Transcript Applications - Killingly Public Schools

Molecular Diagnosis
Applications
Molecular Diagnostics
• Excellent sensitivity and specificity
– Minimal residual disease
• Genotype not phenotype
– Prior to appearance of symptoms
• Classification of related diseases
– Critical treatment decisions
• Diagnostic
• Prognostic
• Predictive
Molecular Pathology
A Universal Discipline of Laboratory Medicine
INFECTIOUS
DISEASE
HEMATOLOGY
Molecular
Pathology
SOLID
TUMORS
IDENTITY
TESTING
GENETIC
DISEASE
The Fight Against Disease
• The human population is growing and people
are living longer
• Medical and technical knowledge is always
increasing
• More diseases can be predicted, diagnosed
and properly treated than ever before in
history
• Still, cancer and infectious diseases kill
millions per year worldwide
The Cancer Dilemma
• Even with tests for early detection, the ability to
change one’s lifestyle, the availability of treatments
or even cures, this disease still causes immense
suffering
The newest DNA-based
techniques are making
great headway in
studying cancer to find
new treatments for
improving and prolonging
quality life for patients.
Chronic Myelogenous Leukemia
• A slowly progressing disease in which too many white blood cells
(not lymphocytes) are made in the bone marrow. Too many WBCs
crowd the healthy cells and consume the nutrients/resources they
need to develop properly. Also called chronic granulocytic leukemia,
chronic myeloid leukemia, and CML.
• Most people with CML have a gene mutation (change) called the
Philadelphia chromosome.
– Part of the DNA from one chromosome moves to another
chromosome. This change is called the “ Philadelphia chromosome.” It
results in the bone marrow making an enzyme, called tyrosine kinase,
that causes too many stem cells to become white blood cells
(granulocytes or blasts).
– The Philadelphia chromosome is not passed from parent to child.
CML
Philadelphia chromosome. A piece of chromosome 9 and a piece of chromosome 22
break off and trade places. The bcr-abl gene is formed on chromosome 22 where the
piece of chromosome 9 attaches. The changed chromosome 22 is called the
Philadelphia chromosome.
Fluorescence in situ hybridization
(FISH)
• Test that “maps” the genetic material in a person’s
cells
• Can be used to visualize specific genes or portions of
genes
• Done on breast cancer tissue removed during biopsy
to see if the cells have extra copies of the HER2 gene
– The more copies of the HER2 gene that are present, the
more HER2 receptors the cells have. These HER2 receptors
receive signals that stimulate the growth of breast cancer
cells.
FISH: Fluorescence in situ Hybridization in Cancer
Diagnosis
Her2/neu gene amplification is found in some breast cancers.
Amplification of genes can contribute to the development of cancer and
can be detected using FISH among other assays.
Disease Causing Microorganisms
• The immune system is constantly under siege and
can at times be overcome by viruses, bacteria, and
fungi
• Molecular testing is more sensitive and specific than
traditional assays, so diagnosis is improved
• It is now possible to know which drugs a particular
bacterium will be resistant to and how many
organisms compose an infection
Mycobacteria tuberculosis
Present-day, worldwide
vaccination programs have
greatly reduced suffering from
Mycobacteria tuberculosis.
Molecular Tests for TB
• The hsp65 gene is present in all Mycobacteria
organisms
• Restriction enzyme digestion of the gene with
BstEII and HaeIII allows identification of
particular species
• Treatment is species-specific
• Molecular assays are rapid while most
traditional lab tests are not
Branched DNA Technology
This method of signal amplification is widely used to
measure HIV viral load
http://www.youtube.com/watch?v=-bIJt7zsx2k
Viral Isolation Methods
• Viral Isolation (HSV) – see handout
– Positive result: average 108 hours
– Negative result: average 154 hours
Hybrid Capture® II
HPV DNA Test
• Human Papilloma
Virus (HPV)
• Primary cause of
cervical cancer
• Over 70 specific types
• Assay is simple but
sensitive
Release and
denature
nucleic acids
Digene®
Hybridize RNA
probe with
target DNA
Digene®
Capture RNA:DNA
hybrids onto a solid
phase (in tube or
microplate format)
Digene®
React captured
hybrids with multiple
antibody conjugates
Digene®
Detect amplified
chemiluminescent
signal
Digene®
Genetic Disorders
• Single gene, one mutation
– Sickle cell anemia
• Single gene, multiple mutations
– Cystic fibrosis
• Multiple genes
– Hereditary thrombophilia
– Cancer
Sickle Cell Anemia
• Serious disorder in which the body makes sickle-shaped red
blood cells
• Normal RBCs contain an iron-rich protein called hemoglobin
which carries oxygen from the lungs to the rest of the body
• Sickle cells contain abnormal hemoglobin which causes the
cells to develop a sickle, or crescent, shape
– stiff and sticky
– tend to block blood flow in the blood vessels of the limbs
and organs causing pain and organ damage – can also raise
risk of infection
• Detection = blood test
Sickle Cell Anemia
Sickle Cell Anemia
Cystic Fibrosis (CF)
• Inherited chronic disease that affects the lungs and digestive
system of about 30,000 children and adults in the U.S. (70,000
worldwide)
– Defective gene and its protein product cause the body to
produce unusually thick, sticky mucus
• 1950s: few children with CF lived to attend elementary school
– Today, advances in research and medical treatments have
further enhanced and extended life for children and adults
with CF (live into 30s, 40s, +)
Testing for CF
• Newborn Screening
– checks for a chemical (immunoreactive
trypsinogen – IRT) made by the pancreas
– normally found in small amounts in the body
• Genetic Carrier Testing
– blood test
– more than 10 million Americans are symptomless
carriers of the defective CF gene
– To have CF, a child must have 2 alleles for CF
Cystic Fibrosis
Multiple Genes
• Hereditary Thrombophilia
– group of inherited or acquired disorders that
increase a person’s risk of developing thrombosis
(abnormal “blood clotting”) in the veins or
arteries
• Cancer
– the uncontrolled growth of abnormal cells in the
body
Multiplex PCR
M B N A 1 2 3 4 5 6 7 8 9 10 11 12 13
Factor II
Factor V
Mutated genes for some coagulation factors contribute to Hereditary
Thrombophilia which has high morbidity and moderate mortality. Multiplex
PCR can rapidly detect deletions or duplications in a large gene.
Future of Medicine
http://www.ted.com/talks/daniel_kraft_medicine_s_future.html
(Video: 18:21)