hematology chapter 17: bone marrow examination

aspiration

1. In a separate location from the biopsy, the physician inserts a 14- to 18-gauge aspiration needle such as the University of Illinois needle, with obturator, through the skin and cortex of the bone 2. The obturator is removed, and a 10- to 20-mL syringe is attached. The physician withdraws the plunger to create negative pressure and aspirates 1.0 to 1.5 mL of marrow into the syringe 3. Collecting more than 1.5 mL dilutes the hematopoietic marrow with sinusoidal (peripheral) blood 4. The physician detaches the syringe and passes it immediately to the laboratory scientist, who expels the material onto a series of clean and sterile microscopic slides or coverslips 5. The physician may attach a second syringe to aspirate an additional specimen for cytogenetic analysis, molecular diagnosis, or immunophenotyping using flow cytometry. The needle is then withdrawn, and pressure is applied to the wound

crush smears

1. MLS expels a portion of the aspirate to a Petri dish or watch glass covered with a few milliliters of K3EDTA solution and spreads the aspirate over the surface with a sterile applicator 2. Individual bony spicules are transferred using applicators, forceps, or micropipettes (preferred) to several ethanol-washed glass slides 3. The scientists places additional glass slides directly over the specimens at right angles and presses gently to crush the spicules 4.The slides are separated laterally to create two rectangular smears, which the scientist may fan to encourage rapid drying 5. Some scientists prefer to transfer aspirate directly to the slide, subsequently tilting the slide to drain off peripheral blood while retaining spicules. Once drained, the spicules are then crushed with a second slide as described earlier 6. The scientist may add one drop of 22% albumin to the EDTA solution, particularly if the specimen is suspected to contain prolymphocytes or lymphoblasts, which tend to rupture. The albumin reduces the occurrence of "smudge" or "basket" cells often seen in lymphoid marrow lesions 7. procedure may also be performed using ethanol-washed coverslips in place of slides. The coverslip method demands adroit manipulation but may yield better morphologic information, because the smaller coverslips generate less cell rupture during separation. Use of glass slides offers the opportunity for automated staining, whereas coverslip preparations must first be affixed smear side up to slides and then stained manually

BM aspiration

1. MLS receives the aspirate syringe from the physician at the bedside and immediately transfers drops of the marrow specimen onto six to eight ethanolwashed microscope slides. Marrow clots rapidly 2. Using spreader slides, the scientist spreads the drop into a wedge-shaped smear 1⁄2 to 3⁄4 the length of the slide, similar to a peripheral blood film 3.Bony spicules 0.5 to 1.0 mm in diameter and larger fat globules follow behind the spreader and become deposited on the slide 4. In the direct smear preparation the scientist avoids crushing the spicules. The scientist may lightly fan the smears to promote rapid drying in an effort to preserve cell morphology 5. In the syringe, the specimen consists of peripheral blood with suspended light-colored bony spicules and fat globules 6. The scientist evaluates the syringe blood for spicules: more spicules mean a specimen with more cells to identify and categorize. If the specimen has few fat globules or spicules, the scientist may alert the physician to collect an additional specimen

BM specimen collection sites

1. Posterior superior iliac crest (spine) of the pelvis: has the same advantages as the posterior superior iliac crest, but the cortical bone is thicker. This site may be preferred for a patient who can only lie supine 2. Sternum, below the angle of Lewis at the second intercostal space: adults, the sternum provides ample material for aspiration but is only 1 cm thick and cannot be used for core biopsy 3. Anterior medial surface of the tibia in children younger than age 2. This site may be used only for aspiration 4. Spinous process of the vertebrae, ribs, or other red marrow- containing bones. These locations are available but are rarely used unless one is the site of a suspicious lesion discovered on a radiograph

anticoagulated aspirate smears

1. The scientist expresses the aspirate from the syringe into a vial containing K3EDTA and subsequently pipettes the anticoagulated aspirate to clean glass slides, spreading the aspirate using the same approach as in direct smear preparation 2. All anticoagulants distort cell morphology, but K3EDTA generates the least distortion

Low-Power (100x) Examination

1. bone marrow aspirate direct smear or imprint is prepared and stained 2. scientist or pathologist begins the microscopic examination using the low-power (103) dry lens, which, when linked with 103 oculars, provides a total 1003 magnification 3. most BM examinations: performed using a teaching report format that employs projection or multiheaded microscopes to allow observation 4. microscopist locates the bony spicules, aggregations of bone and hematopoietic cells, which stain dark blue 5. imprints, spicules are sparse or absent, and the search is for hematopoietic cells in the absence of spicules. Within these areas the microscopist selects intact and nearly contiguous nucleated cells for examination, avoiding areas of distorted morphology or areas diluted with sinusoidal blood 6. Near the spicules, cellularity is estimated by observing the proportion of hematopoietic cells to adipocytes (clear fat areas).

High-Power (500X) Examination

1. microscopist places a drop of immersion oil on the specimen and switches to the 503 objective, providing 5003 total magnification 2.All of the nucleated cells are reviewed for morphology and normal maturation. megakaryocytes, cells of the myelocytic and erythrocytic (rubricytic, normoblastic) series should be present, along with eosinophils, basophils, lymphocytes, plasma cells, monocytes, and histiocytes 3.The microscopist searches for maturation gaps, misdistribution of maturation stages, and abnormal morphology

how the examine BM core biopsy specimen?

1. microscopist first examines the core biopsy specimen preparation using the 103 objective (1003 total magnification) to assess cellularity. the sample is larger, the core biopsy specimen provides a more accurate estimate of cellularity than the aspirate 2.microscopist compares cellular areas with the clear-appearing adipocytes, using a method identical to that employed in examination of aspirate smears to assess cellularity 3.All fields are examined because cells distribute unevenly

Concentrate (Buffy Coat) Smears steps

1. scientist transfers approximately 1.5 mL of K3EDTA-anticoagulated marrow specimen to a narrow-bore glass or plastic tube such as a Wintrobe hematocrit tube 2. The tube is centrifuged at 2500 g for 10 minutes and examined for four layers 3. The top layer is yellowish fat and normally occupies 1% to 3% of the column 4. The second layer, plasma, varies in volume depending on the amount of peripheral blood in the specimen 5. The third layer consists of nucleated cells and is called the myeloid-erythroid (ME) layer. The ME layer is normally 5% to 8% of the total column 6. The bottom layer is RBCs, and its volume, like that of the plasma layer, depends on the amount of peripheral blood present 7. The scientist records the ratio of the fat and ME layers using millimeter gradations on the tube 8. Once the column is examined, the scientist aspirates a portion of the ME layer with a portion of plasma and transfers the suspension to a Petri dish or watch glass 9. Marrow smears are subsequently prepared using the crush smear technique 10. The concentrated buffy coat smear compensates for hypocellular marrow and allows for examination of large numbers of nucleated cells without interference from fat or RBCs 11. cell distribution is distorted by the procedure. Therefore, the scientist does not estimate numbers of different cell types or maturation stages on a buffy coat smear

BM specimen

1.Red marrow is gelatinous and amenable to sampling. 2.Most bone marrow specimens consist of an aspirate (obtained by bone marrow aspiration) and a core biopsy specimen (obtained by trephine biopsy), both examined with light microscopy using 1003 and 5003 magnification

BM differential count

-300 to 1000 nucleated cells -reached in a well-prepared bone marrow smear at 5003 magnification and compensate statistically for the anticipated uneven distribution of spicules and hematopoietic cells - many microscopists choose not to differentiate the four nucleated erythrocytic maturation stages and others may combine three of the four: basophilic, polychromatophilic, and orthochromic normoblasts—in a single total, counting only pronormoblasts separately -In normal marrow, most erythrocytic precursors are either polychromatophilic or orthochromic normoblasts, and differentiation yields little additional information -differentiation may be helpful in megaloblastic, iron deficiency, or refractory anemias

stromal cells

-Adipocytes, endothelial cells that line blood vessels, and fibroblast- like reticular cells complete the bone marrow stroma

BM in adults

-accounts for 3.4% to 5.9% of body weight, contributes 1600 to 3700 g or a volume of 30 to 50 mL/kg, and produces roughly 6 billion blood cells per kilogram per day in a process called hematopoiesis -

Megakaryocytes in BM core biopsy

-are easily recognized by their outsized diameter and even distribution throughout the biopsy - They exhibit the characteristic lobulated nucleus, although nuclei of the more mature megakaryocytes are smaller and more darkly stained in H&E preparations than on a Wright-stained aspirate -Their cytoplasm varies from light pink in younger cells to dark pink in older cells -Normally there are 2 to 10 megakaryocytes per 103 field, the same as in an aspirate smear or imprint

Marrow smear dyes

-are stained with Wright or Wright- Giemsa dyes using the same protocols as for peripheral blood film staining -some MLS managers increase staining time to compensate for the relative thickness of marrow smears compared with peripheral blood films

indication for BM examination

-decision to collect and examine a bone marrow specimen requires clinical judgment and the application of inclusion criteria -cytogenetic chromosome studies, flow cytometry, immunohistochemistry, and molecular diagnostics, peripheral blood may often provide information historically available only from bone marrow, reducing the demand for marrow specimens. -examinations may be used to diagnose and stage hematologic and nonhematologic neoplasia, to determine the cause of cytopenias, and to confirm or exclude metabolic or infectious conditions suspected on the basis of clinical symptoms and peripheral blood findings -bone marrow examination is most likely unnecessary in anemia when the cause is apparent from red blood cell (RBC) indices, serum iron and ferritin levels, or vitamin B12 and folate levels -Multilineage abnormalities, circulating blasts in adults, and unexpected pancytopenia usually prompt marrow examination -Bone marrow puncture is prohibited in patients with coagulopathies such as hemophilia or vitamin K deficiency, although thrombocytopenia (low platelet count) is not an absolute contraindication

hematopoietic tissue

-enmeshed in spongy trabeculae (bony tissue) surrounding a network of sinuses that originate at the endosteum (vascular layer just within the bone) and terminate in collecting venules -

Megakaryocytes

-evaluated in lower power -are the largest cells in the bone marrow, 30 to 50 mm in diameter, with multilobed nuclei -three maturation stages: megakaryoblast, promegakaryocyte, and megakaryocyte (MK-I to MK-III) -In a well-prepared aspirate or biopsy specimen, the microscopist observes 2 to 10 megakaryocytes per low-power field: reported as decreased or increased megakaryocytes

marrow aspirate smears and core biopsy specimens stained

-ferric ferricyanide (Prussian blue) solution to detect and estimate marrow storage iron or iron metabolism abnormalities -a number of cytochemical dyes may be used for cell identification or differentiation

prussian blue iron stain examination

-iron stain is commonly used on the aspirate smear -shows normal iron, absence of iron, and increased iron stores in aspirate smears -iron stain may be used for core biopsy specimens, but decalcifying agents used to soften the biopsy specimen during processing may leach iron, which gives a false impression of decreased or absent iron stores

the most difficult cells to recognize in the core biopsy specimen are?

-lymphocytes unless they occur in clusters -Mature lymphocytes exhibit speckled nuclear chromatin in a small, round nucleus, along with a scant amount of blue cytoplasm -Immature lymphocytes (prolymphocytes) have larger round or lobulated nuclei but still only a small rim of blue cytoplasm

50x oil immersion objective in BM biopsy core

-microscopist next observes cell distribution relative to bone marrow stroma -in people older than age 70, normal lymphocytes may form small aggregates in nonparatrabecular regions, whereas malignant lymphoma cell clusters are often paratrabecular. In addition, normal lymphocytes remain as discrete cells, whereas lymphoma cells are pleomorphic and syncytial

wright giemsa-stained biopsy

-myeloblasts and promyelocytes have oval or round nuclei with cytoplasm that stains blue -Neutrophilic myelocytes and metamyelocytes have light pink cytoplasm -Mature segmented neutrophils (SEGs) and neutrophilic bands (BANDs) are recognized by their smaller diameter and darkly stained C-shaped nuclei (BANDs) or nuclear segments (SEGs). The cytoplasm of BANDs and SEGs may be light pink or may seem unstained

osteoclasts

-nearly the diameter of megakaryocytes, but their multiple, evenly spaced nuclei distinguish them from multilobed megakaryocyte nuclei -appear to derive from myeloid progenitor cells and are responsible for bone resorption, acting in concert with osteoblasts -ecognized more often in core biopsy specimens than in aspirates

are difficult to distinguist from myelocytes in H&E-stained sections but recognized in Wright-Giemsa dye in BM core biopsy?

-plasma cells -recognized as: as cells with eccentric dark nuclei and blue cytoplasm and a prominent pale central Golgi apparatus -are located adjacent to blood vessels.

osteoblasts

-responsible for bone formation and remodeling, and they derive from endosteal (inner lining) cells -resemble plasma cells with eccentric round to oval nuclei and abundant blue, mottled cytoplasm, but they lack the prominent Golgi apparatus characteristic of plasma cells -usually found in clusters resembling myeloma cell clusters. Their presence in marrow aspirates and core biopsy specimens is incidental; they do not signal disease, but they may create confusion

histologic sections (cell block) specimen

-specimen is suspended in 10% formalin, Zenker glacial acetic acid, or B5 fixative for approximately 2 hours -fixed specimen is subsequently centrifuged, and the pellet is decalcified and wrapped in an embedding bag or lens paper and placed in a paraffin-embedding cassette -histotechnologist sections the embedded specimen, applies hematoxylin and eosin (H&E) dye, and examines the section

bone marrow core biopsy specimen

-standard dye: H&E -advantage: preservation of bone marrow architecture so that cells, tumor clusters and maturation stages may be examined relative to stromal elements -disadvantage: individual hematopoietic cell morphology is obscured

10x objective is used best when?

-the microscopist searches for abnormal, often molded, cell clusters (syncytia) of metastatic tumor cells or lymphoblasts -Tumor cell nuclei often stain darkly (hyperchromatic), and vacuoles are seen in the cytoplasm. Tumor cell clusters are often found near the edges of the smear -myelocytic cells and erythrocytic cells are best examined using 5003 magnification, they may be more easily distinguished from each other using this objective -The erythrocytic maturation stages stain more intensely, and their margins are more sharply defined, features more easily distinguished at lower magnification

Polychromatophilic and orthochromic normoblasts,

-two most common erythrocytic maturation stages, have centrally placed, round nuclei that stain intensely -Their cytoplasm is not appreciably stained, but the plasma membrane margin is clearly discerned, which gives the cells of these stages a "fried egg" appearance -erythrocytic cells have a tendency to cluster in small groups, they are more easily recognized using the 103 objective, although their individual morphology cannot be seen

when is BM core biopsy specimen and imprint examinations essential?

-when the aspiration procedure yields a dry tap, which may be the result of hypoplastic or aplastic anemia, fibrosis, or tight packing of the marrow cavity with leukemic cells

imprints (touch preparations)

1. Core biopsy specimens and clotted marrow may be held in forceps and repeatedly touched to a washed glass slide or coverslip so that cells attach and rapidly dry 2. The scientist lifts directly upward to prevent cell distortion 3. Imprints are valuable when the specimen has clotted or there is a dry tap: the cell morphology may closely replicate aspirate morphology, although few spicules are transferred

cellularity in patients

1. For anterior or posterior iliac crest marrow, 50% cellularity is normal for patients aged 30 to 70 years 2. childhood, cellularity is 80%, and after age 70, cellularity becomes reduced 3.older than age 70, a rule of thumb is to subtract patient age from 100% and add 610%. for a 75-year-old, the anticipated cellularity is 15% to 35% -by comparing with the age-related normal cellularity values, the microscopist classifies the observed area as hypocellular, normocellular, or hypercellular

core biopsy collection

1.collected first: because aspiration may destroy marrow architecture 2. After the incision is made, the physician inserts a Jamshidi outer cannula with the obturator in place through the skin and cortex of the bone. The obturator prevents coring of skin or bone 3. Reciprocating rotation promotes the forward advancement of the cannula until the resistance weakens, which indicates penetration through the cortex to the medullary cavity of the bone 4. physician removes the obturator, inserts the biopsy needle through the cannula and slowly advances the needle 2 to 3 cm with continuous reciprocating rotation along the long axis 5. physician changes the needle angle slightly to separate the core cylinder specimen from its marrow cavity attachments, and withdraws the biopsy needle and cannula from the bone, taking the core cylinder with them. core cylinder is 1 to 1.5 cm long and 1 to 2 mm in diameter and weighs about 150 mg 6. biopsy needle is placed over an ethanol-cleaned slide and the stylus is pushed through to dislodge the core cylinder onto the slide 7. Using sterile forceps, the laboratory scientist prepares imprints (touch preparations) and transfers the core cylinder to the chosen fixative, Zenker, B5, or formalin 8. Westerman-Jensen needle is used, the physician removes the obturator, inserts the cutting blades through the cannula, and advances the blades into the medullary cavity 9. cutting blades are pressed into the medullary bone, with the outer cannula held firmly in a stationary position 10. physician slowly withdraws the blades so that the cannula entraps the tissue, then withdraws the entire unit. The core cylinder is removed by inserting the probe through the cutting tip and extruding the specimen through the hub of the needle to the selected slide and fixative-containing receptacles

BM preparation

1.less than 24 hr prior BM collection 2. Peripheral blood collection is often accomplished immediately before bone marrow specimen collection 3. peripheral blood specimen is seldom collected after bone marrow collection to avoid stress-related white blood cell (WBC) count elevation 4.patient is asked to lie supine, prone, or in the right or left lateral decubitus position (lying on the right or left side). 5. the skin is shaved if necessary, disinfected, and draped. 6.physician infiltrates the skin, dermis, and subcutaneous tissue with a local anesthetic solution, such as 1% or 2% lidocaine or procaine, through a 25-gauge needle, producing a 0.5- to 1.0-cm papule (bubble). 7. 25-gauge needle is replaced with a 21-gauge needle, which is inserted through the papule to the periosteum (bone surface). 8. With the point of the needle on the periosteum, the physician injects approximately 2 mL of anesthetic over a dime-sized area while rotating the needle, then withdraws the anesthesia needle 9. physician makes a 3-mm skin incision over the puncture site with a No. 11 scalpel blade to prevent skin coring during insertion of the needle

histologic sections (cell block)

After the scientist has prepared aspirate smears and has distributed aliquots of marrow for cytogenetic, molecular, and immunophenotypic studies, the remaining core biopsy specimen, spicules, or clotted specimen is submitted for histologic examination

patient care

Subsequent to bone marrow biopsy or aspiration, the physician applies a pressure dressing and advises the patient to remain in the same position for 60 minutes to prevent bleeding

chronic blood loss or hemolytic anemia

Yellow marrow reverts to hematopoiesis, increasing red marrow volume

fifth to seventh year

adipocytes (fat cells) begin to replace red marrow in the long bones of the hands, feet, legs, and arms, producing yellow marrow, and by late adolescence hematopoietic marrow is limited to the lower skull, vertebrae, shoulder, pelvic girdle, ribs, and sternum

birth

all the bones contain red hematopoietic marrow

basophils in marrow biopsy

cannot be recognized on marrow biopsy specimens fixed with Zenker glacial acetic acid solution

Which specimen provides more accurate estimate of cellularity a core biopsy or a aspirate smear BM ?

core biopsy provides more accurate estimate of cellularity. because in aspirates there is always some dilution of hematopoietic tissue with peripheral blood. In the absence of leukemia, lymphocytes should total fewer than 30% of nucleated cells; if more are present, the marrow specimen has been substantially diluted and should not be used to estimate cellularity

BM megakaryocyte estimates are important because?

estimates are essential to the evaluation of peripheral blood thrombocytopenia and thrombocytosis; for instance, in immune thrombocytopenia, marrow megakaryocytes proliferate markedly

langerhans cells

giant cells with "palisade" nuclei found in granulomas, signal chronic inflammation

BM aspirate

is examined to identify the types and proportions of hematologic cells and to look for morphologic variance

Abnormal Megakaryocytes

may be small, lack granularity, or have poorly lobulated or hyperlobulated nuclei. Indications of abnormality may be visible using low power; however, conclusive descriptions require 5003 or even 10003 total magnification

adipocytes

occupy approximately 50% of red hematopoietic marrow space in a 30- to 70-year-old adult, and fatty metamorphosis increases approximately 10% per decade after age 70

myeloid-to-erythroid (M:E)

once differential completed -ratio is computed from the total of myeloid to the total of nucleated erythroid cell stages -exluded form these ration: lymphocytes, plasma cells, monocytes, histiocytes, nonnucleated erythrocytes, and nonhematopoietic stromal cells

stromal cells and their extracellular matrix

provide the suitable microenvironment for the maturation and proliferation of hematopoietic cells but are seldom examined for diagnosis of hematologic or systemic disease

core biopsy

specimen demonstrates bone marrow architecture: the spatial relationship of hematologic cells to fat, connective tissue, and bony stroma -important for evaluating diseases that characteristically produce focal lesions, rather than diffuse involvement of the marrow. Hodgkin lymphoma, non- Hodgkin lymphoma, multiple myeloma, metastatic tumors, amyloid, and granulomas produce predominantly focal lesions -Granulomas, or granulomatous lesions, are cell accumulations that contain Langerhans cells—large, activated granular macrophages that look like epithelial cells. Granulomas signal chronic infection -allows for morphologic evaluation of bony spicules, which may reveal changes associated with hyperparathyroidism or Paget disease

If no marrow is obtained

the physician returns the obturator to the needle, advances the needle, attaches a fresh syringe, and tries again. The syringe and needle are retracted slightly and the process is repeated. If this attempt is unsuccessful, the physician removes the needle and syringe, applies pressure, and begins the procedure at a new site. If the marrow is fibrotic, acellular, or packed with leukemic cells, the first and second aspiration may be unsuccessful, known as a dry tap. In this case, a biopsy is necessary, and the laboratory scientist may observe cell morphology using a slide imprint, or touch preparation.

Concentrate (Buffy Coat) Smears

useful when there are sparse nucleated cells in the direct marrow smear or when the number of nucleated cells is anticipated to be small, as in aplastic anemia

BM tray

• Surgical gloves. • Shaving equipment. • Antiseptic solution and alcohol pads. • Drape material. • Local anesthetic injection, usually 1% lidocaine, not to exceed 20 mL per patient. • No. 11 scalpel blade for skin incision. -Disposable Jamshidi biopsy needle: provide an obturator, core biopsy tool, and stylet. A Snarecoil biopsy needle also is available (Kendall Company, Mansfield, MA). The Snarecoil has a coil mechanism at the needle tip that allows for capture of the bone marrow specimen without needle redirection -Disposable 14- to 18-gauge aspiration needle with obturator • Microscope slides or coverslips washed in 70% ethanol. • Petri dishes or shallow circular watch glasses. • Vials or test tubes with closures. • Wintrobe hematocrit tubes. • Anticoagulant liquid tripotassium ethylenediaminetetraacetic acid (K3EDTA). • Zenker fixative • Gauze dressings