EOC - Medical Interventions

What is a Medical Intervention?

Anything that helps us prevent, diagnose, and treat our patients

Types of Medical Interventions

1) Prevention

2) Therapy & Rehab

3) Diagnostics

4) Surgery

5) Medical Devices

6) Pharmacology

7) Immunology

8) Genetics

Examples of Prevention

Screenings like colonscopies

Tests like lipid panels

(Anything that helps prevent a disease)

Examples of Therapy & Rehab

Physical Therapy

Rehabilitation

Recovery from injury

Occupational Therapy

Improve mental health

(treatment of recovery from disease/injury)

Examples of Diagnostics

CT Scans

PET Scans

Endoscopy

Biopsies

(Any test used to help confirm/rule out a disease/condition)

Examples of Surgeries

Appendectomy

Transplants

(Procedures to remove/repair parts of the body to fight off disease)

Examples of Medical Devices

Pacemaker

Catheters

Prosthetics

(Any instrument/machine that is intended for the usage of medical purpose)

Examples of Pharmcology

Ibuprofen

Acetaminophen

Adderall

(Drugs & Chemical that aid/affect the biological system)

Examples of Immunology

Vaccines

Allergy shots - reduces the sensitivity of immune system to certain allergens

(Treatments that use the immune system to fight off disease)

Examples of Genetics

Genology

(Studying how genes and traits are passed from one generation to the next)

1st step of sequencing

Isolate Target DNA

2nd Step of sequencing

Do PCR

Process: Polymerase Chain Reaction (PCR)

Materials:

Primers

"Start flag" for polymerase

Prepares target DNA for the rest of PCR

Thermal Cycler

Machine that does PCR

DNA Polymerase

Enzyme that adds nucleotides to target DNA

Builds DNA

Isolated Target DNA (From Step 1)

Product: Millions of target DNA

3rd step of sequencing

PCR again

Process: PCR repeated

Materials:

Same materials (From Step 2)

Fluorescently tagged free Nucleotides

Products: Differently sized fluorescently tagged strands of target DNA

4th step of sequencing

Gel electrophoresis to get data into computer graph

Process: Gel electrophoresis

Materials:

Automatic sequencer

Machine that does gel electrophoresis

Target DNA (From Step 3)

Fragments with fluorescent tags

Product: Graph with colored peaks

Auto-sequencer separates fragments and sends those fragments through a tube

Tube is positive at top and negative at bottom

Tube has lasers on bottom that reads fragments and uploads a peak on a computer graph

5th step of sequencing

Analyze Graph Data

Materials:

Graph (From Step 4)

Products: Complementary sequence of Target DNA

What base pair is blue?

Cytosine (C)

What base pair is green?

Adenine (A)

What base pair is black?

Guanine (G)

What base pair is red?

Thymine

RULES OF GRAPH ANALYSIS

1) One peak, one color = one base

2) Overlapped peaks = "N"

3) Bigger peaks between two is the one you read

4) Sections with a lot of "N's" get taken out (mostly at start or end)

(If a section with a lot of "N's" is in the middle of data, redo lab)

Bioinformatics

The application of computer technology to collect, store, and analyze biological data and information

BLAST

Basic Local Alignment Search Tool

What do BLAST results show?

Shows a database of similar sequences to that of the input sequence such as the patient sample to help figure out what organism is in the patient sample and, from that, find the disease the patient has

What are the several result values we're given from a BLAST?

Max Score

Total Score

Query Coverage

Expect Value

Identity Value

Max Score

Highest local alignment score from query/input sequence to a sequence in the database

(Reflects the best match as a higher max score means better alignment)

Total Score

Sum of alignment score from query/input sequence to a sequence in the database

(Overall similarity between query/input sequence and aligned database sequence)

Query Coverage

Percentage of query/input sequence that overlaps with reference/database sequence

Expect Value ( E Value)

Number of hits we expect to see by chance

Helps to see if hits are unique or not

Lower the E-value, the more precise the hit is

Identity Value (Ident Value)

Percentage of base pairs that are the same between the query/input sequence versus the reference/database sequence

Query Coverage vs Identity Value Example

CCCCCCCCCC target

CCCACCACCC query/input

might be 100% query coverage 80% identity

Steps of an ELISA Test

1) Put in patient sample

2) Primary antibodies

3) Secondary antibodies

4) Substrate

5) Color Change (if pateint had antigen present)

What does ELISA stand for?

Enzyme Linked Immunosorbent Assay

Steps of an ELISA Test if testing for antibodies

1) Antigen

2) Patient sample

3) Secondary antibodies

4) Substrate

5) Color Change (If patient sample has antibody present)

Innate/Nonspecifc Immune system

First line of defense against invaders

Includes external barriers

(Skin, mucous membrane)

Includes internal defenses

(Phagocytes, antimicrobial proteins, attack cells)

Phagocytes

Surrounds and kills any invading cells

Types of phagocytes include:

Neutrophils

Most abudant type of WBC

Self-destruct after ingesting a pathogen

Natural Killer Cells

Can kill own cells if they're infected or are cancerous

Normal cells contain a special protein called MHC1

If a cell stops making MHC1, it's become infected

Natural Killer Cells detect this and then "poke" the infected cell

From that, the cell will go into apotosis or "programmed cell death"

Inflammatory Response

Redness

Swelling

Heating up

Pain

Histamine

Causes vasodilation (redness & heat at site of injury)

Heat increases metabolic rate of repairing

Causes swelling through increasing permability of blood vessels

Causes nearby capillaries to realease protein-rich fluids

Leukocytosis

Release of neutrophils from bone marrow

Adaptive/Acquired Immune System

Slow to act

Must first recognize pathogen before attacking it

Has ability to remember specific pathogens

Can fight throughout whole body at once rather than be restrictive

Deploys Humoral Immunity & Cellular Defenses

Humoral Immunity

Sends out antibodies

Antibodies

Patrol through blood & lymph

Combat viruses & bacteria moving around the space between cells

(Body has to be able to detect pathogens like bacteria, viruses, and fungi to be able to defend against them)

Antigens

Large molecules not normally found in body

B-Lymphocyte

Originates & matures in bone marrow

As it matures, it develops the ability to identify pathogens clearly

Also learns how to bind to a specific antigen

Develops self-tolerance (ability to not attack body's own cells)

What happens when a B-cell detects an antigen it has antibodies for?

It recognizes it and binds to it

Memory Cells

Cells that remember the antigen and stay in your immune system so that, if that specific antigen invades your body again, it can quickly identify it and fight it

Neutralization

Antibodies block binding sites on viruses/bacterial toxins, inhibiting them to attach to tissues

Agglutination

Antibodies binding to multiple antigens

Inhibits movement of antigen (helps macrophages)

What does Humoral response do?

Humoral response helps the body achieve immunity

Can occur naturally or artificially (vaccination)

Babies' passive humoral immunity

Babies obtain passive humoral immunity while in the womb

However, baby's own system won't remember an antigen if it gets infected again

T-Lymphocyte

Goes after infected body cells

Causes inflammation, activates macrophages, increase response of other T-cells, regulate much of immune response

MHC protein

Presents short chains of amino acids based on endogenous proteins

(proteins synthesized in cell)

Several different kinds

(Only need to know about Helper Ts & Cytotoxic Cells)

Helper T-Cells

Can help activate cells that kill invaders

"Calls the shots" in the immune system

Cytotoxic Cells

Kills infected cells

How do Helper T-cells work?

Helper T-cells will have specific receptors that'll only bind to a specific combination of a class 2 MHC & a specific antigen

If combination is met, Helper T-Cell bonds to MHC antigen and gets activated

Replicates & makes memory T-cells

Releases cytokines

Boosts signal that something is wrong in body

Tube Dilution

sample added/ total sample

Final Dilution

(current tube dilution) times (previous final dilution)

Concentration

(previous concentration)times (previous tube dilution)

Single Gene

Caused by changes(mutational) in a DNA sequence. This leads to a denatured

Cystic Fibrosis(Recessive - aa)

• Mutation in the CFTR gene

• Causes Lung Issues/Infertility

Huntington’s(Dominant - AA)

• CAG mutation

• Cognitive Problems

Muscle Dystrophy(Sex Linked - XaXb)

• a group of diseases that cause progressive weakness and loss of muscle mass

Multifactorial

Caused by both environmental(causes genes to malfunction) and mutations in your genes. This affects multiple genes

Breast Cancer

Heart Disease

Alzheimer’s Disease

• Caused by environmental factors and mutations

• Cognitive decline and behavioral changes

• Life expectancy decreases

Chromosomal Mutation

Either missing or having extra copies of a gene. It could also be a damage to a chromosome

• Deletions or breaks or rejoining of a chromosome

• Either adding 1 or deleting 1 chromosome

Down Syndrome

• Trisomy(3 copies) of the #21 chromosome

• Comes with intellectual disabilities and face features are deformed

Huntington’s Disease

• A single #4 chromosome

• Uncontrollable movements and muscle mass loss

Mitochondrial

Mutations in chromosomal DNA(in the mitochondria)

• Passed from the mother gene

Optic Neuropathy

• Is caused by mitochondria DNA mutations

• Causes vision loss which is permanent

Leber Hereditary optic neuropathy

A genetic condition that leads to vision loss due to the degeneration of the optic nerve, often caused by mutations in mitochondrial DNA.

Difference between Diagnostic and Screening Test

Screening tests are used to detect potential health issues or diseases in asymptomatic individuals, aiming for early identification and prevention. These tests are typically applied to large populations, prioritizing sensitivity to catch as many potential cases as possible, even at the risk of false positives. Examples include mammograms, cholesterol checks, and newborn screenings. In contrast, diagnostic tests are used to confirm or rule out a disease or condition in individuals who are symptomatic or who have tested positive in a screening. These tests are more specific and accurate, minimizing false results to ensure a definitive diagnosis, which then informs treatment decisions. Screening tests often lead to follow-up diagnostic testing if abnormalities are detected.

What is Quad Screening

a prenatal blood test done during the second trimester of pregnancy (around 15-22 weeks) to assess the risk of certain birth defects in the fetus

4 things tested..

• HCG

• AFP

• Estriol

• Inhibin A

• Blood drawn → goes to lab

• Look for these proteins/hormones

• If levels too high/low → risk of abnormalities

• Can also check for risk of neural defects

First Trimester Screening

• Occurs between weeks 10-15

• Screen for a protein and a hormone : HCG Hormone or PAPP-A protein

• Done by a blood draw → will be sent to lab, where they measure range of hormone/range (check if levels are too high/low) → if yes, then risk for potential abnormalities

• Done simultaneously with the NT ultrasound (done at same time)

• 85% accurate – 5% false positive

• Early on in pregnancy – making educated guesses to help parents make choices regarding their pregnancies, but it is not 100%

Cell-free fetal (CFF) DNA Analysis(Non-invasive Prenatal Screening)

• Week 10 onwards (can get done anytime later in the future)

• Mom will get blood drawn → sent to lab

• CFF DNA isolated away from mom’s blood sample → testing just the fetus

• Help determine risk of baby having abnormalities, 

  • For pregnancies, separates baby and mother’s blood ( ensures mom doesn’t get rid of 

• Don’t have full picture / all DNA, not a full diagnosi

Chrionic Villus Sampling

• Weeks 10-13

• Diagnose baby with chromosomal abnormalities

• Invasive test → mom comes in , needle put through her transcervical cervix, cut into placenta, CVS is anchor of placenta, where mom/baby info talks to each other, can aspirate (vacuum) up cells that belong to baby, can now pull apart, extract DNA, and analyze baby’s health

  • Transabdominal, needle inserted through abdomen instead

• Next thing that can be determined is paternity (who is the father)

• Risk of miscarriage

• Risk of development issues

Amniocentesis

• What is it, and what does it tell us?

  • A diagnostic test that takes a sample of amniotic fluid from the mother and using the cell DNA that is contained to diagnose certain chromosomal or genetic conditions, such as Down’s Syndrome or Cystic Fibrosis

• When does it get performed?

  • Between weeks 14-20 of pregnancy

  • It isn’t a necessary test, and usually is ordered if a abnormality is detected by an ultrasound. Generally, Amniocentesis is offered if there’s a higher risk that the baby has a genetic condition

    • This could be from prior screenings, a previous pregnancy with a genetic condition, or a family history of a genetic condition

• What happens during Amniocentesis?

  • First, an ultrasound is established to be able to locate the fetus, and gain an image of the amniotic sac(where the baby is located)

  • Then, a thin, hollow needle is inserted through the abdomen and uterus into the amniotic sac, but keeping it away from the fetus

  • The needle then extracts a small amount of the fluid from the sac(called a sample), to be tested for genetic and chromosomal defects

  • The needle is then removed from the abdomen

  • This process only takes 30-45 minutes on average, and the part with the needle usually lasts only 2-5 minutes long!

  • A sample is then sent to the lab for testing, and the results will typically arrive in about two weeks
    

• Risk of miscarriage

• Risk of development issues

NT Ultrasound

• Nuchal Translucency Ultrasound

• Weeks 10-15

• Mom lies on back, gel is applied to stomach and wand is used to display images on a screen

• Looking at (1) size of nasal bone, and (2) fluid volume of the back of the baby’s neck

• Too high/low → indicates risk of chromosomal abnormalities | same if nasal bone not developing properly

Anatomy Ultrasound

• Three names: fetal ultrasound, anatomy ultrasound, obstetric ultrasound

• Mom lies on back, gel is applied to stomach and wand is used to display images on a screen

• Looking at organ formation, size → can determine a baby's age, developing at proper rate, “too small or too big?”, checks heartbeat as well, also to confirm pregnancy

• Occurs throughout pregnancies (confirmation of pregnancies, gestational age, continue through second / third trimester to keep track of development

Karyotype vs Pedigree

• Karyotype:

  • A visual representation of an organism's chromosomes, arranged by size and shape.

  • It shows the number and structure of chromosomes, helping detect abnormalities like extra/missing chromosomes (e.g., Down syndrome).

  • Read by examining chromosome pairs for size, banding patterns, and the presence of 46 total chromosomes (in humans).

• Pedigree:

  • A chart that tracks the inheritance of traits or genetic disorders through a family.

  • It shows relationships and whether individuals are affected, carriers, or unaffected.

  • Read by identifying symbols (circles for females, squares for males), shading (affected individuals), and lines connecting relatives to follow inheritance patterns (e.g., autosomal dominant, recessive, or X-linked traits).

Details on Micropipettes

How to Choose the Right Pipette

1. Determine the Volume You Need: Select a micropipette that covers the range of your desired volume. For example:

   • Use a P10 for volumes between 0.5–10 µL.

   • Use a P20 for volumes between 2–20 µL.

   • Use a P200 for volumes between 20–200 µL.

   • Use a P1000 for volumes between 100–1000 µL.

2. Ensure Precision: Using a pipette close to the middle or upper limit of its range ensures better accuracy than working at its extreme minimum.

Pipette Sizes and Names

• P10: Measures 0.5–10 µL. Commonly used for DNA or RNA samples.

• P20: Measures 2–20 µL. Ideal for small reagent volumes.

• P200: Measures 20–200 µL. Common for general lab solutions.

• P1000: Measures 100–1000 µL. Used for larger liquid volumes.

Steps for Using a Micropipette

1. Set the Desired Volume:

   • Rotate the dial to adjust the volume displayed in the pipette’s window. Ensure the volume is within the pipette’s range.

2. Attach the Correct Tip:

   • Select a tip that fits securely onto the pipette and prevents leakage.

   • Use small tips (yellow or clear) for P10/P20/P200 and larger tips (blue) for P1000.

3. Draw Up the Liquid:

   • Press the plunger to the first stop and insert the tip into the liquid.

   • Slowly release the plunger to draw the liquid up into the tip.

4. Dispense the Liquid:

   • Place the tip into the receiving container and press the plunger to the second stop to fully expel the liquid.

5. Eject the Tip:

   • Press the ejector button to discard the tip into a waste container.

Best Practices:

• Hold the pipette vertically when drawing liquid to ensure precision.

• Avoid blowing air bubbles or over-pipetting.

• Always change tips between samples to prevent cross-contamination.

Details on Gel Electrophoresis

What Is It and What Does It Do?

Gel electrophoresis is a laboratory technique used to separate DNA, RNA, or proteins based on their size. It allows visualization of genetic material to analyze the presence, size, or quantity of specific fragments.

How Do You Properly Load a Well?

1. Prepare the Gel:

   • Use agarose gel with a buffer (e.g., TAE or TBE) to provide an electrically conductive medium.

2. Mix Your Sample:

   • Add a loading dye to your sample to increase its density and make it visible in the gel.

3. Load the Wells:

   • Insert the micropipette tip into the well without puncturing it.

   • Slowly dispense the sample, keeping the liquid steady to avoid overflow or air bubbles.

4. Run the Gel:

   • Place the gel in the electrophoresis chamber, ensuring the DNA moves toward the positive electrode (red).

How Does It Work?

• DNA is negatively charged due to its phosphate backbone. When an electric current is applied, DNA migrates toward the positive electrode.

• Smaller fragments move faster and farther through the gel, while larger fragments move slower, creating bands.

What Do the Bands Represent?

• Position: The distance traveled reflects the fragment size; smaller fragments move farther.

• Intensity: Brighter bands indicate a higher concentration of DNA.

Applications of Gel Electrophoresis:

• Identifying genetic markers.

• Analyzing PCR products for specific gene amplification.

• Detecting mutations or verifying DNA extraction success.

What is gene therapy?

Gene Therapy seeks to modify one or more genes to alleviate the symptoms caused by a genetic disorder. However, the approach varies. 

• INSERT: A functional gene is given to a patient, providing their body with the means to make a functional protein. The defective gene is still present but its effects are masked by the functional gene.

• DISABLE: A dysfunctional gene is disabled, eliminating the impact of the protein.              

• REPAIR: A dysfunctional gene is repaired so that it is able to produce a functional protein.

What is CRISPR?

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a revolutionary genome-editing technology that allows scientists to precisely modify DNA within an organism. It is derived from a natural defense mechanism found in bacteria, where it is used to identify and destroy invading viral DNA

Repeats in CRISPR

Short segments of DNA that are palindromes(read the same left to right)

Has Spacers between each strand of DNA(matches with viral DNA and is unique)

CAS genes

• CAS Genes

  • Makes CAS Proteins

    • Helicases: Proteins that unwind DNA

    • Nucleases: Cut DNA

CRISPR Process

• When Bacteriophage injects DNA…

  • DNA hijacks the cell and becomes embedded in the genome

  • Normally, the cell would kill the internal cell with the bacteria DNA

• Through the Help of CRISPR…

  • CAS transcribes and translates proteins

  • Transcribes proteins(with the help of spacers) to make CRSIPR RNA(crRNA)

  • This combination of crRNA+CAS protein allows for the killing of the bacteria DNA

  • If we don’t have a spacer which can transcribe proteins..

    CAS makes a CAS 1 class protein which..

    • Takes DNA, breaks it down, and copies the DNA into the CRISPR system

CRISPR CAS 9 solves…

• Sickle Cell

• Cystic Fibrosis

• Muscular Dystrophy 

• HIV

What is IVF?

IVF, also known as in vitro fertilization, is a set of procedures that can lead to pregnancy

Process of IVF

1. A patient takes fertility drugs(hormone injections) that allow them to create an excess amount of eggs. The donor then undergoes follicular aspiration to remove the eggs.

2. These eggs are then fertilized with sperm in a laboratory(initial fertilization takes a few hours) and will take 3-5 days overall to complete. 

3. They are then transferred to a patient’s uterus, which will undergo preparation medication for transferring the embryos.

4. Doctors will transfer multiple embryos through a catheter, in hopes of having one of these embryos developing in the uterus.

Why do we use IVF?

1. Infertility: Inability for couples to get pregnant naturally

   1. Blocked Fallopian Tubes 

      1. IVF finds a way to insert embryos into a mother’s uterus while avoiding fallopian tubes 

2. Surrogacy among LGBTQ members 

3. Prevents risk of abnormalities in offspring

What is the purpose of PGT?

Tests to ensure the embryo selected has the correct amount of chromosomes → reducing chance of failed IVF cycle or miscarriage

Types of PGT

PGT-A: preimplantation genetic testing for aneuploidy (extra/missing chromosomes)

PGT- M: preimplantation genetic testing for monogenic disorder (single-gene defect)

PGT-A(How does testing work?)

- Evaluates embryos chromosomes

- Screens for presence of any missing/extra chromosome material

- Cells removed from trophectoderm – (cells that will become placenta )

- Embryo biopsy of blastocysts

PGT-M(How does testing work?)

- Available when there is a known genetic condition in a person/family

- Targets specific gene

- Cells removed from trophectoderm 

- Embryo biopsy of blastocysts

Risks/Advantages of PGT-A

Risks:

• Possible misdiagnosis

• Costly

• Embryo damage (cells removed)

Advantages:

• Improves ability to choose healthier embryos in cohort →success rate↑for single embryo transfer

• Sex selection

• Reduce stressful decisions

What is PGT-M

Purpose: To test embryos for specific inherited genetic diseases caused by single gene mutations (monogenic disorders).

What it detects: Diseases like: Cystic fibrosis Tay-Sachs disease Huntington’s disease Sickle cell anemia BRCA1/2 mutations (in some cases)

Who it's for: Couples who are known carriers or affected by a specific genetic disease Often requires prior genetic testing of the parents and affected family members

Cost of PGT-A and PGT-M

PGT-A;

$4,000-$10,000 ($1,500 for embryo biopsy, $150 for each embryo tested)

PGT-M:

$7,000-$12,000 per IVF cycle

Plasmid

Cell Specificity

Capabilities/Limitations

None

Capabilities

• Can carry genes of any size

• Unlikely to cause an immune response

Limitations

• Doesn’t uptake a plasmid

• Less levels of therapeutic protein production due to the fact that it can’t enter the cell easy

Liposome

Cell Specificity

Capabilities/Limitations

None

Capabilities

• Can carry gene of any size

• Unlikely to cause immune response

Limitations

• Less levels of therapeutic protein production due to the fact that it can’t enter the cell easy

• Does not integrate into cell genome, so the affects are temporary

Herpes Virus

Cell Specificity

Capabilities/Limitations

Nervous Cells

Capabilities

• Can carry a large amount of base pairs

• Carries DNA

Limitations

• Will only effect cells of the nervous system

• Will not retain in the host cells genome, and will be prevalent for long periods of time

• Less levels of therapeutic protein production due to the fact that it can’t enter the cell easy

Adreno-associated- virus

Cell Specificity

Capabilities/Limitations

Dividing and Non-Dividing Variety

Capabilities

• Carries a large amount of base pairs

• Can enter a variety of cells, making it versatile

• Unlikely to cause immune response

• Will not retain in the host cells genome, and will be prevalent for long periods of time

• Less levels of therapeutic protein production due to the fact that it can’t enter the cell easy

Limitations

• Needs a helper virus to replicate

Adenovirus

Cell Specificity

Capabilities/Limitations

Dividing and Nondividing Variety

Capabilities

• Carries large amount of base pairs

• Is efficient at entering cells which causes high levels of therapeutic proteins

• Can enter both dividing and non-dividing

Limitations

• Doesn’t integrate into a cells genome

• Can cause an immune response(deleting the virus)

Retrovirus

Cell Specificity

Capabilities/Limitations

RNA Dividing Cells

Capabilities

• Carries large amounts of bp

• Carries RNA

• Integrates into host cells genomre

Limiations:

• Only infects dividing cells

• Integrates into host cells randomly, which can potentially cause cancer

• Causes an immune response

Lentivirus

Cell Specificity

Capabilities/Limitations

Dividing and Non-Dividing Cells

Capabilities

• Carries large amount of bp

• Can enter both dividing and non dividing

• Enters cells which allow for release of therapeutic proteins

• Will integrate into host cell genome making it long lasting

• Doesn’t cause immune response

Limitations

• Integrates into host cells randomly, which can cause cancer

PCR Materials

DNA Polymerase

Templete DNA

Buffer

DNA Primer

DNA Nucleotides

Thermal Cycler: Amplifies target DNA into millions of copies using PCR

Denaturation

Happens at 95 Degrees Celsius

Template DNA is first separated apart into two strands after exposure to 95 degrees Celsius

• This temp causes the hydrogen bonds in the strand to break

• Is done in 30 seconds the first time around, 10 seconds for any other strands

Annealing

Happens at 55-60 Degrees Celsius

Primers will first target a region to be amplified

• Primers will find a place on opposite ends of a split strand of DNA

• Primers are 20 base pairs long

Primers will anneal(settle) to the denatured strand as the temperature reduces drastically

• Hydrogen bonds start to form