Shashi A et al.

, IJSID, 2012, 2 (2), 274-289

ISSN:2249-5347

IJSID

International Journal of Science Innovations and Discoveries

Research Article

An International peer Review Journal for Science

Available online through www.ijsidonline.info

*Department of Zoology, Punjabi University, Patiala 147002, Punjab, India Shashi A *, Meenakshi G
ABSTRACT

SCANNING ELECTRON MICROSCOPIC STUDY OF HUMAN RED BLOOD CELL ABNORMALITIES IN FLUORIDE TOXICITY

Received: 19.12.2011 Accepted: 30.04.2012

*Corresponding Author

endemic fluoridated areas, the cross sectional study was conducted at Bathinda region of Punjab, India. The used for scanning electron microscopic examination. The SEM analysis revealed multiple discrete blisters on the surface of red blood cells, and formation of hypochromic red cells, leptocytes, stomatocytes, spherocytes, schistocytes, keratocytes, degmacytes, and dacrocytes in patients afflicted with fluorosis. The red blood cells spherocytosis. The majority of red blood cells of fluorotic patients revealed presence of echinocytes and

concentration of fluoride was 8.25 - 10.25 ppm. EDTA anticoagulated venous blood of fluorotic patients was were irregularly shaped with multiple cytoplasmic projections. Morphologic abnormalities caused by fluoride

To elucidate morphological changes in red blood cells in patients of skeletal fluorosis living in

ingestion included marked poikilocytosis, echinocytosis, acanthocytosis, ovalocytosis, elliptocytosis, and projections. Acanthocytes have spherical shapes bearing multiple spicules. There was accumulation of erythrocytes with multiple protuberances, processes, perforations, and crypt like excavations. Spherical and

crenated erythrocytes which were characterized by numerous, short, equally spaced blunt to sharp surface atypical erythrocytes showed the processes of physiological aging and destruction. The surface of some erythrocyte looked granular, roughly folded with microprominences in the cell membrane. A regularly spaced regions and thin collapsed regions. The eccentrocytosis may be associated with the excess oxidative stress to

Address: Name: Dr. Shashi Aggarwal Place: Patiala, India E-mail: Shashiuniindia @yahoo.com

cluster of four red cells adhering side to side in a stack were observed in rouleaux formation. Erythrocytes the erythrocyte membrane which induces cross linking of membrane proteins. The pyknocytes developed separate from cell surface. The shape of erythrocyte changes from biconcave disc to more spherical forms, from eccentrocytes after the loss of much of the fused membrane, and have small membrane tag along one vary in shape, and possess some degree of surface irregularity with large membrane indentations. The destruction.

with increased surface areaINTRODUCTION to volume ratio appeared as codocytes. Eccentrocytes had contracted to spherical side of the cell. These lesions demonstrate beginning of hemolysis, and irregular fragment of cell membrane

and these flat forms consistent with collapsed ghosts were observed with increasing frequency. Ghost cells

INTRODUCTION

surface of many red cells was studded with small spherical bodies. Superimposed red cells were also Skeletal fluorosis.

prominent. The rapidity of hemolysis suggests that action of fluoride toxicity itself lead to red cell Keywords: Micromorphology; Poikilocytosis; Red blood cell abnormalities; Scanning electron microscope;

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Shashi A et al., IJSID, 2012, 2 (2), 274-289 22 states of the Indian republic. Punjab is one of the northern states affected with hydrofluorosis. There is a close correlation between the distribution of fluoride bearing minerals and prevalence of endemic fluorosis (Rao and Mala, 2009). Fluoride is including damage to hematopoietic organs (Eren et al., 2005). known to affect the dental and skeletal systems (Shashi et al., 2008). Earlier findings from our laboratory demonstrated al., 2009, 2010) and in humans (Shashi and Kumar, 2008, 2009). Adverse hematological effects of fluoride have been reported glucose metabolism in erythrocytes have been related to the structural and functional alterations of red cells during Several studies have shown that excessive ingestion of fluoride hampers haemopoiesis, alters blood parameters, and detrimental effects of fluoride on impairment of soft tissue functions in experimental animals (Shashi, 2002, 2003; Shashi et Fluorosis caused by intake of fluoride has been recognized in India for several decades. It is wide spread in as many as INTRODUCTION

affects absorption, excretion, distribution, and retention of several minerals (Bharti et al., 2007). The biochemical changes in erythropoiesis by accumulated fluoride in bones. The decreased hematocrit levels are attributed to a decrease in size of erythrocytes due to stressful conditions. It is now known that when fluoride is ingested, it accumulate on the erythrocyte echinocyte (Rawlani et al., 2010). Echinocytes undergo phagocytosis and are eliminated from circulation. In addition, amount of fluoride. membrane, besides other cells, tissues and organs. The erythrocyte membrane in turn looses calcium content. The RBC development of anemia has also been demonstrated in human and in experimental animals chronically exposed to toxic influence of bioaccumulation of fluoride level in blood and its impact on human red blood cell abnormalities has not yet been sufficiently investigated. MATERIALS AND METHODS age 42.12 11.94). The patients were selected randomly from high fluoride area, Sivian, of district Bathinda, Punjab, India university, Patiala.

membrane, which is deficient in calcium content, is pliable and is thrown into folds and attains the shape of an amoeba called Although environmental fluoride pollution has been linked to increased morbidity from hematological diseases, the The study was conducted on 140 patients affected with dental and skeletal fluorosis (68 males, 72 females, and mean

(water fluoride levels 8.05 to 10.25 mg/L, mean 9.15 1.55 mg/L). EDTA anticoagulated venous whole blood samples were used for scanning electron microscope examination. The study was approved by the Institutional ethics committee, Punjabi SCANNING ELECTRON MICROSCOPY

glutraldehyde prepared in 2 molar cacodylate buffer for one and half hour at room temperature for primary fixation. Red and in distilled water three times for 15 minutes at room temperature.

blood cells were separated from white blood cells by centrifugation. After primary fixation, the cells were washed with 1 molar cacodylate rinsing buffer three times for 15 minutes at room temperature. After washing, the samples were fixed in 1% After secondary fixation, the cells were dehydrated in graded series of ethanol. The samples were vaccum dried in osmium tetraoxide for one hour for secondary fixation. The post-fixed cells were washed in 0.1 molar cacodylate rinsing buffer dessicator for overnight. After drying, samples were mounted on aluminium stubs with double adhesive carbon tapes and International Journal of Science Innovations and Discoveries, Volume 2, Issue 2, March-April 2012

Red cells were prepared for SEM examination by fixing the sequestrenated whole blood in 2.5% buffered

coated with gold for 20 seconds in a gold ion sputterer (HITACHI-E-1010) to render their surfaces conducting. Red cells were

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visualized and photographed in a HITACHI-S-3400 N scanning electron microscope. Microphotographs were taken at direct magnifications between 3,000 and 15,000 X. were common. The SEM images of various types of dysmorphic red blood cells in fluorotic patients promoted the use of a separate word to describe them (Kimzey et al., 1975). characterized by numerous short, more or less evenly spaced, blunt to sharp surface projections, and have a serrated outline characterized by six to seven irregularly spaced projections. The individual spicules have knobby ends (Fig. 6). and irregular edges. The cells have deformed and angulated cell periphery with spicule formation. Spiny knobs were regularly where the forms of spiculae are limited to different degrees of spiny character. Acanthocytes in fluorotic patients were Spiny cells showed various stages of crenation. Echinocytes were morphologically altered crenated red blood cells, The study revealed high levels of fluoride in serum samples of the patients. Mottling of teeth and skeletal deformities RESULTS

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dispersed over the cell surface (Figs. 1-5). Acanthocytic forms were determined by a structural pathologic membrane defect,

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Schistocytes were fragmented red blood cells formed by fragmentation of abnormal cells that have different size and shape (Figs. 10-12).

Ultrastructural study revealed many Tear-drop poikilocytes and the length of tail vary from cell to cell (Figs. 7-9).

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of a vacuole formed near the red cell surface (Figs. 13-14). Spherocytes were spherical red cells with reduced surface area-tovolume ratio. Spherocytes lack the normal central pallor. The spherocyte is formed when there is a defect in the membrane degmacytes with single (Fig. 24) or double (Fig. 25) peripheral bites like appearances on the red cell membrane surface. International Journal of Science Innovations and Discoveries, Volume 2, Issue 2, March-April 2012 function (Figs. 15-17). SEM examination of red blood cell from fluorotic patients exhibited presence of many poikilocytes such as ovalocytes (Figs. 18-19), elliptocytes (Fig. 20-21), bell- shaped (Fig. 22), and triangular cells (Fig, 23). Degmacytes were morphologically altered red blood cells with multiple arcuate bites on peripheral cell membrane. SEM analysis revealed

Keratocytes horned cells were spiculated red cells with one or two knob-like projections. It results from the rupture

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28). Three-dimensional appearance of a red cell showed, characteristic central V- shaped hemoglobinized thick rim surrounded by an area of pallor (Fig. 29). Rouleaux formation (Pseudoagglutination) was a linear arrangement of red blood cells (coinstack) caused by an increased blood concentration of fibrinogen, globin, or paraproteins, were prominent in blood smear of fluorotic patients. A regularly spaced cluster of four red cells adhering side to side in a stack was observed (Fig. 30). Leptocytes were thin, flattened red cells with excessive surface area compared to its content (Fig. 31). These cells tend International Journal of Science Innovations and Discoveries, Volume 2, Issue 2, March-April 2012

Codocytes (Target cell) were thin, bell-shaped, hypochromic cells with a round area of central pigmentation (Figs. 26-

to fold and appear as folded bowl- shaped cells (Fig. 32). Hypochromic red cells with narrow rim of hemoglobin and extended

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central concavity were prominent in blood of fluorotic patients (Figs. 34-36). Torocytes were ring- shaped red cells with a redistribution of haemoglobin (Figs. 37-38). Stomatocytes were uniconcave, cup- shaped red blood cells with a slit like area of 50). central pallor (Figs. 39- 41). A few stomatocyte have a deep pit near the border of red cell surface (Fig. 42). Knizocytes

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sharply defined clear central area and a thickened peripheral ring of haemoglobin, that result from the peripheral

Pinched cells were triconcave red cells that have a central bar of haemoglobin and clear spaces on either sides (Figs. 33, 43-

were considerably larger than mature erythrocytes. As a consequence of reduced hemoglobin concentration, they were less dense. They have an irregular, multilobed surface. Various forms of reticulocytes have been presented in Figs. 51-53. International Journal of Science Innovations and Discoveries, Volume 2, Issue 2, March-April 2012

Reticulocytes were young red cells, newly released from the bone-marrow, that still contain ribosomal RNA. They

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thin membrane of the eccentrocyte get ruptured, a small cell which lacks central pallor was formed, this contacted spherocytic cell with a projection was termed Pyknocyte (Figs. 54-56). Pyknocyte were red cells in which the haemoglobin is concentrated in one half of the cell with the other half mostly empty of haemoglobin giving a blister appearance.

Eccentrocytes were red cells with condensed hemoglobinized fringe of cytoplasm along one side of the cell. When the

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damage to the membrane. Pyknocyte revealed the small membrane tag along one side of the cell (Fig. 57). Blister cell is formed when the cell is injured and a portion of the haemoglobin leaks out. A red cell with discrete and ill defined areas of cell membrane exhibited blister like structure protruding from the cell surface (Fig. 58). During the phase of accelerated hemolysis, irregular fragments of membranous material, presumably cell membrane, appeared to separate from the cell (Fig. 60). The surface of many red cells was studded with small spherical bodies. The cells showed small protrusions on the International Journal of Science Innovations and Discoveries, Volume 2, Issue 2, March-April 2012 surface (Fig. 59). Ghost cells vary in shape, and possess some degree of surface irregularity with large membrane indentations

Pyknocytes represent one of many red cell shapes that occur as a result of irregular contraction of red cells due to

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cell surface (Fig. 61). Superimposed red cells were observed in blood of fluorotic cases. These cells were more thicker than other red cells (Fig.62).

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intoxication. Structural alterations correlated well with the kinetics of the fluoride toxin. Reports have also shown that fluoride induced disorders in hematopoitic organs in mice (Machalinska et al., 2002) and human hematopoietic progenitor cells (Machalinski et al., 2000). studies, which revealed that aluminium fluoride perturbed the structure of dimyristoylphosphatidylcholine, class of lipids phagocytosis and are eliminated from circulation. This would mean that RBCs in individuals exposed to fluoride poisoning hemoglobin levels in patients chronically ill due to fluoride toxicity. Echinocyte forms infact are determined by a structural pathologic membrane defect. It was found that aluminium fluoride altered the shape of erythrocytes inducing the formation of echinocytes. This effect was explained by X-ray diffraction located in the outer monolayer of the erythrocyte membrane confirmed by fluorescence spectroscopy on dimyristoylphosphatidylcholine large unilamellar vesicles (Suwalsky et al., 2004). The echinocytes were present in large numbers, depending upon the extent of fluoride poisoning and duration of exposure to fluoride. Echinocytes undergo Highly crenated red cells (burr cells) have also been found in uremia, bleeding peptic ulcer and carcinoma of stomach shall not live their entire life span of 120-130 days, but are likely to be eliminated as echinocytes. This would lead to low (Rao and Friedman, 1975). The most striking was the separation of large fragments of cell membrane from red blood cell surfaces. These alterations produce finger like protrusions during the period of accelerated lysis and forms various type of disc to more spherical forms, and were observed with increasing frequency. deformed red cells such as keratocytes, schistocytes and degmacytes. The shape of erythrocytes was changed from biconcave

The present study provides evidences that the erythrocyte membrane is the primary site of action of fluoride DISCUSSION

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increase in lipid peroxidation, and membranes cholesterol and phospholipids. Additionally, electrophoretic patterns of ghost protein and predominace of bands of =93Kd and =20 Kd. The activities of sodium, potassium, magnesium, calcium ions and ATPase were significantly decreased in the red cell ghosts of fluorotic patients (Kumari and Rao, 1991).

membrane proteins revealed the presence of a new band in the range of =66Kd and increase in the high molecular weight that lead to alterations in erythrocytes cell membrane function and structure in humans (Ailani et al., 2009). The observations presented here exhibit morphologic alterations in shape of human red blood cells during fluoride toxicity. Ovalocytes and horizontal red cell membrane protein interactions. Qualitative and quantitative abnormalities of spectrin and membrane protein band 4.1 have been associated with elliptocytosis in humans (Salsbury and Clarke, 1967). Shape changes and human red blood cells. Cells lost their membrane material and became smaller (Strunecka et al., 1991). Fluoride causes disruptive effect on erythropoiesis, enhanced production of superoxide radicals and lipid peroxidation

Red cells from humans exposed chronically to toxic levels of fluoride through drinking water showed significant

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elliptocytes and microspherocytes are characteristic red blood cells from cases of fluorosis. Oval shape attributes to a defect in disorganization of spectrin network were observed after addition of 1mM sodium fluoride and 10 M aluminium chloride in erythrocytes which induce cross linking of membrane proteins. An eccentrocyte is an erythrocyte in which the hemoglobin is oxidant damage to erythrocyte membranes. An eccentrocyte forms when opposing areas of the cytoplasmic face of the bonding of erythrocyte membranes requires both an alteration in membrane skeletal proteins to make them adhesive and a force that will bring opposing sides of the membrane together (Fischer, 1986). Pyknocytes developed from eccentrocytes and remaining. as a result of irregular contraction of red cells due either to damage to the membrane or shape changes that occur as a result of the spleen, the red cell shape changes to that of a keratocyte (horn cell). were contacted distorted erythrocytes with spiny projections and irregularly spheroid, with only a tag of fused membrane

located eccentrically, leaving a hemoglobin-poor area in the remaining part of the cell. Eccentrocytes develop secondary to

During present investigation, the formation of eccentrocytes may be associated with the excess oxidative stress to the

erythrocyte membrane adhere together, concentrating the hemoglobin in the remaining volume of the erythrocyte. The cross-

the removal of red cell inclusion (e.g. Heinz bodies, unstable hemoglobin) mostly by spleen. The blister cells formed as a result of fusion of the inner red cell membrane on the end that was devoid of hemoglobin. When the blister ruptures or is removed by cells. The most striking was the separation of large fragments of cell membrane from red blood cell surfaces. These alterations produce finger like protrusions during the period of accelerated lysis and forms various type of deformed red cells such as pyknocytes, keratocytes, schistocytes and degmacytes. The shape of erythrocytes was changed from biconcave disc to more spherical forms, and were observed with increasing frequency. Moreover, high fluoride concentration may disturb the anion channel of the erythrocytes membrane, which leads to hemolysis and swelling of cells (Grabowska et al., 1991). The rapidity of 50 to 500 g/mL cause destabilization of red blood cell membrane leading to influx of water into the cells thereby causing hemolysis (Verma et al., 2006). International Journal of Science Innovations and Discoveries, Volume 2, Issue 2, March-April 2012 hemolysis suggests that the action of the fluoride toxin itself lead to red cell destruction. Ghosts can evidently maintain some In the present study, scanning electron microscopy revealed multiple discrete, blisters on the surface of red blood

During present investigation, scanning electron microscopy reveals the presence of many red cells shapes that occur

shape due to an increase in surface rigidity induced by the hemolysis. Various concentration of sodium fluoride in the range of

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seen when there is a severe demand for red blood cells to be released by the bone marrow in the anemic conditions. It is known that chronic fluoride ingestion leads to elevation and accumulation of fluoride in bone. Elevated numbers of haemolytic anemia. A number of deformed red blood cells commonly seen in patients with various anemias, were also noted in fluorosis. Hence, it is possible to assume that a relation between presence of fluoride and pathological changes in erythrocytes exists. formation. Tear drop cells are pathologic and indicate significant bone marrow dysfunction. CONCLUSION

reticulocytes is associated with hypoxia, red blood cell destruction, glucose-6-phosphate dehydrogenase deficiency and

In the present study, reticulocytes were visible in the blood of fluorosed patients. There are immature red blood cells

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This syndrome of fluoride toxicity and erythrocyte membrane injury secondary to the erythrocyte oxidative stress has not been reported previously in animals or humans. Fluoride could cause hypochromic microcytic anemia due to bioaccumulation The results of scanning electron microscopy give morphological confirmation of increased destruction of erythrocytes We wish to thank the Management and Staff of Electron Microscopy and Nanoscience Laboratory (EMN), Punjab ACKNOWLEDGEMENT REFERENCES

The present study showed a significant positive correlation between blood fluoride level and erythrocyte morphology.

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