formation of Zinc-Cysteine-Acetic acid complex when a section pre-exposed with ionic zinc is treated with a solution of acetic acid and Na2SO3 which may appear as visible dot or granule. Thus this method could localize only those zinc binding sites which were bound with cysteine such as polymerase, and other transcription factors. In consequence of this test result, it was adopted for intranuclear co-localization of zinc and silver binding sites in nuclei. When dots due to AgNOR were compared with co-localized Zn-AgNOR dots, in these co-localized dots, there was presence of markedly more number of fine visible discrete dots dispersed throughout the nuclei along with few large Zn-AgNOR dots closely resembling to AgNOR dots formed due to AgNOR staining alone. A consistent appearance of these small fine AgNOR dots along with large Zn-AgNOR dots indicated sites of transcription of non-NOR genes as well as replication sites of DNA. Such sites might have failed to be visualized by AgNOR staining due to very poor silver reaction there and treatment of zinc on these sites made them strongly reactive and clearly visible. Thus increase in their number and density indicated enhanced transcriptional or replicational activities in cells. When co-localized Zn-AgNOR dots were further stained with Dithizone, the fine granules were found to be more distinct because of coloured reaction as well as due to mounting and refractive index used in this procedure. This staining reaction due to combination of Zn-AgNOR-Dithizone staining could be able to indicate whether Zn- AgNOR dots are rich in zinc or silver, red dots (Dithizone stained Zinc) rich in zinc Abstract……… Page 4 indicated active sites of transcription or replication whereas black or brown dots might be representing presence of silver binding transcriptional or replicational proteins. A thorough and critical study of morphologic pattern of Zn-AgNOR-Dithizone, Zn-AgNOR dots as well as AgNOR dots revealed variable pattern in different cells. Nuclear proteins are highly dynamic and such proteins from nucleolar compartment are migrated to different replicating and transcribing sites in nuclei. Moreover, during process of replication almost all the clustered genes are unwound and loosened. These might be responsible for dispersion of nucleoli in S-phase and formation of small dots dispersed throughout the nuclei. Due to doubling of DNA and aggregation of transcription proteins like polymerases and upstream binding factors (UBF) with actively transcribing genes to be packaged in chromosomes; dots in G2-phase were comparatively larger than that found in S-phase. In M-phase Zn-AgNOR dots were arranged in forms of mitotic figures showing clumps or aggregation of granules. G1 phase was characterized by presence of one or few nucleoli with variation in their degree of organisation represented by varying size of large NORs with fine well dispersed in Zn-AgNOR dots in nuclei. In highly proliferative cells there may be more number of irregular compound dots along with focal presence of features of S or G2 phases indicative of fast replicative and transcriptive activity in the cells. These cells in this study were graded as irregular or aberrant cells. In this way this system of morphologic pattern of Zn-AgNOR dots enabled us to identify and count the relative proportion of cells in different phases of cell cycle. Counting of cells by this system revealed a major relative proportion of cells in each phase of cell cycle and consequently it also indicated the timing of each phase and thus it can give an estimation of cell cycle velocity and proliferating fraction of the tumour. Abstract……… Page 5 The higher count in S or G2 phase with lower count in M-phase indicated that the majority of cells are in synthetic phase (S-phase) with slow rate of proliferation. Likewise, higher cell count in S-phase as well as in M-phase both indicated higher rate of proliferation and represent a fast or newly developing tumour. This above mentioned system of phases of cell cycle was further applied to different tumours of mammary gland origin (tubular, solid, anaplastic, mucinous, myoepithelioma and osteosarcoma) and seminoma to assess their proliferative behaviour. In order to see the accuracy of this assessment it was further tested for positive correlation with count of mitotic figures (H&E staining) as well as histological grading of these tumours. A general trend of variation in mean count of cells in S/G2+M (taken as proliferation fraction) in almost all tumours was consistently found to be either significantly or non-significantly higher in Grade III followed by Grade II and lowest in Grade I lesions of their tumours. This clearly indicated highly proliferative population of neoplastic cells in tumour of Grade III followed by Grade II and thereafter in Grade I. These results clearly showed an association between assessment of proliferation made by our system of cell count and histological grading of tumour suggesting that both are positively correlated. In majority of cases assessment of histological grading of tumour was also found positively correlated with cell count in different phases of cell cycle. Zinc-AgNOR-Dithizone staining showed best result while identifying, differentiating and counting the number of cells in different phases of cell cycle followed by Zinc-AgNOR co-localization while AgNOR could reveal these feature in case of one or two tumours. In this way, this system could stand in association with histopathological grading of tumours as well as their morphological behaviour. It was also able to explain number of aspects pertaining to developmental and biological behaviour of different tumours thus it could be concluded that this system is able to identify cells in different phases of Abstract……… Page 6 cell cycle on the basis of pattern of Zn-AgNOR dots and can help in assessing proliferative fraction as well as proliferative behaviour of tumours which has got immense importance in determining prognostic and therapeutic decisions on them. It could be concluded that use of histochemical staining of sections using Zn-AgNORDithizone or Zinc-AgNOR for identification and counting of number of cells in different phases of cell cycle has potency to evolve as important histochemical technique to assess the proliferative behaviour of tumours which can be helpful in taking therapeutic and prognostic decision for them.
Open Access
LOCALIZATION OF ZINC BINDING SITES IN NUCLEI TO BE USED AS PROLIFERATION MARKER FOR NATURALLY OCCURRING MESENCHYMAL TUMOURS
(Birsa Agricultural University, Kanke, Ranchi, Jharkhand, 2012) Kumari, Rekha; Singh, K. K.
Therapeutic decisions are determined by proliferative behaviour of a tumour and this is measured by determining the mean growth fractions and mean cell cycle time. AgNORs are only parameter available on formalin fixed tissues to measure proliferation speed of tumours. They have the advantage that their numbers are increased only in actively and fastly dividing cells. The major problem in using AgNOR counts as marker of proliferative speed are great variation in count, size, shape and cluster of these NORs as well staining intensity in different metabolic and proliferative states of the cell. Most of the sites of the NOR proteins reactive with silver are their zinc binding motifs. These motifs remain bound with zinc particularly in their active states. The chemical reaction and mechanism involved in impregnation of silver at these sites of NORs are such that they can be greatly influenced by presence or absence of zinc in these zinc motifs. The process of cell division is initiated by replication of DNA alongwith increased rate of transcription. Both these processes are initiated and mediated by a number of Zinc binding replication and transcription proteins. These zinc binding proteins are highly dynamic and transportable to different nuclear compartment engaged in transcription or replication of genes. Thus, we hypothesised that presence of zinc bound proteins at different levels and their distribution in different compartments of nuclei as well as nucleoli in different metabolic and proliferative states of the cells have definite impact on AgNOR number and clusters which need to be explored.This warranted localization of zinc binding sites in nuclei and their further co-localization with silver by the method of AgNOR in order to throw more insight on mechanism involved in formation of AgNORs and variation in their number, size, shape and clusters in varying metabolic and proliferating states of the cell as well as in different phases of cell cycle. Keeping these facts in mind the present study aimed at development and standardization of some new protocol for co-localization of zinc and silver binding sites in nuclei and to devise a system of morphologic pattern of these granules to identify and count the cells in different phases of cell cycle, which can evolve as a more perfect marker of speed of proliferation . For localization of zinc, three methods were tested which were zinc impregnation, zinc precipitation and zinc-cysteine complex formation methods. Attempt to localize zinc by impregnation method (hypothesized on basis of principles previously used for localization of argyrophilic proteins in nuclei) at their binding sites using citrate buffer, hydroquinone and citric acid as reducing agent as well as zinc precipitation method (based on principle of chromate precipitation technique used previously for demonstration of fine histologic structures like synapses of axons and dendrites) using thioglycolloic acid, sodium sulphide as zinc precipitants caused deposition of very fine ,indiscrete and nonspecific deposits when examined either by light microscope or phase contrast microscope. Further use of Timm’s or AgNOR methods to co-localize silver on these fine particles improved their visibility seen as large round, black, non specific deposits in nuclei as well as cytoplasm making intranuclear specific deposition of zinc obscured . The problem of nonspecific deposition of zinc was removed by treating the section with solution of ionic zinc, sodium sulphite, acetic acid which caused deposition of fine amorphous to crystalline, well discernible and discrete granules or dots mostly restricted in nuclei. This reaction was based on mechanism of previously known reaction during which there is formation of rich insoluble copper cysteine complex compound when solution of copper sulphate and cysteine were treated with solution of sodium sulphite (Ahmed et al., 2011). Keeping this reaction in mind, it was hypothesized that cysteine bound with zinc may also lead to formation of zinc-cysteine-acetic acid complex when a section pre-exposed with solution of ionic zinc is treated with a solution of acetic acid and sodium sulphite and may appear as dots or granules. The size of granules were further enhanced by treating such sections with AgNOR, which resulted in increased number of fine granules in nucleoplasm along with more distinct presence of large AgNOR dots. Thus this method could localize only those zinc binding sites which were bound with cysteine such as polymerase, and other transcription factors. Moreover, this solution could also remove zinc which were not bound with cysteine and were deposited in nonspecific way since acetic acid in this solution removes such zinc. In consequence of tests applied, most favourable results were adopted for intranuclear co-localization of Zn and silver binding sites in nuclei. Three methods of staining were proposed on the basis of these tests i.e. Zn-AgNOR-Dithizone, Zn-AgNOR and AgNOR alone. When dots due to AgNOR were compared with co-localized Zn-AgNOR dots, these co-localized dots presented markedly more number of fine visible discrete dots dispersed throughout the nuclei along with few large Zn-AgNOR dots, closely resembling to AgNOR dots formed due to AgNOR staining alone. A consistent appearance of these small fine AgNOR dots along with large Zn-AgNOR dots indicated transcription sites of non-NOR genes as well as replication sites of DNA. Such sites might have failed to be visualized by AgNOR staining due to very poor silver reaction there. But initial treatment with zinc made these sites strongly reactive to silver and clearly visible in case of Zn-AgNOR staining. Thus, increase in number and density of fine Zn-AgNOR dots indicated enhanced transcriptional or replicational activities in cells. Also, co-localized large Zn-AgNOR dots presented an important feature of cluster of fine, discrete and distinct dots within them in case of controlled stained sections; suggestive of multiple transcriptional sites. Further Zn-AgNOR-Dithizone staining caused co-localized fine Zn-AgNOR dots to be more distinct because of coloured reaction given by dithizone as well as due to mounting media and refractive index used in this procedure. Some dots were red while many others were black or brown.It should to be noted that zinc gives red staining with dithizone while silver gives black. Such coloured reaction due to combination of Zn-AgNOR-Dithizone staining could be able to indicate whether Zn-AgNOR dots are rich in zinc or silver; red dots rich in zinc indicated active sites of transcription or replication whereas black or brown dots might be representing presence of silver binding transcriptional or replicational proteins. Though counting and assessment of Zn-AgNOR dots in light of coloured reaction could not be included in this study. However, further improvement of this technique regarding this aspect can enable us to recognize active transcriptional and replicational sites in nuclei as well as to differentiate between active and inactive NORs. Zn-AgNOR dots not only clearly and distinctly demonstrated even weakly reactive NORs, but also protected large dots from overstaining and strongly reactive NORs from confluence. Varying morphological and distribution pattern of Zn-AgNOR dots as well as AgNOR dots in nuclei was thoroughly studied and cells in different phases of cell cycle were recognized. Cells presenting fine, innumerable dots distributed throughout nuclei without any large NOR dots resembling nucleoli were considered to be in S phase of replication. Though transition between S phase and G2 phase has been difficult to delineate. But critical observation revealed that number of small fine dots in S and G2 phase remained almost constant. There was variation in size of dots, which were found to be larger in G2 phase. In M phase, zinc and silver binding proteins packaged in chromosomes might be responsible for arrangement of Zn-AgNOR dots in form of mitotic figures showing clumps or aggregation of granules. G1 phase was characterized by presence of single or few dots within the nucleus presenting nucleolus along with presence of numerable small dots in nucleoplasm. Presence of more number of such large dots indicated early or immature phase of G1 while their decreased number indicated towards maturity in G1 phase. Some aberrant or irregular cells were having more number of irregular compound dots along with focal presence of S or G2 phase indicating high and independent replicative and transcriptive activity in the cells. Such system of morphologic pattern of Zn-AgNOR dots enabled us to identify and count the cells in different phases of cell cycle. Counting of cells by this system revealed a major relative proportion of cells in each phase of cell cycle; consequently indicating the timing of each phase along with estimation of cell cycle velocity and proliferating fraction of tumours. Counting of number of cells in different phases of cell cycle in serial sections using Zn-AgNOR-Dithizone, Zn-AgNOR and AgNOR stains separately; along with counting of mitotic figure and grading of tumours in respective areas of other serial sections stained with H & E was done to assess the proliferative behaviour of mesenchymal tumours like venereal sarcoma, osteosarcoma, mammary osteosarcoma, mixed interstitial seminoma and synovioma. The mean count of cells in S, G2 as well as S+G2+M taken as proliferative fraction of tumour along with mitotic figure were significantly higher in grade III followed by grade II and grade I in most of the tumour, suggesting their impact on morphological behaviour of the tumour. The higher count of S+G2 phase (46%) and lower count in M phase (0.7%) in venereal sarcoma indicated that the major part of the cells were in synthetic or replicative phase but the timing of S phase was relatively longer and the rate of proliferation of cells were comparatively slow. In osteosarcoma of lower grades, S+G2 phase (84%) was longer having low proliferating activity while in case of higher grades S/G2phase (25%) was shorter resulting into increased proliferative speed and behaviour of the tumour. Whereas mammary osteosarcoma revealed comparatively lower S+G2 count (grade 1 : 20%; grade 2 : 43%) with almost equal number of cells in M phase (grade 1 : 9%; grade 2 : 12%) in both grades of tumour, indicating small but highly proliferating pool of neoplastic cells. In higher grades of osteosarcoma, intracytoplasmic Zn-AgNOR dots were found in Zn-AgNOR staining or AgNOR alone. Mixed interstitial seminoma during this study revealed comparatively lower S+G2 cells (11%) almost equal count of cells in M phase (9%), which indicated small but highly proliferative pool of neoplastic cells. Grossly tumour was of large size but histopathologically there was no marked degree of anaplasia. This small but highly proliferative pool might be responsible for its large size, though it failed to show marked anaplastic change. In synovioma of lower grade, high number of cells contained more than five large, irregular compound dots (89%) in areas showing lower grade of tumour while higher count of cells in S+G2+M phases ( 98%) in areas of higher grades of lesions. Both these features clearly indicated this tumour to be highly and fastly proliferating. In most of the mesenchymal tumours mean cell counts were found to be positively correlated with tumour grading as well as count of mitotic figure suggesting accuracy of these count for different phases of cell division . A general trend of variation in mean count of cells in S, G2 and M (proliferating fraction) of tumour in almost all the tumour was consistently found to be either significantly higher in grade 3 followed by grade 2 and lowest in grade 1 lesions of their tumour. This clearly indicated highly proliferative population of neoplastic cells in tumours of grade 3 followed by grade 2 and thereafter in grade 1. These results clearly showed an association between assessment of proliferation made by our system of cell count and histological grading of tumour suggesting that both are positively correlated. Qualitatively, co-localization of zinc and AgNORs with dithizone staining was found to give best result followed by Zn-AgNOR staining as compared to AgNOR alone in identifying and counting of cells in different phases of cell cycle. The superiority of Zn-AgNOR-Dithizone in assessing the proliferative aggressiveness of tumour was established on the basis of its ability to count maximum number of cells in S, G2 or M phases of cell division in correlation with histologic grading and mitotic figure count of that tumour. Thus, it could be concluded that use of histochemical staining of sections using Zn-AgNOR-Dithizone or Zn-AgNOR for identification and counting of number of cells in different phases of cell cycle has potency to evolve as an important histochemical technique in assessing proliferative fraction as well as prolferative behaviour of tumour which have got immense importance in determining prognostic and therapeutic decisions on them. Moreover, these staining protocols and system of counting of cells has still many scopes for improvement as well as exploitation to make it more accurate, reliable and usable in other areas of tumour and cell biology.
Open Access
LOCALIZATION OF ZINC BINDING SITES IN NUCLEI TO BE USED AS PROLIFERATION MARKER FOR NATURALLY OCCURRING HAEMOPOIETIC TUMOURS
(Birsa Agricultural University, Kanke, Ranchi, Jharkhand, 2012) Soren, Punam; Singh, K.K.
Therapeutic decisions are determined by proliferative behaviour of a tumour and this is measured by determining the mean growth fractions and mean cell cycle time Ag-NORs are only parameter available on formalin fixed tissues to measure proliferation speed of tumours. They have the advantage that their numbers are increased only in actively and fastly dividing cells. The major problem in using Ag-NOR counts as marker of proliferative speed are great variation in count, size, shape and cluster of these NORs as well staining intensity in different metabolic and proliferative states of the cell. Most of the sites of the NOR proteins reactive with silver are their Zinc binding motifs. These motifs remain bound with zinc particularly in their active states . The chemical reaction and mechanism involved in impregnation of silver at these sites of NORs are such that they can be greatly influenced by presence or absence of zinc in these zinc motifs. The process of cell division is initiated by replication of DNA along with increased rate of transcription. Both these processes are initiated and mediated by a number of Zinc binding replication and transcription proteins. These zinc binding proteins are highly dynamic and transportable to different nuclear compartments engaged in transcription or replication of genes .Thus we hypothesised that presence of zinc bound proteins at different levels and their distribution in different compartments of nuclei as well as nucleoli in different metabolic and proliferative states of the cells have definite impact on Ag-NOR number and clusters which need to be explored .This warranted localization of zinc binding sites in nuclei and their further colocalization with silver by the method of Ag-NOR in order to throw more insight on mechanism involved in formation of Ag-NORs and variation in their number ,size ,shape and clusters in varying metabolic and proliferating states of the cell as well as in different phases of cell cycle .Keeping these facts in mind the present study aimed at development and standardization of some new protocols for colocalization of zinc and silver binding sites in nuclei and to devise a system of morphologic pattern of these granules to identify and count the cells in different phases of cell cycle, which can evolve as a more perfect marker of speed of proliferation . For localization of zinc, four methods were tested which were zinc impregnation, zinc precipitation and zinc-cysteine complex and Zinc fixation in tissue smear. Attempt to localize zinc by impregnation method at their binding sites using citrate buffer, hydroquinone and citric acid as reducing agent as well as zinc precipitation method using Thioglycolic acid DTT ,sodium sulphide (Na2S) as zinc precipitants caused deposition of very fine ,indiscrete and nonspecific deposits when examined either by light microscope or phase contrast microscope. Further use of Timm’s or Ag-NOR methods to co-localize silver on these fine particles improved their visibility but were seen as large round, black, nonspecific deposits in nuclei as well as cytoplasm making intranuclear specific deposition of zinc obscured . The problem of nonspecific deposition of zinc was removed by treating the section with solution of ionic zinc with Na2SO3 and acetic acid which caused deposition of fine amorphous to crystalline, well discernible and discrete granules or dots mostly restricted in nuclei. This reaction was based on previously known reaction for Cu during which there is formation of insoluble Cu-Cysteine-Acetic Acid complex when solution of Cu and Cysteine were treated with solution of Na2SO3 and acetic acid . Keeping this reaction in mind, it was hypothesised that cysteine bound with zinc may lead to formation of Zinc- Cysteine-Acetic acid complex when a section pre-exposed with ionic zinc is treated with a solution of acetic acid and Na2SO3 which may appear as visible dots or granules. These granules were taken as specific since this staining aimed at formation of insoluble complex of cysteine and cysteine bound zinc. Moreover, these granules or dots were further histochemically conformed for presence of Zinc with Dithizone method. Thus this method could localize only those zinc binding sites which were bound with cysteine such as polymerase, and other transcription factors. Moreover, this solution could also remove zinc which were not bound with cysteine and were deposited in nonspecific way since acetic acid in this solution removes such zinc. On the basis of these findings two protocols were developed for co-localization Zinc and Silver in nuclei which are 1)Zn-AgNOR –Dithizone. 2) Zn-AgNOR method. Another method developed during this study for colocalization of Zinc by zinc fixative in tissue during preservation. It was based on fact that, Most of the site of NOR proteins are reactive with silver and their Zn -binding motifs that is Zn finger. These Zn finger remain bound with Zn particularly in their active state. Impregnations of silver in these sites are such that they can be greatly influenced by presence or absence of Zn in these fingers. The proteins that bind Zn are easily denatured in water or any other liquid containing fixative with loss of Zn ions from their fingers. It may alter the pattern of Ag-NOR reaction because Zn bound and Zn free motifs had different kinetic features for this reaction and may fill to revealed real pattern of NOR formation. Moreover, such alteration might be responsible for lack of reproducibility as well as variation in pattern of Ag-NOR formed by existing methods of their staining. Keeping these facts in mind, after trial and standardization of number of Zn precipitant, we formulated new composition of formalin fixative which could precipitate Zn at their original motifs during the process of fixation. After fixation tissue were washed, processed and embedded in paraffin. Three to four micron thickness were cut and stained with AgNOR staining as described earlier for co-localization of Zn-AgNOR dots. Zn fixed AgNOR staining showed best result while identifying differentiating and counting the number of cells in different phases of cell cycle followed by Zinc-AgNOR-Dithizone, zinc-AgNOR co-localization while AgNOR could reveal these feature in case of one or two tumours. When dots due to AgNOR were compared with co-localized Zn- AgNOR dots, there were presence of markedly more number of fine visible discrete dots dispersed throughout the nuclei along with few large Zn-AgNOR dots closely resembling to AgNOR dots formed due to AgNOR staining alone. A consistence appearance of these small fine AgNOR dots along with large Zn- AgNOR dots indicated sites of transcription of non-NOR genes as well as replication sites of DNA. Such sites might have failed to be visualized by AgNOR staining due to very poor silver reaction there and treatment of zinc on these sites made them strongly reactive and clearly visible. Thus increase in their number and density indicated enhanced transcriptional or replicational activities in cells. Another important feature of co-localized Zn-AgNOR dots was that even in large dots resembling in AgNOR dots, there were presence of cluster of fine discrete and distinct dots within them in case of controlled stained sections suggesting them to be of multiple transcriptional sites. Qualitative assessment of morphological characteristics of zinc binding and argyrophilic sites appearing as dots were assessed by all the 4methods described above was made and a system was devised on the basis of these characteristic patterns to identify the different stages of cell cycle. i) S-phase- replicating cells in s-phase were identified on the basis of presence of small sized innumerable dots in nuclei with no large dots appearing as nucleoli ii)G2- phase –cells in G2 phase were identified by their pale-brown–orange condensed background with innumerable small to medium sized dots having no large dots appearing as nucleoli comparatively lower in number than in S-phase iii)M-phase – The cells in mitotic phase were showing presence of dots and granules regularly or irregularly aggregated in the form of mitotic clot or mitotic hairs or mitotic figures were classified as cells in M-phase. iv)G1-phase – The cells in G1- phase showed presence of single or few dots within the nucleus presenting nucleolus along with presence of numerable small dots in nucleoplasm .Presence of more number of such large dots indicate early or immature phase of G1 while their decreased number indicated towards maturity in G1 phase. V) In highly proliferative cells there were more number of irregular compound dots along with focal presence of features of S or G2 phase indicative of high and fast replicative and transcriptive activity in the cells taking within short cycle time of G1.These cells in this study were graded as irregular cell or fast G1. The literature about the use of system of classification of lymphomatous lesions in birds for their typing their grading as used in mammals appears to be very scarce. Any system of typing and grading aims at evolution of proliferative behaviour, prediction of prognosis and assessment of survival rate. Hence attempt for typing and grading of lymphomatous lesions of birds using these systems may give important feedback for improvement of these systems of classification in their purpose. So in this study we have attempted to classify lymphomas of birds in different organs on basis of WHO system of classification for lymphomas and to assess the proliferative behaviour of these tumours with our system of identification and counting of cells in different phases cell cycle. On the basis of WHO system, the lymphomatous tumour found in birds were classified as lymphoblastic and lymphocytic lymphoma (Grade1, Grade2 and Grade 3). A general trend of variation in mean count of cells in S, G2 and M (proliferating fraction) of tumour in almost all the tumour was consistently found to be either significantly higher in grade 3 followed by grade 2 and lowest in grade 1 of their tumour. This clearly indicated highly proliferative population of neoplastic cells in tumours of grade 3 followed by grade 2 and thereafter in grade 1. These results clearly showed an association between assessment of proliferation made by our system of cell count and histological grading of tumour suggesting that both are positively correlated. Further mitotic figure counts were found to be positively correlated with number of cells in different phases of cycle(S, G2 & M) in different grades of tumour validing the accuracy of identification of cells in different phases. It is so because increase in number of mitotic figures will associated with atleast one or more of the M,S,G2 or M+S+G2. Zinc fixed AgNOR stains was found to be superior than others because count of cells in different phases of cycle was consistently more(S,M,G2). It also enabled to differentiate between S & G2 phases in lymphomatous lesions. It was concluded that use of histochemical staining of sections using Zn fixed AgNOR or Zn-AgNOR-Dithizone or Zinc-AgNOR or AgNOR alone for identification and counting of number of cells in different phases of cell cycle has potency to evolve as important histochemical technique to assess the proliferative behaviour of tumours which can be helpful in taking therapeutic and prognostic decision for them .

Birsa Agricultural University, Ranchi

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