Manual Staining of Bone Marrow Preparations

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The page below is a sample from the LabCE course Bone Marrow Aspiration: Normal Hematopoiesis and Basic Interpretive Procedures. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Manual Staining of Bone Marrow Preparations

Both prepared bone marrow slides and coverslip smears are stained using a Romanowsky staining method, which uses aqueous-based solutions of Azure blue, Eosin Y, and Methylene Blue to produce the desired morphological staining characteristics. Slides and coverslips are dipped in sequential solutions that first fix and then sequentially apply stain. Slides or coverslips can be dipped singly or in batches using slide or coverslip holders. Once the smears are rinsed and air-dried, they can be mounted or coverslipped as required.

It is important to ensure that the staining solutions used are fresh to achieve good stain quality. It is also important that the necessary contact time in each solution is met, since bone marrow samples are much more cellular than peripheral smears. This causes bone marrow samples to require maximum time in each solution to obtain the preferred stain quality. Bone marrows will need at least 10 - 15 minutes in each solution rather than the 5-10 minutes that peripheral smears require. Bone marrows that are extremely cellular may need a second trip through the staining solutions (without the fixative as this would decolorize the sample and prevent additional staining).

Even with attention to stain timing and solution quality, it is not uncommon for quick staining-type (diff quick) stains of bone marrows to have a slightly muddy quality without the sharp, crisp detail that can be found in the methanol-based Wright or Wright-Giemsa stains. This is a trade-off for the much shorter staining times needed for the quick stains; 3-5 minutes versus the longer 20-30 minutes required for a good Wright stain.