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					                                            Shashi A et al., IJSID, 2012, 2 (2), 274-289



                                                                                                               ISSN:2249-5347
                                                                                                                         IJSID
                        International Journal of Science Innovations and Discoveries                                An International peer
                                                                                                               Review Journal for Science


 Research Article                                                            Available online through www.ijsidonline.info
   SCANNING ELECTRON MICROSCOPIC STUDY OF HUMAN RED BLOOD CELL ABNORMALITIES IN FLUORIDE TOXICITY


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




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


                             endemic fluoridated areas, the cross sectional study was conducted at Bathinda region of Punjab, India. The
                             concentration of fluoride was 8.25 - 10.25 ppm. EDTA anticoagulated venous blood of fluorotic patients was
                             used for scanning electron microscopic examination. The SEM analysis revealed multiple discrete blisters on
Received: 19.12.2011


                             the surface of red blood cells, and formation of hypochromic red cells, leptocytes, stomatocytes, spherocytes,
Accepted: 30.04.2012

                             schistocytes, keratocytes, degmacytes, and dacrocytes in patients afflicted with fluorosis. The red blood cells
                             were irregularly shaped with multiple cytoplasmic projections. Morphologic abnormalities caused by fluoride
*Corresponding Author

                             ingestion included marked poikilocytosis, echinocytosis, acanthocytosis, ovalocytosis, elliptocytosis, and
                             spherocytosis. The majority of red blood cells of fluorotic patients revealed presence of echinocytes and
                             crenated erythrocytes which were characterized by numerous, short, equally spaced blunt to sharp surface
                             projections. Acanthocytes have spherical shapes bearing multiple spicules. There was accumulation of
                             erythrocytes with multiple protuberances, processes, perforations, and crypt like excavations. Spherical and
                             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
                             cluster of four red cells adhering side to side in a stack were observed in rouleaux formation. Erythrocytes
                                                         to volume ratio appeared as codocytes. Eccentrocytes had contracted to spherical
                             with increased surface areaINTRODUCTION
                             regions and thin collapsed regions. The eccentrocytosis may be associated with the excess oxidative stress to
Address:


                             the erythrocyte membrane which induces cross linking of membrane proteins. The pyknocytes developed
Name: Dr. Shashi
Aggarwal
                             from eccentrocytes after the loss of much of the fused membrane, and have small membrane tag along one
                             side of the cell. These lesions demonstrate beginning of hemolysis, and irregular fragment of cell membrane
Place: Patiala, India

E-mail: Shashiuniindia       separate from cell surface. The shape of erythrocyte changes from biconcave disc to more spherical forms,
                             and these flat forms consistent with collapsed ghosts were observed with increasing frequency. Ghost cells
@yahoo.com                                               INTRODUCTION

                             vary in shape, and possess some degree of surface irregularity with large membrane indentations. The
                             surface of many red cells was studded with small spherical bodies. Superimposed red cells were also
                             prominent. The rapidity of hemolysis suggests that action of fluoride toxicity itself lead to red cell
                             destruction.
                             Keywords: Micromorphology; Poikilocytosis; Red blood cell abnormalities; Scanning electron microscope;
                                              Skeletal fluorosis.




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         Fluorosis caused by intake of fluoride has been recognized in India for several decades. It is wide spread in as many as
                                                        INTRODUCTION


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
known to affect the dental and skeletal systems (Shashi et al., 2008). Earlier findings from our laboratory demonstrated
detrimental effects of fluoride on impairment of soft tissue functions in experimental animals (Shashi, 2002, 2003; Shashi et
al., 2009, 2010) and in humans (Shashi and Kumar, 2008, 2009). Adverse hematological effects of fluoride have been reported
including damage to hematopoietic organs (Eren et al., 2005).
         Several studies have shown that excessive ingestion of fluoride hampers haemopoiesis, alters blood parameters, and
affects absorption, excretion, distribution, and retention of several minerals (Bharti et al., 2007). The biochemical changes in
glucose metabolism in erythrocytes have been related to the structural and functional alterations of red cells during
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
membrane, besides other cells, tissues and organs. The erythrocyte membrane in turn looses calcium content. The RBC
membrane, which is deficient in calcium content, is pliable and is thrown into folds and attains the shape of an amoeba called
echinocyte (Rawlani et al., 2010). Echinocytes undergo phagocytosis and are eliminated from circulation. In addition,
development of anemia has also been demonstrated in human and in experimental animals chronically exposed to toxic
amount of fluoride.
         Although environmental fluoride pollution has been linked to increased morbidity from hematological diseases, the
influence of bioaccumulation of fluoride level in blood and its impact on human red blood cell abnormalities has not yet been
sufficiently investigated.


         The study was conducted on 140 patients affected with dental and skeletal fluorosis (68 males, 72 females, and mean
                                                 MATERIALS AND METHODS


age 42.12 ± 11.94). The patients were selected randomly from high fluoride area, Sivian, of district Bathinda, Punjab, India
(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
university, Patiala.


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


glutraldehyde prepared in 2 molar cacodylate buffer for one and half hour at room temperature for primary fixation. Red
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%
osmium tetraoxide for one hour for secondary fixation. The post-fixed cells were washed in 0.1 molar cacodylate rinsing buffer
and in distilled water three times for 15 minutes at room temperature.
         After secondary fixation, the cells were dehydrated in graded series of ethanol. The samples were vaccum dried in
dessicator for overnight. After drying, samples were mounted on aluminium stubs with double adhesive carbon tapes and
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.


        The study revealed high levels of fluoride in serum samples of the patients. Mottling of teeth and skeletal deformities
                                                           RESULTS


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).
         Spiny cells showed various stages of crenation. Echinocytes were morphologically altered crenated red blood cells,
characterized by numerous short, more or less evenly spaced, blunt to sharp surface projections, and have a serrated outline
and irregular edges. The cells have deformed and angulated cell periphery with spicule formation. Spiny knobs were regularly
dispersed over the cell surface (Figs. 1-5). Acanthocytic forms were determined by a structural pathologic membrane defect,
where the forms of spiculae are limited to different degrees of spiny character. Acanthocytes in fluorotic patients were
characterized by six to seven irregularly spaced projections. The individual spicules have knobby ends (Fig. 6).




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         Ultrastructural study revealed many ‘Tear-drop’ poikilocytes and the length of tail vary from cell to cell (Figs. 7-9).
Schistocytes were fragmented red blood cells formed by fragmentation of abnormal cells that have different size and shape
(Figs. 10-12).




        Keratocytes “horned” cells were spiculated red cells with one or two knob-like projections. It results from the rupture
of a vacuole formed near the red cell surface (Figs. 13-14). Spherocytes were spherical red cells with reduced surface area-to-
volume ratio. Spherocytes lack the normal central pallor. The spherocyte is formed when there is a defect in the membrane
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
degmacytes with single (Fig. 24) or double (Fig. 25) peripheral ‘bites’ like appearances on the red cell membrane surface.
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        Codocytes (Target cell) were thin, bell-shaped, hypochromic cells with a round area of central pigmentation (Figs. 26-
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
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
sharply defined clear central area and a thickened peripheral ring of haemoglobin, that result from the peripheral
redistribution of haemoglobin (Figs. 37-38). Stomatocytes were uniconcave, cup- shaped red blood cells with a slit like area of
central pallor (Figs. 39- 41). A few stomatocyte have a deep pit near the border of red cell surface (Fig. 42). Knizocytes
“Pinched” cells were triconcave red cells that have a central bar of haemoglobin and clear spaces on either sides (Figs. 33, 43-
50).




        Reticulocytes were young red cells, newly released from the bone-marrow, that still contain ribosomal RNA. They
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.
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        Eccentrocytes were red cells with condensed hemoglobinized fringe of cytoplasm along one side of the cell. When the
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.




        Pyknocytes represent one of many red cell shapes that occur as a result of irregular contraction of red cells due to
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
surface (Fig. 59). Ghost cells vary in shape, and possess some degree of surface irregularity with large membrane indentations
(Fig. 60). The surface of many red cells was studded with small spherical bodies. The cells showed small protrusions on the



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


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).
        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
studies, which revealed that aluminium fluoride perturbed the structure of dimyristoylphosphatidylcholine, class of lipids
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
phagocytosis and are eliminated from circulation. This would mean that RBCs in individuals exposed to fluoride poisoning
shall not live their entire life span of 120-130 days, but are likely to be eliminated as echinocytes. This would lead to low
hemoglobin levels in patients chronically ill due to fluoride toxicity.
        Highly crenated red cells (burr cells) have also been found in uremia, bleeding peptic ulcer and carcinoma of stomach
(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
deformed red cells such as keratocytes, schistocytes and degmacytes. The shape of erythrocytes was changed from biconcave
disc to more spherical forms, and were observed with increasing frequency.



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        Red cells from humans exposed chronically to toxic levels of fluoride through drinking water showed significant
increase in lipid peroxidation, and membranes cholesterol and phospholipids. Additionally, electrophoretic patterns of ghost
membrane proteins revealed the presence of a new band in the range of =66Kd and increase in the high molecular weight
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).
        Fluoride causes disruptive effect on erythropoiesis, enhanced production of superoxide radicals and lipid peroxidation
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
elliptocytes and microspherocytes are characteristic red blood cells from cases of fluorosis. Oval shape attributes to a defect in
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
disorganization of spectrin network were observed after addition of 1mM sodium fluoride and 10 µM aluminium chloride in
human red blood cells. Cells lost their membrane material and became smaller (Strunecka et al., 1991).
        During present investigation, the formation of eccentrocytes may be associated with the excess oxidative stress to the
erythrocytes which induce cross linking of membrane proteins. An eccentrocyte is an erythrocyte in which the hemoglobin is
located eccentrically, leaving a hemoglobin-poor area in the remaining part of the cell. Eccentrocytes develop secondary to
oxidant damage to erythrocyte membranes. An eccentrocyte forms when opposing areas of the cytoplasmic face of the
erythrocyte membrane adhere together, concentrating the hemoglobin in the remaining volume of the erythrocyte. The cross-
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
were contacted distorted erythrocytes with spiny projections and irregularly spheroid, with only a tag of fused membrane
remaining.
         During present investigation, scanning electron microscopy reveals the presence of many red cells shapes that occur
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 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
the spleen, the red cell shape changes to that of a keratocyte (horn cell).
         In the present study, scanning electron microscopy revealed multiple discrete, blisters on the surface of red blood
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
hemolysis suggests that the action of the fluoride toxin itself lead to red cell destruction. Ghosts can evidently maintain some
shape due to an increase in surface rigidity induced by the hemolysis. Various concentration of sodium fluoride in the range of
50 to 500 µg/mL cause destabilization of red blood cell membrane leading to influx of water into the cells thereby causing
hemolysis (Verma et al., 2006).


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             In the present study, reticulocytes were visible in the blood of fluorosed patients. There are immature red blood cells
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
reticulocytes is associated with hypoxia, red blood cell destruction, glucose-6-phosphate dehydrogenase deficiency and
haemolytic anemia. A number of deformed red blood cells commonly seen in patients with various anemias, were also noted in
fluorosis.
         The present study showed a significant positive correlation between blood fluoride level and erythrocyte morphology.
Hence, it is possible to assume that a relation between presence of fluoride and pathological changes in erythrocytes exists.
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
of fluoride ions on erythrocyte membrane. These lesions demonstrate beginning of hemolysis, cellular collapse, and ghost
formation. Tear drop cells are pathologic and indicate significant bone marrow dysfunction.


         The results of scanning electron microscopy give morphological confirmation of increased destruction of erythrocytes
                                                            CONCLUSION


in fluorosis and indicate the great importance of fragmentation and sequestration in this process.


         We wish to thank the Management and Staff of Electron Microscopy and Nanoscience Laboratory (EMN), Punjab
                                                       ACKNOWLEDGEMENT


Agricultural University, Ludhiana, India for providing SEM facilities.


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