PROCESSING TISSUE FOR EM BUFFERS Fixatives are buffered at pH ____ ____ equivalent to blood plasma or can be slightly hypertonic. ____ are the buffers of choice. ____ improve the fixation process

- 7.2 to 7.6 - Osmolarity - Phosphate buffers - Buffered fixatives

PROCESSING TISSUE FOR EM - Dehydration ____ an embedding material, is not miscible with water Increasing concentration of an ____ just like in routine tissue processing to make sure that the embedding process after dehydration will be okay.

- Epoxy resin - organic solvent

PROCESSING TISSUE FOR EM - most frequently used dehydrating agents - Avoid this dehydrating agent if tissue will be stained with uranyl acetate to prevent precipitation of uranium salts

- Acetone and ethanol - Acetone

[F] Week 15: Electron Microscopy & Molecular Pathology

Provides improved resolution compared to light microscopy
Used by higher laboratories

Electron Microscopy

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It reveals the substructure or ultrastructure of individual cells
- Resolves object around 100 nm apart (200 nm in light microscopy)
- Nuclear features and cytoplasmic structures like mitochondria, golgi apparatus, etc. can be visualized.

Electron Microscopy

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Electron Microscopy

For EM specimen handling, tissues should be?

fixed ASAP

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Electron Microscopy - Specimen Handling

the most sensitive indicators of postmortem change
- This happens when there is no longer a blood supply to a particular tissue

Mitochondria and endoplasmic reticulum

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Electron Microscopy - Specimen Handling

The specimen must immediately be immersed in fixative that is?

pre-cooled to 4C

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Electron Microscopy - Specimen Handling

Tissue to be process in EM should have a size of

1 mm3

milimeter cube

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PROCESSING TISSUE FOR EM

The volume of the fixative should be at least ____ tissue volume, which means that the tissue must be immersed in the fixative.

Fixation

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PROCESSING TISSUE FOR EM

Factors that would affect the fixation of tissues:

Familliarize nalang

Fixing agent buffer (In routine histopathology, we prefer fixatives that are buffered. )
Concentration
Temperature
Duration

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PROCESSING TISSUE FOR EM

Primary fixation with an aldehyde is to?

To stabilize proteins

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PROCESSING TISSUE FOR EM

Secondary fixation in osmium tetroxide is to?

To retain lipids

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PROCESSING TISSUE FOR EM

What is the standard protocol for fixation of tissues in electron microscopy?

Primary fixation with an aldehyde
Secondary fixation in osmium tetroxide

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PROCESSING TISSUE FOR EM

Osmium tetroxide is a fixative that does not free from disadvantages, it must be handled appropriately because the sediments that can be form with osmic acid/tetroxide can lead to ____ of the one utilizing this type of fixative

blindness

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PROCESSING TISSUE FOR EM

What are the fixative concetrations for Glutaraldehyde and Osmium tetroxide

Glutaraldehyde: effective at 1.5% to 4% (Used for EM)
Osmium tetroxide: 1% or 2%

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PROCESSING TISSUE FOR EM

This temperature:
- improves penetration rate, particularly the aldehyde
- reduces fixation time

HOWEVER, increases the risk of autolytic changes

Room temperature

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PROCESSING TISSUE FOR EM

Osmium tetroxide or osmic acid is generally used at what temp?

Room temperature

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PROCESSING TISSUE FOR EM

Time required for optimal fixation depends on:

Familliarize nalang

○ Tissue type
○ Size
○ Fixative
○ Buffer system

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PROCESSING TISSUE FOR EM

A tissue measuring 0.5 to 1 mm3 and fixed in 2.5% glutaraldehyde requires fixation with duration of?

2 to 6 hours

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PROCESSING TISSUE FOR EM

Secondary fixation in 1% osmium tetroxide lasts for (give duration)

60 to 90 minutes

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PROCESSING TISSUE FOR EM

BUFFERS

Fixatives are buffered at pH ____
____ equivalent to blood plasma or can be slightly hypertonic.
*____ are the buffers of choice.
____ improve the fixation process

7.2 to 7.6
Osmolarity
Phosphate buffers
Buffered fixatives

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PROCESSING TISSUE FOR EM

The next step after fixation

Dehydration

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PROCESSING TISSUE FOR EM - Dehydration

____ an embedding material, is not miscible with water
Increasing concentration of an ____ just like in routine tissue processing to make sure that the embedding process after dehydration will be okay.

Epoxy resin
organic solvent

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PROCESSING TISSUE FOR EM

most frequently used
dehydrating agents
Avoid this dehydrating agent if tissue will be stained with uranyl acetate to prevent precipitation of uranium salts

Acetone and ethanol
Acetone

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PROCESSING TISSUE FOR EM

TOF

Clearing step is not required

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PROCESSING TISSUE FOR EM - Embedding

There is infiltration with the use of

Liquid Resin

In embedding in routine processing, molten paraffin wax is most commonly used.

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# PROCESSING TISSUE FOR EM - Embedding * Placed in a mold filled with ____ and polymerized with heat * ____ are the embedding medium of choice but we can also use acrylic resins

- Resin - Epoxy resins

# PROCESSING TISSUE FOR EM - Ultramicrotomy used exclusively for electron microscopy

ultrathin microtome

# PROCESSING TISSUE FOR EM - Ultramicrotomy what blades/knives are used for ultrathin microtome

glass knives and diamond knives to cut semi-thin sections prior to thin sectioning.

# PROCESSING TISSUE FOR EM - Ultramicrotomy * Through fluids: used in section collecting ____ or ____ (not with diamond knife) * Block trimming: ____ or ultramicrotome

- 10-15% ethanol or acetone - Manual

# PROCESSING TISSUE FOR EM - Ultramicrotomy - Needed if larger viewing areas are required - Plastic films from collodion, Formvar, Butvar

Support films

# PROCESSING TISSUE FOR EM - Ultramicrotomy Ultrathin sectioning - Sections are cut with optimal resolution and specimen contrast (TOF) - ____ sections (80 nm) are recommended

- T - Silver to straw-colored

# PROCESSING TISSUE FOR EM - Ultramicrotomy * Collect sections with a grid immerse in the ____ * Grid placed on filter paper in a open container, dry completely before staining (TOF)

- floatation fluid - F (Lidded not open)

# PROCESSING TISSUE FOR EM To **increase capacity** of selected structural elements to *scatter electrons* and *give contrast*

Staining

# PROCESSING TISSUE FOR EM - Staining It is not like the routine H&E staining, where you get colors of pink, bright eosinophilic color, purple, or blue. This one involves only this color

shades of gray.

# PROCESSING TISSUE FOR EM - Staining Staining can be done at several points, what are those?

- During **secondary** fixation - When **uranyl acetate** is used during the post-fixation - By staining the sections with **lead and uranium salts**

# Diagnostic Application of Electron Microscopy * ____ immune complex deposits * Diagnosing ____ tumors * Detection of ____

- **Renal disease** (immune complex deposition is demonstrated using electron microscopy) - malignant - Non-neoplastic diseases

What are the molecular test for?

* DNA and RNA Test/Assays * Evaluation of Human Disease Conditions * Part of Therapeutic Decisions

# MOLECULAR BIOLOGY DEVELOPMENT coined the double-stranded DNA and the helix pattern, to which according to them, each DNA strand has discrete units known as bases.

1953: James Watson & Francis Crick

# MOLECULAR BIOLOGY DEVELOPMENT verified the discovery of restriction enzymes by Werner Arbe

Late 1960s: Hamilton Smith:

# MOLECULAR BIOLOGY DEVELOPMENT developed the southern blot system. It is not because of an area (e.g. western blot), but it is actually discovered by him. The succeeding blot procedures are named after it.

1975: Edwin Southern

# MOLECULAR BIOLOGY DEVELOPMENT developed the polymerase chain reaction (PCR)

1985: Kary Mullis

What are the molecualr pathology application

* Tumor pathology * Microbiology * Pharmacogenomics * Prenatal diagnosis * Carrier testing * HLA typing * Identity assessment

# MOLECULAR PATHOLOGY APPLICATIONS Detection of tumors, be it benign or malignant, molecular pathology is used.

Tumor pathology

# MOLECULAR PATHOLOGY APPLICATIONS Detect bacterial (tuberculosis) and viral (COVID) infections

Microbiology

# MOLECULAR PATHOLOGY APPLICATIONS Molecular pathology is also utilized in treatment and management of patients to develop medications (e.g. chemotherapeutic agents)

Pharmacogenomics

# MOLECULAR PATHOLOGY APPLICATIONS To see whether a congenital anomaly is expected or a genetic disease will be observed in the baby

Prenatal diagnosis

# MOLECULAR PATHOLOGY APPLICATIONS Organ transplantation and other disease processes

HLA typing

# MOLECULAR PATHOLOGY APPLICATIONS Paternity testing (identity of mother or father)

Identity assessment

It is the flow of information from DNA to RNA to proteins

Central Dogma

is the process by which a molecule of DNA is duplicated, afterwards, the message is transcribed into RNA.

DNA replication

the process of copying information from DNA into new strands of messenger RNA (mRNA)

Transcription ## Footnote From nucleus, it will now go to the cytoplasm wherein translation

the process of forming a protein molecule at a ribosomal site of protein synthesis from information contained in messenger RNA

Translation

# Basic Component of Genetic Information The unit of DNA or RNA consisting of sugar, Phosphate, Base

Nucleotide

# Basic Component of Genetic Information What are the components of nucleotide

- Sugar - Phosphate - Base

# Basic Component of Genetic Information What are the nitrogenous bases?

- Purine - Pyrimidines

# Basic Component of Genetic Information Nitrogenous Bases - Adenine and Guanine - Cytosine, Thymine (DNA), Uracil (RNA)

- Purine - Pyrimidine

What are the pairings of Nitrogenous bases

A = T or U G = (triple bond) C

* The hereditary material in humans and almost all other organisms * located in the **cell nucleus**

DNA (DEOXYRIBONUCLEIC ACID)

A small amount of DNA can also be found in the?

mitochondria (mitochondrial DNA or mtDNA)

- Usually of maternal inheritance is the main contributor - Important because there are certain genetic diseases that are inherited only from the mother and not the father,

mitochondria

Human DNA consists of about how many bases?

**3 billion bases** and 99.9% of those bases are the same in all people

DNA bases pair up with each other (A with T and C with G) to form units called?

base pairs

Nucleotides are arranged in two long strands that form a spiral called a

double helix.

* Single- stranded molecule similar to DNA * Comprised of the same nitrogenous bases except, thymine is replaced with uracil (Adenine (A), uracil (U), cytosine (C), guanine (G))

RNA (Ribonucleic acid)

# STANDARD PROTOCOL FOR FIXATION OF TISSUES IN ELECTRON MICROSCOPY - Carry genetic information from chromosomes to ribosomes. - With ribosomal polypeptide to form ribosomes - Deliver amino acids to the ribosomes in order for proteins to develop.

- Messenger RNA (mRNA) - Ribosomal RNA (rRNA) - Transfer RNA (tRNA)

* It aids in diagnosis & treatment of patients * Utilizes **nucleic acid** (DNA or RNA) probes to assess intact cells * Applications: ○ Detect cancer cells in cytology specimens ○ Detect chromosomal alteration that predict prognosis

In situ hybridization (ISH)

Typically, ISH is most commonly performed with?

fluorescently labeled probes (FISH)

# IN SITU HYBRIDIZATION (ISH) There are also some with probes labeled with a chromogen, known as

Chromogenic in situ hybridization (CISH)

# IN SITU HYBRIDIZATION (ISH) PRINCIPLE: - DNA is broken down into two through ____. Then, a probe is applied. - If the sample DNA is specific with the base pairing of the known DNA, the probe will ____. - Using FISH, the sample DNA will be visualized through a microscope. However, when the base pairings are not compatible, it will not hybridize. Thus, it will not form any ____.

- denaturation - hybridize - Reaction

# IN SITU HYBRIDIZATION (ISH) defined as breaking apart DNA or DNA-RNA hybrid strands using heat. It is also known as the “melting” of DNA.

Denaturation

# IN SITU HYBRIDIZATION (ISH) is a process known to bring two complementary strands of DNA or DNA-RNA back together.

Renaturation/Annealing/Hybridization

# IN SITU HYBRIDIZATION (ISH) labeled complementary single strand where nucleotides anneal or hybridize.

Probe

What are the advantages of ISH over immunohistochemistry

1. High degree of specificity 2. DNA and RNA are not sensitive to formalin fixatives 3. Probe target hybrid is stronger than Ab-Ag complex 4. Provides alternative means of detection when Abs are not available 5. Provides diagnosis at the molecular level

# ADVANTAGES OF ISH OVER IMMUNOHISTOCHEMISTRY IHC is about antigen-antibody interaction; base pairing is superior to Ag-Ab attachment; ISH is more specific

High degree of specificity

# ADVANTAGES OF ISH OVER IMMUNOHISTOCHEMISTRY - They can collect tissues or materials and extract DNA from them without being sensitive to formalin fixative - Duration of fixation in IHC would affect Ab and Ag

DNA and RNA are not sensitive to formalin fixatives

# ADVANTAGES OF ISH OVER IMMUNOHISTOCHEMISTRY In certain disease processes (infection), there are instances of very low concentration of antibodies, therefore, it will not be detected using IHC even if there are a few antibodies; this will produce a false positive result.

Provides alternative means of detection when Abs are not available

What are the forms of ISH?

* Chromogenic ISH (CISH) * Fluorescent ISH (FISH) * Polymerase chain reaction ISH (PISH)

what are the phases of ISH that can be applied to?

metaphase and interphase

# ISH REAGENT PREPARATION * Mostly purchased ____ or ____ for easy mixing (in a hospital setting) ○ Convenient and safer to use ○ Lesser clerical or human errors * If in a budget, you can ____ reagents to save money

- pre-mixed or in a kit - manually prepare

# PROBES USED IN ISH * A ____ used to find its complementary sequence * The choice is based on the type of ____ to be detected * ____ depends on the degree of substitution and the size of labeled fragments * Probe with ____ has the highest sensitivity

- labeled bit of DNA or RNA - sequence - sensitivity - 25-32% substitution

what are the types of Probes used in ISH?

- Oligonucleotide probes - Single-stranded DNA probes - Double-stranded DNA probes - RNA probes

# TYPES OF PROBES (ISH) * 20-50 bases * Resistant to RNAses * Small as it easily penetrates tissues or cells * Covers fewer targets only * Single stranded

Oligonucleotide Probes

# TYPES OF PROBES (ISH) * Larger (200-500 bp) * Prepared by primer extension on single stranded templates by RT-PCR of RNA or by amplified primer extension of a PCR generated fragment

Single Stranded DNA Probes

# TYPES OF PROBES (ISH) * Denatured prior to hybridization * Less sensitive than single stranded probes * Tendency to rehybridize to each other * Not widely used

Double Stranded DNA Probes

# TYPES OF PROBES (ISH) * Thermostable and resistant to digestion by RNAse * Single stranded * Most widely used

RNA Probes

# RNA PROBE * A stretch of RNA that can detect a target sequence in the ____ * Probe and target base sequences must be non- complementary to each other (TOF)

- Genome - F (non-complementary)

# PROBE PREPARATION AND LABELING FOR ISH Attach a detectable label to the probe before?

hybridization

# PROBE LABELING (ISH) What are the method of Labeling

1. **Direct**: reporter molecules are **directly** attached to the DNA or RNA. 2. Indirect: a **hapten** (connector) is attached to the probe and detected by a labeled binding protein then attached to the DNA

# PROBE LENGTH - Weaker signals - Penetrate less effectively - Non-selective binding

Longer

# ISH SAMPLE PREPARATION can be an initial step or intermediate step

Fixation

It an important step since we want to get rid of unnecessary protein that might interfere in the detection of DNA/ RNA

ISH Proteolytic Digestion

# ISH PROTEOLYTIC DIGESTION * Improves probe penetration by increasing ____ * Minimal tissue ____ is observed * Commonly used proteolytic agents are ____ and ____ * ____ is the negative control

- permeability - degradation - Proteinase K and pepsin - Nuclease digestion

# ISH PROTEOLYTIC DIGESTION * ____ for bone, cartilage, kidney and brain * Post fixation in ____ prevents tissue loss, prevents leaching, inhibit RNAse activity

- Proteoglycan digestion - 4% paraformaldehyde

This is the cooling process after denaturization in the presence of complementary probe

Hybridization Denaturization → apply probe → hybridization process in a lower temperature

Results in H bonding of two strands of nucleic acid

Hybridization

# HYBRIDIZATION * RNA and DNA hybrids are formed optimally at ____ below the melting point * Longer probes and ____ content are more stable * *Increased temperature and formamide concentration are ____

- 25C - higher G+C - destabilizing factors

# ISH CONTROLS TOF Positive and hybridization control should always be included

F "no hybridization control"

# EQUIPMENT AND REAGENT PREPARATION * ____ in skin degrade naked DNA and RNA * Wear gloves, additional precautions to ensure absence of ____ in solutions used for hybridization

- Nucleases - nucleases

* Analysis of human DNA, RNA, chromosomes, proteins or metabolites * To detect inherited or disease related genotypes, mutations, phenotypes, or karyotypes | clue: fluoresces

FISH UTILIZED IN GENETIC TESTING

what are the methodolog of FISH

1. **Fixation** of DNA (metaphase or interphase) 2. **Denaturation** in situ >> single stranded 3. This target **DNA is hybridized** to specific DNA probe sequences labeled with fluorochromes to allow detection

FISH Probes, what are the categories included?

1. Repetitive sequences 2. Whole chromosome sequences 3. Unique sequences

Probe labeling for FISH are generally directly labeled as?

Green, red, blue, gold

# TISSUE TYPES * Can be ____ * Advantage is that it does not require ____ or cultured cells * Can be applied to ____ or non-dividing cells * Can be performed on paraffin block sections, disaggregated cells from peripheral blood, touch preparation from LN (lymph node) or solid tumors. (TOF)

- Varied - metaphase spreads - interphase - True

What are the Clinical Applications of FISH

* Used for both **constitutional** and **acquired chromosomal analyses** * Microdeletion syndromes, sub telomeric rearrangements, prenatal studies, tumors * Denaturation in situ = **single stranded**

# GENERAL SCORING ANALYSIS CRITERIA Metaphase: 1. Only complete ____ scored 2. Analysis of ____ o Having only 1 may result in inaccurate results 3. Record coordinates and results of at least ____ spreads (2 images captured for abnormal; 1 image if normal) 4. In ____, additional metaphase cells are counted.

1. metaphases 2. 10 metaphase cells 3. 2 representative metaphase 4. mosaicism

# GENERAL SCORING ANALYSIS CRITERIA Interphase scoring: 1. At least ____ cells (monolayer) 2. Discrete ____ 3. A signal is counted as ____ (fusion of orange and green) if they are on top of each other.

1. 200 individual interphase 2. signals 3. Yellow

[F] Week 15: Electron Microscopy & Molecular Pathology - Part 1 Flashcards

(106 cards)