HCT Lab Lesson 8-11 Study Notes
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What is a key benefit of using microwave ovens specifically designed for tissue processing?

  • They are more energy-efficient than conventional ovens.
  • They are easier to maintain than kitchen-type microwaves.
  • They provide better controls and even energy distribution. (correct)
  • They reduce the time required for tissue fixation.

Which fixative combination is stated to allow for the recovery of DNA, RNA, and proteins for molecular analyses?

  • Formaldehyde with microwave-based processing
  • Methanol with polyethylene glycol
  • Traditional formalin-based fixations
  • Alcohol-based fixative with formalin-free processing (correct)

What is a notable effect of using the universal molecular fixative (UMFIX) on tissue morphology?

  • It allows for better preservation of low-molecular weight RNA.
  • It completely prevents any staining artefacts in tissues.
  • It significantly alters the structural integrity of tissues.
  • It makes tissue morphology comparable to formalin-fixed tissues. (correct)

Which temperature range is optimal for metallic staining methods when using UMFIX?

<p>750C to 950C (D)</p> Signup and view all the answers

What is one of the significant advantages of using UMFIX instead of formalin?

<p>Improved antigen preservation in tissue sections. (A)</p> Signup and view all the answers

What is the purpose of preparing bone marrow smears immediately after aspiration?

<p>To reduce storage artifact (D)</p> Signup and view all the answers

Which fixative is considered standard for trephine core biopsies?

<p>Neutral Buffered Formalin (B)</p> Signup and view all the answers

What additional investigation can be performed alongside bone marrow aspirate?

<p>Flow cytometric immunophenotyping (B)</p> Signup and view all the answers

Why must core biopsies be at least 2 cm long?

<p>To assess overall marrow architecture (D)</p> Signup and view all the answers

Which method is NOT used for preparing bone marrow smears?

<p>Decalcification (C)</p> Signup and view all the answers

What is a safety concern associated with using B5 fixative?

<p>It contains mercuric chloride (C)</p> Signup and view all the answers

What is the purpose of using a mounting medium that hardens and dries rapidly for bone marrow smears?

<p>To preserve cellular detail (D)</p> Signup and view all the answers

What is the first step in preparing Giemsa's stain?

<p>Weigh out 1 g of the powdered dye (B)</p> Signup and view all the answers

What temperature should the mixture for Giemsa's stain be warmed to?

<p>50 degrees Celsius (A)</p> Signup and view all the answers

Which dyes are components of Giemsa's stain?

<p>Eosin Y and methylene blue (D)</p> Signup and view all the answers

What is the color outcome of cytoplasm when using Giemsa's stain?

<p>Red-pink (B)</p> Signup and view all the answers

What is the role of methyl alcohol in Wright's stain preparation?

<p>It fixes the sample (C)</p> Signup and view all the answers

For how long should Giemsa's stain be allowed to act?

<p>3 minutes (A)</p> Signup and view all the answers

What type of staining does Giemsa's stain produce?

<p>Differential staining (D)</p> Signup and view all the answers

What is the main characteristic of Jenner's stain preparation?

<p>Preparation of a 5g/l solution in methanol (C)</p> Signup and view all the answers

What is a key step involved in the Wright's stain procedure?

<p>Dilute the stain with equal volume of water after application (D)</p> Signup and view all the answers

What is the purpose of adding aqueous solutions of methylene blue and eosin?

<p>To improve staining of cytoplasmic granules (D)</p> Signup and view all the answers

How long should the air-dried smear be covered with Wright stain?

<p>For 1 minute (C)</p> Signup and view all the answers

What should be done if distilled water does not provide adequate differentiation?

<p>Use a phosphate buffer with a specific pH (D)</p> Signup and view all the answers

What does Giemsa stain poorly on its own?

<p>Red cells (B), Neutrophil granules (D)</p> Signup and view all the answers

Which step is essential after applying Wright stain to ensure proper washing?

<p>Flood with distilled water (D)</p> Signup and view all the answers

What should be used for decolorization in the staining process?

<p>0.5% aqueous acetic acid (B)</p> Signup and view all the answers

How does increasing the stain temperature affect the staining time?

<p>It shortens the staining time (B)</p> Signup and view all the answers

Which combination improves the staining of cytoplasmic granules?

<p>Wright stain and Giemsa stain (D)</p> Signup and view all the answers

What happens during the washing step after using the diluted stain?

<p>The thinner parts of the film appear pinkish red (A)</p> Signup and view all the answers

What is the primary fixation agent in the staining method described?

<p>Methanol (C)</p> Signup and view all the answers

What is one advantage of rapid microwave processing in immunohistochemical reactions?

<p>It results in more flexible work hours for histotech and pathologists. (A)</p> Signup and view all the answers

What must be considered when preparing sections for rapid tissue processing?

<p>Sections must be thin enough for adequate penetration of fixatives. (A)</p> Signup and view all the answers

What is a potential disadvantage of continuous flow processing?

<p>It requires ongoing attention to the instrument. (C)</p> Signup and view all the answers

Which of the following tissues may require additional steps before rapid tissue processing?

<p>Brain tissues (A)</p> Signup and view all the answers

How does rapid tissue processing impact surgical pathology reports?

<p>It can enhance the speed of obtaining diagnostic results. (A)</p> Signup and view all the answers

What aspect of paraffin blocks is highlighted in terms of molecular analyses?

<p>They can be analyzed for DNA, RNA, and proteins. (D)</p> Signup and view all the answers

What is one disadvantage of the rapid tissue processing method?

<p>It can increase grossing time due to thin section requirements. (B)</p> Signup and view all the answers

What leads to enhanced processing times in tissue specimens?

<p>Rapid tissue processing techniques. (C)</p> Signup and view all the answers

Which factor can negatively affect the quality of immunohistochemical reactions?

<p>Inadequate attention to the processor (B)</p> Signup and view all the answers

Which component is critical for adequate penetration during processing of specimens?

<p>Thinly sliced sections (A)</p> Signup and view all the answers

Flashcards

Microwave Fixation

A technique for tissue preservation using microwave energy instead of traditional chemical fixatives like formalin.

Non-formaldehyde Fixatives

Fixatives that don't use formaldehyde, often preferred in microwave fixation because they preserve better for molecular analysis.

Universal Molecular Fixative (UMFIX)

A mixture of methanol and polyethylene glycol that offers an excellent and cost-effective alternative to formaldehyde for tissue preservation.

Microwave Slides

Slides prepared using tissue processed with microwave energy, offering better contrast and staining compared to traditional methods.

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Optimum Temperature

The specific heat range required for different types of stains, depending on whether they are metallic or non-metallic.

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Wright Stain

A type of Romanowsky stain used to stain blood smears, typically for microscopic examination.

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Giemsa Stain

A stain used in microscopy, often in combination with Wright stain, to visualize cell structures like granules and nuclei.

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Romanowsky Stain

A category of stains used in hematology to differentiate blood cell components.

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Metallic Scum

A visual indication that the Wright stain has been diluted to the correct concentration for optimal staining.

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Phosphate Buffer

A solution used to adjust the pH of the staining process, ensuring proper cell differentiation.

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Fixation (in Microscopy)

The process of preserving biological specimens to maintain their structure for observation.

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Dehydration (in Microscopy)

Removing water from a specimen after staining to prevent damage during mounting.

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Differentiation (in Staining)

The process of adjusting staining intensity to reveal distinct cellular components.

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Azurophil Granules

Specific granules found in certain white blood cells, stained red by Giemsa stain.

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Cytoplasmic Granules

Small particles found within the cytoplasm of cells, often stained by Wright-Giemsa combination.

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Jenner's Stain

A staining solution made with a 5g/l concentration of dye in methanol, prepared similarly to May-Grünwald stain.

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Giemsa's Stain Preparation: Step 1

Start by weighing 1 gram of the powdered dye.

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Giemsa's Stain Preparation: Step 2

Transfer the measured dye into a conical flask with 200-250 mL capacity.

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Giemsa's Stain Preparation: Step 3

Add 100 mL of methanol and heat the mixture to 50 degrees Celsius.

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Giemsa's Stain Preparation: Step 4

Maintain the solution at 50 degrees Celsius for 15 minutes while shaking occasionally.

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Giemsa's Stain Preparation: Step 5

Filter the solution.

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Wright's Stain: Principle

Eosinates of polychromed methylene blue are dissolved in absolute methyl alcohol, which functions as a fixative when applied to a dried blood smears.

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Rapid Tissue Processing

A method used to prepare tissue samples for microscopic examination, significantly reducing the traditional processing time.

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Benefits of Rapid Tissue Processing

Advantages include quicker turnaround times for diagnoses, improved immunohistochemical reactions, and compatibility with molecular analysis from paraffin blocks.

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Reduced Tissue Processing Time

Rapid tissue processing reduces the typical processing time from 4.5 to 5 hours to a much shorter duration.

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Immunohistochemical Reactions

These reactions, used for identifying specific molecules within tissues, are not negatively affected but rather improved by rapid tissue processing.

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Molecular Analysis from Paraffin Blocks

Rapid processing allows for molecular analysis of DNA, RNA, and proteins directly from paraffin blocks, comparable to fresh or frozen tissues.

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Disadvantages of Rapid Tissue Processing

Limitations include the need for thinner tissue sections, additional steps for certain tissue types, and continuous attention to the processing instrument.

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Thin Tissue Sections

Rapid tissue processing requires thinner tissue sections (around 1.5 mm) to allow for proper penetration of fixatives and dehydrating solutions.

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Special Tissue Handling

Some tissues, like brain and large blocks, may require extra steps before being placed in the rapid processor.

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Continuous Flow Processing

Rapid processing involves a continuous flow method, requiring ongoing attention to the instrument as samples complete the cycle.

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Batching of Specimens

Rapid processing eliminates batching of specimens, meaning each sample is processed individually and requires continuous monitoring.

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Bone Marrow Aspirate: Additional Investigations

Beyond basic analysis, further techniques like immunophenotyping (cell identification), cytochemistry (enzyme activity), FISH (chromosome analysis), and molecular genetics (gene mutations) can provide detailed information about cells in the bone marrow.

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Bone Marrow Smear Preparation

Bone marrow smears should be prepared immediately after aspiration to ensure accurate representation of the cells. EDTA samples should be prepared rapidly to reduce storage artifacts.

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Bone Marrow Smear Staining

Bone marrow smears are stained with standard methods for blood smears, such as Wright's or Giemsa stains, to visualize cells and their features.

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Bone Marrow Smear Mounting

All bone marrow smears should be mounted using a mounting medium that dries quickly and hardens, preserving the smear for long-term study.

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Bone Marrow Core Biopsy: Purpose

The core biopsy, also called a trephine biopsy, provides information about the overall structure and cellularity of the bone marrow. It can also detect focal lesions, which might not be evident in the aspirate.

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Bone Marrow Core Biopsy: Fixation

Core biopsies are commonly fixed in neutral buffered formalin, which preserves tissue structures well. B5 fixative, containing mercuric chloride, can also be used but raises safety concerns.

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Bone Marrow Decalcification

If bone tissue is a subject of study, a small piece of bone marrow attached to hard bone is decalcified for 24-48 hours. This process removes calcium from the bone, allowing for better visualization of cells and other structures.

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Study Notes

HCT (Lab) - Lesson 8-11 Study Notes

  • Bone Marrow Preparation: Bone marrow can be processed as a smear, touch prep, or biopsy. Romanowsky technique is a standard diagnostic tool. Trephine biopsy needles provide distortion-free bone marrow tissue suitable for staining. Zenker's solution is often used as a fixative.
  • Smear Preparation: Bone marrow aspirates are expelled into a dish, then spread onto a slide using a Pasteur pipette. Squash smears involve pushing marrow particles onto a slide. Spreading smears involve carefully placing marrow material at one end of the slide and spreading it out.
  • Bone Marrow Aspirate Examination: Essential for diagnosing bone marrow and malignant hematologic disorders. Additional investigations include flow cytometry, immunophenotyping, cytochemistry, FISH, and molecular genetics.
  • Bone Marrow Smears: Prepared immediately after aspiration. EDTA samples should be prepared as soon as possible to avoid storage artifacts. Air-drying, fixation, and staining are standard procedures using stains like Wright's or Giemsa.
  • Core Biopsy: Also called Bone Marrow Trephine Biopsy, this procedure is useful for overall marrow architecture assessment and in detecting focal lesions. Needs a core length of at least 2 centimeters in an adult.
  • Neutral Buffered Formalin: A standard fixative for trephine core biopsies.
  • Decalcification: Required for bone marrow samples to be studied. Both organic and mineral acids are used for decalcification. EDTA, formic acid, acetic acid, picric acid, or nitric acid are common solutions.
  • Fixation: Protects cellular and fibrous elements of bone during decalcification procedures. Commonly done before the start of the decalcification step. EXTENDING fixation time for bone specimens is often a practice.
  • Romanowsky Stains: Use methylene blue, azure B, and eosin Y in an acetone-free methanol solution. These stains are widely used in hematology and cytology to differentiate cells from blood and bone marrow samples.
  • May-Grünwald Stain: A two-step staining technique, followed by Giemsa stain. Used to stain blood and bone marrow samples for microscopy.
  • Jenner's Stain: A solution in methanol used for staining, similar to may-grünwald stain.
  • Giemsa Stain: A stain used in hematology and cytology to produce a blue-purple coloration of cell nuclei and a red-pink coloration of the cytoplasm.
  • Wright's Stain: A stain used in cell morphology analysis especially for blood cells. This stain is composed of oxidized methylene blue, azure B, and eosin Y dyes.
  • Rapid Tissue Processing: Aims to minimize the time it takes to process tissue specimens by shortening the various steps, such as using microwave technology for faster fixation and also incorporating improved computer systems. Allows for shorter turn-around time for laboratory reports to be processed.
  • Universal Molecular Fixative (UMFIX): A cost-effective alternative to formalin. Preserves tissue morphology and high-molecular weight RNA well.
  • Microwaves in Processing: Microwave energy is used to speed up tissue processing, allowing for quicker dehydrations and impregnation with paraffin or other medium. This also improves antigen preservation.
  • Enzyme Histochemistry: A crucial technique for detecting specific enzymes using substrates to produce colored products in tissue specimens. This technique aids in the identification of cellular enzyme activity.
  • Immunohistochemistry: A technique used for identifying cellular or tissue components using antibody-antigen interactions that leads to a visible reaction. Used in many medical analyses.
  • Immunofluorescence: A method used to visualize specific proteins or antigens in cells or tissues through antibody-antigen binding, employing specially conjugated fluorescent antibodies.
  • In-Situ Hybridization: A technique for detecting specific nucleic acids in tissue sections or cells. It involves using labeled probes (DNA or RNA) that hybridize with complementary targets in tissue sections, facilitating their detection and visualization.
  • Cytology: The microscopic study of cells.
    • Exfoliative Cytology: Examination of cells exfoliated from epithelial surfaces, and used for screening health and for detecting abnormal cells.
    • Fine Needle Aspiration Cytology (FNAC): A sampling of tissue by inserting a fine needle in a mass to acquire tissue material which will be studied.
    • Gynecological Cytology: Study of specimens from female genital tracts for diagnosis or screening purposes.
    • Pap Smears: A sample taken from the cervix that is smeared onto a slide. Used to detect abnormal cell changes in the cervix.
    • Liquid-Based Preparations: An alternative to conventional smears to preserve the cells in liquid media. This method helps in minimizing cell overlap, allowing for better abnormal cell identification.
  • Staining Methods: Methods used for differentiating cell structures and elements in tissue samples, such as Papanicolaou method using hematoxylin, eosin, and other stains.

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Description

This quiz covers key concepts from Lessons 8 to 11 of the HCT Lab, focusing on bone marrow preparation and examination techniques. It includes standard practices for smear preparation and diagnostic investigations. Understanding these methods is crucial for diagnosing hematologic disorders.

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