Cytological Screening of Conjunctiva Changes Among Sudanese

Home , Conjunctiva

ﺑﺴﻢ ﺍﷲ ﺍﻟﺮﲪﻦ ﺍﻟﺮﺣﻴﻢ

University of Khartoum

Faculty of Medical Laboratory Sciences

Department of Histopathology and Cytology

Cytological Screening of Conjunctiva Changes among

Sudanese Patients Attending Ophthalmic Clinic

A thesis submitted in partial fulfillment of degree of BSc (Honour) in Medical

Laboratory Sciences (Histopathology and cytology)

2014

Prepared and Submitted by: Sajda Yagoub Abd Alla

Supervised by: Amel Al Tayeb Ahmed, (MSc, BSc U of K)

اﻵﯾﺔ

ﻗﺎل ﺗﻌﺎﱃ :

ﻳﺄﻳﻬﺎ ﺍﻟﹼﺬﻳﻦ ﺁﻣﻨﻮﺍﹾ ﺍﺗﻘﹸﻮﺍﹾ ﺍﻟﻠﹼﻪ ﻭﻟﹾﺘﻨﻈﹸﺮ ﻧﻔﹾﺲ ﻣﺎ ﻗﹶﺪﻣﺖ ﻟﻐﺪ ﻭﺍﺗﻘﹸﻮﺍﹾ ﺍﻟﻠﹼﻪ ﺇﹺﻥﹼ ﺍﻟﻠﹼﻪ ﺧﺒﹺﲑ ﺑﹺﻤﺎ ﺗﻌﻤﻠﹸﻮﻥ)

ﺳﻮﺭﻩ ﺍﳊﺸﺮ ١٨

. Abstract

Introduction: The conjunctiva eye cytology is a part of the diagnostic eye cytology provide a tool of a diagnosis of many ocular diseases and conditions with using of a suitable tool of collection of sample. This kind of cytological procedures doing as screening for population ,or specify the disease under study also for comparison of accuracy and usefulness of the different types of tools use in collection of samples.

Objectives: The aim of this study was to screen the conjunctival eye cytology in the population under study ,and to describe the cellular pattern in different eye condition to find out the different factors affect cellular components of the conjunctiva and subsequently the eye health status, and to evaluate the usefulness of the conjunctiva eye cytology as an important diagnostic procedures in the diagnosis of different eye condition .

Materials and Methods: This was a descriptive cross sectional hospital base study, done in university of Khartoum, faculty of medical laboratory sciences in department of histopathology and cytology ,to screen conjunctiva cytological changes of ophthalmic clinic patients using swabbing and scrapping cytology for different ocular diseases in period from January 2014 to May 2014.

Conjunctiva swabbing and scrapping smears were collected from Abd Alfadeel centre for medicine and eye surgery, Noor Alauoon military eye hospital, Alneelin university eye hospital (collage of optometry) and Maka charity foundation for eye medicine (Ridhy division) ophthalmology clinic. Ninety two92patients participated in this study,56were females and 36were males, conjunctiva smears were fixed by 95% alcohol and air then stained by Pap , Giemsa and hematoxylin stains for cytological evaluation.

Results: (89.4%) of cytological finding among adequate samples shown matching with the clinical ocular diagnosis. Scrapping method had the best role in getting adequate samples( 90%),with marked increase of adequacy of sample obtained from elder age patients(34.2%from 41- 60year, 31.5%from 61-80year) and in cases of infection(48.7%). Bacteria was the major cause of infections (86.2%).

Conclusion: Conjunctiva cytology is an impressive tool to get information about ocular tissue health status , and can be a useful complementary diagnostic method ,get its role in epidemiological surveys of endemic ocular diseases such as trachoma in our population and for better understand of diseases spreading manner.

Key Words: Conjunctiva, cytology, ocular, ophthalmic, trachoma, Pap stain , Giemsa stain.

ﻣﻠﺨﺺ اﻟﺪراﺳﺔ

اﻟﻤﻘﺪﻣﺔ :

ﻋﻠﻢ ﺧﻼﯾﺎ ﻣﻠﺘﺤﻤﺔ اﻟﻌﯿﻦ ھﻮ ﺟﺰء ﻣﻦ ﻋﻠﻢ ﺧﻼﯾﺎ اﻟﻌﯿﻦ اﻟﺘﺸﺨﯿﺼﻲ اﻟﺬي ﯾﻮﻓﺮ أداة ﻟﺘﺸﺨﯿﺺ ﻛﺜﯿﺮ ﻣﻦ أﻣﺮاض و ﺣﺎﻻت اﻟﻌﯿﻮن ، ﻣﻊ إﺳﺘﺨﺪام اداة ﻣﻨﺎﺳﺒﺔ ﻟﺠﻤﻊ اﻟﻌﯿﻨﺔ . ھﺬا اﻟﻨﻮع ﻣﻦ اﻹﺟﺮاءات اﻟﺨﻠﻮﯾﺔ ﯾُﻌﻤﻞ ﻛﻤﺴﺢ ﻟﻤﺠﻤﻮﻋﺔ ، أو ﺑﺘﺤﺪﯾﺪ اﻟﻤﺮض ﻗﯿﺪ اﻟﺪراﺳﺔ ، و اﯾﻀﺎ ﻟﻌﻤﻞ ﻣﻘﺎرﻧﺔ ﻟﺪﻗﺔ و ﻓﺎﺋﺪة اﻷدوات اﻟﻤﺨﺘﻠﻔﺔ اﻟﻤﺴﺘﺨﺪﻣﺔ ﻟﺠﻤﻊ ھﺬا اﻟﻨﻮع ﻣﻦ اﻟﻌﯿﻨﺎت اﻟﺨﻠﻮﯾﺔ.

اﻷھﺪاف:

اﻟﮭﺪف ﻣﻦ ھﺬه اﻟﺪراﺳﺔ ھﻮ إﺟﺮاء ﻣﺴﺢ ﺧﻠﻮي ﻟﻤﻠﺘﺤﻤﺔ اﻟﻌﯿﻦ ﻟﻠﻤﺠﻤﻮﻋﺔ اﻟﺴﻜﺎﻧﯿﺔ ﺗﺤﺖ اﻟﺪراﺳﺔ ، و وﺻﻒ اﻟﻨﻤﻂ اﻟﺨﻠﻮي ﻓﻲ اﻟﺤﺎﻻت اﻟﻤﺨﺘﻠﻔﺔ ﻟﻠﻌﯿﻦ ﻟﻤﻌﺮﻓﺔ اﻟﻌﻮاﻣﻞ اﻟﺘﻲ ﺗﺆﺛﺮ ﻋﻠﻰ اﻟﻤﻜﻮﻧﺎت اﻟﺨﻠﻮﯾﺔ ﻟﻠﻤﻠﺘﺤﻤﺔ و ﺑﺎﻟﺘﺎﻟﻲ ﻋﻠﻰ ﻣﺴﺘﻮى ﺻﺤﺔ اﻟﻌﯿﻦ ، و ﺗﻘﯿﯿﻢ ﻓﺎﺋﺪة إﺳﺘﺨﺪام ﺧﻼﯾﺎ ﻣﻠﺘﺤﻤﺔ اﻟﻌﯿﻦ ﻛﻮﺳﯿﻠﺔ ﺗﺸﺨﯿﺼﯿﺔ ﻣﮭﻤﺔ ﻓﻲ ﺗﺸﺨﯿﺺ أﻣﺮاض اﻟﻌﯿﻮن اﻟﻤﺨﺘﻠﻔﺔ.

اﻟﻄﺮﯾﻘﺔ و اﻷدوات:

ھﺬه دراﺳﺔ اﻟﻮﺻﻔﯿﺔ أﺟﺮﯾﺖ ﺑﺠﺎﻣﻌﺔ اﻟﺨﺮﻃﻮم ، ﻛﻠﯿﺔ ﻋﻠﻮم اﻟﻤﺨﺘﺒﺮات اﻟﻄﺒﯿﺔ ، ﻗﺴﻢ اﻷﻧﺴﺠﺔ و اﻟﺨﻼﯾﺎ اﻟﻤﺮﯾﻀﺔ ، و ﺗﮭﺪف ﻟﻤﻌﺎﯾﻨﺔ اﻟﺘﻐﯿﺮات اﻟﺨﻠﻮﯾﺔ ﻟﻤﻠﺘﺤﻤﺔ اﻟﻌﯿﻦ ﻟﺪى ﻣﺮﺿﻰ ﻋﯿﺎدة اﻟﻌﯿﻮن ﺑﺈﺳﺘﺨﺪام اﻟﻤﺴﺢ و اﻟﻜﺤﺖ اﻟﺨﻠﻮي ﻓﻲ أﻣﺮاض اﻟﻌﯿﻮن اﻟﻤﺨﺘﻠﻔﺔ ﻓﻲ اﻟﻔﺘﺮة ﻣﻦ ﯾﻨﺎﯾﺮ ٢٠١٤ و ﺣﺘﻰ ﻣﺎﯾﻮ ٢٠١٤.

ﺗﻢ ﺟﻤﻊ ﻣﺴﺤﺎت ﺧﻠﻮﯾﺔ ﻟﻤﻠﺘﺤﻤﺔ اﻟﻌﯿﻦ ﻣﻦ ﻋﯿﺎدة اﻟﻌﯿﻮن ﻓﻲ ﻣﺮﻛﺰ ﻋﺒﺪ اﻟﻔﻀﯿﻞ اﻟﻤﺎظ ﻟﻄﺐ و ﺟﺮاﺣﺔ اﻟﻌﯿﻮن ، ﻣﺴﺘﺸﻔﻰ ﻧﻮر اﻟﻌﯿﻮن اﻟﻌﺴﻜﺮي ، ﻣﺴﺘﺸﻔﻰ اﻟﻌﯿﻮن اﻟﺠﺎﻣﻌﻲ ﺟﺎﻣﻌﺔ اﻟﻨﯿﻠﯿﻦ (ﻛﻠﯿﺔ ﻋﻠﻮم اﻟﺒﺼﺮﯾﺎت) و ﻣﺆﺳﺴﺔ ﻣﻜﺔ اﻟﺨﯿﺮﯾﺔ ﻟﻄﺐ اﻟﻌﯿﻮن ﻓﺮع اﻟﺮﯾﺎض.

٩٢ ﻣﺮﯾﺾ ﺷﺎرﻛﻮا ﻓﻲ ھﺬه اﻟﺪراﺳﺔ ، ٥٦ أﻧﺜﻰ و ٣٦ ذﻛﺮ . ﻣﺴﺤﺔ اﻟﻤﻠﺘﺤﻤﺔ ﺣﻔﻈﺖ ﺑﺈﺳﺘﺨﺪام ٩٥%اﻟﻜﺤﻮل و اﻟﺘﻌﺮﯾﺾ ﻟﻠﮭﻮاء ﺛﻢ ﺻﺒﻐﺖ ﺑﺈﺳﺘﺨﺪام ﺻﺒﻐﺔ ﺑﺎﺑﺎ ﻧﯿﻜﻮﻻ و ﺟﻤﺴﺎ و اﻟﮭﯿﻤﺎﺗﻮﻛﺴﻠﯿﻦ ﻟﺘﺤﻀﯿﺮھﺎ ﻟﻠﺘﻘﯿﯿﻢ اﻟﺨﻠﻮي.

اﻟﻨﺘﺎﺋﺞ: (٨٩.٤%) ﻣﻦ اﻟﻨﺘﺎﺋﺞ اﻟﺨﻠﻮﯾﺔ ﻟﻠﻌﯿﻨﺎت اﻟﻜﺎﻓﯿﺔ ﺗﺸﺨﯿﺼﯿﺎ أﻇﮭﺮت ﺗﻄﺎﺑﻖ ﻣﻊ اﻟﺘﺸﺨﯿﺺ اﻟﻤﺮﺿﻲ ﻟﻠﻤﻠﺘﺤﻤﺔ ﻓﻲ اﻟﻌﯿﺎدة.

ﻃﺮﯾﻘﺔ اﻟﻜﺤﺖ ﻟﮭﺎ أﻓﻀﻞ دور ﻓﻲ اﻟﺤﺼﻮل ﻋﻠﻰ ﻋﯿﻨﺎت ﻛﺎﻓﯿﺔ ﻛﻤﺎً ﻟﻠﺘﺸﺨﯿﺺ ، ﻣﻊ إزدﯾﺎد واﺿﺢ ﻓﻲ ﻛﻤﯿﺔ اﻟﻌﯿﻨﺔ اﻟﻤﺄﺧﻮذة ﻣﻦ اﻟﻔﺌﺎت اﻟﻌﻤﺮﯾﺔ اﻷﻛﺒﺮ و ﻓﻲ ﺣﺎﻻت اﻟﻌﺪوى. اﻟﺒﻜﺘﯿﺮﯾﺎ ﻣﺜﻠﺖ اﻟﺴﺒﺐ اﻟﺮﺋﯿﺴﻲ ﻟﻌﺪوى أﻣﺮاض اﻟﻌﯿﻮن.

اﻟﺨﻼﺻﺔ:

إﺳﺘﺨﺪام ﺧﻼﯾﺎ اﻟﻤﻠﺘﺤﻤﺔ ھﻲ ﻃﺮﯾﻘﺔ ﻣﻨﺎﺳﺒﺔ ﻟﻠﺤﺼﻮل ﻋﻠﻰ ﻣﻌﻠﻮﻣﺎت ﻋﻦ ﺻﺤﺔ اﻟﻨﺴﯿﺞ اﻟﺴﻄﺤﻲ ﻟﻠﻌﯿﻦ و ﺑﺎﻹﻣﻜﺎن إﺳﺘﺨﺪاﻣﮭﺎ ﻛﻄﺮﯾﻘﺔ ﺗﺸﺨﯿﺼﯿﺔ ﺗﻜﻤﯿﻠﯿﺔ ﺗﻘﻮم ﺑﺪورھﺎ ﻓﻲ اﻟﻤﺴﻮﺣﺎت اﻟﻮﺑﺎﺋﯿﺔ ﻷﻣﺮاض اﻟﻌﯿﻮن اﻟﻤﺴﺘﻮﻃﻨﺔ ﻣﺜﻞ اﻟﺘﺮاﻛﻮﻣﺎ و ﻟﻤﻌﺮﻓﺔ أﻓﻀﻞ ﻷﻧﻤﺎط أﻧﺘﺸﺎر أﻣﺮاض اﻟﻌﯿﻮن.

اﻟﻜﻠﻤﺎت اﻟﻤﻔﺘﺎﺣﯿﺔ : اﻟﻤﻠﺘﺤﻤﺔ ، ﻋﻠﻢ اﻟﺨﻼﯾﺎ ، ﺑﺼﺮي ، ﺗﺮاﻛﻮﻣﺎ ، ﺻﺒﻐﺔ ﺑﺎﺑﺎ ﻧﯿﻜﻮﻻ ، ﺻﺒﻐﺔ ﺟﻤﺴﺎ.

DeDication

To my Parents, Sister and Brother

Acknowledgment

-The first and lastly thanks for GOD.

-I want to thanks my Supervisor Amel Al Tayeb Ahmed (BSc, MSc u of k) the director for this work for his support, and patience.

-To who believe the idea of my project and encourage me all the time and make me till now proud to this work and put the first steps and skeleton to my project the special thanks for him Emmanuel Edward.

-I would like to express my deep gratitude and appreciation to

Mr. Abdulla Hassab alnabe for his brilliant and outstanding ideas which inspired me, and for his help in conformation and evaluation of cytological smears.

-Special thanks to O.D. Sulma Yagoub for being the source of knoweldgment and engorgement.

-To staff of Abd alfeedel almagh Eye Center , Alneelin Eye hospital (collage of optometry) , Noor aleyoon Military Eye Hospital , Makah Eye Hospital for their tenderness and collaboration.

-To lecturer Kawther Mohammed who help me to find the tools of this project.

-To all gave me an inspiration ..., thank you.

List of contents

Contents Page Title Alaya I Abstract II-III Abstract in Arabic V Dedication VI Acknowledgement VII List of tables IX List of figures X List of abbreviations XI

List of Tables

Table number Title page

Table (1) Distribution of the population according to occupation 37 Table (2) Distribution of the infectious organisms 43 Table (3) Infection and washing occasion of face per day 47

List of figures

No. of figure Title Page

(1) Matching of cytological findings with ophthalmic diagnosis 34

(2) Distribution of gender 35

(3) Distribution of age 36

(4) The residence distribution 38

(5) Distribution according to original area 39

(6) Disease according to the education level 40

(7) The involved eye in disease 41

(8) Distribution of different eye conditions 43

(9) Distribution according to types of eye diagnosis 44

(10) Distribution of eye diagnosis according to job 45

(11) Occasions involve face washing per day 46

(12) Duration of exposure to direct sun rays per day 48

(13) Duration of contact with visual media per day 49

(14) Family history of ocular diseases 50

(15) Potential allergen types in the population environment 51

List of abbreviations

PAS Period acid shiff IgA Immunoglobulin Alpha KCS Keratoconjunctivitis sicca IOP Intra ocular pressure Pap stain Papanicolaou stain HIV Human immunodeficiency virus LPG Liquefied petroleum gas W.S Wound swab N.S Nylon swab

Chapter one

1.1.Introduction 1-2 1.2. Scientific Background 3 1.2.1. Accessory structures of the eye 3 1.2.1.1. Eyebrows 3 1.2.1.2. Eyelids 3 1.2.1.3. Conjunctiva 4 1.2.1.3.1. Anatomy and physiology of conjunctiva 4-7 1.2.1.3.2. Histology of conjunctiva 7 1.2.1.3.3. Cytology of conjunctiva 7-8 1.2.1.4.The lacrimal apparatus 8 1.2.2. Tear film 8-9 1.2.3. The main structures of the eye 9 1.2.3.1. The eye ball 9 1.2.3.2. The cornea 10-11 1.2.3.3. The sclera 11-12 1.2.3.4. The ueava 12 1.2.3.4.1. The iris 12 1.2.3.4.2. The ciliary body 13 1.2.3.4.3. The choroid 13 1.2.3.5. The lens 13 1.2.3.6. The retina 14 1.2.4. Abnormalities of the eye 14 1.2.4.1. Neoplasms 14-15 1.2.4.2. Physical and chemical eye injuries 16 1.2.4.3 Eye ball abnormalities 16 1.2.4.4. Eye lids abnormalities 17 1.2.4.5. Conjunctiva abnormalities 17-19 1.2.4.6. Inner structures abnormalities 19-20 1.3 Literature review 21-24 1.4.Justification 25 1.5. Objectives of the study 26 1.5.1. General objective 26 1.5.2. Specific objectives 26

Chapter two

2.1. Study design 27 2.2 Study area 27 2.3. Study population 27 2.4. Sample size 28 2.5. Study duration 28 2.6. Materials 28 2.7. Methods 28 2.7.1. Data collection 28 2.7.2. Sample collection 28 2.7.2.1. Conjunctiva swabbing and scrapping collection 28 2.7.3. Sample preparation 29 2.8. Staining assessment 29 2.9. Interpretation of slides 29 2.10. Statistical assessments 29 2.11. Ethical consideration 30

Chapter three

3. Results 31 3.1. Introduction 31-33 3.2. The analysis 34-51

Chapter four

4.1 Discussion 52-54 4.2 Conclusion and recommendations 55

References Appendixes

Chapter one

Introduction

1.1 Introduction

The conjunctival eye cytology is a part of the diagnostic eye cytology provide a tool of a diagnosis of many ocular diseases and conditions. It is a developing part of cytology in the developed countries and many studies is done in this field ,other studies carryout in the most epidemic and endemic areas with infectious eye diseases such as India(6) ,Taiwan ,Nepal that spotted mainly in Africa and Asia show the quite benefits and exhibit the histological and cytological status of conjunctiva of the patients as follow up ,prognosis ,involve grading of certain ocular condition (dry eye syndrome ,vitamin deficiencies ) (1).

The cellular pattern of the conjunctival cytological sample express a useful information about : the epithelium reaction ,nature of cells ,and any infested matter such as foreign bodies and micro-organisms also the use of the tissue markers and molecular technology give more field of research and understand of causes ,events of many ocular diseases .(4)

The conjunctiva ,cornea, vitreous, aqueous and puncum are parts of the eye which comprise the sites of cytological specimens for diagnosis of specific eye conditions such as infection ,inflammation, traumas ,and tumors ,choose of type of sample depend on case under investigation and what goal of diagnosis. Samples from eyelid and eyelashes consider as skin samples.(15) Tools and methods used to get conjunctival cytology smears are :The eye spatula(scrapping),a sterile polyester tipped swab(swabbing/scrapping)(5),eye microbrush (brushing )(8),cellulose acetate filter paper (disc or strip ) (6) and Eye prim device from Opia company (France) for impression cytology method(4), imprint method by applying a clean slide on conjunctival surface , the old method of pipetting samples in certain cases(ocular benign mucous membrane pemphigoid) (7),fine needle aspiration for eyelid lesions.(24)

Stains had been used for demonstration of conjunctival eye cytology are: Periodic Acid- Schiff (PAS) and counter-stained with haematoxylin and eosin (1), a routine Papanicolaou method (12),Giemsa(14) or May-Grunewald Giemsa (13), haematoxylin and eosin, some bioassays markers.(4)

Cells of conjunctiva epithelium obtained from cytological smears are: goblet cells , polyhedral or long columnar cells with microvilli and small cubidal basal cells. Other cell types may be present according to the eye condition .(21)

Certain ocular tumors express conjunctival cytological changes, the most common benign are hemangiomas. Samples from eyelid and eyelashes consider as skin samples.(21)

1.2. Scientific Background

1.2.1 The accessory structures of the eye:

1.2.1.1 Eyebrows:

The eyebrows consist of thick skin covered by characteristic short, prominent hairs extending across the superior orbital margin, usually arching slightly but sometimes merely running horizontally. Generally, in men the brows run along the orbital margin, whereas in women the brows run above the margin. The first body hairs produced during embryologic development are those of the eyebrow. The muscles located in the forehead: the frontalis,procerus, corrugator superciliaris, and orbicularis oculi, produce eyebrow movements, an important element in facial expression .(19)

1.2.1.2 Eyelids:

The eyelids, or palpebrae, are folds of skin and tissue that, when closed, cover the globe. The eyelids have four major functions: (1) cover the globe for protection,(2) move the tears toward drainage at the medial canthus on closure, (3) spread the tear film over the anterior surface of the eye on opening, and (4) contain structures that produce the tear film.

On closure the upper eyelid moves down to cover the cornea, whereas the lower eyelid rises only slightly. When the eyes are closed gently, the eyelids should cover the entire globe.(19)

1.2.1.3 Conjunctiva :

1.2.1.3.1 Anatomy and physiology of conjunctiva:

The conjunctiva is a transparent mucous membrane lining the posterior surface of the eyelids and the anterior aspect of the eyeball It is richly vascular, supplied by the anterior ciliary and palpebral arteries. There is a dense lymphatic network, with drainage to the preauricular and submandibular nodes corresponding to that of the eyelids. It has a key protective role, mediating both passive and active immunity. The name conjunctiva (conjoin: to join) has been given to this mucous membrane owing to the face that it joins the eyeball to the lid .It stretches from the lid margin to the limbus and enclosed a complex space called conjunctival sac. (22)

The conjunctiva plays a critical role in maintaining ocular health by forming a smooth , flexible , and protective sac that covers the pericorneal surface of the front of the eye . The conjunctiva Is responsible for tear film synthesis and distribution and aids in providing adequate wetting of globe. The conjunctiva secretes mucus and maintains the tear film , thus permitting smooth movement of the eyelid over the cornea. An intact conjunctiva forms a barrier for entrance of infectious organisms and provide immune surveillance and immune reactivity for antigenic stimuli .The vessels in the conjunctiva provide nourishment to the cornea .

In addition the conjunctiva participates in the lymphatic drainage from the eyelid. It is subdivided into the following: 1.Palpebral conjunctiva: marginal , tarsal and orbital.

2.Bulbar conjunctiva: Scleral and limbal

3.Conjunctival fornix: superior , inferior , lateral and medial. (19)

1.Palpebral conjunctiva:

It lines the lids and can be subdivided into marginal ,tarsal and orbital conjunctiva. a-Marginal conjunctiva: extend from the lid margin to about 2mm on the back of lid up to shallow groove—the sulcus subtarsalis .It is actually a transitional zone between skin and conjunctiva proper . b- Tarsal conjunctiva: is thin, transparent and highly vascular. It is firmly adherent to the whole tarsal plate in the upper lid . In the lower lid ,it is adherent only to half width of tarsus . c- Orbital part: of palpebral conjunctiva lies loose between the tarsal plate and fornix. Orbital conjunctiva of the upper lid is loose and lies over the Muller`s muscle. The bulbar conjunctiva contains a specialization known as the plica semilunaris. This is a crescent –shaped fold of bulbar conjunctiva that has the greatest concentration of goblet cells. It is loose and redundant so that there is no limitation of the globe during abduction.

2.Bulbar conjunctiva:

It is thin, transparent and lies loose over the underling structures and thus can be moved easily .It is separated from the anterior sclera by episcleral tissue and Tenon`s capsule .Subconjunctival vessels and the anterior ciliary arteries forming the periconeal plexus can be seen in the loose tissue under the bulbar conjunctiva. A 3 mm ridge of bulbar conjunctiva around the cornea is called limbal conjunctiva. In the area of limbus, the conjunctiva , Tenon`s capsule and the episcleral tissue are fused into a dense tissue which is strongly adherent to the underlying coreo-scleral junction . At the limbus , the epithelium of conjunctiva becomes continuous with that of cornea.

The fornical conjunctiva is the cul-de-sac region and houses the ducts of the main lacrimal gland .

3- Conjuncival fornix:

Conjunctival fornix is a continuous circular cul-de-sac , which is broken only on the medial side by caruncle and plica semilunaris. Conjunctiva fornix joins the bulbar conjunctiva with the palpebral conjunctiva .It can be subdivided into superior ,inferior ,medial lateral fornix.

i. Superior fornix. It is extend from slightly above the upper border of the tarsal plate to a distance about 10 mm from the upper limbus and is thus located at the level of superior orbital margin . ii. Inferior fornix. It is extends from slightly below the lower border of the lower tarsal plate to a distance about 8 mm from the lower limbus and is located near the inferior orbital margin .The extension of the fascial sheath of the inferior rectus and inferior oblique muscles is attached to the conjunctival fold in the lower fornix ,It helps in maintaining the recess of the inferior fornix during movements of the lower lid . iii. Lateral fornix . It is a small cul-de-sac which extend to just behind the equator of the eyeball . iv. Medial fornix. It is a shallow cul-de-sac in which lie the caruncle and plica semilunaris dipped in the pool of tears called the `lacus lacrimalis ` or `tear lake`. As one progresses toward the cornea , the epithelial layer thickens ;dendritic langerhans cells become more prominent ; and goblet cells disappear . The caruncle is a fleshy ,pink specialization located in the medial portion of the eye. The caruncle functions to direct tear flow , and excision of this structure should be avoided because dry eye syndrome may result .(22)

1.2.1.3.2. Histology of conjunctiva:

The conjunctiva is composed of non keratinized stratified squamous and columnar epithelium with goblet cells .Squamous differentiation is most prominent at the limbus and palpebral margins. Columnar epithelium is located in the bulbar, palpebral , and fornical regions. The goblet cells are present in the middle and superficial layers of the epithelium.(16)

The conjunctival epithelial cell surface has microvilli , which function to help mucus remain on the ocular surface . Henle’s crypt which are present in the palpebral conjunctiva ,are thought to function in an active role in trapping and neutralizing pathogenic organisms.(16) Dendritic melanocytes are scattered in the basal epithelium of the limbal and bulbar conjunctiva. The substantia propria beneath the stroma is composed of loose fibrovascular connective tissue and is thinnest in the palpebral conjunctiva , over the tarsus , and in the perilimbal region .

In addition, the substantia propria contains vessels , lymphatics , peripheral nerves , and a meshwork of collagen and elastic fibers. The inherent population of plasma cells , in the substantia propria , produces immunoglobulins that can initiate the inflammatory cascade. Other immunocompetent cells in the substantia propria include mature lymphocytes and mast cells.(16)

1.2.1.3.2. Cytology of conjunctiva:

The normal cell population in scrape smears consists of squamous cells of corneal origin and cuboidal to columnar epithelial cells of conjunctival origin. Goblet cells may be present. Squamous cell nuclei may display a central chromatin bar as that observed in the oral cavity.(24) Also observed nuclear protrusions in the form of nipples in conjunctival cells, a common finding in endocervical cells. These nuclear variants may occur in health and disease and have no diagnostic significance.(24) 1.2.1.4 The lacrimal apparatus:

The lacrimal apparatus includes the main lacrimal gland, the accessory lacrimal glands, meibomian and Zeis glands, and the conjunctival goblet cells. The main lacrimal gland is located on the temporal side of the orbital plate of the frontal bone, just posterior to the superior orbital margin. The lacrimal gland is divided into two portions, palpebral and orbital. The lacrimal gland consists of lobules made up of numerous acini. Each acinus is an irregular arrangement of secretory cells around a central lumen surrounded by an incomplete layer of myoepithelial cells. Histologically, the main lacrimal gland is identical to the accessory lacrimal glands.) A network of ducts connects the acini and drains into one of the main excretory ducts. There are approximately 12 of these ducts, which empty into the conjunctival sac in the superior fornix. The accessory glands are located in the Subconjunctival tissue from the fornix area to near the tarsal plate. Basic secretion maintains the normal volume of the aqueous portion of the tears, and reflex secretion increases the volume in response to a stimulus.(19)

1.2.2. Tear Film:

The tear film, which covers the anterior surface of the globe, has several functions: (1) keeps the surface moist and serves as a lubricant between the globe and eyelids;(2) traps debris and helps remove sloughed epithelial cells and debris; (3) is the primary source of atmospheric oxygen for the cornea; (4) provides a smooth refractive surface necessary for optimum optical function; (5)contains antibacterial substances (lysozyme, beta- lysin, lactoferrin, IgA) to help protect against infection; and (6) helps to maintain corneal hydration by changes in tonicity that occur with evaporation. The tear film is composed of three layers. The outermost is a lipid layer containing waxy esters, cholesterol, and free fatty acids, primarily produced by the meibomian glands with a small contribution from the Zeis glands. The lipid layer retards evaporation and provides lubrication for smooth eyelid movement. The middle or aqueous layer contains inorganic salts, glucose, urea, enzymes, proteins, glycoproteins, and most of the antibacterial substances. It is secreted by the main and accessory lacrimal glands. The innermost or mucous layer is absorbed by the glycocalyx of the ocular surface cells and acts as an interface that facilitates adhesion of the aqueous layer of the tears to the ocular surface. It is produced and secreted by the conjunctival goblet cells.(19)

Tear film PH: The PH of tear is nearly 7.4 , variations have been found (5.2-8.3) .Corneal injuries tend to produce an alkaline PH. Inflammatory condition of conjunctiva and cornea tend to shift to a more acidic PH. Age ,sex ,time of examination and presence of pterygium have a little effect.(22)

1.2.3 The main structures of the eye:

1.2.3.1.The eyeball:

The eyeball is a cystic structure kept distended by the pressure inside it. It is an ablate spheroid, consisting of two spheres fused together. Cornea is a part of the anterior smaller sphere with a radius of 7.8mm and sclera is part of the posterior larger sphere with a radius of about 12mm.

The eyeball comprises of three coats: outer (fibrous coat), middle (vascular coat), and inner (nervous coat). Fibrous coat: anterior one sixth of this coat is transparent and is called cornea. Posterior five sixth opaque part is called sclera.

a. Vascular coat: it supplies nutrition to the various structures of the eyeball. b. Nervous coat ( retina): it is concerned with visual functions.(22)

1.2.3.2. The cornea:

The cornea is part of the outer coat and it is continuous with the conjunctiva and ultimately the skin. It is an avascular structure bathed in fluid- tears anterior and aqueous posteriory. Traditionally the cornea is described as a five-layered structure, epithelium (outermost layer), anterior limiting lamina (Bowman’s layer), stroma, posterior limiting lamina (Descemet’s membrane), endothelium (innermost layer)

Histologically, the cornea consists of five distinct layers as mentioned above.

a. Epithelium: Corneal epithelium is of stratified squamous type. It is about 50-90µm thick and consists of 5-6 layers of cells.

The deepest (basal) layer is made up of columnar cells, next 2-3 layers of wing or umbrella.

b. cells and the most superficial two layers are of flattened cells.

The wing cells form 2-3 layers of the polyhedral shaped cells. Their nuclei are flattened parallel to the surface . The flattened cells constitute two most superficial cell layers, the anterior cell wall of the most superficial cells has many microvilli .

b. Bowman’s membrane (layer): this layer consists of a cellular mass of condensed collagen fibrils. Once destroyed, it does not regenerate as epithelium. c. Stroma (substantia propria): It consists of collagen fibrils lamella, and cells embedded in hydrated matrix of proteoglycan. The lamellae are arranged in many layers( 200-250). d. Descemet’s membrane: It is homogenous layer which binds the stroma posteriory. It is made up of collagen and glycoprotein with no elastic fibres . e. Endothelium: It consist of a single layer of flat polygonal( mainly hexagonal) cells. With increasing age the number of cells is reduced .

The two primary physiologic functions of the cornea are: To act as a powerful refracting lens of fixed focus for proper image formation and protect the intraocular contents.(22)

1.2.3.3. The sclera : Anatomically The sclera forms the posterior opaque five-sixths part of the external fibrous tunic of the eyeball. Its whole outer surface is covered by Tenon’s capsule and also by the bulbar conjunctiva in the anterior part. The sclera maintains the shape of the globe, offering resistance to internal and external forces, and provides an attachment for the extraocular muscle insertions. The thickness of the sclera varies from 1 mm at the posterior pole to 0.3 mm just behind the rectus muscle insertions.

Histologic features: a. Episclera : is a loose, vascularized, connective tissue layer that lies just outer to the sclera. b. Sclera: The sclera is a thick, dense connective tissue layer that is continuous with the corneal stroma at the limbus. The diameter of the collagen fibrils in this tissue varies from 25 to 230 nm. These fibrils are arranged in irregular bundles that branch and interlace. Bundle widths and thicknesses vary, with the external bundles narrower and thinner than the deeper bundles. The orientation of the bundles is very irregular compared with bundles in the cornea. c. Scleral spur: The scleral spur is a region of circularly oriented collagen bundles that extends from the inner aspect of the sclera. In its entirety the scleral spur is actually a ring, although on cross section it appears wedge shaped, resembling a spur d. Scleral formina and canals: The sclera contains a number of foramina and canals. The anterior scleral foramen is the area occupied by the cornea. The optic nerve passes through the posterior scleral foramen.(22)

1.2.3.4. The Uvea :

The middle layer of the eye, the uvea (uveal tract), is composed of three regions: from front to back, the iris, ciliary body, and choroid. The uvea sometimes is called the vascular layer because its largest, structure, the choroid is composed mainly of blood vessels, which supply the outer retinal layers.(22)

1.2.3.4.1. The iris:

Iris is a thin, circular structure located anterior to the lens. The center aperture, the pupil, actually is located slightly nasal and inferior to the iris center. Pupil size regulates retinal illumination.

The iris can be divided into four layers: (1) the anterior border layer, (2) stroma and sphincter muscle, (3) anterior epithelium and dilator muscle, and (4) posterior epithelium.

Anterior border layer is a thin condensation of the stroma It is composed of fibroblasts, pigmented melanocytes, and collagen fibrils. The sphincter muscle lies within the stroma and is composed of smooth- muscle cells joined by tight junctions. Anterior epithelium composed of unique myoepithelial cell The apical portion is pigmented cuboidal epithelium joined by tight junctions and desmosomes, whereas the basal portion is composed of elongated, contractile, smooth muscle processes. Posterior epithelium it is the anterior continuation of the non-pigmented epithelium of the ciliary body which in turn is the continuation of the sensory retina.(22)

1.2.3.4.2. The Ciliary body:

It is triangular in shape, the anterior side of triangle forms the part of the angle of anterior and posterior chamber. Ciliary body consists of five layers, supraciliary lamina, stroma ,layer of pigmented epithelium, layer of non-pigmented epithelium, and internal limiting membrane. Ciliary muscle, it is a non–striated muscle which occupies most of the outer part of the ciliary body. The main action of it is to slacken the suspensory ligaments of the lens.(22)

1.2.3.4.3. The choroid:

Choroid is the posterior most part of the vascular coat of the eyeball. Its inner surface is smooth, brown and lies in contact with pigment epithelium of the retina. The outer surface is rough and lies in contact with the sclera .

The choroid consists of four layers, suprachoroidal lamina which is a thin membrane of condensed collagen fibres, melanocytes and fibroblasts. Stroma of the choroid, consists of loose collagenous tissue with some elastic and reticular fibres. Choriocapillaris, it consists of a rich capillary network which receives most of its blood from the medium and large vessels of the stroma. Basal lamina, is the innermost layer of choroid, it is a multilayered structure.(22)

1.2.3.5. The lens:

The lens is a transparent, biconvex structure suspended from the ciliary body by the zonular fibres and situated between the iris and the vitreous. The lens is about 9mm in diameter and about 4mm thick at the center. The anterior surface of the lens is less convex than posterior surface. The most anterior part of the lens is called the anterior polar, the periphery is called the equator and the most posterior axial area is the posterior pole. The lens consists of capsule, epithelium and lens fibres.(22)

1.2.3.6. The retina:

Retina is a thin , delicate and transparent membrane .It is the most highly developed tissue of the eye . It appears purplish-red due to visual purple of the rods . After death of a person , the retina appears white opaque.

Retina consists of three types of cells and their synapses arranged in the following layers: Retinal pigment epithelium, layer of rods and cones, external limiting membrane ,outer nuclear layer, outer molecular layer(plexiform),inner nuclear layer ,inner molecular layer(plexiform), ganglion cell layer, nerve fiber layer and internal limiting membrane.(22)

1.2.4 The abnormalities of the eye:

1.2.4.1.Neoplasms:

Metastatic lesions are the most common neoplasms that affect the eye. Of the neoplasms that arise within the eye, the majority arises from immature retinal neurons (retinoblastoma) and uveal melanocytes(melanoma).(24) a. Malignant melanoma : Malignant melanoma represents the most common primary intraocular malignancy and originates from melanocytes or nevi in the uvea. These lesions often appear circumscribed and invade Bruch’s membrane, forming a mushroom-shaped mass. Microscopically, these melanomas may contain spindle-shaped cells without nucleoli (spindle A cells), spindle-shaped cells with nucleoli (spindle B cells) or polygonal cells with distinct cell borders and prominent nucleoli (epithelioid cells). Variable amounts of necrosis are present. Melanomas that arise in the ciliary body and iris may extend circumferentially around the globe and are termed ring melanomas. Because the eye does not contain lymphatics, spread of melanoma occurs via extension through the sclera and hematogenous spread. Treatment often involves enucleation of the eye, but radiotherapy or local excision may be of use. Over half of all patients with melanoma may survive for 15 years.(23) b. Retinoblastoma: Retinoblastoma is the most common intraocular malignant neoplasm of childhood and arises from immature neurons of the retina. Although the majority of retinoblastomas are unilateral and sporadic, approximately 6% of retinoblastomas are inherited and more commonly bilateral. Grossly, retinoblastomas are cream-colored tumors that contain scattered, chalky white, calcified flecks within yellow necrotic zones. Microscopically, these lesions are cellular and a variety of patterns may be present, including sheets of round cells with hyperchromatic nuclei and scant cytoplasm, or cells arranged around a central cavity (Flexner-Wintersteiner rosettes). Necrosis and calcification may be seen. Spread of retinoblastomas occurs by way of direct extension along the optic nerve to the brain or by hematogenous spread, often to the bone marrow. Patients present with a white pupil (leukocoria), squint, poor vision, spontaneous hyphema, or a red, painful eye. Secondary glaucoma may occur. Some retinoblastomas grow toward the vitreous body (endophytic retinoblastoma), whereas others growth between the sensory retina and the retinal pigment epithelium, detaching the retina (exophytic retinoblastoma). Early diagnosis and treatment results in a high survival rate, although these patients are at risk for other malignancies, including osteogenic sarcoma,Ewing sarcoma, and pinealoblastoma.(23) Key features of intraocular neoplasias

• Melanoma: spindle, epithelioid with enlarged nuclei and prominent nucleoli with accompanying pigment filled macrophages

• Retinoblastoma: small cell tumor with areas of necrosis and calcification with rare rosettes

• Lymphoma: atypical lymphoid cells with prominent nucleoli.(13)

1.2.4.2.Physical and chemical eye injuries:

A variety of manifestations of ocular injury may occur:

1. Black eye: result of ecchymosis of the vascular eyelids caused by physical trauma

2. Superficial disruptions of the corneal epithelium: result of traumatic abrasion, contact lens wear, foreign bodies, ultraviolet light, caustic chemicals.

3. Blowout fracture: fracture of the bones of the orbital floor into the maxillary sinus caused by blunt trauma; inferior rectus muscle may be trapped in the fracture leading in the fracture leading the eye into the orbit (enophthalmos).

4.Injury caused by foreign bodies: can result in acute inflammation, granulomatous inflammation, retinal degeneration, cataracts, retinal detachment, glaucoma.(21)

1.2.4.3. Eye ball (Orbit) abnormalities:

An abnormal forward protrusion of the eyeball is termed exophthalmos when bilateral and proptosis when unilateral. A variety of causes, including thyroid disease, orbital dermoid cysts, hemangiomas, inflammation, lymphomas, and neoplasms, result in forward protrusion of the eyeball. The most common cause of exophthalmos is Graves disease, a form of hyperthyroidism that most commonly affects middle-aged women. Exophthalmos may precede or follow other manifestations of Graves disease and may be accompanied by retraction of the upper eyelid (due to increased sympathetic tone) and a characteristic stare appearance. Ocular complications of this disease include corneal exposure with ulceration and optic nerve compression, both of which may lead to eventual blindness if uncorrected.(23)

1.2.4.4. Eyelids abnormalities: Various conditions that affect the eyelids include:

-Blepharitis: inflammation of the eyelids that produces an acute, red, tender mass.(23)

- Hordeolum (sty): acute, inflammatory, focal lesion of the eyelids that may involve the meibomian or sebaceous glands.(23)

- Chalazion: granulomatous inflammation centered around the meibomian or sebaceous glands; probable reaction to extruded lipid secretions; results in a painless swelling of the eyelid.(23)

- Xanthelasma: yellow plaque of lipid-containing macrophages often on the nasal aspect of the eyelid; affects older persons and persons with elevated lipids.(23)

1.2.4.5. Conjunctiva abnormalities: a. Sub conjunctival hemorrhage: Conjunctiva may undergo following blunt trauma, anoxia, severe bouts of coughing, or spontaneously on first arising after sleep. (21)

b. Conjunctivitis:

is any inflammation of the conjunctiva and may be caused by a variety of factors. Among the common causative agents are bacterial or viral invasion and allergic. Acute bacterial conjunctivitis is most frequently caused by S. aureus, Staphylococcus epidermidis,H. influenzae , Streptococcus pneumoniae, Streptococcus viridans, Moraxella catarrhalis and Gram-negative intestinal bacteria(16). Viral conjunctivitis is most frequently caused by an adenoviruses.

Acute hemorrhagic conjunctivitis caused by enterovirus , Herpes simplex virus can cause follicular conjunctivitis . An HIV conjunctivitis is also recognized.(21)

c. Pterygium: often associated with pinguecula; represents a fold of vascularized conjunctiva that grows horizontally onto the cornea in the shape of an insect wing. (21)

d. Trachoma is the leading cause of preventable irreversible blindness in the world. It is related to poverty, overcrowding, and poor hygiene, the morbidity being consequence of the establishment of re-infection cycles within communities. Whereas an isolated episode of trachomatous conjunctivitis may be relatively innocuous, recurrent infection elicits a chronic immune response consisting of a cell-mediated delayed hypersensitivity (type IV) reaction to the intermittent presence of chlamydial antigen and can lead to loss of sight. The family childcare group is the most important re-infection reservoir, and consequently young children are particularly vulnerable. The fly is an important vector, but there may be direct transmission from eye or nasal discharge. Trachoma is associated principally with infection by serovars A, B, Ba, and C of Chlamydia trachomatis, but the serovars D-K conventionally associated with adult inclusion conjunctivitis, and other species of the Chlamydiaceae family such as Chlamydophila psittaci and Chlamydophila pneumoniae have also been implicated. (23)

e. Concretions :

Concretions are extremely common and are usually associated with aging, although they can also form in patients with chronic conjunctival inflammation such as trachoma. And signs appear as multiple tiny cysts containing yellowish-white deposits of epithelial debris including keratin, usually located subepithelially in the inferior tarsal and forniceal conjunctiva, can become calcified and ,particularly if large ,may erode the overlying epithelium and cause irritation.(21)

f. Dry eye syndrome: occurs when there is inadequate tear volume or function resulting in an unstable tear film and ocular surface disease. Inflammation in the conjunctiva and accessory glands is present in 80% of patients with KCS and may be the cause and consequence of, amplifying and perpetuating disease. The presence of inflammation is the rationale for steroid therapy. Hyper osmolarity of tears is also a key mechanism of disease and may be the major pathway for epithelial cell damage.(21)

g. Vitamin A deficiency : Vitamin A deficiency has been associated with a loss of goblet cells and keratinization of the epithelium. In dry-eye disorders showing a decrease in the number of goblet cells, treatment with vitamin A therapy can induce the reappearance of goblet cells.(19)

1.2.4.6.Inner structures abnormalities:

a. Glaucoma:

Glaucoma affects up to 2% of those over the age of 40 years globally, and up to 10% over the age of 80; 50% may be undiagnosed . Glaucoma is not a single disease process but a group of disorders characterized by a progressive optic neuropathy resulting in a characteristic appearance of the optic disc and a specific pattern of irreversible visual field defects that are associated frequently but not invariably with raised intraocular pressure (IOP).(23) b. Cataracts

Cataracts are one of the most common diseases of the lens. Cataracts are opacifications of the crystalline lens that may occur secondary corticosteroids, ultraviolet light, and trauma, among other causes. The most common cataract occurs with aging and begins when clefts accumulate between the lens fibers. Over time, degenerated lens material accumulates in these spaces and absorbs water, resulting in a swollen lens that may obstruct the pupil and cause glaucoma. In a mature cataract, the lens may degenerate and decrease in volume. Cataracts may be surgically removed.(23)

c. Myopia:

Myopia is a refractive ocular abnormality in which light from the visualized object focuses at a point in front of the retina because of a longer than usual anteroposterior diameter of the eye. Myopia usually begins in young persons and varies in severity. Treatment involves corrective lenses or refractive surgery (LASIK).(23)

1.3.literature review

Koss et al, (1992) concluded that the external surface of the eye and the eyelids are accessible to simple Cytologic procedures, within the reach of any ophthalmologist and any laboratory of cytology. Scraping the surface of the lesion under local anesthesia with an appropriate small instrument and preparation of alcohol fixed or air-dried smears is sufficient for the diagnosis of inflammatory disorders and some malignant lesions, such as carcinoma of the cornea and conjunctiva, or carcinomas of the eyelid . Also the use of a modified small endocervical brush with short, soft bristles (S-Brush) for securing material from the conjunctiva may increase the yield of sampling . Another technique prefer use of a cytocentrifuge instead of smears and, more recently ThinPrep for processing of the samples . Another brush with a spherical tip was used for the same purpose . An ingenious technique named “impression cytology” for the study of conjunctival samples was described. Following local anesthesia, a cellulose acetate strip is placed on the surface of the cornea using gentle pressure, carefully peeled off, fixed in alcohol, and stained with Papanicolaou stain.(24)

Kobayashi TK, et al (1997) , prepared conjunctival brush cytology using the ThinPrep method was applied in ocular surface disorders especially for dry eye status. To assess its diagnostic value in cellular samples, 17 patients with Keratoconjunctivitis sicca (KCS) and 10 normal volunteer patients were examined using this technique.

Conjunctival cells from normal controls revealed fine chromatin and polyhedral cytoplasm without having keratinized cytoplasm. On the other hand, the cellular samples from KCS revealed increased keratinized cells with pyknotic nuclei. They also contained extremely elongated cells. In KCS patients, the mean number of keratinized cells was significantly higher (34.1 cells/300 cells) than that of the normal control group (0.2 cells/300 cells). In patients with KCS, inflammatory cell counts were also higher than those of normal controls. Conjunctival cytology by means of the ThinPrep method obviously deserves additional trials as an adjunct in the cytology of dry eye states, especially in quantitative ocular evaluation for various ocular lesions.(9)

Satici, A et al(2003) concluded that chronic smoking has a negative effect on the ocular surface and affects some tear characteristics. The chronic ocular irritative effects of cigarette smoking may lead to defects in ocular surface defence.(26)

Somnath M .et al( 2013) assessed the health of ocular surface in a defined urban population, conjunctival goblet cell density and degree of surface squamous metaplasia were utilized as study tools .Two thousand of those aged between 20 and 79 years were initially selected. Normal healthy human volunteers without any history of ocular surface disorder were recruited. Impression cytology samples were obtained from interpalpebral part of bulbar conjunctiva from all the participants fixated and stained by a single observed . A variation in the number of goblet cells according to gender (women having less cells) and age (20– 30 years group having the highest number of cells) was found . Those working outdoors were found to have fewer goblet cells compared to those who stay indoors. The majority of the people had grade 1 cytological appearance in both males and females. People using coal and kerosene to cook were found to have a smaller goblet cell density than those who cooked on LPG or those who did not cook at all. Besides age and sex, environmental factors like the method of cooking and occupational variables (like outdoor activity, prolonged period of computer use, etc.) modify the health of the ocular surface.(2)

Thiel M et al ,(1997) concluded that for epidemiological and therapeutic reasons early diagnosis of superficial viral infections is crucial. Conventional microbiological techniques are expensive, time consuming, and not sufficiently sensitive. In their study impression cytology techniques were evaluated to analyze their diagnostic potential in viral infections of the ocular surface. A biopore membrane device instead of the original impression cytology technique was used to allow better quality and handling of the specimens. The impressions were processed, using monoclonal antibodies and immunoperoxidase or immunofluorescence techniques to assess the presence of herpes simplex virus, varicella zoster virus, or adenovirus antigens. Ocular surface specimens from healthy individuals(nine)and from patients with suspected viral surface disease(ten) were studied. Infected and non- infected cell cultures served as controls. Immunocytological staining of these samples provided diagnostic results for all three viruses in patients with viral surface disease. these membrane devices can provide virological results within 1-4 hours. This contributes considerably to the clinical management of patients with infectious diseases of the ocular surface.(10)

Koss et al, (1992) found that certain conjunctival involved infections exhibits a characteristic cytological changes such as Trachoma (Chlamydia) ,Adenovirus and Vaccinia virus.

Trachoma (Chlamydia): Cytoplasmic multiple small basophilic inclusions with halos , remarked appear in necrotic cells.

Adenovirus : Intranuclear multiple small eosinophilic becoming coalescent basophilic with halos in small cells and few cells involved.

Vaccinia virus: Cytoplasmic single large Eosinophilic inclusions.

Harpers simplex and Zoster viruses: Intranuclear often in multinucleated cells with nuclei molding to enlarge “ground-glass” nuclei forming Eosinophilic inclusions.

Measles: In multinucleated giant cells, appear as multiple eosinophilic cytoplasmic inclusions with sharp halos.(24)

Crick and Khaw(2003) mentioned that malaria has relatively few ocular complications and anti-malarial treatment will usually prevent their occurrence although increasing drug resistance is a problem.(27)

1.4. Justification

In Sudan according to my search and asking of eye health care works ,there is no eye cytology procedures were done as a tool of diagnosis . But the routine protocol of medical laboratory diagnostic procedures include : eye swab for microbiology culture ,tissue biopsy ,or may be the hole eye in certain cases (e.g. retinoblastoma).

The conjunctival cytological samplings is non invasive ,taking of specimens not required special experience, and the tools use for collection are cheap , easy to handle . The procedures doesn’t need hospital administration , and practicable for simple ocular clinics . This techniques can be involve in eye diagnostic protocol to see the hole image by additional cytomophological pattern of the particular eye conditions.

This study have priority to exhibit the conjunctiva different aspects and cellular morphology in normal conditions as those of pathological cases . It seek to overcome the specification done in similar studies that had studied certain ocular conditions and to describe the cytological appearance of conjunctiva involved in. It provides wide frame for naïve description of different conjunctiva element exhibit by samples involve in the study, allow to comparison , sharing pictures of cytological pattern to take place .

1.5. Study Objectives

1.5.1.General Objective:

- to screen conjunctiva cytological changes of ophthalmic clinic patients

1.5.2. Specific objectives:

1- To describe the cellular pattern in different eye condition.- 2- To find out the different factors that affect cellular components of the eye and subsequently the eye health status . 3- To evaluate the usefulness of the conjunctiva eye cytology as an important diagnostic procedures in the diagnosis of different eye conditions.

Chapter two Materials and methods

Chapter two 2. Materials and Methods 2.1. Study Design:

This was a descriptive cross sectional hospital base study , aimed to screen conjunctiva cytological changes of ophthalmic clinic patients using swabbing

and scrapping for different ocular diseases in period from January 2014 to May 2014.

2.2. Study Area:

The study was conducted at Abd Alfadeel centre for medicine and eye surgery,

Noor Alauoon military eye hospital, Alneelin university eye hospital (collage of optometry) and Maka charitiy foundation for eye medicine (Ridhy division).

2.3. Study Population:

The study population grouped into the following categories:

-Gender

-Level of education

-The diagnosis type

-The type of potential allergen

-Family history of diseases (ocular and non ocular diseases)

-Duration of exposure to direct sun rays.

-Duration of contact with visual media during day

-Washing occasions involve face (per day). 2.4. Sample size:

The available samples (patients) in period from January to May in 2014 conducted to clinic were ninety two(92) patients.

2.5. Study Duration:

The study was conducted during the period of January - May 2014.(five months).

2.6. Materials: see (Appendix II)

2.7.Methods: see (Appendix III)

2.7.1.Data Collection:

The data were taken from the patients by questionnaire asking them about their personal information(Age, occupation, residence area, original area and education level.),sources of potential allergens in their environments ,family history of diseases(ocular and non ocular diseases) and about their behaviors that may affect conjunctiva (duration of exposure to direct sun rays, hours of contact with visual media per day, their times of washing face per day, smoking and their using cosmetic creams or soaps on their faces).

2.7.2.Sample Collection:

Swabbing and scrapping materials was obtained by nyilon tipped and cotton wound swab from conjunctiva of patient ,fixed and stained and examined.

2.7.2.1.Conjunctiva Swabbing and scrapping Collection:

Conjunctival smears were obtained by anesthetizing the conjunctiva with topical 0.4% Benoxinate hydrochloride for about 5 minutes (this optional and not required when use cotton wound swab for collection), by using a sterile swab, the bottom eye lid conjunctiva was rubbed horizontally two times .

2.7.3. Sample preparation:

The swabbed and scrapped material smeared in spotted manner on clean slide and immediately fixed by 95% ethyl alcohol for 15 minute then air dried , prepared for Papanicolaou stain , slides for hematoxylin and eosin or Giemsa stain were fixed by air drying . All slides were put in slide storage box for transport to the laboratory . On arrival in the laboratory, the slide is placed in 95% ethanol prior to staining to rehydrate the sample cells.

Note : Trachoma smeared slides were fixed by 95% and air dry ones ,two slides each patient for Pap and Giemsa stains.

2.8. Staining Assessment:

The staining were assessment by an experience cytologist .

In Pap stained smears: Nuclei are stained blue while cytoplasm displays varying shades of pink , orange , gray and green. In hematoxylin stained smears nuclei are stained blue while cytoplasm stain red. In Giemsa stained smears nuclei are stained dark blue while cytoplasm stain gray.

2.9. Interpretation of results:

The adequacy of sample is reported , the type of cells , presence or absence of inflammatory process , other materials on smear like bacteria , crystals , mucous were reported

. 2.10. Statistical Analysis:

Data were obtained and processed using Statistical Package for Social Sciences (SPSS) version sixteen to calculate results.

2.11.Ethical Consideration:

The aims of the study were explained to health eye service providers and verbal consent were obtained , and discussed with department of histopathology and cytology. The objectives of the study were explained to participants, agreement of patients and ethical clearance were obtained.

Chapter three Results

Chapter three 3. Results 3.1. Introduction

The matching of conjunctiva cytological finding with the ophthalmology clinical diagnosis within adequate samples shows high specificity( 89.40%). (figure 1)

Ninety two (92) patients were screened using conjunctival cytology (swabbing and scrapping), 56(61%) were female and 36(39%) were male. (figure 2) Their ages were ranged from 1 to 80 years with the age mean 43 years. The study population were classified into age groups, large number of them were within the age group 41 to 60 years age (34%) and least number fill in group range 1to 20 years. (figure 3)

The most frequent groups according to occupation/ job were house wives 29(31.5%), then students17(18.5%), the workers10(10.9%) and the drivers 7(7.6%),(Table 1) show the population according to types of job activities.

They distributed according to resident area as 16(17.4%) from out of state and the rest of them distributed in the Khartoum state in the three cities (Khartoum 44(47.8%) ,Omdurman12(13.1%) and Bahri 20(21.7%))(figure 4) .

The most frequents patients from the middle states(31.5%) and the northern states(29.3%) in origin.(figure 5)

Most of patients had a primary level of education(40.2%). (figure 6)

The most eye involved was the right one (70.7%). (figure 7)

Infectious conjunctivitis(19.6%) and infection with Chlamydia trachomatis (trachoma)(13%) show marked frequency as a diagnosis.(figure 8), in infection cases the most common cause was bacteria (86.2%) include cases of Chlamydia trachomatis infection.(Table2 ) According to the study typing of diagnosis the category of (other) show highest frequency of occurrence ( 32.6%) , which include(refractive errors ,cataract, glaucoma and who diagnosed as normal ), and vitamin deficiency category had least percentage of cases(1.1%.) as type of diagnosis.(figure 9) and the infections Show the highest percentages among house wives(40%) and students(35%) ,while the category named(other) shows the highest percentages among drivers(40%) and workers(50%) groups.(figure10)

The most common cause of traumatic conjunctiva cytological smear among patients were surgery (80%), while (20%) from an accident.

According to washing occasion involve face per day population distributed as (64.1%) in range of -more than five times- involved (Wado), (30.4%) in range- 3 to 5- times occasion and least group fill in range of -less than three times- events.(figure 11). The association between frequency of infection and different ranges of number of washing times occasion involve face per day shows in (Table 3 ).

According to exposure to direct sunrays ( 39.1%) exposed to sunrays in a range more than 9hours per day , (33.7%) in a range of( 5 to 9)hours per day and( 27.2%) had less than 5hours per day.(figure 12)

Population distribution according to duration of contact with visual media shows in (figure 13)

(78.3%) of population answered (yes) and (21.7%) answered (no) about their using cosmetic creams /soaps on their faces. Smoking behavior distributed within population as( 12%) were smokers while (88%)were non smokers. (66.3% )of population answered (yes) about their wearing glasses and( 33.7%) answered (no).

According to general medical status of patients (80.4%) answered they were healthy. (52.2%) of population had no family history of eye diseases and (21.7%) had a family history of cataract. (figure14) Distribution of family history of non ocular diseases within population shows that (56.5%) had no family history of diseases while blood hypertension(15.2%)and diabetes(16.3%).

The types of potential allergen among population under study shows in.(figure15) The hot air and dust had highest percentages of distribution within different jobs environments with minimum variation , house wives (50.9%)hot air(23.5%)dust, students(52.3%)hot air(28.5%)dust, workers (43.75%)hot air (25%)dust and drivers ( 66.6%)hot air (33.3%)dust.

The house wives group had the highest number of the involved potentional allergens in their environments (five factors).

Cotton wound swab was used as a tool of collection of sample in( 51.1%) of population and show adequacy (19.5%),Nylon fiber tip flocked swab in (38%)(swabbing) had adequacy (50%) and in( 10.9% ) as scrapping tool of sampling and show the highest adequacy percentage (90%).

The frequency of getting adequate samples in different types of diagnosis shows the highest percentage( 48.70%) in infections diagnosed patients.

Also the elder age groups appear to get more adequate samples and exfoliated cells ,( 41-60)group had highest percentage (34.20%) and (61- 80)group had(31.5%) of adequate samples in cytology smears preparations.

3.2.The analysis

89.40%

90.00%

80.00%

70.00%

60.00%

50.00% Matching Non matching

40.00%

30.00%

20.00% 10.50%

10.00%

0.00%

Figure (1): matching of cytological findings with ophthalmic diagnosis

39%

Male 61% Female

Figure (2): distribution of gender

40%

34% 35%

30% 30%

25% 22%

20%

15% 14%

10%

0% ٦١ _ ٨٠ ٤١ _ ٦٠ ٢١ _ ٤٠ ١ _ ٢٠

Figure (3): distribution of age

Table (1) : The population according to types of job activities Types of jobs Frequency Percent

Office jobs 27 29.3% Out office jobs 22 24% Mix of in and out office activities 41 44.6% Others 2 2.1% Total 92 100%

47.8% 50.0%

45.0%

40.0%

35.0%

30.0%

25.0% 21.7%

17.4% 20.0%

13.1% 15.0%

10.0%

5.0%

0.0% Khartoum Omdurman Bahri Out of state

Figure (4): the residence distribution

35.0%

31.5% 29.3% 30.0%

25.0%

20.0%

15.2% 15.0% 13%

10.0% 7.6%

5.0% 3.3%

0.0% Estran Northan Kurdufan Darfor Khartoum Middle

Figure (5):distribution according to original area

45% 40.2% 40%

35%

30% 25% 23.90% 25%

20%

15% 10.90%

10%

0% Illiterate Primary Secondary Tertiary

Figure (6):distribution according to the education level

80.0% 70.7%

70.0%

60.0%

50.0%

40.0%

30.0%

14.1% 15.2% 20.0%

10.0%

0.0% RE LE Both eyes

Figure (7): the involved eye in disease

Vitamin Deficiency 1.1%

Truma 5.4%

Tracuma 13%

Spring catarh 1.1%

Refractive error 5.4%

Normal 4.3%

Muscle problem 1.1% 16.3% Inflammation

Infectin 19.6%

Glaucoma 5.4% Foregin body 3.3%

Facial problem 1.1%

Dry eye syndrome 2.2%

Concrision 1.1%

Chalizion 2.2%

Cataract 8.7%

Allergic conjunctivitis 8.7%

0.0% 5.0% 10.0% 15.0% 20.0% 25.0%

Figure (8): distribution of different eye conditions

Table (2): distribution of the infectious organisms

Infection cause frequency Percentages

Bacteria 25 86.20%

Bacteria+Virus 2 5.40%

Virus 1 4.20%

Parasite 0 0%

Fungal 1 4.20%

Total 29 100%

Type of eye diagnosis 35.0% 32.6%

30.0% 29.4%

26.1% 25.0%

20.0%

15.0%

10.9% 10.0%

5.0%

1.1% 0.0% Infection Inflamma Trauma Vitdefic Other

Figure (9): distribution according to types of eye diagnosis

50% 50%

45%

40% 40% 40%

35% 35% 33% 33% 30% 30% 30% 30% Infection Inflamation 25% Trauma Vitamin deficiency 20% Other 17% 17%

15% 10% 10% 10% 10% 10%

5% 5%

0% 0% 0% 0% 0% House wife Student Driver Worker

Figure (10): distribution of eye diagnosis according to job

70.0% 64.1%

60.0%

50.0%

40.0% 30.4%

30.0%

20.0%

5.4% 10.0%

0.0% Less than 3 mes 3 _ 5 mes More than 5 mes

Figure (11): occasions involve face washing per day

Table (3) : infection and washing occasions of face per day:

Washing occasion Infection Percent

Less than 3 times 3 10%

3 _ 5 times 13 45%

More than 5 times 13 45%

Total 29 100%

45.0%

39.1% 40.0%

35.0% 33.7%

30.0% 27.2%

25.0%

20.0%

15.0%

10.0%

5.0%

0.0% Less than 5 hours 5 _ 9 hours More than 9 hours

Figure (12): duration of exposure to direct sun rays per day

١٨%

Less than 3 hours ٥٩% Between 3-5 hours ٢٣% More than 5 hours

Figure(13): duration of contact with visual media per day

Vitamin Deficiency 1.1%

Trachoma 2.2%

Spring Catarrh 1.1%

Myopia 2.2%

Lacrimal Gland Disturbance 1.1%

Keratoconous 1.1%

Glaucoma 2.2%

Corneal Opsity 1.1%

Chalaizion 1.1%

Cataract/Glaucoma 3.3%

Cataract 21.7%

Allergic Conjunctivitis 9.8%

No family history of eye disease 52.2%

0.0% 10.0% 20.0% 30.0% 40.0% 50.0% 60.0%

Figure (14): family history of ocular diseases

None 15.4%

Hot air 34.1%

Tree 1.1%

Tree dust chemicals 1.1%

Cold air 5.5%

Dust 10.9%

Dust + others 10.9%

Dust chemicals +other 5.4%

Dust chemicals 1.1%

Chemicals + others 4.4%

Chemicals 4.3%

Animal Tree Dust Chemicals + others 1.1%

Animal 3.3%

Animal Tree Dust Chemicals 1.1%

Animal Dust 1.1%

0.0% 5.0% 10.0% 15.0% 20.0% 25.0% 30.0% 35.0% 40.0%

Figure (15): potential allergen types in the population environment

Chapter four Discussion , Conclusions and Recommendations

Chapter Four 4.1 Discussion

This was a descriptive study which aimed to screen the conjunctiva cytological changes of ophthalmic clinic patients using swabbing and scrapping cytology .

In this study the population have many factors suggested to make an effect on the conjunctival cytology smear picture ,the occupation or job determined their range of exposure hours to direct sun rays and involvement of particular potential allergen. In Somnath M .et al study they sub grouped the job into outerdoors and indoors , while in our study the population was grouped according to their occupation and the most frequent jobs were (house wives , students, workers and drivers. Each group studded for their common eye diagnosis, the particular potential allergens and counting numbers of the allergens in their environments, infection as a type of diagnosis shown the highest percentage among house wives and students groups , while in groups of workers and drivers the most frequent diagnosed in typing of (others) that included normal cases and intra ocular diseases such as glaucoma , cataract and refractive errors. Subsequently the particular potential allergens in each job group was determined by asking the participants and the results that no wide variation between groups as the category named( other )that included (hot weather and cold air of condition devices ) as source of allergic reaction, had the highest percentage of answers ,and( dust) get secondly. The house wives group shown the highest number of involved allergens factors and according to their data results we agree with them. Koss et al 1992 used the scrapping cytology for diagnosis of the malignant lesions on conjunctiva ocular surface, in our study this not expectable by ophthalmologists in the clinic because they are not familiar with the procedures.(24)

Kobayashi TK, et al (1997) studied the conjunctiva ocular cytological changes in dry eye status in patients with Keratoconjunctivitis sicca(KCS) , in our study the frequent of conduction of patient with this disease was 1.08% from total population, and the dry eye status appear as a secondary involvement in conjunctiva ocular disease so not include in typing of eye diagnosis done by the ophthalmologist.(9) Thiel M et al ,(1997) in their study impression cytology techniques were evaluated to analyze their diagnostic potential in viral infections of the ocular surface. A biopore membrane device instead of the original impression cytology technique was used to allow better quality and handling of the specimens. The impressions were processed, using monoclonal antibodies and immunoperoxidase or immunofluorescence techniques. Immunocytological staining of these samples provided diagnostic results for all three viruses in patients with viral surface disease.(10) However , and based on this study we found that conjunctival cytology as a diagnostic and screening tool for patient of different ocular diseases is simple , safe , cost-effective a virtually quick for the vast majority of infectious agents and non infectious ones such as Candida ,bacteria , degenerative changes due to eye drops prolonged uses as in glaucoma patients, crystals appear due precipitation of environmental matter, and complementary to the clinical ophthalmology diagnosis as the swabbing and scrapping collection method used in our study exhibited high percentage of matching of cytological finding with the clinical ocular diagnosis(89.40%), with scrapping method had impressive percentage of yield adequate samples (90%).

Charles Gerald Connor(2003) described that dry eye disease includes Keratoconjunctivitis (KCS), age-related dry eye, Stevens- Johnson syndrome, Sjogren's syndrome, occular cicatrical pemphigoid, blepharitis, Riley-Day syndrome, and congenital alacrima. Dry eye disease can also be caused by nutritional disorders or deficiencies (including vitamins), pharmacologic side effects, eye stress and glandular and tissue destruction, environmental exposure to smog, smoke, excessively dry air, airborne particulates, autoimmune and other immunodeficient disorders, and comatose patients who are unable to blink.(25) And also Satici, A et al(2003) concluded that chronic smoking has a negative effect on the ocular surface and affects some tear characteristics. The chronic ocular irritative effects of cigarette smoking may lead to defects in ocular surface defence.(26) In our study there was no facilitates of time and resources to detect the specific effects of factors include smoking , cosmetics creams or soaps used on face and the wearing glasses, on cytology of the conjunctiva ocular surface, although the information about events of these factors were collected from population under study by quesnnaire and plotted in frequency figures as results. In this study the relation between the general medical status of the patient , the presence of family history of ocular diseases or non ocular ones and conjunctiva cytology finding in certain eye diagnosis type is limited . As the majority of the population as filled in healthy , with no family history of eye disease or other diseases.

4.2. Conclusion and Recommendations

Application of conjunctival cytology in the diagnosis of ocular diseases , can help us to get information about effect of eye drops or systemic administrated drugs on conjunctiva cells ,observation of the type of tissue reaction against different environmental factors and to reveal evidence of infestation allow subsequently submitted for microbiological examination. So we can carry out it as complementary examination. conjunctival scraping is preferred to swabbing because it yields more epithelial cells and causes less contamination due to inflammatory debris from the ocular surface.

Also I recommend by :

- The involve of the malignant eye patients in the future studies. - The use of high applicable and more specific tools in collection of cytological samples . - Increase the knowledge and practice of the ophthalmologists and cytologists about this type of cytological procedures.

References

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(7) Bandyopadhyay R, Nag D, Mondal SK, Gangopadhyay S, Bagchi K, and Bhaduri G. (2010). Ocular surface disorder in pterygium: Role of conjunctival impression cytology. Indian J Pathol Microbiol; 53:692-5.

(8) Norn M. S., and Kristensen E. B. (1974). Benign mucous membrane pemphigoid II. Cytology. Acta Ophthalmologica; 52: 282– 290.

(9) Kobayashi TK, Ueda M, Nishino T, Ishida Y, Takamura E, and

Tsubota K. (1997). Cytologic evaluation of conjunctival epithelium using Cytobrush-S: value of slide preparation by ThinPrep technique. Cytopathology. Dec ;8(6):381-387.

(10) M Thiel, W Bossart, and W Bernauer. (1997). Improved impression cytology techniques for the immunopathological diagnosis of superficial viral infections. The British Journal of Ophthalmology.

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(11) R Singh, A Joseph, T Umapathy, N L Tint, and H S Dua .(2005). Impression cytology of the ocular surface. Br J Ophthalmol ;89:1655– 1659.

(12) Glenda R. Nolan, Lawrence W. Hirst, and B. Josephine Bancroft. (2001) The Cytomorphology of Ocular Surface Squamous Neoplasia by Using Impression Cytology Cancer (Cancer Cytopathol); 93:60–67.

(13) Anagnostopoulou-Fotinopoulou, I. and Rammou-Kinia, R. (1993), Cytobrush sampling in conjunctival cytology. Diagn. Cytopathol., 9: 113–115.

(14) C.Voneda, C.R.Dawson ,T.Daghfous, I.Hoshiwara , P.Jones ,M.Messadi and J.Schachter. (1975). Cytology as a guide to the presence of Chlamydia inclusion in Giemsa –stained conjunctival smears in severe endemic trachoma .Br J Ophthalmol;59:116-124.

(15) Marluce Bibbo, and David C Wilbur. (2008). Comprehensive cytopathology .3rd edition chapter17:section B, page454-470.

(16) Gadkari ,S., Adrianwala, S. D., Prayag, A. S., Khilnani, P., Mehta, N. J., and Shaha, N. A. (1992). Conjunctival impression cytology a study of normal conjunctiva. J Postgrad Med 22 April;38:3-21. (17) Qi-hua Le, Wen-tao Wang, Jia-xu Hong, Xing-huai Sun , Tian-yu Zheng , Wen-qing Zhu and Jian-jiang Xu. (2010). An In Vivo Confocal Microscopy and Impression Cytology Analysis of Goblet Cells in Patients with Chemical Burns. Invest. Ophthalmol. Vis. Sci. March vol. 51 no. 3 1397-1400.

(18) Høvding, G. (2008). Acute bacterial conjunctivitis. Acta Ophthalmologica, 86: 5–17.

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(24) Leopold G. Koss., and Myron R. Melamed . (2006). Koss’ diagnostic cytology and its Histologic bases . volume one .5th edition chapter 41 pages:3165-3187.

(25) Charles Gerald Connor. (2003). Method to use transdermal administration of androgens to the adnexa of the eye. Southern College

Of Optometry. Publication date :Dec 9 in same year.

(26) Satici, A., Bitiren, M., Ozardali, I., Vural, H., Kilic A., and Guzey, M. (2003). The effects of chronic smoking on the ocular surface and tear characteristics: a clinical, histological and biochemical study. Acta Ophthalmologica Scandinavica, 81: 583–587.

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Appendixes

Appendix I

Materials and instruments

- Absolute ethanol

- Coplin jars

- Samsung Glassy Note 2 phone

-Microsoft Office Picture Manger program

- Disposable gloves

-Sterile wound swabs

-Sterile swab applicator tip flocked with nylon fiber

-Filter papers

-Glass slides

-Cytology cover glass

-Microscope :Olympus

-Pasteur pipette

-Wooden stick

-Plastic slide storage box

-Distilled water

-DPX

-Xylene

-Stains solutions :Giemsa , Harri’s hematoxylin , Eosin ,OG6 and EA 50.

-Buffer

-Ammonia Appendix II Preparation of reagents

Ethanol 95%:

Ethyl alcohol ……………………95ml.

Distiller Water …………………..05ml.

70% alcohol:

Ethyl alcohol………………..……70ml.

Distilled Water……………..…….30ml.

90% alcohol:

Ethyl alcohol……………….……90ml.

Distilled Water…………….…….10ml.

Acid alcohol:

Conc. Hcl………………………….1ml.

70% alcohol……………………...90ml.

Ammonical water:

Ammonia……………….………0.1ml

Distilled water…………...... ….99.9ml.

Pap staining:

Nuclear stain Harri’s hematoxylin:

Hematoxylin …………….….…….5g.

Ethanol………..….………..…...50ml.

Potassium alum……..….………100g.

Distilled Water..…………….1000ml.

Mercuric oxide...………………….3g. Glacial Acetic Acid…………….40ml.

Cytoplasm staining:

Orange G6

Orange G (10%)……..…………50ml.

Alcohol………………...……….95ml.

Phosphotungestic acid ……..…….5g.

Eosin Azure 50

0.04M Light Green …………….10ml.

0.3M Eosin Y…...………..…….20ml.

Phosphotungestic acid……...…….2g.

Ethyl alcohol…...…….……....250ml.

Appendix III Methods of staining

Papanicolaou Staining procedures as follow:

1.Rehydrate the sample by put it in 95% ethyl alcohol 15 minutes

2.Transfer to 70% alcohol for 2 minutes

3.Wash in running tape water for 2 minutes

4.Stain in Harri’s hematoxylin for 10 minutes

5.Differentiate in 1% acid alcohol by just one dip

6.Blue for one minute in ammonical water

7.Bring smear to 70%,two changes of 95% rinse in each

8.Stain with O.G 6 for 2 minutes

9.Transfer to two changes of 95% three rinses in each

10.Stain with E.A.50 for 3 minutes

11.Trasfer to two changes of 95% alcohol three rinses in each

12.Dehydrate by air

13.Clear in Xylene and mounting in D.P.X.

Hematoxylin staining procedures :

1.Rehydrate the smear by put it in 95% alcohol for 15 minutes

2.Transfer to 70% alcohol 2 minutes

3.Wash in running tape water 2 minutes

4.Stain in Harri’s hematoxylin for 10 minutes

5.Differentate in 1% acid alcohol

6.Blue in ammonical water for 1 minute 7.Stain by eosin as counter stain 10 seconds

8.wash in water

9.Dehydrate by air

10.Cleain Xylene and mounting in D.P.

Giemsa staining procedures :

1.Trasfer to absolute methanol for 5 minutes

2.Apply cellulose filter paper on smear on pore stock Giemsa stain on it for 3mins

3.Rinse in sprite and let the paper drain out

4.Rinse in Sorensen phosphate buffer PH 6.8

5.Dry by air

6.Clear in Xylene and mounting in D.P.X.

Appendix IV Data collection tool

Personal information

Date: ………… Serial number……………..

Age: …… Gender: …………………..

Occupy: …………………… Original area:……………..

Residence:………………….

Level of education: Primary Secondary Tertiary

Ocular history

Eye condition(Diagnosis): …………………….………………………….

Family history of ocular disease:……………………………………………..

Is it one/two eye(s) is(are) involved:

Right Left

The diagnosis type :

(1)Infection (2)Inflammation

(3)Truma (4)Vitamin A deficiency

(5)Others

If infection :cause is /are

Virus Bacteria Parasite Fungus

If truma: is it result from:

Surgery Accidence Medicine abuse Others

Did he/she use any type of glasses: Yes No

General history

Medical condition:………………………………………………………

Family history of non ocular disease:………………………………….

The potential allergen type (in around environment)

Animal Trees/flowers Dust Chemicals

Others

Duration of exposure to direct sun rays (per day):

Less than5hrs 5-9hrs More than 9hrs

Duration of contact with visual media (per day):

Less than 3hrs 3-5hrs More than 5hrs

Washing occasions involve face (per day):

Less than 3times 3-5times More than 5times

Use cosmetic (creams/soaps)on face: Yes No

Smoking behavior : Yes No

Signature of the doctor…………..

Appendix V

Picture (1): inflammatory process include bacteria

Picture (2) :cluster of conjunctiva columnar cells

Picture (3): conjunctiva columnar cell in plasid arrangement