Cytochemical studies
Acid ferrocyanide (ACF) stain for iron
Blood cells did not display any types of reaction (Table 1) when blood smears were stained with ACF stain (Fig 1a).
Toluidine blue (TBO) stain for mucopolysaccharide
The packed granules of basophils displayed a strong positive reaction and stained metachromatically in the form of intense violet colour with the 1% TBO stain (Table 1). Mucopolysaccharides reactivity was absent in the remaining leukocytes (Fig 2a).
Periodic Acid Schiff’s (PAS) stain for glycogen
The granules of neutrophils, eosinophils and basophils displayed a positive reaction and pink colour granules were observed in the cytoplasm. The cell membrane of lymphocytes also showed an intensely positive reaction when blood smears were treated with PAS stain (Fig 1b,c,d). The neutrophils and basophils granules were recorded as moderately positive, whereas the granules of eosinophils showed a stronger reaction(Fig 1c);(Table 1).
Sudan black B (SBB) stain for lipid
The eosinophils and basophils displayed a strong positive reaction which revealed darkly stained bluish-black granules (Fig 2c,d). SBB staining was weakly positive in neutrophils (Fig 2b) but lymphocytes and monocytes did not show any reaction to lipids (Table 1).
Cytoenzymatic studies
Acid phosphatase (ACP)
The eosinophils showed a strongly positive reaction and formed brown-colored granules in the cytoplasm (Fig 3a) but the neutrophils, basophils and lymphocytes did not exhibit any reaction when blood smears were stained for ACP activity (Table 2).
Alkaline phosphatase (ALP)
The eosinophils exhibited a strongly positive reaction and formed brown-coloured granules (Fig 3c), whereas a few cytoplasmic granules of neutrophils and basophils were stained light brown (Fig 3b,d). The lymphocytes and monocytes did not show any reaction to ALP activity (Table 2).
Peroxidase (POX)
The neutrophils (Fig 4a), basophils, lymphocytes and monocytes did not show any reaction to POX activity. The granules of eosinophils exhibited a strong positive reaction for POX (Table 2) in the form of brick-red coloured granules (Fig 4b).
Arylsulphatase (ARS)
The eosinophils and basophils granules showed a positive reaction as dark brown colour granules in the cytoplasm (Fig 4d, 5a). But other leukocytes were non-reactive (Fig 4c) for ARS activity (Table 2).
Cytochrome oxidase (CYO)
The eosinophils and basophils showed a strongly positive reaction in the shades of blue coloured granules of various intensities and some granules were darkly stained (Fig 5c,d). The granules of neutrophils elucidated negative reactivity (Fig 5b) but the monocytes and lymphocytes were non-reactive for CYO (Table 2).
Beta-glucuronidase (GUSB)
The neutrophils, basophils, eosinophils, monocytes and lymphocytes showed no reactivity for GUSB activity (Table 2).
Succinate dehydrogenase (SDH)
The cytoplasm of granulocytes and agranulocytes did not exhibit any reaction when blood smears were treated for SDH activity (Table 2).
Cytochemical studies
There was no cytochemical reactivity in the erythrocytes when blood smears were stained with ACF stain. It could be because of the absence of non-heame iron in the blood. Similar findings were also reported by
Mehta et al., (2012) in the blood samples of pigs. However,
Singh (2000) in buffalo calves,
Weiss (2005; 2006) in dog,
Mrigesh (2011) in donkey and
Yadav et al., (2015) in fowl recorded some siderocytes with their small blue colour granular structure inside the erythrocytes.
The packed granules of basophils demonstrated a strong positive reaction and stained a metachromatically intense violet colour with 1% TBO stain. It could be due to the presence of heparin in basophils because heparin is a good example of a mucopolysaccharide. The present findings were similar to that of
Singh (2000) in buffalo calves,
Yokohama et al., (2002) in chronic myeloproliferative disorders,
Mrigesh (2011) in donkey,
Mehta et al., (2012) in pig and
Yadav et al., (2015) in fowl.
Yokohama (2002) reported that the intensity of TBO increased in chronic myeloproliferative disorders, whereas decreased in myeloid leukemia.
The granules of neutrophils, eosinophils and basophils displayed a positive reaction because of glycogen in the cells and observed pink colour granules in the cytoplasm when blood smears were treated with PAS stain. Similar findings were reported by
Santos et al., (2003) in roadside hawk,
Mrigesh (2011) in donkey,
Mehta et al., (2012) in pig and
Yadav et al., (2015) in fowl. In the present study, some granules of lymphocytes were recorded as positive reactivity. The same result was also observed by
Feldman et al., (2000) in horse for lymphocytes. The pattern of cytoplasmic PAS reactivity can be used to characterize lymphocytic leukemia
(Schwarze,1980).
The eosinophils and basophils demonstrated a strong positive reaction in the form of darkly stained bluish-black granules with an SBB stain. It might be because of the higher amount of lipid materials inside the cell granules. The present investigations also closely agreed with the findings of
Santos et al., (2003) in roadside hawk,
Salakij et al., (2007) in Palm civet cat,
Mehta et al., (2012) in pig for eosinophils and
Yadav et al., (2015) in fowl. SBB staining was weakly positive for neutrophils but lymphocytes and monocytes did not show any reaction for lipids as reported by
Mrigesh (2011) in donkey and
Mehta et al., (2012) in pig. The affinity of SBB stain in neutrophils and eosinophils increased in myeloblastic leukemia without maturation, myeloblastic leukemia with maturation and monocytic leukemia whereas lymphoid malignancies lack positive reaction
(Jain, 1993).
Cytoenzymaticstudies
The eosinophils showed a strong positive reaction in the form of brown-coloured granules in the cytoplasm. It could be because of the ACP enzymatic activity of the granules. In the eosinophils,
Feldman et al., (2000) and
Shigdar et al., (2009) recorded positive reactions in the granules of eosinophils for ACP activity in horse and Murray cod, respectively. In the current study, the neutrophils, basophils and lymphocytes did not show any reaction when the blood smear was stained for ACP activity, as also reported by
Mehta et al., (2012) in pig. ACP activity within the lymphocytes was correlated with various types of lymphoproliferative disorders
(Schwarze, 1980; Savage et al., 1981). The highest levels of ACP were found in sickle cell disease or multiple myeloma or lysosomal disorders, such as Gaucher’s disease, which showed moderately increased levels
(Moul, 1998).
The eosinophils recorded a strongly positive reaction in the form of brown-coloured granules. It may be because of the ALP enzymatic activity of the granules of eosinophils. A similar result was also noticed by
Singh (2000) in buffalo calves and
Feldman et al., (2000) in horse. In the current investigation, a few cytoplasmic granules of neutrophils and basophils were stained light brown colour as also recorded by
Mehta et al., (2012) in pig. The lymphocytes and monocytes did not show any reaction which was also recorded by
Singh (2000) in buffalo calves and
Mrigesh (2011) in donkey for ALP activity. Higher levels of leukocyte ALP were seen in polycythemia vera, essential thrombocytosis, primary myelofibrosis and the leukemoid reaction and lower levels were found in chronic myelogenous leukemia and paroxysmal nocturnal hemoglobinuria. The ALPactivity of circulating neutrophils can facilitate the differentiation of chronic myelogenous leukemia from leukemoid reactions or neutrophilic leucocytosis associated with non-malignant causes
(Okum and Tanaka, 1978).
The neutrophils, basophils, lymphocytes and monocytes did not show any types of reaction for POX enzyme activity as also reported by
Singh (2000) in buffalo calves,
Santos et al., (2003) in roadside hawk,
Mrigesh (2011) in donkey and
Mehta et al., (2012) in pig. It was observed that the granules of eosinophils exhibited a strong positive reaction for POX in the form of brick-red coloured granules. It could be because of the POX enzymic activity of the granules.
Feldman et al., (2000) in horse and
Salakij et al., (2007) in Palm civet cat eosinophils also found similar types of results for POX activity. The negative POX activities of lymphocytes help in the differentiation of lymphocytic leukemia from granulocytic leukemia
(Mehta et al., 2012).
The granules of basophils and eosinophils showed a positive reaction and recorded dark brown colour granules in the cytoplasm. It could be because of the ARSenzymatic activity of the granules of basophils and eosinophils. The current findings were also closely similar to the findings of
Singh (2000) in buffalo calves,
Mehta et al., (2012) in pig. In the present study, the remaining leukocytes showed no reaction when stained for ARS activity, as also noticed by
Mrigesh (2011) in donkey.
The basophils and eosinophils showed a strong positive reaction as shades of blue coloured granules. It may be due to the presence of the CYO enzymatic activity of the granules of basophils and eosinophils. The present investigations were also similar to the findings of
Singh (2000) in buffalo calves,
Gupta and Singh (2008) in
Guinea fowl and Mrigesh (2011) in donkey. In the current findings, the granules of neutrophils recorded moderate reactivity, but the reaction was absent in monocytes and lymphocytes.
Mehta et al., (2012) in the neutrophils of pig also noticed a positive reaction while the reaction was absent in monocytes and lymphocytes for CYO.
Neutrophils, basophils, eosinophils, monocytes and lymphocytes did not show any reaction to the GUSB enzyme. It could be the absence of beta-glucuronidase enzymatic activity in the cells. However,
Singh (2000) in buffalo calves,
Salakij et al., (2000) in Asian wild dog,
Salakij et al., (2005a) in Asiatic black bear and
Salakij et al., (2005b) in Asiatic elephant recorded positive reactions for GUSB activity. The absence of GUSB activity in the present study might be due to variations in species and age. Deficiencies in GUSB result in the non-recessive inherited metabolic disease known as Sly syndrome or mucopolysaccharidosis.GUSB is used in veterinary medicine primarily to detect focal staining in T lymphocytes and acute undifferentiated leukemia
(Jain, 1993).
The cytoplasm of granulocytes and agranulocytes remained non-reactive for SDH enzyme activity. It may be due to the absence of SDH enzymatic activity of the granulocytes and agranulocytes. However,
Singh (2000) in buffalo calves,
Mehta et al., (2012) in pig,
Mrigesh (2011) in donkey observed positive reactions to SDH activity. The absence of SDH activity in the present study might be due to variations in species and age.
