Leukocytosis D. S. CHABOT-RICHARDS, T. I. GEORGE SUMMARY INTRODUCTION. International Journal of Laboratory Hematology REVIEW

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1 International Journal of Laboratory Hematology REVIEW The Official journal of the International Society for Laboratory Hematology INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY Leukocytosis D. S. CHABOT-RICHARDS, T. I. GEORGE Department of Pathology, University of New Mexico, Albuquerque, NM, USA Correspondence: Devon Chabot-Richards, Tricore Reference Laboratories, Department of Hematopathology, 1001 Woodward Pl NE, Albuquerque, NM 87102, USA. Tel.: ; Fax: ; unm.edu doi: /ijlh Received 17 January 2014; accepted for publication 5 February 2014 SUMMARY An increased white blood cell count, or leukocytosis, is a common laboratory finding. Appropriate specimen evaluation depends on which lineages are increased and the morphologic findings on peripheral blood smear review to guide further testing. The presence of blasts is concerning for acute leukemia and may require bone marrow biopsy. Lymphocytosis may be morphologically divided into polymorphic and monomorphic populations. Polymorphic lymphocytosis is most consistent with a reactive process, while monomorphic populations are concerning for lymphoproliferative neoplasm. The differential can be further narrowed based on morphologic findings. Myeloid leukocytosis can occur in a number of reactive conditions as well as myeloid malignancies. The types of cells present and morphology can help to guide additional workup. This study provides guidance for the appropriate evaluation and further workup of leukocytosis. Keywords Leukocytosis, lymphocytosis, neutrophilia, morphology, lymphoma INTRODUCTION Leukocytosis, defined as an increase in white blood cell (WBC) count, is a common finding with a broad differential diagnosis, encompassing both benign and malignant entities. Careful evaluation of complete blood cell count (CBC) data and morphologic features are key steps necessary to characterize the nature of the process and guide further workup. Reference intervals for WBC counts and relative percentages and absolute cell counts vary by patient age and hospital population. Each hospital laboratory must determine reference ranges during the validation process of their hematology analyzers [1]. Total WBC counts are higher in infants, with newborns having the highest WBC count and absolute neutrophil count of any age [2]. By 1 2 weeks of age through early adolescence, lymphocytes become the predominant WBC. This gradually shifts and neutrophils are the predominant WBC in teenagers and adults [3]. Race is also associated with differences in total WBC count and differential, with individuals of black African descent having lower absolute neutrophil counts [4]. Laboratories should determine automated CBC criteria that trigger a peripheral blood smear review [5]. Common WBC flags include numeric flags such as overall leukopenia or leukocytosis or abnormalities of the differential counts, as well as morphologic criteria such as immature granulocytes, atypical or variant lymphocytes, or blasts [5]. 279

2 280 D. S. CHABOT-RICHARDS AND T. I. GEORGE LEUKOCYTOSIS EXAMINATION OF THE PERIPHERAL BLOOD SMEAR When appropriate CBC criteria are met, a peripheral blood smear should be examined. Slides may be prepared using anticoagulated blood or fresh specimen. The smear may be made by an instrument or manually by placing a drop of blood at one end of a slide and then smearing it over the surface of the slide with a second slide or a coverslip. After air drying, the slide is typically stained with a Romanowsky stain [6]. The smear should first be examined at low power to identify overall cellularity and types of cells. The findings should be correlated with the automated CBC report. Large cells or aggregates of cells or platelets are often deposited at the edges of the smear. Assessment of WBC morphology is most commonly carried out in the thin areas of the slide, where cell crowding and over staining do not interfere. Cells should be examined to determine the appropriate classification, the level of maturity, the morphology, and the presence of inclusions [7]. PRESENCE OF BLASTS The leading differential in a peripheral blood smear with blasts is acute leukemia; however, other conditions may be associated with circulating blasts. While blast counts of >20% are diagnostic for acute leukemia, a lower blast count in the peripheral blood does not exclude acute leukemia [8]. Acute leukemias are often associated with bone marrow failure and are accompanied by anemia, neutropenia, and thrombocytopenia, in addition to leukocytosis with circulating blasts. Clinical history is important to evaluate for progression of a previously diagnosed chronic disorder such as chronic myelogenous leukemia. The first step in evaluating blast cells is to examine for lineage-specific features. It is important to realize that there is considerable morphologic overlap between lymphoblasts and myeloblasts and that morphology alone may not be definitive. Lymphoblasts show a range of appearances, from small- to intermediate-sized cells with scant cytoplasm and condensed nuclear chromatin to larger cells with moderate blue or blue gray cytoplasm and dispersed chromatin with prominent nucleoli. The nuclei may be smooth and round or irregular and convoluted. There may be cytoplasmic vacuoles or, rarely, granules. Although there is a considerable range of morphology, myeloblasts tend to be larger, with more abundant cytoplasm. Blasts with minimal differentiation may have a similar appearance to lymphoblasts. The presence of Auer rods and cytoplasmic granules strongly suggests myeloid differentiation; however, lymphoblasts may occasionally contain azurophilic granules [9]. Cytochemical stains can be helpful to confirm lineage, with myeloperoxidase staining granules in myeloblasts and nonspecific esterase staining cells with monocytic differentiation. Subtypes of acute myeloid leukemia are associated with specific morphologic findings. Most importantly, acute promyelocytic leukemia with t(15;17) (APL) is associated with hypergranular cells with coalescing granules and Auer rods, with cells containing multiple Auer rods highly specific for APL. The nuclei are often folded, bilobed, or kidneyshaped. The hypogranular variant shows similar nuclear features with agranular cytoplasm. APL may be associated with schistocytes due to disseminated intravascular coagulation (Figure 1). Lower circulating blast counts may be seen in chronic myeloid neoplasms, including myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), and overlap MDS/MPN. MDS with excess blasts may have up to 19% circulating blasts. The blasts in MDS are associated with cytopenias and dysplasia; blasts in MDS may be smaller in size with less differentiation compared with typical myeloblasts. Patients with MPN generally have <10% blasts and increased cell counts in one or more myeloid lineage. Ten to nineteen percent blasts are seen in the accelerated phase of MPNs. Patients with MDS/MPN syndromes have dysplasia and a combination of increased and decreased cell counts in different lineages. Circulating blasts can be seen in the absence of hematologic malignancy. Iatrogenic or endogenous excess granulocyte colony-stimulating factor (G-CSF) stimulation can cause a left shift of the myeloid lineage to the blast stage [10]. Bone marrow damage or infiltration by fibrosis, malignancy, or infection can be associated with circulating immature cells (leukoerythroblastosis), including blasts and nucleated red blood cells. LYMPHOCYTOSIS In the patient with an increased lymphocyte count, the differential diagnosis depends on patient age,

3 D. S. CHABOT-RICHARDS AND T. I. GEORGE LEUKOCYTOSIS 281 (a) (b) (c) (d) Figure 1. Myeloblasts. (a) Large blasts with irregular nuclei, prominent nucleoli, and moderate cytoplasm. (b) Myeloblast with cytoplasmic Auer rod. (c) Monoblasts with abundant blue cytoplasm. (d) Cytoplasmic nonspecific esterase positivity in monoblasts. (e) Acute promyelocytic leukemia (APL) showing multiple Auer rods. (f) Bright myeloperoxidase staining in APL. (e) (f) clinical history, and morphologic findings. Absolute lymphocyte counts are higher in children, and pediatric lymphocytosis is most commonly benign. Benign causes of lymphocytosis commonly include infection, particularly viral, autoimmune disorders, transient stress, and polyclonal B-cell lymphocytosis. Lymphocytosis in adults requires a diligent workup to exclude a neoplastic process. Lymphocytes are fragile, and both clonal and reactive lymphocytes may appear as smudge cells on peripheral blood smear. Albumin preparations preserve lymphocytes to allow morphologic examination when many smudge cells are present in the initial blood smear [11] (Figure 2).

4 282 D. S. CHABOT-RICHARDS AND T. I. GEORGE LEUKOCYTOSIS Monomorphic Lymphocytosis Pleomorphic Suspect Neoplastic Suspect Reactive Differen al Diagnosis Ancillary Tests Small, round nuclei CLL MBL PBL Burkitt MCL T-PLL FISH Folded or cleaved nuclei FL MCL Atypical CLL T-cell Pertussis* FISH CCND1, BCL2 Tissue biopsy Convoluted nuclei Sezary syndrome Adult T-cell leukemia T-cell clonality Villous cytoplasm Plasmacytoid Granules Prominent nucleoli Large cells HCL SMZL HCLV T-PLL LPL LPL Plasma cell myeloma Plasma cell leukemia T-LGL NK cell leukemia T-PLL B-PLL HCLV MCL Burkitt Leukemia DLBCL MCL ALCL SPEP/UPEP T-cell clonality KIR profile Cytogenetics FISH MYC Figure 2. Diagnostic algorithm for the workup of lymphocytosis. Pleomorphic lymphocytosis favors a reactive etiology. Correlation with clinical and laboratory testing is required. Monomorphic lymphocytes are concerning for a neoplastic process, and further workup including flow cytometric immunophenotyping and appropriate ancillary testing is required. *Pertussis infection is a reactive cause of monomorphic lymphocytosis, most often seen in pediatric populations. LYMPHOCYTOSIS WITH PLEOMORPHIC MORPHOLOGY Pleomorphic lymphocytosis is most commonly associated with a reactive process. Reactive lymphocytoses rarely exceed /L and show a range of lymphocyte size and shapes, often best appreciated at low power [12]. The nuclei show mature chromatin and inconspicuous nucleoli. Many cells are large with abundant clear to light blue cytoplasm with a basophilic rim. The cytoplasm may partially wrap around adjacent red blood cells. There may be large granular lymphocytes (LGLs) with azurophilic cytoplasmic granules. Immunoblasts and plasma cells may also be present. These cells are large with deeply basophilic cytoplasm, and round to oval nuclei, and prominent nucleoli. Pleomorphic lymphocytosis with activated morphology is most commonly associated with viral infection. Epstein Barr virus, or infectious mononucleosis, is the classic example, but other causes include CMV, influenza, adenovirus, and HIV. Some bacterial or parasitic infections may be associated with pleomorphic lymphocytosis. Noninfectious causes include medication, stress, trauma, vaccination, postsplenectomy, hypersensitivity reaction, smoking, and autoimmune disease [6]. LYMPHOCYTOSIS WITH MONOMORPHIC MORPHOLOGY Monomorphic lymphocytoses are much more concerning for lymphoproliferative neoplasm; however, there are some reactive processes associated with a

5 D. S. CHABOT-RICHARDS AND T. I. GEORGE LEUKOCYTOSIS 283 homogenous appearance. It is important to distinguish a monomorphic population of neoplastic lymphocytes in a background of normal lymphocytes from a polymorphic lymphocytosis. Monomorphic lymphocytosis with small cells with small, round nuclei Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. The incidence increases with age, and it is important to consider the diagnosis when evaluating lymphocytosis in an older adult. Flow cytometric analysis shows B-lymphocytes with aberrant expression of CD5 and weak surface immunoglobulin. Typical lymphocytes in CLL are round and small, with condensed, clumped nuclear chromatin and scant cytoplasm. Nucleoli are inconspicuous. Cases with atypical morphology can show a range of morphology including cleaved nuclei and larger cells, with up to 55% prolymphocytes. Prolymphocytes are large cells with abundant cytoplasm and a round, central nucleus with a single prominent nucleolus. The diagnosis of CLL can only be made when /L monoclonal lymphocytes are present. Lower numbers of neoplastic cells should be diagnosed as monoclonal B-lymphocytosis, a proliferation of clonal B-lymphocytes found in 7% of healthy subjects that may progress to CLL in a small fraction of patients [13]. Other disorders can occasionally present with lymphocytosis with small cells with round nuclei. Polyclonal B-lymphocytosis may present in this fashion; however, it is more typically associated with nuclear irregularities and binucleated forms. Burkitt cells are more often moderate or large in size and have moderate, deeply basophilic cytoplasm with small vacuoles. Mantle cell lymphoma (MCL) cells usually have irregular or folded nuclei and may include large cells with blastic features. The neoplastic cells in T-prolymphocytic leukemia (T-PLL) are usually larger prolymphocytes; however, small cells with condensed chromatin can be seen. Monomorphic lymphocytosis with folded or cleaved nuclei There are a number of benign and malignant causes of lymphocytosis with folded or cleaved nuclei. Although infectious causes of lymphocytosis are most commonly associated with a polymorphic appearance, Bordetella pertussis is an important exception. Bordetella infection is associated with severe, paroxysmal coughing, or whooping cough, most often seen in children. The bacteria produce a toxin which results in a characteristic severe, monomorphic lymphocytosis characterized by small, mature cells with deeply cleaved nuclei [14]. Patient age and clinical presentation are important in making this diagnosis. Another benign cause of lymphocytosis with deeply cleaved or binucleated cells is polyclonal B-lymphocytosis. This is a benign condition seen in young to middle-aged female smokers and associated with HLA- DR7 [15]. The lymphocytosis is typically moderate with approximately 10% binucleated forms [16]. Although follicular lymphoma only rarely involves the peripheral blood, it is associated with a characteristic appearance with small to intermediate cells with folded, convoluted nuclei and scant cytoplasm. Occasional large cells may be present [17]. The neoplastic cells are monoclonal B-cells, which often express CD10. FISH analysis showing rearrangement of BCL2 can be helpful. Although mantle cell lymphoma is primarily lymph node based, it involves the peripheral blood in almost 50% of cases [18]. Circulating MCL cells often vary in size and shape, but the typical cell is large with a folded or indented nucleus and variably prominent nucleolus. Blastic variants are large with fine chromatin and prominent nucleoli; however, they also often have indented nuclei. reveals a monoclonal B-cell population with expression of CD5. FISH analysis demonstrating IGH-CCND1 can help confirm the diagnosis. Atypical CLL may show predominantly cleaved cells and must be distinguished from MCL when a CD5+ monoclonal B-cell population is identified in the peripheral blood. T-cell prolymphocytic leukemia can occasionally show nuclear irregularity, but the prolymphocytic form usually predominates. Circulating mature T-cell leukemias and lymphomas often show irregular nuclei; however, they are more typically highly convoluted or cerebriform in appearance. Monomorphic lymphocytosis with highly convoluted or cerebriform nuclei Highly convoluted or cerebriform nuclei are most commonly associated with mature T-cell leukemias.

6 284 D. S. CHABOT-RICHARDS AND T. I. GEORGE LEUKOCYTOSIS Peripheral blood involvement by mycosis fungoides (MF) or Sezary syndrome (SS) features medium to large lymphocytes with characteristic dark, cerebriform nuclei. The neoplastic cells are most often CD4-positive T-cells with loss of CD7. T-cell clonality studies can be helpful. While the peripheral blood features overlap, the distinction between MF and SS is made based on the clinical course. In SS, erythroderma and lymphadenopathy with blood involvement are present at diagnosis, while MF does not exhibit peripheral blood involvement at presentation [19]. Adult T-cell leukemia is an aggressive disease caused by chronic infection with human T-cell leukemia virus (HTLV)-1. Only a small percentage of those infected with the virus develop leukemia. Patients often show systemic manifestations, including skin rash, hypercalcemia, and lytic bone lesions. The peripheral blood smear shows a severe lymphocytosis with highly irregular nuclei. Cytopenias are common; however, there may be eosinophilia. The neoplastic cells are typically CD4-positive T-cells with expression of CD25 and loss of CD7. Monomorphic lymphocytosis with villous cytoplasm Hairy cell leukemia (HCL) and splenic marginal zone lymphoma (SMZL) often show peripheral blood involvement with villous lymphocytes with associated splenomegaly. In HCL, there is typically pancytopenia with monocytopenia. The lymphocyte count may be low or normal, and circulating neoplastic hairy cells are rare. These cells show abundant cytoplasm with circumferential spiky projections. The nuclei are round or kidney-bean-shaped. in classic HCL shows a monoclonal B-cell population with bright CD20, CD11c, CD22, CD25, and CD103. In contrast, in SMZL, the lymphocytes show bipolar projections, and the nuclei are usually round. CD25 may be positive, but CD103 is typically negative and CD22 is usually dim. Hairy cell leukemia variant (HCLV) is an uncommon entity with neoplastic cells appearing similar to HCL; however, the lymphocyte count is usually much higher, and cells may be larger with prominent nucleoli. HCLV is positive for CD103, and only rare cases are positive for CD25. Rarely, the neoplastic lymphocytes in T-PLL and plasma cell leukemia may show cytoplasmic projections or cytoplasmic blebbing; however, the more typical forms usually predominate. Monomorphic lymphocytosis with plasmacytoid lymphocytes or plasma cells Lymphoplasmacytic lymphoma is typically a tissuebased disease; however, in 10% of cases, it may involve the peripheral blood with circulating plasmacytoid lymphocytes and occasional plasma cells [18]. The red blood cells may show rouleaux formation. shows a CD5-negative, CD10-negative monoclonal B-cell and plasma cell populations. Small numbers of circulating plasma cells can rarely be seen with plasma cell myeloma. If plasma cells account for >20% of WBCs, a diagnosis of plasma cell leukemia should be made. The circulating plasma cells in plasma cell leukemia may be smaller with less cytoplasm and may be difficult to distinguish from plasmacytoid lymphocytes. Monomorphic lymphocytosis with large granular lymphocytes Large granular lymphocytes are a subset of T-cells expressing CD3, CD8, and CD57. Natural killer (NK) cells can also have a similar appearance. These cells are surface CD3 negative and express cytoplasmic CD3, weak CD56, CD2, TIA-1, and granzyme. Lymphocytosis with a dominant population of LGLs can be seen in reactive disorders, most commonly autoimmune disease, viral infection, other malignancy, chemotherapy, and following bone marrow transplantation [20]. It can be difficult to distinguish these reactive processes from T-cell large granular lymphocytic leukemia or chronic lymphoproliferative neoplasm of NK cells. Clinical and laboratory workup is necessary to exclude infection and autoimmune disease. A positive T-cell receptor gene rearrangement test can be helpful; however, some reactive processes will show a pseudo clone. Demonstration of a clonal NK population requires demonstration of a restricted KIR expression profile [21]. Monomorphic lymphocytosis with prominent nucleoli Both B-Cell Prolymphocytic Leukemia (B-PLL) and T- PLL present with a rapidly increasing, striking lymphocytosis primarily composed of large cells with a central

7 D. S. CHABOT-RICHARDS AND T. I. GEORGE LEUKOCYTOSIS 285 round nucleus, abundant blue cytoplasm, and a single prominent nucleolus. The morphologic appearance of T-PLL is more variable than B-PLL. T-PLL is an aggressive disease. Patients often present with systemic symptoms, including abdominal distention, skin rash, organomegaly, and lymphadenopathy. The neoplastic cells can show a range of morphology including small cells with condensed chromatin and cells with cytoplasmic projections. B-Cell Prolymphocytic Leukemia is a very rare disease which should only be diagnosed when a prolymphocytic transformation of CLL can be excluded. The clinical course is generally extremely aggressive. B-PLL requires >55% prolymphocytes at diagnosis. If the percentage is lower and typical CLL cells are present, a diagnosis of prolymphocytic transformation of CLL is preferred. Occasionally, HCLV will present with a cells showing prominent nucleoli; however, the characteristic cytoplasmic villi are usually also present. Mantle cell lymphoma can also present with circulating lymphoma cells that mimic prolymphocytes. Monomorphic lymphocytosis with large cells Burkitt lymphoma/leukemia (BL) is the most common cause of circulating large lymphocytes. BL is an aggressive disease that is most commonly nodal based. If >25% of bone marrow is involved, the process is classified as leukemia. BL generally shows intermediate to large cells with deeply basophilic cytoplasm, often with vacuoles. The nuclei are oval to round with multiple nucleoli. Diffuse large B-cell lymphoma and B-cell lymphoma with features intermediate between BL and DLBCL can also show circulating neoplastic cells. Very rarely, anaplastic large cell lymphoma may have circulating lymphoma cells. These cells typically have irregular nuclei. Circulating lymphocytes in MCL may be large; these cells usually have irregular nuclei and blastic appearing chromatin. MYELOID LEUKOCYTOSIS Myeloid leukocytoses are elevations of neutrophils, eosinophils, basophils, or monocytes. Causes vary by the type of cell increased, but include infection, medication, autoimmune disorders, metabolic disorders, and other reactive states as well as clonal myeloid neoplasms. Patient history and clinical findings are necessary to ensure appropriate classification (Figure 3). Neutrophilia Neutrophils are the most abundant leukocyte and play a key role in immune defense from bacterial infections. These cells are short-lived, and the bone marrow production rate is astronomical, with an additional reserve pool of cells available. Typically, only the mature, polymorphonuclear forms are present in the peripheral blood, with 5 10% band forms. Immature granulocytes or left-shifted forms can be seen in both reactive and neoplastic conditions. It should be noted that the band count is not reproducible and its use is not recommended [22]. Reactive neutrophilias can be seen with infection, particularly bacterial, inflammation, drugs (particularly steroids, epinephrine, and lithium), colony-stimulating factors such as G-CSF, metabolic disorders, trauma, stress, pregnancy, other malignancy, and smoking [17]. Reactive neutrophilias are associated with activated changes, including toxic granulation, vacuoles, and Dohle bodies. The other lineages are generally unremarkable; however, monocytes may also show toxic changes. There can be significant morphologic overlap between reactive neutrophilias and MPN and MDS/ MPN. Chronic myelogenous leukemia (CML) is often associated with a very high WBC count. There is generally a pronounced left shift with increased myelocytes. Toxic changes are typically absent. An associated basophilia and eosinophilia are usually present. Definitive diagnosis of CML requires demonstration of the BCR-ABL1 translocation by cytogenetics, FISH, or molecular genetic methods. Chronic neutrophilic leukemia (CNL) is a myeloproliferative neoplasm characterized by a mature neutrophilia accounting for >80% of WBC with limited left shift. Definitive diagnosis can be difficult, as a reactive process must be excluded. Recent identification of an oncogenic mutation in CSF3R in CNL may aid in diagnosis [23]. Other MPNs may show neutrophilia, particularly as a part of a leukoerythroblastic picture due to underlying bone marrow fibrosis. Dacrocytes (tear-drop shaped red blood cells) may be present in this case. Atypical chronic myeloid leukemia (acml) is a MDS/MPN with neutrophilia and left shift. There are prominent dysplastic changes with nuclear hypolobation or bizarrely

8 286 D. S. CHABOT-RICHARDS AND T. I. GEORGE LEUKOCYTOSIS Myeloid Leukocytosis Neutrophilia Features supporting neoplastic WBC >50 x 10 9 /L Pronounced left shift Basophilia or Eosinophilia Dacrocytes Dysplasia Features supporting reactive WBC <50 x 10 9 /L Predominantly mature Toxic granulation and vacuoles Döhle bodies Thrombocytosis Features supporting neoplastic Monocytosis Features supporting reactive Persistent Promonocytes and blasts Dysplasia Transient Predominantly mature Reactive changes Features supporting neoplastic Persistent Immature cells present Cytopenias and dysplasia in other lineages Features supporting neoplastic Other lineage abnormalities Eosinophilia Basophilia Features supporting reactive Transient Clinical presence of drugs, allergy or infection Rare Features supporting reactive Figure 3. Diagnostic algorithm for myeloid leukocytosis. Most myeloid leukocytoses are reactive. If there is concern for a neoplastic process, further workup including bone marrow biopsy and appropriate ancillary testing is required. segmented nuclei, abnormal chromatin clumping, and hypogranularity. There may also be dysplasia in red blood cells and platelets. A subset of acml has been associated with CSF3R mutations. Eosinophilia Eosinophilia is most often reactive in nature. The most common causes are infection, particularly parasitic, hypersensitivity reaction, connective tissue disease, and other malignancy, particularly Hodgkin lymphoma and T-cell lymphoproliferative disorders. Pulmonary disease, cardiac disease, gastrointestinal disease, and adrenal insufficiency can also cause eosinophilia. Reactive eosinophilia is typically transient; however, depending on the cause, some may be chronic. Chronic eosinophilia can lead to systemic symptoms due to eosinophil degranulation, including cardiovascular symptoms with cardiac fibrosis. When reactive causes have been excluded, a clonal process may be considered. The WHO classification system recognizes a category of myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDG- FRA, PDGFRB, and FGFR1. Cytogenetics can detect

9 D. S. CHABOT-RICHARDS AND T. I. GEORGE LEUKOCYTOSIS 287 abnormalities of PDGFRB and FGFR1; however, the common FIP1L1-PDGFRA fusion is cryptic and requires FISH or molecular studies for identification [24]. Chronic eosinophilic leukemia is a rare MPN characterized by eosinophilia with either >2% blasts in the blood, >5% blasts in the bone marrow, or a clonal cytogenetic or molecular genetic abnormality. A reactive cause must be excluded. If there is no increase in blasts and a clonal abnormality cannot be identified, the process may be classified as idiopathic hypereosinophilic syndrome if eosinophil-related tissue damage is present or idiopathic hypereosinophilia if no damage is identified [25]. Basophilia Isolated basophilia is extremely uncommon. Reactive basophilia has been linked to hypersensitivity disorders, iron deficiency, chronic inflammation, and rarely infection, including influenza and chicken pox [26]. As reactive causes are rare, a finding of basophilia should prompt further workup to exclude a myeloproliferative neoplasm such as CML. Monocytosis Reactive monocytosis is associated with chronic infection, autoimmune disease, splenectomy, and a range of malignancies, including carcinoma, lymphoma, and plasma cell myeloma. It can also be seen with neutropenia and in regenerating bone marrow following bone marrow transplant or chemotherapy. If monocytosis is persistent and reactive causes are excluded, the differential diagnosis includes chronic myelomonocytic leukemia (CMML), acute myelomonocytic leukemia (AMML), CML, juvenile myelomonocytic leukemia (JMML), atypical CML, and MDS/MPNunclassifiable. Chronic myelomonocytic leukemia is most commonly seen in older adults and is associated with a persistent monocytosis with dysplasia in one or more myeloid lineage. There may be increased blasts, with promonocytes counted as blast equivalents. Although immunophenotypic abnormalities may be seen in reactive monocytosis, flow cytometric identification of aberrant expression of two or more antigens on monocytes is correlated with CMML [27]. Identification of a clonal abnormality is helpful to confirm the diagnosis. Bone marrow evaluation should be performed as AMML may present with mature monocytosis in the peripheral blood while immature forms predominate in the marrow. JMML is a rare disease seen in children with persistent monocytosis with marked hepatosplenomegaly and increased hemoglobin F. There is often left shift of the granulocytes, and dysplasia is minimal. CONCLUSION Accurate classification and diagnosis of leukocytosis require confirmation of automated differential counts and examination of the peripheral blood smear. Increased blasts should prompt a workup for acute leukemia, including flow cytometric immunophenotyping and bone marrow examination with cytogenetic and molecular genetic tests. In cases with lymphocytosis, a pleomorphic population of lymphocytes favors a reactive process. Cases with very high lymphocyte counts or homogenous morphology should be evaluated for a lymphoproliferative disorder. Flow cytometric immunophenotyping can be helpful as an initial ancillary test following morphologic review to prove clonality and guide additional cytogenetic and molecular tests. Myeloid proliferations can be more difficult to classify. Correlation with clinical presentation and persistence of abnormalities can be helpful. Features such as dysplasia, basophilia, prominent left shift, increased blasts, and a very high WBC count (> /L) favor a malignant process. Activated features such as toxic granulation, vacuolization, and Dohle bodies favor a reactive etiology. Flow cytometric immunophenotyping is often less helpful in myeloid disorders, but may demonstrate an aberrant phenotype or abnormal maturation pattern. A combination of CBC data, clinical and laboratory findings, and morphologic evaluation is needed to guide further testing and appropriately classify patients with leukocytosis.

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