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