Report : Literature Review

Ariz Qillah 5536010/SCPA M

1. Target Cell

Target cells can be found in peripheral blood. Predominantly, it caused by a consequence of RBC surface membrane increased. By electron microscope, this abnormal cell can be seen in the form of bell shaped like (14, Figure 1). The marker of target cells or codocyte can be recognized by a large part of hemoglobin displayed at the rim of the cell and then, the part of hemoglobin located in the central as targets (10,13,14). Mechanism of target cell occurs when mature red blood cells have not capabilitiy to synthesize either cholesterol or phospholipid by itself (13,14). Therefore, there is have greatly accumulation of cholesterol in the plasma so that red blood cells extended by elevating of membrane lipid then it gives rise to the osmotic fragility decreased (13. The condition related with mechanism of target cell are when membrane cholesterol and phospholipid exceed as well as cellular hemoglobin reduced (13). Mostly, target cells are available in hemoglobinopathies, thalassemias, liver diseases; sometimes in anemia (14, Figure 2).

Figure 1. Light microscopy of the target cells in the peripheral blood (by Wright and Giemsa Staining

Figure 2. Pathological condition related with target cells

2. Spherocytes

The appearance of spherocytes in peripheral blood is have reducing ratio between surface to volume resulting no central pallor in the red blood cell morphology (14, Figure 3. Another marker to determine this abnormal shape in a peripheral smear is solid color and smaller size than normal red blood cell commonly (11,12,13,14). Mechanism of spherocytes occur when lack or no function between one of membrane proteins gives rise to destabilize the cytoskeleton generating morphology of red blood cells become abnormal and poor lifespan for the red blood cells affected in the circulation (11,13). Thereupon, spherocytes have highly ability to metabolize glucose and easily maintain amount of intracellular sodium extremely. When they achieve surrounding part of the spleen, homeostasis between the active-passive transport system undergoes unbalanced following by sodium elevated and glucose reduced producing the cell to be swelling and hemolysis (11,13,14). Predominantly, spherocytes can be seen in hereditary spherocytosis and autoimmune hemolytic anemia (13,14, Figure 4).

Figure 3. Morphological of spherocyte by Light Microscopy (By using Blood Smear and Wright and Giemsa staining, 12

Figure 4. Pathological condition correlated with appearance spherocyte in the peripheral blood.

3. Stomatocytes

The characteristic of stomatocytes in peripheral blood are having a central pallor as mouth-like, diameter of red blood cell normal but it has no biconcave (13,14, Figure 5). Usually, stomatocytes can be seen in artifactual form than an original morphology from causing of pathophysiologic process. The appearance of stomatocytes occur when permeability of sodium increased as a result osmotic fragility becoming high (13). In general, stomatocytes can be found in patients with hereditary spherocytosis, hereditary stomatocytosis, hemolytic anemia, alcoholic cirrhosis, and acute alcoholism (13,14).

Figure 5. Appearance of stomatocytes in the peripheral blood by Light Microscopy.

4. Ovalocytes (Elliptocytes

The shape of ovalocytes morphology in peripheral blood is more egg shaped, appears in normochromic or hypochromic, normocytic or macrocytic, macroovalocytes size can reach diameter as 9µm or more with lacking of central pallor (13,14). Whereas elliptocytes appear like pencil, rod, cigar shaped and hemoglobin located in the tag end of the cell (13,14, Figure 6).

Generally, they are not seen in hypochromic and still have a normal central pallor (13,14). Mechanism formation of ovalocytes unknown but this abnormal morphology constitutes as a part consequence of mechanical weakness that make unbalanced of fragility of the membrane (13). In most cases, both of these abnormal red blood cells can be seen in microcytic and hypochromic anemia, myelodysplastic syndromes, and myelopthistic anemia (14, Figure 7).

Figure 6. Light Microscopy of the elliptocytes morphology in Hereditary Elliptocytosis (By blood smear, Wright and Giemsa staining

Figure 7. Pathological condition of the ovalocyte and elliptocyte morphology

5. Sickle Cells (Drepanocytes

Recognizing sickle cells or drepanocytes shape in peripheral blood showed by crescent or sickle with pointed projections of one or more the tag end of the cell (13,14). There are two presence morphology of sickle cells equally irreversibly sickled cells (Figure 8) and reversible, oat-shaped sickle cells (Figure 9). Mostly, sickle cells have ability to convert the abnormal shape to discocyte when oxygenated (13,14). This abnormal red blood cell morphology appears when polymerize hemoglobin becoming rigid then generating tubules which draw up in bunches to disfeature the cell and gives rise to form the transformed cell (13,14). Accordingly, these cells have extinct their potential functions to deform and incapable to establish the microvasculature of the tissues resulting in impairment of oxygen surrounding of those areas of the cell (13,14). This abnormal shape can be found in sickle cell anemia (SCA and Hemoglobin C Harlem) (14, Figure 10).

Figure 8. Morphology of irreversibly sickled cells by Light Microscopy

Figure 9. Light microscopy of reversible, oat-shaped sickle cells

Figure 10. Pathological condition connected with sickle cells

6. Schistocytes

This abnormal shape of red blood cell marked by split, cut, cloven cells as consequence from some trauma to the cell membrane (13, 14 Figure 11). The cell membrane is stressful due to trauma leading to form extremely fragmented of red blood cells (13,14). Several factors can be triggered resulting fragmentation into the cell, one of them is fluid alteration. Regularly developing fibrin strands, endhotelium and heart valve prosthesis damaged are some examples of the fluid alteration. By fluid modification, blood flow in the circulation probably blow down red blood cells over the fibrin strands. Then, they dissociate the red cells. Hence, the shape of schistocytes appear as fragmented due to they have been cutted by the fibrins (13. Schistocytes can be posed by patients with microangiopathic hemolytic anemia, disseminated intravascular coagulation (DIC, heart valve surgery, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, renal graft rejections, vasculitis, burn cases, and march hemoglobinuria (13, 14, Figure 12).

Figure 11. Light microscopy of the schistocyte in Renal disease (Labelled by D

Figure 12. Pathological condition related with fragmented cells (Burr cells, Schistocyte and Helmet cells

7. Burr Cells (Echinocytes)

Commonly, the shape of echinocytes show in peripheral blood are a rounded spicules (13,14). Approximately, the rounded spicules of the echinocytes are 10 to 30 spicules located surrounding the surface portion of the red blood cells (13, 14, Figure 13). Beside that, they are normochromic and normocytic in the most cases. Determining these abnormal red cells often can be seen as artifact due to specimen contamination. Mechanism formation of the echinocytes occurs when there is have homeostasis unbalanced in the intravascular fluid leading to change in tonicity

especially in dehydration and azotemia. These abnormal morphology in the most case found in patients with uremia, heart disease, cancer of the stomach, peptic ulcer bleeding, and patients with untreated hypothyroidism (13, 14, Figure 12).

Figure 13. The presence of burr cell in Renal disease by Light microscopy (Labelled by A)

8. Acanthocytes (Spurr cells

The morphology of acanthocytes are marked by possessing approximate 3 to 12 spicules of uneven length dispersed throughout the edge of the cell membrane (13, 14, Figure 14). Particular mechanism relating to form the acanthocytes is still unknown. The component of acanthocytes have containing of cholesterol exceed and have greatly ratio of cholesterol to phospolipid, then resulting in increased their surface area so that they have affecting to reduce the lecithin containing of the acanthocytes. As consequence, the response of red cells towards cholesterol accumulation excessively are very responsively depending on presence of lipid balanced in the membrane. Therefore, it can precipitate to create an acanthocyte or another form equally as target cell. When creating an acanthocytes, there is have responsible to seizure the spleen and fragmented. Hence, the fluidity membrane of the red blood cells is influenced. They can be seen in abetalipoproteinemia, myeloproliferative disorders, microangiopathic hemolytic anemia (MAHA, autoimmune hemolytic anemia, and McLeod Syndromes (13, 14, Figure 15).

Figure 14. Light microscopy of the acanthocytes in peripheral blood

Figure 15. Pathological condition related with acanthocytes

9. Teardrop Cells (Dacrocytes

Hallmark of teardrop in peripheral blood circulation showed by morphology of tear-shaped or pear-shaped (13, 14, Figure 16). Throughly, mechanism physiological formation of these abnormal red blood cells is specifically unknown. On the other hand, another reason shows that presence of teardrops can be caused by formation of inclusion containing red cell (13,14). Its evidence has been kindly documented. These red blood cells consist of high number of inclusion arrange to enter the microcirculation but they can not enter so that they squeezed then forms a tailed end. The red blood cells can not enter the microcirculation because they incompetent to stabilize their normal shape when this event happened. Predominatly, teardrops are the most available in patients with idiopathic myelofibrosis following with myeloid metaplasia, thalassemia syndromes, drug-induced Heinz body formation, iron deficiency and inclusion bodies formed (13, 14).

Figure 16. Light microscopy of tear drop in peripheral blood smear

10. Keratocytes

The appearance of keratocytes in peripheral blood circulation posses two horns which are formed since the vacuole destroyed (13,14). These horn cells constitute as resembling of a helmet (13,14). Keratocytes found surrounding of fibrin strand in circulation then they divided. These abnormal red cells involve the fibrin fusing two sides of the keratocytes together (13,14). After the cells reached the fibrin strands, they appear in the peripheral blood as a red blood cell which have a vacuole that is resembling like a blister then it is called blister cell (12, 13, 14, Figure 17).

Figure 17. Morphology of blister cell/keratocytes by Light Microscopy (12, Labelled by C)

11. Dangocyte or Degmacyte (Bite Cell

Bite cell also it is well known called by a helmet cell (Figure 18, Figure 20). The helmet cell posses two particular projections that are located surrounding in empty area of the red blood cell membrane (12,13, 14). In hematological conditions, these abnormal red blood cells can be seen in patients with large inclusion bodies especially Heinz bodies and Howel-Jelly bodies formed. The mechanism of fragmentation in bite cells occur when hollowing of the spleen (13, 14). The hollowing event of the spleen can eliminate the presence of inclusion bodies in the cells and then, appearance as a bite cell. Predominantly, bite cell can be found in patients with pulmonary emboli, myeloid metaplasi, and DIC (13, 14, Figure 12).

Figure 18. Light microscopy of bite cell/dangocyte morphology in the peripheral blood (12)

Figure 20. Bite cell in acute hemolysis with glucose 6 phosphate dehydrogenase (G6PD deficiency (12)

References :

10. Davis, L. R. 1972. Target cells in hemoglobinopathies. J Clin Path. Volume 25. Pp 169-170.

11. Crosby, W. H. 1951. Analytical Review : The pathogenesis of spherocytes and leptocytes (target cells). Blood Volume 7. Pp 261-274.

12. Bain, B.J. 2005. Review Article : Diagnosis from the blood smear. The New England Journal of Medicine. Volume 353. Pp 498-507. DOI: 10.1056/NEJMra043442.

13. Lynch, E.C. 1990. Peripheral blood smear. In: Walker HK, Hall WD, Hurst JW, editors. Clinical Methods: The History, Physical, and Laboratory Examinations. 3rd edition. Boston: Butterworths. Chapter 155.

14. Samson, A.A., and Ogbenna, AA. Morphologic evaluation of anemia I. 2016. Biol Med (Aligarh). Volume 8. Pp 322. doi:10.4172/0974-8369.1000322.

12. 2 Thalassemia

12.2.1 Introduction of thalassemia : Definition, Epidemology, and Classfication

Based on Santos, et al (2015) mentioned that thalassemia is a multiform group of red blood cell disorders that has the severity ranging from a heavy phenotype to moderate symptomatic chaos (37).Thalassemia is a variant cluster of authenthically phenomenon from inherited alterations represented by the abnormality of expression of globin gene (20).Their globin gene expression induce completely lack or alleviation of α and β globin chain synthesis in human erythroid cells (20).Generally, thalassemia can be classified into two major namely α and β thalassemia (20). Panich, Pornpatkul and Sriroongrueng(1992) mentioned that approximately one per cent of the Thai population have manythalassemicincidences (21). Every year, about 50,000 gestations of women has strongly risk to get affected into fetus equally one fourth leading to thalassemic newborns. Thalassemia either α and β constitute common in Thailand including hemoglobin E and Constant Spring. Roughly 30-40 % of the distribution one region to another regionsor between different ethnic groups from the Thai population are carriers and slightly presence one case from the total population (21).

12.2.2 αThalassemia : Definition, Classification, and Clinical Manifestation

α Thalassemia is a predominantly thalassemia has relationship with no production or lack of α chains resulting in increasingly β chains produced (20). Based on Bernini and Harteveld (1998), α Thalassemia constitutes a genetic malformations highly regular available on some populations and are indicated by diminishing or flawless repression production of α globin polypeptide chains (22). Alpha-thalassemia is inherited chaos of an autosomal recessive group that is signified with hypochromic anemia, and clinical phenotypes varies from almost without symptoms into the deadly hemolytic anemia (26).In accord with Galanello and Cao (2011) stated that α thalassemia is the commonest hemoglobin genetic anomalies and familiar in tropical and subtropical distribution region of the world and also as carriers of hemoglobinopathies (23). The major malformation occurs in reducing or without α globin chains production whivch divided into many portions of several kinds of hemoglobin such as the adult of HbA (Alpha 2 Beta 2), fetal Hb F (Alpha 2 Gamma 2), and the minor portions like Hb A2 (Alpha 2 Delta 2).

Level of severe from α Thalassemia can be approved into four clinical conditions (clinical characteristics each condition can be seen in Table 1 ) such as two carriers portion arenamely alpha+-thalassemia and alpha°-thalassemia (23). Alpha+-thalassemia occurs as consequence of the deletion or impairment in the function of one of the four normal alpha globin genes while alpha°-thalassemia caused by two alpha genes in cis undergo abolishment or their function have impairment. Another two clinical conditions of α Thalassemia are clinically connected with shape such as Hb H disease and Hb Bart hydropsfetalis syndrome. Usually, Hb H disease developed because justone of alpha gene is functioning whereas Hb Bart hydropsfetalis syndrome takes place when alpha genes lost of function (23).

In many times, the silent carrier usually go on appearance of one alpha globin gene abolishment and is reflected in the new birth by a range of severity from a very mild enhancement about 1-2 % of Hb Bart where already formed tetramer structure of globin chains

of gamma 4 and usually available when there is a lot of gamma chains connect to alpha chains (23). In the grown up level, reducing one gene genotype might be absolutely silent or related with a medium level of microcytosisand hypochromia with normal HbA2(23). Furthermore, in thalassemia trait either takes place in cis or trans brightly exhibit the alpha-thalassemia trait can be recognized by increasing in medium level of Hb Bart approximately 5-6 % in the new birth and by red blood cells index with normal HbA2 and F in the adult, and dropping synthesis ratio of alpha or beta globin chain in the range of 0.7–0.8 (23). Non deletion disruption carriers have many kinds of hematologic phenotypes start from alpha trait to silent level (23). Double heterozygote for both of deletion and non deletion of α Thalassemia have each phenotype trait of thalassemia, while homozygote only for non deletion probably available phenotype of the alpha trait and occasionally, it has a mild level of Hb H disease (23).

Hb H disease is a clinical condition that results from the presence of only one function of the rest of the alpha globin genes. As a result, there is have extremely overage of beta globin chain, which establish tetramers beta 4. Hb H is labile and especially found in the sediment in red blood cells that are older where prematurely destroyed in the spleen in the range of severity from moderate to severe hemolysis (23). The most significant characteristics are hypochromic, microcytic, hemolytic anemia, hepatosplenomegaly, jaundice, and sometimes in moderate level of alpha thalassemia like bone modifications. The hemoglobin concentration is mostly started from 7-10 g / dL, and the mean corpuscular volume (MCV) based on different age namely in childhood around 58 fl and in adulthood about 64 fl, while the average corpuscular hemoglobin (MCH) is approximately 18 pginconsiderated by age (24).

The severest clinical condition in alpha thalassemia is Hb Bart's hydropsfetalis syndrome. Generally, it has related with unfunctional of all four alpha globin genes. In gestation period, fetus is labile and uncapable to generate any alpha globin chains to create HbA or HbF. The clinical features are described by extremely severe anemia (Hb level, 3-8 g / dL), labelled by presence of hepatosplenomegaly, hydropsfetalis, and cardiac failure (25). Another abnormalities in congenital are notably associated with the cardiac, skeletal and urogenital system.

Table 1. Clinical Classification of α Thalassemia (23, 27)

12.2.3  β-Thalassemia : Definition, Classification and Clinical Manifestation

Beta thalassemia is the commonest of monogenic disorder in the world which results from decreasing of  β globin chains production followed by a quantitative reduction or absent of  β chain synthesis (28,36). According to Origa (2015) stated that beta thalassemia is indicated by minimizing of hemoglobin beta chain synthesis that precipitate to lead to microcytic hypochromic anemia, abnormally peripheral blood smear is labelled by nucleated red blood cells appearant, and alleviated amounts of hemoglobin A (HbA) when analyzed by hemoglobin typing (29). In pursuance of Cao and Galanello (2010), Beta thalassemia is greatly distributedan autosomal recessive disordersprevalent in the world which is resulted fromdecreasing or without the beta globin chains synthesis of normal hemoglobin structurally. Extremely predominant of beta thalassemia found in the Mediterranean, Middle-East, Transcaucasus, Central Asia, Indian subcontinent, and Far East (30). The great cases of beta thalassemia happening arenotified Cyprus (14%), Sardinia (12%), and South East Asia (31,32).

Three conditions can increase the severity of clinical and hematological of beta thalassemia are divided into the beta thalassemia carrier state, thalassemia intermedia, and thalassemia major (33,34). Beta thalassemia carrier state is characterized by clinical asymptomatic and certain clinical features and also it arises from heterozygosity beta thalassemia. Thalassemia major is marked by presence of heavy anemia that depending on transfusion. Thalassemia intermedia is associated either in clinical or genotype which is more various the group of thalassemia-like disorders by the level of severity starts from the asymptomatic carrier state to the severe transfusion-dependent type (30). The difference between beta thalassemia major and intermedia lies on transfusion because beta thalassemia major usually necessary blood transfusion in early time, while intermedia no need it.

As a result of late handling and monitoring from beta thalassemia major, it gives an heavy impact for  untreated or poorly transfused individuals such as growth retardation, pallor, jaundice, brown pigmentation of the skin, poor musculature, genu valgum, hepatosplenomegaly, leg ulcers, development of masses from extramedullary hematopoiesis, and skeletal changes that effect from expansion of the bone marrow. The examples of skeletal changing are namely a malformation of the long bones of the legs and craniofacial bones. Therefore, person who has positively getting beta thalassemia major and does not get the blood transfusion immediately lead to the death before the third time (30).

Presence of heterogenous clinically features is a marker of beta thalassemia intermedia (35). The fundamental symptoms represented with pallor, jaundice, cholelithiasis, liver and spleen enlargement, skeletal changing in various range of severity from moderate to heavy, leg ulcers, extramedullary masses of hyperplastic erythroid marrow, osteopenia and osteoporosis developed, and hypercoagulable condition as a consequence of thrombotic complications. This condition is effected from existence of excess the contents of the lipid membrane in abnormal morphology of the red blood cells (30,35).

Asymptomatic feature is the major clinical condition of beta thalassemia carriers state. These hematological features such as a reduction volume of red blood cells (RBCs) lead to microcytosis, reducing the component of hemoglobin in RBCs is called hypochromia, enhanced level of HbA as the small portion of hemoglobin in the adult which is formed of two pieces of globin chains (alpha and delta), and quite balance between ratio of alpha/beta and gamma globin chain production in the range of 1.5-2.4 (30).The characteristics of beta thalassemia heterozygotes decribedby the pattern of hemoglobin marked with ranging of HbA as 92-95%, >3.8 HbA2, and approximate amout of HbF from 0.5–4%. Whereas by the peripheral blood smears showed microcytosis, hypochromia, and variations in size and shape of the abnormal red blood cells (30).

Beta thalassemia major has been associated with a heavy microcytic and appearance of hypochromic anemia, number of RBCs increased,and both of mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) decreased, were analyzed by Complete Blood Count (CBC). Additionally by peripheral blood smear exhibits anisocytosis, poikilocytosis (spiculated tear drop and elongated cells), and nucleated red blood cells. The highly number of nucleated red blood cells are related with the severity of anemia and increasingly they usually present in post-splenectomy (30). The severity level of anemia in beta thalassemia intermedia is a moderate and displays a grossly various hematological features starting from the severity of the beta thalassemia carrier state to the beta thalassemia major (30).

Because of lowering amount or lack of beta globin chains lead to overage of unbound alpha globin chains (see in Table 2) and their degradation products that speed up in erythroid precursors in the bone marrow, they give rise to premature apoptotic destruction of erythroblasts giving a consequence to cause ineffective erythropoiesis (37,38). Ineffective erythropoiesis is as the fingerprint of beta thalassemia pathology which is caused by overage of alpha globin chains production (39,46). Beside that, another influence of excessively amount of unbound α globin chains in β-thalassemia lead to degradate and denaturate their products to form heme and hemichromes as the result of the formation of the insoluble precipitates then both of those give rise to occur hemolysis and membrane binding of IgG and C3 quickly by iron mediated toxicity resulting in damaged of cell membrane leading to generate ineffective erythropoiesis (39,46) . Impact of the aggregation of excess α globin chains and their degradation products has been studied by using the red cell membrane and its skeleton then showed that any presence of abnormalities take place in the the ratio of spectrin to band 3 and in the function of band 4.1.

Hemolysis can be occurred when in the red blood cells have a lot of inclusion bodies that are able to destroy their membranes caused by effect of α-globin chains synthesis and their degradation results. Therefore, hemolysis in conjuction with ineffective eythropoiesis also can precipitate to give rise anemia. On the other hand, because of ineffective eryhtropoiesis is leading to anemia then distribution of abnormal red blood cells that would be entering the vessels of the spleen could not capable to change to be the new ones. Consequently, it occurs enlargement of the spleen because the spleen can not able to remove the abnormal red blood cells that enter within its vessels and contributes to form splenomegaly (39,46).

Because of decreased amount of red blood cells in the body, with the result that red blood cells can not able to attach oxygens leading to hypoxia in the tissue and then contributes to increase erythropoietin synthesis (39,46). Extremely production of erythropoietin

synthesis might encourage to create an extramedullary erythropoietic tissue forming. Formation of extramedullary erythropoietic tissue generates erythroid marrow expansion that also constitutes from impact both of anemia and splenomegaly. A presence of marrow expansion contributes within deformities of skeletal organs and lead to highly amount of irons in the red blood cells leading to iron overload (39,46).

.

Figure 1. Consequence of highly amount of free α-globin chains in β-Thalassemia (39,46)

12.2.4 Iron overload and Thalassemia

Iron overload is one of impact of ineffective erythropoiesis in β-Thalassemia that could give rise several complications of thalassemia syndromes that can develop some organs damaged and mortality increased (41). Another side, iron overload comes off when increasing of intake of iron during in the continous time which is an influences from red blood cells donor by blood transfusion or highly iron absorbed pass through gastrointestinal (GI) tract (43). For patients who have iron overload, iron deposition has been presented during a year starting from regular transfusions (41). Iron is an essential containing in the red blood cells which is able to regulate closely. Iron is saved in the body in form of ferritin. Extremely progressive concentration of iron overload can occur when a presence of iron deposition in several organs must be equal to serum ferritin value about more than 1000µg/L (40).

The important thing of iron overload within context of pathophysiological mechanism of disease usually is because of iron has characteristic as reversibly molecule oxidized and reduced (44). These components make iron quite dangerous to able to contribute in

producing of reactive oxygen species (ROS) especially generating hydroxyl radical. The starting point to recognize iron overload in thalassemic patient is a presence of peroxides forming in stead of lipid peroxides that able to interact easily with another molecules to produce cross links compound. Generally, the iron is strongly reactive compound and in any condition, it is easily changed to be in two states such as Iron II and III within process where bring in the development or released some electrons and turn out hazardous free radical as if atoms or unpaired electrons which are connected with another molecules (43).

The iron saving in thalassemics who have been done blood transfusion in many times or another condition where a presence of highly amount of iron absorption ancient in the body, then the patients can have a lot of storage and detoxification loading of ferritin as well as excessively total of iron saturated transferrin entirely (41). As a result, many free irons or well known as non transferrin bound iron (NTBI) present to accumulate much more especially in tissues and blood (42). After that, the iron does not have ability to bind any molecules naturally as transferrin, ferritin, or therapeutic iron chelators. Therefore, these kind of iron molecules precipitate to produce multiform reactive oxygen species (ROS) especially generating hydroxyl radicals. Hence, free iron has capability to catalyze the formation of greatly harmful chemical compounds like as result of the hydroxyl radical (42).

The characteristic of hydroxyl radical is extremely very reactive and can destroy lipids, proteins and DNA (44). This hydroxyl radical is arised from hydrogen peroxide that constitutes as the last result product of normal metabolic through by fenton reaction (42). Usually Oxygen is able to receive four electrons and these electrons changed to water (44). Nevertheless, reducing a half portion of oxygen in sequence gives rise to produce superoxide anion, hydrogen peroxide and water. Superoxide and hydrogen peroxide are the major production from this process. Both of them have a key function within forming of extra oxidants and make the oxidants becoming super reactive, as example is generating extremely reactive hydroxyl radical (44).

On the other side, hydroxyl radicals generated when labile plasma iron have been received and assembled as iron storage have been in the form of ferritin and hemosiderin. Then, ROS produce lipid peroxidation and can destroy the organelle along DNA components as well as disrupt normal mechanisms interrelated in apoptotic cell death and neoplasia risky alleviated like hepatoma (43).

Iron overload excessively present in the body have several harmful effects for all cells and give rise to produce some serious problem and irreversible original damaged especially cirrhosis, diabetes, heart disease and hypogonadism (41,43). As example, to recognize iron overload that present in the liver fibrosis then it can be determined by connecting with age of patient, number of transfusion unit and iron concentration level in the liver. A resume of the mechanism and pathological consequences of iron overload is shown in figure 2.

Figure 2. Mechanism and pathological consequences of iron overload (43)

12.2.5 Splenectomy and Thalassemia

The prevalent of pathophysiological effect from Thalassemia is disfigurement of red blood cells by a spleen which is a part of reticuloendothelial system lead to splenomegaly. Usually, Thalassemic patient need splenectomy. The principal therapeutic of using splenectomy for thalassemic is to reduce blood transfusion and usage more within anticipating iron overload. If the annual red blood cell requirement exceeds 180-200 ml/kg RBC (assuming HCT of red blood cells is about 75%) a splenectomy mechanism is required, for other reasons to increase consumption such as hemolytic reactions excluded. Other indications for splenectomy are symptoms of enlarged spleen, Leukopenia and / or thrombocytopenia and increased excess iron depend on good chelation (45).

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12. 3 Microscopy

12.3.1 Scanning electron microscope (SEM

12.3.1.1 Functional SEM

Electronically, SEM is kind of an electron microscope that magnify the surface portion of the sample where the target of view is illustrated by imaging within the natural history, biology and medicine (47,48). With another word, SEM also is an electron microscope that able to produce images of the specimen through by surface scanned with a focused beam of electrons (49,47). Generally, SEM can be able to supply and prepare detail the information surrounding the surface area of topography, structure of crystalline, chemical compound, and electrical environment of the top 1µm or behavior of the specimen (48,49). The scanning electron microscope itself only needs one electron gun where is located in the top of the column like in the conventional microscope of transmission electron (48).

12.3.1.2 Principle of SEM

Interaction between electrons and atoms in the specimen can precipitate to generate different signals which are consist of the detail informations related with topography of the specimen and their containing. Mostly, the techniques of using SEM is to discovery secondary electrons released by atoms continued by the electron beams. The signals generated from the electron beams interact with atoms used for producing a good image as the result of scanning electron microscope (59). As consequence of emittion the secondary electron is close to the surface of the specimen, SEM is able to create the high resolution images of a specimen surface until knowing the detail of specimen less than 1 nanometer. Back scattered electrons are a signals of beam electron which continued from the sample by elastic scattering. They appear from the profound within the specimen so that the resolution from back scattered electrons imaging is less than secondary electron images (50,56,59).

The electrons are generated by heating by an electron gun (Figure 26), which acts like a cathode. These electrons are pushed toward the anode, in the same direction as the sample, due to the strong electric field. As soon as the electron beam is condensed, the lens enters the objective lens, which is calibrated by the user to a fixed position on the sample. Once electron pills are conductive, two things can happen. First, the main electrons that swallow the sample will pass through the tunnel to a level dependent on the energy level of the electron. Then, the secondary and backscattered electrons will sample and reflect on it. These reflected electrons are then measured well by secondary electron detectors (SE) or backscattered (BS). After a wave emerges, a sample image is formed (53,56,59).

In SE mode, the secondary electrons are attracted by the positive bias on the front of the detector because of their low energy. The intensity of the signal varies with the sample angle. Therefore, SE mode provides highly image topography. On the other hand, in BS mode, the direction of electrons is almost opposite to the e-beam and the detection intensity is proportional to the number of sample atoms. Therefore, this is less topography, but useful for compositional drawings. The BS mode is also less affected by the filling effect on the sample, which is useful for non-conductive samples (53,56).

Figure 26. Generating electron heated by electron guns in SEM (53,59)

12.3.1.3 Processing and image information

Scanning electron microscopy, or SEM, is a powerful microscope that uses electrons to form images. This allows for the imaging of conductive samples at enlargements that can not be achieved using traditional microscopes. Modern light microscopes can achieve 1,000x magnification, whereas ordinary SEMs can achieve magnification of over 30,000 X. Because SEM does not use light to create images, the resulting images are black and white (53,59).

Conductive samples are SEM. As soon as the sample space goes to a vacuum, the user will re-align the electron gun in the system to the right location.The electronic weapon is as strong as high energy, moving through a combination of lens and hole and finally.As the electron gun continues to shoot the electron at the proper position in the example, the secondary electrons will bounce off the sample.These secondary electrons are detectors. Signals found from secondary electrons are amplified and sent to monitors, create 3D images (53,59).

Thermionically, emittion of electron beam is arised from an electron gun completed with a tungsten filament cathode (Figure 21). Fundamentally, tungsten is very good and often used in thermionic electron gun because it has good enough of melting point and vapor pressure is lower than all metals so and so it eases heated electrically for electron emitted. Energy of the electron beams approximate 0.2 keV to 40 keV then focused into one or two condenser lenses to diameter of spot around 0.4nm to 5nm. The beam passes through the screw coil pair or plugs the deflector plate in the electron column, usually in the end lens which switches the rays on the x and y axes so that it is scanned in raster mode over the rectangular area of the surface of the sample (50,55,59).

When the main electron beam interacts with the sample, the electrons lose energy by random random scattering and absorption in teardrop-shaped volume of the specimen known as the interaction volume. The size of the volume of emotion depends on the energy of the electron landing, the number of atomic specimens and the density of the specimen. The energy exchange

between the electron beam and the result. With elastic scattering, secondary electron emissions with injection scattering and emission of electromagnetic radiation. The current of the beam absorbed by the specimen can also be done. Each computer video memory pixel is synchronized to the beam position on the specimen in the microscope, and the resulting image is the signal distribution intensity map emitted from the scanned area of the specimen(50,55,59).

SEM may have a condenser and objective lens, but its function is to focus the beam onto a point, and not for a specimen image. During electron rifles can produce beams with a fairly small diameter, SEM can in principle work at all with no condenser or objective lens, although it may not be very versatile or achieve very high resolution. Raster ratio enlargement results in specimens and raster on display devices. Therefore, higher magnification results reduce the raster size of the specimen, and vice versa (50,55,59).

Figure 21. Electron beam emitted from electron gun of tungsthen filament cathode in SEM (55, 50)

12.3.1.4 Advantages and Disadvantages

Scanning electron microscope (SEM) has many advantages present importantly, are higher resolution of visualization the detail of specimen surface than any imaging techniques approximately 3.5nm and then, it has capability to measure complete and measure detail of the information in three dimensions (56,58,59). On the other hand, scanning electron microscope (SEM) does not require a transparent sample, but a dry subject surface is scanned by secondary electrons that are accelerated by the voltage between the anode to the cathode, which is usually 1.0 to 25 kV (54).

Specifically, SEM imaging requires a high vacuum around 10-8Torr to make its optimal condition by first is chemically fixed, dehydrated, and coated with a conductive material (usually gold) to remove overload from electron beam (59). SEM also needs many preparations of the sample such as glutaraldehyde fixation, negative staining, the Sputter–Cryo technique, and coating with gold or osmium (51).

12.3.2 Light Microscope

12.3.2.1 Introduction of Light Microscope (LM)

Light microscope is a guidance technically within modern cell biology. LM also called optical microscope that uses visible light and lens systems to enlarge a small sample image. Images from optical microscopes can be captured by normal light sensitive cameras to produce micrographs. There are two kinds of this light microscope namely simple and compound microscopes. A simple microscope is microscope using a single lens to magnify the image, whereas a compound microscope is kind of microscope using several lens to magnify the enlargement of the object such as condenser lens, objective lens, and ocular lens (52,57,59).

12.3.2.2 Principle of Light Microscope

By a compound microscope, the lens are close with the visible of the object through collecting the light as called as objective lens by focusing the real image from the object deep in the microscope (Figure 22). The image is then enlarged by a second lens or group of lenses as called an eyepiece that gives the viewer a reversed virtual image of the object (Figure 23). Therefore, a compound microscope easily shows microscope features an exchangable destination lens, which allows the user to adjust the zoom quickly (57,59).

Figure 22. Focusing the real image by objective lens Figure 23. Magnify the object by

eyepiece

12.3.2.3 Components

The components of light microscope (Figure 24) are consist of : 1. ocular lens, 2.revolver, 3. objective lens, 4. coarse adjustment, 5. fine adjustment, 6. stage, 7. light source, 8. Diaphragm and condenser, and 9. Mechanical stage (57,59).

Figure 24. Basic elements of light microscope Figure 25. Objective lens (Left :100x, Right: 40x)

12.3.2.4 Processing and Image Formation

The actual strength or magnification of compound optical microscopes is the product of ocular forces (eyepieces) and objective lenses. Generally, normal enlargement of the ocular and objective respectively 10× and 100×, resulting in a final magnification of 1000×. When using the camera for micrographic images, the image should be in the picture. Last enlargement of the picture is the product of the objective lens magnification, optical zoom camera and the mold film enlargement factor relative to the negative (57,59).

The objective lens is a very high-powered magnifying glass, the lens with a very short focal length. It is very close to the specimen that examined the light from the specimen to a focus of about 160 mm inside the microscope tube. It draws an enlarged subject. This image is rolled over and can be seen with the strength of the eyes and a sheet of tracing paper at the end of the tube (59). By focusing on a brightly lit specimen, a very enlarged image can be seen. This real image is seen by the lens of the eye that provides further enlargement. The eye lens is a compound lens, with one lens component and a close lens tube. It forms a temple separated by air. In many designs, virtual imagery becomes the focus between two lens eyepieces, the first lens brings the original image to the focus and a second lens that allows the eye to focus on the virtual image(57,59).

12.3.2.5 Advantages and Disadvantages

The advantages of using a light microscope are purchasing, maintenance and inexpensive operations; small and portable, the natural color of the specimen can be observed, the preparation is relatively quick and simple it requires little skill, and is not affected by the magnetic field (57,58,59). While the disadvantage is a lens enlargement up to 1500x, the preparation can distort the specimen, the depth of the field is restricted, the completion of the biological specimen's strength is about 1nm and optical microscope limited in resolution by the light wavelength and the gap are limited from the microscope (56,57,58,59).

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