Histology Flashcards
All information that was taught to me while attending Vanier College's "Animal Health Technology" Program, located in St-Laurent Montreal.
What is histology
study of the microscopic anatomy of cells and tissues of plants and animals~sectioned, stained and mounted on a microscope slide~enhanced through the use of histological stains
What is histopathology
the microscopic study of diseased tissueimportant tool in anatomical pathology
What are the two types of histological samples that can be taken
from a dead animal (necropsy)from a live one (biopsy)
What are the components of the histology lab
Fixation of tissuesDehydration, clearing and embeddingSection cuttingParaffinCryostatStaining sections and mounting coverslipsPhotomicrography
What is the goal of histological techniques
evenly sectioned (no thick and thin areas),nicely stained (not over or under stained) with the appropriate stainno artifacts (rips, tears, air bubbles, bits of dirt or crystals of stain)in a condition to last many years (properly fixed, coverslip properly mounted)
Describe fixation of the tissues
To fix the physical state of the cells, as well as the chemical state. Allows for the subsequent treatment of the tissue with minimal damage and alteration of the tissue. Must not interfere with cell components that are active in staining (or they won’t stain)
What are characteristics of a well fixed slide
good nuclear and cytoplasmic morphologyminimal shrinkage clearly defined basement membranes and cell margins.
What are the characteristics of a badly fixed slide
Inferior nuclear and cytoplasmic morphologyexcessive shrinkage and poorly defined cell margins
What is very important for the technician for fixation of cells
Kills microorganisms~prevents tissue deterioration~protects the technician handling the tissue from pathogens which might be present.
What are examples of chemical fixatives
Formalin: most commonSafe-fix: less toxicOther:Special for electron microscopyCryostat
During fixation can tissue deteriorate?
Yes.Tissue can deteriorate very rapidlyE.g. Bone marrowMetabolically active deteriorate fastestKidney, liver, pancreasShould be removed first
What is an alternative form of tissue fixation
An alternative method of tissue preservation is perfusion of the animal (often used in research). Removal of blood from organs by perfusing with physiological salineImmediate entry of the fixative (normally 10% formalin) into all the blood vessels
What are the necessary components of dehydration, clearing and embedding
Gets the tissue ready for sectioning (except for sectioning in a cryostat)We are embedding the tissues in waxMust remove water from the tissues (replacing with a fluid soluble in both water and wax) (dehydration and clearing). Cannot be done all at once (would damage the tissue), so series of steps. Each step is at least one hour, so the process is automated.
How do you dehydrate tissue sample
Remove waterUsing alcohol in increasing concentrationMakes the tissue firm for cuttingPrevents shrinkage in paraffin
How do you clean the tissue sample
Replaces the alcohol with a liquid compatible with paraffinIncreases tissue transparencyToluol and xylene are mostly used
How do you embed the tissue sample
Makes tissue firmer to prepare for actual block preparationThis last step needs to be timed so that the tissue can be removed as soon as it is ready
Why do we trim the tissues before embedding
smaller pieces should be cut for treatment, so that a cut surface suitable for sectioning is prepared. This is also a time when excess fatty and connective tissue can be cleaned off the outside of an organ.
Describe block embedding
Done at the embedding machine, which has a liquid paraffin dispenser. set at 56C-60CA thin layer of paraplast (liquid) is put in plastic embedding mold.The paraplast impregnated tissue is then addedThe paraplast should have cooled slightly, just starting to form a thin skin, when the tissue is placed in it.
What is the microtome and what is it used for
machine equipped with a very sharp knife and a mechanism to advance the tissue in very small increments. Tissues are normally sliced at 6 - 10 microns. We use a rotary microtome, which uses a wheel to advance the tissue block.The knife should be clean, sharp and cold for best sectioning. Sharpen the knife and store it in the freezer (or overnight) before use.
What are the steps for cutting things with a microtome
Fasten block Adjust for straightnessBring forward blade so block is almost or just touching. Cut (advance to tissue) (10-15 microns) until a whole section is being taken. When ready to take good sections, be sure the cutting part of the knife is sharp (reposition it if necessary). Reduce thicknessTurn the wheel slowly and smoothly to get even sections (hard tissues can be cut more quickly). The sections should come off in a ribbon.
How do you float tissues
The ribbon is placed on paper or in box, and the sections are cut apart and transferred to a floating-out bath which contains a very weak gelatin solution at about 45C .Alternately a ribbon can be transferred to the bath and then the sections separated. Ribbons need to be spread out.
Describe staining tissues
The goal of a stain or stains is to allow examination of the various characteristics and relationships of the cells.Different tissues, and different cell components, attract different dyes and stains. Hematoxylin and eosin, the most commonly used pair of stains, are attracted by different cell components,
Describe H/E staining
Slide must be dryStains are in solutionFor paraffin-impregnated tissue sections, the wax is removed by xylene, then the tissue is rehydrated by moving from 100% to 95% to 80 or 70% alcohol. The tissue is then placed in water before staining. After staining the tissue section is dehydrated with 95% and 100% alcohol, and the alcohol is removed by xylene. The slide is now ready to have its coverslip mounted.
what are the 2 main functions of the mounting medium
It has a refractive index close to that of glass so that the tissue and the slide match It protects the tissue from physical and chemical injury.
What are some types of artifacts on a slide
Air bubblesfold of tissuesknife crackshairdebris
What is a laboratory diagnosting test dependant on
Good historyQuality of sampleProper identification of sample
How do you choose what sampling technique to use for histology
Anatomic locationPatient’s overall healthSuspected tumor typeClinician’s preference
What are the pretreatment biopsy types
Needle core biopsyPunch biopsyWedge biopsy
How do you obtain additional information about a tumor
treatment planning (surgical, medical)
What is excisional biopsy
Surgical removal of the tumor
what is a post treatment biopsy method
excisional biopsy
How do you obtain a more complete picture about a growth
GradingLymphatic/vascular invasionMargins
Is a biopsy a good first step?
No
What is the disadvantage to not doing a biopsy first when a lump is removed
can result in incomplete removal, more morbidity and costs
What are the advantages to pre-treatment biopsies
Can help clients make an informed decisionCan consult with oncologist and surgeonCan plan treatment sooner after surgery
When are pre-treatment biopsies not indicated
Treatment or Sx would not change (spleen, testicle)As risky as removal (spinal cord)
What are needle core biopsies done on
external palpable masses (no highly inflamed or necrotic)deep (kidney, liver)
Describe the needle core biopsy punch
manual or spring/pneumatic poweredSmall sample size still enough for pathologic exam
what is the size of the needle core biopsies needle
1 mm wide biopsy1.0 – 1.5 cm long
What does a needle core biopsy require
local anesthesia and sedationsterile preparation
Why do you use a small scalpel incison for the needle core biopsy
Prevents dullingFacilitates tru-cut mechanismCan be sutured
How do you handle the tissue from the needle core biopsy
Tissue can be removed with blade, needle or salineCan be rolled on glass slide for cytologyPlace in formalin (in cassette)
What is a possible risk when you do a needle core biopsy
minimal risk of seeding but you should plan ahead and remove original incision tractconsider hemorrhage and fluid leakage
Why do you use a punch biopsy
Typically for skinSkin, oral, perianalDirect access with laparoscopyLiver, GIT, etc.
What is the size of a punch biopsy
2-8mm
What is required for punch biopsy
local anesthesia and sedationusually no sterile preparation
what is the ideal size of a punch biopsy
6mm4 mm only for nose, footpad8 mm slight more chances of infection
What can cause tissue compression and artifacts when doing a punch biopsy
dull punches
how do you handle a tissue sample from a punch biopsy
handle sample very gentlyplace in formalin, no cassette
what is important for punch biopsies if you’re doing dermatology
draw line in direction of hair
When do you do an incisional biopsy
When cytology and/or biopsy is unsuccessfulFor ulcerated and necrotic lesions (larger sample)
What do you need to do for an incisional biopsy
Surgical preparation + drapesLocal anesthesiaTumors are usually POORLY innervatedSkin is incised and tumor wedge removed
Is it necessary to remove intact skin with the incisional biopsy?
NOT necessary to remove intact skin (next or over)Margins evaluated with removal of tumorCan compromise Careful not to sample just the reactive tissue surrounding the tumorImprint cytology can be done
Describe endoscopic biopsy
Convenient, cost-effective, safeLimited sample, inadequate visualization
Describe laparoscopy, thoracoscopy
Very good, can always convert to laparotomyNeeds specialized tools & skills
How do you properly identify margins
tissue ink is preferred but sutures can also be used.need to write: color = which margin.
when should tissue ink be done
before fixation, to orient the sample and identify areas of concern
describe the tissue inking process
Tissue should be blotted with paper towel beforeInk can be applied with gauze on surface or cotton swab for precisionAllow to dry 20 min before formalin
describe proper tissue fixation
10% buffered neutral formalinSpecial fixative for eyes, testicles1 part tissue to 10 part fixativeIdeally 1 container per lesiondone within 30 minutes
describe containers for fixation
Wide container, secure lidNo glassNo more than 1LIn secure plastic bag (ziploc)With absorbent packing materialInsulated (avoid freezing)
Descriibe large unusual specimen fixation
Amputated limbs, spleenCan overnight on iceCan pre-fix 48-72h with partial parallel incisions approximately 1 cm apart (‘‘bread loafing’’)Then shipped without formalin (double-bag) or in 1:1 formalinCan section and fix/send in individual containersAn annotated digital image or sketch of the original specimen to depict sectioning and orientation should accompany the samples
describe very small unusual specimen fixation
Labeled cassettesDo not use gauze sponges or cardboard because tissue may become compromised upon retrieval
describe luminal organs unusual specimen fixation
Flush the intact lumen with formalinPartial longitudinal incision3 labeled sections (cranial/proximal, mass and caudal/distal) can be submitted
describe thin flat sample fixation
Small: placed in a tissue cassette with a foam pad to minimize tissue curlingLarger samples can be tacked onto a flat piece of cardboard presoaked in formalin or water with suture through edges of tissue not needed for examination
What do you need to write about the lesion in the mass submission form
Lesion-specific clinical history (eg, anatomic site, date first noticed, rate of growth)Potentially lesion-associated clinical signs (eg, lameness, vomiting)Type of lesion (eg, new lesion, recut following incomplete excision, excisional biopsy following previous incisional biopsy, local recurrence)Results of prior lesion-associated diagnostic tests - cytology, prior biopsy reports, imaging (radiographs, ultrasound, MRI, CT); access to radiographs may be especially important for bone and gingival tumors.
What general information do you need to put on the mass submission form
General clinical history—previous neoplastic diseases, previous or current nonneoplastic conditions of relevanceTreatment history—local and systemic, current and previous (eg, chemotherapy, radiation, corticosteroids)Previous unrelated treatments or potential tumor-inducing historical events at tumor site (eg, previous radiation, vaccination, implants)
What other abnormalities should you include on the mass submission form
CBC, biochemical, and hormonal (eg, hyperinsulinemia) abnormalities
What else should be on the submission form
Working clinical diagnosis and/or list of differentialsThorough gross lesion description.Indication whether the submitted sample is an incisional or excisional biopsy. Excisional biopsy indicates assessment of surgical margins is necessary, whereas for incisional biopsies the margin evaluation is null. Anatomic site should be thoroughly describedFeatures appreciated during diagnostic imaging or perioperatively should be describedtissues involved or associated with the mass (eg, thyroid mass invading subjacent skeletal muscle).
What is cytology
examination of cells having exfoliated from tissue or having accumulated in body fluid
what can cytology provide
May provide definitive diagnosisVery dependent on sample quality!
is cytology invasive?
Not invasive
How quickly does cytology need to be processed
rapidly
What should be in the cytology kit
ClippersCleansing & disinfectant wipesSyringes: 6 – 12 ml, up to 20 mlNeedles: 1 – 1.5 in (20g to 22g), 2.5 – 3.5 in spinal needle with styletBone Marrow aspiration needle & core biopsy materialScalpel bladesCulture swabs & applicator sticks for slide preparationBox of slides (frosted)EDTA and red top tubesRigid, flat surface for 6 -10 slides (foam tray)Butterfly catheter, IV extension tubingPencil or slide markerSterile EDTA
What is the skin prep for a FNA
Minimal for cutaneous, subcutaneousShave, clean & disinfect for internal
Where can you do an FNA
Cutaneous, subcutaneous, internal organs
What are the benefits of going needle only, vs aspiration
Should start with no aspirationLess blood contamination
What is the best FNA size needle
22g
What do you do if a FNA sample is liquid
place in edta
what do you do if an FNA sample is solid
prepare slides immediately
Describe imaging guided aspiration
typically ultrasound guidedcomplications are rarethoracic samples can be taken.
Should ascites and pleural effusion be sampled via US
yes
Describe the squash preparation
Most commonFor semi-solid, mucus-like, or pelleted (via centrifugation)Place sample close to frosted edge and use second slide to spread sample
What do you do with fluid cytology samples
Keep in EDTAPrevents clotting (fibrin)Preserves cellular morphologyFacilitates cell countRefrigeratedUp to 24h in generalCSF needs special measuresMake slide as soon as possibleSend unstained slide with EDTA tube
How do you prepare a fluid slide if the sample is cloudy
direct smear (like blood smear)squash
how do you prepare a fluid slide if the sample is clear
If lower cellularityNeed to concentrateSimilar to urine sedimentVia special centrifuge (CSF, BAL)Buffy CoatStill do a direct smear
What hematology methods can you use with cytology fluid
Cell countsCan be automated (hematology machine)Flush with saline afterManuallyTotal proteinsRefractometerConversion tables for low TP
Describe the touch imprint
Permits evaluation of a biopsyWith surface lesions is often of poor diagnostic utilitySuperficial inflammation, secondary bacterial infectionException for fungal diseasesNeed to blot aggressively the sampleUntil tackyThen imprint on slideFibrous lesions can be scrapedScalpel blade
How do you prepare joint-synovial fluid cytology
Surgical preparationNormally viscousIdeally small EDTA tubeTo get cell countTo be able to use hyaluronidazeDirect smear (like seen previously)Culturette for microbiology
What are miscellaneous types for doing cytology
Always air dry – do not use flameDo not freezeDo not expose to formalinLabel properly all tubes & slidesAlways submit an unstained smear with a tube (also for hematology)
When do you do bone marrow evaluation
Bone marrow evaluation is indicated when peripheral blood abnormalities are detectedpersistent neutropenia, unexplained thrombocytopenia, poorly regenerative anemiaTo stage neoplasiaLymphoma, plasma cell tumor, mast cell tumors, other
What is the difference between aspiration and core biopsy
Aspirates are easier, faster, and less expensive to perform than are core biopsies. Bone marrow core biopsies require special needles. Core biopsy sections provide a more accurate way of evaluating marrow cellularity and examining for metastatic neoplasia than do aspirate smears, but cell morphology is more diffcult to assess.
How can you classify inflammation
purulentpyogranulomatousmacrophagiceosinophiliclymphocytic
Describe purulent inflammation
predominance of neutrophils (>85%)Try to say if degenerated or not
Describe pyogranulomatous inflammation
mix of neutrophils and macrophages
Describe macrophagic inflammation
predominance of macrophages (>50%)
Describe eosinophilic inflammation
important component of eosinophils (>10 – 30%)
describe lymphocytic inflammation
need to rule out lymphoma
What does it mean if you have degenerate neutrophils in your purulent inflammation
Degenerate neutrophils:Bacterial infectionsNeed to see to call septic
What are the causes of degenerate neutrophils in purulent inflammation
Degenerate neutrophils:Immune-mediatedNeoplasticSterile irritants (bile, urine)
What are granulomatous lymphocytes associated with
Foreign bodyFungal infectionMycobacterial infectionPanniculitisLick granulomaOther chronic lesions
what is eosinophilic inflammation due to
Eosinophilic granulomasHypersensitivityParasitesFungalMast cell tumorsSome neoplasms
what is lymphocytic inflammation due to
RareImmune reactionViralChronic lesion
What is the infectious agent blastomycosis associated with
Pyogranulomatous or granulomatousDogs mostly“hunting”Nose, legsFound in the environment
What is the infectious agent cryptococcus associated with
GranulomatousDogs & CatsNose
where is the aspergillus fungi found
OpportunisticDog: noseHorse: cornea
Describe mycobacterium
Fairly rareGranulomatousVery slow to grow in microbiology
How do you classify a neoplasm
need to say the cell typeif it is benign or malignant
what are the 4 cell types of neoplasms
epithelial cellsmesenchymal cellsround cellsneuroendocrine cells
how do you classify if a cell is benign or malignant
Set of criteriasCytoplasmicNuclear
what are the cellular features of malignancy
Cellular Crowding Pleomorphism Different shapesAnisocytosisDifferent cell sizeGiant cellsBasophiliaHigh N/C ratio (nuclear/cytoplasmic)Not always
what are the nuclear features of malignancy
Nuclear Nuclear moldingMore than 1PleomorphismeAnisocaryosisWithin a cell alsoMitotic figureNumber and shape
what are the nucleolus features of malignancy
multiplevaried within one nucleusmay be normal
Describe the organization of mesenchymal cells
Weak cohesion, loosely arrangedOften extra-cellular matrix
describe the cellular types of mesenchymal cells
Cellular typesEg. fibroblasts, osteoblasts, chondroblasts…
describe the morphology of mesenchymal cells
Morphology Spindled, stellate, ovalPoorly defined cytoplasmic margins
describe the exfoliation for mesenchymal cells
ExfoliationModerate to weak
describe the organization of epithelial tumors
Cohesive clusters
describe the cell types of epithelial tumors
Glandular and parenchymal tissueSurface liningEg. Basal cells, squamous cells, hepatocytes, tubular cells, renal cells
describe the morphology of epithelial tumors
Variable: round to polygonal +/- elongatedDistinct cytoplasmic borders
describe the exfoliation of epithelial tumors
very easy
What are all the round cell types
LymphomePlasmocytomeMastocytomeHistiocyteSarcome histiocytaire Transmissible veneral tumor
What does it mean when you see lymphoglandular bodies
Cytoplasm fragmentMostly seen with lymphocyteslymphoma
what does it mean when you see collagen breakdown
Mostly seen in mast cell tumorsSome soft tissue sarcoma
what does it mean when you see a hematoidin crystal
Hematoidin crystalHemoglobin breakdownIndicates chronic bleeding
what does it mean when you see cholesterol crystals
Cholesterol crystalsCell membrane damageFrequent inFollicular cysts
what does it mean when you see skeletal muscle
Skeletal muscleNormalIncidental finding
How do you histologically recognize the liver
Solid tissue, characterized by cuboidal cells in linesMay or may not see cortex of thin connective tissueLittle C.T. , so not good for trichrome stains
How do you histologically recognize the spleen
Solid tissue like liver, H and E, stains darkVarious cell types, notice patches of colors at high power differentiating white and red pulp
How do you histologically recognize the brain
Solid tissue, look for characterictic grey and white matter, and Gyri (round, curving areas)Cerebellum is also helpfulDifficult to get a good cut, so keep trying
How do you histologically recognize the kidney
Remember, nephron- look for glomeruliAppear as nest like structures, near cortex (outside)Also easy to ID renal pelvis
How do you histologically recognize the intestines
In general, all should be cross-sectional, and have a lumenDuodenum, jejenum and ileum all resemble each other, just differentiate by looking for peyers patches, length of villi..etc…
How do you histologically identify the colon
Slightly different with larger villi, and more prominent muscularis layer for larger peristaltic action
How do you histologically identify other tubular structures
Differentiate uterus from esophagus/trachea from intestinal structuresUterus has more CT, and large endometrium, myometriumUterus Great for trichrome
How do you histologically recognize esophagus/trachea
Usually can pick up cartilage cells on trachea, large and clear. Esophagus often collapsesUsually the 2 are together
How do you histologically identity lymph nodes
Solid structures, with white and red pulp like spleenDifferentiate by shape, and amount of red pulp in circles
how do you histologically identify glands
All glands resemble each other by having acini filled with somethingDifferentiating salivary versus thyroid versus prostate gland is challenging.
How do you histologically identify ovaries and testis
Look for characterized cells of eachOvaries have ova at different stages, CL is also easy to see Look for the hilus
How do you histologically identify testis
Look for seminiferous tubules which take up most of the structure, and will have sperm in them
How do you histologically identify the esophagus
This is esophagus, longitudinal section. Note the stratified squamous epithelium
how do you histologically identify muscle
Striated muscle at high power- all look the same, lots of elastic fibers
