Coverslips Information and Courses from MediaLab, Inc.

Coverslip smears are made on 22 x 22 mm coverslips using a technique similar to the pull prep method. If done correctly, both coverslips will have quality smears that will appear similar to a thumb print. To create a coverslip smear preparation, a coverslip is picked up by the corner and the point is held between the thumb and forefinger in one hand. The other hand uses a capillary pipette to transfer a small drop of concentrated bone marrow on to the center of the coverslip. The capillary pipette is put down and a second coverslip is placed over the first to form a star shape. When the marrow has spread almost to the edges, the coverslips are slid apart using the protruding corners/points of the star. The motion is a parallel slide with no pushing or rotation of the wrist as the coverslips are slid apart.Making smears on coverslips requires manual dexterity. Not only does the laboratory professional need to be proficient in the use of capillary tubes to pipette bone marrow, he/she must also be able to manipulate fragile coverslips without breakage. Staining also requires modified/adapted methods.A 22x22 mm coverslip can be glued to a standard slide after staining to provide easy evaluation. Since only a minimal amount of bone marrow is needed, many smears can be made from a small bone marrow sample. Another advantage is the small amount of storage space required. Three dozen coverslips will easily fit in an envelope and would take considerably less storage space compared to the same number of regular slides.

Accurate interpretation of bone marrow aspirate specimens is highly dependent on two factors: The quality of the sample obtained by the clinician The quality of the prepared bone marrow smears While the technologists have no control over the quality of the aspirate obtained by the clinician, in most cases they are able to impact the quality of laboratory prepared smears.The method used for preparing slides may be dependent on sample volume, staining techniques, storage space, clinical setting, patient status, and hematopathologist's preference. Bone marrow sample preparations can be made on slides or coverslips from a direct marrow sample or a sample that is manipulated to enhance cellularity (selected for marrow fragments or spicules). In situations where the technologist goes to the bedside, touch-preparation (touch-prep) smears from the biopsy core can be prepared before it is placed in fixative.Regardless of which method is the preferred primary technique at your institution, it is useful to be familiar with other methods, since there may be a situation when the use of an alternate method is the only way to obtain an interpretable smear.When adequate samples are provided, it is desirable to make several slides or coverslips so that there will be enough preps available for all the possible tests/special stains that may be requested. The extra smears can be stored at room temperature and protected from light in an envelope or sleeve for further testing, if required.

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 seconds in each solution rather than the 5-10 seconds 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.

The procedure your laboratory utilizes for bone marrow stains is determined by the type of stainer available to you. The stainer available may also dictate the type of smear preparations that your laboratory makes. There are several types of automated hematology stainers on the market today. Some stainers are simple continuous-feed stainers with limited programmability. Some are batch stainers that can have multiple programs, customizable to the sample type or stain preference of the user. Other stainers are dip stainers that automatically move a slide rack from bucket to bucket, or an inline corkscrew that moves slides down a platen and dispenses stain/solutions at fixed positions. Finally, there are centrifuge stainers that apply stains to a spinning slide tray during programmed intervals.Hema-tek® stainers, with a fixed stain area, require shorter preparations on long slides, so slide pull preps or differential smears would be the laboratory standard. Since the stain time and volume is fixed, bone marrow slides may need to be stained twice and sometimes even three times for extremely cellular bone marrows. When using this type of stainer, always check the stain quality before coverslipping.Automated dipping stainers can be used with either long slides or coverslips,when utilizing a coverslip basket, so the choice of smear type is driven by laboratory and pathologist preference. As with manual staining times, the laboratory should have a separate program for bone marrow staining that reflects the need for longer contact times. Wescor® hematology stainers are quite flexible. They are centrifugal stainers that are pre-programmed for rapid, Wright-Giemsa and May-Grunwald stains, as well as having programmable custom settings. Each stain type can be adjusted for color balance and intensity. They can be used with slides or coverslips when coverslip adapters are utilized. Since it is a centrifugal staining system, stain precipitate is minimized and it is very easy to change programs as you shift from peripheral bloods to fluids cytospins to bone marrows.

Staphylococcus aureus, particularly methicillin resistant strains (MRSA), have represented a likely target for molecular development, particularly in blood cultures. As more institutions implement patient screening protocols for MRSA, replacement of routine culture methods with molecular assays has gained increasing attention.PNA-FISH assays provide for the definitive identification of Staphylococcus aureus from positive blood culture vials. Peptide nucleic acid fluorescent in-situ hybridization is a relatively straight forward procedure that does not involve amplification and has limited equipment requirements. Procedurally it is easy to perform with minimal hands on time.PNA is a synthetic imitator of a nucleic acid sequence in which the backbone is a pseudopeptide rather than a sugar. PNA behaves similarly to DNA and will bind to complementary nucleic acid strands. A PNA probe is constructed, utilizing a complementary, hybridizing sequence for a known nucleic acid target sequence. The probe is typically bound to a fluorescent protein as a means of visualizing/detecting the target. In one commercially available method, once a blood culture vial demonstrates gram-positive cocci in clusters, a drop of the blood culture broth is added to fixation solution on a slide. Heat or methanol is used to fix the smear. After fixation, probe that targets species-specific ribosomal RNA is added to the smear, which is then cover-slipped.Slides are then incubated at 55oC. Post incubation, slides are immersed in a preheated wash solution and coverslips gently removed. After incubation in the wash solution, smears are air dried; a drop of mounting medium is added and the slide is cover-slipped again.The slides are examined with a fluorescent microscope, utilizing specific filters. Green fluorescing cocci in clusters are identified as Staphylococcus aureus. This identification would be available, depending on the routine identification system utilized, potentially 24 hours earlier than the norm.A significant number of blood cultures that demonstrate gram-positive cocci in clusters yield coagulase negative staphylococci (CNS), which represent potential contaminants, rather than significant infection. What is the significance of differentiating blood cultures that contain S. aureus from those that are growing CNS in a much earlier timeframe?Studies have shown that IF the differentiation of CNS from S. aureus is effectively communicated to clinicians and pharmacy/antimicrobial stewardship teams, active assessment can occur utilizing defined exclusion criteria for those patients whose cultures yielded CNS rather than S. aureus. In scenarios where contamination rather than infection is indicated, vancomycin can be discontinued earlier, and length of hospital stay is also shortened. Reduced antibiotic exposure, reduced risk of development of resistance, and reduced cost are all potential benefits.