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 involvement. .In addition, these procedures can identify situations where timely intervention can prevent fractures and cord compression that may cause paralysis.

The natural history of multiple myeloma has been treatment followed by remission followed by relapse. Specific treatment protocols for a multiple myeloma patient depend on the patient’s age and comorbidities. Most treatments are focused on reducing the plasma cell clonal load which in turn will reduce the symptoms associated with the disease. In addition, treatment with bisphosphonates is used to prevent fractures. Transfusions are administered to offset the anemia and bortezomid is a protease inhibitor used primarily for treating relapsed patients.

Historically, conventional treatment protocols for multiple myeloma had resulted in average patient survival periods of less than 3 years. More recently advanced, aggressive, treatments have been developed. The results have been that upwards of 40% of multiple myeloma patients are achieving 5‐7 years survival. These increasingly lengthy remissions are being achieved using combinations of steroids, chemotherapy, protease inhibitors, and immunomodulatory drugs such as thalidomide and lenalidomide. For .younger patients, defined as those less than 65 year old at time of diagnosis, autologous stem cell transplants have

Cell ID Results

Specimen 1 Specimen 2 Specimen 3 Specimen 4 Specimen 5 Code ‐ Result No. Flag Code ‐ Result No. Flag Code ‐ Result No. Flag Code ‐ Result No. Flag Code ‐ Result No. Flag Rouleaux 412 Plasma Cell, any stage 213 Immature Neutrophil 342 Teardrop Cell (dacrocyte) 411 Lymphocyte, normal 381 Erythrocyte, normal RBC 1 *** Abnormal, would refer 51 PMN with Toxic 26 *** Target Cell (codocyte) 3 *** Lymphocyte; atypical, Downey, 10 *** Agglutination 1 *** Myelocyte 34 *** PMN with Dohle Bodies 26 *** Poikilocytosis 1 *** Lymphocyte, reactive 8 *** Poikilocytosis 1 *** Promyelocyte 24 *** Eosinophil, any stage 12 *** Spherocyte 1 *** Hairy Cell 4 *** Plasma Cell, any stage 1 *** Lymphocyte, reactive 18 *** Segmented Neutrophil (PMN, poly) 10 Total: 416 Abnormal Lymphocyte, would refer 3 *** Total: 416 Blast, undifferentiated 14 *** Total: 416 Intended result: Teardrop Cell (dacrocyte) Abnormal, would refer 2 *** Intended result: Rouleaux Abnormal Lymphocyte, would refe14 *** Intended result: Immature Neutrophil Immature WBC, would refer 2 *** Immature WBC, would refer 11 *** Blast, undifferentiated 2 *** Lymphocyte; atypical, Downey, 11 *** Nucleated RBC, any stage 1 *** Myeloblast 8 *** Plasma Cell, any stage 1 *** Lymphocyte, normal 6 *** Platelet, normal 1 *** Monocyte, any stage 5 *** Total: 416 Metamyelocyte 2 *** Intended result: Lymphocyte, normal Abnormal Granulocyte, would refer 1 *** Total: 416 Intended result: Plasma Cell Evaluated on the basis of referees.

22 of 24 referees reporteed Plamsa Cell or Abnormal. Correct responses are defined as those reflecting agreement among 80% or more of all participants or referees. Unacceptable responses are indicated by "***" on the Flagging line of each specimen.

THIRD QUADRIMESTER 2014 Cell ID ‐ Page 2 of 4 EDUCATIONAL CHALLENGES

Specimen 1 No. Basophilic Stippling 194 Target Cell (codocyte) 18 Polychromatophil ic RBC 4 Parasite 4 Abnormal, would refer 3 Stomatocyte 2 Abnormal RBC, would refer 1 Macrocytic 1 Schistocyte (bite, blister, 1 Heinz Bodies (supravital 1 Pappenheimer Bodies 1 Total Population: 231 Intended result: Basophilic Stippling

*To see the original fullsized immages, please refer to the original CD or sign on to your data entry sheet at http://www.aab‐pts.org/

Sample 14Q3‐1: A 10‐month‐old girl is brought to the Emergency Department by her mother. Her mother states she is not feeding well and her abdomen seems “big”. On physical examination, she is small for her age, has hepatosplenomegaly and several petechiae scattered over her abdomen and back. CBC results: WBC 1,200/µL, Hgb 5.9 g/dL, Hct 16.1%, Plts 16,000/μL. Identify the indicated cells.

The automated CBC values indicate pancytopenia, with a reduction in all cell lines. Review of the peripheral blood smear. Review of the peripheral blood smear is somewhat compromised by stain deposits and scratch mark artifact (colorless streaks caused by either dirt particles on the slide when the smear was prepared or wiping off immersion oil with a rough tissue or paper after the slide was viewed). It is important to not confuse the stain deposits with platelets, or with Howell‐Jolly or Pappenhimer bodies if the deposits overlie red cells. The peripheral smear confirms the decrease in WBCs and platelets. Red cells show ansio‐ and poikilocytosis, with hypochromia and microcytosis. Target cells are abundant and are admixed with microspherocytes, red cell fragments, and a rare nucleated RBC. The cells to be identified show basophilic stippling. Basophilic stippling is the result of many small blue‐staining granules that typically fill the red cell (versus one to three blue‐to‐ azurophilic staining inclusions such as would be seen with Howell‐Holly or Pappenheimer bodies). These granules represent precipitated ribosomal RNA which forms aggregates; stippling is characterized as fine or coarse and can be present in mature as well as nucleated RBCs. Fine basophilic stippling can be seen in normal individuals and is associated with increased red cell production; therefore, it is often seen to a slight degree in association with polychromatophilia. Coarse basophilic stippling, however, often indicates impaired hemoglobin synthesis, is classically associated with lead poisoning, although it can also occur with any heavy metal poisoning, including arsenic, bismuth, zinc, silver, and mercury. More commonly, when pronounced basophilic stippling is present, one should suspect conditions such as a thalassemia, hemoglobinopathy, sideroblastic anemia, megaloblastic anemia, myelodysplastic syndrome, liver disease, or a deficiency of the red cell enzyme pyrimidine 5’‐ nucleotidase.

This infant was found to have β‐thalassemia major, an inherited disease resulting in a marked reduction to absence of synthesis of β‐globin chains. Clinically, such individuals show growth retardation and suffer severe anemia, hepatosplenomegaly, and eventual deformity of the skull and jaw. These individuals become transfusion dependent and, if not combined with chelation therapy or red cell exchange, may develop further organ damage as a result of iron overload. The anemia associated with this thalassemia is typically severe and the peripheral smear shows moderate to marked aniso‐ and poikilocytosis with numerous target cells and basophilic stippling. Nucleated RBCs are frequent. The automated CBC will show reduction of the MCV, MCH, and MCHC, with an elevation of the RDW. Although the platelet count can be normal to increased, as the splenomegaly increases, the platelet count falls.

THIRD QUADRIMESTER 2014 Cell ID ‐ Page 3 of 4 Specimen 2 No. Parasite 187 Basophilic Stippling 27 Abnormal, would refer 6 Polychromatophil ic RBC 3 Reticulocyte (supravital stain) 2 Pappenheimer Bodies 1 S/C crystasis 1 Platelet, giant 1 Schistocyte (bite, blister, helmet) 1 Target Cell (codocyte) 1 Total Population: 231 Intended result: Parasite

*To see the original fullsized immages, please refer to the original CD or sign on to your data entry sheet at http://www.aab‐pts.org/ Sample 14Q3‐2: A 5‐year‐old girl is brought to a pediatrician by her relatives. She is visiting from Ghana and two days after arriving in the U.S., developed a fever of up to 101.4F, shaking chills, and vomiting. On physical examination, the patient appears acute ill, pale, and is febrile. CBC results: WBC 1,100/µL, Hgb 5.1 g/dL, Hct 16.1%, Plts 22,000/μL. Identify the indicated cells.

The automated CBC results show pancyopenia. Review of the peripheral blood smear shows mild to moderate ansio‐ and poikilocytosis, characterized by a few spherocytes, macrocytes, and polychromasia. In contrast to the automated platelet count, the platelet count on the peripheral smear appears relatively normal. The “cells” to be identified are gametocytes of the malarial parasite Plasmodium falciparum. These gametocytes are crescent‐to‐sausage shaped and contain a single mass of condensed chromatin. No ring forms are identified.

There are four species of Plasmodium that cause malaria in humans – falciparum, vivax, malariae, and ovale. P. falciparum is the most deadly and is transmitted through the bite of the female Anopheles mosquito. These mosquitoes breed in water, often preferring shallow pools of fresh water (puddles, rice fields, collections of rainwater in shallow roads) and typically bite at night. During the bite, sporozoites are injected into the bloodstream. They travel to the liver where they continue maturation becoming a cyst‐like structure containing thousands of merozoites (the exoerythrocytic stage). The infected liver cells eventually rupture and the merozoites are released into the blood, where they invade the red blood cells. Once in the RBC, the merozoite develops into a trophozoite or “ring form”, followed by coalescence into a schizont. Within 48‐hours of infecting the red cell, the parasite has exhausted the supply of hemoglobin and the infected cell ultimately ruptures, releasing the merozoites to infect adjacent RBCs. The cyclical fever associated with P. falciparum infection is the result of the cycles of red cell rupture; however, in many infected individuals the fever is rather erratic and not at all cyclical (in contrast to infection with P. vivax and P. ovale, where symptoms wax and wane occur at regular intervals). A small number of merozoites develop into gametocytes, which are essential for the parasite to complete its life cycle. The gametocytes will enter the mosquito during a subsequent blood meal, allowing the eventual formation of zygotes in the mosquito gut. Most malaria infections and deaths occur in sub‐Saharan Africa; however, parts of Asia, the Middle East, Europe, and Latin American are also affected. Young children who have not yet developed protective immunity are most at risk of infection and serious complications.

P. falciparum infection causes an acute febrile illness, with symptoms appearing approximately seven to ten days after the mosquito bite. The initial symptoms can be relatively mild –low‐grade fever, headache, abdominal discomfort, chills, muscle aches – and, therefore, not readily recognized as a malarial infection. However, this rapidly progresses to severe illness, particularly in children, leading to severe anemia, respiratory distress, cerebral symptoms, and death. P. falciparum is unique in its ability to cause the red cells to express an atypical adhesive protein, resulting in RBCs abnormally adhering to vessel walls and each other, ultimately obstructing the microcirculation of several organs, notably the brain (“cerebral malaria”). Diagnosis is made by demonstrating the parasite in RBCs on a peripheral blood smear. Usually, with P. falciparum infection, infection is demonstrated by multiple small ring forms within a normocytic red cell. Occasionally, only a single ring form may be present per cell. The ring forms tend to be slightly smaller than with other species of Plasmodium, and schizonts are seldom seen. The presence of a large number of rings in the absence of more mature stages, as well as multiply‐ infected erythrocytes, is highly suggestive of P. falciparum. The crescent‐shaped gametocytes are distinctive for this infection; however they do not appear until late in the disease. Although the gametocytes are within the red cell, often the red cell membrane is distorted or not visible. Occasionally, a thin remnant of the RBC membrane can be seen next to the gametocyte (see Cell 2). This crescent‐shaped bit of membrane is known as “Laveran’s bib”.

THIRD QUADRIMESTER 2014 Cell ID ‐ Page 4 of 4