HTl 2

Three classes of hazards

Biological

Mechanical

Chemical

Universal Precautions

Treating all human blood and fluids like they are infectious to minimize risk

Bloodborne Pathogen Standard

From OSHA, uses universal precautions to protect against blood diseases

Engineering and Work Practice Controls required by CAP

Ventilation, altering techniques to reduce exposure, PPE, decontamination, prevention and monitoring employee exposure

Creutzefeld-Jakob Disease

Prion disease that alters protein in the brain. Rapid progression after diagnosis and fatal.

CAP guideline for sterilizing CJD

Autoclave at 132C

Sodium hydroxide

Formic Acid

Sodium hypochlorite

Fixation for CJD

Brain sections fixed in NBF for 48 hours

Whole brain in NBF for 10-14 days

Post fix in phenol formalin

Process as normal

Formalin waste treated with sodium hydroxide

EPA definition for biological waste

Microbiological or culture material

Pathological material

Blood

Sharp objects

Types of mechanical hazards

Sharps, glass, electrical

Right to Know Law

Employees must be informed of chemical hazards and have access to SDS sheets

Hazard Communication Standard

By OSHA, allows for both employees and nonemployees to be aware of hazards in the lab

Laboratory Standard

By OSHA, does not always apply to every lab, must have a chemical hygiene plan

Chemical Hygiene Plan

Documented plan to minimize exposure to chemical hazards.

Formaldehyde Standard guidelines

Time weighted average for 8 hours is 0.75 ppm

Short term exposure limit is 2.0 ppm for 15 minutes 4 times a day

If TWA is 0.5 employees must be monitored every 6 months

Medical Surveillance Program

By CAP, employees who are exposed to formalin higher than action level or STEL or showing symptoms must be monitored

Fire and extinguisher classifications

Class A- dry combustible fires, water based extinguisher

Class B- flammable liquid or gas fires, carbon dioxide or dry chemical extinguisher

Class C- electrical fires, halon extinguisher

Class D- combustible and reactive metal fires, dry powder media

NFPA

National Fire Protection Agency, has regulations on the storage and warnings for flammable chemicals

Compound microscope

Light microscope with two sets of lens, objectives and oculars

achromic lens

reduces chromic aberration to reflect light in the correct angle

Polarizing microscope

used for identifying crystals, pigments, and birefringence elements. Uses cross optical paths

Phase contrast microscope

uses a condenser to observed unstained living cells

Dark field microscope

blocks directly transmitted light and uses scattered or oblique light to observe unstained microorganisms

fluorescent microscope

uses UV light from mercury or halon lamps to observe fluorescent elements

electron microscope

uses electron beam to make highly magnified images, transmission EM used for diagnosing muscle and kidney disease

cryostat maintance

Cleaning thoroughly at the end of the day. Using silicone lubricant on a routine basis. Defrosting and cleaning with 70% alcohol. Covering when not in use.

sliding microtome

Block is held in place and blade moves across it. Used for large sectioning and celloidin blocks. .

clinical freezing microtome

Portable, uses CO2 to freeze tissue. Mostly replaced by cryostats, can transmit airborne disease from CO2

Shapes of blades

Planar concave (very sharp), wedge (what we use), chisel point (more blunt but stable)

Clearance angle

Knife tilt, the angle the blade is positioned at against the block, 3-8*

wedge angle

physical angle of the bottom of the blade, 15-18*

bevel angle

physical angle of the point of the blade, 27-32*

causes of thick and thin sections

improper knife tilt, loose microtome parts, dense hard tissue

cause of crooked ribbons

horizontal block edges are not parallel to knife

cryostat temperatures

-20*C for most tissue

-25*C for fatty tissue

-15*C for brain, liver, spleen, lymph node

OCT for frozen sectioning

Preferred mounting media, pure formulation of water-soluble glycols and resins

open tissue processor

tissue is moved from one solution to the next, more flexible but slower without heat and vacuum, reagents are more exposed

closed system processing

heat and vacuum speeds up processing, reagents are more contained. small and large tissue must be in seperate runs.

flotation bath temp

distilled water 5-10*C below melting point, too high temp can cause parched earth

coat adhesive slides

albumin, elmers glue, gelatin, Sta-on. Protein adhesives can cause background staining

incubators

kept at human body temp 37*C for enzyme reactions

oven dryers temp

Kept just above melting point, 60*C, too high temp can cause nuclear bubbling

IF or frozen freezer temp

-70*C

refridgerator temp

4 to 9*C

How many patient identifies are required on an artifact?

Minimum 2 by CAP

Surgical number, patient name, date of birth, bar code

Revolving stainer

computerized open system stainer, times in solutions vary for wide range of stains (special stain stainer)

Robotic stainer

Most flexible of stainers, limited to specific batch number of slides (IHC Bond stainer)

Linear stainer

Continiously loaded stainer, slides are moved from containers of solutions, limited on procedures (H&E stainer)

Embedding center paraffin temp

2-4*C above melting point, high temperature can make tissue brittle

hypertonic

Higher concentration on outside, cell shrinks

hypotonic

lower concentration on outside, cell swells

isotonic

equal balance of solution

Hydrate conversion formula

grams of non-hydrate/molecular weight non-hydrate = grams hydrate/molecular weight hydrate

Molar solution formula

weight in grams = Molarity x volume in liters x molecular weight

Normal solution formula

weight in grams = (Normality x volume in liters x molecular weight) / positive valence number

Normal solution for liquids formula

mL of liquid = (Molecular weight of liquid x volume per 1000 mL) / (Valency x specific gravity x concentration x normality)

Gravimetric factor

GF = Old dye % / new dye %

GF x old amount in grams = new amount needed in grams

Fahrenheit to Centigrade

C = (F - 32) x 5/9

Centigrade to Fahrenheit

F = (C x 9/5) + 32

Which way to mix acids and water?

Always add acid to water

How often to check solutions before using?

Twice, once when grabbing the solution, and second right before using

Definitions of in vivo, ex vivo, in vitro, in situ, ex situ

In vivo: cells within an intact living organism
Ex vivo: cells out of a living organism, but in natural conditions (skin explants)
In vitro: (in glass) cells cultured in test tubes or petri dishes
In situ: in the original place
Ex situ: out of the original place

What is a micron equal to

Same as micrometer, one millionth of a meter, equal to 1000 nanometers

What is HIPPA stand for

Health Insurance Portability and Accountability Act of 1996, mandates the privacy and security of patient information

Chain of custody

A function of LIS to have real time tracking of who, when, and were had access to a patient artifact

Function of LIS

Laboratory Information System, processes, stores, and manages patient data related to laboratory processes and testing, manages pathology workflow, allows ordering of additional tests,

Does EPA and CDC allow blood to be disposed down the drain?

Yes if the hospital and local plumbing guidlines allow it

autolysis

The natural cell self destruction by enzymes once the cell is cut off from blood supply and nerve connection

putrefaction

The further breakdown of cells by bacteria, can be prevented by antiseptic techniques

Function of fixation

Stops autolysis and preserves tissue morphology, changes proteins into a stabilized structure, stops enzyme functions, readys tissue for processing, enhances staining

additive fixative

Chemically binds itself onto protein charge points to alter the tertiary shape of the protein

nonadditive fixative

acts by removing bound water molecules from proteins which alters the tertiary structure

coagulant fixative

creates a mesh like network which allows fixative to easily penetrate tissue

noncoagulant fixative

creates a gel around the tissue which makes penetration slow

nonadditive coagulant fixatives

alcohols, acetone, acetic acid

additive coagulant fixatives

mercuric chloride, chromic acid, picric acid, zinc and cupric salts

additive noncoagulant fixatives

formaldehyde, glutaraldehyde, glyoxal, osium tetroxide, potassium dichromate

7 factors that affect fixation

temperature, tissue size, volume ratio, time, choice of fixative, pH, osmolality

glacial acetic acid

the concentrated form of acetic acid, freezing point 16*C, preffered form to making solutions

acetic acid fixative affects

Leaves tissue swollen and soft, preserves nuclear detail

Formalin ingrediant ratio

1 part 10% formaldehyde (37-40% concentration) 9 parts water

Millonig formalin

isotonic and buffered solution, primary use for EM tissue storage

alcoholic formalin use

primarily used in tissue processors, both fixative and starts dehydration process

paraformaldehyde

a white insoluble polymeric powder that will form overtime if methaol is not added to solution. Commonly used in EM

formic acid

a black pigment that can form in formalin if solution is acidic

acid hematin

formalin pigment that forms from acidic formalin and will react stronger with bloody tissue

formalin pigment removal

wash tissue with alcoholic picric acid or alkaline alcohol

glutaraldehyde fixation affects

crosslinks proteins, penetrates and fixes slowly, overhardens if fixed too long, common for EM

Can glutaraldehyde be used to fix tissue for PAS stain?

No, glutaraldehyde has free aldehyde groups that will cause Schiff reaction and create false positives

Glyoxal fixative

smallest aldehyde, common in textile fixation, less toxic

mercury pigment removal

nonpreventable, removed by washing tissue in iodine followed by sodium thiosulfate

mecuric chloride fixative

very toxic, does making for good staining quality

osium tetroxide fixative

only fixative that makes lipids completely insoluble. Primarly used as a post fixative for EM

picric acid fixative

only reagant used as both a fixative and stain. does not preserve DNA. fixes glycogen well. causes shrinkage.

potassium dichromate fixative

dissolves dna but preserves mitochondria. leaves tissue soft but shrunken.

chromate pigment

preventable if tissue is moved into alcohol after fixation without washing. removed with hydrochloric acid in alcohol

zinc salts fixative

replacing mercury. can act as a mordant for H&E stain. zinc chloride can not go in processors as it will make a buildup in tubing

formaldehyde-gluteraldehyde fixative

general duel purpose for both routine and EM. gluteraldehyde levels low enough PAS can be preformed

Davidson fixative

NBF or formaldehyde with ethonal and glacial acetic acid. commonly used for eye retinas.

Orth solution fixative

formaldehyde and potassium dichromate. preferred for demonstrating chromaffin granules of the adrenal medulla.

B-5 fixative

mercuric chloride or zinc with formaldehyde. used for hematopoietic and lymphoreticular tissue and nuclear detail