Preparation of blood samples for electron microscopy: The standard protocol

Choudhary, Om Prakash; Sarkar, Rupan; Choudhary, Priyanka; Chethan, G.E.; Doley, Probal Jyoti; Kalita, P. C.; Kalita, A.

doi:10.1016/j.amsu.2021.102895

Cited by 16 publications

(25 citation statements)

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“…The fixed samples were rinsed again in 0.1 M phosphate buffer saline (pH 7.2) to drain out the remaining primary fixative. These fixed blood samples were (Figure 1a) suspended in 0.1 M phosphate buffer saline (pH 7.2) and sent to the electron microscopy laboratory, that is, Sophisticated Analytical Instrumentation Facility (SAIF), All India Institute of Medical Science (AIIMS), New Delhi, for further processing and analysis (Choudhary et al, 2021).…”

Section: Methodsmentioning

confidence: 99%

“…After washing with 0.1 M phosphate buffer saline (pH 7.2) solution, the sections were placed in another clean Eppendorf tube containing 0.1 M phosphate buffer saline (pH 7.2). These sectioned buffy coat samples were kept in iceboxes and sent to the Sophisticated Analytical Instrumentation Facility (SAIF), All India Institute of Medical Science (AIIMS), New Delhi, for further processing and analysis (Choudhary et al, 2021).…”

Section: Processing Of the Samples For Transmission Electron Microscopymentioning

confidence: 99%

“…The studies on the blood cells are important from the morphological, physiological, clinicopathological, and therapeutic points of view. The blood examination is important for accessing general health and diagnosing various diseases (Choudhary et al, 2021). The blood examination is performed routinely to diagnose the hematological diseases, determine the body's ability to respond to a hematological insult, and to monitor the course of certain diseases besides the assessment of general health (Jain, 1993).…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Observations on the cytomorphology and ultrastructure of the peripheral blood cells of native cattle (Zobawng) of Mizoram, India

Sarkar

Kalita

Choudhary

et al. 2022

Microscopy Res & Technique

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This study aims to characterize the cytomorphology and ultrastructure of blood cells of native cattle of Mizoram. Twelve numbers of blood samples (10 ml) were collected from the Zobawng cattle, irrespective of sex. Blood smears were prepared and stained with different stains for cytomorphological study. The standard protocol has been followed for preparing blood samples for electron microscopy. Under a light microscope, erythrocytes of cattle were non-nucleated and round. The neutrophils were round, and the cytoplasm contained cytoplasmic granules. The eosinophils were rounded in outline with distinct cytoplasmic granules. The presence of basophils was infrequent with distinct blue color cytoplasmic granules. Small, medium, and large types of lymphocytes were recorded. The monocytes were round to oval in outline. Platelets were irregular to round. The reticulocytes were recorded occasionally, like small blue thin rods or granules. The cytoplasm and nucleus of granulocytes fluoresced greenish-yellow and red, respectively, with supravital stain. Under scanning electron microscopy (SEM), the erythrocytes appeared as biconcave discs. Different leukocytes were observed with their finger-like, plate-like, and narrow cell processes on their surface. Platelets were irregular structures. In transmission electron microscopy (TEM), the erythrocytes appeared anucleated biconcave elongated, neutrophils were roughly rounded with small cytoplasmic processes, and eosinophils were roughly circular with small cytoplasmic processes, the basophils were roughly circular with oval to elongated cytoplasmic granules, lymphocytes were roughly circular with centrally placed well-marked oval indented nucleus and some cytoplasmic processes, monocytes appeared spherical with long thick cytoplasmic processes and the non-nucleated platelets appeared roughly round to elongated. Research Highlights• Blood tests are a very good tool for determining animal health and diagnosing hematological illnesses.• This study showed the cytomorphological and ultrastructural features of the blood cells of native cattle (Zobawng) of Mizoram state of India.Rupan Sarkar and Om Prakash Choudhary contributed equally to this manuscript

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Section: Methodsmentioning

confidence: 99%

Section: Processing Of the Samples For Transmission Electron Microscopymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Observations on the cytomorphology and ultrastructure of the peripheral blood cells of native cattle (Zobawng) of Mizoram, India

Sarkar

Kalita

Choudhary

et al. 2022

Microscopy Res & Technique

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“…The ultrathin sections were post-stained in saturated uranyl acetate and lead citrate, and examined by JSM1400 plus-JEOL transmission electron microscope (Alexandria university microscopy unit). Fixed samples that were stored for scanning electron microscopy (SEM) were processed and coated using Sputter Coating Evaporator as described in ( 32 , 33 ). JSM-5300 – JEOL scanning electron microscope (Alexandria university microscopy unit) was used to obtain SEM images.…”

Section: Methodsmentioning

confidence: 99%

Platelets’ morphology, metabolic profile, exocytosis, and heterotypic aggregation with leukocytes in relation to severity and mortality of COVID-19-patients

Yasseen

Elkhodiry²,

El-Messiery³

et al. 2022

Front. Immunol.

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Roles of platelets during infections surpass the classical thrombus function and are now known to modulate innate immune cells. Leukocyte-platelet aggregations and activation-induced secretome are among factors recently gaining interest but little is known about their interplay with severity and mortality during the course of SARS-Cov-2 infection. The aim of the present work is to follow platelets’ bioenergetics, redox balance, and calcium homeostasis as regulators of leukocyte-platelet interactions in a cohort of COVID-19 patients with variable clinical severity and mortality outcomes. We investigated COVID-19 infection-related changes in platelet counts, activation, morphology (by flow cytometry and electron microscopy), bioenergetics (by Seahorse analyzer), mitochondria function (by high resolution respirometry), intracellular calcium (by flow cytometry), reactive oxygen species (ROS, by flow cytometry), and leukocyte-platelet aggregates (by flow cytometry) in non-intensive care unit (ICU) hospitalized COVID-19 patients (Non-ICU, n=15), ICU-survivors of severe COVID-19 (ICU-S, n=35), non-survivors of severe COVID-19 (ICU-NS, n=60) relative to control subjects (n=31). Additionally, molecular studies were carried out to follow gene and protein expressions of mitochondrial electron transport chain complexes (ETC) in representative samples of isolated platelets from the studied groups. Our results revealed that COVID-19 infection leads to global metabolic depression especially in severe patients despite the lack of significant impacts on levels of mitochondrial ETC genes and proteins. We also report that severe patients’ platelets exhibit hyperpolarized mitochondria and significantly lowered intracellular calcium, concomitantly with increased aggregations with neutrophil. These changes were associated with increased populations of giant platelets and morphological transformations usually correlated with platelets activation and inflammatory signatures, but with impaired exocytosis. Our data suggest that hyperactive platelets with impaired exocytosis may be integral parts in the pathophysiology dictating severity and mortality in COVID-19 patients.

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Peripheral blood cells of native pig (Zovawk) of Mizoram, India: Light and scanning electron microscopy analysis

Choudhary

Sarkar

Madkour

et al. 2022

Microscopy Res & Technique

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The literature on the blood cells of Zovawk pigs is scanty; thus, this study was designed to elaborate the light microscopy (LM) and scanning electron microscopy (SEM) characterization of blood cells of Zovawk pigs by LM and SEM. Twelve different blood samples were aseptically obtained from adult healthy Zovawk pigs, irrespective of sex. To determine the cytomorphological and cytochemical structures of the many produced constituents of blood, blood smears were stained with various stains. The blood samples were treated with various substrates for cytoenzymatic research, and the alterations were noted. A 1000× magnification Olympus Trinocular Research microscope was used to examine the smears. The blood samples were prepared for electron microscopy according to the standard procedure. The prepared samples were delivered to the Sophisticated Analytical Instrumentation Facility (SAIF), All India Institute of Medical Sciences (AIIMS), New Delhi, for SEM imaging. On LM, pig erythrocytes were spherical and nonnucleated. The cytoplasm of the neutrophils was spherical and included cytoplasmic granules. The eosinophils had prominent cytoplasmic granules and were round. Basophils were infrequently present and had cytoplasmic granules that were clear blue. The sizes of small, medium and large lymphocytes were noted. The monocytes were oval or circular. The platelets ranged in form from asymmetric to round. The blood samples were stained for cytochemical analyses using acid ferrocyanide stain for iron, Sudan black blue stain for lipids, toluidine blue stain for mucopolysaccharides, and periodic acid Schiff's stain for glycogen. The cytoenzymatic characteristics were evaluated and compared with substrates treated with acid phosphatase, alkaline phosphatase, peroxidase, arylsulfatase, cytochrome oxidase, beta‐glucuronidase, and succinate dehydrogenase. Erythrocytes appeared as biconcave disks under SEM. Two forms of leukocytes were observed, having a rough and pointed cell surface like a flower. It can be concluded that the LM and SEM morphology of blood cells of Zovawk pigs resembled other domestic animals, however, few differences were observed among the discussed animals.

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Preparation of blood samples for electron microscopy: The standard protocol

Cited by 16 publications

References 2 publications

Observations on the cytomorphology and ultrastructure of the peripheral blood cells of native cattle (Zobawng) of Mizoram, India

Observations on the cytomorphology and ultrastructure of the peripheral blood cells of native cattle (Zobawng) of Mizoram, India

Platelets’ morphology, metabolic profile, exocytosis, and heterotypic aggregation with leukocytes in relation to severity and mortality of COVID-19-patients

Peripheral blood cells of native pig (Zovawk) of Mizoram, India: Light and scanning electron microscopy analysis

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