Rodak's Hematology Clinical Principles and Applications

PART V

Leukocyte Disorders

CHAPTER 29

Nonmalignant leukocyte disorders

Steven Marionneaux

OUTLINE

Qualitative Disorders of Leukocytes

Morphologic Abnormalities with and without Functional Defects

Normal Morphology with Functional Abnormalities

Monocyte/Macrophage Lysosomal Storage Diseases

Genetic B and T Lymphocyte Abnormalities

Quantitative Abnormalities of Leukocytes

Neutrophils

Eosinophils

Basophils

Monocytes

Lymphocytes

Qualitative (Morphologic) Changes

Neutrophils

Monocytes

Lymphocytes

Infectious Mononucleosis

Objectives

After the completion of this chapter, the reader will be able to:

  1. Describe the basic genetic defect and the morphologic consequences in Pelger-Huët anomaly.
  2. Discuss how Pelger-Huët cells might be confused with the presence of a neutrophilic left shift.
  3. Compare and contrast two genetic causes of neutrophilic hypersegmentation.
  4. Describe the basic genetic defect and the morphologic consequences in Alder-Reilly anomaly, Chédiak-Higashi syndrome, and May-Hegglin anomaly.
  5. Indicate how inclusions in Alder-Reilly anomaly and May-Hegglin anomaly might be confused with morphologically similar conditions.
  6. Discuss the defect and functional consequences of chronic granulomatous disease.
  7. Describe the cellular deficiencies and functional consequences of leukocyte adhesion disorders.
  8. Describe the characteristic macrophage morphology associated with the mucopolysaccharidoses, Gaucher disease, and Niemann-Pick disease.
  9. Describe the basic defect in genetic disorders leading to decreased T lymphocyte production, decreased B lymphocyte production, and the combined decrease of T, B, and natural killer lymphocytes.
  10. Define what is meant by neutrophilia, neutropenia, lymphocytosis, lymphocytopenia, monocytosis, monocytopenia, eosinophilia, eosinopenia, and basophilia, and give some examples of conditions in which each occurs.
  11. Describe the nonmalignant alterations in granulocyte, monocyte, and lymphocyte morphology that are associated with infection, inflammation, or other causes.
  12. Outline pathogenesis; and clinical/laboratory features of infectious mononucleosis.

Nonmalignant leukocyte disorders

CASE STUDIES

After studying the material in this chapter, the reader should be able to respond to the following case studies:

Case 1

A 5-year-old boy has a long history of recurring infections, including gastroenteritis, pneumonia, severe staphylococcal infections, and a liver abscess. He was treated with antibiotics in each case and responded well, albeit slowly. The CBC was essentially normal, and no morphologic abnormalities were detected. His neutrophils were tested and were shown to migrate normally and to respond to chemotactic agents. His neutrophils also phagocytized normally; however, they were not able to reduce nitroblue tetrazolium to its insoluble formazan.

  1. What is the most likely cause of this child’s recurring infections?
  2. Genetically and biochemically speaking, what is the specific nature of the problem?
  3. What is the prognosis as it relates to treatment?
  4. How is this disorder transmitted genetically in the majority of cases?

Case 2

A 66-year-old retired male professor presents with malaise, weakness, a fever of 102° F, anorexia, and weight loss. Blood cultures ×8 were negative for pathogens. Ova and parasite examinations ×5 produced negative findings. Tuberculosis and fungal serologic testing were negative. The CBC revealed a mild normocytic, normochromic anemia; a WBC count of 4.0 × 109/L; and a platelet count of 130 × 109/L. The differential count showed a neutrophilic left shift, 20% lymphocytes, 28% monocytes, and 1% basophils. Neutrophils contained toxic granulation and large vacuoles. Monocytes were large and highly vacuolated. The patient’s erythrocyte sedimentation rate was 70 mm/hour.

  1. Based on the differential results, what cells should the medical laboratory scientist look for on the patient’s blood film?
  2. Where on the blood film should the examination be made and why?
  3. What is the significance if the suspected cells are found?
  4. What preparation other than a blood film might be helpful in this situation?

This chapter concentrates on nonmalignant disorders of WBCs—etiologies underlying changes in number and morphology of neutrophils, lymphocytes, monocytes, eosinophils, and basophils. Both hereditary and acquired causes are presented. In recent years, the genetic origin of many of these disorders has come to light and the chapter has been updated to reflect these findings.

Qualitative disorders of leukocytes

Morphologic abnormalities with and without functional defects

Pelger-huët anomaly

Pelger-Huët anomaly (PHA), also known as true or congenital PHA, is an autosomal dominant disorder characterized by decreased nuclear segmentation (bilobed, unilobed) and a characteristic coarse chromatin clumping pattern potentially affecting all leukocytes, although morphologic changes are most obvious in mature neutrophils.1 The prevalence of PHA is approximately 1 in 4785 in the United States.1 The disorder is a result of a mutation in the lamin β-receptor gene.2 The lamin β receptor is an inner nuclear membrane protein that combines β-type lamins and heterochromatin and plays a major role in leukocyte nuclear shape changes that occur during normal maturation.1 Mutations in the lamin β-receptor gene result in the morphologic changes characteristic of PHA, although the exact pathological mechanisms are not known.1 The nuclei may appear round, ovoid, or peanut shaped. Bilobed forms—the characteristic spectacle-like (“pince-nez”) morphology with the nuclei attached by a thin filament—can also be seen (Figure 29-1).3 In homozygous PHA, all neutrophils are affected and demonstrate round nuclei, whereas in the heterozygote, 55% to 93% of the neutrophil population are affected, and there is generally a mixture of all of the aforementioned nuclear shapes.4 Neutrophils in Pelger-Huët anomaly appear to function normally.5

 
FIGURE 29-1 Pelger-Huët cell. Pince-nez form with two rounded segments connected by a filament. Notice the dense chromatin pattern.

Pseudo- or acquired pelger-huët anomaly

Neutrophils with PHA morphology can be observed in patients with hematologic malignancies such as myelodysplastic syndromes (MDS), acute myeloid leukemia, and chronic myeloproliferative neoplasms. Pseudo-PHA neutrophils can also be seen in patients with HIV infection, tuberculosis, Mycoplasma pneumoniae and severe bacterial infections. Drugs known to induce pseudo-PHA include mycophenolate mofetil, valproate, sulfisoxazole, ganciclovir, ibuprofen, and chemotherapies such as paclitaxel and docetaxel.6

Laboratory issues in pelger-huët anomaly (true/congenital and pseudo/acquired)

Differentiating between true PHA and pseudo-PHA can be challenging. (1) An important consideration is the number of cells present with PHA morphology. In true PHA, the number of affected cells is much higher than in pseudo-PHA (63% to 93% vs. < 38%, respectively).4,  7 (2) Also in true PHA, all WBC lineages are potentially affected in terms of nuclear shape and chromatin structure. (3) In pseudo-PHA the phenomenon is usually seen only in neutrophils, except for some cases of MDS where monocytes, eosinophils, and basophils may exhibit PHA morphology. (4) Furthermore, if true PHA is suspected, a careful examination of peripheral blood smears of family members may reveal similar findings. (5) Hypogranular neutrophils are a common finding in MDS-related pseudo-PHA. In true PHA, neutrophils exhibit normal granulation.

In both true and pseudo-PHA there are potential challenges for the clinical lab related to cell identification. Because the nuclei of Pelger-Huët neutrophils may appear round, oval, or peanut shaped, the cells may be classified and counted as myelocytes, metamyelocytes, or band neutrophils, mimicking a neutrophilic left shift and triggering a clinical workup to uncover the cause. A careful examination of the chromatin structure can help to differentiate between Pelger-Huët cells, which are mature, and neutrophils, which are less mature.2 Also, immature neutrophils such as metamyelocytes and myelocytes should show some degree of cytoplasmic basophilia. PHA cells are mature, so the cytoplasm is nearly colorless, except for the color imparted by normal cytoplasmic granulation.

Another laboratory challenge is determining the most appropriate label to use for reporting Pelger-Huët cells. PHA neutrophils may be unilobed or bilobed, so “segmented neutrophil” seems inappropriate. “Band neutrophil” is also not suitable for reasons stated above. It is suggested that one label should be applied to all morphologic variants of PHA neutrophils. Laboratories should address this concern and develop standardized labels to be used for all morphologic variants of PHA, the goal being to ensure that the clinician understands that PHA cells are present and that lineage maturity is not left shifted. One suggested approach would be to count Pelger-Huët neutrophils as “others” and then define “others” as Pelger-Huët neutrophils.

Neutrophil hypersegmentation

Normal neutrophils contain three to five lobes that are separated by filaments. Hypersegmented neutrophils have more than five lobes and are most often associated with the megaloblastic anemias, where the neutrophil is also larger than normal (Figure 29-2). Hypersegmented neutrophils can also be seen in the myelodysplastic syndromes and represent a form of myeloid dysplasia. Much less frequently, hypersegmented neutrophils can be found in hereditary neutrophil hypersegmentation. In this disorder, patients are asymptomatic and have no signs of megaloblastic anemia.

 
FIGURE 29-2 Hypersegmented neutrophil.  Source:  (From Carr JH, Rodak BF: Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders.)

Myelokathexis refers to a rare hereditary condition characterized by normal granulocyte production; however, there is impaired release into circulation that leads to neutropenia. Neutrophil morphology is also affected. Neutrophils appear hypermature. There may be hypersegmentation, hypercondensed chromatin, and pyknotic changes. Cytoplasmic vacuoles may also be observed.8 Myelokathexis is a component of an extremely rare inherited disorder, WHIM, a syndrome in which warts, neutropenia, hypogammaglobulinemia, infections, and myelokathexis are common findings.9,  10 Mutations in CXCR4 result in a hyperfunctional CXCR4 receptor and ligand binding, which impairs cellular homeostasis and trafficking, leading to neutropenia, lymphopenia, and hypogammaglobulinemia.11,  12

Alder-reilly anomaly

Alder-Reilly anomaly is transmitted as a recessive trait and is characterized by granulocytes with large, darkly staining metachromatic cytoplasmic granules composed primarily of partially digested mucopolysaccharides. The granules are referred to as Alder-Reilly bodies or Reilly bodies. The morphology may resemble heavy toxic granulation, which is discussed later (Figure 29-3). Neutrophilia, Döhle bodies, and left shift, which are usually associated with toxic granulation, are not seen in Alder-Reilly anomaly. Also, in some patients with Alder-Reilly anomaly, the granules are found in lymphocytes and monocytes, ruling out toxic granulation, which is exclusive for neutrophils. The basic defect is the incomplete degradation of mucopolysaccharides. Reilly bodies are most commonly associated with Hurler syndrome, Hunter syndrome, and Maroteaux-Lamy polydystrophic dwarfism.13 Leukocyte function is not affected in Alder-Reilly anomaly.

 
FIGURE 29-3 Two neutrophils from a patient with Alder-Reilly anomaly. Note the dark granules present in both cells. Such granules may also be seen in eosinophils and basophils.  Source:  (Courtesy Dennis R. O’Malley, MD, US Labs, Irvine, CA.)

Chédiak-higashi syndrome

Chédiak-Higashi syndrome is a rare, fatal, autosomal recessive disease. In 2008, only 800 cases were reported worldwide.14 The disease is characterized by abnormal fusion of granules in most cells that contain granules throughout the body. The fused granules are large and mostly dysfunctional. Hematopoietic cells are affected, but disease manifestations can be found in hair, skin, adrenal and pituitary glands, and nerves. Hematologic findings in Chédiak-Higashi syndrome include giant lysosomal granules in granulocytes, monocytes, and lymphocytes (Figure 29-4). These fused granules result in leukocyte dysfunction and recurrent pyogenic infections. Patients often have bleeding issues due to abnormal dense granules in platelets.15Chédiak-Higashi syndrome is associated with a mutation in the CHS1 LYST gene on chromosome 1q42.1-2 that encodes for a protein involved in vesicle fusion or fission.15

 
FIGURE 29-4 Three cells from a patient with Chédiak-Higashi syndrome. A, Neutrophil with large dark lysosomal granules. B, Monocyte with large azure granules. C, Lymphocyte with one large azure granule.

May-hegglin anomaly

May-Hegglin anomaly is a rare, autosomal dominant platelet disorder characterized by variable thrombocytopenia, giant platelets, and large Döhle body–like inclusions in neutrophils, eosinophils, basophils, and monocytes (Figure 29-5). May-Hegglin anomaly is caused by a mutation in the MYH9 gene on chromosome 22q12-13.16 There is disordered production of myosin heavy chain type IIA which affects megakaryocyte maturation and platelet fragmentation.16 The basophilic Döhle body–like leukocyte inclusions in May-Hegglin anomaly are composed of precipitated myosin heavy chains. Döhle bodies are composed of lamellar rows of rough endoplasmic reticulum. Clinically, the majority of individuals with May-Hegglin anomaly are asymptomatic, but a few have mild bleeding tendencies that are related to the degree of thrombocytopenia.

 
FIGURE 29-5 A neutrophil and a giant platelet from a patient with May-Hegglin anomaly. Note the large, elongated, bluish inclusion in the neutrophil cytoplasm.

A summary of morphologic changes and clinical findings associated with the above disorders is shown in Box 29-1.

BOX 29-1

Morphologic Abnormalities of Neutrophils with and without Functional Defects

Morphologic Abnormality

Morphologic Changes

Clinical Findings

Pelger-Huët anomaly

Decreased nuclear segmentation in neutrophils; sometimes also affects other WBCs

Asymptomatic

Pseudo-Pelger-Huët-anomaly

Decreased nuclear segmentation in neutrophils

Depends on underlying condition

Neutrophil hypersegmentation

> 5 nuclear lobes in neutrophils

Depends on underlying cause

Alder-Reilly anomaly

Granulocytes contain large, darkly staining metachromatic cytoplasmic granules

Normal neutrophil function. Clinical findings, if present, are due to associated condition

Chédiak-Higashi disease

Giant lysosomal granules in granulocytes, monocytes, and lymphocytes

Leukocyte dysfunction and recurrent pyogenic infections; bleeding due to abnormal dense granules in platelets

May-Hegglin anomaly

Thrombocytopenia, giant platelets and large Döhle body–like inclusions in neutrophils, eosinophils, basophils, and monocytes

Usually asymptomatic; sometimes mild bleeding related to the degree of thrombocytopenia

Normal morphology with functional abnormalities

The majority of genetic functional leukocyte disorders, with the exception of some of the storage disorders, are not characterized by specific morphologic alterations in leukocytes. Box 29-2 outlines the causes and clinical findings seen in these disorders.

Box 29-2

Normal Morphology of Neutrophils with Functional Abnormalities

Disorder

Cause(s)

Clinical Findings

Chronic granulomatous disease

Mutation(s) in NADPH oxidase genes leads to failure of neutrophil respiratory burst following phagocytosis of organism

Heterogeneous, however most experience recurrent bacterial and fungal infections; granulomas may obstruct organs (liver, spleen, others)

Leukocyte adhesion disorder – I

Mutation in gene(s) responsible for β2 integrin subunits, leads to decreased or truncated β2 integrin, needed for neutrophil adhesion to endothelial cells, recognition of bacteria, and outside-in signaling

Recurrent infections, neutrophilia, lymphadenopathy, splenomegaly, and skin lesions; variable severity and survival

Leukocyte adhesion disorder – II

Mutation in SLC35C1 which codes for a fucose transporter involved in synthesis of selectin ligands. Results in decreased amount or function of selectin ligands and defective leukocyte recruitment

Physical growth retardation, coarse face, and/or other physical deformities; neurological defects, recurring infections, and absent blood group H antigen

Leukocyte adhesion disorder – III

Mutations in Kindlin-3 and defective protein product Kindlin-3, needed for β integrin activation and leukocyte rolling. Failed response to external signals that would normally result in leukocyte activation.

Recurrent bacterial and fungal infections (less severe than LAD-I). Decreased platelet integrin GPIIbβ3, resulting in bleeding

Chronic granulomatous disease

Chronic granulomatous disease is a rare inherited disorder caused by the decreased ability of phagocytes to produce superoxide and reactive oxygen species. Following phagocytosis ofmicroorganisms, there is no respiratory burst that normally results in the production of these antimicrobial agents. The basic defect is one or more mutations in genes responsible for proteins that make up a complex known as NADPH oxidase (NADPH is the reduced form of nicotinamide adenine dinucleotide phosphate). Under normal conditions, phagocytosis of foreign organism leads to phosphorylation and binding of cytosolic p47phos and p67phos.17 Primary granules containing antibacterial neutrophil elastase and cathepsin G and secondary granules containing the cytochrome complex gp91phox and gp22phox migrate to the phagolysosome. NADPH oxidase forms when p47phos and p67phos along with p40phox and RAC2 combine with the cytochrome complex. Superoxide is generated in the phagolysosome when an electron from NADPH is added to oxygen. NADPH has additional regulatory functions in the generation of other antimicrobial agents. Most cases of chronic granulomatous disease are due to mutations in gp91phox or p47. The majority of cases (approximately 60% to 65%) are X-linked recessive, whereas 35% to 40% are autosomal recessive.18

The disease is heterogeneous, and survival is based on the type of mutation, which in turn determines the level of superoxide produced.17 Most patients experience bacterial and fungal infections of the lung, skin, lymph nodes, and liver. Macrophage-rich granulomas can be found in the liver, spleen, and other organs. These granulomas sometimes obstruct the intestines, urinary tract, and lungs. Advancements in treatment, in particular antifungal agents have greatly increased survival rates, where 90% of patients survive well into adulthood.19 In the nitroblue tetrazolium reduction test, normal neutrophils, when stimulated, reduce the yellow water-soluble nitroblue tetrazolium to a dark blue insoluble formazan. Neutrophils in chronic granulomatous disease cannot perform this reduction (Figure 29-6). The disease can also be diagnosed through flow cytometry, which uses a fluorescent probe, such as dihydrorhodamine-123, to measure intracellular production of reactive oxygen species.18

 
FIGURE 29-6 Two nitroblue tetrazolium preparations. A, Neutrophils from a normal control that have reduced the NBT to a dark formazan. B, Neutrophils from a patient with chronic granulomatous disease.  Source:  (Courtesy Valerie Evans, University of Arizona Medical Center, Tucson, AZ.)

Leukocyte adhesion disorders

Recruitment of leukocytes to the site of inflammation involves capture of leukocytes from peripheral blood, followed by rolling along a vessel wall. This process is mediated through selectins, which interact with their ligands on the surface of leukocytes.20 Ligand binding induces high-affinity binding of integrins with endothelial cell receptors. The cytoskeleton in leukocytes is reorganized, and cell spreading occurs, which ultimately leads to transmigration of the leukocyte out of the blood and into the tissues.

Leukocyte adhesion disorders (LADs) are rare autosomal recessive inherited disorders that result in the inability of neutrophils and monocytes to adhere to endothelial cells and to transmigrate from the blood to the tissues. The consequence is increased and potentially lethal bacterial infections. The basic defect is a mutation in the genes responsible for the formation of cell adhesion molecules (Chapter 12).

Leukocyte adhesion disorders have been subdivided into three subcategories. LAD I is caused by a mutation in exons 5 to 9 in the gene(s) responsible for β2 integrin subunits, resulting in either a decreased or truncated form of the β2 integrin,21 which is necessary for adhesion to endothelial cells, recognition of bacteria, and outside-in signaling.22 In addition to experiencing recurrent infections, patients with LAD I frequently have neutrophilia, lymphadenopathy, splenomegaly, and skin lesions. The clinical severity, including number of infections and survival, depends on the amount of the β2 integrins produced.23

LAD II is considerably rarer than LAD I and presents in a similar manner (recurrent infection and neutrophilia), but the leukocytes have normal β2 integrins. There are molecular defects in SLC35C1, which codes for a fucose transporter that moves fucose from the endoplasmic reticulum to the Golgi region.24 Fucose is needed for posttranslational fucosylation of glycoconjugates, which is required for synthesis of selectin ligands.25 Clinically, LAD II patients have growth retardation, a coarse face, and other physical deformities.26 In LAD II the defective fucose transporter leads to an inability to produce functional selectin ligands and defective leukocyte recruitment, which leads to recurring infections. Other clinical findings related to defective fucose transport are absence of blood group H antigen, growth retardation, and neurological defects.27

LAD III is a very rare autosomal recessive disease. In LAD III, leukocytes and platelets have normal expression of integrins, but there is failure in response to external signals that would normally result in leukocyte activation.23 Mutations in Kindlin-3 have recently been identified as the culprit.28 Kindlin-3 protein along with talin are required for activation of β integrin and leukocyte rolling. Clinically LAD III patients experience a mild LAD I–like immunodeficiency with recurrent bacterial and fungal infections. In addition, in LAD III, there is decreased platelet integrin GPIIbβ3, resulting in bleeding similar to what is seen in Glanzmann thrombasthenia (Chapter 41).

Miscellaneous granulocyte disorders

Myeloperoxidase (MPO) deficiency is characterized by a deficiency in myeloperoxidase in the primary granules of neutrophils and lysosomes of monocyte. Myeloperoxidase normally stimulates production of hypochlorite and hypochlorous acid, which are oxidant agents that attack phagocytized microbes. The disorder is inherited in an autosomal dominant manner with a prevalence of approximately 1 in 2000 individuals.29 The defect originates through mutation in the MPO gene on chromosome 17Most patients do not experience problematic recurring infections because compensatory pathways are utilized for microbe killing that do not involve myeloperoxidase.29 Acquired myeloperoxidase deficiency can present in association with hematologic neoplasms and lead poisoning.30 In the hematology laboratory, MPO deficiency can be easily detected by the Siemens Advia analyzer, which uses myeloperoxidase to identify cells in the automated differential.

Monocyte/macrophage lysosomal storage diseases

Monocyte/macrophage lysosomal storage diseases can be subdivided into mucopolysaccharide (or glycosaminoglycan [GAG]) storage diseases and lipid storage diseases (Table 29-1). As a group, they represent inherited enzyme deficiencies or defects that result in flawed degradation of phagocytized material and buildup of the partially digested material within the phagocyte. All cells containing lysosomes can be affected, including T lymphocytes.31

TABLE 29-1

Variants of Monocyte/Macrophage Lysosomal Storage Disorders

Type

Name

Deficient Enzyme

Substance Stored

Mucopolysaccharidosis

MPS I—severe

Hurler syndrome

α-l-iduronidase

Dermatan sulfate, heparan sulfate

MPS I—attenuated

Scheie syndrome

α-l-iduronidase

Dermatan sulfate, heparan sulfate

MPS II—severe

Hunter syndrome

Iduronate sulfatase

Dermatan sulfate, heparan sulfate

MPS III

Sanfilippo syndrome type A

Heparan N-sulfatase

Heparan sulfate

 

Sanfilippo syndrome type B

α-N-acetylglucosaminidase

Heparan sulfate

 

Sanfilippo syndrome type C

Acetyl–coenzyme A:α-glucosaminide N-acetyltransferase

Heparan sulfate

MPS IV

Morquio syndrome type A

Galactose-6-sulfatase

Keratan sulfate, chondroitin-6-sulfate

 

Morquio syndrome type B

β-Galactosidase

Keratan sulfate

Lipid Storage Diseases

Gaucher disease

β-Glucocerebrosidase

Glucocerebroside

Niemann-Pick disease

Sphingomyelinase

Sphingomyelin

Fabry disease

α-Galactosidase

Ceramide trihexoside

Tay-Sachs disease, Sandhoff disease

Hexosaminidase A

GM2 ganglioside

The mucopolysaccharidoses (MPSs) are a family of inherited disorders of GAG degradation. Each MPS is caused by deficient activity of an enzyme necessary for the degradation of dermatan sulfate, heparan sulfate, keratan sulfate, and/or chondroitin sulfate. The partially degraded GAG builds up in the lysosomes and eventually results in physical abnormality and sometimes mental retardation. The MPSs have been subdivided according to which enzyme is defective, which GAG is being stored, and whether the symptoms are severe or attenuated (Table 29-1).32

The peripheral blood of a patient with MPSs may appear relatively normal; however, metachromatic Reilly bodies may be seen in neutrophils, monocytes, and lymphocytes (Figure 29-7). Bone marrow may reveal macrophages with large amounts of metachromatic material. Diagnosis relies on assays for the specific enzymes involved. Treatment has consisted of enzyme replacement therapy or hematopoietic stem cell transplantation.32

 
FIGURE 29-7 Lymphocyte on the blood film for a patient with a mucopolysaccharide storage disorder known as Hurler disease. Notice the dark cytoplasmic granules.

Lipid storage diseases are inherited disorders in which lipid catabolism is defective (Figure 29-8). Two of these disorders are characterized by macrophages with distinctive morphology and are discussed here.

 
FIGURE 29-8 Pathways and diseases of sphingolipid metabolism.  Source:  (From Orkin SH, Fisher DE, Look AT, et al: Nathan and Oski’s hematology of infancy and childhood, ed 7, Philadelphia, 2009, Saunders.)

Gaucher disease is the most common of the lysosomal lipid storage diseases. It is an autosomal recessive disorder caused by a defect or deficiency in the catabolic enzyme β-glucocerebrosidase (gene located at 1q21), which is necessary for glycolipid metabolism. At least 1 in 17 Ashkenazi Jews are carriers.33 More than 300 genetic mutations have been reported,34 and while some correlations have been found with specific mutations and disease severity and course, the majority of cases (phenotypes) cannot be predicted by genotype. In Gaucher disease there is an accumulation of unmetabolized substrate sphingolipid glucocerebroside in macrophages throughout the body, including osteoclasts in bone and microglia in the brain.

The clinical triad used in diagnosis is hepatomegaly, Gaucher cells in the bone marrow, and increase in serum phosphatase. Gaucher disease has been subdivided into three types based on clinical signs and symptoms (Table 29-2).35 Neurologic symptoms play a key role in differentiating between the three subtypes. The phenotype is quite heterogeneous, with some patients being completely asymptomatic (seen in Type I), while others experience a multitude of clinical problems. Clinical findings are mostly related to the patient’s age and the degree of the enzyme deficiency.

TABLE 29-2

Clinical Subtypes of Gaucher Disease

Clinical Features

Type I: Nonneuropathic

Type II: Acute Neuropathic

Type III: Subacute Neuropathic

Clinical onset

Childhood/adulthood

Infancy

Childhood

Hepatosplenomegaly

+

+

+

Skeletal abnormality

+

+

Neurodegeneration

+++

++

Death

Variable

By 2 years

Second to fourth decade

Ethnic predilection

Ashkenazi Jews

Panethnic

Swedes

Note: Absence and severity of features are indicated by – to +++.

Hematologic features include anemia and thrombocytopenia as a result of hypersplenism that is common in these patients. Bone marrow replacement by Gaucher cells may contribute to peripheral cytopenias. The bone marrow contains Gaucher cells, distinctive macrophages occurring individually or in clusters, that have an abundant fibrillar blue-gray cytoplasm with a striated or wrinkled appearance (sometimes described as onion skin–like) (Figure 29-9).36 A useful test in terms of determining the level of glucocerebroside in storage is chitotriosidase.37 This biomarker can be used in diagnosis and monitoring of the disease. The periodic acid-Schiff stain tests for mucopolysaccharides in Gaucher cells. Polymerase chain reaction is sometimes used in Ashkenazi Jews to screen for the most common mutations, but to confirm a diagnosis of Gaucher disease, gene sequencing is often needed. In all three forms of the disease, there is a fifteenfold increase for developing hematologic malignancies such as plasma cell neoplasm, chronic lymphocytic leukemia, lymphoma, and acute leukemia.38

 
FIGURE 29-9 Characteristic macrophages with cytoplasmic striations found in the bone marrow of a patient with Gaucher disease.  Source:  (From Carr JH, Rodak BF: Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders.)

Treatment of Gaucher disease includes the use of enzyme replacement therapy with recombinant glucocerebrosidase.39,  40 Agents are also available that reduce the amount of the substrate glucocerebroside. Stem cell transplantation offers the potential for cure, but safety is always a concern in allogeneic transplant, where the mortality rate associated with the procedure is high.41 Another treatment approach is the use of pharmacologic chaperones that are active site-specific competitive glucocerebroside inhibitors.42

Pseudo-Gaucher cells can be found in the bone marrow in some patients with thalassemia,43 chronic myelogenous leukemia,44 and acute lymphoblastic leukemia.45 In these diseases, pseudo-Gaucher cells form as a result of excessive cell turnover and overwhelming the glucocerebrosidase enzyme rather than a true decrease in the enzyme. Electron microscopy can distinguish between the two cells because pseudo-Gaucher cells do not contain the tubular inclusions described in Gaucher cells.

Niemann-Pick disease is an autosomal recessive lipid storage disease that has three subtypes: A, B, and C. Types A and B are characterized by recessive mutations in the SMPD1 gene, which leads to a deficiency in the lysosomal hydrolase enzyme acid sphingomyelinase (ASM) and a subsequent buildup of the substrate sphingomyelin in the liver, kidney, and lungs. In type A, the brain is also affected. In types A and B, Niemann-Pick cells are usually found in the bone marrow. These are macrophages with a foamy cytoplasm packed with lipid-filled lysosomes that appear as vacuoles after staining (Figure 29-10). Type A presents in infancy and is associated with a failure to thrive, hepatosplenomegaly, and a rapid neurodegenerative decline that results in death, usually by age 3 years. In type A, there is less than 5% of normal sphingomyelinase activity. Type B patients have approximately 10% to 20% normal enzyme activity,46 and the disease presents later in life with a variable clinical course. These patients have little or no neurological symptoms, but many experience severe and progressive hepatosplenomegaly, heart disease, and pulmonary insufficiency.47

 
FIGURE 29-10 Niemann-Pick cell with eccentric nucleus and bubble-like pattern of storage deposit in the cytoplasm.  Source:  (From Carr JH, Rodak BF: Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders.)

In Niemann-Pick type C disease there is a decrease in cholesterol effluxing from the intracellular endosome/lysosome to the cytosol. In normal conditions this process is under control of two proteins: Niemann-Pick C I and Niemann-Pick C II. Mutations in these genes results in Niemann-Pick type C disease, where cholesterol, bisphosphate, and sphingolipids build up in lysosomal storage organelles of macrophages.48 Patients with type C disease present with systemic, neurologic, and psychiatric symptoms.49 The prognosis in type C is poor. Most patients die before the age of 25 years. The diagnosis can be confirmed through gene sequencing that identifies mutations in NPC1 in 95% of cases. Five percent of patients will have mutations in NPC2.50,  51

Morphology, disease characteristics and associated laboratory tests for Gaucher and Niemann-Pick disease are highlighted in Box 29-3.

Box 29-3

Lipid Storage Disease Characteristics and Associated Laboratory Tests

Disease

Morphologic Changes

Clinical Findings

Laboratory Test

Gaucher

Macrophages with blue-gray cytoplasm appear striated or wrinkled (onion skin–like)

Variable depending on subtype: includes neurologic symptoms, hypersplenism, anemia, thrombocytopenia

Chitotriosidase: determines level of storage glucocerebrosidase 
Periodic-acid-Schiff: stains glucocerebrosidase in macrophages, polymerase chain reaction and gene sequencing: screens for associated genetic mutations

Niemann-Pick type A

Macrophages contain foamy cytoplasm packed with lipid-filled lysosomes that appear as vacuoles after staining

Failure to thrive in infancy, hepatosplenomegaly, rapid neurodegenerative decline that results in death, usually by age 3 years

Acid sphingomyelinase activity level distinguishes type A vs. B

Niemann-Pick type B

Same as above

Presents in first decade to adulthood, hepatosplenomegaly, heart disease, and pulmonary insufficiency, no neurological symptoms

Acid sphingomyelinase activity level distinguishes type A vs. B

Niemann-Pick type C

Same as above

Systemic, neurologic, and psychiatric symptoms, poor prognosis, most patients die before the age of 25 years

Gene sequencing to screen for NPC1 mutations (95% of cases) or NPC2 (5% of cases)

Genetic b and t lymphocyte abnormalities

Functional B and T lymphocyte abnormalities are genetic disorders that generally result in the decreased production of B cells, T cells, or both. They are all associated with an increased risk of infection and secondary malignancy.

T lymphocyte abnormality is best represented by a condition known as DiGeorge syndrome. This syndrome is characterized by the absence or underdevelopment of the thymus and thus markedly decreased numbers of T lymphocytes. It is associated with a microdeletion in chromosome band 22q11.2, which is the most common chromosome deletion and occurs in approximately 1 in 3500 births. Individuals with this deletion have a broad range of abnormalities, including defective parathyroid glands, cardiac abnormalities, abnormal facial development, neurologic disorders, and hypocalcemia. Less than 1% of patients with this deletion are athymic, a condition sometimes referred to as complete DiGeorge syndrome.52 Many of these patients are treated with thymus transplantation, with an approximately 75% survival rate.53

Sex-linked agammaglobulinemia (XLA) is a B cell disease that is caused most frequently by a mutation in the gene encoding Bruton tyrosine kinase (BTK). Such mutations result in decreased production of BTK, which is important for B cell development, differentiation, and signaling. Without BTK, B lymphocytes fail to reach maturity and will die prematurely.54,  55Infants with XLA start to display symptoms after 1 to 2 months, once maternal antibodies have been cleared. Recurring bacterial infections ensue and can be life-threatening.

Combined B lymphocyte/T lymphocyte abnormalities include severe combined immunodeficiency (SCID) and Wiskott-Aldrich syndrome. SCID can be divided into two types: adenosine deaminase deficiency and X-linked SCID. Both result in depletions of T, B, and natural killer (NK) lymphocytes. Adenosine deaminase deficiency results in excess amounts of its natural substrates (adenosine and 2′-deoxyadenosine), which cause lymphocyte depletion through a variety of mechanisms.56

X-linked SCID is the more common of the two and is caused by a mutation in the gene encoding the IL-2 receptor γ chain, which is shared by several interleukins. The mutation results in T cell lymphopenia, B cells that are dysfunctional, and a lack of NK cells.57

Wiskott-Aldrich syndrome is also X-linked and is caused by a mutation in a gene that encodes a protein called WASp. The mutation results in low levels or absence of the protein, and affected individuals have immunodeficiency, eczema, and thrombocytopenia. Absence of WASp affects migration, adhesion, and activation of a variety of leukocytes, including T cells, B cells, and NK cells.58

Quantitative abnormalities of leukocytes

Neutrophils

The age-specific reference range for leukocyte subsets is listed in the cover of this book. An increase in neutrophils above 7.0 × 109/L in adults and 8.5 × 109/L in children is referred to asneutrophilia. The normal relative neutrophil count is approximately 50% to 70%; however, neutrophilia should always be evaluated using absolute values. The absolute neutrophil count (ANC) is determined by adding the number of segmented and band neutrophils. Some laboratories calculate the ANC differently and include metamyelocytes, or metamyelocytes and myelocytes in the count. An increase in neutrophils can be related to several factors, including catecholamine-induced demargination or a shift in neutrophils from the marginal pool (cells normally adhering to vessel walls) to the circulating pool. This can occur from strenuous exercise, emotional stress, shock, burns, trauma, labor, or an increase in epinephrine. Neutrophilia also occurs in conditions that result in an increase in bone marrow production or in the shift of neutrophils from the bone marrow storage pool to the peripheral blood. The latter is almost always accompanied by a shift to the left (presence of immature neutrophils). Table 29-3 lists the major causes of neutrophilia.

TABLE 29-3

Causes of Nonmalignant Neutrophilia and Neutropenia

Neutrophilia

Neutropenia

• Emotional

• Strenuous exercise

• Trauma/injury

• Pregnancy: labor and delivery

• Postsurgery

• Acute hemorrhage

• Infections: bacterial, some viral

• Burns

• Surgery

• Myocardial infarction

• Pancreatitis

• Vasculitis

• Colitis

• Autoimmune disease

• Acute hemorrhage/ hemolysis

• Steroids

• Lithium

• Colony-stimulating factors (G-CSF)

• Smoking

• Chronic blood loss

• Metabolic ketoacidosis

• Drugs

Analgesic/Antiinflammatory: acetaminophen, ibuprofen, indomethacin

Antibiotics: cephalosporins, chloramphenicol, clindamycin, gentamicin, penicillin, vancomycin, tetracycline

Anticonvulsants: carbamazepine, mephenytoin, phenytoin

Antidepressants: amitriptyline, amoxapine, doxepin

Antihistamines: cimetidine, ranitidine

Antimalarials: chloroquine, dapsone, quinine

Cardiovascular: methyldopa, propranolol, captopril

Diuretics: chlorothiazide, hydrochlorothiazide

Antianxiety/hypnotics: benzodiazepines, meprobamate

Hypoglycemics: chlorpropamide, tolbutamide

Phenothiazides: chlorpromazine, phenothiazines

Others: allopurinol, clozapine, levamisole

• Radiation

• Toxins

• Immune mediated

• Alloimmune neonatal neutropenia

• Autoimmune neutropenia

• Lupus erythematosus

• Rheumatoid arthritis

• Uremia

• Eclampsia

• Malignancy

• Leukocyte adhesion deficiency

• Familial cold urticaria

• Hereditary neutrophilia

• Sjögren syndrome

• Felty syndrome

• Overwhelming infections

• Splenomegaly

• Hemodialysis

• Decreased or ineffective hematopoiesis

• Copper deficiency

• Alcoholism

• Babies born from hypertensive mothers

• Constitutional

• Shwachman-Diamond syndrome

• Severe congenital neutropenia

• Cyclic neutropenia

• Fanconi anemia

• Dyskeratosis congenita

• Common variable immunodeficiency syndrome

• Hyper-IgM syndrome

• X-linked agammaglobulinemia

• Reticular dysgenesis

• Glycogen storage disease, type Ib

• Chediak-Higashi syndrome

• Griscelli syndrome, type 2

• Hermansky-Pudlak syndrome, type 2

• Myelokathexis/WHIM syndrome

• Barth syndrome

• Cohen syndrome

Adapted from Kaushansky K: Williams hematology, ed 8, New York, 2010, McGraw-Hill, pp 951-950; and Foucar K: Bone marrow pathology, ed 3, Chicago, 2010, ASCP Press, p 209.

The term leukemoid reaction refers to a reactive leukocytosis above 50 × 109/L with neutrophilia and a marked left shift (presence of immature neutrophilic forms). Leukemoid reactions are mostly a result of acute and chronic infection, metabolic disease, inflammation, or response to a malignancy. Leukemoid reaction most often refers to neutrophils, but the increased count may be due to an increase in other types of leukocytes. A neutrophilic leukemoid reaction may be confused with chronic myelogenous leukemia. There are several distinguishing features between the two that are listed in Box 29-4.

BOX 29-4

Distinguishing between Chronic Myelogenous Leukemia and Leukemoid Reaction

Chronic myelogenous leukemia (malignant)

Increases in all granulocytes, including eosinophils and basophils. Occasional blast may be seen.

Dyspoietic morphology such as mixed eosinophil/basophil granulation and pseudo-Pelger-Huët cells may be encountered.

Involvement of platelets (may be increased), including giant, hypogranular, and/or bizarre forms.

Leukocyte alkaline phosphatase score is markedly decreased.

Leukemoid reaction (reactive)

Increase in neutrophils, including immature forms. Blasts are only rarely seen.

Reactive morphologic changes, including toxic granulation, Döhle bodies, and, less commonly, cytoplasmic vacuolization, are present.

Normal platelet morphology and number.

Increased leukocyte alkaline phosphatase score.

The term leukoerythroblastic reaction refers to the presence of immature neutrophils, nucleated red blood cells, and teardrop RBCs in the same sample. Leukoerythroblastic reactions are often accompanied by neutrophilia, but not always. Leukoerythroblastic reactions point to the possibility of a space-occupying lesion in the bone marrow. A space-occupying lesion can be a metastatic tumor, fibrosis, lymphoma, leukemia, or simply a marked increase in one of the normal marrow cells (e.g., erythroid hyperplasia seen in hemolytic anemia). A leukoerythroblastic reaction is also strongly associated with primary myelofibrosis.

Neutropenia refers to a decrease in the absolute neutrophil count (ANC) below 2.0 × 109/L in white adults and 1.3 × 109/L in black adults. The risk of infection increases as the ANC lowers, especially below 1.0 × 109/L. Severe neutropenia (< 0.5 × 109/L) further increases the risk. Agranulocytosis refers to neutrophil counts of less than 0.5 × 109/L. Some causes of neutropenia are an increased rate of removal/destruction of neutrophils from the blood; fewer neutrophils being released from the bone marrow to the blood as a result of decreased production or ineffective hematopoiesis, where neutrophils are present in the bone marrow but are not released into the blood because they are defective; changes in ratio of circulating versus marginal pool of neutrophils; or a combination of the above.

Neutropenia can also be classified as inherited or acquired. Table 29-3 lists the causes of neutropenia. Acquired causes of neutropenia are much more common than inherited causes. Immune-mediated neutropenia is caused by antibody binding to neutrophil antigens. In alloimmune neonatal neutropenia, maternal IgG crosses the placenta and binds to neutrophil-specific antigens inherited from the father, such as FcγRIIIb, NB1, or HLA.59,  60 Alloimmune neonatal neutropenia occurs in approximately 1 in 2000 births. The severity of the neutropenia varies. Neutrophil counts rise after a few months, consistent with the half-life of the maternal antibody. Autoimmune neutropenia in children is a primary illness, with moderate to severe neutropenia developing as a result of antibodies to HNA-1. The disease tends to be self-limiting, with resolution of neutrophil counts after 7 to 24 months.61Secondary autoimmune neutropenia is associated with autoimmune disorders such as rheumatoid arthritis and associated Felty syndrome, systemic lupus erythematosis, and Sjogren syndrome. In addition to antineutrophil antibodies, other factors may also induce neutropenia in secondary autoimmune neutropenia, including immune complex deposition, granulopoiesis-inhibiting cytokines, and splenomegaly. Over 100 drugs in use today are associated with neutropenia. Table 29-3 contains a partial listing. The annual rate of occurrence is 3 to 12 cases per million.6263 The mechanism of drug-induced neutropenia may be related to a dose-dependent toxicity on cell replication in hematopoiesis. Another cause may be immunologic and occurs when a particular drug is given subsequent to the initial exposure that resulted in antibody formation.64

Neutropenia may also result from infection, such as viral infection of hematopoietic progenitor cells, suppression of hematopoiesis by inflammatory cytokines, and increased usage of neutrophils due to overwhelming infection.

Intrinsic (constitutional/congenital) neutropenias are a relatively rare group of inherited disorders that usually present at birth. They are due to either decreased production from marrow hypoplasia or proliferation defect. Clinical presentation can be quite heterogeneous, but bacterial infections are the biggest risk. These infections can be life-threatening and/or diminish quality of life. However, antibiotic prophylaxis and therapy and the use of colony-stimulating factors such as G-CSF have lowered this risk and improved the quality of life for the majority of these patients.65 All of the congenital neutropenias have an increased risk for developing secondary hematologic neoplasms, and G-CSF has been linked with an increased risk for secondary leukemia.66,  67 The intrinsic causes of neutropenia are listed in Table 29-3.

Shwachman-Diamond syndrome, or Shwachman-Bodian-Diamond syndrome, is an autosomal recessive disorder characterized by marrow failure, pancreatic insufficiency, and skeletal abnormalities. Intermittent neutropenia that fluctuates from severely low to near normal is the most common hematologic finding, affecting 88% to 100% of patients.68 Mild normocytic to macrocytic normochromic anemia and reticulocytopenia is seen in approximately 80% of patients.69,  70 Thrombocytopenia occurs in 24% to 88% of patients.71,  72 Dysplastic changes involving all three granulocyte lineages are not uncommon. There is an increased risk for transformation to myelodysplasia and acute myeloid leukemia, which for many patients is a terminal event.

Congenital severe neutropenia consists of Kostmann syndrome and related diseases. Kostmann syndrome, or infantile genetic agranulocytosis, is an autosomal recessive disease characterized by severe neutropenia (often < 0.2 × 109/L) that presents shortly after birth and bone marrow granulocyte hypoplasia with maturation arrest at the promyelocyte stage. In cyclic neutropenia, approximately 50% of patients have mutations in ELANE/ELA2, the gene that codes for neutrophil elastase.73 Patients with cyclic neutropenia have periods of severe neutropenia every 21 days, during which time there is increased risk for fevers, bacterial infections, mouth ulcers, and sometimes gangrene, bacteremia, and septic shock. Administration of G-CSF has greatly reduced these neutropenia-associated events.74

Chronic idiopathic neutropenia in adults predominantly affects women 18 to 35 years of age. The bone marrow is quite variable between patients but generally shows more immature neutrophils than mature neutrophils, suggesting that cells are lost during maturation. Clinical severity is related to the degree of neutropenia. G-CSF has been shown to be a very useful treatment in these patients.

Fanconi anemia is a rare autosomal recessive or X-linked inherited disease characterized by variable degrees of bone marrow failure, peripheral cytopenias, and an increased risk for hematologic malignancies and other cancers.75 Chapter 22 contains more information about Fanconi anemia.

Dyskeratosis congenita is a sex-linked recessive, autosomal dominant or autosomal recessively inherited disorder with a heterogeneous presentation.76 In the classic form of the disease, patients have mucocutaneous abnormalities, abnormal skin pigmentation, nail dystrophy, and leukoplakia. Most patients also have bone marrow failure and increased risk for malignancy. Chapter 22 has more information about dyskeratosis congenita.

Eosinophils

Several factors influence the number of eosinophils in circulation: bone marrow proliferation rate and release into the bloodstream, movement from the blood into the extravascular tissues, and cell survival/destruction once the eosinophils have moved into the tissues. Eosinophilia is defined as an absolute eosinophil count above 0.4 × 109/L. Nonmalignant causes of eosinophilia are generally a result of cytokine stimulation, especially from interleukin-3 and interleukin-5 (IL3 and IL5).77,  78 Most causes of eosinophilia can be divided into two broad categories, depending on geography. In underdeveloped areas of the world, increased peripheral blood eosinophils are seen in patients with parasite infestation, especially helminthes and protozoa. A major function of eosinophils is degranulation, where substances are released that damage an offending organism (i.e., parasites) or target cell.79 In developed countries eosinophilia is most often associated with allergic conditions, including asthma, hay fever, urticarial, and atopic dermatitis.80,  81 Eosinophilia is also seen in scarlet fever, HIV, fungal infections, autoimmune disorders, and hypersensitivity to antibiotics and antiseizure medications. In addition, abnormalities in cytokine regulation and expression in some neoplasms result in a reactive eosinophilia. For example, reactive eosinophilia is seen in acute lymphoblastic leukemia, subtype t(5; 14).82 In some cancers, eosinophils are able to penetrate solid tumors, allowing tumoricidal cytokines to bring about tumor necrosis.83 If an individual is found to have eosinophilia (> 1.5 × 109/L) lasting more than 6 months without an identifiable cause, the diagnosis is most likely hypereosinophilic syndrome, or HES.84 HES is considered to be a myeloproliferative neoplasm and will therefore not be discussed further in this chapter.

Eosinopenia is defined as an absolute eosinophil count of less than 0.09 × 109/L and can be difficult to detect because the normal eosinophil reference range is very low. Eosinopenia is most often associated with conditions that result in marrow hypoplasia, specifically involving leukocytes. Another common cause of decreased eosinophils is infection or inflammation that is accompanied by neutrophilia. Eosinophils move into the tissues under these circumstances, and marrow release of eosinophils may be inhibited. Absolute eosinopenia has also been reported in autoimmune disorders, steroid therapy, stress, sepsis, and acute inflammatory states.85,  86

Basophils

Basophilia is defined as an absolute basophil count greater than 0.15 × 109/L. The most common cause of basophilia is the presence of a malignant myeloproliferative neoplasm such as chronic myelogenous leukemia, which is covered in Chapter 33. Nonmalignant causes of basophilia are rare and include allergic rhinitis, hypersensitivity to drugs or food, chronic infections, hypothyroidism, chronic inflammatory conditions, radiation therapy, and bee stings.87,  88

Monocytes

Monocytosis is defined as an absolute monocyte count greater than 1.0 × 109/L in adults and greater than 3.5 × 109/L in neonates. Monocytosis is associated with a wide range of nonmalignant conditions because of their participation in acute and chronic inflammation and infections, immunologic conditions, hypersensitivity reactions, and tissue repair. Monocytosis is frequently the first sign of recovery from acute overwhelming infection or severe neutropenia (most commonly after cancer chemotherapy). Monocytosis in these conditions is considered a positive sign of recovery. Monocytosis when due to administration of G-CSF or GM-CSF may be accompanied by reactive changes in monocyte morphology. Monocytosis is associated with a number of neutropenic disorders. In cyclic neutropenia, monocytosis occurs inversely with neutropenia in the 21-day cycle. A listing of the conditions associated with nonmalignant monocytosis is provided in Table 29-4.89-97

TABLE 29-4

Reactive Causes of Monocytosis

Monocytosis

Infection

•Tuberculosis

•Viral

•Malaria

•Brucellosis

•Leishmaniasis

•Fungal

•Subacute bacterial endocarditis

•Syphilis

•Protozoal

Recovery from acute infection

Recovery from neutropenia

Immunologic/Autoimmune

•Systemic lupus erythematosus

•Rheumatoid arthritis

•Autoimmune neutropenia

•Inflammatory bowel disease

•Myositis

•Sarcoidosis

Hematologic

•Acute/chronic neutropenia

•Cyclic neutropenia

•Wiskott-Aldrich syndrome

•Drug-induced neutropenia

•Hemolysis

•Immune thrombocytopenia

Drugs

•Colony-stimulating factors

•Olanzapine

•Carbamazepine

•Phenytoin

•Phenobarbital

•Valproic acid

Cancer

•Carcinoma

•Sarcoma

•Plasma cell dyscrasias

•Lymphoma

Stress

•Trauma

•Myocardial infarction

•Intense exercise

Splenectomy

Gastointestinal disease

•Alcoholic liver disease

•Sprue

Adapted from Foucar K: Bone marrow pathology, ed 3, Chicago, 2010, ASCP Press, p 199.

Monocytopenia, defined as an absolute monocyte count of less than 0.2 × 109/L, is very rare in conditions that do not also involve cytopenias of other lineage(s), such as aplastic anemia or chemotherapy-induced cytopenias. However, absolute monocytopenia has been found in patients receiving steroid therapy98 or hemodialysis, or in sepsis.99 Viral infections, especially those due to the Epstein-Barr virus (EBV), can cause monocytopenia (Table 29-4).100

Lymphocytes

The definition of lymphocytosis varies with the age of the individual. Children older than 2 weeks and younger than 8 to 10 years normally have higher absolute lymphocyte counts than adults. Lymphocytosis in young children is defined as an absolute lymphocyte count greater than 10.0 × 109/L, whereas in adults it is defined as a count greater than 4.5 × 109/L. As can be seen from the tables inside the front cover of this book, newborn infants have lymphocyte counts very similar to those of adults. The reference range for relative lymphocytes is approximately 20% to 40%. This number, however, should not be used to define lymphocytosis. Blood smear review criteria should be based on the absolute numbers rather than the relative percentage of lymphocytes present.

Lymphocytoses can be subdivided into those with and those without reactive morphologic alterations. See Table 29-5 for a listing of disorders that result in benign lymphocytosis.

The definition of lymphocytopenia is age-dependent. Lymphocytopenia in young children is defined as an absolute lymphocyte count below 2.0 × 109/L, whereas in adults it is defined as a count below 1.0 × 109/L. Nonmalignant causes of lymphocytopenia can be subdivided into inherited and acquired and are listed in Table 29-5.

TABLE 29-5

Causes of Nonmalignant Lymphocytosis and Lymphocytopenia

Lymphocytosis

Lymphocytopenia

Reactive Morphology

Inherited

Infection

•Infectious mononucleosis

•Cytomegalovirus Infection

•Hepatitis

•Acute HIV infection

•Adenovirus

•Chickenpox

•Herpes

•Influenza

•Paramyxovirus (mumps)

•Rubella (measles)

•Roseola

•Mumps

•ß-Hemolytic streptococci

•Brucellosis

•Paratyphoid fever

•Toxoplasmosis

•Typhoid fever

•Listeria

•Mycoplasma

•Syphilis

Miscellaneous

•Idiosyncratic drug reactions

•Postvaccination

•Sudden onset of stress from myocardial infarction

•Allergic reaction

•Hyperthyroidism

•Malnutrition


Nonreactive Morphology

•Bordetella pertussis (whooping cough)

•Acute infectious lymphocytosis

•Polyclonal B-lymphocytosis

Congenital immunodeficiency diseases

•Severe combined immunodeficiency disease

•Common variable immune deficiency

•Ataxia-telangiectasia

•Wiskott-Aldrich syndrome

•Others


Acquired 

Aplastic anemia

Infections

•Acquired immunodeficiency syndrome

•Severe acute respiratory syndrome

•West Nile

•Hepatitis

•Influenza

•Herpes

•Measles

•Tuberculosis

•Typhoid fever

•Pneumonia

•Rickettsiosis

•Ehrichiosis

•Sepsis

•Malaria

Iatrogenic

•Immunosuppressive agents

•Stevens-Johnson syndrome

•Chemotherapy

•Radiation

•Platelet or stem cell apheresis collection

•Major surgery

Systemic disease

•Autoimmune diseases

•Hodgkin lymphoma

•Carcinoma

•Primary myelofibrosis

•Protein-losing enteropathy

•Renal failure

Nutritional/dietary

•Ethanol abuse

•Zinc deficiency

Adapted from Kaushansky K: Williams hematology, ed 8, New York, 2010, McGraw-Hill, pp. 1141-1151; and Foucar K: Bone marrow pathology, ed 3, Chicago, 2010, p 450.

Qualitative (morphologic) changes

Neutrophils

Neutrophil reaction to infection, inflammation, stress, or administration of recombinant colony-stimulating factor (CSF) therapy may affect the number and types of circulating neutrophils (left shift), induce morphologic change, or both. While these changes may be considered “abnormal,” they usually reflect a normal, reactive response. Depending on the severity of the infection, inflammation, or dose/reaction to CSF, the left shift can range from mild (an increase in band neutrophils and metamyelocytes) to moderate (metamyelocytes, myelocytes, and an occasional promyelocyte) to marked (myelocytes, promyelocytes, and an occasional blast form).

Reactive morphologic changes in neutrophils include toxic granulation, Döhle bodies, cytoplasmic vacuoles, hypersegmentation, and pyknosis. Toxic granulation of neutrophils appears as dark, blue-black granules in the cytoplasm of neutrophils: segmented, bands, and metamyelocytes. Toxic granules are peroxidase positive and reflect an increase in acid mucosubstance within primary, azurophilic granules of neutrophils.101 The result is a lowered pH in phagolysosomes that enhances microbial killing.101 There is a positive correlation between levels of C-reactive protein (acute phase protein) and the percentage of neutrophils with toxic granulation;102 therefore, the intensity of toxic granulation is a general measure of the degree of inflammation.103 In addition, toxic granulation can be seen in various infections as well as in patients who have received CSF. Toxic granulation, especially when intense, can mimic the granulation found in the mucopolysaccharidoses and Alder-Reilly anomaly. One helpful defining characteristic of toxic granulation is that in most patients not all neutrophils are equally affected (Figure 29-11). Box 29-5A highlights reactive neutrophil morphologic changes and associated conditions.

 
FIGURE 29-11 Toxic granulation. Note that one neutrophil contains toxic granulation and the other does not. Also note that the toxic granules are clustered in some areas of the cytoplasm. Both of these findings help in distinguishing toxic granulation from poor staining or from the dark granules seen in Alder-Reilly anomaly.

Box 29-5A

Reactive Morphologic Changes in Neutrophils

Reactive Change

Morphology

Associated with

Toxic granulation

Dark, blue-black cytoplasmic granules

Inflammation, infection, administration of granulocyte colony stimulation factor (G-CSF)

Dohle bodies

Intracytoplasmic pale blue round or elongated bodies between 1 and 5 μm in diameter, usually adjacent to cellular membranes.

Nonspecific finding, or associated with bacterial infections, sepsis, and pregnancy

Cytoplasmic vacuolization of neutrophils

Small to large circular clear areas in cytoplasm, rarely may contain organism

Septicemia or other infection; autophagocytosis secondary to drug ingestion, acute alcoholism, or storage artifact; vacuoles are sometimes seen in conjunction with toxic granulation.

Döhle bodies are cytoplasmic inclusions consisting of remnants of ribosomal ribonucleic acid (RNA) arranged in parallel rows.104 Döhle bodies are typically found in band and segmented neutrophils (Figure 29-12) and often in cells containing toxic granulation. They appear as intracytoplasmic, pale blue round or elongated bodies between 1 and 5 μm in diameter. They are usually located in close apposition to cellular membranes. A delay in preparing the blood after collection in EDTA tube may affect Döhle body appearance in that they are more gray than blue and in some cases may not be visible. Döhle bodies are relatively nonspecific. Their presence has been associated with a wide range of conditions, including bacterial infections, sepsis, and normal pregnancy.104,  105

 
FIGURE 29-12 Neutrophil containing a bluish cytoplasmic inclusion known as a Döhle body.

Cytoplasmic vacuolation of neutrophils is seen less often than toxic granules and Döhle bodies. Vacuoles generally reflect phagocytosis, either of self (autophagocytosis) or of extracellular material. Autophagocytic vacuoles tend to be small (approximately 2 μm) and distributed throughout the cytoplasm. In addition, autophagocytosis can be induced by drugs such as sulfonamides and chloroquine,100 storage in EDTA (artefactual) for more than 2 hours, autoantibodies,106 acute alcoholism,107 and exposure to high doses of radiation.108 Phagocytic vacuoles caused by either bacteria or fungi are often seen in septic patients. Phagocytic vacuoles are large (up to 6 μm) and are frequently accompanied by toxic granulation (Figure 29-13). When vacuoles are seen, especially when not accompanied by toxic granulation, Döhle bodies, or both, artefactual causes should be suspected. The blood collection time should be compared with when the smear was made. As stated above, this time should not exceed 2 hours.

 
FIGURE 29-13 Cytoplasmic vacuoles. A, Band neutrophil with autophagocytic vacuoles. Note their small size. B, Neutrophil with phagocytic vacuoles. Note their larger size. Other evidence of toxicity in this cell is the pyknotic nucleus.

When phagocytic vacuoles are seen, a careful examination should be made for bacteria or yeast within the vacuoles. Cases of ehrlichiosis and anaplasmosis have been increasing in the United States over the past decade.109 Ehrlichia and Anaplasma are small, obligate, intracellular bacteria transmitted by ticks to humans and other vertebrate hosts. These organisms grow as a cluster (morula) in neutrophils (A. phagocytophilum and E. ewingii) (Figure 29-14A) and in monocytes (E. chaffeensis) (Figure 29-14B). Leukopenia, thrombocytopenia, and elevated liver enzymes are common laboratory findings, and anemia may develop in about half the cases of human monocytic ehrlichiosis.110 Intracellular aggregates in neutrophils or monocytes may occasionally be detected in the first week of infection on a Wright-Giemsa–stained peripheral blood film or a buffy coat preparation. Immunofluorescent antibody titers or polymerase chain reaction testing may help to confirm the diagnosis.110 Early diagnosis is essential because antibiotic treatment with doxycycline is very effective and can prevent serious complications.

 
FIGURE 29-14 A, Anaplasma phagocytophilum in a neutrophil. B, Ehrlichia chaffeensis in a monocytic cell.  Source:  (Courtesy J. Stephen Dumler, MD, Division of Medical Microbiology, The Johns Hopkins Medical Institution, Baltimore, MD.)

Pyknotic nuclei in neutrophils generally indicate imminent cell death. In a pyknotic nucleus, nuclear water has been lost and the chromatin becomes very dense and dark; however, filaments can still be seen between segments.111,  112 Pyknotic nuclei should not be confused with necrotic nuclei found in dead cells. Necrotic nuclei are rounded fragments of nucleus with no filaments and no chromatin pattern (Figure 29-15).

 
FIGURE 29-15A, Upper cell is a neutrophil whose nucleus is dehydrated, which makes it very dark and dense. Note that there is still a filament between the segments. This is referred to as a pyknotic cell. The cell is also highly vacuolated. B, Neutrophil that has died. Note that the nucleus has disintegrated into numerous rounded spheres of DNA with no filaments. This is referred to as a necrotic or necrobiotic cell.  Source:  (B from Carr JH, Rodak BF: Clinical hematology atlas, ed 3, St. Louis, 2009, Saunders.)

Degranulation is a common finding in activated neutrophils and eosinophils (Figure 29-16). Both primary and secondary granules are emptied into phagosomes, and secondary granules are also secreted into the extracellular space.113 In vitro degranulation in eosinophils often occurs when cellular membranes are disrupted during the process of making the blood film. Eosinophils are fragile.

 
FIGURE 29-16Partially degranulated eosinophil. This cell was found on the blood film for a patient with trichinosis.

Cytoplasmic swelling may be caused by actual osmotic swelling of the cytoplasm or by increased adhesion to the glass slide by stimulated neutrophils. Regardless of the cause, the result is a variation in neutrophil size or neutrophil anisocytosis (Figure 29-17).

 
FIGURE 29-17 Neutrophil anisocytosis. The neutrophil to the left is larger than the other neutrophil. This is often caused by cytoplasmic swelling.

Monocytes

Occasional immature monocytes may be seen in the peripheral blood in response to infection or inflammation, but this is not as common as a neutrophilic left shift (Figure 29-18). Reactive changes may be seen in monocytes in infections, during recovery from bone marrow aplasia, and after GM-CSF administration. The nucleus can become thin and band-like in areas and may appear to be segmenting (Figure 29-19). Reactive changes also include increased cytoplasmic volume, increased numbers of cytoplasmic granules, and evidence of phagocytic activity (cytoplasmic vacuolation, intracellular debris, and irregular cytoplasmic borders) (Box 29-5B). Large numbers of immature monocytes occur most often in hematologic neoplasms involving the monocytic series.

 
FIGURE 29-18 Immature monocyte.  Source:  (From Carr JH, Rodak BF: Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders.)

 
FIGURE 29-19 Reactive monocyte with contorted nucleus. Other evidence of toxicity is the several vacuoles in the cytoplasm.

Box 29-5B

Reactive Morphologic Changes in Monocytes

Morphology

Associated with

Thin and band-like, or segmentation of nucleus; increased cytoplasmic volume and granulation, and/or evidence of phagocytic activity (cytoplasmic vacuolation, intracellular debris, and irregular cytoplasmic borders)

Infection, recovery from bone marrow aplasia, and granulocyte monocyte colony stimulating factor (GM-CSF) administration

Lymphocytes

Over the years, reactive morphologic changes in lymphocytes have been described using various terms, including variant lymphocytes, reactive lymphocytes, effector lymphocytes, transformed lymphocytes, Turk cells, Downey cells, immunoblasts, and atypical lymphocytesAtypical is commonly used, but it is probably the least suitable of all because it implies that the cells are abnormal when in fact the lymphocytes are reacting to antigen in a normal manner. Also, the term atypical, when used by the cytology lab in a Pap smear result, suggests a suspicious, possibly precancerous lesion. Regardless of the labels that are applied, it is very important that the clinical staff understand the meaning behind the terms the lab uses to describe the reactive and malignant-appearing lymphocytes reported in the WBC differential.

Reactive lymphocytes are the result of complex morphologic and biochemical events that occur as lymphocytes are stimulated when interacting with antigens in peripheral lymphoid organs (Figures 29-20 and 29-21). B and T lymphocyte activation results in the transformation of small, resting lymphocytes into proliferating larger cells. These lymphocytes spill into peripheral circulation, which is what is encountered upon smear review. Reactive lymphocytes often present as a heterogeneous population of various shapes and sizes. There is variation in the nuclear/cytoplasmic ratio, nuclear shape, and the chromatin pattern, which is generally clumped, but some cells may demonstrate chromatin patterns that are less mature (less clumped). Nucleoli may be visible. Most obvious in reactive lymphocytes is an increase in basophilic cytoplasm that may vary in intensity within and between cells. The cytoplasm may be indented by surrounding RBCs, but it is important to realize that other cells, including blasts, may also show similar indentation. A plasmacytoid lymphocyte is a type of reactive lymphocyte that has some of the morphologic features of plasmacytes (Figure 29-22). However, because reactive lymphocytes may be activated T or B cells, it is important to understand that plasmacytoid is a morphologic term and does not imply lineage. Features of reactive lymphocytes are summarized in Box 29-5C.

 
FIGURE 29-20 Reactive (variant) lymphocyte.

 
FIGURE 29-21 Reactive (variant) lymphocytes from a patient with infectious mononucleosis.

 
FIGURE 29-22 Reactive (variant) lymphocyte (plasmacytoid).

Box 29-5C

Morphologic Changes in Reactive Lymphocytes

  • Heterogeneous population of various shapes and sizes.
  • Cells exhibit increased amount of variably basophilic cytoplasm.
  • Lymphocyte population exhibits variation in nuclear/cytoplasmic ratio and/or nuclear shape.
  • Chromatin is usually clumped however some cells may demonstrate less mature (less clumped) pattern.
  • Nucleoli may be visible.
  • The cytoplasm may be indented by surrounding RBCs

Epstein-barr virus (ebv)–related infections

Most humans are subclinically infected with EBV, which has been associated with several benign and malignant diseases but has only been proven to be the causative agent in a few, including infectious mononucleosis.

Infectious mononucleosis (im)

When primary infection with Epstein-Barr virus occurs in childhood, it often goes unnoticed. Infectious mononucleosis is the symptomatic illness that ensues whenever adolescents and adults are infected. The incubation period of infectious mononucleosis is about 3 to 7 weeks, and during this time the virus preferentially infects B lymphocytes through attachment of viral envelope glycoprotein 350/220 to CD21 (C3d complement receptors).114 The oropharynx epithelial cells are also infected, but the mechanism is unclear because these cells do not express CD21. The cellular response in IM is important in the control of the infection and is characterized by proliferation and activation of natural killer (NK) lymphocytes, CD4+ T cells, and CD8 + memory cytotoxic T cells (EBV-CTLs) in response to B cell infection. Most of the circulating reactive lymphocytes seen in circulation represent activated T cells.

Common clinical manifestations include sore throat, dysphagia, fever, chills, cervical lymphadenopathy, fatigue, and headache. Hematologic findings resemble those seen in many viral disorders. The WBC count is usually elevated to a range of 10—30 × 109/L or more with an absolute lymphocytosis. There is a wide variation in lymphocyte morphology, with up to 50% or higher exhibiting reactive features. Complications that may occur are generally mild and include hepatosplenomegaly (and elevated transaminases), hemolytic anemia, and moderate thrombocytopenia. In rare cases patients may develop aplastic anemia, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, Guillain-Barré syndrome, or other neurologic complications.115 The incidence of IM in the United States is 500 cases per 100,000 annually. It has its highest frequency in young adults, aged 15 to 24 years,116 although infections have been reported in patients 3 months to 70 years of age.

Testing for infectious mononucleosis includes rapid screening tests for the detection of heterophile antibodies, antibodies stimulated by the EBV that cross-react with antigens found on sheep and horse red cells. However, not everyone with infectious mononucleosis will produce heterophile antibodies, especially children. Definitive testing for EBV infection includes a panel of antigen and antibody tests for VCA, EBNA, and IgG/IgM antibodies against VCA and EBNA. Cytomegalovirus is capable of causing a mononucleosis syndrome with similar clinical features.

Clinical and laboratory findings associated with infectious mononucleosis are summarized in Box 29-6.

Box 29-6

Infectious Mononucleosis: Clinical and Lab Findings

Clinical Manifestations

Laboratory Test Results

• Common

• Sore throat

• Dysphagia

• Fever

• Chills

• Cervical lymphadenopathy

• Fatigue

• Headache

• Less common

• Hepatomegaly

• Elevated transaminases

• Splenomegaly

• Hemolytic anemia

• Thrombocytopenia

• WBC: 10–30 x 109/L due to an absolute lymphocytosis

• Reactive lymphocyte morphology

• Positive heterophile antibody test

• Positive EBV specific antigen & antibody tests

Summary

  • Pelger-Huët anomaly is a genetic disorder resulting in hypolobulated mature leukocytes. These cells can be confused with immature neutrophils. An acquired form of hyposegmentation called pseudo-PHA can be seen in some malignant myeloproliferative neoplasms.
  • Alder-Reilly anomaly is a manifestation of the mucopolysaccharidosis characterized by metachromatic granules in leukocytes, which can be confused with toxic granulation.
  • Chediak-Higashi syndrome is an inherited lethal disorder characterized by giant lysosomes in granular cells and dysfunctional leukocytes.
  • May-Hegglin anomaly is characterized by thrombocytopenia, giant platelets, and Döhle body–like inclusions in leukocytes. Most affected individuals are asymptomatic.
  • Chronic granulomatous disease is an inherited disorder of the NADPH oxidase system resulting in neutrophils that are incapable of killing many microorganisms due to a failure in the respiratory burst, which is necessary to produce antibacterial agents.
  • Leukocyte adhesion disorders are a group of disorders caused by mutations in the genes for adhesive molecules required for cells to migrate from the blood into the tissues.
  • The mucopolysaccharidoses are a group of disorders, each of which is associated with a specific defect in an enzyme necessary for the degradation of GAGs such as heparan sulfate, keratan sulfate, dermatan sulfate, and chondroitin sulfate. The result is the buildup of partially digested GAGs within macrophages and leukocytes and clinical symptoms.
  • The lipid storage diseases are a group of disorders, each of which is associated with a specific defect in an enzyme necessary for the degradation of lipids. The two lipid storage diseases with characteristic macrophage morphology are Gaucher disease and Niemann-Pick disease.
  • Inherited lymphocyte disorders include DiGeorge syndrome, in which the lack or underdevelopment of the thymus results in decreased T cells; sex-linked agammaglobulinemia, in which the lack of a kinase results in blocked B cell development; and two types of severe combined immunodeficiency. Wiskott-Aldrich syndrome is a third inherited disorder affecting both T and B cells.
  • Reactive changes in granulocytes include a left shift, Döhle bodies, toxic granulation, vacuoles, degranulation, and cytoplasmic swelling.
  • Reactive changes in monocytes include occasional immature forms, contorted nuclei, and the presence of more immature forms.
  • Reactive changes in lymphocytes include cell enlargement, increased basophilic cytoplasm, and morphologic heterogeneity.

Now that you have completed this chapter, go back and read again the case studies at the beginning and respond to the questions presented.

Review questions

Answers can be found in the Appendix.

  1. Which of the following inherited leukocyte disorders is caused by a mutation in the lamin B receptor?
  2. Pelger-Huët anomaly
  3. Chédiak-Higashi disease
  4. Alder-Reilly anomaly
  5. May-Hegglin anomaly
  6. Which of the following inherited leukocyte disorders is one of a group of disorders with mutations in nonmuscle myosin heavy-chain IIA?
  7. Pelger-Huët anomaly
  8. Chédiak-Higashi disease
  9. Alder-Reilly anomaly
  10. May-Hegglin anomaly
  11. Which of the following inherited leukocyte disorders might be seen in Hurler syndrome?
  12. Pelger-Huët anomaly
  13. Chédiak-Higashi disease
  14. Alder-Reilly anomaly
  15. May-Hegglin anomaly
  16. Which of the following lysosomal storage diseases is characterized by macrophages with striated cytoplasm and storage of glucocerebroside?
  17. Sanfilippo syndrome
  18. Gaucher disease
  19. Fabry disease
  20. Niemann-Pick disease
  21. The neutrophils in chronic granulomatous disease are incapable of producing:
  22. Hydrogen peroxide
  23. Hypochlorite
  24. Superoxide
  25. All of the above
  26. Individuals with X-linked SCID have a mutation that affects their ability to synthesize:
  27. Deaminase
  28. Oxidase
  29. IL-2 receptor
  30. IL-8 receptor
  31. An absolute lymphocytosis with reactive lymphocytes suggests which of the following conditions?
  32. DiGeorge syndrome
  33. Bacterial infection
  34. Parasitic infection
  35. Viral infection
  36. What leukocyte cytoplasmic inclusion is composed of ribosomal RNA?
  37. Primary granules
  38. Toxic granules
  39. Döhle bodies
  40. Howell-Jolly bodies
  41. The expected complete blood count (CBC) results for women in active labor would include:
  42. High total white blood cell (WBC) count with increased lymphocytes
  43. High total WBC count with a slight shift to the left in neutrophils
  44. Normal WBC count with increased eosinophils
  45. Low WBC count with increased monocytes
  46. Which of the following is true of an absolute increase in lymphocytes with reactive morphology?
  47. The population of lymphocytes appears morphologically homogeneous.
  48. They are usually effector B cells.
  49. The reactive lymphocytes have increased cytoplasm with variable basophilia.
  50. They are most commonly seen in bacterial infections.

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