Bone marrow Information and Courses from MediaLab, Inc.

The presence of blast cells or immature stages of hematopoietic cell lines are significant abnormal findings in any spinal fluid sample. However, the presence of nucleated red blood cells is the result of bone marrow contamination during the spinal tap.

A sideroblast, shown at the arrow, is a nucleated red cell containing siderotic granules. However, these granules are generally not clustered around the nucleus, but are found in the periphery of the cell. Siderocytes are present normal bone marrow.

Although the nucleus has been extruded, the reticulocyte is still considered immature because it retains numerous organelles needed for hemoglobin production, such as ribosomes, mitochondria, and fragments of the Golgi apparatus. The reticulocyte is slightly larger (10 microns) than the mature erythrocyte. A reticulocyte normally remains in the bone marrow for one or two days before entering the circulation and its final 24 hours of maturation. The red cell is mature when hemoglobin production is complete and the organelles have disintegrated. Reticulocytes normally make up 0.5 - 1.5% of the peripheral blood red cells. They appear blue/gray on the Wright's stained smear. The residual RNA in the cytoplasm causes the blue/gray color. The terms, polychromasia or polychromatophilic, are used to describe these cells on a Wright's stained preparation. A supravital stain such as new methylene blue N or brilliant cresyl blue is used to stain reticulocytes for an actual count.

Erythrocyte production (including reticulocytes) is increased when the tissues are not receiving sufficient oxygen and the bone marrow is able to respond in a positive manner.Erythrocyte production (including reticulocytes) is decreased when the bone marrow is unable to respond to the signal for increasing production.

Bone marrow samples are obtained from the patient by a physician. The technologist is responsible for examining the sample macroscopically to ensure that it is adequate, making slides on the unanticoagulated sample and processing the remaining portions of the sample as required for procedures ordered.

The steps involved in collecting a bone marrow sample are:prepare the patientremove the sampleprepare the specimen for examination

Particle smears are also made from the unanticoagulated sample. The bone marrow particles are removed from the watchglass and placed on a coverslip. One of the following items: Pasteur pipet, capillary tube or broken end of a wooden applicator stick, may be used to transfer the particles. A second coverslip is placed over the first and the particles are crushed between the coverslips as they are pulled apart. Some practice is needed to perfect this technique. As mentioned previously, this type of preparation provides a more accurate assessment of marrow architecture and cellularity than the direct smear. Morphological detail is preserved on well made slides. The remaining sample may be added to a tube containing EDTA anticoagulant and additional smears may be made if needed.

The physician ordering the bone marrow is responsible for providing information about the procedure to the patient or parent or guardian, if the patient is a child. In order to reduce the patient's anxiety about the procedure, the physician may prescribe a mild sedative to be administered about an hour before the bone marrow is scheduled. The site is aseptically prepared by shaving, if necessary, washing with soap and water, applying antiseptic and draping the area with sterile towels. A local anesthetic, such as 2% xylocaine, is injected into the bone, penetrating the covering of the bone called the periosteum. Since a number of nerve endings are located near the surface of the bone, it is important to be sure that this area is anesthetized.

Before learning to examine bone marrow microscopically, it is important to understand the basic structure and function of the bone marrow. The bone marrow is one of the largest organs in the body. The normal adult marrow on a daily basis produces approximately 2.5 billion red cells, 2.5 billion platelets and 1.5 billion granulocytes per kilogram of body weight. The main function of this organ is the formation and development of blood cells. Hematopoiesis begins in the yolk sac in the first weeks of embryonic life; stem cells from the yolk sac travel first to the liver and then to the spleen. These organs are the only blood forming sites during the first three months of fetal life. At the beginning of the fourth month the bone marrow begins its life-long function of cell production.

Lymphocytes are often located in nodules and these nodules are unevenly distributed throughout the marrow so the lymphocyte count may vary in bone marrow samples from different sites. Plasma cells are often found clustered around blood vessels. Monocytes seem to congregate about arterioles in the center of the cord.

The site of iron storage in the bone marrow is the macrophage. This is a bone marrow smear showing a macrophage containing near the top of the smear showing clumps of blue-staining material, which is iron. Notice the number of young red cells (erythroid precursors) clustered around the iron in the lower portion of the slide.

"Romanowsky stain" is a general term which can include several specific stains or stain combinations. Wright-Giemsa is a Romanowsky type stain combination frequently used to stain bone marrow smears.

This field under high magnification shows an increased number of megakaryocytes (megakaryocytic hyperplasia). This patient had thrombotic thrombocytopenic purpura. He was therefore consuming increased numbers of platelets, and his bone marrow was responding by increasing the number of megakaryocytes, which of course break up into platelets.

Another example of a cell rarely seen in the bone marrow is an osteoblast. Osteoblasts are cells which are similar in appearance but somewhat larger than plasma cells or tumor cells. The nucleus is eccentric and the "hoff" area is sometimes located away from the nucleus. The cytoplasm appears rather foamy when compared to a plasma cell. The size of an osteoblast is 20-25 microns. Osteoblast produce bone.

A thin area of a slide taken from a patient who has chronic lymphocytic leukemia, which is characterized by an increased number of small lymphocytes in the bone marrow. At this power, numerous small dark cells similar in appearance to immature red cells are seen, but can be quickly confirmed as lymphocytes when viewed under oil. The actual M:E ratio is normal, since lymphocytes are not included in the final ratio. The arrows show several cell most likely representing small lymphocytes. Some small lymphocytes are normal in the bone marrow.

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.

Examples of conditions in which examination of the bone marrow may provide diagnostic information include:Erythrocyte Disordersanemiamegaloblasticsideroblasticiron deficiencyerythrocytosispolycythemia veraLeukocyte DisordersneutropenialeukemialymphomaPlatelet DisordersthrombocytopeniathrombocytosisMiscellaneous Disordersprotein abnormalitiesmultiple myelomaWaldenstrom's Macroglobulinemiadiseases of the RE systemhypersplenismmetastatic carcinomagranulomatous infectionsstorage diseasesGaucher's diseaseNiemann-Pick disease

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 is another view of the same slide showing increased cellularity and decreased fat.

It is also important to note that in addition to red cells, ABO antigenic determinants (epitopes) are found in many tissues, body fluids, and other cells including endothelial cells and platelets. Because ABO antigens are so widely expressed, ABO antigens are also a major consideration in solid organ and bone marrow transplants.

Specialists in microbiology perform testing to diagnose and stop the spread of infectious organisms, including bacteria, viruses, and parasites. Specialists should be able to isolate and identify a wide variety of these organisms. Testing procedures include direction examination and antigen detection methods. Specialists in serology and immunology measure antibodies to infectious organisms. Specialists should be familiar with all serology techniques (except those specific to immunohematology). This specialty includes all lab procedures performed in the specialty of histocompatibility. Specialists in hematology must be able to identify and evaluate cells in blood and bone marrow and identify disorders of these cell. Specialists should be familiar with routine and special tests to determine the number, morphology, and function of cells in body fluid.

Platelets are derived from the cytoplasm of megakaryocytes, giant cells in the bone marrow. At any given time, two thirds of the total platelets are in the circulation and one third are present in the spleen. In persons with enlarged spleens 80-90% of the platelets are in the spleen resulting in a decreased concentration of circulating platelets. In individuals who have had a splenectomy all of the platelets will be in the circulating blood. The life span of the platelet is 8-10 days.

Antibody - A modified type of serum globulin synthesized by lymphoid tissue in response to antigenic stimulus. By virtue of specific combining sites each antibody reacts with only one antigen. Anucleate - Having no nucleus. Azurophilic granules - The well-defined large reddish granules (lysosomes) which may be present in large lymphocytes. They are called "azurophilic granules" because they stain blue with the azure stains which were originally used. Basophilic granules - Specific granules present in the cytoplasm of basophils. These granules are large and stain purple-black due to their strong affinity for basic stain. B-cell - Bone marrow derived lymphocytes which produce humoral antibodies. Biconcave - Having two concave surfaces. Cellular Immunity - The capacity of a small proportion of lymphoid population to exhibit response to a specific antigen. Chromomere - The centrally located granular portion of the platelet. Clone - A population of cells descended from a single cell. Delayed Hypersensitivity - (part of cellular immunity) that develops slowly over a period of 24-72 hours after an antigenic stimulus. It consists of an accumulation of cells around small vessels and/or nerves. Example: Tuberculin skin test reaction. Digestive Enzyme - A substance that catalyzes or accelerates the process of digestion. Eosinophilic Granules - Specific granules present in the cytoplasm of eosinophils. These granules are large, refractile spheres which stain reddish-orange due to their strong affinity for acid stain. Erythrocyte (red blood cell, RBC) - One of the elements found in peripheral blood. Normally the mature form is a non-nucleated, circular, biconcave disk adapted to transport respiratory gases. Fixed Macrophage - A phagocyte that is non-motile. Free Macrophage - An ameboid phagocyte present at the site of inflammation. Graft Rejection - A transplanted tissue that is rejected by the body's antibodies. Graft vs. Host Reaction - A complication that occurs when an implanted piece of tissue, which contains antibodies, rejects the host's tissue. Granulocyte - A leukocyte which contains granules in its cytoplasm, i.e., neutrophilic, eosinophilic, or basophilic granules. Half-life - is the length of time it takes for half of the cells circulating at a given time to leave the blood for the tissues. Hemocyte - Any blood cell or formed element of the blood. Hemostasis - A mechanism of the vascular system to arrest an escape of blood. It involves an interaction between blood vessels, platelets, and coagulation. Heparin - A mucopolysaccharide acid which, when present in sufficient amounts, functions as an anticoagulant by inhibiting thrombin. Histamine - A powerful dilator of capillaries and a stimulator of gastric secretions. Humoral Immunity - Acquired immunity produced after response to an antigenic stimulus in which B cells produce circulating antibodies. Hyalomere - the clear, blue non-granular zone surrounding the chromomere of a platelet. Immune Response - The interaction of a cell and an antigen that results in a proliferation of the cell and a capacity to produce antibodies. Isotonic Fluid - A fluid whose elements have an equal osmotic pressure. Leukocyte (white blood cell, WBC) - One of the formed elements of the blood; involved primarily with the body's defense. Lysosome - A microscopic body within cell cytoplasm; contains various enzymes, mainly hydrolytic, which are released upon injury to the cell. Megakaryocyte - A giant cell of the bone marrow from which platelets are derived. Mononuclear - A cell having a single nucleus.

Eosinophils have a circulating half-life of approximately 18 hours and a tissue life span of at least 6 days.They are capable of locomotion and phagocytosis and can enter inflammatory sites, but do so less readily than neutrophils.In tissues the primary location for eosinophils is in the epithelial barriers to the outside world such as, lungs, skin and GI tract.They are capable of returning to the circulating blood and bone marrow after they enter the tissues.

Reticulocytes are red blood cells prematurely released from the bone marrow. On a Wright-Giemsa stained blood smear, they appear as polychromatic macrocytes. Their presence in the peripheral blood may suggest hemolysis or bleeding. Their presence is expressed as a percentage of the red cell count: newly born= 3-7%; up to one week of age=1-3%; >one week =0.3-1.8%. Automated or manual methods may be used to enumerate reticulocytes. In clinical context, retics must be separated from debris, precipated stain, Pappenheimer bodies, Howell-Jolly bodies, and Heinz bodies.

As previously mentioned, tear drop cells are present in disorders with altered splenic or bone marrow structure. Disrupted splenic cords and myelofibrosis with myeloid metaplasia are examples. Tear drop cells appear in the peripheral blood as a response to red cell alterations by thalassemia when red cell inclusions are expelled by a stripping process through splenic cords. A marrow disrupted by malignant cells may also set the stage for release of teardrop cells into the peripheral blood. Importantly, teardrop cells may arise as an artifact of improper smear preparation, identified by their uniformity in pointing in the same direction. In contrast, teardrops noted in the photograph are irregularly arranged and oriented in various directions. Teardrops always have pointed ends and disappear after splenectomy.

The photograph represents peripheral blood smear findings in another patient with hereditary spherocytosis. The red cells vary in size (anisocytosis)with a mixture of microcytes (red cells with central pallor) and microspherocytes (red cells with central staining). Macrocytes are conspicuous, some staining light blue. They are immature erythrocytes (reticulocytes)released from the bone marrow early. The bone marrow, geared up for rapid cell release in response to severe hemolysis, expels young red blood cells into the circulation before completing their 24 hour maturation cycle. Hemolysis, jaundice, and gall stone formation disappear following splenectomy. Gallbladder and stone removal eliminate the right upper quadrant pain. A serious consideration, especially in children with hereditary spherocytosis, is hemolytic crisis. A viral infection may allow red blood cell destruction to continue unabated. Anemia of such sudden onset and severity may become catastrophic, with death as the outcome. Splenectomy removes this possibility.

Another example of macrocytes is seen in this slide. This patient had pernicious anemia, which results from an inability to absorb the vitamin B12 needed for DNA synthesis. Since many cells are destroyed in the bone marrow, decreased numbers of red cells are present in the circulating blood causing low hemoglobin(anemia).

The large cell in the center of this field is slightly blue/gray and is an example of a polychromatophilic red cell.Increased numbers of these cells, (approximately 2 or greater per oil immersion field,) indicate increased red cell output by the bone marrow. Polychromatophilic cells are larger and younger than mature red cells, and may be larger than 9 micron in diameter. Under normal conditions, these young red cells remain in the bone marrow one or two days before release into the bloodstream. However, when the bone marrow is stressed due to blood loss or other conditions, these cells are prematurely released into the blood, resulting in a blood smear with polychromasia. These red cells are often referred to as shift cells. If stained with a supravital stain, they would be identified as reticulocytes.

Alder Reilly Anomaly is a rare autosomal recessive hereditary disorder in which the basic defect involves protein-carbohydrate complexes called mucopolysaccharides. The accumulation of partially degraded (broken down) protein-carbohydrate complexes within the lysosomes account for the larger than normal purple-staining granules seen in the granulocytes, monocytes and/or lymphocytes. The granules may occur in clusters, rather than diffusely, throughout the cytoplasm as in toxic granulation. These inclusions may be seen in the bone marrow more frequently than in peripheral blood. The physical characteristics associated with this disorder include gargoylism and dwarfism. The function of the cells involved is not affected. This morpholical change would be classified as pathological since the body is responding abnormally even though the function is not affected.

A normal bone marrow smear stained with Wright/Giemsa stain is captured in this photograph.Note the normal maturation sequence beginning with myelocytes (the two large cells in the left upper corner)through metamyelocytes, band neutrophils,and multi-lobed segmented neutrophils.The small cells with darkly staining, centrally placed nuclei are normoblasts (three are clustered in the left lower field).Absent in this field are eosinophils, basophils and megakaryocytes.A normal M:E ratio of 2.4:1 is calculated from the twelve myeloid cells and five normoblasts. Two lymphocytes are identified, one left center, the other left upper.

The large cell illustrated in this photograph of a Wright/Giemsa-stained bone marrow smear is a megakaryocyte. This megakaryocyte appears mature. The nucleus has at least 8 lobes and the nuclear chromatin is coarse and distinct. Clusters of young platelets are being released from distinct platelet territories at the periphery of the cytoplasm (blue arrows). When mature, each megakaryocyte produces approximately 4000 platelets/day. Production can expand by 8-fold during times of increased demand and under the stimulus of thrombopoietin.

Illustrated in the photograph is a phagocyte devouring several erythrocytes.This uncommon phenomenon occurs in the bone marrow and in the spleen as part of the process of erythrocyte destruction. Erythrophagocytosis is found in histological sections of the spleen in cases of hemolytic anemia.This phenomenon appears also in splenic sections in lupus erythematosis, and in rheumatoid arthritis.Our example is from a patient with a myeloproliferative disorder and is a rare example of a circulating erythrophagocytic cell in the peripheral blood.