An update on the etiology and diagnostic evaluation of a leukemoid reaction

      Abstract

      Persistent neutrophilic leukocytosis above 50,000 cells/μL when the cause is other than leukemia defines a leukemoid reaction. The diagnostic work-up consists of the exclusion of chronic myelogenous leukemia (CML) and chronic neutrophilic leukemia (CNL) and the detection of an underlying cause. The major causes of leukemoid reactions are severe infections, intoxications, malignancies, severe hemorrhage, or acute hemolysis. The present article points out the difficulties in the differential diagnosis of a leukemoid reaction and suggests an algorithm for a rational clinical and laboratory evaluation of this problematic entity.

      Keywords

      1. Introduction

      A leukemoid reaction (LR) is a hematological disorder, defined by a leukocyte count greater than 50,000 cells/μL, caused by reactive causes outside the bone marrow [
      • Nimieri H.S.
      • Makoni S.N.
      • Madziwa F.H.
      • Nemiary D.S.
      Leukemoid reaction response to chemotherapy and radiotherapy in a patient with cervical carcinoma.
      ,
      • Curnutte J.T.
      • Coates T.D.
      Disorders of phagocyte function and number.
      ]. It is characterized by a significant increase in mature neutrophils in the peripheral blood and a differential count showing marked left shift [
      • Curnutte J.T.
      • Coates T.D.
      Disorders of phagocyte function and number.
      ]. The diagnosis of LR is based on the exclusion of chronic myelogenous leukemia (CML) and chronic neutrophilic leukemia (CNL). The absence of immature cells, basophilia or monocytosis, increased leukocyte alkaline phosphatase (LAP), and the absence of the bcr/abl translocation distinguishes LR from CML. CNL is a rare, distinct myeloproliferative syndrome with a poor prognosis [
      • Bohm J.
      • Kock S.
      • Schaefer H.E.
      • Fisch P.
      Evidence of clonality in chronic neutrophilic leukaemia.
      ]. The differential diagnosis between LR and CNL may be difficult or even impossible because both conditions share identical morphological features, including a raised LAP score and the absence of the bcr/abl translocation [
      • Bohm J.
      • Kock S.
      • Schaefer H.E.
      • Fisch P.
      Evidence of clonality in chronic neutrophilic leukaemia.
      ].
      The current review discusses the different causes of LR, pointing out the difficulties in the differential diagnosis. Moreover, a diagnostic approach to a rational clinical and laboratory evaluation that mainly refers to an isolated LR is proposed.

      2. Etiology

      The major causes of LRs are severe infections, intoxications, malignancies, severe hemorrhage, or acute hemolysis (Table 1). A variety of infections, such as colitis due to Clostridium difficile, disseminated tuberculosis [
      • Au W.Y.
      • Ma S.K.
      • Kwong Y.L.
      Disseminated hepatosplenic mycobacterial infection masking myeloproliferative diseases as leukemoid reaction: a diagnostic pitfall.
      ], and severe shigellosis, have been associated with an LR. C. difficile colitis with an LR appears to be associated with a much higher mortality rate (∼50%) [
      • Marinella M.A.
      • Burdette S.D.
      • Bedimo R.
      • Markert R.J.
      Leukemoid reactions complicating colitis due to Clostridium difficile.
      ]. A study of 111 children with Shigella dysenteriae type 1 infection revealed 25 patients with LR. Its presence indicates a poor prognosis because it may accompany or precede hemolytic–uremic syndrome [
      • Azim T.
      • Qadri F.
      • Ahmed S.
      • Sarker M.S.
      • Halder R.C.
      • Hamadani J.
      • et al.
      Lipopolysaccharide-specific antibodies in plasma and stools of children with Shigella-associated leukemoid reaction and hemolytic–uremic syndrome.
      ]. A leukemoid reaction can also follow exposure to a drug (e.g., corticosteroids, minocycline, recombinant hematopoietic growth factors [
      • Ganti A.K.
      • Potti A.
      • Mehdi S.
      Uncommon syndromes and treatment manifestations of malignancy: case 2. Metastatic non-small-cell lung cancer presenting with leukocytosis.
      ]) or to a toxin (e.g., ethylene glycol [
      • Mycyk M.B.
      • Drendel A.
      • Sigg T.
      • Leikin J.B.
      Leukemoid response in ethylene glycol toxication.
      ]). Other rare conditions that have been described as causes of a LR are mesenteric inflammatory pseudotumor [
      • Kutluk T.
      • Emir S.
      • Karnak I.
      • Gaglar M.
      • Buyukpamukeu M.
      Mesenteric inflammatory pseudotumor: unusual presentation with leukemoid reaction and massive calcified mass.
      ], alcoholic steatohepatitis [
      • Juturi J.V.
      • Hopkins T.
      • Farhangi M.
      Severe leukocytosis with neutrophilia (leukemoid reaction) in alcoholic steatohepatitis.
      ,
      • Arguelles-Grande C.
      • Leon F.
      • Matilla J.
      • Dominguez J.
      • Montero J.
      Steroidal management and serum cytokine profile of a case of alcoholic hepatitis with leukemoid reaction.
      ], and retroperitoneal hemorrhage [
      • Marinella M.A.
      Extreme leukemoid reaction associated with retroperitoneal hemorrhage.
      ].
      Table 1Major causes of leukemoid reaction
      1. Infections (some major examples are listed)
      a. Clostridium difficile colitis
      b. Disseminated tuberculosis
      c. Severe shigellosis
      2. Malignancies
      a. Carcinomas (lung, oropharyngeal, gastrointestinal, genitourinary)
      b. Hodgkin's lymphoma
      c. Melanoma
      d. Sarcoma
      3. Drugs
      a. Corticosteroids
      b. Minocycline
      c. Recombinant hematopoietic growth factors
      4. Intoxication
      a. Ethylene glycol
      5. Severe hemorrhage or acute hemolysis
      a. Retroperitoneal hemorrhage
      6. Miscellaneous
      a. Mesenteric inflammatory pseudotumor
      b. Alcoholic steatohepatitis
      Granulocytosis with a modest elevation of the white blood cell count above 15,000 cells/μL, without infection or leukemia, is common in neoplasms and can be observed in several other conditions, such as smoking. Levels above 50,000 cells/μL, or even above 100,000 cells/μL, constitute a rare, but well-documented, paraneoplastic syndrome. Leukemoid reactions can present simultaneously with the malignancy, late in the course of the disease, or precede the diagnosis by as many as 4 years [
      • Nimieri H.S.
      • Makoni S.N.
      • Madziwa F.H.
      • Nemiary D.S.
      Leukemoid reaction response to chemotherapy and radiotherapy in a patient with cervical carcinoma.
      ]. Malignancy-associated LRs are commonly observed in a variety of carcinomas, most notably lung and kidney cancer [
      • Ganti A.K.
      • Potti A.
      • Mehdi S.
      Uncommon syndromes and treatment manifestations of malignancy: case 2. Metastatic non-small-cell lung cancer presenting with leukocytosis.
      ,
      • McKee Jr., L.C.
      Excess leukocytosis (leukemoid reactions) associated with malignant diseases.
      ,
      • Kasuga I.
      • Makino S.
      • Kiyokawa H.
      • Katoh H.
      • Ebihara Y.
      • Ohyashiki K.
      Tumor-related leukocytosis is linked with poor prognosis in patients with lung carcinoma.
      ]. In a study of 227 patients with carcinoma of the lung, 33 patients (14.5%) were diagnosed with tumor-related leukocytosis and 6 patients (2.6%) with LRs [
      • Kasuga I.
      • Makino S.
      • Kiyokawa H.
      • Katoh H.
      • Ebihara Y.
      • Ohyashiki K.
      Tumor-related leukocytosis is linked with poor prognosis in patients with lung carcinoma.
      ]. Only occasionally is an LR seen in association with other neoplastic conditions, such as Hodgkin's lymphomas, melanomas, and sarcomas. A number of case reports have described an LR associated with melanoma [
      • Schniewind B.
      • Christgen M.
      • Hauschild A.
      • Kurdow R.
      • Kalthoff H.
      • Klomp H.J.
      Paraneoplastic leukemoid reaction and rapid progression in a patient with malignant melanoma: establishment of KT293, a novel G-CSF-secreting melanoma cell line.
      ], oropharyngeal [
      • Horii A.
      • Shimamura K.
      • Honjo Y.
      • Mitani K.
      • Miki T.
      • Takashima S.
      • et al.
      Granulocyte colony-stimulating factor-producing tongue carcinoma.
      ,
      • Saussez S.
      • Heimann P.
      • Vandevelde L.
      • Bisschop P.
      • Jortay A.
      • Schandene L.
      • et al.
      Undifferentiated carcinoma of the nasopharynx and leukemoid reaction: report of case with literature review.
      ], gastrointestinal [
      • Endo K.
      • Kohnoe S.
      • Okamura T.
      • Haraguchi M.
      • Adachi E.
      • Toh Y.
      • et al.
      Gastric adenosquamous carcinoma producing granulocyte-colony stimulating factor.
      ,
      • Ferrer A.
      • Cervantes F.
      • Hernández-Boluda J.C.
      • Alvarez A.
      • Montserrat E.
      Leukemoid reaction preceding the diagnosis of colorectal carcinoma by four years.
      ], and genitourinary [
      • Nimieri H.S.
      • Makoni S.N.
      • Madziwa F.H.
      • Nemiary D.S.
      Leukemoid reaction response to chemotherapy and radiotherapy in a patient with cervical carcinoma.
      ,
      • Tachibana M.
      • Murai M.
      G-CSF production in human bladder cancer and its ability to promote autocrine growth: a review.
      ] tumors. The exact mechanism of the generation of an LR in association with a neoplasm has not been fully elucidated. It is likely that various cytokines produced irregularly by the tumor cells, including granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin 6 (IL-6), may underlie the pathogenesis of LR in such conditions [
      • Kasuga I.
      • Makino S.
      • Kiyokawa H.
      • Katoh H.
      • Ebihara Y.
      • Ohyashiki K.
      Tumor-related leukocytosis is linked with poor prognosis in patients with lung carcinoma.
      ,
      • Schniewind B.
      • Christgen M.
      • Hauschild A.
      • Kurdow R.
      • Kalthoff H.
      • Klomp H.J.
      Paraneoplastic leukemoid reaction and rapid progression in a patient with malignant melanoma: establishment of KT293, a novel G-CSF-secreting melanoma cell line.
      ,
      • Horii A.
      • Shimamura K.
      • Honjo Y.
      • Mitani K.
      • Miki T.
      • Takashima S.
      • et al.
      Granulocyte colony-stimulating factor-producing tongue carcinoma.
      ,
      • Endo K.
      • Kohnoe S.
      • Okamura T.
      • Haraguchi M.
      • Adachi E.
      • Toh Y.
      • et al.
      Gastric adenosquamous carcinoma producing granulocyte-colony stimulating factor.
      ,
      • Tachibana M.
      • Murai M.
      G-CSF production in human bladder cancer and its ability to promote autocrine growth: a review.
      ,
      • Watanabe M.
      • Ono K.
      • Ozeki Y.
      • Tanaka S.
      • Aida S.
      • Okuno Y.
      Production of granulocyte-macrophage colony-stimulating factor in a patient with metastatic chest wall large cell carcinoma.
      ]. Therapeutic strategies for the neoplasm, like surgical excision [
      • Mycyk M.B.
      • Drendel A.
      • Sigg T.
      • Leikin J.B.
      Leukemoid response in ethylene glycol toxication.
      ,
      • Endo K.
      • Kohnoe S.
      • Okamura T.
      • Haraguchi M.
      • Adachi E.
      • Toh Y.
      • et al.
      Gastric adenosquamous carcinoma producing granulocyte-colony stimulating factor.
      ,
      • Ferrer A.
      • Cervantes F.
      • Hernández-Boluda J.C.
      • Alvarez A.
      • Montserrat E.
      Leukemoid reaction preceding the diagnosis of colorectal carcinoma by four years.
      ], chemotherapy, and radiotherapy [
      • Nimieri H.S.
      • Makoni S.N.
      • Madziwa F.H.
      • Nemiary D.S.
      Leukemoid reaction response to chemotherapy and radiotherapy in a patient with cervical carcinoma.
      ,
      • Watanabe M.
      • Ono K.
      • Ozeki Y.
      • Tanaka S.
      • Aida S.
      • Okuno Y.
      Production of granulocyte-macrophage colony-stimulating factor in a patient with metastatic chest wall large cell carcinoma.
      ], frequently result in a decrease in the white blood cell count. Nevertheless, in such patients, LRs have been reported to correlate with an aggressive clinical course, lower survival time, occurring shortly before death [
      • McKee Jr., L.C.
      Excess leukocytosis (leukemoid reactions) associated with malignant diseases.
      ].

      3. Diagnostic evaluation

      An LR is, by definition, diagnosed after the exclusion of a malignant hematological disorder. In the case of concomitant polycythemia vera or thrombocytosis,a diagnostic approach concerning general myeloproliferative diseases should be made. However, the underlying cause of an LR is usually obvious. The clinician should obtain a clear history which, together with a good physical examination and limited imaging procedures, may provide significant clues to a diagnosis, such as a neoplasm or an infection. In this regard, it is important not to forget to ask about potentially important exposures to drugs or toxins.
      Nevertheless, there are cases where the clinician may not be able to exclude other significant causes of a LR without specific testing. The diagnostic work-up of an LR consists of the exclusion of a clonal disorder and special testing to identify other, more obscure, causes. White blood cell count and differential count, a peripheral blood smear, the leukocyte alkaline phosphatase (LAP) score, serum vitamin B12 levels, a bone marrow aspiration and biopsy, cytogenetic studies, and immunophenotyping of peripheral blood and bone marrow, as well as serum levels of hemopoietic growth factors, may help in distinguishing LR from CML. Since CNL and LRs share identical morphological features, clonality studies of blood neutrophils can help to demonstrate the monoclonality of neutrophils in CNL in contrast to a polyclonal LR (Table 2). Further clinical evaluation and laboratory and imaging tests for an underlying infection or an occult tumor may be necessary.
      Table 2Differential diagnosis of leukemoid reaction, chronic myelogenous leukemia, and chronic neutrophilic leukemia
      Leukemoid reactionChronic myelogenous leukemiaChronic neutrophilic leukemia
      Peripheral blood
      • Curnutte J.T.
      • Coates T.D.
      Disorders of phagocyte function and number.
      Mature neutrophils, marked “left shift”Immature cells, basophils, and eosinophilsMarked neutrophilia, no immature cells
      LAP score
      • Nimieri H.S.
      • Makoni S.N.
      • Madziwa F.H.
      • Nemiary D.S.
      Leukemoid reaction response to chemotherapy and radiotherapy in a patient with cervical carcinoma.
      ,
      • Reilly J.T.
      Chronic neutrophilic leukaemia: a distinct clinical entity?.
      HighLowHigh
      Serum vitamin B12
      • Vlasveld L.T.
      • Bos G.M.
      • Ermens A.A.
      • Bakker J.A.
      • Lindemans J.
      Hyperhomocysteinemia in patients with CML and peripheral stem cell donors treated with G-CSF: functional cobalamin deficiency due to granulocytosis-induced alterations in the cobalamin-binding proteins.
      Variable?, highHighHigh
      Bone marrow
      • Nimieri H.S.
      • Makoni S.N.
      • Madziwa F.H.
      • Nemiary D.S.
      Leukemoid reaction response to chemotherapy and radiotherapy in a patient with cervical carcinoma.
      ,
      • Curnutte J.T.
      • Coates T.D.
      Disorders of phagocyte function and number.
      Myeloid hyperplasia, orderly maturation, normal morphologyBasophilia, eosinophilia, monocytosis, slight increase in blasts and reticulin fibrosisSimilar morphology with LR, packed bone marrow, slight increase in reticulin
      Cytogenetic analysis
      • Reilly J.T.
      Chronic neutrophilic leukaemia: a distinct clinical entity?.
      No cytogenetic abnormalitiesbcr/abl translocationCytogenetic abnormalities in 37% of cases
      Immuno-phenotyping
      • Chianese R.
      • Brando B.
      • Gratama J.W.
      European Working Group on Clinical Cell Analysis. Diagnostic and prognostic value of flow cytometric immunophenotyping in malignant hematological diseases.
      CD13 (+++), CD15 (+++), CD34 (−) HLA-DR (−)CD13 (+++), CD15 (+++), CD34(−) HLA-DR (+)CD13 (+++) CD15(+++), CD34 (−) HLA-DR (+)
      Serum G-CSF
      • Kasuga I.
      • Makino S.
      • Kiyokawa H.
      • Katoh H.
      • Ebihara Y.
      • Ohyashiki K.
      Tumor-related leukocytosis is linked with poor prognosis in patients with lung carcinoma.
      ,
      • Schniewind B.
      • Christgen M.
      • Hauschild A.
      • Kurdow R.
      • Kalthoff H.
      • Klomp H.J.
      Paraneoplastic leukemoid reaction and rapid progression in a patient with malignant melanoma: establishment of KT293, a novel G-CSF-secreting melanoma cell line.
      ,
      • Horii A.
      • Shimamura K.
      • Honjo Y.
      • Mitani K.
      • Miki T.
      • Takashima S.
      • et al.
      Granulocyte colony-stimulating factor-producing tongue carcinoma.
      ,
      • Endo K.
      • Kohnoe S.
      • Okamura T.
      • Haraguchi M.
      • Adachi E.
      • Toh Y.
      • et al.
      Gastric adenosquamous carcinoma producing granulocyte-colony stimulating factor.
      ,
      • Tachibana M.
      • Murai M.
      G-CSF production in human bladder cancer and its ability to promote autocrine growth: a review.
      ,
      • Watanabe M.
      • Ono K.
      • Ozeki Y.
      • Tanaka S.
      • Aida S.
      • Okuno Y.
      Production of granulocyte-macrophage colony-stimulating factor in a patient with metastatic chest wall large cell carcinoma.
      ,
      • Reilly J.T.
      Chronic neutrophilic leukaemia: a distinct clinical entity?.
      High
      Only in G-CSF-producing tumors.
      LowLow
      Clonality studies
      • Bohm J.
      • Kock S.
      • Schaefer H.E.
      • Fisch P.
      Evidence of clonality in chronic neutrophilic leukaemia.
      PolyclonalMonoclonalMonoclonal
      (+++): high expression of the surface antigen.
      a Only in G-CSF-producing tumors.

      3.1 White blood cell count, differential count, and peripheral blood smear

      In LR, leukocyte counts are, by definition, greater than 50,000 cells/μL and consist mostly of mature neutrophils. The differential count discloses a marked left shift, as evidenced by the presence of myelocytes and metamyelocytes [
      • Curnutte J.T.
      • Coates T.D.
      Disorders of phagocyte function and number.
      ]. An expert's review of the peripheral smear is necessary to exclude a myeloproliferative syndrome. In CML, there are more immature cells, basophils, and eosinophils. In CNL, there is marked neutrophilia with no immature cells and, in contrast to an LR, myelocytes, metamyelocytes, and nucleated red cells are infrequent. The peripheral smear may, in addition, disclose toxic granulation, Doëhle bodies, and cytoplasmic vacuoles in the neutrophils of patients with an LR attributed to an infection [
      • Curnutte J.T.
      • Coates T.D.
      Disorders of phagocyte function and number.
      ].

      3.2 Leukocyte alkaline phosphatase (LAP) score

      LAP is an enzyme present in the cytoplasmic microsomes of neutrophils, bands, metamyelocytes, and myelocytes, but not in lymphocytes or monocytes. Immature neutrophils, such as those observed in CML, have decreased LAP scores. The stimulated neutrophils of an LR have increased LAP scores [
      • Nimieri H.S.
      • Makoni S.N.
      • Madziwa F.H.
      • Nemiary D.S.
      Leukemoid reaction response to chemotherapy and radiotherapy in a patient with cervical carcinoma.
      ]. In CNL, low LAP scores are the exception [
      • Reilly J.T.
      Chronic neutrophilic leukaemia: a distinct clinical entity?.
      ].

      3.3 Serum vitamin B12 and vitamin B12-binding capacity

      Vitamin B12 levels are usually elevated in CML and CNL, but they do not seem to be useful in differentiating these conditions from an LR. In fact, a recent observation of an LR due to G-CSF supplementation showed B12 levels that were just as high as the ones observed in CML [
      • Vlasveld L.T.
      • Bos G.M.
      • Ermens A.A.
      • Bakker J.A.
      • Lindemans J.
      Hyperhomocysteinemia in patients with CML and peripheral stem cell donors treated with G-CSF: functional cobalamin deficiency due to granulocytosis-induced alterations in the cobalamin-binding proteins.
      ].

      3.4 Bone marrow aspiration and biopsy

      Increased cellularity with myeloid hyperplasia is the principle picture of an LR. In contrast to acute leukemia, there is marked proliferation and orderly maturation of all normal myeloid elements with normal morphology. No fibrosis is present [
      • Curnutte J.T.
      • Coates T.D.
      Disorders of phagocyte function and number.
      ]. Similar morphological features are present in CNL, but a packed bone marrow biopsy, together with a slight increase in reticulin fibrosis, may help differentiate it from a reactive process [
      • Nimieri H.S.
      • Makoni S.N.
      • Madziwa F.H.
      • Nemiary D.S.
      Leukemoid reaction response to chemotherapy and radiotherapy in a patient with cervical carcinoma.
      ]. In CML, basophilia, eosinophilia, monocytosis, or even a minimum percentage of blasts and reticulin fibrosis are characteristic features.

      3.5 Cytogenetic testing and molecular analysis of peripheral blood and bone marrow granulocytes

      The presence of Philadelphia 1 chromosome in the karyotype or the detection of t(9;22) translocation (creating the bcr/abl oncogene) by molecular techniques is the hallmark for CML and excludes CNL. Still, in less than 10%, an atypical myeloproliferative disorder may be present. In such a case, the erythroid and megacaryocytic series may be involved. In a review, cytogenetic abnormalities occurred in 37% of cases of CNL, the most frequent of which appeared to be involvement of chromosome 20, whereas the majority of the cases recorded a normal karyotype [
      • Reilly J.T.
      Chronic neutrophilic leukaemia: a distinct clinical entity?.
      ]. No karyotypic abnormalities are expected in LRs.

      3.6 Immunophenotyping of peripheral blood and bone marrow

      Immunophenotyping has not been validated as a differential diagnostic tool in LRs, but it may exclude the presence of AML or the progression of CML to a blast crisis [
      • Chianese R.
      • Brando B.
      • Gratama J.W.
      European Working Group on Clinical Cell Analysis. Diagnostic and prognostic value of flow cytometric immunophenotyping in malignant hematological diseases.
      ]. It should be stressed, however, that acute leukemias usually present completely differently and the presence of blasts above 20% is what is characteristic of these conditions compared to the other entities discussed. The immunophenotype shows characteristic findings in leukemias. Gating should comprise granulocytes in FS/SS scattergrams of peripheral blood and granulocytes in SS/CD45 scattergrams of bone marrow. Mature neutrophils express surface antigens CD13 and CD15 at a level higher than 95% while, on the other hand, they are absolutely negative for CD34 and HLA-DR. Expression of CD34 would seem to imply the presence of acute leukemia or of a myelodysplastic syndrome. Cells in CML stain may stain positive for HLA-DR, a finding not observed in LR.

      3.7 Serum levels of hematopoietic growth factors

      Although G-CSF, GM-CSF, and IL-6 are not included in the routine diagnostic work-up, their determination by an enzyme-linked immunosorbent assay (ELISA) have been used by several physicians to demonstrate an association between a cytokine-producing tumor and the development of an LR [
      • Kasuga I.
      • Makino S.
      • Kiyokawa H.
      • Katoh H.
      • Ebihara Y.
      • Ohyashiki K.
      Tumor-related leukocytosis is linked with poor prognosis in patients with lung carcinoma.
      ,
      • Schniewind B.
      • Christgen M.
      • Hauschild A.
      • Kurdow R.
      • Kalthoff H.
      • Klomp H.J.
      Paraneoplastic leukemoid reaction and rapid progression in a patient with malignant melanoma: establishment of KT293, a novel G-CSF-secreting melanoma cell line.
      ,
      • Horii A.
      • Shimamura K.
      • Honjo Y.
      • Mitani K.
      • Miki T.
      • Takashima S.
      • et al.
      Granulocyte colony-stimulating factor-producing tongue carcinoma.
      ,
      • Endo K.
      • Kohnoe S.
      • Okamura T.
      • Haraguchi M.
      • Adachi E.
      • Toh Y.
      • et al.
      Gastric adenosquamous carcinoma producing granulocyte-colony stimulating factor.
      ,
      • Tachibana M.
      • Murai M.
      G-CSF production in human bladder cancer and its ability to promote autocrine growth: a review.
      ,
      • Watanabe M.
      • Ono K.
      • Ozeki Y.
      • Tanaka S.
      • Aida S.
      • Okuno Y.
      Production of granulocyte-macrophage colony-stimulating factor in a patient with metastatic chest wall large cell carcinoma.
      ]. These glycoproteins are normally secreted by monocytes, macrophages, endothelial cells, and fibroblasts. Their functions include regulation of the growth and differentiation of hematopoietic progenitor cells and functional activation of mature neutrophils and macrophages [
      • Nimieri H.S.
      • Makoni S.N.
      • Madziwa F.H.
      • Nemiary D.S.
      Leukemoid reaction response to chemotherapy and radiotherapy in a patient with cervical carcinoma.
      ]. LR due to a GM-CSF-producing tumor is characterized by marked eosinophilia [
      • Watanabe M.
      • Ono K.
      • Ozeki Y.
      • Tanaka S.
      • Aida S.
      • Okuno Y.
      Production of granulocyte-macrophage colony-stimulating factor in a patient with metastatic chest wall large cell carcinoma.
      ]. CML and CNL patients have significantly low G-CSF levels, suggesting that the neoplastic granulopoiesis can exert a suppressor effect on G-CSF synthesis [
      • Reilly J.T.
      Chronic neutrophilic leukaemia: a distinct clinical entity?.
      ].

      3.8 Human androgen receptor gene assay (HUMARA)

      Bohm et al. have studied this method for the analysis of clonality in tissues in female patients. The assay examines the inactivation patterns of the human androgen receptor gene on the X chromosome. In myeloproliferative syndromes, including CML and CNL, the neutrophils show monoclonality, whereas in reactive situations there is a polyclonal pattern [
      • Bohm J.
      • Kock S.
      • Schaefer H.E.
      • Fisch P.
      Evidence of clonality in chronic neutrophilic leukaemia.
      ].

      3.9 Cultures

      Searching for an underlying infection includes taking cultures of several fluids and tissues, especially blood, sputum, and bone marrow for common bacteria and mycobacteria. Stool cultures should not be overlooked since colitis due to Shigella spp. or C. difficile is one of the classic examples of an infection-associated LR.

      3.10 Imaging studies and biopsies

      Ultrasound and computerized tomographic (CT) scans or magnetic resonance images (MRI) of the chest and abdomen should be performed in order to reveal an occult infection or malignancy. As soon as a solid tumor is diagnosed, a biopsy should be accompanied by special immunohistochemical staining to detect the possible expression of G- or GM-CSF receptors that will confirm a cytokine-producing tumor.

      4. Conclusions

      A LR is a rare condition that can be challenging and that may require a careful diagnostic work-up. The diagnosis is verified by a combination of the following: (i) a complete blood count with a peripheral blood smear that shows marked mature neutrophilia with a left shift; (ii) a high leukocyte alkaline phosphatase (LAP) score; (iii) hypercellular bone marrow with intact maturation and morphology of all the elements; (iv) the absence of cytogenetic abnormalities by cytogenetic-molecular studies; (v) a mature granulocyte pattern by immunophenotyping of peripheral blood and bone marrow; (vi) a high serum level of hemopoietic growth factors, in the case of a cytokine-producing tumor; and (vii) a polyclonal pattern of blood neutrophils in clonality studies.
      The most important tool in discriminating between CML and LR is molecular diagnostics and the absence of t(9;22) translocation (creating the bcr/abl oncogene). The diagnostic evaluation is completed with the revelation of the underlying disease, which will help in planning the optimal therapeutic strategy. Yet, the approach presented in the current review is mainly considered an expert opinion and should be further evaluated in everyday clinical practice and, possibly, in carefully designed research protocols.
      Learning points
      1. A leukemoid reaction (LR) is defined by a leukocyte count greater than 50,000 cells/μL.
      2. By definition, it is diagnosed by the exclusion of a malignant hematological disorder, CML or CNL.
      3. The major causes of LRs are severe infections, malignancies, intoxication, and severe hemorrhage or acute hemolysis.
      4. Careful history, good physical examination, and limited imaging studies may assist in revealing the underlying cause.
      5. Diagnostic characteristics:
      a. marked mature neutrophilia with a left shift
      b. a high leukocyte alkaline phosphatase (LAP) score
      c. hypercellular bone marrow with intact maturation and morphology of all the elements
      d. the absence of cytogenetic abnormalities in cytogenetic-molecular studies
      e. a mature granulocyte pattern by immunophenotyping of peripheral blood and bone marrow
      f. a high serum level of hemopoietic growth factors in the case of a cytokine-producing tumor
      g. a polyclonal pattern of blood neutrophils in clonality studies.

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