HCT Lab - Lesson 8 & 9 - Merged PDF

HCT (Lab) - Lesson 8 1st Semester | A.Y. 2024-2025 transcribed by Iverson Dence Oraa / template by @adrenaliane ◆ should not be sprayed with or dipped in a fixative. P...

HCT (Lab) - Lesson 8 1st Semester | A.Y. 2024-2025 transcribed by Iverson Dence Oraa / template by @adrenaliane ◆ should not be sprayed with or dipped in a fixative. PREPARATION OF BONE MARROW SECTION ◆ usually made at the bedside. (1ml of aspirate added to 1.8% EDTA) Introduction ◆ How to prepare a bone marrow ➔ Bone Marrow and Blood Elements smear: ◆ may be processed as a TOUCH, 1. Expel the aspirate into a SMEAR, or in a BIOPSY form. plastic or siliconized glass ➔ Romanowsky Technique dish. ◆ currently used as a main 2. Draw up the particles using diagnostic tool by a Pasteur pipette. hematopathologists. 3. Place on a glass slide. 4. Smear… ➔ Trephine Biopsy Needles ◆ developed to produce a good NOTE: A minimum of 6 smears and 2 particle length core of distortion-free bone squash slides should be made. marrow tissue that can be stained with hematoxylin and eosin. ➔ Bone Marrow Biopsies ◆ Zenker’s solution is recommended for fixing. Used in Trephine biopsy TYPES OF SMEAR PREPARATION ➔ Squash Smear ◆ Squash the marrow particles with another slide and pull to the end of the marrow slide. ◆ often causes cellular artifact BONE MARROW PREPARATIONS ➔ Examination Of Bone Marrow Aspirate and Trephine Biopsy ◆ essential for the diagnosis of bone ➔ Spread Smear marrow and malignant ◆ marrow particles are placed at one hematologic disorders. end of the slide and spread with ◆ additional investigations including another slide. flow cytometric ◆ allows excellent details of immunophenotyping, individual cell morphology cytochemistry, FISH, and molecular genetics can also be performed. BONE MARROW ASPIRATE ➔ Bone Marrow Smears ◆ should be prepared immediately following aspiration. ◆ smears prepared from EDTA samples should be made as soon as possible to reduce storage artifact. ◆ air-dried, fixed and stained using NOTE: All bone marrow smears should be standard methods for blood cover slipped using a mounting medium that smear, such as Wright’s or hardens and dries rapidly. Giemsa. LABORATORY #2 | HCT – TISSUE FIXATION CORE BIOPSY ➔ Neutral Buffered Formalin ◆ standard fixative for trephine core biopsies ➔ Core Biopsy ◆ also called Bone Marrow ➔ B5 (mercuric chloride, sodium acetate Trephine Biopsy and formalin) ◆ can be performed either before or ◆ gives good morphology with short after the aspirate. TAT ◆ useful in the assessment of overall ◆ contains mercuric chloride causing marrow architecture and cellularity safety and environmental and provide greater sensitivity for concerns the assessment of focal lesions. ◆ length of the core from an adult NOTE: If bone is to be studied, a small piece should be at least 2 cm. of bone marrow attached to a thin hard bone is decalcified for 24-48 hours. ➔ Decalcification ◆ both organic and mineral acids are commonly used for decalcification of bone marrow specimens ◆ tissue suspension, mild agitation, and gentle mixing will ensure an even distribution of decalcifying acid around the bone sample ◆ SOLUTIONS USED: EDTA, formic acid, acetic acid, picric acid or nitric acid. ◆ decalcification of EDTA results in better preservation of nucleic acids Note: Recommended thickness of section – 2- 3 microns. ➔ Fixation ◆ protects the cellular and fibrous elements of bone from damage caused by the acids used as decalcifying agents. ◆ most important step prior to decalcification ◆ it is a common practice to EXTEND fixation time for bone specimens before commencing decalcification ROMANOWSKY STAIN ➔ Romanowsky Stains ◆ consists of methylene blue / azure B and eosin, dissolved in acetone- free methanol. ◆ are the stains that are used in hematology and cytological studies, to differentiate cells in microscopic examinations of blood and bone marrow samples ◆ works principally in its ability to produce a variety of hues which makes it possible to differentiate various cellular components. LABORATORY #2 | HCT – TISSUE FIXATION TYPES OF ROMANAWSY STAIN MAY-GRUNWALD STAIN ➔ It is a two-step staining procedure whereby the first staining is done with May-Grünwald stain and a second stain of Giemsa stain which produces the Romanowsky effect (wide range of hue/color) ➔ PROCEDURE: 1. Weigh out 0.3 g of the powdered dye 2. Transfer to a conical flask of 200-250 mL capacity 3. Add 100 mL of methanol and warm the mixture to 50 degrees Celsius 4. Allow the flask to cool to 20 deg. Celsius and shake several times during the day 5. Stand for 24 hours 6. Filter the solution JENNER’S STAIN ➔ Prepare a 5g/l solution in methanol in exactly the same way as described earlier for the May-Grünwald stain. GIEMSA’S STAIN ➔ It is made up of acidic and basic dyes of eosin Y and methylene blue hence it is an azure stain. ➔ produces blue-purple colored staining of the nuclear and red-pink coloration of the cytoplasm and cytoplasmic granules. ➔ PROCEDURE: 1. Weigh out 1 g of the powdered dye 2. Transfer to a conical flask of 200- 250 mL capacity 3. Add 100 mL of methanol and warm the mixture to 50 degrees Celsius 4. Keep at 50 degrees Celsius for 15 mins. With occasional shaking 5. Filter the solution WRIGHT’S STAIN ➔ PRINCIPLE: ◆ Eosinates of polychromed methylene blue are dissolved in absolute methyl alcohol. The methyl alcohol serves as a fixative if the solution is placed on a dried LABORATORY #2 | HCT – TISSUE FIXATION blood smear. After 1-3 mins. The GIEMSA STAIN stain is diluted with an equal volume of distilled water. This ➔ DEFINITION differentially stains the ◆ Member of the Romanowsky cytoplasmic granules and is group of stains. allowed to act for about 3 mins. It ◆ The black precipitate formed from is then poured off and the the addition of aqueous solutions preparation is washed in tap water of methylene blue and eosin, then dry. dissolved in methanol. ➔ METHOD ◆ Best purchased commercially in 1. Place the air-dried smear, film side solution up, on a staining rack. ➔ METHODS 2. Cover the smear with undiluted 1. Bring sections to distilled water. Wright stain and leave for 1 2. Stain with diluted Giemsa's stain minute. (The methyl alcohol fixes made up fresh. the smear) 3. Rinse in distilled water. 3. Dilute with distilled water (approx. 4. Differentiate with 0.5% aqueous equal volume) until a metallic scum acetic acid. appear. (allow this diluted stain to 5. Dehydrate rapidly. act for 2 ½ to 5 minutes) 6. Clear and mount. 4. Without disturbing the slide, flood with distilled water and wash until the thinner parts of the film are pinkish red. 5. If distilled water does not give adequate differentiation, a phosphate buffer (pH 6.4 to 6.5) should be used, at least for washing, but possibly also for diluting the stain (step 3). RESULTS RESULTS NOTES TO REMEMBER 1. The staining is usually performed at room temperature overnight. Increasing the stain temperature shortens staining time. 2. By itself, Giemsa stains red cells and neutrophil granules poorly, but azurophil granules (red) are well stained. WRIGHT-GIEMSA ➔ DEFINITION LABORATORY #2 | HCT – TISSUE FIXATION ◆ This combination of a Romanowsky stain with another stain (such as Giemsa) improves the staining of cytoplasmic granules. ◆ It is advisable to keep a separate stock of Wright's stain for bone marrow staining at least 6 months before use to allow it to ripen. ◆ Composed of oxidized methylene blue, azure B and eosin Y dyes. ➔ METHODS 1. The smears are first dipped in methanol to FIX the specimens. 2. Then placed in Wright’s or Wright- Giemsa Stain for 10-15 mins to NOTE: stain. 3. Move the smears to a mixture of ➔ A Prussian blue stain should be stain and 6.8 pH phosphate buffer performed on a bone marrow smear for (usually 1:2-3 buffer ratio) and the evaluation of storage iron and allowed to stain at 20-30 mins. sideroblasts. 4. Give a quick rinse in distilled water. ➔ A bone marrow smear with increased iron 5. Allow to air dry before mounting or stores should be included as a positive cover-slipping. control. RESULTS ➔ Core biopsy sections are less reliable than the aspirate for the assessment of storage iron, since decalcification removes storage iron MYELOPEROXIDASE STAIN ➔ DEFINITION ◆ helpful in identifying cytoplasmic granules characteristic of myeloid cells. ◆ useful when there are large, immature white blood cells in the PERLS’ PRUSSIAN BLUE IRON STAIN peripheral blood, to differentiate myeloid leukemia cells from those ➔ The detection of hemosiderin in a Perls’ of lymphoid origin. stained bone marrow is regarded as the ➔ METHODS gold standard by which other tests for 1. Use fresh smears of blood or bone iron deficiency are assessed. marrow imprints. Activity may be ➔ The section is treated with dilute preserved for as long as 3 weeks if hydrochloric acid to release ferric ions preparations are stored in the dark. from binding proteins. (These ions then Venous or peripheral blood is react with potassium ferrocyanide to equally satisfactory. Heparin, produce an insoluble blue compound oxalate and EDTA are not inhibitory. A.K.A The Prussian Blue Reaction) 2. Fix slides for 60 seconds at room temperature in 10 per cent formal- ethanol. (10 ml. of 37 per cent formaldehyde and 90 ml. of absolute ethyl alcohol). Wash for 15 to 30 seconds with gently running tap water. Shake off excess water. 3. Place wet slides in incubation mixture in a Coplin jar for 30 seconds at room temperature. 4. Wash briefly (5 to 10 seconds) in running tap water, dry and examine. 5. If greater nuclear detail is desired, the stained preparations may be re- LABORATORY #2 | HCT – TISSUE FIXATION counterstained in 1% aqueous cresyl violet acetate for 1 minute, or in freshly prepared Giemsa stain for 10 minutes. (Wright’s stain is less satisfactory as a counterstain.) RESULTS MASSON’S TRICHROME STAIN ➔ DEFINITION ◆ Masson's trichrome is a classical method of visualization with several variants. The nuclear dye can be Masson's hemalum or several hematoxylin solutions. ◆ The cytoplasmic dye is fuchsine. ◆ An aniline-derived dye allows the differentiation of collagen fibers ➔ METHODS 1. Place in modified Weigert’s iron hematoxylin for 5 minutes. 2. Wash briefly in running tap water and rinse in two changes of distilled water. 3. Decolorize with 0.5% hydrochloric acid in 70% alcohol for 5 seconds. Wash in running tap water for 30 seconds and rinse in two changes of distilled water. 4. Place in Biebrich scarlet-acid fuchsin solution for 30 minutes. 5. Rinse in three changes of distilled water. 6. Place in phosphomolybdic- phosphotungstic acid for 10 minutes. 7. Rinse in two changes of distilled water. 8. Place in aniline blue solution for 7 minutes. 9. Rinse in two changes of distilled water. 10. Place in 1% acetic acid for 1 minute. 11. Rinse in three changes of distilled water and air dry. Dip in xylene and mount with synthetic resin. LABORATORY #2 | HCT – TISSUE FIXATION RAPID PROCESSING TECHNIQUE RAPID TISSUE PROCESSOR ➔ Done when there is an urgent need for an ➔ Enclosed system similar in configuration immediate histopathologic evaluation. to modern vacuum assisted conventional ➔ Done when some specimens and some tissue processor. diagnoses for that matter are not ➔ Reduces processing time to 3 hours amenable to frozen section study either when combined with microwave fixation due to: ➔ May also be used in formalin-free 1. nature of the specimen environment. 2. complexity of the diagnoses ➔ Run in batch modes 3. So accelerated specimen ➔ Large blocks require special procedure or processing methods were handling. developed. ➔ How? By shortening the time for every step in the processing. ➔ Applicable to: small specimens only ➔ Improvements: ◆ Microwave incorporation in manual, semiautomated, and fully automated systems Incorporating computer control of the microwave ➔ allows for the addition of new specimens “dosage” to the process every 15 minutes as a Temperature reaction chamber becomes available. Vacuum paraffin ➔ Utilizes a computer controlled microwave impregnation delivery and a temperature control in a ➔ improved tissue penetration and fixation formalin-free environment and preservation of tissue antigens of ➔ Advantages: interest. ◆ Reagent volumes are small ➔ Size of tissue: up to 1.5 mm thick ◆ All reagents are inexpensive ➔ Beyond: uneven penetration of ◆ Reagents are non-toxic reagents>> ➔ Equipment: ◆ Kitchen knife ◆ Microwave oven ➔ To sophisticated semi-automated or automated instruments ◆ Basic principles - the same, but with distinct variations depending on the laboratory needs and practice patterns ➔ Specifically designed microwave ovens developed ◆ utilizes removable reaction vessel (to hold cassettes) with manual reagent exchanges according to a predetermined schedule. ➔ Xylene has been eliminated. ➔ Vacuum-assisted paraffin impregnation allows for a complete processing cycle of about 60 – 70 minutes. LABORATORY #2 | HCT – TISSUE FIXATION RAPID TISSUE PROCESSING RAPID TISSUE PROCESSING ➔ Advantages: ➔ Positive outcome based on studies: 1. Early diagnosis for patient (more ◆ Tissues processed are time for discussion and planning) comparable, and in some cases ◆ Urgent cases: superior to the conventional Transplant patients and method. others benefit from ◆ Immunohistochemical stains can prompt adjustments on be performed w/o antigen retrieval the treatment based on in some cases, and with more early biopsy report. diluted primary antibodies in 2. Elimination of toxic chemicals others. (formalin and xylene) from the work ➔ Most dramatic impact: environment, and elimination of ◆ Turnaround time for completion of requirements to monitor the fumes the surgical pathology reports. in the environment. (elapsed time from 3. Immunohistochemical reactions are obtaining the biopsy until not adversely affected, instead, the surgical pathology enhanced after rapid tissue report is finalized) processing ➔ Specimens are processed and diagnosed 4. With appropriate fixatives, throughout the day when they are molecular analyses for DNA, RNA, received from the surgical suites. proteins can be performed from ➔ Tissue processing reduced: 4 ½ to 5 paraffin blocks, rivaling those hours obtained from fresh or frozen tissues. 5. Rapid microwave processing results in more “family-friendly” work hours for histotech and pathologists. ➔ Disadvantages: 1. Sections must be thin enough so fixative and dehydrating solutions can penetrate completely (about 1.5 mm) which increases time required in grossing of specimens. 2. Certain types of tissues (brain, large tissue blocks) may require additional steps before placing into the rapid tissue processor. 3. Continuous flow processing eliminates batching of specimens and necessitates ongoing attention to the instrument, since as samples complete the processing cycle, they must be removed from the instrument to accommodate the next basket of cassettes. 4. Microwave ovens specifically designed for tissue processing are now preferred (since they have better controls and can provide an even distribution of the energy to the tissues. MICROWAVE FIXATION ➔ Non-formaldehyde fixatives are better in this fixation. ➔ Alcohol based fixative when combined with formalin-free microwave based tissue processing permits recovery of *DNA, RNA and proteins for molecular analyses. LABORATORY #2 | HCT – TISSUE FIXATION UNIVERSAL MOLECULAR FIXATIVE – UMFIX with temp probes and air-bubble agitation device producing a consistent or even heating not oberved with the kitchen- ➔ mixture of methanol and polyethylene type. glycol ◆ excellent and cost-effective alternative of formalin. MICROWAVE SLIDES ➔ Tissues exposed to UMFIX have their morphology comparable to formalin-fixed tissues. ➔ Better contrast ◆ The high-molecular weight RNA is ➔ More intense staining preserved in tissues fixed ➔ less-non-specific staining immediately and stored up to 8 ◆ Compared with conventional weeks at rm temperature. metal-staining methods ➔ Advantages: ➔ Optimum temperature: 1. Improved antigen preservation in ◆ Metallic stains - 750C to 950C tissue sections ◆ Non-metallic stains – 550C to 600C 2. Elimination of noxious formalin fumes from the workplace, and expensive ambient formalin monitoring program. MICROWAVE PROCESSING ➔ Based on the principle of using the microwave energy to speed up the diffusion of fluids into and out of the tissues. ➔ It also allows the use of alcohol during dehydration using one step only ( No need of increasing conc). ➔ Paraffin must be added to the microwave in liquid form ◆ Melted with the intermedium at: 82 0C for ethyl alcohol 78 0C for isopropanol ➔ Purpose: “Flash evaporation of the alcohol or propanol. ◆ Alcohol quickly heats and dissipates while the paraffin remains inert, allowing paraffin to fully infiltrate the specimen, eliminating the use of xylene. ➔ Microwave processing oven has thermocouple temperature probe and controller ◆ This is calibrated, to maintain a very narrow range of temperature range +/- 1 °C producing consistent and reproducible results. ➔ Microwave Antigen Retrieval ◆ Used to unmask or retrieve antigens using 10 mm. citrate buffer (pH 6). ◆ Lower temperature – longer heating time ➔ Higher temperature – shorter heating time to achieve strong intensity of staining. MICROWAVE STAINING ➔ Microwave reduced significantly the time required for staining of tissue, particularly when using heavy metals (metallic stains) such as : methenamine silver O Equipped  HCT (Lab) - Lesson 10 1st Semester | A.Y. 2024-2025 transcribed by Iverson Dence Oraa / template by @adrenaliane ENZYME HISTOCHEMISTRY Phosphatases ➔ Enzymes – catalysts for most biological ➔ Alkaline phosphatase (Gomori calcium reactions method) ➔ Frozen sections are recommended ◆ Fixation: formol calcium at 4°C ◆