Technical faults in histopathology lab

Technical faults in histopathology Lab

Technical problems and troubleshooting

•Specimen receive and registration• Grossing•Fixation•Processing•embedding

•staining

Specimen receive and registration

•The specimen name is identical to that written on the request form

•All the patient details are properly written mentioned

•Date of biopsy/procedure • Time of specimen receive

•Register the specimen with a lab number

Grossing

The specimen should be completely covered with the fixation fluid .

If not , change with 10 % formalin solution. Or other fixative according to your lab protocol.Hint :

FixationThe major problems related to fixation are delayed or incomplete fixation

•Autolysis is caused by delayed fixation .

•In H& E section, the tissue may show loss or total disappearance of nuclear chromatin. Some cells may disappear (intestinal mucosa), shrink and leave artifactual space around.

•Prevention: add fixative to the specimen as soon

as possible •Open uterus for endometrial fixation

•Open GIT specimen for mucosal fixation

•Slice solid organ eg breast kidney, thyroid

•Bisect lymph nodes

FixationIncomplete fixationThe cells characterized by smudge nuclei (indistinct nuclear

pattern), nuclear bubblingPrevention:

Prolong fixation timeThin gross sectionFresh formalin solutionCassete should not be tightly backedAgitation of cassettes in the fixative

processing

Most problem encountered in processing is related to either over processing or under processingOverdehydrationDue to excessive dehydration, which results in microchatter around the edges of the tissue on H & Stain.Prevention:

Small biopsies precessed separetelyShorten the dehydration time

processing

Poor processing :due to improper dehydration (water in tissue), impaired clearing, clearing agent in paraffin, too much heat during processing.Prevention:No cassettes condensation Absolute alcohol is fresh, free of

WaterFluid are changed according to the scheduleHeat is used only for paraffin& E Stain.

Embedding and specimen orientation

•It refers to casting or blocking (paraffin blocking)Hints:

•Specimen orientation•Proper pressure force applied

to the entire specimen during orientation and initial solidification to obtain flat

tissue

Hints:•Hard tissue such as bone, can

will section more easily if they are embedded diagonally

•Tissue with wall, such as cyst, gall bladder, and GIT must be

embedded on the edge .

Embedding and specimen orientation

Troubleshooting in Embedding and specimen orientation

Soft Mushy Tissue•The most common cause is

thick sections at gross examination and have been compressed between the top and bottom of the cassette.

•Prevention:•The tissue section should be

thin•Adequate time for fixation•Fluid change according to

schedule

Incorrect orientation•Section may be incorrectly

oriented at the embedding station if the correct method

was not indicated .•Prevention:

•Marking the side of tissue to be embedded facing up with ink

Troubleshooting in Embedding and specimen orientation

Tissue carryover•Small pieces or fragments of

tissue may be carried from 1 tissue to the next at the embedding table, resulting in cross-contamination

•Prevention:•Carefully clean forceps used

at the embedding table between specimens

•Open only the cassette with the tissue to be embedded

Tissue not embedded at the same level

•If the tissue is not properly flattened by pressing it down uniformly when it is placed in the embedding mold, or multiple tissues to be palced in the same mold are embedded at different levels

•Prevention:•Press the tissee uniformly

•Keep the paraffin molten enough to get all pieces embedded at the same level.

•Work very fast when embedding multiple pieces

Troubleshooting in Decalcification

Bone dust•When obtaining sections of bone

with the saw commonly used for the process, bone dust is pressed into the surface of the bone

•Prevention:•Use a saw with diamond blade

•Trim the bone surfaces after decalcification

Underdecalcification•The tissue still hard and sectioning is

difficultOverdecalcificationOccur when the end point of decalcification is not carefully checked, result in poorly stained sections

Prevention Choose decalcification agent that fits the need of the labDevelop good method for detecting the end point of decalcification.

Troubleshooting in frozen sectionFreezing artifact

•If ice crystals were formed due to improper freezing of tissue.

•Prevention:•Snap freezing

•Make sure the tissue was not immersed in saline before freezing

Troubleshooting in frozen sectionBlock loosens from the chuck while sectioning

•Occur if the chuck was too cold when the embedding medium was applied Prevention

Reattach the tissue block to a clean chuck with additional embedding mediumAvoid storing the chuck without

•If the tissue is not embedded flat on the chuck, then sectioning will have to be deeper into the block, and some important parts of the tissue may be wasted.

•Prevention:•Place the tissue on a slide,

surround it with the medium, when the medium begin to turn white, coat the chuck with embedding medium and . invert over the tissue. Then remove the slide.

Tissue not embedded flat

Troubleshooting in microtomy

•Result when the horizontal edges (to and bottom) of the block are

not parallel .•If the lower block edge is not

parallel to the knife edge .•Prevention:

•The upper and lower edge •should be parallel

•the lower block edge is parallel to the knife edge .

•No problem in the blade edge

Crooked ribbons


•Occur when the block holder is not parallel to the blade. One side of the block is exhausted while attempting to get a complete section of the block face

•Prevention:•Ensure at the beginning of the

sectioning that the block holder is adjusted so that the block face and the blade are perfectly parallel.

Block face unevenly sectioned


•Prevention:•Adjust the thickness

Thick section


•Caused by defect in the blade edge, calcium, bone or hard material in the specimen

•Prevention:•Ensure at the beginning of

the sectioning that the block holder is adjusted so that the block face and the blade are perfectly parallel.

Vertical scratches


•Occur when block is faced too aggressively.

•The specimen is either excessively dehydrated or improperly processed.

•Prevention:•Ensure to chill the block with ice

before cutting and discard ribbons until the hole disappear

•Facing the block less aggressively, with smaller micrometer advances of the block for eaah section removed

Holes in the section


•Commonly caused by dull blade. Could result from too hard paraffin, too much blade tilt

•Prevention:•Paraffin with lower melting

point•Decrease blade tilt

•Change room temperature

Failure of ribbon to form


•Commonly occurs in very hard tissue such as uerus or in over fixed tissue.

•It is the macroscopic type of chatter commonly caused by loose clamping of blade or block.

•Prevention:•Proper clamping of blade and block•Ensure the block holder shaft is not

over extended•Ensure the microtome is in good

working order•Decrease the blade tilt

Washboarding or Undulation in the section


•Commonly caused by over dehydration or lack of moisture in the tissue. It could also result from dull blade or too much blade tilt which cause the section to be scrabed rather than cut. Or cutting too rapidly.

•Prevention:•Proper processing

•Restore moisture by facing the block down on an ice tray

•Decrease the blade tilt •Decrease cutting speed: one wheel

per second is considered reasonable speed.

Chatter or microscopic Vibration

Troubleshooting in H& E staining

•White spots may be seen in tissue sections after the deparaffinization step. Usually caused by water left in he tissue, incomplete drying or not leaving the slides in xylene long enough

•Prevention:•Dry section properly before beginning

deparaffinization•Allow sufficient time in xylene for

complete deparaffinization•Avoid contaminated xylene, change

fluids according to the schedule•If the slides have been stained,

decolorize and restain.

Incomplete Deparaffinization


•The Nuclei is too light due to: •Slide not exposed to the

hematoxylin long enoughExhausted (over oxidized or depleted) hematoxylin

•Over differentiation•Prevention:

•Leave the slide longer•Use fresh hematoxylin•Time the differentiation

Pale nuclear staining


•The nuclei is too dark due to: Slide exposed to the hematoxylin too long

•Section are too thick•Differentiation step is too short

•Prevention:•Thin sections

•Decrease hematoxylin exposure•Increase time for differentiation

Dark nuclear staining


•If the nuclei is stained red or reddish brown instead of blue, either the hematoxylin is breaking down or the blueing step was not properly done

•Prevention:•New hematoxylin

•Prper blueing

Red or red-brown Nuclei


•Hematoxylin precipetate•Prevention:

•Filter the hematoxylin

Blue black precipitate


•Eosin pH is over 5, high ph may result from carryover of the blueing agent. The section may be too thin, or left long in the dehydration

•Prevention:•Check Eosin pH

•Completely remove blueing agent before transferring the slides to eosin

•To allow stained slides to stand in the lower concentration of alcohols after the eosin. The more water in the alcohol, the more eosin that will be removed

Pale Cytoplasmic staining


•If the cytoplasm is overstained, and the differentiation is poor, the contrast between the nucleus and the cytoplasm is lost

•Prevention:•Avoid overconcentration of eosin,

dilute eosin solution•Do not leave sections in eosin for long

•Allow sufficient time in dehydration solution, specially 70% alcohol, to allow good eosin differentiation

•Check section for proper thickness

Dark Cytoplasmic staining


•Laser and electrocautry techniques denature macromolecules and produce heat artifact, generally marked by dark basophilic staining in nuclei and cytoplasm

•Prevention:•Nothing to be done

Dark basophilic staining of nuclei and cytoplasm, especially around tissue edges

Thank you

Technical faults in histopathology lab

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