scanning election microscopic study of human red blood cell abnoinormalities in fluoride toxicity

7/31/2019 Scanning Election Microscopic study of Human Red Blood Cell Abnoinormalities in Fluoride Toxicity

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

International Journal of Science Innovations and Discoveries, Volume 2, Issue 2, March-April 2012

274

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

Shashi A *, Meenakshi G

*Department of Zoology, Punjabi University, Patiala 147002, Punjab, India

INTRODUCTION

INTRODUCTION

ISSN:2249-5347

IJSID

International Journal of Science Innovations and Discoveries An International peerReview Journal for Science

Research Article Available online through www.ijsidonline.info

Received: 19.12.2011

Accepted: 30.04.2012

*Corresponding Author

Address:

Name: Dr. Shashi

Aggarwal

Place: Patiala, India

E-mail: Shashiuniindia

@yahoo.com

ABSTRACT

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

endemic fluoridated areas, the cross sectional study was conducted at Bathinda region of Punjab, India. Th

concentration of fluoride was 8.25 - 10.25 ppm. EDTA anticoagulated venous blood of fluorotic patients wa

used for scanning electron microscopic examination. The SEM analysis revealed multiple discrete blisters onthe 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 cell

were irregularly shaped with multiple cytoplasmic projections. Morphologic abnormalities caused by fluoride

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 surfac

projections. Acanthocytes have spherical shapes bearing multiple spicules. There was accumulation o

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. Erythrocyte

with increased surface area to volume ratio appeared as codocytes. Eccentrocytes had contracted to spherica

regions and thin collapsed regions. The eccentrocytosis may be associated with the excess oxidative stress to

the erythrocyte membrane which induces cross linking of membrane proteins. The pyknocytes developed

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 membran

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 cell

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 cel

destruction.

Keywords: Micromorphology; Poikilocytosis; Red blood cell abnormalities; Scanning electron microscope

Skeletal fluorosis.


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INTRODUCTION

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

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.

MATERIALS AND METHODS

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

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.

SCANNING ELECTRON MICROSCOPY

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

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.

RESULTS

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

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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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 periphera

redistribution of haemoglobin (Figs. 37-38). Stomatocytes were uniconcave, cup- shaped red blood cells with a slit like area o

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 cel

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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DISCUSSION

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

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 cell

(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 cel

surfaces. These alterations produce finger like protrusions during the period of accelerated lysis and forms various type o

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 significan

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 weigh

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 observation

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 M 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 th

erythrocytes which induce cross linking of membrane proteins. An eccentrocyte is an erythrocyte in which the hemoglobin i

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

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

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 membran

remaining.

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

as a result of irregular contraction of red cells due either to damage to the membrane or shape changes that occur as a result o

the removal of red cell inclusion (e.g. Heinz bodies, unstable hemoglobin) mostly by spleen. The blister cells formed as a resul

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 a

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 anio

channel of the erythrocytes membrane, which leads to hemolysis and swelling of cells (Grabowska et al., 1991). The rapidity o

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 o

50 to 500 g/mL cause destabilization of red blood cell membrane leading to influx of water into the cells thereby causin

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 cell

seen when there is a severe demand for red blood cells to be released by the bone marrow in the anemic conditions. It i

known that chronic fluoride ingestion leads to elevation and accumulation of fluoride in bone. Elevated numbers o

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 no

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 ghos

formation. Tear drop cells are pathologic and indicate significant bone marrow dysfunction.

CONCLUSION

The results of scanning electron microscopy give morphological confirmation of increased destruction of erythrocyte

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

ACKNOWLEDGEMENT

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

Agricultural University, Ludhiana, India for providing SEM facilities.

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scanning election microscopic study of human red blood cell abnoinormalities in fluoride toxicity

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