Preparation of Bone Marrow Specimen
Spread smear
Allows excellent detailsof individual cell morphology
EDTA-rinsed Pasteur pipette
An aliquot of the aspirate is transferred to a watch glass using what?
Bur
An excellent stain for inclusion bodies
Touch and smear preparation Air-dried Fixed Stained by Wright's stain or Giemsa stain
BM preparation
Negative
Basophils, lymphocytes and erythroblasts are (positive or negative?) for peroxidase reaction for myeloid cells
Bedside
Because he marrow smear preparation is usually done at?
Giemsa stain
Best purchased commercially in solution
Aplastic anemia Myelofibrosis
Cases wherein no specimen is aspirated
Methylene blue/ azure b and eosin
Composition of Romanowsky stains?
Romanowsky stains
Composition: * Methylene blue/ azure B * eosin Solvent: acetone-free methanol
Myeloid cells (leukocytes)
Contain peroxidase responsible for the oxidation of benzidine by H2O
Neutral fast red
Counter stain used in modified gomori
Freshly diluted Giemsa
Counterstain for May-Grunwald-Giemsa stain
Giemsa stain diluted with 1 in 10 buffered distilled water (pH 6.8)
Counterstain in Peroxidase reaction for myeloid cells
Manner and method of polychroming methylene blue
Difference of modern Romanowsky techniques
Giemsa stain
Employs a various azure compounds (thionine and methyl derivatives) with eosin and methylene blues
14-24 hours; 3-6 hours
Fixation time with zenker's fluid and acetic acid then washed with running water for _________
10% neutral buffered formalin
Fixative for modified gomori's method
Methyl alcohol
Fixative used for May-Grinwald-Giemsa stain
Methanol
Fixative used for Wright-Giemsa or Jenner-Giemsa stain
40% formaldehyde and 95% ethanol
Fixative used for peroxidase
Zenker's solution
For BM biopsies use _________________ since it is also a decalcifying agent to decalcify bone spicules and mixed with the blood clot for 1-2 hours
Reticulin stain
For the detection of myelofibrosis
Leishman's stain
Further ripening is allowed for 10 days before it is mixed with equal volume of 0.1% eosin B
Monocytes
Has slight peroxidase activity
Neutrophils and eosinophils
Have rods and needle like projections emanating from the cell margins
1 mL
How much is added to snap cap vial containing 1-8% EDTA?
24-48 hours
If bone is to be studied, a small piece is taken and decalcified for how many hours?
Glycol methacrylate sections
If the stain is heated and used at high alkaline pH, it easily penetrates the plastic and stains various tissue components a blue color of differing shades and intensifies. The embedding medium is not stained
Wright's stain
In general, these stains are somewhat empirical mixtures of polychrome dyes, one batch may differ from one another
Peroxidase
Iron-porphyrin containing enzymes
Leishman's stain
MB is polychromed by heating 1% solution with 0.5% sodium carbonate at 65*C for 12 hours
Romanowsky technique
Main diagnostic tool used by hematologists
BM biopsies and. H&E staining
Method used for cytological details for diagnosis of hematological disorder (except for the demonstration of iron and reticulin)
Wright's stain Leishman's stain
Modern Romanowsky techniques:
Basophils Lymphocytes Erythroblasts
Negative for peroxidase
Water
Poor results are obtained if the stock solution is contaminated with water
Smear, direct Particle crush techniques Touch imprint preparation Biopsy specimen
Preparations:
Myeloid cells are positive except basophils
Presence of green to dark blue granules in the cytoplasm
Modified Gomori's method for Reticulum
Principle: includes the oxidation and sensitization of tissues with reticulum fibers using potassium permanganate and 2% ferric ammonium sulfate respectively
Perl's Prussian blue
Principle: when strong acids, like HCL and potassium ferricyanide are combined, their union releases ferric iron, which is tightly bound to proteins
Ferric iron
Produced by the union of strong acids like HCl and potassium ferricyanide
Buffered phosphate buffered water
Rinsing is done by what solution I'm Wright-Giemsa or Jenner-Giemsa stain?
Wright's stain
Ripening or polychroming continuous as the solution is stored
Neutral fast red
Should not be overused because it may mask the iron pigments that are delicately stained
May-Grunwald-Giemsa stain
Slightly better result than Jenner-Giemsa
Acetone-free methanol
Solvent for Romanowsky stains?
Giemsa stain for 15 minutes to 1 hour
Stain for Giemsa stain?
Equal volume of 20% HCl and 10% potassium ferricyanide
Stain for modified gomori
Glycol methacrylate sections
Staining of the granulopoietic precursor granules in glycol methacrylate sections then stained with toluidine blue and eosin
Jenner Giemsa May grunwald Leishman
Stains under Romanowsky
Neutral Fast Blue
The counterstain for Perl's Prussian blue
Ferric ferrocyanide
The ferric iron merges with potassium ferricyanide to form what?
Red cells and neutrophils
They are stained poorly with Giemsa stain
Azurophilic granules
They are well stained with Giemsa stain
Histotechnologist/ histotechnocian
They need to be present while the physician extracts the bone marrow specimen
Wright-Giemsa or Jenner-Giemsa stain
This combination improves the staining of cytoplasmic granules
Copper sulfate
This forms blue-green compound with the oxidized benzidine and renders the granules visible
New and improved trephine biopsy
To produce a good length core of distortion-free bone marrow tissue for H&E or Romanowsky stain
Methyl-green pyronin stain
Used to demonstrate plasma cells
Distilled water
Used to wash the slides
Bone marrow smear preparation
Usually done at bedside, directly after aspiration
1-8% EDTA
What is contained in the snap cap vial?
Reticulum
What is stained black by the modified gomori stain?
Nuclei; Basophils and mast cell granules
What is stained blue by the rapid toluidine-eosin stain for methacrylate section?
Cytoplasm
What is stained blue/pink by the rapid toluidine-eosin stain for methacrylate section?
Cytoplasm
What is stained pink/blue by the Wright-Giemsa or Jenner-Giemsa stain?
Eosinophils
What is stained pink/red by the Wright-Giemsa or Jenner-Giemsa stain?
Nuclei
What is stained purple/blue by the Wright-Giemsa or Jenner-Giemsa stain?
Background
What is stained red by the modified gomori stain?
Eosinophilic granules
What is stained red by the rapid toluidine-eosin stain for methacrylate section?
Red
What is the color of the background in modified gomori stain ?
Blue
What is the color of the basophils and mast cell granules when stained with rapid toluidine-eosin stain for methacrylate section?
Blue/pink
What is the color of the cytoplasm when stained with rapid toluidine-eosin stain for methacrylate section?
Pink/blue
What is the color of the cytoplasm when stained with the Wright-Giemsa or Jenner-Giemsa stain?
Red
What is the color of the eosinophilic granules when stained with rapid toluidine-eosin stain for methacrylate section?
Pink/red
What is the color of the eosinophils when stained with the Wright-Giemsa or Jenner-Giemsa stain?
Blue
What is the color of the nuclei when stained with rapid toluidine-eosin stain for methacrylate section?
Purple/blue
What is the color of the nuclei when stained with the Wright-Giemsa or Jenner-Giemsa stain?
Black
What is the color of the reticulum in modified gomori stain ?
Methyl alcohol for 2-3 minutes after air drying
What is the fixative used in Giemsa stain?
Coplin jars
What is used for staining to minimize the possibility or precipitation on the slides
