PROFICIENCY TESTING SERVICE AMERICAN ASSOCIATION OF BIOANALYSTS 205 West Levee St. Brownsville, TX 78520-5596 800-234-5315 281-436-5357 Fax 713-781-5008 PARTICIPANT STATISTICS CELL IDENTIFICATION THIRD QUADRIMESTER 2014 Q3‐2014 Multiple Myeloma Multiple Myeloma, a cancer of plasma cells, is the second most frequently diagnosed hematologic malignancy, representing 1% of all cancers diagnosed in the United States. It is diagnosed at a rate of 6 per 100,000 of the population and is diagnosed slightly more often in men than women. Currently, median age at diagnosis is 67, but more recent data suggests that the median age at diagnosis may be trending lower than 67 and that the incidence in the population is trending higher than 6 per 100,000. It has been observed that the frequency of occurrence of multiple myeloma in the African American population is twice the rate seen in the European‐Americans. Although there is no specifically identified cause(s) of multiple myeloma, there are occupational, environmental and genetic factors associated with increased risk of developing multiple myeloma. Plasma cells are the final developmental step in a process that begins in the bone marrow, when a portion of stem cells begin to differentiate into pre B cell lymphocytes. B cell lymphocytes are the cells that produce the immunoglobulins we identify as antibodies. These cells undergo a complicated, multi‐step maturation process dependent on transcription factors and rearrangements, of both the heavy chain and light chain genes, that allow the cell to express surface immunoglobulins. Once the B cells have matured to this point they move from the bone marrow to the peripheral blood where they remain in a resting phase until they are exposed to foreign antigens. Once antigen exposure occurs, the cells change again and some evolve into long lived “memory cells” and take up residence in the primary follicles of the lymph nodes. If subsequently challenged by the initiating antigen, the lymph node primary follicles transform into secondary follicles with germinal centers. The B memory cells in the germinal centers undergo more complicated multistep changes and begin to generate “high affinity” specific antibodies. At this point the B cell lymphocytes have become what we identify as plasma cells. From the germinal centers these plasma cells return to the bone marrow and begin expressing serum immunoglobulin. Immunoglobulins are composed of two identical heavy chains and two identical light chains. These chains are held together by disulfide bonds. The immunoglobulin (antibody) manufactured by each plasma cell has one light chain, either kappa or lambda, and one heavy chain. The five types of heavy chains are designated as IgG, IgA, IgM, IgD and IgE. Each of these five types of immunoglobulin performs a specific, function in the immune system. IgM is the initial antibody produced in response to infection, followed by the production of IgG. Rapid escalation of IgG production from memory cells takes place when re‐ exposure to the initial antigen re occurs. IgA is associated with protecting the cell surfaces that line both the GI and respiratory tracts. Allergic reactions trigger IgE production. IgD immunoglobulin performs some as yet unidentified function. Normally, the immune system keeps the proliferation of B cells and the secretion of antibodies under tight control. When chromosomes and genes are damaged, often through rearrangement, the control is lost. Multiple myeloma develops in B lymphocytes after they have left the part of the lymph node called the germinal center. It is presumed that this disease we call multiple myeloma is the results of a single B cell precursor being somehow damaged in such a way as to no longer be susceptible to control by normal immune system mechanisms. The single cell then multiplies without constraints, resulting in the formation of a malignant, immunoglobulin‐producing clone of plasma cells. At present, the reason(s) B cells undergo this malignant transformation is not understood. Once regulatory control is lost, neoplastic plasma cells proliferate in nodules or diffusely throughout the bone marrow. They also invade liver, spleen and lymph nodes. As the malignant plasma cell clone expands, it replaces normal bone marrow. The bone marrow space cannot accommodate both the normal bone marrow tissue as well as the expanding clone. Therefore, the normal tissue is gradually replaced. Also the expanding clone causes destruction of the rigid bone surface cortex. With the rigid cortex damaged, the periosteum, nerve rich tissue covering the bone surface, stretches as the clone volume expands. The stretching is what causes the initial bone pain experienced by most multiple myeloma patients. Often the plasma cell clone will expand outside of the bone space and compress nearby structures such as the vertebrae, both in the space where the nerve roots exit the spine and in the spine itself, resulting in pain and possible paralysis. The replacement of normal bone marrow with plasma cells causes a reduction in normal production of first RBCs, then platelets and finally, neutrophils. Thus, these patients develop a progressive pancytopenia, experiencing the symptoms associated with anemia, thrombocytopenia and neutropenia. On examination of the blood smear, the most characteristic finding is rouleaux. Rouleaux is caused by increased immunoglobulins which cause the red blood cells to stack together, like coins. The elevated immunoglobulins also cause an increased ESR. Occasionally circulating plasma cells are seen in the peripheral blood of multiple myeloma patients. Their presence in the peripheral blood is a poor prognostic sign. Our case study is an example of such a case. The myeloma cells in the bone marrow attach themselves to the stromal cells that provide the internal spongy matrix of the bone marrow. This adhesion induces the stromal cells to secrete proteins, termed cytokines, which have multiple effects on cell function. Cytokines have been shown to induce bone destruction, inhibit RBC production, promote tumor proliferation, induce drug resistance and contribute to additional genetic changes in plasma cells. The breakdown of bone results in elevated calcium levels. Hypercalcemia can lead to the formation of kidney stones and can damage renal cells. Additional renal damage occurs when, high levels of light chains passing thru the kidneys precipitate in renal tubules. Even though the malignant clone of plasma cells is producing high levels of immunoglobulins, they are specific to the malignant clone and as such are unlikely to be the right kind of immunoglobulin needed to fight infections. That, coupled with the neutropenia that results from bone marrow replacement by malignant plasma cells, means that multiple myeloma patients are often essentially immune deficient and very susceptible to infections. Total serum protein is primarily a combination of albumin and immunoglobulin. Small amounts of other proteins, alpha 1‐antitrypsin, alpha 2‐macroglobulin, transferrin and beta‐lipoprotein are also included in the total serum protein. By subtracting the albumin fraction from the total serum protein one can approximate the amount of immunoglobulin present. Immunoglobulin overproduction is the signature laboratory finding in multiple myeloma. Since the immunoglobulin is manufactured by a clone of plasma cells that originated from a single malignant plasma cell, the immunoglobulins are monoclonal. In contrast, a normal response to infection will result in production of polyclonal immunoglobulins. In order to differentiate between polyclonal and monoclonal immunoglobulin, serum protein electrophoresis (SPEP) must be done. Although multiple myeloma malignant clones can produce all 5 classes of immunoglobulins, IgG is the most commonly seen, produced in more than 50% of cases. IgA is produced in about 20% of cases and another approximately 20% of cases produce light chains only. If SPEP demonstrates the presence of a monoclonal immunoglobulin, then additional testing, in the form of immunofixation, is performed to further characterize the immunoglobulin . In addition to multiple myeloma, there are other plasma cell disorders potentially responsible for the presence of elevated monoclonal immunoglobulin among which are,monoclonal gammopathy of undetermined significance (MGUS), Waldenstrom’s macroglobulinemia, heavy chain disease, non‐secretory myeloma and amyloidsis. In addition to SPEP, urine protein electrophoresis is typically also part of the normal work‐up in a suspected case of multiple myeloma. Normally light chains and heavy chains are produced in about equal amounts. In this disease, this is no longer the case, and there is usually an overproduction of free light chains that show up in the urine as Bence‐Jones protein. Bone marrow biopsy examination is performed primarily to enable estimation of the percentage of the bone marrow that has been replaced by plasma cells. This percentage is used to differentiate between myeloma (clonal plasma cells >10%) and monoclonal gammopathy of undetermined significance ( clonal plasma cells <10%). Special stains and cytogenetics can be performed on bone marrow and are used to characterize the malignant plasma cell clone. In the future, treatment protocols may be customized depending on the specific characteristics of the patients particular plasma cell clone. Radiological procedures are used to make the diagnosis and establish the degree and location of lytic bone disease and soft tissue