Leukocytosis

Neutrophilia

The most common type of leukocytosis is neutrophilia (an increase in the absolute number of mature neutrophils to greater than 7,000 per mm³, which can arise from infections, stressful conditions, chronic inflammation, medication use, and other causes.

• Infection
Fever, system-specific symptoms
DISTINGUISHING FEATURES:
Physical examination findings
EVALUATION:
Obtain system-specific cultures and imaging (e.g., sputum cultures, chest radiography)
Consider empiric antibiotics
Consider use of other biomarkers, such as CRP and procalcitonin

• Reactive neutrophilia
DISTINGUISHING FEATURES:
Exercise, physical stress (e.g., postsurgical, febrile seizures), emotional stress (e.g., panic attacks), smoking
EVALUATION:
Confirm with history

• Chronic inflammation
DISTINGUISHING FEATURES:
Rheumatic disease, inflammatory bowel disease, granulomatous disease, vasculitides, chronic hepatitis
EVALUATION:
Obtain personal and family medical history
Consider erythrocyte sedimentation rate and CRP levels, specific rheumatology laboratories
Consider subspecialist consultation (e.g., rheumatology, gastroenterology)

• Medication induced
DISTINGUISHING FEATURES:
Corticosteroids, beta agonists, lithium, epinephrine, colony-stimulating factors
EVALUATION:
Confirm with history; consider discontinuation of medication, if warranted

• Bone marrow stimulation
DISTINGUISHING FEATURES:
Hemolytic anemia, immune thrombocytopenia, bone marrow suppression recovery, colony-stimulating factors
EVALUATION:
Complete blood count differential; compare with baseline values (if available)
Examine peripheral smear
Consider reticulocyte and lactate dehydrogenase levels
Consider flow cytometry, bone marrow examination, hematology/oncology consultation

• Splenectomy
DISTINGUISHING FEATURES:
History of trauma or sickle cell disease
EVALUATION:
Confirm with history

• Congenital
DISTINGUISHING FEATURES:
Hereditary/chronic idiopathic neutrophilia, Down syndrome, leukocyte adhesion deficiency
EVALUATION:
Obtain family, developmental history
Consider hematology/oncology, genetics, and immunology consultations

Hyperviscosity syndrome

Very rarely, in people with leukemia, extremely high levels of immature neutrophils (more than 100,000 cells per microliter of blood) can cause the blood to become too thick and cause breathing problems, stroke, and death.
This condition is a medical emergency and requires hospitalization so fluids can be given by vein and drugs to reduce the white blood cell count (hydroxyurea and chemotherapy drugs) can be given.
Sometimes, a type of blood-filtering treatment (leukapheresis) is used to remove the white blood cells from the blood.

Neutrophilia

Neutrophils may increase in response to a number of conditions or disorders, including
• Infections
• Injuries
• Inflammatory disorders
• Certain drugs
• Certain leukemias

The most common cause of an increased number of neutrophils is
• The normal response of the body to an infection
In many instances, the increased number of neutrophils is a necessary reaction by the body, as it tries to heal or ward off an invading microorganism or foreign substance. Infections by bacteria, viruses, fungi, and parasites may all increase the number of neutrophils in the blood.

• The number of neutrophils may rise in people who have an injury, such as a hip fracture or burn.
• Inflammatory disorders, including autoimmune disorders such as rheumatoid arthritis, can cause an increase in the number and activity of neutrophils.
• Some drugs, such as corticosteroids, also lead to an increased number of neutrophils in the blood.
• Myelogenous leukemias can lead to an increased number of immature or mature neutrophils in the blood.

A high number of neutrophils does not cause symptoms. However, people often have symptoms of the disorder that is causing the increased number of neutrophils.

Lymphocytosis

CAUSES:
Infections (viral, pertussis)
Hypersensitivity reaction
Acute or chronic leukemia

Lymphocytosis is more likely to be benign in children than in adults.

Monocytosis

Monocytes make up more than 8% of the WBC count or the absolute count is greater than 880 per mm³.

CAUSES:
Infections: Epstein-Barr virus infection, tuberculosis or fungal disease, splenectomy, protozoan or rickettsial infections
Autoimmune disease
Malignancy
Splenectomy

Eosinophilia

Eosinophilia (eosinophil absolute count greater than 500 per mm³, although uncommon, may suggest allergic conditions such as asthma, urticaria, atopic dermatitis or eosinophilic esophagitis, drug reactions, dermatologic conditions, malignancies, connective tissue disease, idiopathic hypereosinophilic syndrome, or parasitic infections, including helminths (tissue parasites more than gut-lumen parasites).

CAUSES:
Allergic conditions
Dermatologic conditions
Eosinophilic esophagitis
Idiopathic hypereosinophilic syndrome
Malignancies
Medication reactions
Parasitic infections

Basophilia

Isolated basophilia (number of basophils greater than 100 per mm³ is rare and unlikely to cause leukocytosis in isolation, but it can occur with allergic or inflammatory conditions and chronic myelogenous leukemia.

CAUSES:
Allergic conditions
Leukemias

Leukocytosis

Nonmalignant Etiologies
A reactive leukocytosis, typically in the range of 11,000-30,000/mm³, can arise from a variety of etiologies.

Any source of stress can cause a catecholamine-induced demargination of WBCs, as well as increased release from the bone marrow storage pool.
Examples include surgery, exercise, trauma, burns, and emotional stress.
One study showed an average increase in WBCs of 2,770 per mm³ peaking on postoperative day 2 after knee or hip arthroplasty.

During the recovery phase after hemorrhage or hemolysis, a rebound leukocytosis can occur.

Medications known to increase the WBC count include corticosteroids, lithium, colony-stimulating factors, beta agonists, and epinephrine.

Leukocytosis is one of the hallmarks of infection.
In the acute stage of many bacterial infections, there are primarily mature and immature neutrophils.
The release of less-mature bands and metamyelocytes into the peripheral circulation results in the so-called “left shift” in the WBC differential.
Of note, some bacterial infections paradoxically cause neutropenias, such as typhoid fever, rickettsial infections, brucellosis, and dengue.
Sometimes, as the infection progresses, there is a shift to lymphocyte predominance.

Viral infections may cause leukocytosis early in their course, but a sustained leukocytosis is not typical, except for the lymphocytosis in some childhood viral infections.

An elevated WBC count is a suggestive, but not definitive, marker of the presence of significant infection.
For example, the sensitivity and specificity of an elevated WBC count in diagnosing acute appendicitis are 62% and 75%, respectively.
For diagnosing serious bacterial infections without a source in febrile children, the discriminatory value of leukocytosis is less than that of other biomarkers, such as C-reactive protein or procalcitonin.
Although a WBC count greater than 12,000 per mm³ is one of the criteria for the systemic inflammatory response syndrome (or sepsis when there is a known infection), leukocytosis alone is a poor predictor of bacteremia and not an indication for obtaining blood cultures.

Other acquired causes of leukocytosis include functional asplenia (predominantly lymphocytosis), smoking, and obesity.
Patients with a chronic inflammatory condition, such as rheumatoid arthritis, inflammatory bowel disease, or a granulomatous disease, may also exhibit leukocytosis.
Genetic causes include hereditary or chronic idiopathic neutrophilia and Down syndrome.

Leukocytosis

Malignant Etiologies
Leukocytosis may herald a malignant disorder, such as an acute or chronic leukemia, or a myeloproliferative disorder, such as polycythemia vera, myelofibrosis, or essential thrombocytosis.

A previous article on leukemia in American Family Physician reviewed the features and differentiation of malignant hematopoietic disorders.
Chronic leukemias are most commonly diagnosed after incidental findings of leukocytosis on complete blood counts in asymptomatic patients. Patients with features suggestive of hematologic malignancies require prompt referral to a hematologist/oncologist (Table 5).

Many solid tumors may lead to a leukocytosis in the leukemoid range, either through bone marrow involvement or production of granulocyte colony-stimulating or granulocyte-macrophage colony-stimulating factors.

Table 5: Findings Suggestive of Hematologic Malignancies in the Setting of Leukocytosis

• Symptoms
Fatigue, weakness
Fever > 100.4°F (38°C)
Night sweats
Unintentional weight loss
Bruising/bleeding tendency
Immunosuppression

• Physical examination findings
Lymphadenopathy
Splenomegaly or hepatomegaly
Petechiae

• Laboratory abnormalities
Anemia
White blood cell count > 30,000 per mm³, or > 20,000 per mm³ after initial management
Increased or decreased platelet count
Predominantly immature cells on peripheral smear
Monomorphic lymphocytosis on peripheral smear

Evaluation of Leukocytosis

Figure 5: Evaluation of Leukocytosis

Evaluation of Leukocytosis

Approach to Evaluation
A systematic approach to patients with leukocytosis includes identifying historical clues that suggest potential causes (Figure 5).
Fever and pain may accompany infections or malignancies; other constitutional symptoms, such as fatigue, night sweats, weight loss, easy bruising, or bleeding, might suggest malignancy.
Previous diagnoses or comorbid conditions that cause chronic inflammation should be noted, as well as recent stressful events, medication use, smoking status, and history of splenectomy or sickle cell anemia.
A history of an elevated WBC count is important, because duration will help determine the likely cause. Leukocytosis lasting hours to days has a different differential diagnosis (e.g., infections, acute leukemias, stress reactions) than a case that persists for weeks to months (e.g., chronic inflammation, some malignancies).

The physical examination should note erythema, swelling, or lung findings suggestive of an infection; murmurs suggestive of infective endocarditis; lymphadenopathy suggestive of a lymphoproliferative disorder; or splenomegaly suggestive of chronic myelogenous leukemia or a myeloproliferative disorder; petechiae or ecchymoses; or painful, inflamed joints suggestive of connective tissue disease or infection.

Initial laboratory evaluation should include`a repeat complete blood count to confirm the elevated WBC level, with differential cell counts and a review of a peripheral blood smear. The peripheral smear should be examined for toxic granulations (suggestive of inflammation), platelet clumps (which may be misinterpreted as WBCs), the presence of immature cells, and uniformity of the WBCs. On evaluation of a leukocytosis with lymphocyte predominance, a monomorphic population is concerning for chronic lymphocytic leukemia, whereas a pleomorphic (varying sizes and shapes) lymphocytosis is suggestive of a reactive process. With all forms of leukocytosis, concurrent abnormalities in other cell counts (erythrocytes or platelets) suggest a primary bone marrow process and should prompt hematology/oncology evaluation.

As indicated by the history and examination findings, physicians should consider performing cultures of blood, urine, and joint or body fluid aspirates; rheumatology studies; a test for heterophile antibodies (mononucleosis spot test); and serologic titers. Radiologic studies may include chest radiography (to identify infections, some malignancies, and some granulomatous diseases) and, as indicated by history, computed tomography or bone scan. If hematologic malignancy is suspected, additional confirmatory testing may include flow cytometry, cytogenetic testing, or molecular testing of the bone marrow or peripheral blood.

Malignant or benign leukocytosis

Abstract
Leukocytosis is a commonly encountered laboratory finding. Distinguishing malignant from benign leukocytosis is a critical step in the care of a patient, which initiates a vastly different decision tree.
Confirmation of the complete blood cell count and the WBC differential is the first step.
Examination of the PB smear is essential to confirming the automated blood cell differential or affirming the manual differential performed on the PB smear.
Next is separation of the leukocytosis into a myeloid versus a lymphoid process.
Distinguishing a reactive lymphoid proliferation from a lymphoproliferative disorder requires examination of lymphocyte morphology for pleomorphic lymphocytes versus a monomorphic population, with the latter favoring a lymphoproliferative neoplasm.
Samples suspicious for lymphoproliferative disorders can be confirmed and characterized by flow cytometry, with molecular studies initiated in select cases; precursor lymphoid neoplasms (lymphoblasts) should trigger a BM examination.
Myeloid leukocytosis triggers a differential diagnosis of myeloid leukemoid reactions versus myeloid malignancies. The manual differential is key, along with correct enumeration of blasts and blast equivalents, immature granulocytes, basophils, and eosinophils and identifying dysplasia to identify myeloid malignancies.
Confirmation and characterization of myeloid malignancies should be performed with a BM examination and the appropriate ancillary studies.
Myeloid leukemoid reactions commonly result from infections and show activated neutrophil changes on morphology; these should prompt evaluation for infection.
Other causes of reactive myeloid leukocytoses are also discussed herein.

Diagnostic algorithm for the workup of leukocytosis.

Diagnostic algorithm for the workup of leukocytosis.
• If increased blasts are present, then evaluation for an acute leukemia or precursor neoplasm should begin with BM examination including appropriate ancillary studies.
• If myeloid cells are present, the leukocytosis should be stratified into neutrophilia, monocytosis, basophilia, or eosinophilia; more than one type of leukocytosis may be present.
• Neutrophilia should prompt examination for left shift, signs of activated neutrophils, basophilia, dysplasia, and degree of leukocytosis. Most neutrophilias are reactive in nature. A WBC count < 50 × 109/L, but usually < 30 × 109/L, is typical. Signs of activated neutrophils, mild left shift, and an absence of basophilia all suggest a reactive process. A marked leukocytosis of > 50 × 109/L, marked left shift, dysplasia, or basophilia should prompt a BM examination to evaluate for a myeloid malignancy.
• Most monocytoses are reactive in nature. However, if reactive causes have been excluded, a persistent monocytosis of more than 3 months or the findings of dysplasia, blast cells, or significant left shift should trigger a BM examination to evaluate for malignancy.
• Most eosinophilias are reactive in nature and these should be evaluated as outlined by Gotlib.30 Once reactive eosinophilias are excluded, myeloid and lymphoid neoplasms with eosinophilia and PDGFRA, PDGFRB, and FGFR1 should be searched for by performing a BM examination, cytogenetic studies, and FISH or PCR for the PDGFRA mutation.
• Basophilia, although rare, is most suggestive of a MPN, especially CML. PCR for BCR-ABL1 and JAK2 mutational studies can be performed in blood, but a BM examination with cytogenetic studies should also be performed.
• If a lymphocytosis is present, the lymphoid cells should be examined for pleomorphism or monomorphism.
A pleomorphic lymphocytosis favors a reactive lymphocytosis. Correlation with clinical findings is necessary; a monospot test for EBV or viral serologies can also be performed.
If monomorphic lymphocytosis is present, a lymphoproliferative disorder should be searched for using flow cytometric immunophenotyping. Depending on these results, select molecular genetic tests will be helpful. A BM biopsy or extramedullary tissue biopsy may be necessary for a final diagnosis of lymphoma.

Note: MPN, myeloproliferative neoplasm; MPN eos, myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1; and FL, follicular lymphoma

Introduction
Leukocytosis is defined as an elevation of the WBC count for the patient's age. Using an appropriate reference interval is critical, because a WBC count of 30 × 109/L is considered elevated in an adult, but completely normal in the first few days of life.
Similarly, the normal WBC differential also changes with age, with greater absolute numbers of neutrophils and lymphocytes seen in newborns and the normal “elevation” of lymphocytes persisting throughout childhood.
Hospital laboratories are required to validate reference ranges in their respective patient populations as part of the validation process of automated hematology instruments.

Automated differentials
The newer generation of hematology analyzers can examine thousands of leukocytes using flow cytometry–based methodology, some in combination with cytochemistry or fluorescence or conductivity, to elucidate different types of WBCs, including neutrophils, lymphocytes, monocytes, basophils, and eosinophils (ie, the 5-part differential). Nucleated RBCs are also detected routinely, and some analyzers also quantitate immature granulocytes.

Older analyzers: Spurious elevations of the WBC count can also be seen, including platelet clumps, nucleated RBCs, incomplete lysis of RBCs, cryoglobulins, and cryofibrinogen.

Examination of the PB smear
Beginning at low power, the slide should be examined to determine abnormalities in cell number, cell type, and aggregation.
In manual prepared smears, larger WBCs tend to collect at the edges of the smear.
Good practice for slide review requires assessment of leukocytes, erythrocytes, and platelets in both quantity and quality, correlating with automated cell counts.
For RBCs, the blood film is examined in the area of the smear called the “feathered” edge, where the erythrocytes are barely overlapping or just touching.
WBCs may be viewed in this area or in slightly thicker areas of the smear.

Newer techniques for leukocyte classification include digital microscopy, in which computer algorithms classify cells and more leukocytes can be examined.

With regard to leukocytes specifically, neoplastic cells such as blasts and the lymphoid cells of lymphoproliferative disorders such as chronic lymphocytic leukemia (CLL) are extremely fragile and crush easily with mechanical pressure, resulting in increased numbers of “smudge” or “basket” cells.

Leukocytes may also undergo apoptosis and other changes in morphology when aged samples are used, so smears must be prepared promptly from fresh specimens.

Workup of leukocytosis

If increased blasts are present, then evaluation for an acute leukemia or precursor neoplasm should begin with BM examination including appropriate ancillary studies.

Myeloid lineage cells: neutrophilia, monocytosis, basophilia, or eosinophilia; more than one type of leukocytosis may be present.
• Neutrophilia should prompt examination for left shift, signs of activated neutrophils, basophilia, dysplasia, and degree of leukocytosis. Most neutrophilias are reactive in nature. A WBC count < 50 × 109/L, but usually < 30 × 109/L, is typical. Signs of activated neutrophils, mild left shift, and an absence of basophilia all suggest a reactive process. A marked leukocytosis of > 50 × 109/L, marked left shift, dysplasia, or basophilia should prompt a BM examination to evaluate for a myeloid malignancy.
Neutrophils include segmented and band forms. Bands constitute approximately 5%-10% of the nucleated cells in the blood under normal conditions; increased bands may be seen in certain pathologic states. However, with blood smear method, the interobserver reproducibility of the band count is poor, rendering the band count of questionable value in most patients.
• Most monocytoses are reactive in nature. However, if reactive causes have been excluded, a persistent monocytosis of more than 3 months or the findings of dysplasia, blast cells, or significant left shift should trigger a BM examination to evaluate for malignancy.
• Most eosinophilias are reactive in nature and these should be evaluated as outlined by Gotlib.30 Once reactive eosinophilias are excluded, myeloid and lymphoid neoplasms with eosinophilia and PDGFRA, PDGFRB, and FGFR1 should be searched for by performing a BM examination, cytogenetic studies, and FISH or PCR for the PDGFRA mutation.
• Basophilia, although rare, is most suggestive of a MPN, especially CML. PCR for BCR-ABL1 and JAK2 mutational studies can be performed in blood, but a BM examination with cytogenetic studies should also be performed.

Lymphocytosis
Blood smear: the lymphoid cells should be examined for pleomorphism or monomorphism.
A pleomorphic lymphocytosis favors a reactive lymphocytosis. Correlation with clinical findings is necessary; a monospot test for EBV or viral serologies can also be performed.
If monomorphic lymphocytosis is present, a lymphoproliferative disorder should be searched for using flow cytometric immunophenotyping. Depending on these results, select molecular genetic tests will be helpful.
A BM biopsy or extramedullary tissue biopsy may be necessary for a final diagnosis of lymphoma.

Myeloid leukocytosis

Myeloid leukocytosis may represent granulocytosis (ie, neutrophilia, eosinophilia, and basophilia) or monocytosis.

Neutrophilia
Neutrophilia is defined as an elevated circulating neutrophil count ( >7.7 × 109/L in adults). The absolute neutrophil count can be determined by multiplying the total WBC count by the percentage of polymorphonuclear cells (segmented neutrophils) and band forms.
Reactive neutrophilia (neutrophilic leukemoid reaction) results from an increased production of neutrophils, demargination (process of neutrophils entering the peripheral circulation from areas of intravascular marginated neutrophil pools), or a decreased egress of neutrophils from the peripheral circulation to the tissues.
Neutrophilia is seen commonly with infections (bacterial, viral, mycobacterial, or treponemal), but any strong stimulus to the BM can trigger this reaction.
Causes of neutrophilia include malignancy, inflammation, drugs (eg, glucocorticoids or lithium), myeloid growth factors, hemorrhage, and splenectomy.
Neutrophilic leukemoid reactions show morphologic overlap with chronic myelogenous leukemia (CML), acute myeloid leukemia, chronic neutrophilic leukemia, and myelodysplastic/myeloproliferative neoplasms. Whereas a neutrophilic leukemoid reaction rarely has a WBC count > 50 × 109/L, CML may far exceed that value; however, a lower WBC does not preclude a consideration of CML.
Immature granulocytes (ie, promyelocytes, myelocytes, and metamyelocytes), which are indicative of a left shift in the PB, may be seen with either reactive neutrophilias or myeloid neoplasms such as CML.
A more pronounced left shift is typical of CML accompanied by absolute increases in the numbers of basophils and eosinophils, but a leukemoid reaction must be excluded (Figure 3).

Figure 3: Myeloid leukocytosis

(A) Neutrophilia in a patient with a perforated tumor and infection.
(B) G-CSF effect with immature granulocytes.
(C) Neutrophils with toxic granulation and vacuoles are seen in a patient with a bacterial infection.
(D) CML, BCR-ABL1+, chronic phase, shows many immature granulocytes and occasional blasts.
(E) CMML with abnormal monocytes and a large hypogranular platelet.
(F) Myeloid neoplasm with eosinophilia and PDGFRA shows 2 eosinophils with eosinophilic granules that do not completely fill the cytoplasm and occasional hypogranular platelets.

FISH or molecular studies for BCL-ABL1 can be performed in PB to confirm the diagnosis of CML, although BM examination is recommended in new diagnoses with accompanying metaphase-based cytogenetic studies.

Activated neutrophils (eg, toxic granulation, Dohle bodies, and cytoplasmic vacuoles) are more characteristic of a reactive neutrophilia such as that seen in patients with bacterial infections or those receiving growth factors.
Cytoplasmic vacuoles alone must be interpreted with caution because this can also be seen in old samples.
Other features favoring a reactive neutrophilia include circulating fragments of neutrophil cytoplasm, thrombocytosis, and a lack of basophilia; eosinophilia and monocytosis.

If the left shift is accompanied by nucleated RBCs, a leukoerythroblastic smear may suggest a BM-infiltrative process such as tumor or fibrosis. In the latter process, this is typically accompanied by increased numbers of dacrocytes (teardrop-shaped cells).
Leukoerythroblastic smears can also be seen in patients receiving growth factors.

Monocytosis
An absolute monocytosis is defined as >1 × 109/L monocytes.
A reactive monocytosis may be seen with malignancy (ie, carcinoma, plasma cell myeloma, or lymphoma), chronic infections, autoimmune disorders, and splenectomy.
It is also seen in a regenerating BM such as after BM transplantation or chemotherapy.
The persistence of an absolute monocytosis should prompt examination of the BM with flow cytometric immunophenotyping and cytogenetic studies, because persistent monocytosis raises a differential diagnosis, including chronic myelomonocytic leukemia (CMML), acute monoblastic/monocytic leukemia, CML, juvenile myelomonocytic leukemia, atypical (BCR-ABL negative) CML, and myelodysplastic/myeloproliferative neoplasms, unclassifiable.
Molecular genetic testing, although not now routinely available, may be used in the future to help distinguish the various MPNs/MDSs (eg, PTPN11, RAS, NF1, TET2, and RUNX1).
It is not uncommon for patients with acute monoblastic/monocytic leukemia to present with more differentiated monocytes in the PB, so such cases of “CMML” should always be confirmed with BM examination. CML, CMML, and atypical CML can be distinguished as shown in Table 4 (and Figure 3).

Table 4: PB examination in the differential diagnosis of CMML, CML, and atypical CML

The WBC count is higher in CML than either CMML or atypical CML, including WBCs greater than 200 × 109/L.
The percentage of monocytes is generally greater than 10% in CMML, higher than in either CML or atypical CML.
CML shows a prominent basophilia compared with CMML and atypical CML.
Large numbers of immature granulocytes are typical of CML, with fewer numbers in atypical CML and even less in CMML.
Greater numbers of circulating blasts are seen in atypical CML than in either CMML or chronic-phase CML.
Finally, atypical CML has the most granulocytic dysplasia, which is generally absent in CML and may be present or absent in CMML.

Eosinophilia
Most eosinophilias, typically defined as >1.5 × 109/L eosinophils, are reactive in nature and should be investigated for a reactive cause of eosinophilia; these include: infections (especially parasites), allergy/hypersensitivity diseases, connective tissue diseases, pulmonary diseases, cardiac diseases, dermatologic disorders, malignancy (eg, lymphomas including Hodgkin lymphoma and carcinomas), gastrointestinal disease, and adrenal insufficiency.

Once a reactive cause is excluded, investigations into primary eosinophilias should be performed, including BM examination, cytogenetic studies, and FISH or PCR studies for the FIP1L1-PDGFRA fusion gene (this is a cryptic deletion not seen by standard cytogenetic karyotyping) (Figure 3).
Cytogenetic studies will detect abnormalities indicative of PDGFRB and FGFR1. Immunohistochemistry can be performed on the trephine biopsy to identify mast cell proliferations that may be seen in those myeloid neoplasms with eosinophilia. Those myeloid neoplasms with eosinophilia associated with PDGFRA mutations are sensitive to imatinib mesylate.
Both hypereosinophilic syndrome and chronic eosinophilic leukemia, not otherwise specified will have an eosinophilia ≥1.5 × 109/L; clonality or increased blasts indicates chronic eosinophilic leukemia once the myeloid neoplasms with eosinophilia and PDGFRA, PDGFRB, and FGFR1 are excluded.
An absence of clonality, no increase in blasts, no evidence for a reactive cause of eosinophilia, and a persistent eosinophilia for at least 6 months with organ involvement and dysfunction are diagnostic of hypereosinophilic syndrome.
T-cell phenotyping and T-cell clonality studies are also recommended in the workup of eosinophilia to identify the lymphocyte-variant hypereosinophilia.

Basophilia
Basophilia, defined as >0.3 × 109/L in adults, is extremely uncommon and, if confirmed, should prompt a BM examination given its strong association with myeloproliferative neoplasms (although rare reactive basophilias have been reported).
Distinguishing basophils from mast cells may also be difficult.

Conclusion
In conclusion, leukocytosis in a patient should prompt confirmation of the CBC and WBC differential. Examination of the blood smear should be performed to establish a manual differential or to confirm the automated differential. This will allow the distinction of myeloid from lymphoid disorders. Distinguishing myeloid leukemoid reactions from myeloid malignancies is difficult, with features such as dysplasia, basophilia, WBC count > 50 × 109/L, a pronounced left shift, and increased blasts favoring a myeloid malignancy with recommended BM examination and appropriate ancillary testing. Myeloid leukemoid reactions may be seen with a variety of stimuli, but markers of infection such as activated neutrophils can be helpful features to look for in conjunction with the appropriate laboratory testing. With respect to lymphocytoses, pleomorphic lymphocytosis in the appropriate clinical context favors a reactive lymphocytosis, whereas a homogenous population of lymphoid cells favors a lymphoproliferative disorder. Flow cytometric immunophenotyping is typically recommended for further classification of lymphoproliferative disorders, with molecular genetic testing as appropriate. Regardless of whether a myeloid or lymphoid leukocytosis is favored, the presence of increased blasts should prompt BM examination with appropriate ancillary studies.
An algorithmic approach as shown in Figure 4 may be helpful. Specific details for evaluation of eosinophilia are best addressed by Gotlib.

Malignant Etiologies

Blast cells --- Blood Smear
Distinguishing a myeloid process from a lymphoid process is essential.
Morphologic features alone are unreliable in distinguishing blast type, so immunophenotypic studies are necessary to distinguish myeloid from lymphoid blasts.
Such patients should have a BM examination with tissue obtained for aspirate smears, trephine biopsy, flow cytometric immunophenotyping, and cytogenetic and molecular genetic studies to classify acute leukemias/precursor neoplasms properly according to the World Health Organization classification.
In some cases, cytochemical stains may also be useful.
If an adequate aspirate smear is unable to be performed (a “dry tap”), touch preparations should be prepared from the fresh trephine biopsy and additional fresh BM cores should be sent for flow cytometric phenotyping and cytogenetic/molecular genetic studies.

A sample containing blast cells requires careful examination for features suggesting a myeloid or lymphoid lineage.
Auer rods or granules suggest a myeloid lineage, although lymphoblasts with granules may also be seen.
Additional blast features such as large salmon-colored granules, prominent hofs, butterfly-shaped nuclei, and background dysplasia may suggest a certain type of acute myeloid leukemia.
Acute promyelocytic leukemia (APL)
In particular, acute promyelocytic leukemia (APL) shows a characteristic morphology often accompanied by changes of microangiopathic hemolytic anemia (eg, schistocytes), because these patients may present with bleeding due to disseminated intravascular coagulopathy.
• The more common hypergranular variant of APL shows hypergranular blasts with many fine cytoplasmic granules and Auer rods; bundles of Auer rods may be seen in a small percentage of cells.
• The hypogranular variant of APL on morphology shows agranular blasts, but will show the typical nuclear features of APL with folded nuclei.
Flow cytometry typically shows a characteristic phenotype with lack of expression of HLA-DR and usually a lack of CD34.
Rapid FISH or RT-PCR testing for the PML-RARA fusion gene is typically performed.

Left: The hypergranular variant of APL shows hypergranular blasts with many fine cytoplasmic granules and Auer rods
Right: The hypogranular variant of APL on morphology shows agranular blasts, but will show the typical nuclear features of APL with folded nuclei

Left: Acute myeloid leukemia with t(8;21)(q22;q22); RUNX1-RUNX1T1 with a small myeloblast containing a thin, delicate Auer rod.
Middle: Recurrent acute myeloid leukemia with associated microangiopathic hemolysis (schistocytes). The 2 myeloblasts contain partially condensed chromatin and sparsely granular cytoplasm, features sometimes found in blasts seen in myeloid neoplasms with myelodysplasia.
Right: Acute monoblastic leukemia with abundant lightly granular cytoplasm containing vacuoles.

Left: Precursor B-lymphoblastic leukemia with numerous blasts and smudge cells and with 2 large lymphoblasts.
Right: Precursor T-lymphoblastic leukemia with a lymphoblast containing a moderate amount of basophilic cytoplasm.

Lymphoid leukocytosis

Reactive lymphocytosis
Lymphocytosis is defined as an increase in the absolute lymphocyte count above that expected in an individual of the same age. Absolute lymphocyte counts are higher in children and infants compared with adults, so the appropriate reference intervals must be used. In adults, an absolute lymphocyte count of > 3.5 × 109/L can be considered lymphocytosis.

Distinguishing reactive lymphocytes from lymphoma cells can be challenging, but several key features should be kept in mind.
First, the age of the patient helps to focus the differential diagnosis. Many of the lymphomas involving the PB are much more common in middle-aged to elderly adults than in children or infants. Certain lymphomas (ie, Burkitt lymphoma) are seen more commonly in children.
Second, accurate clinical history can be critical, because any prior diagnosis of lymphoma can aid in the identification of these cells.
Third, a well-prepared and stained PB smear is important. Old blood and poorly prepared samples can lead to misdiagnoses.

When looking at the peripheral smear, a scan should be performed at 10× examining the overall slide before going to higher magnification.
Reactive lymphocytoses will show a wide range of cellular sizes and shapes.
The classic example of reactive lymphocytosis is infectious mononucleosis (eg, EBV infection), in which a variety of lymphocytes are seen, ranging from small lymphocytes with round nuclei and condensed chromatin to reactive lymphocytes with an abundant pale blue cytoplasm, round to oval nuclei, and moderately condensed chromatin (Figure 2).
The cytoplasm of these reactive lymphocytes may “hug” adjacent RBCs and show a basophilic rim at their margins.
Immunoblasts are often present, seen as large lymphocytes with round to oval nuclei containing one or more prominent nucleoli. The cytoplasm of an immunoblast is abundant and deeply basophilic.
Plasmacytoid lymphocytes may also be seen.

Figure 2: Lymphoid leukocytosis

(A) Infectious mononucleosis with a reactive lymphocytosis including an immunoblast at top. Note the pleomorphism of the lymphocytes.
(B) A reactive lymphocytosis is seen in this patient with massive trauma due to a vehicular accident.
(C) Large granular lymphocytosis, reactive.
(D) CLL with characteristic small, round lymphocytes containing coarse, blocky chromatin.
(E) Prolymphocytoid transformation in CLL.
(F) Splenic marginal zone lymphoma with villous lymphocytes containing bipolar cytoplasmic projections.
(G) Blastic MCL.
(H) Lymphoplasmacytic lymphoma.
(I) Circulating follicular lymphoma with clefted lymphoma cells.

Reactive lymphocytosis may be seen with several different viral infections, other infections, drug effects, stress, and secondary to malignancy (Table 1).

Table 1: Causes of reactive lymphocytosis


There are some exceptions to the rule of a pleomorphic lymphocytosis equating to reactive lymphocytosis.
Infection by Bordetella pertussis gives a very characteristic reactive lymphocytosis composed of small, mature lymphocytes with deep nuclear clefts with a monomorphic appearance that can raise concern for lymphoma; however, clinical history will help with this diagnosis.
Polyclonal B-lymphocytosis can also show lymphocytes with distinct nuclear clefts, but will demonstrate a spectrum of morphologic changes, including nuclear lobation and binucleate forms. This disorder is typically found in young to middle-aged women who smoke, have a high association with HLA-DR7, and show a longstanding lymphocytosis that is polyclonal by flow cytometry, but some genetic abnormalities have been documented.
In addition, large granular lymphocytosis, an increased number of similar appearing large granular lymphocytes (a normal component of the blood), may be seen with viral infections, malignancy, after BM transplantation, and after chemotherapy (Figure 2).
The persistence of a large granular lymphocytosis with accompanying neutropenia and variable anemia should trigger an evaluation for T-cell large granular lymphocytic leukemia (T-LGL), including flow cytometry and TCR gene rearrangements.
Typical immunophenotypic findings of T-LGL include expression of CD3, CD8, CD57, and TCRα/β; TCR gene rearrangement is positive, which is helpful in excluding reactive large granular lymphocytes. Confirmatory testing for infections can be performed if the clinical history and physical examination are consistent with a specific type of infection; most commonly, this is a heterophilic Ab test (ie, a monospot test) for EBV infection.

Lymphoproliferative neoplasms
Whereas reactive lymphocytes are heterogenous, lymphoma cells tend to be homogenous.
A PB smear may contain a subset of lymphoma cells amidst a background of normal lymphocytes, but those lymphoma cells will typically resemble one another.

Lymphoma cells can exhibit a variety of morphologic appearances with various sizes and shapes, ranging from 8-30 μm in size and variable amounts of cytoplasm, depending on the type of lymphoma (Figure 2).
The overall frequency of PB involvement by BM lymphoma was shown to be almost 30% in a study using a combination of morphology and flow cytometry, although it should be noted that this study specifically excluded chronic leukemias (Table 2).

Table 2: Frequency of PB involvement by BM lymphoma


Chronic lymphocytic leukemia, prolymphocytoid leukemia
CLL is the most common leukemia in adults in the Western world and therefore it is the most common neoplasm seen in the PB. Whether a patient has CLL or a monoclonal B-cell lymphocytosis (< 5 × 109/L of clonal B cells in a healthy person without symptoms, cytopenias, or lymphadenopathy), the morphology of the lymphocytes in the blood will be the same. Monoclonal B-cell lymphocytosis occurs in 3% of healthy persons, usually shows a CLL immunophenotype, and will progress to CLL in a small number of patients; it is important to be aware of the criteria for this clonal benign lymphocytosis to avoid overdiagnosis of lymphoproliferative disorders. Typical CLL morphology shows round, small nuclei with coarse blocky chromatin and frequent smudge cells; an albumin preparation may be prepared in the laboratory to prevent the formation of smudge cells. Atypical CLL morphology includes cleaved nuclei or larger lymphoid cells with slightly less condensed chromatin and those with a lower nuclear/cytoplasmic ratio. Up to 55% prolymphocytes may now be seen with CLL. Prolymphocytes are larger than normal lymphocytes, ranging from 10-18 μm in size, with a central oval to round nucleus, abundant blue cytoplasm, and, typically, a single prominent nucleolus. More than 55% prolymphocytes in the blood at initial diagnosis is seen in B-cell prolymphocytic leukemia (B-PLL) patients. Large cell transformation of CLL may show large cell lymphoma cells in the PB similar to those described below under diffuse large B-cell lymphoma. T-cell PLL (T-PLL) is similar to B-PLL in that both present primarily in elderly men with a striking leukocytosis and anemia and thrombocytopenia also commonly present. Morphology in T-PLL can range from lymphoid cells similar to the prolymphocytes of B-PLL to small lymphoid cells with cytoplasmic protrusions, occasional nuclear irregularities, condensed chromatin, and prominent nucleoli. Immunophenotyping shows a mature T-cell phenotype, including expression of CD7 and TCL1 expression by immunohistochemistry; a characteristic inversion (14)(q11q32) is present in the majority of cases as shown by cytogenetic studies.

Splenic marginal zone lymphoma, hairy cell leukemia
A high frequency of blood involvement by splenic marginal zone lymphoma has been described frequently. Splenomegaly, often with anemia or thrombocytopenia, is often accompanied by PB containing lymphoma cells with “villous lymphocytes” in which bipolar cytoplasmic projections are present. However, not all cases of splenic marginal zone lymphoma in the blood will contain lymphoma cells with prominent “villi.” These morphologic features often overlap with hairy cell leukemia. Hairy cells, in contrast, have spiky cytoplasmic projections extending from the entire periphery of the cell.

Mantle cell lymphoma
Similarly, a high frequency of PB involvement by mantle cell lymphoma (MCL) is now recognized.15,18 In MCL, lymphoid cells may range from small to intermediate-sized lymphoid cells with irregular nuclear contours to large blast-like cells resembling acute leukemia. Typical MCL cells are larger than lymphocytes with folded nuclei and a small amount of basophilic cytoplasm. Although nuclei are condensed, they contain more reticular chromatin and may contain a single prominent nucleolus, thus resembling prolymphocytes. Blastic or blastoid variants of MCL are the size of large cell lymphoma cells (20-30 μm) and have a moderate amount of basophilic cytoplasm; although nuclei are round to oval, they may be indented or convoluted. Chromatin is less condensed, resembling blastic chromatin, although these lymphoma cells are typically more variable than blasts seen in acute leukemia.

Burkitt lymphoma
Burkitt lymphoma cells are morphologically identical to the cells seen in Burkitt leukemia, with the distinction resting on whether there is less than or greater than 25% BM involvement, respectively. The Burkitt cell is moderate in size (10-25 μm) with an oval to round nucleus, moderately coarse chromatin, and 1-3 prominent nucleoli. A moderate amount of deeply basophilic cytoplasm is present and typically contains multiple small vacuoles. Surprisingly, a relatively high rate of diffuse large B-cell lymphoma may be seen in the blood, although this is likely underrecognized.

Large cell lymphoma
Large cell lymphomas show some of the most abnormal morphology of the lymphomas. They may resemble a proliferation of immunoblasts with 1-2 prominent nucleoli or may present with deep convolutions with occasional nucleoli. These cells are large with small to moderate amounts of deeply basophilic cytoplasm, are occasionally vacuolated, and often show angulated and folded nuclear contours. Although these cells can be confused with blasts, large cell lymphoma cells vary in size more than blasts and lack the smooth, even chromatin found in blasts. “Double-hit” B-cell lymphomas, defined as B-cell lymphomas with break points involving MYC in combination with another recurrent break point (typically BCL2), show features of diffuse large B-cell lymphoma and Burkitt lymphoma and are associated with higher-stage disease, including frequent BM involvement.

Follicular lymphoma
Follicular lymphoma, when it involves the blood, shows a characteristic morphology. These lymphoma cells are slightly bigger than normal lymphocytes with a cleft appearance and moderately coarse chromatin; occasionally, large neoplastic cells may also be seen. Cytoplasm is typically scant, but may be moderate, and the majority of nuclei show folds and convolutions. Nucleoli may be present.

Other types of lymphoma cells also show angulated nuclei, including MCL, Sézary syndrome, and adult T-cell leukemia/lymphoma (ATLL). MCL cells lack the coarse chromatin of follicular lymphoma and are usually larger in size with gentle nuclear folds, not the deep clefts of follicular lymphoma. Sézary cells, characteristic of Sézary syndrome, show convoluted nuclear membranes with a cerebriform appearance and contain dark, hyperchromatic chromatin typically lacking nucleoli. Patients with Sézary syndrome have a generalized erythroderma with blood involvement by malignant T cells typically expressing CD4 and other pan-T-cell antigens with absent or diminished expression of CD7. The malignant T cells of ATLL are present in high number in the PB, with prominent nuclear irregularities and nuclear lobations; other cytopenias may also be present. ATLL is an aggressive disease caused by HTLV-1, and is more commonly seen in Japan, Africa, the Caribbean, and the southeastern United States. The immunophenotype is that of a mature T-cell lymphoma with expression of CD2, CD3, CD4, CD5, and CD25 with diminished to absent expression of CD7. Serologic or molecular studies can confirm HTLV-1 infection when ATLL is suspected. Molecular genetic studies can be helpful in the differential diagnosis of cleaved lymphocytes. FISH for CCND1/IGH@ is useful for confirmation of circulating MCL. FISH or RT-PCR can detect the BCL2-IGH@ rearrangement representing the t(14;18)(q32;q21) of follicular lymphoma. However, the BCL2-IGH@ rearrangement has also been reported in the blood of healthy donors. The malignant T cells of Sézary syndrome show TCR gene rearrangements; T-cell gene rearrangements are also found in other T-cell lymphomas, including ATLL and T-LGL. In lymphoplasmacytic lymphoma, plasmacytoid lymphocytes, small to intermediate-sized lymphocytes, and even frank plasma cells may be seen. The distinction among the lymphoma types requires flow cytometric immunophenotyping, as shown in Table 3 for the small B-cell lymphoproliferative disorders.

Table 3: Common marker expression patterns in small B-cell lymphoproliferative disorders involving the blood

NOS indicates not otherwise specified; and NK, natural killer.
* 8 of 9 cases of splenic marginal zone lymphoma showed PB involvement.
† A few cases with limited material were classified as high-grade, not otherwise specified and low-grade, not otherwise specified.
‡ Not meeting the criteria for CLL.

Myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB or FGFR1

Abstract
The 2008 World Health Organization (WHO) classification of tumors of hematopoietic and lymphoid tissues introduced a new category for myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB or FGFR1.
Many of these cases present as a myeloproliferative neoplasm, usually with eosinophilia. However, neoplasms associated with rearrangement of PDGFRA can present as acute myeloid leukemia or as T lymphoblastic lymphoma with eosinophilia. Neoplasms associated with rearrangement of FGFR1 even more frequently present as acute myeloid leukemia or T lymphoblastic lymphoma, in both instances with eosinophilia, and both T lymphoblastic and B lymphoblastic transformation of chronic eosinophilic leukemia have also been described. Because of the prominent lymphoid component these disorders have been assigned, in the WHO classification, to a specific category rather than being categorized as a myeloproliferative neoplasm. However, it should be noted that BCR-ABL1-positive chronic myelogenous leukemia is accepted as a bona fide myeloproliferative neoplasm and yet it too can undergo lymphoblastic transformation and even present as acute lymphoblastic leukemia with the underlying chronic myelogenous leukemia being revealed only after remission has been achieved.

Conclusion
Hematologic neoplasms associated with rearrangement of PDGFRA or PDGFRB are rare but nevertheless important to diagnose because of their responsiveness to imatinib. Their recognition can be difficult so that the generic quantitative polymerase chain reaction developed by Erben and colleagues is a diagnostic advance. The diagnosis of hematologic neoplasia associated with FGFR1 rearrangement is also important since only a trial of midostaurin or an allogeneic transplant offers the hope of avoiding an early death.

Myeloid and Lymphoid neoplasms with eosinophilia
(Myeloid and lymphoid neoplasms with PDGFRA rearrangement)

Myeloid and lymphoid neoplasms with abnormalities of PDGFRA are neoplasms in which eosinophilia is highly typical, although not required.
For years, most patients with eosinophilia and abnormalities of PDGFRA were diagnosed with hypereosinophilic syndrome (HES) as karyotype was frequently normal and no secondary cause was identified.
However, in 2001, a subset of patients with HES were found to show responsiveness to tyrosine kinase inhibitors (TKI), specifically imatinib.
Investigation of the tyrosine kinases within these patients revealed a cryptic deletion resulting in fusion of PDGFRA to FIP1L1.
After this groundbreaking research, the entity was first formally accepted in the 2008 edition of the World Health Organization's Classification of Tumours of Haematopoietic and Lymphoid Tissues along with abnormalities of PDGFRB and FGFR1.
In the WHO 2016 edition, myeloid and lymphoid neoplasms with eosinophilia and t(8;9)(p22;p24.1) PCM1 / JAK2 is now recognized as a provisional entity.

Phenotype / cell stem origin
The cell of origin is a hematopoietic cell with commitment to eosinophilic differentiation. These clonal eosinophils may show evidence of activation by immunohistochemistry, with CD23, CD25, and/or CD69 expression.
Clinics
Patients with hematopoietic neoplasms involving PDGFRA often present with fatigue or pruritic rash.
Multi-organ tissue damage, including respiratory, gastrointestinal, or cardiac sequelae, is well reported due to tissue infiltration by clonal eosinophils.
Physical examination reveals splenomegaly in most patients and hepatomegaly within a minority.
Peripheral blood smear traditionally shows features suggestive of chronic eosinophilic leukemia (CEL).
Although abnormalities of PDGFRA usually resemble CEL, rarely, patients may display characteristics of acute myeloid leukemia (AML) or T-cell lymphoblastic leukemia/lymphoma (T-ALL/LBL). However, eosinophilia traditionally remains a consistent feature.
Pathology
In patients with abnormalities of PDGFRA, eosinophilic atypia may be present (Figure 1), but is variable and not required.
This includes atypical nuclear segmentation, hypogranular cytoplasm with puddling of eosinophilic granules, more variability in granule texture/color, and cytoplasmic vacuolation.
Eosinophils are typically mature with usually only rare eosinophilic precursors noted (Figure 2).
Anemia and thrombocytopenia are common, while monocytosis and basophilia are infrequent.
Bone marrow trephine biopsies are typically hypercellular with increased eosinophils and their precursors (Figure 3) (Bain et al., 2008).
Fibrosis may also be increased and can be highlighted with a reticulin histochemical stain (Figure 4).
Mast cells are often increased as well, either in loose or cohesive clusters, and may have atypical spindled morphology. Moreover, they may have an aberrant immunophenotype, frequently expressing CD25 or even CD2 and CD25. However, the findings usually fall short of criteria for systemic mastocytosis as KIT D816V mutation is not present and serum tryptase levels are typically less than 20 ng/mL.
Treatment
Patients with abnormalities of PDGFRA are exquisitely sensitive to imatinib. An initial dose of 100 mg per day leads to complete hematologic response in most patients. If imatinib is discontinued secondary to adverse reactions or very rarely due to resistance, it can be replaced by second or third generation TKI.
Prognosis
Prior to identification of this neoplasm with response to TKI, prognosis was poor. However, now while on maintenance therapy, >90% of patients will achieve complete molecular response. Prognosis is excellent particularly if therapy is initiated prior to organ damage especially cardiac sequela.

Gene Name: PDGFRA (platelet-derived growth factor receptor, alpha polypeptide)
Location: 4q12
Note: Platelet Derived Growth Factor Receptor Alpha (PDGFRA) is located on chromosome 4, band q12. It contains 23 exons and spans approximately 65 kb. The encoded protein is 1089 amino acids with molecular weight of 122670 Da. It is inactive as a monomer in the absence of bound ligand. It is a member of the type III class of tyrosine kinase receptors. It contains 5 immunoglobulin-like extracellular domains, a single transmembrane domain, and an intracellular split kinase domain. The protein plays an essential role in the regulation of many biological processes which include cell proliferation, differentiation, migration, and survival.

Cytogenetics Morphological
Although a number a fusion partners for PDGFRA have been identified, the most common by far is FIP1L1. The FIP1L1-PDGFRA fusion gene is created by an interstitial deletion on chromosome 4q12); this deletion includes cysteine-rich hydrophobic domain 2 ( CHIC2). The deletion is most often cryptic resulting in a normal karyotype, necessitating FISH for identification (Figure 4). However, rarely it may be caused by chromosomal rearrangement.
Fusion partner genes and locations:
FIP1L1 4q12   BCR 22q11   ETV6 12p13   STRN 2p22   CDK5RAP2 9q33   KIF5B 10p11   FOXP1 3p13

Figure 1,2,3,and 4 : GoTo Atlas of Genetics and Cytogenetics in Oncology and Haematology April 2017 Myeloid/Lymphoid neoplasms with abnormalities of PDGFRA