Indian Journal of Animal Research

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Diagnostic Importance of Cytochemical and Cytoenzymatic Patterns of Peripheral Blood Cells in Native Cattle (Zobawng) of Mizoram, India

Rupan Sarkar1, Arup Kalita1, Pranab Chandra Kalita1, Probal Jyoti Doley1, Swarup Debroy1, Monalisha Debbarma1, Om Prakash Choudhary1,*
1Department of Veterinary Anatomy and Histology, College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Selesih, Aizawl-796 015, Mizoram, India.
Background: The cytochemistry and cytoenzymatic profile of the blood cells is an important tool in the hands of clinical veterinary practitioners. It has also applications in various fields of veterinary sciences. Therefore based on its clinical importance present study was conducted on the blood cells of native cattle (Zobawng) of Mizoram to examine the cytochemical and cytoenzymatic characteristics.

Methods: For this study, a total of 12 numbers of blood samples (5 ml) were taken aseptically from adult local cattle of Mizoram, irrespective of sex. The blood smears were prepared and stained with acid ferrocyanide (ACF) for the detection of non-heame iron, toluidine blue (TBO) stain for acid mucopolysaccharides, Periodic Acid-Schiff (PAS) stain for observation of glycogen and Sudan black B (SBB) stain for demonstration of lipid. Acid phosphatase (ACP), alkaline phosphatase (ALP), peroxidase (POX), arylsulphatase (ARS), cytochrome oxidase (CYO), beta-glucuronidase (GUSB) and succinate dehydrogenase (SDH) enzymes were used for cytoenzymatic features of blood cells. The smears were observed under 1000X magnification in an Olympus Trinocular Research microscope.

Result: In the cytochemical studies, basophils showed a positive reaction for mucopolysaccharides in the toluidine blue stain and neutrophils, eosinophils and basophils recorded positive for glycogen in the PAS stain. Eosinophils and basophils also displayed positive for lipids in the SBB stain. The cytoenzymatic studies of eosinophils and basophils recorded positive reactions for ACP, ALP, POX, ARS and CYO activity.
Blood tests are required to determine overall health status and to diagnose various hematological diseases. Blood is a fluid connective tissue in which plasma is suspended together with produced elements such as erythrocytes, granulocytes (neutrophils, eosinophils and basophils), agranulocytes (monocytes and lymphocytes) and platelets (Atkins et al., 2017; Choudhary et al., 2021). Blood is tested regularly to detect hematological problems and diagnose illnesses (Choudhary et al., 2022). In some disorders, blood cell identification and awareness of species differences in terms of morphological distinction are also required. Blood cell cytochemistry can be used to diagnose and classify different kinds of leukemia, as well as to investigate different myeloproliferative illnesses (Cline, 1981). The use of cytochemistry in the diagnosis of many types of hematological malignancies is becoming more common. Blood cells are sensitive to changes in the animal’s internal physiological states and stimuli from the external environment, the number, morphology and chemical components of different types of blood cells could reflect the health status of the animals (Khan et al., 2011; Peng et al., 2018; Sarkar et al., 2022) and their abnormal alterations may be related to inflammation, pathogenic microorganism infection or other diseases (Salakij et al., 2000; Fang et al., 2014).

Earlier some cytochemical studies of blood leucocytes have been investigated by Menaka and Singh (2002) in goat, Weiss (2005; 2006) in dog, Mrigesh (2011) in donkey and Mehta et al., (2012) in pig. To comprehend the body’s reaction to any form of stress, proper cell identification and localization of the numerous cytoenzymes in them are critical. Some cytoenzymic characteristics of blood cells were observed by Feldman et al., (2000) in horse, Salakij et al., (2000) in Asian wild dog, Singh (2000) in buffalo calves, Santos et al., (2003) in roadside hawk, Gupta and Singh (2008) in guinea fowl, Salakij et al., (2005a) in Asiatic black bear, Salakij et al., (2005b) in Asiatic elephant, Mrigesh (2011) in donkey and Mehta et al., (2012) in pig. With these considerations in mind, the present study was designed to assess comprehensive cytochemical and cytoenzymatic characteristics of the blood cells of native cattle of Mizoram.
Animals and ethical approval
 
The present study was conducted on blood cells of native cattle of Mizoram state of India from October 2020 to September 2021. The blood samples of Zobawng cattle were collected from the cattle farm, College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Selesih, Aizawl, Mizoram, India. The animals used for the experiment were ethically approved by the Institutional Animal Ethics Committee (IAEC) vide Approval No.CVSC/CAU/IAEC/20-21/P-3 dated 24.09.2021.
 
Sample collection
 
The present study was conducted with twelve numbers of blood samples collected from the adult native cattle of Mizoram, irrespective of sex. From each animal, approximately 10ml of blood was collected from the external jugular vein and was transferred to a sterile siliconized tube filled with heparin or ethylenediaminetetraacetic acid (EDTA).
 
Cytochemical analysis
 
For cytochemical studies, the blood smears were prepared immediately after collection on grease-free slides and stained for acid ferrocyanide stain for non-heame iron (Bover, 1964), toluidine stain for acid mucopolysaccharides (Bover, 1964), Periodic Acid Schiff’s stain for glycogen (Bover, 1964) and Sudan black- B for lipid (Bover, 1964) (Table 1).

Table 1: Cytochemical intensity of different blood cells in local cattle of Mizoram.


 
Cytoenzymatic analysis
 
For cytoenzymatic studies, the blood smears were prepared freshly after collection on grease-free slides and stained samples were treated for acid phosphatase (Bover, 1964), alkaline phosphatase (Bover, 1964), peroxidase (Bover, 1964), arylsulphatase (Bover, 1964), cytochrome oxidase (Bover, 1964), Beta-glucuronidase (Bover, 1964) and succinate dehydrogenase (Bover, 1964), (Table 2).

The stained blood smears and treated slides were examined under oil immersion to record the cytochemical and cytoenzymatic activity of different blood cells with the help of a light microscope (Olympus BX 51, Japan). Critical images were captured with a ProgRes C5 Cool CCD camera (D-07739 Jena, Jenoptik, Germany) for a typical demonstration.

The staining intensity was analyzed as per the activity of different blood cells for the particular chemical or enzyme recorded and graded them using the score from 0-3 (0= no stating/ negative, 1= weak staining, 2= moderate staining, 3= strong staining) (Table 1, 2).

Table 2: Cytoenzymic intensity of different blood cells in local cattle of Mizoram.

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).

Fig 1: Photomicrograph (a) showing erythrocytes (R) in ACF and (b) neutrophil (N). (c) eosinophils (E). (d) basophil (B) in PAS stain, blood smears of native cattle of Mizoram. (X1000).


 
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).

Fig 2: Photomicrograph (a) showing basophil (B) in TBO and (b) neutrophil (N). (c) eosinophils (E). (d) basophil (B) in SBB stain, blood smears of native cattle of Mizoram. (X1000).


 
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).

Fig 3: Photomicrograph (a) showing eosinophils (E) in ACP, and(b) neutrophil (N). (c) eosinophils (E). (d) basophil (B) in ALP, blood smears of native cattle of Mizoram. (X1000).


 
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).

Fig 4: Photomicrograph (a) showing neutrophil (N), (b) eosinophils (E) in POX and(c) neutrophil (N), (d) eosinophils (E) in ARS, blood smears of native cattle of Mizoram. (X1000).



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).

Fig 5: Photomicrograph (a) showing basophil (B) in ARS and (b) neutrophil (N), (c) eosinophils (E), (d) basophil (B) in CYO, blood smears of native cattle of Mizoram. (X1000).


 
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.
This present study exposed the cytochemical profile and showed a positive reaction for mucopolysaccharides of basophils in TBO stain; glycogen and lipids recorded positive for PAS stain of neutrophils, eosinophils and basophils; eosinophils and basophils also displayed positive for SBB stain. Thecytoenzymatic studies of eosinophils and basophils recorded positive reactions for ACP, ALP, POX and ARS enzyme activity.
The authors are thankful to the Dean, College of Veterinary Sciences and  Animal Husbandry, Central Agricultural University (I), Aizawl, Mizoram for providing all the necessary facilities to carry out the research work.
None

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