Bone Marrow Aspiration: Normal Hematopoiesis and Basic Interpretive Procedures Media Lab

Bone Marrow Biopsy

A bone marrow biopsy is obtained by a similar technique but rather than aspirating liquid marrow, a core of bone is allowed to enter the biopsy needle and is then removed within the needle. The image* illustrates the body site where the bone marrow is normally obtained. The core can be rolled between two slides to make touch preparations (touch preps), which are then stained and reviewed. The bony core is fixed and sent to pathology, where it will be decalcified and prepared for sectioning and staining.

Long Slide Preparation Techniques: Pull Prep

A long slide pull preparation, or pull preparation, is a variation of the T-preparation. This method results in shorter smears than the T-preparation but produces two smears for each drop of bone marrow. In this method, a drop of bone marrow is placed in the center of a slide while a second slide is placed directly on top with as much overlap as possible. The idea is to leave only enough of the edges revealed to allow the preparer to grip the slides for the pulling of the smear. Once the bone marrow has spread toward the edges, the two slides are slowly slid apart, forming a 1-2 inch smear on both the top and bottom slides. This method provides more smears per volume of bone marrow used but is a bit more difficult to use without regular technique practice. The placement of the drop of marrow is critical to obtaining two usable smears from each pulled smear. Pull preps can be made at the patient bedside from a syringe or from an anticoagulated bone marrow sample.

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.

Myelocytes: Eosinophils and Basophils

As stated previously, the myelocyte stage is the first stage where cell-specific maturation appears in the granulocyte lineage. When you look at a promyelocyte, you cannot yet identify if it will mature into a neutrophil, eosinophil, or basophil. It is only when secondary granules are produced that the endpoint of maturation can be identified. The size and shape of these initial secondary granules help with this identification. While the cytoplasm and granules may look different depending on the lineage of the cell, the progression of nuclear maturation is the same for all granulocytes. When eosinophil secondary granules are first produced, they may not show the same bright orange color found in the mature eosinophil. It is the larger, spherical shape of the granules that identifies the early eosinophil myelocyte, not necessarily the eosinophilic color. In fact, early eosinophil granules may appear somewhat basophilic in color. The larger, three-dimensional, spherical shapes of the granules help to identify cells as eosinophil precursors (see red arrows in the top image). Immature basophils (see blue arrow in the lower image) can be hard to distinguish from promyelocytes as well. It is important to note the differences in color and shape between primary neutrophil granules and basophil secondary granules. The primary granules in promyelocytes appear as red/purple grains of sand - think of red/purple cubes with defined edges. Basophil granules have a purple /black color and look more like splinters - think of a purple/black sheet of glass that is shattered into the cytoplasm of a cell. The cytoplasmic color in an early basophil myelocyte may be similar to a promyelocyte since there are no fine granules in the basophilic cytoplasm. Remember that mature basophils are not as fully granulated as neutrophils and have a clear/uncolored cytoplasmic background rather than the pink/tan background of neutrophils.

Introduction to Bone Marrow Aspirates and Biopsies

Bone marrow aspiration and biopsy are standard tools used in the hematology laboratory to aid in the evaluation and diagnosis of peripheral blood abnormalities. Some of these abnormalities include cytopenias such as neutropenia, thrombocytopenia, and anemias. Bone marrow aspiration and biopsies are also used by hematology/oncology specialists in the diagnosis of leukemias, dysplastic syndromes, and proliferative syndromes. A bone marrow aspiration and biopsy may also be a part of the evaluation of some metabolic and genetic disorders, assessment of fever of unknown origin (FUO), as well as when assessing failure to thrive (FTT) in the pediatric setting. A bone marrow aspirate sample is obtained by inserting a needle into the bone marrow space and withdrawing using a syringe. A portion of this liquid marrow is smeared for staining and evaluation under light microscopy. Liquid marrow samples are also transferred to evacuated blood collection tubes containing the anticoagulants required for the types of assays desired. It can be sent for various types of laboratory assessment including immunophenotyping, cytogenetic evaluation, and molecular analysis. While bone marrow aspirations and biopsies are usually obtained by the hematologist or oncologist, they are evaluated and interpreted by a hematopathologist with the assistance of the laboratory technologists who prepare and stain the smears. In many laboratory settings, the technologists also perform bone marrow differentials.

Rules for Bone Marrow Differentials

Bone marrow differentials have significant differences from peripheral blood differentials that need to be considered as they are reviewed and counted. One of the most important facts to consider is the large variability in cellularity and cell distribution depending on the type of preparation that is used. Choosing where to count and when to use which of the smear types available to you, takes time and experience and can be directed by a pathologist's preference. Regardless of how many, or what types of smears you have available to choose from, you will always start with a simple visual inspection of your smears. -Begin by recording the patient identification information as well as the date of sample, and any other mandatory patient identifying information necessary for your laboratory. -Record aspiration site information when provided. Some patients will have bilateral bone marrow aspirates performed as part of a diagnostic or staging workup. -Standard aspiration sites are: posterior iliac crest (PIC), anterior iliac Crest (ANT), sternum (S), spinous process (SP), and sometimes in very young children, bone marrow is obtained from the tibia (T). -Be aware, that while a bilateral bone marrow aspirate usually involves an aspirate of the same site from opposite sides of the body, e.g., L-PIC and R-PIC, in some situations, a bilateral staging aspirate will be from two different compartments on the same side, e.g. R-AIC, R-PIC. Observe the appearance of the bone marrow smears. Do any have feather edges? Are there fragments or spicules present on any of the smears available? If so, they should be your first choice to view, since they are more representative of what the biopsy will show if one was obtained. Once you select your smears, scan using 10X magnification on the microscope. Are some of the fragments/smears so thick that you cannot see good morphology? If so, reject these areas/slides. Are some of the fragments/smears so thin that everything is smashed? These areas/smears cannot be used either. Are there areas in the vicinity of any of the fragments that have good staining characteristics as well as readable morphology? This is where you should begin your differential.

Rules for Bone Marrow Differentials, continued again

Bone marrow smears can be very cellular and it can be difficult to keep track of where you are on the smear while keeping your correct hand position on the keyboard. Having a good strategy to use when counting cells and performing differentials can make this less difficult. On peripheral blood differentials, it is easy to observe and count each cell individually as the stage is moved to bring the next field into view. However, with bone marrows, the total number of keys that need to be used on the differential counter is greater than the number that needs to be used with a peripheral blood smear, and the number of cells per field is also increased dramatically, making it easy to lose track of the cells on the smear or one's hand/keyboard placement. It can be simpler and less stressful to work on the quadrant system. There are two different ways to do this: 1.)Divide the field into quadrants. Count the individual cells in each quadrant separately. This decreases the number of cells into more manageable bites. However, you still have an increased number of cell types to deal with and possible keyboard frame shifts. 2.)Divide your keyboard into quadrants. Search your field for a limited number of cell types and tally all you see before moving on to the next grouping of cell types. Once you tally all your groups then move on to the next field (e.g., lymphocytes, monocytes, macrophages, eosinophils, basophils, plasma cells, erythroid, segmented neutrophils, bands, etc). You can make these small groupings for any cells as long as you cover the entire list of cell types that your laboratory reports in its bone marrow differential protocol. Remember that blasts are identified by cell type and there will usually be a separate key for pronormoblasts, myeloblasts, lymphoblasts, and possibly monoblasts and plasmablasts. Depending on the laboratory's protocol, promyelocytes, myelocytes, and metamyelocytes may be quantified together. All erythroid precursors may also be reported together. Eosinophils and precursors & basophils and precursors may also be reporting categories. Because of the variations in methods of cell type reporting in bone marrow differentials, it's paramount to refer to your laboratory's reporting procedure. It is possible to combine both methods, using the keyboard quadrant technique with a restricted portion of the total microscope field. This is useful when you are getting close to your total tally and do not want to alter the balance by only counting one cell type for the last few cells.

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.

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 thumbprint. 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 an 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.

Cellularity and Additional Information

Depending on the institution and laboratory protocol, comments on the degree of cellularity, presence of megakaryocytes, and presence of tumor cells may be added to the report by the technologist who performs the differential count. The terms used to describe these features will be determined by the hematopathologist. Cellularity is usually rated as normal, increased, or decreased. However, other terms may be used as well, such as "slightly decreased," "markedly decreased," or "markedly increased," etc. When spicules/fragments are not present, terms like "hemodilute" can be used to note very dilute bone marrows or, it may simply be marked as "not evaluable". For megakaryocytes the common terms of quantitation may be: None Seen Rare Decreased Present Normal Increased The presence of tumor cells should be noted as well as the slide or site in which they were observed. The top image on the right demonstrates a bone marrow that is markedly hypocellular. Only fragments of the bone marrow structure are present, with very few bone marrow precursor cells observed. The amount of fatty tissue is increased. Compare this to the bottom image that depicts a bone marrow that is normocellular.

Which of the following smear techniques can be utilized when processing bone marrow aspirate samples? (Choose all that apply) Differential smear Touch prep Coverslip smear Pull prep T-prep

Differential smear Coverslip smear Pull prep T-prep Coverslips, differential smears, pull preps and t-preps are all techniques used when making bone marrow aspirate smears. Touch preps are made from bone marrow biopsy samples.

In which of the following situations would a bone marrow aspirate and biopsy possibly be used as an aid for diagnosis?(Select all that apply) Evaluation of anemia and thrombocytopenia Evaluation of cytopenias Diagnosis of leukemias Evaluation of fever of unknown origin (FUO) Screening for hematologic issues during routine check-ups

Evaluation of anemia and thrombocytopenia Evaluation of cytopenias Diagnosis of leukemias Evaluation of fever of unknown origin (FUO) Bone marrow biopsies are helpful diagnostic tools in: -Evaluation of anemia and thrombocytopenia -Evaluation of cytopenias Diagnosing leukemias Can be part of evaluation for FUO Bone marrow biopsies are NOT used as screens for hematologic issues during routine check-ups.

The same staining protocol can be used for bone marrow smears as is used for peripheral blood smears. T/F

False Bone marrow smears are much more cellular and require additional contact time in both stain and buffer. They should not be processed in exactly the same way as peripheral bloods.

When performing bone marrow differentials it is not necessary to distinguish the precursor forms of the erythroid sequence. T/F

False In the bone marrow differential, all stages of the erythroid sequence are counted independently. This differs from a peripheral blood differential, where the term "nucleated red blood cells" ("NRBCs") is used to describe all stages of circulating normoblasts.

Bone marrow Differentials

For the clinical laboratory professionals who are only familiar with peripheral blood morphology, the first few observations of bone marrow aspirate smears can be overwhelming. The difference in cellularity between the two sample types, not to mention the wider variety of cell types, can lead to mental and visual overload. It is important to step back and break it down into more manageable pieces, starting on low power. Use low power (10x) to look at the distribution on the slide and the quality of the stain. Find areas where the spread/distribution of cells are thin enough (monolayer) to read easily and where you like the color balance and intensity of the stain. Next, add oil and move up to 50x and/or 100x power on the microscope.* Remember that there are several different cell types that are normally present and develop in the bone marrow before heading out into the peripheral blood. Most hematology technologists are familiar with the myeloid maturation sequence from peripheral differentials, even if immature cells are less commonly seen. However, there are additional cell types that are not seen on the peripheral blood differential, since they reside only in the bone marrow. Becoming more familiar with these cell types and the maturation sequences of the myeloid, erythroid, and megakaryocytic cells found in normal bone marrows will make performing these differentials less intimidating. One important concept to grasp is the continuum of cellular maturation sequences. There is no such thing as a magical switch that flips causing cells to jump to the next "textbook photo stage" as cell lines mature. Rather, each cell matures at its own pace. The maturation and morphology will vary from cell to cell and bone marrow to bone marrow. Understanding both nuclear and cytoplasmic normal morphology can aid in the identification of cells.

Hematogone

Hematogone is a term applied to a subset of early B-lymphocytes, found in normal bone marrow, whose morphology greatly resembles that of leukemic lymphoblasts, but are actually benign. These immature B-lymphocytes can be difficult to differentiate from other blast cells or even prolymphocytes. These cells are larger than the average mature lymphocyte, have scant cytoplasm, and a fine, soft chromatin texture; however, they are not quite as immature in appearance as a true leukemic lymphoblast. Hematogones are more common in younger children but can be found in bone marrow samples of patients at any age. They tend to be found in increased numbers within the bone marrows of patients recovering from bone marrow suppression. Common causes of increased concentrations of hematogones include viral illness, chemotherapy recovery, and immune-mediated cytopenias, such as idiopathic thrombocytopenic purpura (ITP). Hematogones are also common in patients with neuroblastoma. With experience, knowledge of the patients underlying clinical condition, and the ability to review a patient's bone marrow, it is possible to distinguish hematogones from blasts. When necessary, a hematogone flow cytometry panel can be obtained to distinguish these benign cells from lymphoblasts. Notice the size of these blast-like hematogones (see red arrows). They are larger than the few background lymphocytes present in these images. Notice the fine chromatin and scant cytoplasm. They are usually found mixed in with the full range of bone marrow cellular lineages but can cluster with other lymphocytes within the spicules.

Bone Marrow Delivery

In some institutions, the laboratory technologist does not assist the clinician at the bedside with the bone marrow aspiration procedure. Instead the clinician delivers the bone marrow sample to the laboratory, similarly to other laboratory specimens. When this is the case, the bone marrow sample may be delivered in one of two manners with the laboratory's responsibilities dependent on which method is used. 1.) A clinician may deliver to the laboratory a specified number of smears, made at the bedside, along with the bone marrow sample. Samples may also be designated for flow cytometry, cytogenetics, or molecular diagnostics. 2.) A clinician may deliver a standard package of bone marrow aspirate to the laboratory in various evacuated blood collection tubes. In this situation, the laboratory will usually have a standard order set that directs the distribution of the marrow samples based on the diagnosis. The hematology laboratory will use these samples to prepare the bone marrow smears, while the other tubes would be distributed for flow cytometry, cytogenetics, molecular diagnostics, etc. based on the direction of the hematopathologist.

Metamyelocyte

In the metamyelocyte stage, the cytoplasm and nucleus continue to decrease in size. The cytoplasm achieves full secondary granule content. The chromatin becomes denser, knotted, and compact, while the nucleus begins to indent and acquire the familiar "kidney bean" shape.By the end of the stage, the cell will be similar in size to a mature neutrophil with similarly cytoplasmic granularity. The top image to the right shows a fairly classic metamyelocyte. Observe the indented kidney bean-shaped nucleus and neutrophil-colored cytoplasm. Notice the clumped aggregates of chromatin in each pole of the nucleus. The vacuolated cytoplasm in this cell is an indication of toxic stress. In the bottom image to the right, notice the metamyelocytes (see red arrows) and their chromatin patterns. The patterns become denser and clumped as the metamyelocytes continue to mature to the band neutrophil stages (see blue arrows).

Polychromatophilic Normoblast

In the polychromatophilic normoblast stage, the cytoplasm has begun to produce hemoglobin and, as a result, the color starts to shift from deep basophilic to a slate blue/gray shade. The cell continues to slowly shrink in size while the chromatin becomes much more knotted and clumped. The spoke-like pattern of the chromatin accentuates the nuclear membrane and the nuclear pores. The top image on the right shows a clump of polychromatophilic normoblasts. Notice they are all very similar in size, shape, and stage of maturation. This is a classic pattern in erythroid development and these clusters are frequently associated with macrophages or histocytes in the marrow as they are the RBC precursors' source of iron. Note that the cytoplasm color is now a blue/gray rather than the deep midnight blue of the basophilic pronormoblast stage. The lower image on the right shows a range of RBC precursors. At the bottom is a cluster of basophilic normoblasts (see red arrow), one of which is binucleate. There are also two cells above the cluster: the top cell is an early polychromatophilic normoblast (blue arrow) while the lower is a late basophilic normoblast (green arrow). Note the difference in cytoplasm color. The polychromatophilic normoblast is slate blue/gray while the basophilic normoblast still maintains the midnight blue hue. Observe the nuclei and the chromatin pattern: the chromatin is much more condensed in the polychromatophilic normoblast.

Promonocyte

In the promonocyte stage of development, the nucleolus is still visible while the nucleus begins to indent and fold. This may be observed as pleated or creased-looking chromatin or as a definite flattening or indenting of the nucleus. The chromatin will begin to condense but will still be finer and more "lacy" than what is found in a mature monocyte. The cytoplasm of the promonocyte will begin to mature and the color begins to shift toward the blue-gray, grainy texture found in mature monocytes. The fine pink granules found in mature monocytes will also begin to appear. The image on the right is from a patient with monoblastic leukemia. This slide permits the observation of several promonocytes in one image. These cells would only rarely be seen in the normal bone marrow. Notice the folded and indented nuclei of the promonocytes (see red arrows). Note that as the promonocyte matures, the cell size decreases and the complexity of the nucleus increases. Notice the fine pink granules, which increase in number as the cell size decreases.

Iron Staining

Iron staining on bone marrow aspirate smears is commonly part of the standard order protocol for bone marrows aspirates. The iron staining procedure utilizes the Prussian Blue stain for ferric iron to assess bone marrow iron stores. This procedure is particularly helpful when evaluating patients with anemia, iron overload, myelodysplasia, etc. In the adult setting, it is commonly performed on the bone marrow biopsy, but can be requested on the aspirates as well. In the pediatric setting, it is less likely to be part of the standard order set since young children rarely have stainable iron stores. However, iron staining may be requested on patients with congenital anemia and possible mitochondrial defects to look for sideroblastic anemia. In this technique, iron will stain blue and will normally be found in bone marrow stromal/ macrophages, which are found in the spicules. On aspirate smears, without fragments/spicules, it is not possible to evaluate for iron stores. However, if there are nucleated red blood cells (NRBCs) present, it is still possible to look for the ringed sideroblasts, common in sideroblastic anemias. The image on the right is a field from a bone marrow slide from a patient with congenital sideroblastic anemia. The NRBC indicated by the red arrow is a normal siderocyte with few granules of hemosiderin scattered through the cytoplasm. The NRBC that is indicated by the blue arrow has a large number of hemosiderin granules concentrated in the mitochondria that surround the nucleus. This is a pathologic ringed sideroblast.

Rules for Bone Marrow Differentials, continued

It is important to note that not all smears will have good areas to perform a differential in the vicinity of the bone marrow fragment. When this occurs, you must keep looking at additional smears. This is one of the reasons that several smears are stained and prepared for possible review. It can take time to recognize from 10x magnification what will be countable on 50x magnification. The best tip is to be patient and does not fail to keep on looking! In fact, sometimes it may be necessary to stain additional smears/slides, if available, to obtain enough readable material. While you are checking the smears on 10x magnification for readable areas, you should take the time to evaluate and record the following: -The cellularity of the bone marrow sample -Presence and number of megakaryocytes -Presence of tumor cells -Anything else out of the ordinary, which should be noted on the report (such as evidence of hemophagocytosis, storage disease etc.). Once you have decided where to count the marrow, you will perform the differential count. Usually a 200-cell bone marrow differential is the minimum acceptable count. However, more cells may be required depending on your laboratory/pathology protocol. Remember, unlike peripheral differentials, all nucleated cells are included in the total count, including all maturation stages of the erythroid cell series. Cell counts are performed on 40 - 50x magnification with oil depending on the optics of your scope, moving up to 100x magnification with oil as needed for fine detail. Once the oil is added to the smear, move systematically through your chosen area until the morphology/cellularity/stain quality is no longer acceptable, then move back to 10x power to find another good area in the vicinity of the fragment to continue your count. You may need to progress from one slide to the next to accumulate enough cells for your differential. In fact, if there is variability in cell distribution from one smear or fragment/spicule to the next, then the count should be split between more than one smear/fragment to avoid a biased final count. If there are no spicules, then the differential should be performed in any portion of the slide that demonstrates readable morphology. In pull preps and coverslip preps, this will usually be in the thin area near the edge of the smear. If differential-type (wedge) smears are available, then the usual feathered-edge area should be used. On any of these smears, be sure that you are in deep enough from the thin edge so that the numbers of stripped cells are kept at a minimum to avoid skewing the count, as some cell types are more fragile than others. The pathologist is ultimately responsible for the final sign-out and will change/adjust/return smears for recount if there is any disagreement over numbers and cell types.

Lymphocyte

Lymphocytes undergo two phases of maturation: first in the bone marrow or thymus, and then in the lymph nodes or other lymph tissue. Many hematology texts describe the maturation series as lymphoblast, prolymphocyte, and lymphocyte. This lymphocyte is the one seen in peripheral blood smears and is considered "naive" because it has not yet been stimulated by antigen. Also, mature lymphocytes are normally present in the bone marrow and, when clustered in a lymphoid follicle, can be very prominent. Lymphocytes can be found scattered throughout the bone marrow and must be distinguished from early erythroid precursors, which they can closely resemble. Lymphocytes are frequently found in and around early NRBC clusters. In the top image on the right, notice the medium-sized lymphocyte (red arrow) next to the two basophilic normoblasts (blue arrow). The color and texture of the scant lymphoid cytoplasm are almost identical to the NRBC, which can be a bit confusing. However, observe the differences in the nuclei between the two cell types. The lymphocyte has a less distinct chromatin clumping pattern than the basophilic normoblasts and the lymphocyte does not have any "nuclear pores." Also, the lymphocyte has an irregularly-shaped nucleus that is hugging the cytoplasmic border, while the NRBC has a round and regular, centrally-placed nucleus. Identify the three lymphocytes circling the NRBCs in the second image (see red arrows). Notice the chromatin of the lymphocytes; the lymphoid smudgy/clumpy pattern is certainly not as dense and clumped as what is noted in the NRBCs. This nuclear difference becomes more pronounced as the erythroids mature. The cytoplasmic differences should be more apparent as well since lymphocytes will never produce hemoglobin.

The role of the laboratory technologist in processing bone marrow aspirates can vary depending on laboratory and clinician protocols. Which of the following roles may be performed by a laboratory technologist?(Select all that apply) Make smears from samples delivered by clinicians. Makes smears at bedside. Perform bone marrow aspirate and biopsy. Interpret bone marrows.

Make smears from samples delivered by clinicians. Makes smears at bedside. The laboratory technologist's role may include: making smears, either at the patient bedside or in the laboratory and staining the smears. The clinician or pathologist obtains the bone marrow samples and interprets the results. In some laboratories, the technologist may also be responsible for performing the bone marrow differential count.

Monoblast

Monocytes progress through maturational stages in a similar fashion to the myeloid series before entering the peripheral blood circulation. The final stage of monocyte maturation into macrophages occurs after they have migrated out of the peripheral blood and into the surrounding tissues via diapedesis. Mature macrophages are also found in the bone marrow. The monocyte lineage does not maintain a maturational pool in the bone marrow as large as the myeloid pool. As a result, the monoblast stage is infrequently noted in most normal bone marrows. Monoblasts are the largest blasts of all the hematopoietic cell lines present in the bone marrow. They have a large, round, centrally-placed nucleus with soft, fine-stranded chromatin. They normally have a single, large, prominent nucleolus. The cytoplasm is very generous and has a fine, grainy texture. In the monoblast stage, the cytoplasm will be basophilic, similar to other blasts, but will possess a slightly lighter shade of blue. In the monoblast, the color will shift to blue-gray as the cell matures into a monocyte The top image on the right shows a single monoblast. Notice the large, round nucleus, the single large nucleolus, and the generous blue, grainy cytoplasm. The second image shows a group of monocyte precursors. The large cell at the top is a monoblast (see red arrow). Notice the round and flat look of the nucleus in the blast compared to the other stages. Observe the nuclear shape becoming more folded and three-dimensional as the cell matures.

Bone marrow Smear Preparation: Selecting Fragments

Most bone marrow slides are made simply by placing a drop of bone marrow on a slide and using a smear preparation technique. However, in order to obtain consistently high-quality smears, it is necessary to select or concentrate the fragments on these smears. Selecting or concentrating fragments can be performed with different methodologies. At the patient bedside, some clinicians will use the touch-preparation or pull-preparation method, while tilting the slide to allow excess blood to roll off. This leaves more of the bone marrow spicules on the slide. This can be wasteful and rather messy but does not require a high level of skill. A less wasteful method is to pour a portion of the marrow aspirate into a small petri dish and swirl it about, then tilt the dish to reveal the marrow spicules. These can then be extracted using a capillary pipette with a micro-pipette bulb and transferred to the slide for use in making smears. This technique allows the laboratory professional to make numerous smears containing fragments rather than relying on the random luck of the drop. Any excess marrow can be saved and returned to the EDTA tube for further testing. This capillary pipette concentration technique can be coupled with any of the smear preparation techniques but does require practice to perfect and maintain proficiency. When coupled with the coverslip method, it is possible to make 2-3 dozen quality smears from as little as a 0.25 - 0.50 mL of marrow aspirate, making it ideal in small sample volume situations.

Place the following stages of neutrophilic granulocyte cellular development in the correct order of maturation, beginning with the earliest recognizable form:

Myeloblast=Stage 1 Promyelocyte=Stage 2 Myelocyte=Stage 3 Metamyelocyte=Stage 4 Band neutrophil=Stage 5 Segmented neutrophil=Stage 6

Calculating and Reporting the Myeloid:Erythroid (M:E) Ratio

Once the bone marrow cell count is completed and recorded, the M: E ratio should be assessed. This is performed by calculating the total myeloid precursors in proportion to the total erythroid precursors. Remember that this does not use the total white blood cell tally; the myeloid cells alone are counted, excluding lymphocytes, monocytes, macrophages, plasma cells, megakaryocytes, osteoclasts, osteoblasts, and other non-myeloid cells. In most circumstances, it is quite simple to divide the myeloid total by the erythroid total to find the ratio. This is always reported as a whole number ratio and is normally around 3:1 (reference range= 2:1 to 4:1). In some situations where the erythroid portion is increased or the myeloid series is decreased, the M:E ratio is reversed. This would still be expressed as a whole number ratio (example: 1:2). A simple way to perform the calculation is to always divide the larger value by the smaller one. Which side of the colon the 1 is placed on depends on which cell type was larger. The 1 always belongs on the side of the cell type found in lower numbers. For example: Myeloid total 120 : Erythroid total 40 M:E ratio =120 ÷ 40 = 3 or 3:1 So, the M:E ratio is 3:1 Another example: Myeloid total 30 : Erythroid total 150 Divide the larger number by the smaller (notice that the placement is reversed). 150 ÷ 30 = 5 So, the M:E ratio is 1:5

Plasma Cell

Once the naive B lymphocyte leaves the bone marrow, it will enter the circulation and then travel to the lymph nodes. In the lymph nodes (or other lymph tissue) if a B lymphocyte's antigen receptor recognizes its specific antigen and attaches to it, a series of intracellular signals are sent to the nucleus to begin dividing and cloning itself, as well as starting to synthesize immunoglobulin (antibody). This process often needs the help of cytokines from T Helper Cells. Once the daughter cells of that original stimulated B lymph start secreting antibodies, they are known as Plasma Cells. Under normal circumstances, plasma cells are not a large percentage of the lymphoid cells found in a marrow. They are usually placed in a separate category in the differential, unlike viral/atypical lymphs. There can be a relative increase in plasma cells in reactive marrows, and both plasma cells and their early precursors will be markedly increased in plasma cell disorders. While mature plasma cells somewhat resemble lymphocytes, there are a few important differences. The size of the cell is usually larger with a more abundant cytoplasm. The nucleus is eccentrically placed and the overall shape of the cell generally resembles a wedge or comet with the nucleus leading the cytoplasm. The chromatin is just as thick and clumpy as a lymphocyte's but is aligned in a more "spokey" or "clockwork" pattern. The cytoplasm is usually more basophilic than the cytoplasm of a normal lymphocyte and will have a well-defined perinuclear halo or noticeable clearing in the golgi area. Vacuoles may or may not be present. Notice the size of the single plasma cell in the top image (see red arrow). It is larger than the neutrophil precursors surrounding it and is almost rectangular in shape. Observe that the nucleus leads the cytoplasm, causing the wedge or comet shape. Sometimes, this distinct morphology is described as a "fried egg" appearance. Notice the prominent perinuclear halo. Find the two plasma cells in the upper left corner of the second image. There is much more cytoplasm in these plasma cells compared to the occasional lymphocyte present in the field. Notice the eccentric nuclear placement as well as the characteristic clearing in the golgi area.

Hemophagocytosis

One of the normal roles of the marrow macrophage is to remove cellular debris. In normal bone marrow, this includes the engulfment of extruded RBC nuclei and old, non-nucleated RBCs at the end of their lifespan. In some patients, macrophages lose their ability to distinguish self from non-self (i.e., invader/ pathogen) and good cells from old/senescent cells. This can happen because of an inborn error in the macrophages or by an infection-mediated transformation. When this change occurs, any cell in the vicinity of a defective macrophage becomes a target for engulfment. This term is called hemophagocytosis The top image on the right shows two macrophages that have ingested several different cell types (see red arrows). There is the normally ingested non-nucleated RBC, but also the abnormally ingested segmented neutrophil and at least one early nucleated RBC precursor. Viable bone marrow precursors are not the usual diet of macrophages. The lower image on the right shows an even more impressive macrophage with at least a dozen or more ingested RBCs as well as three-segmented neutrophils and a lymphocyte.

Orthochromic Normoblast

Orthochromic normoblasts are the last nucleated stage of erythroid maturation. In this stage, the nuclei of the cells completely shrink to a pyknotic remnant. The cytoplasm color approaches the color of a peripheral RBC as it becomes fully hemoglobinized. This is the stage that is most commonly seen when NRBCs are found in the peripheral blood. In the top image on the right there are many orthochromic normoblasts scattered across this section of bone marrow. Note the pyknotic-appearing nuclei which make them easy to spot, even at lower magnification. It is also evident that the cytoplasm is well hemoglobinized and the color is just slightly more blue than the non-nucleated red blood cells present. In the higher magnification (second image), notice the orthochromic normoblast (blue arrow) to the right of the basophilic normoblasts. The color of the cytoplasm of the orthochromic normoblast is almost identical to the background RBCs. Notice how condensed the nucleus has become as well. You can actually observe the nucleus in the early stages of extrusion/elimination from the cell. Once the nucleus has been extruded, the slight blue color, also known as polychromasia, will begin to fade and the now non-nucleated RBC will be indistinguishable from any other circulating RBC.

Osteoblast

Osteoblasts are the cells responsible for the production and deposition of bone. They may not be apparent in normal cellular bone marrow, since they appear in low frequency. In situations where the total bone marrow cellularity is decreased, they become more visible. Osteoblasts are individual cells but tend to travel in small groups or clusters. They are quite large compared to the normal background blood cells and resemble giant plasma cells. They are oval-shaped cells and tend to have quite basophilic cytoplasm. An osteoblast has a single round nucleus with a fairly open chromatin texture. Notice in the images to the right how the nucleus of the osteoblast is eccentrically placed. On some smears, it will almost appear as if the nuclei are in the process of being extruded from the cells. This effect is more commonly seen on extremely hypocellular bone marrows and is less pronounced in bone marrows with higher cellularity. Notice the large size of the osteoblasts in comparison to the background bone marrow elements.

Osteoclast

Osteoclasts are the cells responsible for bone resorption. They work in conjunction with osteoblasts. Under normal circumstances, both cells are constantly in the process of rebuilding/reshaping/repairing bone to ensure strength and function. Osteoclasts are only infrequently seen in bone marrow aspirates. They become more obvious when the cellularity is depressed. Osteoclasts are large multinucleate cells somewhat similar in appearance to megakaryocytes, which can cause confusion. Notice in the images to the right that the nuclei of the osteoclasts are flat, even in number, oval or round, uniform in size, and well separated in the cytoplasm. In contrast, megakaryocyte nuclei are segmented and clump in three-dimensional clusters. The cytoplasm of an osteoclast is grainy and paler in color than a megakaryocyte. Observe how fluid and irregular the cytoplasmic borders are in the osteoclast.

Promyelocyte

Promyelocytes are generally larger than myeloblasts, measuring approximately 12 to 20 microns. The nucleus is similar in size to the myeloblast but the cytoplasm is more abundant at this stage. The nucleoli will begin to close and become less prominent than in the blast stage. The chromatin strand texture in promyelocytes tends to become slightly more coarse and clumped than the chromatin pattern present in a myeloblast. Promyelocyte cytoplasm will have a gritty basophilic color and texture; however, there will also be prominent PRIMARY granules. These granules will look like red/purple grains of sand. With careful observation, one can note the cuboid nature of the granules. In the top image to the right notice the size of the promyelocyte on the right-hand edge (red arrow), versus the other myeloid cells in the frame. Notice how basophilic the cytoplasm is compared to the more mature myelocytes that are present. Observe the prominent, red, primary granules, which stand out against the basophilic background. In the bottom image on the right, the promyelocyte (blue arrow) has matured a bit more, giving it an appearance closer to an early myelocyte. Though the overall size of the cell has not decreased noticeably (as a general rule for what happens as blood cells mature), the depth of the basophilia is not as prominent, nor are the primary granules as obvious as they were in the cell shown in the top image. While the nucleoli are obvious in both cells, the chromatin texture in the cell indicated by the arrow in the bottom image is a bit more clumped and coarse. Also, notice the clearing/ lighter color in the Golgi (perinuclear) zone of the bottom cell (indicated by the green arrow). This is where the first development of neutrophil secondary granules will become evident as the cell progresses to the next stage of maturation.

Which of the following is an important technique in order to obtain consistently high quality bone marrow smears? Use the largest drop of marrow possible for each slide. Do not prepare the slides at the patient bedside. Select or concentrate bone marrow spicules, ensuring they are transferred to the slide. Only make one slide.

Select or concentrate bone marrow spicules, ensuring they are transferred to the slide. In order to obtain consistently high quality smears, it is necessary to select or concentrate the bone marrow spicules (fragments). Selecting or concentrating fragments can be performed with different methodologies. At the patient bedside, some clinicians will use the touch-preparation or pull-preparation method, while tilting the slide to allow excess blood to roll off. This leaves more of the bone marrow spicules on the slide. This can be wasteful and rather messy but does not require a high level of skill.A less wasteful method is to pour a portion of the marrow aspirate into a small petri dish and swirl it about, then tilt the dish to reveal the marrow spicules. These can then be extracted using a capillary pipette with a micro-pipette bulb and transferred to the slide for use in making smears. This technique allows the laboratory professional to make numerous smears containing fragments rather than relying on the random luck of the drop. You would not want to use a very large drop of marrow as this would probably not smear out to a readable layer of cells. Slides can be prepared at the patient bedside if the work area is prepared sufficiently to ensure slides can be easily made. Making slides at the patient bedside is also dependent on hospital policy. It is important to prepare as many slides as possible from the obtained sample to provide the opportunity for choosing the best quality slides and for special stains, if needed.

Bone Marrow Aspirate and Biopsy Collection

Some pathologists prefer their bone marrow smears to be made fresh at the bedside without the use of any anticoagulants. This however limits the number of smears that can be made before the sample clots. Using a syringe, that has been rinsed with preservative-free heparin, to pull the marrow during the procedure will prevent clotting but will introduce morphology changes and staining artifacts. It is preferable to make smears as soon as possible after sample collection. However, when stored in the refrigerator, acceptable smears can be made from an EDTA tube until about 8-10 hours after sample aspiration without introducing excessive amounts of artifacts. This is useful when marrows are collected at times when staffing trained in marrow smear preparation may not be available.

Stromal Cells

Stromal cells are the cells that comprise the backbone of a bone marrow fragment. They provide the support matrix as well as some of the nutrients necessary for the growth of all cellular precursors found in the bone marrow. Stromal cells appear similar to macrophages and tend to be found in sheets. Usually, they are deep in the heart of a fragment. When the differential is counted in the areas adjacent to the fragments, these cells may be improperly identified and should not be counted. While stromal cells may be present in clusters, it is important to recognize that these cells are normal bone marrow elements and are not tumor cells. In addition to stromal cells is adipose tissue or adipocytes (fat cells). In aplastic bone marrow or bone marrow with decreased cellularity, they may be more apparent. However, since they are considered tissue cells, they are not included in the differential. The top image to the right demonstrates a typical stromal clump. The bottom image shows stromal cells mixed with phagocytic macrophages.

Long Slide Preparation Techniques: T-prep

The T-prep technique is a simple pull preparation method, which produces one readable long slide for each drop of marrow used. It does not require much manual dexterity or practice to obtain usable smears. Since this method only produces one usable smear per drop and requires a moderate size drop of marrow, it is not a preferred technique for small samples. However, it is easy to learn and is frequently used by clinicians. To perform this procedure, a drop of bone marrow is placed in the center of a slide (crossbar), and a second slide (post) is placed over it; oriented so the combination looks like the letter t. The marrow is allowed to spread between the two slides while the slide that is on the top (post slide) is pulled across the bottom slide (crossbar). This produces one slide with the bone marrow smear on the top slide. The bone marrow smear should cover approximately 3-4 inches in length. This technique can be performed sequentially with a series of 5-6 slides in a row with a drop of marrow quickly placed on each slide. The bone marrow drops can originate directly from the aspirate syringe at the patient bedside or from a transfer pipette, collecting samples from an anticoagulated bone marrow tube. Once the first smear is made, the slide that initially had the drop of marrow becomes the top (post) slide for the next prep. By reusing the bottom slide, which no longer has any sample on it, one can minimize the amount of workspace required at the bedside as well as reduce material wastage. Because the bone marrow is allowed to spread between two slide surfaces before the prep is pulled/smeared, any spicules present will be spread in a monolayer permitting good cellular identification. However, since only a limited number of smears are usually made, it is less useful for certain leukemia patients where many slides are required for special stains in addition to the normal morphology smears.

Basophilic Normoblast

The basophilic normoblast is slightly smaller in size than the pronormoblast. The chromatin is a bit more condensed, while just beginning to clump. At this stage, the nucleoli will have closed completely. The absence of nucleoli is the major feature that distinguishes a basophilic normoblast from a pronormoblast. The midnight-blue, velvety-look of the cytoplasm is still very prominent, which makes this cytoplasm morphology indistinguishable from that found in a pronormoblast. As a basophilic normoblast continues to mature, the overall cell size will decrease and the chromatin will condense. The cytoplasm will gradually begin to lighten as globin chain synthesis begins. The first image to the right shows three early basophilic normoblasts (red arrows), including one that is binucleate. Notice the grainy, reticular texture of the chromatin. The chromatin has clumped where the nucleoli have closed. The nuclear pores are more prominent. The deep basophilia is starting to lighten in the golgi area, which is normal as globin synthesis progresses. Binucleated red blood cells are normal so long as the two nuclei are of even size. They can be observed most commonly in bone marrows with increased erythroid production. The second image shows a group of basophilic normoblasts (red arrow) maturing toward the polychromatophilic normoblast stage. Notice that the size of the cell continues to shrink. The chromatin is becoming more condensed. Also, notice that the cytoplasm remains quite basophilic.

Macrophage (Histiocyte)

The macrophage is the final stage of development in the monocyte lineage. It is a phagocyte whose roles include the removal of dead and dying tissue and the destruction and ingestion of invading organisms. Macrophages (histiocytes) act as immune modulators as they will present antigens from ingested pathogens to helper T-cells. Their primary role in the bone marrow is the removal of cellular debris, including old red blood cells (RBCs). As a result, they become a source of iron for maturing RBC precursors. A histiocyte is a less phagocytic form of a macrophage with fewer lysosomal granules. Histiocytes may form clusters, or even fuse together into multinucleated giant cells. These giant cells are particularly evident on bone marrow biopsy from a patient with a marrow granuloma. The top image on the right shows the early transformation of a monocyte into a macrophage (see red arrow). Notice the increase in the amount of cytoplasm present as the cell begins to ingest debris in the bone marrow. This is demonstrated by the increasing vacuolization present in the cytoplasm. The larger the debris ingested, the larger the vacuoles will be. The lower image on the right shows a macrophage with large vacuoles (red arrow) adjacent to an RBC cluster (blue arrow). This is a common placement since the macrophage is the iron source for these developing RBCs in the bone marrow.

Megakaryocyte: Mature

The mature megakaryocyte is the largest cell found in the bone marrow. It can easily reach more than ten times the size of the other cells and usually has as much, if not more, cytoplasmic volume than it has nucleus. Once nuclear/DNA replication found in the early megakaryocyte has halted, the amount of cytoplasm will begin to increase until the megakaryocyte reaches its maximum mature size. As platelet granule production increases, the cytoplasm color will shift from basophilic to the grainy pink texture and light lavender color we are familiar with in peripheral platelets. When the cytoplasm has matured enough to produce platelet granules, the process of shedding platelets can begin. This can occur either as an ongoing continuous process or as a single complete release leaving a naked megakaryocyte nucleus. Notice the extremely large sizes of the megakaryocytes in the images to the right. The megakaryocytes tend to be found in the heart of the bone marrow fragments rather than loosely scattered throughout the smear. Observe the proportion of nucleus to cytoplasm. Notice the foamy, shaggy, irregular cytoplasmic border of the single megakaryocyte that is circled in the bottom image. This megakaryocyte is in the process of releasing platelets and small clusters of platelets can be seen in its vicinity.

Megakaryocyte: Immature

The megakaryocyte lineage is the cell line responsible for the production of platelets found in the peripheral blood. Unlike the other bone marrow lineages that decrease in size as they mature, the megakaryocyte starts smaller and increases in size as it matures. The megakaryocyte begins as a mononuclear cell that has the same physical size and nuclear/cytoplasmic proportions as a lymphoblast. This stage is often referred to as the megakaryoblast. Eventually, the megakaryocyte matures into a multinucleated giant cell with vast amounts of cytoplasm. As this cell matures, it can actually increase to more than ten times the size of other nucleated cells found in the bone marrow; it is also larger than the original blast. In the early stages of development, the cytoplasm of a megakaryocyte is basophilic without any obvious platelet granules. The cytoplasm will be darker near the edges of the cell and may have a "foamy" look in the golgi area adjacent to the nucleus. Cytoplasmic granule development is not usually noticeable until the cell's cytoplasm color begins to lighten. Notice the sizes of the early-intermediate stage megakaryocytes (red arrows) in comparison to the background bone marrow cells present in the two images to the right. The megakaryocyte nucleus makes up the largest part of the cell at this early stage. Notice the increasing lobulation as the cell increases in size and how the cytoplasm becomes foamier and slightly more granular as well.

Monocyte

The monocyte is the final stage of monocyte maturation found in the peripheral blood before it migrates into tissues and further develops into a macrophage (histiocyte). When seen in the bone marrow, a mature monocyte will look identical to its peripheral counterpart. It will have fine, lacy chromatin pattern with varying degrees of nuclear folding and condensation. The cytoplasm will be blue-gray in color with a slightly grainy texture. The cytoplasm may have a light sprinkling of fine pink cytoplasmic granules. The mature monocyte will be larger than mature segmented neutrophils, but not quite as large as promyelocytes or early myelocytes. The top image to the right shows several monocytes with varying degrees of nuclear folding (see red arrows). Notice that the chromatin clumping is not as dense as that found in neutrophils. Notice also that the cytoplasm is blue-gray and grainy, not the pink/tan of a neutrophil. Observe that the mature monocytes are slightly smaller than the promyelocytes in the image. The lower image to the right shows a monocyte (red arrow) adjacent to a segmented neutrophil (blue arrow). The monocyte is clearly larger. Notice the increase in size of the two monocytes below (green arrows) as they begin to transform into macrophages (histiocytes). The vacuolation is an indication of this transformation occurring.

Myelocyte

The next stage of the myeloid maturation sequence is the myelocyte. The cytoplasm of this cell begins to produce specific, SECONDARY granules. If the cell is destined to be a neutrophil these secondary granules will be pink/tan and will cause the basophilic color to lighten and break up. At the "dawn" of neutrophilia, these secondary granules are most obvious in the Golgi area. As the cell matures closer to a metamyelocyte, they fill the entire cytoplasm. While the cytoplasm shifts to producing secondary granules it also loses the prominence of its primary granules. In situations where the bone marrow is stressed or forced to make neutrophils quickly, as in sepsis or during certain therapeutic injections, some of these primary granules may persist as "toxic granules". At the same time the secondary granule production begins, the nucleus is shrinking and condensing. The nucleoli close and disappear, the chromatin gets coarser/denser and more clumped, and the chromatin gets tighter darker, and more compact. The very early myelocyte (red arrow) in the top image to the right still displays its immature features. While the chromatin is not as condensed as in the intermediate and late-stage myelocytes in the bottom image, notice how the cytoplasm no longer has the darker basophilic color of a promyelocyte. There are clusters of neutrophil secondary granules that are changing and breaking up the solid basophilic color. Notice too, that you can no longer see any red/purple primary granules. In this cell, the cytoplasm is leading the maturational dance and the nucleus is lagging. The bottom image to the right shows two myelocytes (blue arrows): one intermediate in maturity, one a bit more mature, as well as a metamyelocyte (green arrow). Notice how the size of the cell continues to shrink as the cell matures. It is apparent that both the nucleus and the cytoplasm of the metamyelocyte adjacent have decreased in size and the chromatin has condensed/clumped as the cell matured toward a metamyelocyte.

Automated Stainers

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 plate 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 blood to fluids cytospins to bone marrow.

Pronormoblast (Proerythroblast)

The pronormoblast, or erythroblast, is the earliest stage in erythroid maturation. It is a very round cell that is about the same size as a myeloblast. It has a distinctive deeply basophilic, velvety cytoplasm that does not have the fine background grittiness found in the myeloblast. A pronormoblast typically has a round, centrally-located nucleus, unlike a myeloblast that typically has an eccentric nucleus. The chromatin texture is coarser than myeloid chromatin and is more reticular and bumpy, almost like beads on a string. The pronormoblast will have multiple prominent nucleoli. The nuclear membrane appears highlighted compared to other cell types and there will be small breaks in the membrane that are known as nuclear pores. The erythroid lineage is the only cell line that has nuclear pores, which can help to distinguish intermediate erythroid precursors from lymphocytes. The upper image on the right shows a pronormoblast (red arrow) adjacent to a few monocytes (blue arrows). Notice that the pronormoblast is round and regular and the cytoplasm is intensely basophilic. Observe the central placement of the round nucleus and the nucleoli. Notice the coarse and grainy chromatin texture as well. The lower image on the right shows a late pronormoblast (red arrow) with a few later-stage erythrocyte precursors (blue arrows). While the overall size of the late pronormoblast shown in this image is similar to the cell in the upper image, notice the less prominent nucleoli with the classic reticular grainy pattern of the chromatin. The cytoplasm still has the midnight-blue, velvety look of a pronormoblast.

Segmented Neutrophil

The segmented neutrophil is the end stage of maturation in the myeloid lineage. The cell is similar in size to the band neutrophil and has a well-granulated cytoplasm with a deeply condensed, knotted, and clumped chromatin pattern. The chromatin pinches into several segments, usually separated by visible filaments. In some segmented neutrophils, this filament is inferred by the folding and shape of the nucleus. The top image on the right shows the classic morphology of a segmented neutrophil. The nucleus of a normal segmented neutrophil has two to five lobes, connected by thin filaments. Six or more lobes are an indication of abnormal development, usually related to B12 or folate deficiency. The bottom image shows the progression from band neutrophil (red arrows) to early segmented neutrophil (blue arrow) and finally to fully-mature segmented neutrophil (green arrow). Take a close look at the cell closest to the promyelocyte. You can see a drumstick-like projection arising from the end terminal segment. This can be seen in smears on female patients and is a Barr body or inactivated X-chromosome.

Long Slide Preparation Techniques: Wedge Smear

The simplest way to prepare a bone marrow smear is to treat it similarly to a peripheral blood smear. A drop of well-mixed marrow sample is placed near one end of the slide and a second slide is used to draw the length of the slide until a feathered edge is produced. The slide should be air-dried and labeled with the patient's identification, date of collection, sample site, or lab accession number (depending on the laboratory's standards). If the marrow is rich in spicules or fragments, there will be "clumps" of marrow fragments at the end of the feathered edge where these fragments are deposited. Fragments, also known as spicules, are aggregates of bone marrow cells that are pulled from the bony matrix of the marrow space (trabecula) during the aspiration process. They represent what will be seen on the bone marrow biopsy between the bony trabecula, once the biopsy is decalcified and sectioned. Since these spicules are cohesive aggregates of cells, they will not spread well if a wedge smear technique is used, which may make it difficult to identify cell types present in the thicker parts of the spicules. Another disadvantage of using the wedge technique to make smears of bone marrow is the fact that the distribution of cells will not be uniform or representative of how the cells are distributed in the marrow. This technique tends to skew the spread, based on the size of the cells present. Just as the larger bone marrow spicules wind up at the end of the feathered edge, so do cells like megakaryocytes and other aggregates, such as tumor clumps. However, since there tends to be more of a feathered edge in this type of preparation, it can be preferable to other methods when the marrow to be evaluated has no fragments. Long slide prepared smears are particularly useful when evaluating leukemic patients at days 8 and 15 of therapy when the cellularity is greatly reduced and clear morphology is needed to differentiate between treated lymphocytes and treated blasts. When this smear technique is used, it is easier to distinguish blasts from lymphocytes.

Myeloblast

Under normal circumstances, the segmented neutrophil is the most common nucleated cell in the peripheral blood. These bacterial-infection-fighting cells are produced in the bone marrow and arise from their precursor cell, the myeloblast. The myeloblast is the youngest cell in the myeloid lineage. It is approximately 12-20 microns in size with a very basophilic cytoplasm. The nucleus takes up around 2/3 of the total cell volume with soft, finely-stranded chromatin with very little clumping. The nucleus is eccentrically placed and ovoid, but can also be slightly flattened. Myeloblasts will typically have two or more nucleoli with well-defined nucleolar membranes. In a well-stained preparation, you should be able to observe the outline and blue color of the nucleoli. The myeloblast's cytoplasm is basophilic and can have a hint of background "ground glass" graininess. This graininess is separate from any primary granules that develop as the cell progresses toward the progranulocyte stage. The cytoplasmic membrane tends to be regular without much denting, bumps, pseudopods, or shredding. Within the cytoplasm of myeloblasts, Auer rods may be present. Auer rods are needle-like cytoplasmic inclusions that result from an abnormal fusion of the primary (azurophilic) granules. The cell in the first image on the right shows the relative size, nucleus, and gritty basophilic cytoplasm of a classic myeloblast. Note that there is a small cluster of red primary granules present which, in addition to its other features, help to identify this cell as a myeloblast. While the myeloid sequence tends to be the predominant cell type found in normal bone marrows, myeloblasts should make up less than 5% of the bone marrow's nucleated cells.

Band Neutrophil

When a neutrophil reaches the band stage, the cytoplasm is normally fully granulated and should be identical to its fully mature segmented neutrophil counterpart. The chromatin has continued to condense and should appear as knotted and clumped as a mature neutrophil. When a cell matures from a metamyelocyte to a band, the condensation and indentation of the nucleus reach the point where all parts of the nucleus are of uniform width. While a U-shaped nucleus is the most classic shape, the band nucleus can be curled or coiled as well. The cell in the top image to the right is an early band. It has a U-shaped nucleus, but still has a bit of openness to the chromatin texture. As the band moves on to the mature segmented neutrophil stage, the entire nucleus will become quite condensed. In the second image, there is a late band neutrophil just above the promyelocyte (see red arrow). Observe the increased amount of clumped and condensed chromatin present here. The cytoplasm appears similar to the segmented neutrophils in the same frame. It is also important to note how much more condensed the chromatin becomes in the mature segmented neutrophils, also in this frame. When it becomes difficult to see if the band has pinched enough to be considered a mature segmented neutrophil, the degree of chromatin condensation and clumping can be used as an additional deciding factor.

Erythrocytic Cells: Introduction

When performing bone marrow cell identification, it is necessary to differentiate the stages of erythrocyte development. This differs from a peripheral blood differential, where the term "nucleated red blood cells" ("NRBCs") is used to describe all stages of circulating normoblasts. As with the myeloid sequence, there is a continuum in the erythroid maturation process in terms of nuclear and cytoplasmic morphology. Becoming familiar with the range of variation in each nucleated erythrocyte stage will make bone marrow differentials less intimidating. The image to the right shows several different stages of erythroid maturation with several clusters of NRBCs all maturing together.

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, microcapillary tubes or Pasteur pipettes, micro-pipette bulbs, 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 samples for additional assays.

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 a 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

Clinical Laboratory's Role: Bone Marrow Aspirates and Biopsies

While the role of the hematopathologist in the interpretation of bone marrow samples is well defined, the role of the technologist can vary greatly depending on the laboratory and the clinical setting. In some cases, physicians deliver prepared bone marrow smears to the laboratory that they have been prepared at the patient's bedside. In other settings, the technologist assists in the bone marrow collection procedure by making bone marrow smears at the bedside. There are also situations where a physician will bring anticoagulated bone marrow in specimen tubes to the laboratory for the technologist to smear, stain, and distribute as the hematopathologist requires. Once the marrow smears are prepared and stained, the next steps will vary depending on the laboratory. In some institutions, it may be the hematology or oncology fellows/attending physicians who are responsible for counting and evaluating the aspirate smears, while the biopsy samples go to pathology. In other settings, it may be the technologists who perform the differentials while hematopathology completes the evaluation and interpretation.

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 artifacts that can occur on humid days or with aged stains. 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-Giemsa 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 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 occurring, 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.