a study on size and shape of erythrocytes of normal and malarial blood using laser diffraction...

A STUDY ON SIZE AND SHAPE OF ERYTHROCYTES OF

NORMAL AND MALARIAL BLOOD USING LASER

DIFFRACTION TECHNIQUE

Amar Alansi

Osmaina University

Master Of Science

In

Physics

A STUDY ON SIZE AND SHAPE OF ERYTHROCYTES

OF NORMAL AND MALARIAL BLOOD

USING LASER DIFFRACTION TECHNIQUE

Author:Amar Alansi

Supervisor:Dr. Kaleem Jaleeli

May, 2015

Contents

Certificate iii

Declaration vi

Dedication vii

Acknowledgements viii

Abstract ix

Preface x

1 Introduction 1

1.1 Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.1.1 Properties of Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.2 Composition of Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.3 Blood Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

1.4 Plasma Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

1.4.1 Plasma Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

1.4.2 Properties of Plasma Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

1.4.3 Red Blood Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

1.4.4 Normal Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

1.4.5 Properties of red blood cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

1.4.6 Function of Red Blood Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

1.4.7 Pathological Variations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

1.5 White Blood Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

1.5.1 Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

1.5.2 Properties of White Blood Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

1.5.3 Function of White Blood Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

1.6 Platelets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

1.6.1 Size of Platelets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

1.6.2 Shape of Platelets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

1.6.3 Strucyure and Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1.6.4 Properties of Platelets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1.6.5 Functions of Platelets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1.7 Malaria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1.7.1 Plasmodium vivax . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1.7.2 Plasmodium malariae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1.7.3 Plasmodium ovale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1.7.4 Plasmodium falciparum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

1.8 Survey of Literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

2 Theoretical aspects of diffraction 13

2.1 Diffraction and the Wave Theory of Light . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

2.2 Diffraction by a Circular Aperture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

2.2.1 Resolvability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

2.2.2 Babinet Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

i

3 Materials and Methods 193.1 Samples collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193.2 Preparation of a thin blood film on the slide . . . . . . . . . . . . . . . . . . . . . . . . . . 193.3 Experimental set up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203.4 Procedure and Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

4 Results and Discussion 234.1 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Appendix of Mathematica Programs 47

Bibliography 55

List of Figures 57

List of Tables 59

ii

Declaration

The Author declares that he carried out the project entitled “A Study on Size and Shapeof Erythrocytes of normal and malarial blood using Laser Diffraction Technique”under the supervision of Dr. Kaleem Ahmed Jaleeli at Biophysics unit, Department ofPhysics Nizam College (Autonomous), Osmania University, Hyderabad, India.

Amar Yahya Yahay Alansi

Dedicated to

my parents

my wife

my son

my brothers and my sisters

Amar alansi

Acknowledgements

The author solemnly offers his sincere gratitude to the almighty, the most beneficent andmerciful for bestowing the opportunity to complete this project. Peace and blessing of Allahbe upon his last Messenger MOHAMMED (Sallallah Alaihe Wasallam ), who guided us toThe right path.

The author declares that he carried out the project entitled A Study on Size and Shapeof Erythrocytes of normal and malarial blood using Laser Diffraction Technique under thesupervision of Dr. Kaleem Ahmed Jaleeli at Biophysics unit, Department of Physics NizamCollege, Osmania University, Hyderabad, India.

The author wishes to thank Dr. Kaleem Ahmed Jaleeli for co-operation, constantencouragement and guidance and useful discussion. Further the author thanks Prof.Adeel Ahmed, former Head, Department of Physics, Nizam College Osmania University,for useful discussion in completing the project. Special thanks to Dr. N.V. Prsad, HeadDepartment of physics, and Prof. T.L.N. Swamy, Principle, Nizam college for the interestthey have shown during the program.

The author would like to thank in particular his lovely classmates.

Amar Yahya Yahay Alansi

Abstract

The subtle changes in the physiology of erythrocytes at the cellular level are documented inthe present laser diffraction study. Using this technique one can differentiate the morphologiesof erythrocytes. The size and shape of blood cells are determined usually by microscope. Thismethod, besides being tedious, cannot be extended to a large number of cells and samples. Inview of this, a simple and quick method has been developed for determining the average sizeand shape of blood cells by employing laser diffraction technique. Blood samples were collectedfrom normal healthy persons and patients suffering from malaria. The laser diffraction methodis very rapid and simple for assessing the average size of the cells. This could also be usedwith advantage as a diagnostic tool for assessing the variation in the size of human RBC.

Preface

The blood serves as the principle transport medium of the body , carrying oxygen, nutrients messagesto tissues and waste product , synthesized metabolites and carbon dioxide to the organs of excretion. In other words , Blood is described as a fluid connective tissue . The blood plays many importantroles in coordinating the individual cells into whole complex organisms. Human blood is characterizedby a number of physicochemical properties in disease analysis. With modern electronic instruments it ispossible to detect changes in the chemistry of blood cells that are responsible for severe disorders. Thechange in the sublet interaction between erythrocytes and plasma may be termed as disease . this subtleinteraction perturbs the membrane physiology of erythrocytes because the proteins are very sensitivedetectors of environment and these are present in plenty on the erythrocyte membrane. Hence, the bloodis being studied extensively by the physiologists, biochemists and biomedical engineers , but it has notdrawn much attention of physicists. However , some reports are available on ultrasonic, dialectic anddieelctrophoretic properties of human blood and its erythrocytes.The application of concepts, principles and techniques of physics for the selection of problems in biologyat different levels of complexity to get an integral picture is drawing the attention of many researches.The main aim of this project is to present the size of normal blood and malarial blood by using laserdiffraction technique.This project contains four chapters namely

1. Introduction

2. Theoretical Aspects of diffraction

3. Materials, and methods

4. Results and Discussion

This project starts with the first chapter which presents a brief description of blood and its components,functional properties of blood (erythrocytes, leukocytes, platelets) ,and malaria and its types .The second chapter is concerned with materials and methods of the present investigation. A detaileddescription of collection of blood, the procedure of preparation of sample and Theoretical aspects ofdiffraction has been discussion.The third chapter describes the theory of optical diffraction.The forth Chapter reports results on size of normal human erythrocytes and malarial human erythrocytes. A detailed discussion is mentioned.

Chapter 1

Introduction

1.1 Blood

Blood is a connective tissue in fluid form. It is considered as the ‘fluid of life’ because it carries oxygenfrom lungs to all parts of the body and carbon dioxide from all parts of the body to the lungs. It is knownas ‘fluid of growth’ because it carries nutritive substances from the digestive system and hormones fromendocrine gland to all the tissues.

The blood is also called the ‘fluid of health’ because it protects the body against the diseases and getsrid of the waste products and unwanted substances by transporting them to the excretory organs likekidneys.

1.1.1 Properties of Blood

Color

Blood is red in color. Arterial blood is scarlet red because it contains more oxygen and venous blood ispurple red because of more carbon dioxide.

Volume

Average volume of blood in a normal adult is 5 L. In a newborn baby, the volume is 450 ml. It increasesduring growth and reaches 5 L at the time of puberty. In females, it is slightly less and is about 4.5 L.It is about 8% of the body weight in a normal young healthy adult, weighing about 70 kg.

Reaction and pH

Blood is slightly alkaline and its pH in normal conditions is 7.4.

Specific gravity

Specific gravity of total blood : 1.052 to 1.061Specific gravity blood cells : 1.092 to 1.101Specific gravity of plasma : 1.022 to 1.026

Viscosity

Blood is five times more viscous than water. It is mainly due to red blood cells and plasma proteins.

1.2 Composition of Blood

Blood contains the blood cells which are called formed elements and the liquid portion known as plasmaFigure 1.1.

1

1.3. BLOOD CELLS

Figure 1.1: Composition of Blood

1.3 Blood Cells

Three types of cells are present in the blood:

• Red blood cells or erythrocytes

• White blood cells or leukocytes

• Platelets or thrombocytes.

1.4 Plasma Proteins

1.4.1 Plasma Proteins

Plasma proteins are:

• Serum albumin

• Serum globulin

• Fibrinogen

1.4.2 Properties of Plasma Proteins

Molecular Weight

Albumin : 69,000Globulin : 1,56,000Fibrinogen : 4,00,000

Specific Gravity

Specific gravity of the plasma proteins is 1.026.

Buffer Action

Acceptance of hydrogen ions is called buffer action. The plasma proteins have 1/6 of total bufferingaction of the blood.

1.4.3 Red Blood Cells

Red blood cells (RBCs) are the non-nucleated formed elements in the blood. Red blood cells are alsoknown as erythrocytes (erythros = red). Red color of the red blood cell is due to the presence of thecoloring pigment called hemoglobin. RBCs play a vital role in transport of respiratory gases. RBCs arelarger in number compared to the other two blood cells, namely white blood cells and platelets.

2

CHAPTER 1. INTRODUCTION

1.4.4 Normal Size

Diameter : 7.2 µ (6.9 to 7.4 µ).Thickness : At the periphery it is thicker with 2.2 µ and at the center it is thinner with 1 µ Figure 1.2.This difference in thickness is because of the biconcave shape.Surface area : 120 sq µ.Volume : 85 to 90 cu µ.

Figure 1.2: Shape and dimensions of a red blood cell

1.4.5 Properties of red blood cells

Rouleaux Formation

When blood is taken out of the blood vessel, the RBCs pile up one above another like the pile of coins.This property of the RBCs is called rouleaux (pleural = rouleau) formation Figure 1.3. It is acceleratedby plasma proteins globulin and fbrinogen.

Figure 1.3: Rouleau formation

Specific Gravity

Specific gravity of RBC is 1.092 to 1.101.

Paked Cell Volume

Packed cell volume (PCV) is the proportion of blood occupied by RBCs expressed in percentage. It isalso called hematocrit value. It is 45% of the blood and the plasma volume is 55% .

3

1.4. PLASMA PROTEINS

Suspension Stability

During circulation, the RBCs remain suspended uniformly in the blood. This property of the RBCs iscalled the suspension stability.

1.4.6 Function of Red Blood Cells

Major function of RBCs is the transport of respiratory gases. Following are the functions of RBCs:

Transport of Oxygen from the Lungs to the Tissues

Hemoglobin in RBC combines with oxygen to form oxyhemoglobin. About 97% of oxygen is transportedin blood in the form of oxyhemoglobin .

Transport of Carbon Dioxide from the Tissues to the Lungs

Hemoglobin combines with carbon dioxide and form carbhemoglobin. About 30% of carbon dioxide istransported in this form.RBCs contain a large amount of the carbonic anhydrase. This enzyme is necessary for the formation ofbicarbonate from water and carbon dioxide. Thus, it helps to transport carbon dioxide in the form ofbicarbonate from tissues to lungs. About 63% of carbon dioxide is transported in this form.

Buffering Action in Blood

Hemoglobin functions as a good buffer. By this action, it regulates the hydrogen ion concentration andthereby plays a role in the maintenance of acidbase balance .

In Blood Group Determination

RBCs carry the blood group antigens like A antigen, B antigen and Rh factor. This helps in determinationof blood group and enables to prevent reactions due to incompatible blood transfusion .

1.4.7 Pathological Variations

Variations in size of red blood cells

Under physiological conditions, the size of RBCs in venous blood is slightly larger than those in arterialblood. In pathological conditions, the variations in size of RBCs are:

• Microcytes (smaller cells)

• Macrocytes (larger cells)

• Anisocytes (cells with different sizes)

MICROCYTES Microcytes are present in:

• Iron-defciency anemia

• Prolonged forced breathing

• Increased osmotic pressure in blood

MACROCYTES Macrocytes are present in:

• Megaloblastic anemia

• Decreased osmotic pressure in blood

ANISOCYTES Anisocytes occurs in pernicious anemia.

4


Variations in Shape of Red Blood Cells

Shape of RBCs is altered in many conditions including different types of anemia.

1. Crenation: Shrinkage as in hypertonic conditions

2. Spherocytosis: Globular form as in hypotonic conditions

3. Elliptocytosis: Elliptical shape as in certain types of anemia

4. Sickle cell: Crescentic shape as in sickle cell anemia

5. Poikilocytosis: Unusual shapes due to deformed cell membrane. The shape will be of fask, hammeror any other unusual shape

Variations in Structure of Red Blood Cells

PUNCTATE BASOPHILISM Striated appearance of RBCs by the presence of dots of basophilicmaterials (porphyrin) is called punctate basophilism. It occurs in conditions like lead poisoning.RING IN RED BLOOD CELLS Ring or twisted strands of basophilic material appear in the peripheryof the RBCs. This is also called the Goblet ring. This appears in the RBCs in certain types of anemia.HOWELL-JOLLY BODIES In certain types of anemia, some nuclear fragments are present in theectoplasm of the RBCs. These nuclear fragments are called HowellJolly bodies.

1.5 White Blood Cells

White blood cells (WBCs) or leukocytes are the colorless and nucleated formed elements of blood (leukois derived from Greek word leukos = white). Alternate spelling for leukocytes is leucocytes Figure 1.4.

Figure 1.4: WBC

Compared to RBCs, the WBCs are larger in size and lesser in number. Yet functionally, these cells areimportant like RBCs because of their role in defense mechanism of body and protect the body frominvading organisms by acting like soldiers.

1.5.1 Classification

Some of the WBCs have granules in the cytoplasm. Based on the presence or absence of granules in thecytoplasm, the leukocytes are classifed into two groups:

• Granulocytes which have granules

• Agranulocytes which do not have granules

5

1.6. PLATELETS

Granulocytes

Depending upon the staining property of granules, the granulocytes are classifed into three types:

• Neutrophils with granules taking both acidic and basic stains

• Eosinophils with granules taking acidic stain

• Basophils with granules taking basic stain

Agranulocytes

Agranulocytes have plain cytoplasm without granules. Agranulocytes are of two types:

• Monocytes

• Lymphocytes

1.5.2 Properties of White Blood Cells

Diapedesis

Diapedesis is the process by which the leukocytes squeeze through the narrow blood vessels.

Ameboid Movement

Neutrophils, monocytes and lymphocytes show amebic movement, characterized by protrusion of thecytoplasm and change in the shape.

Chemotaxis

Chemotaxis is the attraction of WBCs towards the injured tissues by the chemical substances releasedat the site of injury.

Phagocytosis

Neutrophils and monocytes engulf the foreign bodies by means of phagocytosis .

1.5.3 Function of White Blood Cells

Generally, WBCs play an important role in defense mechanism. These cells protect the body frominvading organisms or foreign bodies, either by destroying or inactivating them. However, in defensemechanism, each type of WBCs acts in a different way.

1.6 Platelets

Platelets or thrombocytes are the formed elements of blood. Platelets are small colorless, non-nucleatedand moderately refractive bodies. These formed elements of blood are considered to be the fragments ofcytoplasm Figure 1.5.

1.6.1 Size of Platelets

Diameter : 2.5 µ (2 to 4 µ)Volume : 7.5 cu µ (7 to 8 cu µ).

1.6.2 Shape of Platelets

Normally, platelets are of several shapes, viz. spherical or rod-shaped and become oval or disk-shapedwhen inactivated. Sometimes, the platelets have dumbbell shape, comma shape, cigar shape or any otherunusual shape. Inactivated platelets are without processes or flopodia and the activated platelets developprocesses or flopodia .

6


Figure 1.5: Platelets

1.6.3 Strucyure and Composition

Platelet is constituted by:

• Cell membrane or surface membrane

• Microtubules

• Cytoplasm

1.6.4 Properties of Platelets

Platelets have three important properties (three As):

• Adhesiveness

• Aggregation

• Agglutination

1.6.5 Functions of Platelets

Normally, platelets are inactive and execute their actions only when activated. Activated plateletsimmediately release many substances. This process is known as platelet release reaction. Functionsof platelets are carried out by these substances.Functions of platelets are:

Role in Blood Clotting

Platelets are responsible for the formation of intrinsic prothrombin activator. This substance is responsiblefor the onset of blood clotting .

Role in Clot Retraction

In the blood clot, blood cells including platelets are entrapped in between the fbrin threads. Cytoplasmof platelets contains the contractile proteins, namely actin, myosin and thrombosthenin, which areresponsible for clot retraction .

Role in Prevention of Blood Loss(Hemostasis)

Platelets accelerate the hemostasis by three ways:

• Platelets secrete 5-HT, which causes the constriction of blood vessels

• Due to the adhesive property, the platelets seal the damage in blood vessels like capillaries

• By formation of temporary plug, the platelets seal the damage in blood vessels

7

1.7. MALARIA

Role in Repair of Ruptured Blood Vessel

Platelet-derived growth factor (PDGF) formed in cytoplasm of platelets is useful for the repair of theendothelium and other structures of the ruptured blood vessels.

Role in Deffnse Mechanism

By the property of agglutination, platelets encircle the foreign bodies and destroy them.

1.7 Malaria

Malaria is one of the most successful parasites ever known to mankind. After thousands of years, itremains the world’s most pervasive infection, affecting at least 91 different countries and some 300 millionpeople. The disease causes fever, shivering, joint pain, headache, and vomiting. In severe cases, patientscan have jaundice, kidney failure, and anemia, and can lapse into a coma.It is ever-present in the tropics and countries in sub-Saharan Africa, which account for nearly 90 percentof all malaria cases. The majority of the remaining cases are clustered in India, Brazil, Afghanistan, SriLanka, Thailand, Indonesia, Vietnam, Cambodia, and China. Malaria causes 1 to 1.5 million deathseach year, and in Africa, it accounts for 25 percent of all deaths of children under the age of five.

Plasmodium spp., which cause malaria, remain endemic throughout the world in tropical and subtropicalcountries with an estimated 300 million to 500 million cases annually. Plasmodium vivax, Plasmodiumovale, Plasmodium malariae, and Plasmodium falciparum are the etiologic agents of human malaria.Along with schistosomiasis and amebiasis, malaria is a major cause of mortality in people inunderdeveloped countries. Between 1 and 2 million deaths worldwide are caused by malaria each year,primarily due to infection with P. falciparum. Mortality in children who have malaria is also signifcantlyassociated with infection by P. falciparum. In the United States, approximately 1000 cases are reportedannually, with P. falciparum being the etiologic agent in more than 50% of the cases. Most of these casesare in travelers to endemic areas.Plasmodium vivax has the widest geographic distribution and is the one most likely to be found intemperate climates. P. vivax and P. falciparum cause the majority of infections. P. ovale is confned toAfrica; P. falciparum and P. malariae have similar distributions throughout Africa and tropical countries.In general, infections caused by P. vivax, P. ovale, and P. malariae are less severe than those caused byP. falciparum (12).

1.7.1 Plasmodium vivax

Plasmodium vivax has a tertian lifecycle patternthat is, it takes approximately 48 hours for the life cycleto be completed. The invasion of a new group of RBCs begins on the third day. P. vivax usually invadesyoung RBCs (reticulocytes) and therefore is characterized by enlarged infected RBCs, often 1 1

2 to 2times normal size. A fne pink stippling known as Schffners stippling (or dots) may be present in thecell. The young trophozoite is characterized by its ameboid appearance; by maturity, it usually fills theRBC, and golden brown malarial pigment is present. The mature schizont contains 12 to 24 merozoites,with an average of 16. Gametocytes are rounded and fill the cell. Macrogametocytes are often difficultto differentiate from mature trophozoites. Figure 1.6 shows several stages of P. vivax.

1.7.2 Plasmodium malariae

Plasmodium malariae usually invades older RBCs, perhaps accounting for the occasional darkerappearance of the invaded RBC. The life cycle is characterized as quartan, with reproduction occurringevery 72 hours and invasion of new RBCs every fourth day. The trophozoite is compact and may assumea characteristic band appearance, in which it stretches across the diameter of the RBCs (Figure 1.7,A).Notice the presence of dark, coarse, brown-black pigment in the band form. Occasionally, a few pinkcytoplasmic dots, called Ziemanns dots, may be seen.The mature schizont contains 6 to 12 merozoites(Figure 1.7,B), with an average of 8. Merozoites may be arranged in a characteristic loose daisy petalarrangement around the clumped pigment; however, they may also be randomly arranged.

1.7.3 Plasmodium ovale

Plasmodium ovale, the least commonly seen species, resembles P. vivax. In P. ovale infections, the RBCis enlarged and may assume an oval shape with fimbriated or fringelike edges. Schffners dots are lesscommonly seen than with P. vivax. The parasite remains compact, has golden-brown pigment, and has

8


Figure 1.6: A, Plasmodium vivax trophozoite. B, P. vivax mature schizont. C, P. vivax macrogametocyte.D, P. vivax microgametocyte.

Figure 1.7: A, Plasmodium malariae band form trophozoite. B, P. malariae schizont

a range of 6 to 12 merozoites in the mature schizont. It also exhibits a tertian life cycle. Figure 1.8,A,shows trophozoites of P. ovale. The Schffners stippling in Figure 1.8,B, has stained almost a bluish pink,but the compact organism and fimbriated cell are characteristic.

Figure 1.8: A, Plasmodium ovale trophozoite. B, Schffners stippling of P. ovale clearly visible

1.7.4 Plasmodium falciparum

Although identifed as having a tertian life cycle, P. falciparum often demonstrates an asynchronous lifecycle with rupture of the RBCs taking place at irregular intervals ranging from 36 to 48 hours. The lifecycle stages seen in peripheral blood are usually limited to the ringform trophozoite and the gametocyte.Other stages mature in the venules and capillaries of the major organs. P. falciparum invades RBCs ofany age and, for this reason, often exhibits the highest parasitemiareaching 50% in some cases. The ringforms of P. falciparum (Figure 1.9,A) are more delicate than those of other species and often have two

9

1.8. SURVEY OF LITERATURE

chromatin dots. Appliqu forms, parasites at the edge of the RBC, and multiple ring forms in a single RBCare common. In this figure, the appliqu form is seen in the lower left and upper right of the photograph.The mature trophozoite is small and compact and may have dark-brown pigment. The schizont has 8 to36 merozoites, with an average of 20 to 24. Gametocytes have a characteristic banana or crescent shape(Figure 1.9,B).

Figure 1.9: A, Plasmodium falciparum ring-form trophozoites. B, P. falciparum gametocyte

1.8 Survey of Literature

Rajendra Kumar et al (2003)(8) investigated the heat shock protein (Hsp-90) of Plasmodium Falciparumand antimalarial activity of its inhibitor-galdanamycin and suggested that an active and essential Hsp-90chaperone cycle exist in plasmodium and the favourable pharmacology of benzoquinone ansamycincompound .Warhurst et al (2003)(18) studied the relationship of physicochemical properties and structure of thedifferential antiplasmodial activity of the cinchoma alkaloids and examined ionization constant, octomalwater distribution and haematin interaction for eight alkaloids to explain the influence of small structuraldifferences on activity.Shiff (2002)(15), Najera (2002)(7), and Hyde (2002)(6) focused on finding a potent and reliable antiparasitic drug that would inhibit plasmodium infection and growth and suggested that in nearly allthe malarial endepic populations , plasmodium has developed resistance against the hall mark drugchloroquine and its derivatives.Mohammed et al (2006)(1) studied various viscometric parameters , such as blood velocity, volume flowrate and viscosity , of malarial blood , and suggested that the viscosity is high and velocity and flow ratesreduced.Ramakrishna rao et al (2009)(13) studied size and shape of blood cells of cancer by using laser diffractiontechnique ,and suggested that mean diameter of RBC of normal human is 7.12 µ and where as for cancercells is 9.35 µ.Scanlon, Sanders (2007)(14) introduced some composition and properties of red blood cells and plasmaand white blood cells.Omolade Okwa (2012)(11) described the malaria and its types. David Halliday, Robert Resnick, JearlWalker (2010)(4) described diffraction of light by a Circular Aperture and The Fresnel Bright Spot andRayleighs criterion.Bruce Torrence, Torrence (2009)(16) reported introduces commands and procedures pertinent to thelinear algebra . The size and shape of Red blood cells (RBC) are of clinical importance not only tocharacteristic different cells but also in differentiating abnormal from normal cells . For example the sizeof red blood cells from one individual are not all equal but are distributed about mean value and hencethe average size has to be determined . Bernard oser (1954)(9) reported that in case pernicious anemia, the mean size of RBC is greater than the normal and there is larger than normal variation among thecells.Hartell (1970)(5) mentioned that there exist to variation not only in size but also in the shape of yeastcells during their cell cycle , leading to periodic fluctuation in density . Longhurst (1964)(10), Charlesmeyer (1934)(3) and Calthrope (1952)(2) reported that average size of the cells can be measure by young’seriometer employed in the determination of the average size of lycopodium particles. This method basedon the babinets principle , given fraunhofer diffraction pattern on the retina of the observer .Anwar Ali (1983)(17) developed a simple and rapid method for the determining the average size andshape of biological cells by Appling laser diffraction , which is found to be free from practical definite ofthe eriometer technique .A Perusal of literature revals different methods and investigation for the control of malarial parasites but

10


not much alteration has been given to study the changes in RBC physiology of the blood drawn from thepatients suffering from malaria at the membrane level. Therefor an attempt has been made to study thealteration in RBC physiology of malarial blood by employing Laser diffraction technique .

11

1.8. SURVEY OF LITERATURE

12

Chapter 2

Theoretical aspects of diffraction

2.1 Diffraction and the Wave Theory of Light

The light produces an interference pattern called a diffraction pattern. For example, when monochromaticlight from a distant source (or a laser) passes through a narrow slit and is then intercepted by a viewingscreen, the light produces on the screen a diffraction pattern like that in Figure 2.1. This pattern consistsof a broad and intense (very bright) central maximum plus a number of narrower and less intense maxima(called secondary or side maxima) to both sides. In between the maxima are minima. Light flares intothose dark regions, but the light waves cancel out one another.

Figure 2.1: This diffraction pattern appeared on a viewing screen when light that had passed through anarrow vertical slit reached the screen. Diffraction caused the light to flare out perpendicular to the longsides of the slit. That flaring produced an interference pattern consisting of a broad central maximumplus less intense and narrower secondary (or side) maxima, with minima between them

Such a pattern would be totally unexpected in geometrical optics: If light traveled in straight lines asrays, then the slit would allow some of those rays through to form a sharp rendition of the slit on theviewing screen instead of a pattern of bright and dark bands as seen in Figure 2.1.Diffraction is not limited to situations when light passes through a narrow opening (such as a slit orpinhole). It also occurs when light passes an edge, such as the edges of the razor blade whose diffractionpattern is shown in Figure 2.2. Note the lines of maxima and minima that run approximately parallelto the edges, at both the inside edges of the blade and the outside edges. As the light passes, say, thevertical edge at the left, it flares left and right and undergoes interference, producing the pattern along

13

2.2. DIFFRACTION BY A CIRCULAR APERTURE

the left edge. The rightmost portion of that pattern actually lies behind the blade, within what wouldbe the blade’s shadow if geometrical optics prevailed.

Figure 2.2: The diffraction pattern produced by a razor blade in monochromatic light. Note the lines ofalternating maximum and minimum intensity

A common example of diffraction when one looks at a clear blue sky and see tiny specks and hairlikestructures floating in the view. These floaters, as they are called, are produced when light passes theedges of tiny deposits in the vitreous humor, the transparent material filling most of the eyeball. Whatis seen when a floater is in the field of vision is the diffraction pattern produced on the retina by one ofthese deposits. If it is sight through a pinhole in a piece of cardboard so as to make the light entering theeye approximately a plane wave, individual maxima and minima in the patterns can be distinguished.Diffraction is a wave effect. That is, it occurs because light is a wave and it occurs with other types ofwaves as well. For example, one might have probably seen diffraction in action at football games. Whena cheerleader near the playing field yells up at several thousand noisy fans, the yell can hardly be heardbecause the sound waves diffract when they pass through the narrow opening of the cheerleader’s mouth.This flaring leaves little of the waves traveling toward the fans in front of the cheerleader. To offset thediffraction, the cheerleader can yell through a megaphone. The sound waves then emerge from the muchwider opening at the end of the megaphone. The flaring is thus reduced, and much more of the soundreaches the fans in front of the cheerleader.

2.2 Diffraction by a Circular Aperture

Diffraction is consider by a circular aperture - that is, a circular opening, such as a circular lens, throughwhich light can pass. Figure 2.3 shows the image formed by light from a laser that was directed ontoa circular aperture with a very small diameter. This image is not a point, as geometrical optics wouldsuggest, but a circular disk surrounded by several progressively fainter secondary rings. Comparison withFigure 2.1 leaves little doubt that we are dealing with a diffraction phenomenon. Here, however, theaperture is a circle of diameter rather than a rectangular slit.

The (complex) analysis of such patterns shows that the first minimum for the diffraction pattern of acircular aperture of diameter d is located by

sinθ = 1.22λ

d(2.1)

The angle θ here is the angle from the central axis to any point on that (circular) minimum. Comparethis with a sin θ = λ

sinθ =λ

d(2.2)

which locates the first minimum for a long narrow slit of width a . The main difference is the factor 1.22,which enters because of the circular shape of the aperture.

14

CHAPTER 2. THEORETICAL ASPECTS OF DIFFRACTION

Figure 2.3: The diffraction pattern of a circular aperture. Note the central maximum and the circularsecondary maxima. The figure has been overexposed to bring out these secondary maxima, which aremuch less intense than the central maximum

2.2.1 Resolvability

The fact that lens images are diffraction patterns is important when one wishs to resolve (distinguish)two distant point objects whose angular separation is small. Figure 2.4 shows, in three different cases,the visual appearance and corresponding intensity pattern for two distant point objects (stars, say) withsmall angular separation. In Figure 2.4a, the objects are not resolved because of diffraction; that is,their diffraction patterns (mainly their central maxima) overlap so much that the two objects cannot bedistinguished from a single point object. In Figure 2.4b the objects are barely resolved, and in Figure 2.4cthey are fully resolved.

Figure 2.4: At the top, the images of two point sources (stars) formed by a converging lens. At thebottom, representations of the image intensities. In (a) the angular separation of the sources is too smallfor them to be distinguished, in (b) they can be marginally distinguished, and in (c) they are clearlydistinguished. Rayleigh’s criterion is satisfied in (b), with the central maximum of one diffraction patterncoinciding with the first minimum of the other

In Figure 2.4b the angular separation of the two point sources is such that the central maximum of thediffraction pattern of one source is centered on the first minimum of the diffraction pattern of the other, acondition called Rayleigh’s criterion for resolvability. From (2.3), two objects that are barely resolvable.by this criterion must have an angular separation θR of

15


θR = sin−1(1.22λ

a) (2.3)

Since the angles are small, we can replace sin θR with θR expressed in radians

θR = 1.22λ

a(2.4)

Applying Rayleigh’s criterion for resolvability to human vision is only an approximation becausevisual resolvability depends on many factors, such as the relative brightness of the sources and theirsurroundings, turbulence in the air between the sources and the observer, and the functioning of theobserver’s visual system.Experimental results show that the least angular separation that can actually be resolved by a person isgenerally somewhat greater than the value given by (2.4). However, for calculations here, taking (2.4)as being a precise criterion: If the angular separation θ between the sources is greater than θR , we canvisually resolve the sources; if it is less, we cannot.Rayleigh’s criterion can explain the arresting illusions of color in the style of painting known as pointillism(Figure 2.5). In this style, a painting is made not with brush strokes in the usual sense but rather with amyriad of small colored dots. One fascinating aspect of a pointillistie painting is that when one changehis distance from it, the colors shift in subtle, almost subconscious ways. This color shifting has to dowith whether he can resolve the colored dots. When one stands close enough to the painting, the angularseparations θ of adjacent dots are greater than θR and thus the dots can be seen individually. Theircolors are the true colors of the paints used. However, when one stands far enough from the painting,the angular separations θ are less than θR and the dots cannot be seen individually. The resulting blendof colors coming into the eye from any group of dots can then cause the brain to ”make up” a color forthat group-a color that may not actually exist in the group. In this way, a pointillistic painter uses theviewers visual system to create the colors of the art.

Figure 2.5: The pointillistic painting The Seine at Herblay by Maximilien Luce consists of thousands ofcolored dots. With the viewer very close to the canvas, the dots and their true colors are visible. Atnormal viewing distances, the dots are irresolvable and thus blend

When anybody wishs to use a lens instead of our visual system to resolve objects of small angularseparation, it is desirable to make the diffraction pattern as small as possible.According to (2.4), this can be done either by increasing the lens diameter or by using light of a shorterwavelength. For this reason ultraviolet light is often used with microscopes because its wavelength isshorter than a visible light wavelength.

2.2.2 Babinet Principle

Two pupil functions, A1(x, y) and A2(x, y) are complementary to each other if

16

CHAPTER 2. THEORETICAL ASPECTS OF DIFFRACTION

A1(x, y) +A2(x, y) = constant (2.5)

If the amplitude is constant over the whole x, y plane, then it is say that two diffracting apertures arecomplementary if the transparent parts in one are the opaque parts in the other, and vice versa. Forexample, the complementary aperture of a circular aperture is the whole x, y plane, excluding only theopaque disc of the same size as the circular aperture.assuming that there are two arbitrary complementary apertures, giving diffraction pattern U1(P ) andU2(P ). If U(P ) is the amplitude of the illumination (Figure 2.6) produced without any diffractionapertures, the Babinet principle says that

U2(P ) + U2(P ) = U(P ) (2.6)

which can be very easily shown to be true.Applying this principle to the Fraunhofer diffraction patterns produced by a circular aperture and anopaque disc, it can see that U(P ) is a very high and narrow peak at the origin, because all the light willtravel in a single direction if there is no diffracting aperture.Thus, except for the point at the origin, the Fraunhofer diffraction patterns of complementary aperturesare identical in shape but with amplitudes of opposite sign.

Figure 2.6: Fraunhofer diffraction pattern for four different apertures

When the diffraction pattern is the equation for the first and second order minima can be written asaccording to Rayleighs criterion

sinθ1 = 1.22λ

d(2.7)

sinθ2 = 1.22λ

d(2.8)

For small angles of diffraction , equations (2.7) and (2.8) may be approximated as

tanθ1 =r

D1= 1.22

λ

d(2.9)

17


tanθ2 =r

D1= 1.22

λ

d(2.10)

Where θ1 and D1 are the angle of diffraction and the sample to screen distance respectively for thefirst order minima, θ2 and D2 are the corresponding quantities for the second order diffraction minima ,the mean diameter of the cells was calls was calculated using the equations (2.9) and (2.10) taking intoaccount the wavelength of the laser light 6328× 10−8cm .

18

Chapter 3

Materials and Methods

This chapter describes the procedure of collection and preparation of blood samples for theexperimentation.This experiment is based on Babinet principle, its theorem concerning diffraction that states that thediffraction pattern from an opaque body is identical to that from a hole of the same size and shape exceptfor the overall forward beam intensity , that gives Fraunhofer diffraction pattern.There are methods to determine of size of blood

• Microscope

• Eriometer

• Diffraction of laser beam

3.1 Samples collection

Fresh samples of normal human blood and samples from the patients suffering from malaria (Plasmodiumvivax) were collected from fever hospital and apollo hospital Hyderabad.Shape and size of blood was determined ,using the technique of laser diffraction in the biophysicslaboratory.

3.2 Preparation of a thin blood film on the slide

Clean the finger with cotton wool dampened with alcohol (Figure 3.1).

Figure 3.1: Clean the Finger

Using a sterile lancet and a quick rolling action, puncture the ball of the finger or toe (Figure 3.2).Apply gentle pressure to the finger and collect a single small drop of blood about this size on the middleof the slide. his is for the thin film (Figure 3.3).Using another clean slide as a spreader and with the slide with the blood resting on a flat, firm surface,touch the small drop of blood with the edge of the spreader, allowing the blood to run right along the edge.Firmly push the spreader along the slide, keeping it at an angle of 45 ◦. the edge of the spreader mustremain in even contact with the surface of the other slide while the blood is being spread (Figure 3.4).

19

3.3. EXPERIMENTAL SET UP

Figure 3.2: Puncture the Finger

Figure 3.3: Collect the Blood

Figure 3.4: Spread of the Blood on the Slide

3.3 Experimental set up

Figure 3.5 is the schematic experimental arrangement of the laser diffraction . A He-Ne laser (L) of power2mw was employed for the diffraction purpose .Figure 3.6 gives the experimental set up to obtain laser diffraction . The specimen slide B, was introducedbetween the laser and the screen S , such that the smeared surface facing the screen .A well defineddiffraction pattern was obtained on the screen. Circular diffraction patterns were observed with malarialcells and disc shaped RBC .

3.4 Procedure and Analysis

In this part of the experiment we will determine the size of blood d by passing a laser beam throughslide and examining the diraction pattern projected on a screen. The laser beam is parallel andmonochromatic. Experimental set up consist of He-Ne laser and slide with blood smear and screen .here, both screen and slide surface are perpendicular to the incident beam. The distance D between the

20

CHAPTER 3. MATERIALS AND METHODS

Figure 3.5: Schematic Experimental Arrangement of Laser Diffraction

Figure 3.6: Experimental Arrangement of Laser Diffraction

slide and the screen was fixed. The laser beam is passing through the slide. A diffraction pattern is seenin the screen, produced by RBC. Then the radius of first order diffraction was measured using meterscale of least count 0.1cm.for distance between screen and slide to be D.

The radius r of the first order diffraction minima was measured for different values of the sample toscreen distance, D. A plot was drawn between radius of the diffraction minima r and the sample toscreen distance D, the slope of which gives the angle of diffraction . using the equation (2.9) to calculated for first order of diffraction.

21

3.4. PROCEDURE AND ANALYSIS

22

Chapter 4

Results and Discussion

The forth chapter concerns with the result of the present investigation on size of erythrocytes of normalpersons and also of patients suffering from malaria. In this chapter, results are presented for ten bloodsamples each of normal and malarial blood.Tables 4.1-4.10 present data on radius (r) of first order diffraction ring and corresponding distance (D)for the erythrocytes of blood collected from 10 healthy donors. These parameters are required for thecalculation of size of erythrocytes. Figures 4.1 - 4.10 show the plots drawn between D on x axis andr on y - axis for 20 samples. Plots are straight lines passing through origin and the slopes of which areused to calculate average size of erythrocytes. The parameters are concerned with first order diffraction.Similarly, Tables 4.11 - 4.20 present data on D and r for the erythrocytes of blood of 10 patients sufferingfrom Malaria. Figures 4.11 - 4.20 show the plots drawn between D on x axis and r on y - axis for 20samples. Here also plots are straight lines passing through origin, The slope of which gives the anglediffraction.Table 4.21 reveals data on average size of erythrocytes of normal and malarial blood for 10 samples each.It is evident from Table 4.21 that size of malarial erythrocytes is more that of normal. In the presentstudy, the size of normal erythrocytes is in the range of 7.70 - 8.16 µm, while it is in the range of 9.46 -10.04 mum for malarial erythrocytes. The same is represented by bar graphs to have at a glance picturefor the comparison purpose (Figure 4.21).Figure 4.22 shows micrographs of normal and malarial erythrocytes taken from light microscope ofL. C. 10 m interfacing with computer. Significantly more size in the case of malarial erythrocytes incomparison with the normal is evident. In the care of malaria still the erythrocytes are circular in shape.No morphological abnormalities can be seen in malarial erythrocytes. Figure 4.23 presents diffractionpatterns of both normal and malarial, when Red laser light is used. The diffraction patterns are circularrings, because of the fact that human erythrocytes are of biconcave disc type. First and second orderdiffraction can be seen. Malarial erythrocytes of relatively larger size give diffraction rings with smallerradius in comparison with normal erythrocytes. This aspect is confirmed with the formula obtained forthe size of the erythrocytes (ie circular aperture).

23

Table 4.1: Data on the distance r of first order of diffraction as a function of distance D between theslide and the screen for sample 1 (Normal sample)

Figure 4.1: The radius r of first order of diffraction ring as a function of distance D between the slideand the screen for sample 1 (Normal sample)

24

CHAPTER 4. RESULTS AND DISCUSSION



25



26




27



28




29



30




31



32




33

Table 4.11: Data on the distance r of first order of diffraction as a function of distance D between theslide and the screen for sample 11 (Malarial sample)

Figure 4.11: The radius r of first order of diffraction ring as a function of distance D between the slideand the screen for sample 11 (Malarial sample)

34




35



36




37



38




39



40




41



42




43

4.1. CONCLUSIONS

Table 4.21: Size (d) of normal and malarial erythrocytes

Figure 4.21: A comparison of Normal and Malarial erythrocytes

The parasite exists on the surface of RBC as a result the size gets affected. The size of human erythrocytesof malaria patients is more, when compared with the normal (Table 4.21), as the parasite enters into theblood. The blood gets affected so also the erythrocytes (Figure 4.22). The diffraction pattern of normaland malarial bloods as seen in the Figure 4.23.The study reveals that the size of the malaria is more in comparison with the normal blood.The mean diameter of RBC of normal human obtained by this method is 7.83 µm and where as formalaria cells is 9.75µm (Figure 4.21). In the case of malaria , the size obtained is drastically increaseddue to changes taken place on the RBC cell membrane, because of high metabolic activity in the caseof malaria and this leads to the changes in the cell morphology and size. The width of the diffractionpattern is a function of the variation in the size of the particles. similarly the sharpness of the minimadepends upon the consistency in the cellular size and shape. All human blood cells are of uniform shapeand hence the diffraction pattern produced by these cells are very sharp and clear. In the case of malarialblood cells the variation in the shape is considerably more and hence the sharpness of the pattern.

4.1 Conclusions

1. Laser diffraction method is very rapid and simple for assessing the average size of the cells.

2. At a glance picture of diffraction pattern gives an insight into the diffraction of light by smallparticles.

44


Figure 4.22: Micrograph of RBCs (A) normal B) malaria

Figure 4.23: Diffractograms of human erythrocytes (A) normal (B) malaria

3. The method is not only simple but also elegant.

4. It can be readily handled and can be easily demonstrated to a large gathering at a time especiallyto moderately sophisticated health science students which would provide them to look at physicaloptics as a set of relevant phenomena.

5. This could also be used with advantage as a diagnostic tool for assessing if there is more thannormal variation in the size distribution of human RBC from the width of the diffraction ring.

6. It is inexpensive, besides being accurate.

7. Size of malarial is significantly large when compared with normal erythrocytes.

8. There is variation in size of malarial erythrocytes to some extent within the same blood.

45

4.1. CONCLUSIONS

46

Appendix of Mathematica Programs

In this appendix the commands present some Mathematica programs aimed at the numerical andgraphical investigation of discrete dynamical systems.

Sample 1

datan1={{5,0.45},{6,0.55},{7,0.7},{8,0.8},{9,0.95},{10,1},{11,1.1},{12,1.25},{13,1.35},{14,1.4},{15,1.45}};

Text@Grid[Prepend[datan1,{”D(cm)”,”r(cm)”}],Frame→All,Dividers→{Center,{False,True}},Spacings→2,FrameStyle→Red]

gp=ListPlot[datan1,PlotStyle→{Black},Frame→True,FrameLabel→{”D(cm)”,”r(cm)”},PlotRange→{{0,20},{0,1.5}},AxesOrigin→{0,0},LabelStyle→Directive[Black,10]];

be=Fit[datan1,{0,x},x]

gbe=Plot[be,{x,0,15},PlotStyle→{Blue},Frame→True,FrameLabel→{”D(cm)”,”r(cm)”},PlotRange→{{0,20},{0,1.5}},AxesOrigin→{0,0},LabelStyle→Directive[Black,10]];

Show[gp,gbe,Epilog→Text[Style[”r=0.100 D”, Black,10],{5,1}]]

Sample 2

datan2={{5,0.45},{6,0.55},{7,0.65},{8,0.75},{9,0.9},{10,1},{11,1.1},{12,1.3},{13,1.35},{14,1.4},{15,1.45}};






Sample 3

datan3={{5,0.45},{6,0.55},{7,0.65},{8,0.7},{9,0.85},{10,1},{11,1.1},{12,1.3},{13,1.35},{14,1.4},{15,1.45}};


47

4.1. CONCLUSIONS





Sample 4

datan4={{5,0.5},{6,0.55},{7,0.65},{8,0.75},{9,0.9},{10,1},{11,1.2},{12,1.25},{13,1.3},{14,1.4},{15,1.45}};






Sample 5

datan5={{5,0.6},{6,0.65},{7,0.7},{8,0.75},{9,0.9},{10,1},{11,1.1},{12,1.2},{13,1.35},{14,1.4},{15,1.45}};






Sample 6

datan6={{5,0.45},{6,0.5},{7,0.7},{8,0.75},{9,0.85},{10,0.9},{11,1.15},{12,1.25},{13,1.35},{14,1.4},{15,1.45}};



48





Sample 7

datan7={{5,0.4},{6,0.55},{7,0.7},{8,0.8},{9,0.85},{10,0.9},{11,1.1},{12,1.2},{13,1.25},{14,1.3},{15,1.35}};






Sample 8

datan8={{5,0.45},{6,0.55},{7,0.7},{8,0.85},{9,0.95},{10,1},{11,1.1},{12,1.25},{13,1.3},{14,1.35},{15,1.4}};






Sample 9

datan9={{5,0.55},{6,0.6},{7,0.65},{8,0.85},{9,0.9},{10,1},{11,1.1},{12,1.2},{13,1.3},{14,1.35},{15,1.45}};






49

4.1. CONCLUSIONS

Sample 10

datan10={{5,0.5},{6,0.65},{7,0.7},{8,0.85},{9,0.9},{10,1},{11,1.1},{12,1.2},{13,1.3},{14,1.35},{15,1.4}};






Sample 11

datam1={{5,0.35},{6,0.4},{7,0.5},{8,0.55},{9,0.6},{11,0.7},{12,0.8},{13,0.9},{14,1.1},{15,1.2}};

Text@Grid[Prepend[datam1,{”D(cm)”,”r(cm)”}],Frame→All,Dividers→{Center,{False,True}},Spacings→2,FrameStyle→Red]

gp=ListPlot[datam1,PlotStyle→{Black},Frame→True,FrameLabel→{”D(cm)”,”r(cm)”},PlotRange→{{0,20},{0,1.5}},AxesOrigin→{0,0},LabelStyle→Directive[Black,10]];

be=Fit[datam1,{0,x},x]



Sample 12

datam2={{5,0.4},{6,0.45},{7,0.5},{8,0.65},{9,0.7},{10,0.75},{11,0.8},{12,0.95},{13,1.1},{14,1.2},{15,1.3}};






Sample 13

datam3={{5,0.35},{6,0.4},{7,0.5},{8,0.6},{9,0.65},{10,0.8},{11,0.9},{12,1},{13,1.1},{14,1.2},{15,1.3}};


50






Sample 14

datam4={{5,0.4},{6,0.45},{7,0.5},{8,0.65},{9,0.7},{11,0.8},{12,0.95},{13,1},{14,1.1},{15,1.2}};






Sample 15

datam5={{5,0.35},{6,0.45},{7,0.55},{8,0.65},{9,0.75},{10,0.7},{11,0.8},{12,1},{13,1.1},{14,1.2}};






Sample 16

datam6={{5,0.4},{6,0.45},{7,0.55},{8,0.6},{9,0.75},{10,0.8},{11,0.9},{12,0.95},{13,1},{14,1.1},{15,1.3}};





51

4.1. CONCLUSIONS


Sample 17

datam7={{5,0.4},{6,0.45},{7,0.5},{8,0.55},{9,0.65},{10,0.7},{11,0.9},{12,1},{13,1.1},{14,1.2},{15,1.3}};






Sample 18

datam8={{5,0.35},{6,0.5},{7,0.55},{8,0.6},{9,0.65},{10,0.8},{11,0.9},{12,1},{13,1.1},{14,1.2}};






Sample 19

datam9={{5,0.35},{6,0.4},{7,0.55},{8,0.6},{9,0.7},{10,0.8},{11,0.85},{12,0.9},{13,1.1},{14,1.2},{15,1.3}};






Sample 20

datam10={{5,0.3},{6,0.4},{7,0.5},{8,0.6},{9,0.7},{10,0.8},{11,0.85},{12,0.9},{13,1},{14,1.1},{15,1.2}};

52







Table of size for all samples

dataa={{1,10.73,7.96},{2,9.53,7.73},{3,9.46,7.79},{4,9.93,7.73},{5,9.61,7.7},{6,9.6,7.83},{7,9.54,8.16},{8,9.48,7.8},{9,9.55,7.79},{10,10.04,7.82}};

Text@Grid[Prepend[dataa,{”sample”,”sizeofmalarialblood”,”sizeofnormalblood”}],Frame→All,Dividers→{Center,{False,True}},Spacings→4,FrameStyle→Red]

Bar graph

Needs[”BarCharts‘”]

BarChart[{7.83,9.75}, BarLabels→{”normal blood”, ”malarial blood”},AxesLabel→size]

53

4.1. CONCLUSIONS

54

Bibliography

[1] Mohammed Gulam Ahamad, Kaleem Ahmed Jaleeli, and Adeel Ahmad. J.Pure & Appl. Phys.,18(4), 2006.

[2] J. Calthrope. Advanced experiments in practical optics. William Heinemann Ltd.,London, 1952.

[3] Charles and F Mayer. The diffraction of light x-ray and material particles. University of ChicagoPress, Chicago, Illinois, 1934.

[4] David Halliday, Robert Resnick, and Jearl Walker. Fundamentals of Physics. Wiley, 10th edition,2010.

[5] Leland H. Hartwell. Periodic density fluctuation during the yeast cell cycle and the selection ofsynchronous cultures. Journal of Bacteiology, 104(3):1280–1285, December 1970.

[6] John E. Hyde. Mechanisms of resistance of plasmodium falciparum to antimalarial drugs. Microbesand Infection, 4(2):165–174, February 2002.

[7] Njera JA. Malaria control: achievements, problems and strategies. Parassitologia, 43(1–2):1–89, Jun2001.

[8] Rajinder Kumar, Alla Musiyenko, and Sailen Barik. The heat shock protein 90 of plasmodiumfalciparum and antimalarial activity of its inhibitor, geldanamycin. Malaria Journal, 2(30),September 2003.

[9] Bernard Levussove and Oser. Hawk’s Physiological Chemistry. McGraw-Hill, 14th edition, 1965.

[10] R.S. Longhurst. Geometrical and Physical Optics. Longman, 3th edition, June 1974.

[11] Omolade O. Okwa. Malaria Parasites. InTech, 1st edition, March 2012.

[12] World Health Organization. Basic malaria microscopy,Part I. Learners guide. WHO press, 2ndedition, 2010.

[13] Ramakrishna Rao, Kaleem Ahmed Jaleeli, Bellubbi BS, and Adeel Ahmad. J. Pure & Appl. Phys.,21(2):201–203, 2009.

[14] Valerie C. Scanlon and Tina Sanders. Essentials of Anatomy and Physiology. F. A. Davis Company,5th edition, 2007.

[15] Clive Shiff. Integrated approach to malaria control. Clinical Microbiology Reviews, 15(2):278–293,April 2002.

[16] Bruce F. Torrence and Eve A. Torrence. The Student’s Introduction to MATHEMATICA. CambridgeUniversity Press, 2nd edition, 2009.

[17] Anwar Ali A K W. ultrasonic and dielectrophoresis studies in biological media. PhD thesis, Osmaniauniversity, Hyderabad, india, 1983.

[18] David C Warhurst, John C Craig, Ipemida S Adagu1, David J Meyer1, and Sylvia Y Lee. Therelationship of physico-chemical properties and structure to the differential antiplasmodial activityof the cinchona alkaloids. Malaria Journal, 2(26), September 2003.

55

BIBLIOGRAPHY

56

List of Figures

1.1 Composition of Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

1.2 Shape and dimensions of a red blood cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

1.3 Rouleau formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

1.4 WBC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

1.5 Platelets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1.6 A, Plasmodium vivax trophozoite. B, P. vivax mature schizont. C, P. vivaxmacrogametocyte. D, P. vivax microgametocyte. . . . . . . . . . . . . . . . . . . . . . . . 9

1.7 A, Plasmodium malariae band form trophozoite. B, P. malariae schizont . . . . . . . . . . 9

1.8 A, Plasmodium ovale trophozoite. B, Schffners stippling of P. ovale clearly visible . . . . . 9

1.9 A, Plasmodium falciparum ring-form trophozoites. B, P. falciparum gametocyte . . . . . 10

2.1 This diffraction pattern appeared on a viewing screen when light that had passed througha narrow vertical slit reached the screen. Diffraction caused the light to flare outperpendicular to the long sides of the slit. That flaring produced an interference patternconsisting of a broad central maximum plus less intense and narrower secondary (or side)maxima, with minima between them . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

2.2 The diffraction pattern produced by a razor blade in monochromatic light. Note the linesof alternating maximum and minimum intensity . . . . . . . . . . . . . . . . . . . . . . . 14

2.3 The diffraction pattern of a circular aperture. Note the central maximum and the circularsecondary maxima. The figure has been overexposed to bring out these secondary maxima,which are much less intense than the central maximum . . . . . . . . . . . . . . . . . . . . 15

2.4 At the top, the images of two point sources (stars) formed by a converging lens. Atthe bottom, representations of the image intensities. In (a) the angular separation ofthe sources is too small for them to be distinguished, in (b) they can be marginallydistinguished, and in (c) they are clearly distinguished. Rayleigh’s criterion is satisfied in(b), with the central maximum of one diffraction pattern coinciding with the first minimumof the other . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

2.5 The pointillistic painting The Seine at Herblay by Maximilien Luce consists of thousandsof colored dots. With the viewer very close to the canvas, the dots and their true colorsare visible. At normal viewing distances, the dots are irresolvable and thus blend . . . . . 16

2.6 Fraunhofer diffraction pattern for four different apertures . . . . . . . . . . . . . . . . . . 17

3.1 Clean the Finger . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

3.2 Puncture the Finger . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

3.3 Collect the Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

3.4 Spread of the Blood on the Slide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

3.5 Schematic Experimental Arrangement of Laser Diffraction . . . . . . . . . . . . . . . . . . 21

3.6 Experimental Arrangement of Laser Diffraction . . . . . . . . . . . . . . . . . . . . . . . . 21

4.1 The radius r of first order of diffraction ring as a function of distance D between the slideand the screen for sample 1 (Normal sample) . . . . . . . . . . . . . . . . . . . . . . . . . 24





57

LIST OF FIGURES






4.11 The radius r of first order of diffraction ring as a function of distance D between the slideand the screen for sample 11 (Malarial sample) . . . . . . . . . . . . . . . . . . . . . . . . 34










4.21 A comparison of Normal and Malarial erythrocytes . . . . . . . . . . . . . . . . . . . . . . 444.22 Micrograph of RBCs (A) normal B) malaria . . . . . . . . . . . . . . . . . . . . . . . . . . 454.23 Diffractograms of human erythrocytes (A) normal (B) malaria . . . . . . . . . . . . . . . 45

58

List of Tables

4.1 Data on the distance r of first order of diffraction as a function of distance D between theslide and the screen for sample 1 (Normal sample) . . . . . . . . . . . . . . . . . . . . . . 24










4.11 Data on the distance r of first order of diffraction as a function of distance D between theslide and the screen for sample 11 (Malarial sample) . . . . . . . . . . . . . . . . . . . . . 34










4.21 Size (d) of normal and malarial erythrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . 44

59

a study on size and shape of erythrocytes of normal and malarial blood using laser diffraction...

Documents

properties of blood

blood film

properties of red blood

function of white blood

function of red blood

normal size

size of platelets61

kaleem ahmed jaleeli