This document provides an overview of various diagnostic techniques used in pathology. It discusses light microscopy, electron microscopy, special staining, immunohistochemistry, flow cytometry, and molecular pathology/cytogenetics which are used to assist in diagnosis. It also discusses techniques used to diagnose chronic kidney disease, hepatitis C, bone cancer, peptic ulcer, and diabetes including blood tests, imaging tests, biopsies, and other assays.

1. Presented by : Group B-5
INTRODUCTION TO
PATHOLOGICAL TECHNIQUE’S
AND THEIR USE AND
ADVANTAGE IN VARIOUS
PATHOLOGICAL CONDITIONS

2. CONTENT
• Introduction to pathology
• Methods Used In Diagnosis
• Chronic Kidney Disease
• Hepatitis C
• Bone Cancer
• Peptic Ulcer
• Diabetes

3. Presented by : Abdul waris
Roll NO.06331313070
Pathology and
Techniques used
In Diagnosis

4. Pathology and Techniques used
• Pathology literally is the study (logos) of suffering
(pathos)
• Pathology is the scientific study of disease
• Techniques in pathology are the systematic
procedures done in Laboratories to evaluate, identify
and differentiate between normal and abnormal
structure of tissue, cells or cell components in body.
4

5. Techniques Used in Diagnosis
• Following are some methods of analysis to assist in
diagnosis
• Light Microscopy & Electron microscopy
• Special Staining
• Immunohistochemistry
• Flow cytometry
• Molecular pathology/ Cytogenetics

6. .
• Light Microscopy: the structure of tissues & cells in health &
disease.
• Electron microscopy (magnify up to 2 million times)
• The usual microscopes used by pathologists
are not powerful enough to see the
smallest parts that make up a cell.
• Some diseases can only be
diagnosed at this subcellular level.
• Examples:Glomerulonephritis (kidney disease)
Aggressive cancers
• In these cases a very powerful type of microscope is used
called the electron microscope. This utilises beams of
electrons rather than visible light to magnify the cells in a
tissue sample.

7. • Special stains:
• Pathologists use the chemical properties of components
of the tissues being studied in their choice of different
stains.
• Stains( haematoxylin and eosin)
• React with acidic and basic cell components
• Purple and pink colors to the tissues.
• Other stains available
• highlight fats,
• different tissue fibers,
• different types of mucus,
• microorganisms, proteins etc.

8. .
• Immunohistochemistry
• Process of detecting antigens (e.g. proteins)
in cells of a tissue section by exploiting
the principle of antibodies
binding specifically to antigens
in biological tissues.
• Used in the diagnosis of
abnormal cells ( cancerous tumors)
• Specific molecular markers
are characteristic of particular cellular events
proliferation or cell death (apoptosis).
• Visualising an antibody-antigen interaction
( Immunoperoxidase staining) (Immunofluorescence)
rhodamine
an antibody is conjugated to an
enzyme
, such as peroxidase,
that can catalyse a colour-
producing reaction).
antibody can also be tagged to a
fluorophore, such as fluorescein
or
rhodamine

9. • Flow cytometry
• This technique is used to diagnosis of cancers of the
blood cells (leukemia and myelomas).
• Cells are suspended in a liquid
• passed laser beam
(single wave length light beam).
• Scattered
• A detector measures
• Fluorescent light is emitted
from excited particles on the cells.
• This is interpreted by a computer as a number of cells/
particles/ proteins (whatever substance is being
examined for) and is shown on a graph.
• This can be used to give the quantities and relative
proportions of different types of cells in the blood and
identify any abnormal cells (e.g. leukemia).

10. Molecular Pathology and Cytogenetics
1. Fluorescence in situ hybridisation (FISH)
• stain chromosomes to reveal areas where genes may
have been
• deleted, duplicated or broken.
• Fluorescent labels are attached to specific DNA
sequence which allow faulty genes to be seen when
examining the cells under a special type of microscope
2. Direct sequencing of DNA:
• It is a way of looking at individual genes or groups of
genes, to detect and characterise which mutation is
present in a particular patient’s tumour.
A. Sanger sequencing,
B. capillary electrophoresis

11. Chronic Kidney
Disease
Presented by : Umama Abdul Qader
Roll NO.06331213113

12. Introduction
• Chronic kidney disease also
called chronic kidney failure
• It describes the gradual loss of kidney
function ( CKD )
• Doesn’t usually cause symptoms until it
reach advanced level in which fluid ,
electrolytes and wastes build up in body .
• Most common cause is diabities and it also
may be cause of hypertension ,

13. Diagnostic Tests of Chronic kidney disease
1. Detecting Kidney Damage
 Direct evidence: which is found on imaging or
histopathological examination of a renal biopsy.
 Indirect evidence : for kidney damage inferred from
urinalysis .
2. Measuring renal function

14. Detecting Kidney Damage
1. Proteinuria :
Detection of an increase in measurement of protein
excretion in a timed urine collection over 24 hours .
 Urine dipstick testing .
 Protein/ creatinine ratio : Protein/creatinine ratio
measured predicting the rate of loss of GFR in
patients
 Albumin/creatinine ratio .

15. 2. Haematuria :
It indicate significant pathology including infection,
malignancy and other forms of kidney damage.
3. Renal tract ultrasound :
Ultrasound is the optimal first line test for imaging
the renal tract in patients with CKD. No evidence was
identified on the usefulness of renal ultrasound
alone in the diagnosis of CKD.

16. Measuring renal function
1. Glomerular filtration rate : it is the volume of plasma
which is filtered by the glomerular per unit time and is
usually measured by estimating the rate of clearance of
a substance from the plasma.
2. Creatinene : measurement of urinary excretion of
creatinine by means of a timed urine collection allows
estimation of creatinine clearance

17. 3. Prediction equations : improve the inverse
correlation between serum creatinine and GFR by
taking into account confounding variables such as
age and body weight.
4. Cystatin : Serum concentrations of the low
molecular weight protein cystatin C correlate
inversely with GFR. The conc. of cystatin C is
independent of weight and height, muscle mass,
adult age or sex .
5. Other Markers : Various other markers have been
used to estimate clearance, including inulin, iohexol
and radioisotopic markers such as 51Cr-
ethylenediaminetetraacetic acid (EDTA).

18. HEPATITIS C
Presented by : Shakeela
Roll NO.06331313073

19. Introduction
• Inflammation of the liver due to
the hepatitis C virus (HCV), which is
usually spread via blood transfusion
(rare), hemodialysis, and needle sticks.
• The damage hepatitis C does to the
liver can lead to cirrhosis and its
complications as well as cancer.
Transmission of the virus by sexual
contact is rare.
• There are 5 main hepatitis viruses,
referred to as types A, B, C, D and E.

20. Diagnostic Tests For Hepatitis C
A variety of different tests are used to diagnose
hepatitis C. These include:
• HCV antibody test
• HCV RNA (Viral Load) Tests
• Genotype / Subtype Test
• Liver Biopsy
• Fibroscan
• Fibrometer
• Non-Invasive Liver Tests
• Staging Liver Disease

21. HCV Antibody Test
• When a person is exposed to HCV, the immune system
produces proteins called antibodies against the virus.
• There are two commercial antibody tests used to detect HCV
antibodies.
• HCV EIA, (HCV ELISA)
• CIA
• The signal-to-to-cut-off (s/co) ratio was developed to give
confidence that an HCV antibody test result is truly positive.
• OraQuick Rapid Antibody Test (finger prick and whole blood
draw)

22. OraQuick Rapid Antibody Test

23. HCV RNA (Viral Load) Tests
A viral load test measures the amount of HCV RNA (genetic
material) in the blood. This test is used to confirm active HCV
infection and it is also used to guide treatment. There are two
types of viral load tests:
• Qualitative
Measures the presence of the virus in the blood. This type of test
is usually used to confirm initial and chronic infection with HCV
• Quantitative
Measures the amount of virus in the blood. This test generally is
used for
HCV treatment to determine if a patient is responding or has
responded to treatment

24. Liver Biopsy
• Liver biopsies are used to measure the
extent of liver damage, including the
degree of inflammation the extent of
fibrosis (fibrous tissue), and the general
health of the liver.
• The most common type of liver biopsy is
the percutaneous biopsy (through the
skin).

25. Liver Biopsy

26. • Fibroscan
The Fibroscan is an approved
imaging test that is used to
evaluate the amount of scarring (if
any) of the liver.
• Fibrometer
A blood test that measures certain
blood markers

27. Non-Invasive Liver Tests
There are other non-invasive tests used to stage the
level of damage in the liver including the
MP3, Fibrotest, Hepascore, Forns score, and APRI that
combine a variety of blood tests to ascertain the
level of damage in the liver.

28. Bone cancer
Presented by : Ramsha
Roll NO.06331213083

29. What is bone cancer
• Uncontrolled and
uncoordinated growth of
bone cells
• Many types of bone cancers
on the basis of their type of
originating cells
• Can be primary or secondary

30. BLOOD TESTS
• detect the presence of bone cancer
• Level of lactate dehydrogenase and alkaline
phosphatase are measured in blood sample
• High level suggest presence of cancer cells

31. BIOPSY
• Removal of small amount of bone
tissues to be observed under a
microscope
• Two types
needle biopsy : small hole in bone
through a needle to take a sample
Incisional biopsy: cut into the tumor is
made to remove sample
• confirms the presence of bone cancer

32. IMAGING TESTS
• Variety of such tests are available which
determine if the cancer is malignant or not .
• Bone scans , CAT and MRI are most common

33. BONE SCANS
• Radioactive material is injected in blood stream
• When it reaches bone cells it is detected by a scanner
• Healthy cells appear gray
• Cancerous cells appear dark in color

34. Computed tomography scan CAT
• Series of detailed images are taken
from different angles created by a
computer and it is connected to an x
ray machine
• Special dye like Contrast medium is
given to patient before scan to get
detailed image
• This test is also use to detect the
tumor size

35. Magnetic Resonance Imaging ( MRI )
• Similar to CAT but magnetic
rays are use instead to X rays
• Dye ( contrast medium ) is
either given IV or as a pill

36. Positron Emmision Tomography (PET)
Scan
• Radioactive glucose is injected in vein and
scanner is used to get the detailed image of bone
• cancer cells are more active than normal cells
therefore they take up more radioactive glucose
than normal cell

37. Peptic Ulcer
Presented by : Maria nisar
Roll NO.06331313083

38. Diagnostic techniques :
• Upper endoscopy
• Barium swallow
• MRI
• PET
• Breath test
• Biospy

39. Upper endoscopy :
• A thin flexible tube with small camera is inserted into
the stomach via throat.

40. Barium swallow :
• Also known as upper gastrointestinal series
• Procedure :
patient drinks chalky liquid containing barium.The
fluid coats the stomach so inside is more visible on X-
rays.

41. Magnetic resonance imaging
• MRI produces detail image using magnetic field.
• Contrast medium ,a special dye is used before scan.

42. PET
• Positron emission tomography
• Creates pictures of organs inside the body
• Procedure :
tracer injection
positrons release
detection by the camera
location of diseased area

43. Breath Test :
• Specific for detection of Helicobacter pylori
• Procedure :
1.Food or drink containing radioactive carbon taken .
2. H.pylori break substance in stomach
patient blows the bag which is sealed later on
3. If patient is infected ,the breath sample contains
carbon in the form of CO2

44. Biopsy
biopsy for stomach is done using
endoscope having needle
Procedure :
small piece of tissue from
Stomach is taken for
Microscopic examination.

45. Diabetes
Presented by : MaHNOOR Javed
Roll NO.06331313019

46. • Diabetes is a complex group of diseases with a
variety of causes.
• People with diabetes have high blood glucose, also
called high blood sugar or hyperglycemia.
• Diabetes is a disorder of metabolism—the way the
body uses digested food for energy.

47. Types
• The three main types of diabetes are type 1,
type 2, and gestational diabetes:
• Type 1 diabetes
• Type 2 diabetes
• Gestational diabetes is a type of diabetes that
develops only during pregnancy.

48. DIABETES BIOMARKERS
• Glycated albumin
• HbA1c

49. Tests for diagnosis:
• Any one of the following tests can be used for
diagnosis:
• an A1C test, also called the hemoglobin A1c, HbA1c,
or glycohemoglobin test
• a fasting plasma glucose (FPG) test
• an oral glucose tolerance test (OGTT)

50. Major HbA1c testing methods:
• Regulatory agencies have widely embraced HbA1c as
the biomarker of choice for proving efficacy in clinical
trials for antidiabetes drugs.
• There are 3 major HbA1c testing methods currently
available to clinical laboratories. They are:
• Chromatography based HPLC assay
• Antibody based immunoassay
• Enzyme based enzymatic assay(Glycated albumin
test)

51. How do these methods work?
1. Chromatographic method:
• The chromatographic assay uses an HPLC
instrument and ion exchange or affinity column to
separate HbA1c molecules from other hemoglobin
molecules.
• The HbA1c content is calculated based on the ratio
of HbA1c peak area to the total hemoglobin peak
areas as shown in the figure below:

52. 2. Latex enhanced Immunoassay method:
• The latex enhanced immunoassay for HbA1c is
based on interactions between antigen molecules
(HbA1c) and HbA1c specific antibodies coated on
latex beads.
• This cross-link reaction results in changes in the
solution turbidity which is proportional to the
amount of the antigen in the samples as depicted
in the scheme below:

53. 3. Enzymatic HbA1c assay method
(glycated hemoglobin):
• The assay’s unique enzymatic methodology directly
measures glycated hemoglobin.
• Assay Principle
1. Oxidizing agents in the lysis buffer react with the whole
blood sample to eliminate low molecular weight and
high molecular weight signal interfering substances.
2. After lysis, the whole blood samples are subjected to
extensive proteolytic digestion.
3. This process releases amino acids, including glycated
valines, from the hemoglobin beta chains.

55. Cont.
• The Direct Enzymatic HbA1c Assay™ glycated valines
serve as substrates for a specific recombinant
fructosyl valine oxidase (FVO) enzyme.
• The recombinant FVO specifically cleaves N-terminal
valines and produces hydrogen peroxide in the
presence of selective agents.
• This, in turn, is measured using a horseradish
peroxidase (POD) catalyzed reaction and a suitable
chromagen(dye).
• The signal produced in the reaction is used to
directly report the %HbA1c of the sample using a
suitable linear calibration curve expressed in
%HbA1c.

56. • Assay Advantages The Direct Enzymatic HbA1c
Assay™ has all the advantages of both the
HPLC and immunoassays methods in the areas
of accuracy, specificity and yet is cost effective,
simpler and has less interferences.

57. References
• http://www.diazyme.com/Websites/diazyme/images/pro
ducts/pdf/data_sheets/MK066-
%20Diabetes%20Biomarkers.pdf
• http://www.diazyme.com/direct-enzymatic-hba1c-
methods
• http://www.ngsp.org/A1ceAG.asp
• http://www.niddk.nih.gov/health-information/health-
topics/Diabetes/diagnosis-diabetes-
prediabetes/Pages/index.aspx
• http://www.diabetesselfmanagement.com/diabetes-
resources/definitions/fasting-plasma-glucose-test/