Marrow Information and Courses from MediaLab, Inc.

The marrow aspiration is usually performed before a biopsy is done. A syringe is attached to the needle, the plunger is pulled and 1.0-1.5 ml. of marrow particles and blood from marrow sinuses is withdrawn. If additional bone marrow samples are needed, a separate syringe must be used each time. If more than 2 cc. per syringe is taken out, the blood to marrow ratio will be too high and the preparations will not accurately reflect the marrow contents. As the marrow is aspirated into the syringe the patient will feel some pain and pressure even though local anesthetic has been administered.

In some laboratories the anticoagulated sample is used to prepare concentrated smears. Placing the fluid in a Wintrobe tube and centrifuging it separates the sample into four layers:fat and perivascular cellsplasmabuffy layer - myeloid and nucleated erythroid cellserythrocytesThe volume of each layer is measured using the scale on the Wintrobe tube and then the percentage of each layer is calculated. Next the plasma is removed and a smear is made from the buffy coat and top of the red cell layer. Either the manual push method or cytospin technique may be used to make the smears. They may be stained with a variety of cytochemical stains. Concentrated smears are used to examine cell morphology and demonstrate the presence of abnormal cells when the marrow is hypocellular. The smears cannot be used for differential counts or evaluation of cellularity.

Special studies, particulary flow cytometry and cytogenetics, may be requested by the physician on bone marrow aspirates, and fresh marrow should be submitted in appropriate media for these studies if requested.

The sample in the first syringe is quickly delivered into a watchglass or onto a slide. After the technologist verifies the presence of white-gray marrow particles in the sample, push smears and/or coverslip smears from this unanticoagulated sample are made immediately. All films should be rapidly air dried. The appearance of fat as irregular holes in the films also give the assurance that marrow and not just blood has been obtained. This type of smear is referred to as a direct smear and is usually used to evaluate morphology. Although some evaluation of cellularity and M:E ratio is possible, particle smears or biopsy sections provide a more accurate representation of these factors.

A touch prep is made by holding the biopsy plug with a forceps and touching the plug to one or more clean slides in several places. Imprints of the marrow remain on the slide. The slides are quickly air dried, fixed with methanol and stained with Wright-Giemsa or other cytochemical stains. Morphologic details of the cells are preserved with this type of preparation.

Fat cells make up about 50% of the total marrow volume in adults. By the age of 65-70 the amount of fat increases to about 66%.

Changes in the distribution of cells in the marrow are most apparent in the first month of life. At birth, granulocyte cells predominate. The myeloid to erythroid (M:E) ratio is somewhat higher in newborns and during infancy than it is later on in childhood and in adults.

Markely increased stainable iron is present in this biopsy. Iron stores may be increased in sideroblastic anemia, chronic infections, hemochromatosis, hemosiderosis due to numerous blood transfusions, chronic hepatitis, cirrhosis, and uremia.

It is also important to scan the slide for the presence of other large cells which are not usually seen in normal marrow. An osteoclast is an example of this type of cell. Osteoclasts are large multiinucleated cells (up to 100 microns) which may be confused with megakaryocytes. One striking difference is that an osteoclast has multiple nuclei which are separate from each other. The multiple nuclei in the megakaryocyte are joined together. The cytoplasm, although somewhat finer in texture, could be mistaken for platelets.

A normal M:E ratio is depicted in this slide. Notice that the area shown is a portion of the slide near a particle or spicule of marrow where the cells are numerous. The morphology can still be clearly differentiated. The small dark cells scattered throughout the slide are erythroid cells, while the larger, lighter staining cells are myeloid cells. The normal M:E ratio varies from 1.2 to 5 myeloid cells for each erythroid cell.

Although an occasional plasma cell is normal in the marrow, the presence of sheets of plasma cells, as seen here, is consistent with multiple myeloma.

High power (40x objective) examination can be used to estimate the myeloid-to-erythroid ratio. The erythrocytes are nucleated, immature erythrocytes. Under high power, nucleated red cells appear to have a dark purple nucleus as opposed to the lighter staining nucleus of the myeloid or granulocyte series. Lymphocytes also have a dark staining nucleus and some may be erroneously included in the erythroid estimate. In the normal marrow these numbers are insignificant.

After the marrow is evaluated, the diagnosis is established and extent of the disease is determined. Follow up bone marrow examinations may be needed to monitor changes in the marrow following treatment or when signs and symptoms of relapse occur. To summarize, a bone marrow examination can provide valuable information to aid in the diagnosis of a variety of disorders. Due to the expense involved and the discomfort to the patient, clear indications of need should be present before this examination is undertaken.

This biopsy section was taken from a patient who has very few cellular elements in the marrow. Notice that over 90% of the marrow is composed of fat. If all of the cellular elements are decreased, the patient's condition is said to be pancytopenic or aplastic. There are numerous causes for aplasia, including drugs such as chloramphenicol, chemotherapy and inheritance (Fanconi's Anemia).

This low power view of a hematoxyln and eosin stained bone marrow biopsy shows fat cells as clear circles, and the darker intervening areas as blood cell precursors. This biopsy is about 25% cellular, or mildly hypocellular. A normal marrow in a middle aged adult is about 50% cellular.

Illustrated in this field is a normoblast and a myelocyte, representing leukoerythroblastosis, a term associated with the release of immature cells from a disrupted marrow. Metastatic disease in the bone marrow, particularly in patients with primary breast or prostate cancer, is usually the culprit. Leukoerythroblastosis in the absence of anemia or thrombocytopenia is a signal to search for cancer metastic to the marrow. Nucleated RBCs were not identified on the blood smear seen here but were detected by an automated analyzer.The mortality rate of elderly patients with increased NRBCs, especially following accidents or general surgery, is greater.

Two photographs of a peripheral blood smear are submitted for review . The smears are from a 9-month-old baby with a heart valve replacement. In the upper photograph is a nucleated RBC and platelets are decreased. Nucleated red cells and occasional giant platelets indicate an active marrow response. In the process of forcing blood cells through the heart valve, erythrocytes are damaged, schistocytes are formed, and platelets are destroyed leading to thrombocytopenia. In the lower field are schistocytes, acanthocytes, echinocytes (burr cells), spherocytes, and the absence of platelets. The presence of burr cells could represent an artifact of smear preparation, but with the history of valve replacement, the red cell changes are likely the result of red cell damage as the cells circulate through the new valve. This situation is described as Waring Blender Effect because of damage to blood cells passing through the new valve, looking as if they had suffered the onslaught of a blender. Target cells and mild hypochromia may reflect iron deficiency through the loss of iron from destruction of RBC's. Iron loss through red cell destruction may be reflected in some hypochromia.

Jaundice was recognized in a day-old infant. Notice particularly the size variation (anisocytosis) of the erythrocytes on the infant's peripheral smear. What does this observation mean? Does it provide immediate information that might serve as guidance in expediting diagnosis and treatment? Note that normal-sized red blood cells, microcytes, microspherocytes, macrocytes, and nucleated red blood cells are all present. Red cell variations are expected findings in healthy neonates, but the variations here are exaggerated. Hyposplenic functional features may appear, including acanthocytes, spherocytes, and possibly Howell-Jolly bodies, especially if hemolysis is particularly vigorous. A high (3-7%) reticulocyte count is not unusual during the first three or four days after birth, however, the marrow in this jaundiced infant is proliferating vigorously in response to hemolysis. A call for more red cells is urgent. Immature red cells (in the form of nucleated red cells) and red cells with stippling of RNA (basophilic stippling) are readily identified. Red cell maturation sequence has not been totally processed in the marrow nor is all residual red cell debris removed by the spleen. In the lower photograph are reticulocytes stained by supravital stain (new methylene blue). Basophilic stippling (specks of RNA) stains with both supravital stains and with routine Wright-Giemsa stain.

In this photograph of blood cells from yet another submitted slide, we find cells resembling lymphoblasts with increased nuclear/cytoplasmic ratios and dense, finely meshed nuclear chromatin. In addition, note the extrusion of delicate strands of cytoplasm from the outer cell membranes (blue arrow). These are cells connoting hairy cell leukemia (HCL). Under scanning electron microscopy, the cytoplasmic extensions appear to be either slender microvilli or delicate pseudopods. The most helpful confirmatory finding is the detection of acid phosphatase isoenzymne 5 in the cytoplasm of suspected hairy cells by staining. The enzyme concentrates primarily in golgi bodies and in the nuclear membrane and its staining is not inhibited by the addition of tartrate. Stated in another way, hairy cells on the peripheral smears are detected by their staining positively for tartrate-resistant acid phosphatase. Be suspicious of HCL if marrow resists aspiration-a consequence of reticulin fibrosis of the marrow in HCL.