Training Report

Internship Report
Submitted to the Life Sine Lab
in partial fulfillment of requirements for the award of training

Internship
in
Clinical Pathology
by
Md. Sanaul Haque Shanto

DEPARTMENT OF PATHOLOGY
Life Sine Lab
Medical Road, Sipaipara, Rajpara 6000, Bangladesh
October 2022
 DECLARATION

I Md. Sanaul Haque Shanto hereby declare that the Training Report, submitted for
partial fulfillment of the requirements for the award of internship of Life Sine Lab,
Medical Road, Sipaipara, Rajpara is a bonafide work done by me under supervision of
Khandker Md. Faisal Alam , Md. Ismail Hossain, and Nadeem Hossain
This submission represents my ideas in my own words and where ideas or words
of others have been included, I have adequately and accurately cited and referenced
the original sources.
I also declare that I have adhered to ethics of academic honesty and integrity
and have not misrepresented or fabricated any data or idea or fact or source in my
submission. I understand that any violation of the above will be a cause for disciplinary
action by the institute and/or the University and can also evoke penal action from the
sources which have thus not been properly cited or from whom proper permission has
not been obtained. This report has not been previously formed the basis for the award
of any degree, diploma or similar title of any other University.

Rajshahi Md. Sanaul Haque Shanto

9-10-2022
Acknowledgement

I take this opportunity to express my deepest sense of gratitude and sincere thanks to
everyone who helped me to complete this work successfully. I express my sincere
thanks to Md. Ismail Hossain, Head of Clinical Technologist, Clinical Pathology,
Life Sine Laboratory Rajshahi for providing me with all the necessary facilities and
support.
I would like to express my sincere gratitude to Nadeem Hossain, department of
Hormone Test, Life Sine Laboratory, for their support and co-operation.
I would like to place on record my sincere gratitude to my training guide Khandker
Md. Faisal Alam, Assistant Professor, Shaheed Ziaur Rahman Medical College
Bogura, Bangladesh, Life Sine Laboratory for the guidance and mentorship through-
out the internship.
Finally I thank my family, and friends who contributed to the succesful fulfilment
of this training work.

Md. Sanaul Haque Shanto

i
ABBREVIATION

Table 1: ABBREVIATION

Sl. No Word Full Meaning

1 ESR Erythrocyte sedimentation rate
2 Hb Hemoglobin
3 WBC White blood cell
4 RBC Red blood cells
5 Het Hematocrit
6 BUN Blood urea nitrogen
7 HIV Human immune deficiency virus
8 RFT Renal function test
9 LFT Liver function test
10 KFT Kidney function test
11 ASO Anti Streptolysin-O
12 VDRL Venereal disease research laboratory
13 WIDAL Widely investigated disease assay laboratory
14 SGOT Serum Glutamate Oxaloacetate Transaminase
15 SGPT Serum glutamate Pyruvate Transaminase
16 ALP Alkaline Phosphatase
17 G6PD Glucose 6 peroxidase
18 CBC Complete blood counts
19 mm Millimeter
20 IU International Unit
21 L Low value
22 H High value

ii
Summary Of Training Report

This report describes a brief description of the work that has been carried out by me
in the laboratory during training at Life Sine Lab. I have been working in laboratory
during my training period from 1 August 2022 to 30 of September 2022. There were
7 department where I have worked and these department where Clinical pathology,
CLIA, Biochemistry, Hematology. Serology. Microbiology, and Histopathology.
In Clinical Pathology I have learn how to operate (Micro Lab RX-50V) a semi-
automated urine chemistry analyzer instrument which gives numbers of the tests
Glucose, Bilirubin Ketone, Protein, Urobilinogen, Nitrite, Leukocytes, colure of the
urine and pH etc. I have also examined stool slides and semen where I observed some
human parasites and abnormal immotile sperms.
In CLIA (CHEMILUMINESENCE) in this section I have gained the knowledge
of how to operate the instrument (Thermo MULTISKAN EX Cubic Spline V2.2)
and conduct hormones test like T3, T4, and TSH. The machine is fully automated only
insert the required amount of serum samples, reagents, and after 3 times of water-bath
I can get the result of the test.
In Biochemistry in section was examine with plasma or serum and specific
reagents at slide in Lab Rotator LRA700 which give the result of Glucose, Uric acid,
Cholesterol and Triglyceride.
In Hematology I have learned how to operate Mythic 22 machine (CBC LH750,
ESR analyzer, Coagulation profile test). The test which conduct in this machines are
RBC, WBC, Platelets and clotting factors.
In Serology there was most of work done by manually but most of I have used
readymade kit which provided by Manufacturer Company. These test were WIDAL,
ASO, VDRL and free testosterone.
In Microbiology I have learned about staining, culture of blood, body fluids and
there was (Olympus CX23 Microscope + Spectrometer + 5 million Pixels+0.5X)

iii
which was microscope and measure the sensitivity of antibiotics and presence of
different bacteria. I have mentioned in this report.
This is the last section where I have worked it was Histopathology where I did
staining and section cutting of various tissues. In this section I examined the tissue and
body fluid for the presence of cancer in the body.

iv
Contents

Acknowledgement i

ABBREVIATION ii

Summary Of Training Report iii

List of Figures viii

List of Tables ix

1 Clinical Pathology 1
1.1 Urine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1.1 Physical Examination Of Urine Determination . . . . . . . . 2
1.1.2 Normal ranges of physical examination . . . . . . . . . . . . 3
1.1.3 CHEMICAL EXAMINATION: . . . . . . . . . . . . . . . . 3
1.1.4 Albumin Protein: . . . . . . . . . . . . . . . . . . . . . . . . 4
1.1.5 Microscopic examination of urine . . . . . . . . . . . . . . . 4
1.2 CHEMICAL EXAMINATION OF STOOL . . . . . . . . . . . . . . 5
1.2.1 BENZIDINE TEST . . . . . . . . . . . . . . . . . . . . . . . 5
1.2.2 MICROSCOPIC EXAMINATION OF STOOL . . . . . . . . 6
1.3 ROUTINE SEMEN EXAMINATION . . . . . . . . . . . . . . . . . 8
1.3.1 PHYSICAL EXAMINATION OF SEMEN: . . . . . . . . . . 8

2 CHEMILUMINESENCE (CLIA) 11
2.1 NAME OF THE TEST: . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.2 T 3 (TRIIODOTHYRONINE) AND T 4 (THYROXINE): . . . . . . . . 12
2.3 TSH (THYROID STIMULATING HORMONE): . . . . . . . . . . . 13

v
3 BIOCHEMISTRY 15
3.0.1 NAME OF INSTRUMENT: . . . . . . . . . . . . . . . . . . 16
3.0.2 Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
3.0.3 Renal function test (RFT) . . . . . . . . . . . . . . . . . . . 16
3.0.4 Liver Function Test (LFT) . . . . . . . . . . . . . . . . . . . 16
3.1 Lipid Profile. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
3.1.1 RFT (RENAL FUNCTION TEST) . . . . . . . . . . . . . . . 18
3.1.2 LIVER FUNCTION TEST (LFT) . . . . . . . . . . . . . . . 20
3.1.3 SGPT (Serum glutamate Pyruvate Transaminase) . . . . . . . 21
3.1.4 SGOT (Serum Glutamate Oxaloacetate Transaminase) . . . . 22
3.2 LIPID PROFILE . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.2.1 CHOLESTEROL . . . . . . . . . . . . . . . . . . . . . . . . 23

4 HEMATOLOGY 26
4.0.1 HEAMATOLOGY . . . . . . . . . . . . . . . . . . . . . . . 26
4.1 Complete blood Count (CBC) . . . . . . . . . . . . . . . . . . . . . 27
4.2 Erythrocyte sedimentation rate . . . . . . . . . . . . . . . . . . . . . 28
4.3 ABO BLOOD GROUPING BY (Slide Method) . . . . . . . . . . . . 30
4.4 GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G6PD): . . . . . 31
4.5 COAGULATION TIME (CT) . . . . . . . . . . . . . . . . . . . . . 34
4.5.1 Procedure:- . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

5 SEROLOGY 36
5.1 WIDAL TEST: - . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
5.2 VDRL (Venereal disease research laboratory) . . . . . . . . . . . . . 38
5.3 ASO (Anti Streptolysin O) . . . . . . . . . . . . . . . . . . . . . . . 38
5.4 HIV Tri- dot test . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

6 MICROBIOLOGY 42
6.1 Microscopy: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
6.1.1 BACTEC . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

7 HISTOPATHOLOGY 46
7.1 Histopathological Instrument and eqquipments:- . . . . . . . . . . . . 47
7.1.1 Steps for the Tissue processing . . . . . . . . . . . . . . . . . 47

vi
 7.1.2 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
7.1.3 Staining of the slides:- . . . . . . . . . . . . . . . . . . . . . 48
7.1.4 Case Study . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

References 50

vii
List of Figures

3.1 Biochemistry Analyzer . . . . . . . . . . . . . . . . . . . . . . . . . 16

4.3 Blood grouping slide . . . . . . . . . . . . . . . . . . . . . . . . . . 31

5.2 ASO Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

7.1 Microscopic examination of tissue . . . . . . . . . . . . . . . . . . . 46

viii
List of Tables

1 ABBREVIATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii

1.1 Determination & Normal Finding of Urine . . . . . . . . . . . . . . . 2

1.2 Abnormal & Pathologic of Urine . . . . . . . . . . . . . . . . . . . . 2
1.3 Normal ranges of physical examination . . . . . . . . . . . . . . . . 3
1.4 Patient Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.5 Patient semen report . . . . . . . . . . . . . . . . . . . . . . . . . . 10

2.1 Unit and Normal ranges . . . . . . . . . . . . . . . . . . . . . . . . . 13

2.2 Patient report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

3.1 Normal ranges of glucose . . . . . . . . . . . . . . . . . . . . . . . . 17

3.2 Various diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

4.1 Patient Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

4.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
4.3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
4.4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
4.5 Blood grouping reaction . . . . . . . . . . . . . . . . . . . . . . . . 32
4.6 Patient Result . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

5.1 Patients positive cases. . . . . . . . . . . . . . . . . . . . . . . . . . 40

6.1 Result of various stain . . . . . . . . . . . . . . . . . . . . . . . . . . 45

7.1 Specimen of Uterus. . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

7.2 Specimen of Uterus . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

ix
Chapter 1

Clinical Pathology

Introduction:
It is a medical specialty that is concerned with the diagnosis of disease based on the
laboratory analysis of bodily fluids, such as urine, semen, and stool.

Types of sample:

1. Urine

2. Stool

3. Semen

Name of Instruments and

Equipment (a) Micro Lab RX-50V

1.1 Urine
Principles:
The reaction of Siemens Multistix 10 SG test strips depends on color development as
an indicator of the concentration of the following test reactions.

Procedure urine examination:

1. Physical Gross Examination.

1
 2. Chemical Examination.

3. Microscopic Examination.

1.1.1 Physical Examination Of Urine Determination

Determination Normal Finding

1. Volume of Urine 50 to 200 ml

2. Color of Urine Pale Yellow

White

3. Appearance of Urine Usually clear

4. Reaction Usually acidic PH 4.88 to 7.5

PH more than 7.5 Alkaline Urine
5. Odor of Urine Aromatic

6. Specific gravity of Urine Varies from 1.003 to 1.060

Table 1.1: Determination & Normal Finding of Urine

Abnormal Pathologic
>500 ml Diabetes insipidus, Polyuria
<20 ml Oliguria, Anuria
Dark yellow Hepatic and post hepatic condition
Redish Chyluria Hematuria
Redish Chyluria Hematuria
Black Urine Alkaptonuria
Dark yellow Biliverdin present
Turbid Presence abnormal Leukocytes.
Milky Chyle
PH less than 4.8 More, acidic Urine Fever, Ketosis.
Sever Vomiting.
Fruity Acidosis, Ketosis.
Ammonical Cystitis.
Foul smelling Urinary tract infection.
Low Sp. Gravity Chronic nephritis, diabetes insipidus.
High Sp. Gravity Diabetes insipidus fever, Acute nephritis.

Table 1.2: Abnormal & Pathologic of Urine

2
1.1.2 Normal ranges of physical examination

TEST ABBREVIATION UNITS Normal ranges

Glucose GLU mg/dL NEGATIVE
Bilirubin BIL NEGATIVE
Ketone KET mg/dL NEGATIVE
Specific Gravity SG 1.016-1.022
pH pH 5.0-8.0
Protein PRO mg/dL NEGATIVE
Urobilinogen URO E.U./dL 0.2-1.0
Nitrite NIT NEGATIVE
Blood BLO NEGATIVE
Leukocytes LEU NEGATIVE

Table 1.3: Normal ranges of physical examination

1.1.3 CHEMICAL EXAMINATION:

SUGAR (GLUCOSE) TEST (”BENEDICT’S QUALITATIVE TEST”)

Principles:
Urine glucose reduces cupric ions present in the reagent to cuprous ion, Alkaline
medium is provided to the reaction by sodium carbonate present in the reagent the
original color change blue to green, yellow, orange and red A/C to concentration
glucose.

Procedure glucose:

1. Take 5ml of Benedict’s reagent in the test tube.

2. Add 8 drops of urine.

3. Boil for 2 minute and allow cooling under tap water.

Observation & Result:

Blueclear − Negative
Green, noppt − T race
Greenwithppt − +
Brownwithcloudy − + +

3
Orangewithcloudy − + + +
Redwithcloudy − + + + +

Disease - Hyperglycemia, Renal glycosuria.

1.1.4 Albumin Protein:

Principle:
Sulphosalicyclic acid solution (3%) precipitates any protein in the urine specimen
irrespective of the type albumin or Bence jones. It is an anion precipitant that works
by the neutralization of the protein cation.

Pathogenic: Nephritic syndrome.

1.1.5 Microscopic examination of urine

In microscopic, I examined the various cells likes Pus cells, RBCs, Epithelial cells,
Triple phosphat Calcium oxalate, Cholesterol and Uric acid.

ROUTINE STOOL EXAMINATION

Collection of stool specimen:

Morning sample is collected in clean dry container.

LABORATORY INVESTIGATIONS

1) Gross and physical examination by visual observation:

• Consistency

• Color

• Mucus

4
 • Blood

• Parasites

2) Chemical Examination:

• Reaction/pH

• Occult blood

3) Microscopic examination:

• Pus cell (WBC).

• RBC.

• Macrophages.

• Starch undigested.

• Vegetable fibril.

• Entamoeba histolytica (EH).

• Giardia

• Trichonomas

• Larvae

• Ova

1.2 CHEMICAL EXAMINATION OF STOOL

1.2.1 BENZIDINE TEST

• Microscopic slides

• Applicator stick

• Glacial acetic acid

5
 • 30% H202 solution

• Benzedrine powder

Specimen:
Stool

PROCEDURE:

• Take pinch of Benzidine powder in a small test tube.

• Acidify it with 2 to 3drops of glacial acetic acid and mix well.

• Add about 1.0 ml of H-02 and mix well.

• Place a small quantity of stool specimen on a clean and dry slide.

• Place one or two drops of the Benzidine, glacial acetic acid, hydrogen peroxide
mixture on the stool specimen on the glass slide.

• Observe change in color.

RESULT:

• No change in color-occult blood absent.

• Color changes green to blue - occult blood present.

1.2.2 MICROSCOPIC EXAMINATION OF STOOL

Requirements:

• Glass slides

• Cover slips (22 mm)

• Normal saline

• Lugol’s iodine solution

• Penicillin bulb

PROCEDURE:

6
Saline preparation:

• Place a drop of normal saline on a glass slide

• Take a little fecal material by using a stick and mix with a drop of normal Saline.

• Place a cover slip.

Result:

1. Cells: Pus Cells, Epithelial cells, Erythrocytes

2. Parasites

3. Crystals

4. Vegetables matter

5. Undigested ingredients.

6. Other findings (Bacteria and yeast)

Date 08/08/2022 08/08/2022

Name xyz xyz
Sex Male Male
Age 28 40
Color Brown Brown
Consistency Semi Formed Semi Formed
Odor Foul Fecal Foul Fecal
Mucus Absent Absent
Blood Absent Absent
WBC’s Not Detected Not Detected
Crystals Not Detected Not Detected
Tesrophozoit Not Detected Not Detected
Cyst Not Detected Not Detected
Ova Not Detected Not Detected
Larva Not Detected Not Detected
Adult Not Detected Not Detected
Occult blood Not Detected Not Detected

Table 1.4: Patient Data

7
1.3 ROUTINE SEMEN EXAMINATION
Introduction: Semen is a gray opalescent fluid which forms at ejaculation.It consists
of a suspension of spermatozoa in seminal plasma. The percentage contribution of
each of the secretions that make up the seminal fluids. The various important purpose
of routine semen analysis is:

• Evaluation of infertility

• Routine follow up of patients who have under gone vasectomy.

• Artificial insemination.

1.3.1 PHYSICAL EXAMINATION OF SEMEN:

Color:
Volume:
Viscosity:
It is observed by taking the specimen in a Pasteur pipette and by allowing it to pour
drop by drop.
The specimen of normal viscosity can be poured drop by drop.

CHEMICAL EXAMINATION OF SEMEN

Determine pH by using pH paper strip and note down the observed pH.
PROCEDURE:
• Pipette 5 ml of resorcinol reagent in a test tube.

• Add 0.5 ml of semen specimen.

• Mix and place in a boiling water bath for 5 minutes.

OBSERVATION:

• No change in color, Fructose absent.

• Red colored precipitate forms within 30 seconds, Fructose present.

8
MICROSCOPIC EXAMINATION OF SEMEN PROCEDURE:

• The semen is dilute with diluting fluid (1:20 dilution) and mixes it well.

• Take a clean glass slide.

• Charge the solution in Neubauer chamber with cover slip.

• Examine the WBC: squares, and count the sperms.

OBSERVATION:

• Abnormally shaped head.

• Abnormally sized head (giant or minute).

• Double head.

• Vacuoles in the chromatin.

• Middle section: absent, bifurcated or swollen.

• Tail: may rudimentary, double or absent.

Normal observation: Color

• Spermatozoa head caps: Light blue.

• Nuclear posterior: Dark blue.

• Bodies and tails: Red or pink.

Size:

• Spermatozoa:50-70µ.

• Head:3-6µ× 2-3µ.

9
 Physical examination Microscopic examination
a) Volume: 2.0 ml Total sperm count: 10 million/ml Motility:
b) Color: greyish white Actively motile :30%
c) pH: alkaline Sluggish motile: 20%
d) viscosity: abnormal
e) Sample collection time: 12:30 pm Normal sperm: 50%
f) Liquefaction time: 4 hr Abnormal sperm: 50%

Table 1.5: Patient semen report

10
Chapter 2

CHEMILUMINESENCE (CLIA)

PRINICPLE:
The principle is based on sandwich method of antigen-antibody reaction. Acredium
Ester binds to the antibody in the presence of a specific antigen and forms a sandwich
Formation (Ag-Ab-Acredium ester), as a result of chemical reaction between Ag. Ab
& Acredium ester emission of light is occurred by Acredium ester, the amount of light
emitted is directly proportional to the antigen present in the sample.

INSTRUMENT NAME: Thermo MULTISKAN EX Cubic Spline V2.2

PARTS OF INSRUMENT:
• Improcess Queue

• Sample probe

• Ancillary probe

• Tip tray

• Cuvette bin

• Cuvette wheel

• Reagent rack

• Reagent probe

• Illuminometer

• Waste keeping container

(a) Thermo MULTISKAN EX Cubic
• Exit Queue Spline V2.2

11
2.1 NAME OF THE TEST:
• T 3 (TRI-IODOTHYRONINE)

• T 4 (THYROXINE)

• TSH (THYROID STIMULATING HORMONE)

2.2 T 3 (TRIIODOTHYRONINE) AND T 4 (THYROX-

INE):
Triiodiodothyronine (T 3 ) and Thyroxin (T 4 ). It is based on essential hormone produced
By the Thyroid gland. Triiodothyronine (T 3 ) is about four times more active in its
biological functions than thyroxin (T 4 ).

FUNCTION OF TRIIODOTHYRONINE (T 3 ) AND THYROXINE (T 4 ):

Thyroid hormones stimulate the metabolic activities.

• It is increases the oxygen consumption in most of the tissues of the body.

• Effect on protein synthesis: Thyroid hormones act like steroid hormones in

promoting protein synthesis.

• Influence on carbohydrate metabolism: Thyroid hormones promote intestinal

absorption of glucose and its utilization.

• Effect on lipid metabolism: Lipid turnover and utilization are stimulated by

thyroid hormone.

CLINICAL SIGNIFICANCE:

• Increase in the size of the thyroid gland is known as Goiter.

• Increase level of thyroid hormone is known as Hyperthyroidism.

• Decrease level of thyroid hormone is known as Hypothyroidism.

12
2.3 TSH (THYROID STIMULATING HORMONE):
TSH is a dimer (a ß) glycoprotein with a molecular weight of about 30,000. The
release Of TSH from anterior pituitary is controlled by feedback mechanism. The
hormones of The thyroid gland (T 3 , and T 4 ) and thyrotrophic releasing hormone (TRH)
of Hypothalamus.

FUNCTIONS:

• It is promotes the uptake of iodine (iodide pump) from the circulation by thyroid
gland.

• Enhance the conversion of iodide to active iodide, a process is known as

organification.

• Increase the proteolysis of thyroglobulin to release T 3 , and T 4 , into the circula-

tion.

CLINICAL SIGNIFICANCE:

• Increase level of TSH: Hypopituitarism.

• Decrease level of TSH: Hypopituitarism.

Case Study 01

Patient Name:
Age/Sex: 31/M
Sample Date: 02/09/2022
Reporting Date: 02/09/2022

TEST RESULT UNIT NORMAL RANGE

T 3 = 188.5(H) µg/dL T 3 =60 − 181η g/dL
T 4 = 3.5(L) µg/dL T 4 = 45-12.6µg/dL
TSH=8.25(H) µIU/L TSH=0.35-5.5 µIU/L

Table 2.1: Unit and Normal ranges

13
 Case Study 02

Patient Name:
Age/Sex: 35/F
Sample Date: 02/09/2022
Reporting Date: 02/09/2022

TEST RESULT UNIT NORMAL RANGE

T 3 = 188.5(H) η g/dL T 3 =60 − 181η g/dL
T 4 = 3.5(L) µg/dL T 4 = 45-12.6µg/dL
TSH=8.25(H) µIU/L TSH=0.35-5.5 µIU/L

Table 2.2: Patient report

14
Chapter 3

BIOCHEMISTRY

INTRODUCTION:
Clinical Biochemistry deals with the biochemistry laboratory applications. To find
out Cause of disease. The chemical constituent of various body fluid such as Blood,
Urine, CSF and other body fluid like are analyzed in clinical biochemistry laboratory.
The Biochemistry test are very useful to determine the severity of disease of many
organ. The Clinical biochemistry tests in relation to the various clinical conditions.

1. The cause of disease

2. Screen assay diagnosis.

3. Suggested effective treatment.

4. Monitoring process of a pathological condition

5. Help in assessing response to therapy

PRINCIPLE:

The Principle of this instrument is based on Lamberts and Beers law. The optical
density (O.D) is directly proportional to the concentration of solution and the thickness
of the Cuvette.

15
 Figure 3.1: Biochemistry Analyzer

3.0.1 NAME OF INSTRUMENT:

1. Plasma or Serum, Reagents, Slides, Incubator, and LRA700.

NAME OF THE TEST:

3.0.2 Glucose

• Fasting blood sugar

• Random blood sugar

• Postprandial blood sugar

3.0.3 Renal function test (RFT)

3.0.4 Liver Function Test (LFT)

• Bilirubin Direct and Total

• SGPT

• SGOT

16
 • ALP

4.

3.1 Lipid Profile.

• Cholesterol

• Triglyceride

Principle:
UV test enzymatic reference method with hexokinase. Hexokinase catalyzes
the phosphorylation of glucose to glucose-6-phosphate by ATP. Glucose + ATP
Hexokinases G-6-P+ ADP Glucose-6-phosphate dehydrogenase oxidizes glucose- 6-
phosphate in the presence of NADP to gluconate-6-phosphate. The rate of NADPH
formation during the reaction is directly proportional to the glucose concentration and
is measured photo metrically.
Procedure:
Separate the serum or plasma sample from the test tube with the help of micro
pipette. Take the sample in a cuvette. Give the command to the analyzer and select
the tests. Press ok. Then place the cuvette in the analyzer. Analyzer gives result
automatically.

Normal ranges of glucose

Fasting Postprandial Random

70-150 mg/dl 70-150 mg/dl 100-150 mg/dl 50

Table 3.1: Normal ranges of glucose

Case Study 01

Name:
Age/sex: 41/male
Sample: Fasting plasma

17
Result obtained: 140 mg/dl

Interpretation: The blood glucose level in the patient is high which indicates
hyperglycemia.

Case Study 02

Name:
Age/sex: 32/male
Sample: Random plasma
Result obtained: 60 mg/dl

Interpretation: The blood glucose level in the patient is low which indicates
hypoglycemia.

Clinical significance

Hyperglycemia
• Diabetes mellitus

• Hyperactivity of thyroid, adrenal, pituitary gland

Hypoglycemia

• Overdose of insulin

• Hypo activity of thyroid, adrenal, or pituitary gland

3.1.1 RFT (RENAL FUNCTION TEST)

Blood urea nitrogen (BUN) Principle: Kinetic test with urease and glutamate
dehydrogenase: Urea is hydrolyzed by urease to form ammonium and carbonate.

Urea + 2HO.REAS E2NH + CO,

18
 In the second reaction 2-oxoglutarate reacts with ammonium in the presence of
glutamate dehydrogenase (GLDH) and the coenzyme NADH to produce L-glutamate.
In this reaction two moles of NADH are oxidized to NAD for each mole of urea
hydrolyzed.
NH + 2 − oxoglutarate + NADHGLDHL − glutamate + NAD + HO

The rate of decrease in the NADH concentration is directly proportional to the urea
concentration in the specimen and is measured photo metrically.
Normal range: 7-10 mg/dl.

Case Study: 01

Name:
Age/sex: 21/Female
Sample: Serum
Result obtained: 6 mg/dL
Interpretation: The blood urea nitrogen level in the patient is low.

Case Study: 02

Name:
Age/sex: 34/Female
Sample: Serum
Result obtained: 26 mg/dL
Interpretation: The blood urea nitrogen level in the patient is high.

Clinical Significance
An abnormally high level of urea nitrogen in the blood is an indication of kidney
function impairment or failure. Some other causes of increased values for urea nitrogen
include perianal azotemia (e.g. shock), post renal azotemia, GI bleeding, and a high
protein diet. Some causes of decreased values for urea nitrogen include pregnancy,
severe liver insufficiency, over hydration and malnutrition.

19
3.1.2 LIVER FUNCTION TEST (LFT)

Introduction
Liver function tests (LFTs) are commonly used in clinical practice to screen for
liver disease, monitor the progression of known disease, and monitor the effects
of potentially hepatotoxic drugs. The most common LFTS include the serum
aminotransferases, alkaline phosphatase, bilirubin, albumin, and prothrombin time.
Aminotransferases, such as alanine aminotransferase (ALT) and aspartate aminotrans-
ferase (AST), measure the concentration of intracellular hepatic enzymes that leaked
into the circulation and serve as a marker of hepatocyte injury.
TOTAL BILIRUBIN Principle:
Diazotized Sulfanilic acid is formed by combining sodium nitrite and sulfanilic
acid at low ph. The sample is diluted in 0.05m Hydrochloric acid. A blank reading
is taken to eliminate interference from non- bilirubin pigments. Upon addition of the
diazotized sulfanilic acid, the conjugate bilirubin is converted to diazo-bilirubin, a red
chromosphere which absorbs at 540nm.

Normal range: 0.20-1.00mg/dl.

Case Study: 01

Name:
Age/sex: 38/Male
Sample: Serum
Result obtained: 1.53 mg/dL
Interpretation: Total Bilirubin level in patient’s serum is high.

Clinical Significance

High levels of bilirubin in the blood may be caused by:

• Some infections, such as an infected gallbladder.

• Some inherited diseases, such as Gilbert’s syndrome, a condition that affects how
the liver processes bilirubin. Although jaundice may occur in some people with

20
 Gilbert’s syndrome, the condition is not harmful.

• Diseases that cause liver damage, such as hepatitis, cirrhosis, or mononucleosis.

• Diseases that cause blockage of the bile ducts, such as gallstones or cancer of
the pancreas.

3.1.3 SGPT (Serum glutamate Pyruvate Transaminase)

(Also called ALT (Alanine Transaminase)

Principle:
Alanine aminotransferase catalyzes the transamination of L-alanine to a-ketoglutarate,
forming L glutamate and pyruvate. The pyruvate formed is reduced to lactate by lactate
dehydrogenase (LDH) with simultaneous oxidation of reduced nicotinamide-adenine
dinucleotide (NADH). The change in absorbance is directly proportional to the ALT
activity and is measured using a dichromatic (340, 700 nm) rate technique.

L − Alanine + a − ketoglutarateALT − pyruvate + L − glutamate

Pyruvate + NADH + H + LDH − L − lactate + NAD+

Normal range: 30-65 U/L.

Case Study

Name:
Age/sex: 21/female
Sample: serum
Result obtained: 116 U/L
Interpretation: The ALT level in patient’s serum is high.

Clinical Significance

High levels of ALT may be caused by:

• Liver damage from conditions such as hepatitis or cirrhosis.

21
 • Lead poisoning.

• Exposure to carbon tetrachloride.

• Decay of a large tumor (necrosis).

3.1.4 SGOT (Serum Glutamate Oxaloacetate Transaminase)

Also called AST (Aspartate Transaminase)

Principle:
Aspartate aminotransferase catalyzes the transamination of L-aspartate to a-Ketoglutarate,
forming L-glutamate and oxaloacetate. The oxaloacetate formed is reduced to
malate by malate dehydrogenase (MDH) with simultaneous oxidation of reduced
nicotinamide-adenine dinucleotide (NADH). The change in absorbance with time due
to the conversion of NADH to NAD is directly proportional to the AST activity and is
measured using a dichromatic (340, 700 nm) rate technique.

L − Aspartate + a − ketoglutarateAS T − oxaloacetate + L − glutamate

Oxaloacetate + NADH + H + MDHL − malate + NAD+

Normal range: 15-37 U/L.

Case study: 01

Name:
Age/sex: 21/female
Sample: serum
Result obtained: 52 U/L
Interpretation: The AST level in patient’s serum is high.

Clinical significance
An increase in AST levels may indicate:

• Acute hemolytic anemia

• Acute pancreatitis

22
 • Acute renal failure.

• Liver cirrhosis

• Heart attack

• Hepatitis

• Infectious mononucleosis

• Liver cancer

• Liver necrosis

3.2 LIPID PROFILE

3.2.1 CHOLESTEROL

Principle:
Cholesterol esters are hydrolyzed by cholesterol ester hydrolase to produce
free cholesterol and fatty acids. The free cholesterol produced and pre-existing
one is oxidized by cholesterol oxidase to cholestenone-4-en-3-one and hydrogen
peroxide. Hydrogen peroxide thus formed is used to oxidize N. N diethylaniline-4-
aminoantipyrine to produce a chromosphere that absorbs at 540 nm. The absorbance
due to oxidized N. N diethyl aniline- 4-amincantipyrine is directly proportional to the
total cholesterol concentration and is measured using a polychromatic (452, 540,700
nm) end point technique.
Normal range: 0-200mg/dl.

Case study

Name:
Age/sex: 21/female.
Sample: serum

Result obtained: 236 mg/dl,

Interpretation: The cholesterol level in patient’s serum is high.

23
 Clinical Significance
Elevated levels of serum cholesterol are associated with atherosclerosis, nephri-
tis, diabetes mellitus, Obstructive jaundice, Biliary cirrhosis, lipoprotenemias, and
myxedema. Decreased level in cholesterol is associated with severe infection, severe
anemia, and malnutrition.

TRIGLYCERIDES

Principle:
T riglycerides+waterGlycerol+AT PGlycerolkinaseLipoproteinLipaseGlycerol−
3 − phosphate + oxygen + Hydrogenperoxide
Glycerol+ f attyacidsHydrogenperoxide+AminoantipyrineGlycerolphosphateoxidaseGlycerol−
3 − phosphate + ADPQuinoncimine + HCL + 4 − Chlorophenol + 4H20
The change in absorbance due to the formation of Quinonimine is directly proportional
to the total amount of glycerol and its precursors in the sample and is measured using
a dichromatic (510, 700 nm) endpoint technique.

Normal range: 15-1000mg/dl

Case study

Name:
Age/sex: 34/male
Sample: Serum
Result obtained: 156 mg/dl.

Interpretation: the triglycerides level in patient is high.

Clinical significance:
TG level decreases in:

• Liver disease

• Cerebral infarction,

24
• Hyper parathyroidism,

• Hyperthyroidism,

• Lactosuria,

TG level increases
Heart disease Renal disease
After severe myocardial infarction Chronic renal failure,
Atherosclerosis, Nephrotic syndrome,
Coronary artery disease, Uremia without nephrosis
Essential hypertension,
Ischemic heart disease
Malignant hypertension,

Table 3.2: Various diseases

25
Chapter 4

HEMATOLOGY

4.0.1 HEAMATOLOGY

INTRODUCTION:

Hematology is the study of blood, blood components, and blood disorders it

involves Studying the anatomy and physiology of blood cells and other cells that
compressed Blood like Red blood cells White blood cells Platelets and hemoglobin.

1. Analysis of blood concentration, structure and function of the cells and their
precursors In the bone marrow.

2. Analysis of chemical constituents of plasma or serum intimately, linked blood

cells Structure and function.

Study of function of the platelets and proteins involved in blood coagulation.

NAME OF THE INSTRUMENT:

26
 • Mythic 22 (For detection of
Hb, Platelets and CBC

• Roll Mixer
(a) Mythic 22 Hematology
• Wintrobe tube Analyzer

NAME OF THE TEST:

1. Complete blood Count (CBC)

2. Erythrocyte sedimentation rate (ESR)

3. Blood grouping

4. Differential leukocyte count (DLC)

5. G-6-PD-1

6. Coagulation profile

4.1 Complete blood Count (CBC)

PRINCIPLE OF CBC ANALYSIS:
The Coulter method accurately counts and sizes cells by detecting and measuring
changes In electrical resistance. When a particle (such as a cell) in a conductive liquid
passes Through a small aperture. Each cell suspended in a conductive liquid (diluent)
acts as an Insulator. As each cell goes through the aperture, it momentarily increases
the resistance of the electrical path between the submerged electrodes on either side
of the aperture. This cause is measurable electronic pulse. For counting, the vacuum
used to pull the diluted suspension of cells through the aperture must be at a regulated
volume.

27
PARTS OF THE INSTRUMENTS:

• Aperture Current

• External electrode.

• Sample beaker.

• Aperture

• Aperture tube.

• Blood cell suspension.

Case Study

Date Name Age/Sex

08/08/2022 23/M
08/08/2022 45/F

Table 4.1: Patient Data

Results

Normal range

WBC = 3.8(L) WBC = 3.8(L) 4-11 cumm

NE = 64.4% NE = 64.4% 40-75%
LY = 14.9%(L) LY = 14.9%(L) 20-45%
MO = 17.2%(H) MO = 17.2%(H) 2-8%
EO = 2.4% EO = 2.4% 1-4%
BA = 1.1% BA = 1.1% 0-1%
RBC = 4.07(L) RBC = 4.07(L) 3-5 Lakh
HGB = 12.2(L) HGB = 12.2(L) 13-17 g/dl
HCT = 37.1(L) HCT = 37.1(L) 42-52%
MCV = 91.1 MCV = 91.1 80-100 fl
MCH = 30.0 MCH = 30.0 27-32 Pg.
MCHC = 32.9 MCHC = 32.9 32-36%
RDW = 15.7%(H) RDW = 15.7%(H) 11-14%

Table 4.2: Results

4.2 Erythrocyte sedimentation rate

PRINCIPLE:

28
 The red cells form Rouleaux, The
settling/sedimentation of RBC’s
occur at a constant rate. The
individual cells also aggregates
due to overcrowding, and get
packed down on the bottom of the
tube. (a) ESR Analyzer

Reagents and Equipment:

1. Automated Analyzer

2. Westergren tube rack

3. Timer

4. 3.8% tri-sodium citrate

5. Test tubes

PROCEDURE:

• Take a clean dry centrifuge tube.

• Add 0.5ml of 3.8% sodium citrate.

• Add 2 ml blood sample into the tube and mix it.

• Fill the Westergren tube up to ’0’ mark.

• Pull the tube in vertical position on the stand.

Clinical Significance

29
 ESR increased in: ESR decreased in:
Chronic inflammations & infections Eg. TB Polycythemia
Acute inflammations & infections Sickle cell disease
Normal Pregnancy (Physiological) Cryoglobinaemia

Table 4.3

Patient Name Age/Sex Result Normal Range

72/F 26/mm/h Female (0-20) mm/h

Table 4.4

Case Study

4.3 ABO BLOOD GROUPING BY (Slide Method)

PRINCIPLE:

Serum of the specimen submitted is reacted with known a cells and B cells. Aggluti-
nation Indicate presence of corresponding antisera in serum.

PROCEDURE:

1. Place 1 drop of anti-A and I drop of anti-B reagent separately on a labeled slide
or tile.

2. Add 1 drop of 20% test red cell suspension to each drop of the typing antiserum
(the Suspension may be prepared by adding 20 parts of red cells to 80 part of
normal saline).

3. Mix the cells and reagent using a clean stick. Spread each mixture evenly on the
slide over an area of 10-15 mm diameter.

4. Tilt the slide and leave the test for 2 minutes at room temperature. Then rock
again and Look for agglutination.

5. Record the results.

Observation:

30
 Figure 4.3: Blood grouping slide

4.4 GLUCOSE-6-PHOSPHATE DEHYDROGENASE

(G6PD):
This test measures the amount of glucose-6-phosphate dehydrogenase (G6PD) in
blood. G6PD is an enzyme in the body. This test is used to evaluate and manage
G6PD enzyme deficiency. This test may also be used if infection is suspected.

PRINCIPLE:

31
 Reaction Monoclonal Monoclonal Monoclonal Result
Antibodies A Antibodies B Antibodies D Blood Group
Agglutination + - + A Positive
Agglutination + - - A Negative
Agglutination - + + B Positive
Agglutination - + - B Negative
Agglutination + + + AB Positive
Agglutination + + - AB - Negative
Agglutination - - + O Positive
Agglutination - - - O Negative

Table 4.5: Blood grouping reaction

Glucose-6-Phosphate Dehygenase present in hemolysate acts on substrate, Glucose

-6- Phosphate (G604) and NADP which in presence of PMS decolorizes blue colored
indophenol dye(DCPIP) leaving behind color only due to hemolysate.The rate of
reaction being proportional to enzyme activity (G6PD) present, time required for
decolonization is inversely proportional to enzyme activity in hemolysate.

KIT REAGENT

1. Lyse agent

2. Buffer

3. Inert oil

PROCEDURE:

1. Take 20ml blood in a vial

2. Put Iml of lysing agent in it.

3. Keep it in refrigerator for 10 mints.

4. Add Sml of buffer to the powdered detergent.

5. Add Iml of inert oil in it

6. Mix well and incubate it for 1 hr.

7. Observe the color change.

32
OBSEVATION:

1. Normal subjects: 30-60 mints.

2. G-6PD deficient subject (Heterozygous male, homozygous female): 140 mints

to 24 hrs.

3. G-6PD carriers (Heterozygous female): Some give result which overlap with
normal males, other decolorizes between 90 mints and several hours.

Case Study

Name Age Sex Result

35 Female Positive

33
4.5 COAGULATION TIME (CT)
PRINCIPLE:

Automated coagulation machines

or Coagulometers measure
the ability of blood to clot
by performing any of several
types of tests including
Partial thromboplastic times,
Prothrombin times (and the
calculated INRs commonly used
for therapeutic evaluation), Lupus
anticoagulant screens, D dimer
assays, and factor.
(a) COAGULATION Analyzer

4.5.1 Procedure:-

1. Take the tube

2. Scan and insert into the its position and select the test

3. Start the test

4. If reagent is not then refill it.

Normal Range of Coagulation studies

PT-11 to 16 sec & APTT-35 to 40 sec
Clinical Significance of PT:

• Prothrombin deficiency

• Vitamine deficiency

• Hemorrhagic diseases of the newborn.

• Liver disease (e.g. Akoholic hepatitis)

• Biliary obstruction

34
 Comparative of Increasing and Decreasing value (APTT)

Increase: Hemophilia deficiency of VIII, IX, XI, V, X and XII.

Decrease: (DIC) Disseminated intravascular coagulation.

Case Study

Name Age/Sex Result

41/Female 17sec PT 42sec APTT

Table 4.6: Patient Result

35
Chapter 5

SEROLOGY

Introduction:
Serology is the study of immune bodies in human blood. These immune bodies are
the product of the defeme mechanisms against disease-causing organisms in the body.
The principle involved with serology is the antibody-antigen response. The antigen
actually comes first, in that the antigen is the substance which ”provokes the body to
produce antibodies.

The tests performed in serology lab:

1. WIDAL test

2. ASO (Anti Streptolysin O)

3. VDRL (Venereal disease research laboratory)

5.1 WIDAL TEST: -

WIDAL slide test provides a simple way of qualitatively and semi-quantitavely
estimating the antibodies to Styphi (O & H) and S.paratyphi (AH & BH). It is based on
the principle of direct agglutination. When the patient’s serum (containing antibodies
to S.typhi & S.paratyphi).

36
KIT CONTENT

Antigen of S. typhi & S.paratyphi.

• S. ’H’.

• S.paratyphy AHS.

• S.’BH’.

• P +ve cont.

• Glass slide. (a) WIDAL Test and Kits

Procedure of WIDAL test:

Take a clean glass slide-add serum 1 drop in each four circle-add a drop of all 4 antigens
in each circle.1,2,3,and 4.

RESULT: If Agglutination titer if 1:80 or more is significant. An increase in titer,

4 to 5 days after the first test is suggestive if active Salmonella infection.

CLINICAL SIGNIFICANCE:

Styphi, Sparatyphi based on their antigenic structure are classified as ’O’ (somatic) and
”H” (FLAGELLAR) Antigens.
’O’ antigens of various species have common antigenic components. Hence only one
antigen S-typhi O’ is used in the routine test’s’ antigen is species specific.

37
5.2 VDRL (Venereal disease research laboratory)
PRINCIPLE:

Patients suffering from syphilis produce antibodies that react with Cardiolipin antigen
in a slide flocculation test, which are read using a microscope.

Procedure:-

Add 50ul serum sample at RT add 20ul antigen and shake it- mix with sticks-rotate for
4min at 150rpm. And see the agglutination.

Results interpretation:

POSITIVE REACTION: Marked and intense visible aggregates are seen. sample is
reactive.
POSITIVE REACTION: Slight but definite small aggregates are seen. Serum sample
weakly reactive.
NEGATIVE REACTION: The mixture remains in a smooth suspension with no
visible aggregates. Serum is non-reactive.

5.3 ASO (Anti Streptolysin O)

It is a rapid latex agglutination test for the qualitative and semi- quantitative determi-
nation of anti-Streptolysin-O antibodies (ASO) in serum. In infections caused by ß-
hemolytic streptococci, Streptolysin-O is one of the two hemolytic exotoxins liberated
from the bacteria that stimulate production of ASO antibodies in the human serum.

PRINCIPLE:

The ASO is a rapid agglutination procedure for the direct detection and semi-
quantitation (on slide) of anti-Streptolysin. The antigen, a latex particles suspension
conted with Streptolysin 0, agglutination in the presence of specific antibodies present

38
in sera of patients with Streptococcal beta- hemolytic infection (Group A and C).

Figure 5.2: ASO Kits

PROCEDURE

1. Place I drop of serum sample on to the slide with the help of disposal serum
dropper,

2. Add 1 drop of ASO- Latex Antigen to the slide

3. Mix properly with the applicator stick

4. Rotate for 2min in a rotator

39
 5. Observe for agglutination

Case Study

Name Age /Sex Result ASO VDRL WIDAL

23/F Agglutination +ve -ve -ve
21/F Agglutination -ve +ve -ve
19/F Agglutination -ve -ve +ve

Table 5.1: Patients positive cases.

5.4 HIV Tri- dot test

Principle

HIV antigens are immobilized on a porous immunofilteration membrane. Sample and

the reagent pass through the membrane and are absorbed into the underlying absorbent.
As the patients sample passes through the membrane, HIV antibodies, if present, bind
to the Immobilized antigens. Conjugate binds to the Fc portion of the HIV antibodies
to give distinct pinkish purple Dot (s) against a white background.

Specimen requirement

Scrum Iml

Procedure

1. Add 3 drops of buffer solution to the center of the device.

2. Hold the dropper vertically and add 1 drop of patient’s sample (serum or plasma).

3. Add 5 drops of buffer solution.

4. Add 2 drops of liquid conjugate directly from the conjugate vial.

5. Add 5 drops buffer solution and read the results

40
Result:

- Report the result positive when both line is appeared control and HIV.

41
Chapter 6

MICROBIOLOGY

Introduction

Clinical microbiology is the branch of medical science that deals with the study of
Microorganisms that infect humans, the disease they cause, their diagnosis prevention,
and Microbiology treatment.
Here microbiology department is divided into three sub departments.:

1. Bacteriology department

2. Mycology department

3. Tuberculosis department

Types of sample received in Laboratory

• Urine (mostly received)

• Sputum sample

• Blood sample

• Stool sample

• Throat swab

• Water

• FNAC smear

42
Instruments:

1. Bactec system

2. Microscan

3. Microscope

4. Hot air even

5. Incubator

Procedure for culture:

Urine, Stool, Body fluid, and CSF etc...

1. Take the sample

2. Inoculate the sample into the media and keep inverted position

3. And keep all equipment in the laminar air flow plate

4. After cold keep the media into the incubator at RT for 24 hrs.

5. Incubator

Result:

-note down the result after 24 hrs. Or 48 hrs..

6.1 Microscopy:
Wet mount is prepared. Single drop of urine is taken in a clean and dry slide, put on
the coverslip and observe under the microscopy.

43
6.1.1 BACTEC

Principle

The sample to be tested is inoculated into the vial which is entered into the BACTEC
instrument for incubation and periodic reading. Each vial contains a sensor which
responds to the concentration of CO2 produced by the metabolism of microorganisms
or the consumption of oxygen needed for the growth of microorganisms.

Anaerobic culture and Aerobic culture.

Procedure:

1. Introduce the blood about 5ml into the both bottle

2. Put the bottle for 24 and 48 and 72hrs

3. Red light shows the positive and negative.

4. Positive culture can take to grow on media.

STAINING

Gram staining Principle

1. Place the slide on the staining glass rods.

2. Cover the smear with crystal violet stain and leave for 1 minute.

3. Wash carefully under running tap water.

4. Flood the smear with the gram’s solution and wait for one minute.

5. Drain off the iodine.

6. Decolorize the smear with alcohol-acetone water. (or rectified spirit) for 20-30
seconds (continue till purple stain just stops coming on the slide).

Acid staining Principle

44
1. Prepare Smear from the sputum specimen on glass slide and fix it by heating.

2. Flood slide with Carbol-fuchsin stain, heat the slide gently with a flame for 5
minutes.

3. Do not over heat the stain if necessary add carbol-fuchsin.

4. Rinse off the over stain under running tap water.

5. Decolorize with acid alcohol or 20% H2SO4 for about 1 minute or until no more
color comes off.

6. Rinse again in running tap water.

7. Counter stain with methylene blue for 30 sec.

8. Examine under microscope with oil immersion objective.

RESULTS

Gram stain Acid fast AF

Yeast cells: Dark purple Acid fast (AF) organisms
Epithelial cells: Pale red
Nuclei of pus cell: Red Bright red bacilli on blue background.
Gram positive bacteria: Dark purple Other organisms - Dark blue
Gram negative bacteria: Pale to dark red.

Table 6.1: Result of various stain

45
Chapter 7

HISTOPATHOLOGY

Introduction

(compound of three Greek words: histos ”tissue”, pathos ”suffering”, and - logia ”study
of) refers to the microscopic examination of tissue in order to study the manifestations
of disease.

(a) Preparation (b) Olympus CX23 Microscope

Figure 7.1: Microscopic examination of tissue

46
 Biopsy FNAC Autopsy Amputed limbs LBC
Utrus Lump From dade body From alive person Vaginal discharge
Placenta Bump
Slivary gland Upper limb Fluids
Cervical Lower limb
Kidney

Table 7.1: Specimen of Uterus.

Types of samples:-

7.1 Histopathological Instrument and eqquipments:-

• Microtome

• Microtome knife

• Timer

• Hot air oven

• Forceps

• Scalpel, dissecting set

• Tissue floatation bath

• Equipment for embedding and vacuum

• Containers for holding specimens

7.1.1 Steps for the Tissue processing

1. Grossing

2. Labeling

3. Fixation

4. Dehydration

5. Clearing

6. Impregnation

47
 7. Embedding

8. Microtomy

9. Staining

10. Microscopic observation

7.1.2 Procedure

Grossing: Tissue cuts into tissue small pieces like 3-4mm.

Labeling: Every tissue need to give an identity for Recognize.
Fixation: It prevent need to decompose give an Exp: Formalin.
from natural Dehydration: Water removed by different grade of Iso by propyl alcohol.
50% Alcohol It used 70%,90%,and with two 95% .
Clearing: Alcohol is removed by Xylene. It used with two changes changes 1 & IInd.
Impregnation: It remove the clearing reagent paraffin. Tissue transfer into P. Wax Ist,
IInd and IIIrd changes. Temperature should be 2-3 degree more than its melting point
Paraffin wax.

7.1.3 Staining of the slides:-

1. Ziehl nelson stain for acid fast bacilli(Acid fast stain)

2. PAP stain

3. Giemsa stain

4. PAS (Periodic Acid Schiff) stain

Mounting: after staining I do mounting with DPX then slide send to the histopathol-
ogist.
Observation:- senior pathologist does all examination of the slides.

Results: If we find the abnormal structure in nucleus and cytoplasm of the cells
then it may report as the cancerous cases or diseases case.

48
 7.1.4 Case Study

Specimen Name Age/sex Result Disease Remark

Uterus 40/F Positive for Cancer Malignancy of
endometrium Cancer Uterine cancer

Table 7.2: Specimen of Uterus

49
References

[1] Life Sine Lab, et al., Internship in Clinical Pathology, 2022 ORG Code:
10029086, License No: HSM4513263

[2] Khandker Md. Faisal Alam, et al., Professor of Microbiology at Shaheed Ziaur
Rahman Medical College, Bogura 5800, Bangladesh, Head of Microbiology
Department at Life Sine Lab. https://www.researchgate.net/profile/Faisal-Alam

[3] Md. Ismail Hossain, Lab Technologist,at Life Sine Lab.

[4] Nadeem Hossain, Hormone Test Specialist, at Life Sine Lab.

50