|Coverslip Smear Preparation Technique|
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.
|Biopsy Touch Preparation Technique|
While smears from the bone marrow aspirate are the most common preparations, touch preparations (touch preps) made from the bone marrow biopsy core may also be useful or necessary. When aspirates are difficult or a dry tap occurs, the only sample available to be evaluated in the hematology laboratory may be the bone marrow biopsy. To create a fresh biopsy touch preparation, the fresh bone marrow core is gently rolled between two slides, then gently rolled between five or six pairs of coverslips. There should be enough cellular elements present when using this method for the laboratory professional to evaluate. The imprints will be wet and cellular at first but as the surface dries it will eventually become less cellular. At this point the core is placed in fixative and sent to pathology for evaluation. The number of touch preps you can make is dependent on how wet/ bloody or fibrotic the core is to begin with, but even one set can be enough to aid in diagnosis.While it is not practical to practice making touch preps from a real biopsy core, it is possible to practice the technique by using a length of applicator stick soaked in either blood or stain to simulate a real biopsy core. To do this, simply break off a short, 0.5 inch piece of a standard plain or cotton tipped applicator stick and soak it in the fluid of your choice. As you roll it between slides or coverslips you will see the pattern it leaves behind. Think of the motion of a teeter-totter (seesaw) as you roll. There should be very little downward force on the core as you coax it to roll. If the core will not roll then you can just touch the slides or coverslips to the surface of the core few times on each slide.Note: If the biopsy is placed directly into fixative and sent to pathology, it must first be decalcified before it can be sectioned and stained. This process will take at least 24 hours depending on the lab and if additional stains are required, it may be at least 48 hours before a result is released.
|Bone marrow Smear Preparation|
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.
|Which of the following smear techniques can be utilized when processing bone marrow aspirate samples? (Choose all that apply)||View Page|
|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 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.
|Manual Staining of Bone Marrow Preparations: Wright's and Wright-Giemsa Stain|
Wright's or Wright-Giemsa stains are usually the preferred staining method for bone marrow aspirate smears. These are methanol-based staining solutions with similar dye composition to the diff-quick stain but require longer stain contact time for adequate staining. The Wright's and Wright-Giemsa stains have a buffer step as well. Since Wright's stains are methanol based they do not require a fixation step prior to staining, although you might prefer to do so first to reduce water artifact that can occur on humid days or with aged stain.In the dip method of staining, the smears are first dipped in methanol to fix the specimens and then placed in Wright's or Wright-Geimsa stain for 10-15 minutes to stain. The smears are next moved to a mixture of stain and 6.8 pH phosphate buffer (usually one part stain to 2-3 parts buffer) and allowed to stain for at 20-30 minutes. After staining, they are given a quick rinse in distilled water and allowed to air dry before mounting or cover-slipping.When using a staining rack, the marrow slides or coverslips are first flooded with enough stain to cover the slide and stained for 10-15 minutes. Then, a 6.8 pH buffer is carefully added without overflowing and gently mixed by blowing until a green metallic sheen forms. This is allowed to stand for 20-30 minutes and then rinsed off with distilled water. The slides or coverslips are then air dried and mounted.Staining times can be extended for extremely cellular marrows; however, care must be taken when using the rack staining method. Extended times can lead to evaporation of the stain and cause excessive precipitation. Both the stain and buffer can be topped up if necessary to prevent this from occuring, while additional rinse time may be needed.Wright's and Wright-Giemsa stains, when performed properly, give sharp and clear nuclear, cytoplasmic, and granule detail. There can be variation in the quality of the stain from batch to batch, dependent on the manufacturer's quality control, storage, and shipping conditions. Many manufacturers age their stains for a minimal amount of time before shipping and assume that there will be additional standing time at the distributor before it reaches your lab. This may work for peripheral blood staining, but it is not ideal for bone marrow staining. It is advisable, if possible, to keep a separate stock of Wright's stain for bone marrow staining which is kept at least 6 months before use. Like a fine wine, the older Wright's stain gets, the better the quality and clarity of the final 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.
|Bone Marrow Collection: Patient Bedside|
When the technologist accompanies the clinician to assist with the bone marrow aspiration procedure to make smears at the bedside, it is necessary to understand the role of the clinician and the technologist.The clinician is responsible for patient positioning and sterile preparation, pain control, and performing the aspirate and biopsy. The clinician often hands off sample syringes to the technologist, once collected. The clinicians are responsible for providing the procedure kit and fixative for the biopsy, all labels, and obtaining the requisitions and a copy of the clinical history for the hematopathologist. The technologist will set up a mini workspace near the bedside where the samples are split into the required tubes. Smears are then prepared from the aspirate as well as biopsy touchpreps before the biopsy is placed in fixative. In this setting the technologist will usually deliver the samples and requisitions to pathology and continue the processing procedure.The kit the technologist brings to the bedside usually contains mini petri dishes, coverslips, slides, microcapilary tubes or Pasteur pipettes, micro-pipette bulb and the various evacuated blood collection tubes and media flasks required for the standard bone marrow draw.Most institutions will have a standard draw and testing protocol designed to ensure that enough sample is obtained to cover all of the usual testing requirements. An example would be a three-syringe-draw with the first two syringes containing no anticoagulant and the third syringe rinsed with preservative-free heparin. The first dry pull would be split between a green and a purple top evacuated blood collection tube and would be used for morphology (EDTA) and flow cytometry and cytogenetics (green) if needed. The second dry pull is split into two additional purple top tubes plus a green top tube and would be used for molecular assays such as SNP array, Flt-3, JAK2, MPL mutation, etc. The final heparinized syringe could be used for other treatment protocol requirements or to provide sample for additional assays.
|Identification of Staphylococcus aureus with Peptide Nucleic Acid (PNA)-Fluorescence In Situ Hybridization (FISH)|
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.