Is it immune-mediated neutropenia?

Neutrophils are a part of the granulocytic cell line and play an essential role in initiating an immune response to invading pathogens, such as bacteria, and releasing chemotactic substances that attract other inflammatory cells, such as monocytes.1 Neutropenia (< 3,000 neutrophils/μl in dogs, < 2,500 neutrophils/μl in cats) as a solitary laboratory finding is often not associated with clinical signs and may be detected on routine screening of otherwise apparently healthy patients. Alternatively, neutropenic dogs may present with a fever or lethargy or other signs associated with infection. Prompt recognition and treatment are vital because if the neutropenia is not resolved, infections may be persistent or recurrent and may result in death.2-6

Immune-mediated neutropenia, also called steroid-responsive neutropenia, is a primary disorder or may occur secondary to infection, neoplasia, or the effects of certain drugs. It results when antibodies directed against neutrophil surface antigens or potentially against growth regulators of granulopoiesis develop.7,8 It can occur concurrently with other immune-mediated diseases, such as rheumatoid arthritis or systemic lupus erythematosis in dogs and people.9-11 Immune-mediated neutropenia is a well-recognized cause of neutropenia in people, primarily affecting infants.12 Much of the available information about immune-mediated neutropenia in animals is extrapolated from the disease in people.12

Immune-mediated neutropenia is a relatively uncommon cause of neutropenia in cats and dogs, and recognizing it is challenging. It can only be identified by understanding the pathophysiology of neutropenia in general and ruling out its differential diagnoses.

PATHOPHYSIOLOGY OF NEUTROPENIA

Several mechanisms of neutropenia must be considered before diagnosing immune-mediated neutropenia.13 Neutropenia can occur because of

• Increased margination or egress of neutrophils from the circulating pool to the marginated pool as may occur with infection, immune-mediated disorders, or hypersplenism

• Decreased production resulting from bone marrow injury, such as that caused by certain drugs, or infiltration as in myelophthisis (normal hemopoietic tissue is replaced with fibrous tissue or a nonhemopoietic tissue such as carcinoma), myeloproliferative disorders, and lymphoproliferative disorders

• Increased consumption or destruction of neutrophils as in immune-mediated destruction or severe infection in which use or destruction is in excess of bone marrow production and release of neutrophils.

These causes of neutropenia are not mutually exclusive, and multiple mechanisms are frequently concurrent.

Immune-mediated neutropenia represents the least common cause of neutropenia and results primarily from increased destruction of neutrophils when they become coated with anti-neutrophil antibodies.14 The spleen also sequesters antibody-coated blood cells, so increased margination may concurrently be present (Felty syndrome: neutropenia, rheumatoid arthritis, and splenomegaly).

DIFFERENTIAL DIAGNOSES FOR NEUTROPENIA

Neutropenia can occur for a variety of reasons including infections, drug reactions, and bone marrow and genetic diseases.

Infections

Overall, infections with bacteria, viruses, and fungi are the most common causes of decreased neutrophils. In the case of endotoxemia, sepsis, or localized infections, increased margination may be noted. If the infectious process is overwhelming, increased destruction may exceed bone marrow production. Some infections, such as canine monocytotropic ehrlichiosis, impair bone marrow proliferation.15 Neutropenia may also be associated with systemic mycosis (histoplasmosis, cryptococcosis) if myelophthisis occurs and results in subsequent cytopenias.16

In dogs, the most common infection consistently associated with neutropenia is parvovirus. In both dogs and cats, parvovirus targets tissues with high mitotic rates such as the bone marrow, leading to cytotoxic hematopoietic cell death as well as ineffective hematopoiesis. Parvovirus infection will also lead to increased neutrophil margination and consumption associated with gastrointestinal damage and endotoxemia.

In cats, feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) infections are the most common infectious causes of neutropenia. FeLV infection causes the destruction of hematopoietic stem cells and ineffective maturation of neutrophils associated with an altered microenvironment. Secondary infections cause increased consumption. A cyclic neutropenia has been reported in association with FeLV infection.17 With FIV infection, neutropenia is also common but is usually mild and transient, occurring in the first few weeks after infection. Neutropenia seems to result from impaired monocyte and granulocyte differentiation.18

Effects of medications

Many medications can cause neutropenia either by a direct toxic effect (at the level of the bone marrow or against circulating cells) or by inducing a secondary immune-mediated disease directed against circulating neutrophils (Table 1).19 The level of cells that are targeted within the bone marrow or circulation will affect the time of onset of the cytopenia and what blood cell lines are affected. If a neutropenic patient has a history of recent drug use, suspect the medication as causative and discontinue it.

Table 1: Drugs That Can Cause Neutropenia in Animals*

Chemotherapeutic agents are known to cause myelosuppression, related to the suppression of cell populations with high mitotic rates. Typically, the myelosuppression is seen five to seven days after treatment. Neutrophils are commonly affected.

Both exogenous (diethylstilbestrol, estradiol cypionate) and endogenous (Sertoli cell tumor) estrogens can cause bone marrow suppression, perhaps because of induction of a myelopoiesis inhibitory substance from the thymus.20 The prognosis for patients may be grave since cytopenias often do not resolve with removal of the excess estrogens.

Griseofulvin may cause an idiosyncratic neutropenia in cats (particularly FIV-infected cats).21

Antithyroid medications, such as methimazole, can cause severe neutropenia. In 1.5% of cats, this occurs within the first three months of therapy. The neutropenia typically resolves within a week of discontinuing methimazole therapy.22

In dogs, anticonvulsants, such as phenobarbital and primidone, can induce neutropenia (as well as thrombocytopenia and anemia) as an idiosyncratic reaction. Phenobarbital can also lead to bone marrow necrosis with long-term use.23

Bone marrow disorders

Neutropenia can also be the result of disorders that affect the bone marrow. Myeloproliferative and lymphoproliferative diseases and myelophthisis are not infrequently encountered and can result in crowding of the bone marrow space with their respective populations and subsequent cytopenias. Bone marrow necrosis and myelofibrosis with resultant cytopenias can be caused by numerous primary disorders (sepsis, lymphoma, drug toxicosis, or systemic lupus erythematosus), or they may be idiopathic.23

Genetic disorders

Infrequently, genetic disorders can also result in neutropenia. Cyclic stem cell proliferations are noted to occur in gray-colored rough collies.24 The neutrophils disappear from peripheral blood at 10 to 12-day intervals, during which time dogs are clinically ill. The syndrome results from a stem cell defect leading to abnormal responses to growth factors or accelerated cell loss through apoptosis.

Malabsorption of vitamin B12 in young giant schnauzers causes neutropenia that is accompanied by anemia and methylmalonic aciduria.25 This disorder responds to parenteral vitamin B12 therapy.

Border collies may present with neutropenia due to a rare disease that causes failure of segmented neutrophils to enter peripheral circulation (myelokathexis).26

Chediak-Higashi syndrome, a form of albinism seen in cats as well as other species, may have a concurrent neutropenia.

DIAGNOSTIC APPROACH

Signalment and clinical signs

No breed, sex, or size predisposition has been identified for immune-mediated neutropenia. However, dogs younger than 4 years old may be 4.1 times as likely to develop idiopathic neutropenia as are dogs older than 4 years.16

Dogs may be asymptomatic or may be presented for nonspecific clinical signs. How long dogs or cats might be neutropenic before showing clinical signs is unknown, but the duration from the onset of illness to the time of referral ranged from three to 180 days (median = 46 days) in one retrospective case series.5 Commonly reported clinical signs include lethargy, lameness (possibly shifting), and anorexia.16 Less commonly reported signs include diarrhea, vomiting, and hypersalivation.2,3,11

History taking and physical examination

Given that infections and drug administration are the most common causes of neutropenia, history taking and physical examinations need to be directed at gathering information relevant to these areas.

A thorough drug history should include over-the-counter products and supplements that the owners might not consider drugs. A vaccine history is essential to assess the possibility of infections such as FeLV or parvovirus.

Physical examinations need to be directed at detecting any nidus of infection. Careful auscultation for murmurs (endocarditis), abdominal palpation, spinal palpation (discospondylitis), and palpation of the lymph nodes are vital.

Persistent fever is the most frequently reported physical examination finding in dogs and cats with immune-mediated neutropenia.6 In case reports, joint effusion, conjunctivitis, facial edema, oral ulcerations, epistaxis, pyoderma, abscessation of a digit with local lymphadenomegaly, abdominal pain, mucoid vaginal discharge, and scrotal inflammation have all been reported.2,3,5,11

Complete blood count

On a complete blood cell count (CBC), dogs with immune-mediated neutropenia have a lower neutrophil count than do dogs with neutropenia attributed to other causes.6 Severe neutropenia (< 500/μl) with a lack of toxic change is common,14 although reports in the literature are limited. Similarly, some reports of immune-mediated neutropenia in cats reveal that the cats commonly have a marked lymphocytosis and mild thrombocytopenia.14 Toxic change of neutrophils strongly indicates concurrent secondary infection and should resolve with appropriate antibiotic therapy.

Concurrent anemia, which can be immune-mediated or from chronic inflammation or bone marrow disease, may be noted. Thrombocytopenia may also be noted, with immune-mediated thrombocytopenia or bone marrow disease being the most likely causes. Multiple cytopenias increase the likelihood of bone marrow disease.

Blood smear cytology may show clumping of neutrophils.3 It has been proposed that a concurrent monocytosis may compensate for the lack of neutrophils; this situation is seen in people. Concurrent lymphocytosis may be related to chronic antigenic stimulation and may lead to hypergammaglobulinemia.

Additional diagnostic testing

Ultimately, the diagnostic approach to a neutropenic dog or cat depends on whether the patient is symptomatic and how severe the neutropenia is.

Febrile patients. If the patient is febrile, look for a septic focus such as a pulmonary, hepatic, or splenic infection or abscess. Use radiography and ultrasonography, and obtain blood, urine, joint fluid, and cerebrospinal fluid samples for bacterial culture.

If the results of these tests are negative, consider using a technetium-99m-labeled neutrophil scan to detect neutrophil accumulation at a focus of infection. Serologic or polymerase chain reaction tests are also indicated to rule out infectious disorders such as ehrlichiosis in dogs and FeLV and FIV infections in cats.

Anemic patients. With the presence of anemia, especially if it is marked, Coombs tests are indicated. An antiplatelet antibody test in dogs with concurrent thrombocytopenia may be of interest. Evidence of concurrent immune-mediated blood cell destruction supports a diagnosis of immune-mediated neutropenia. Similarly, positive antinuclear antibody or rheumatoid factor test results may lend evidence of concurrent immune-mediated disease. False positive results are common if these tests are run indiscriminately (e.g. testing for rheumatoid factor in a dog without evidence of erosive polyarthritis).14 Similarly, restrict antinuclear antibody testing to those patients in which concurrent nonerosive polyarthritis is present or suspected.

Cytopenia. Bone marrow aspiration or core biopsy is a useful diagnostic tool. It is indicated if there are concurrent cytopenias that cannot be explained by immune-mediated mechanisms or if these cytopenias lack a regenerative response. Bone marrow may show a neoplastic population of cells such as leukemia. Hypoplastic marrow would lend evidence to a drug-induced cytopenia, canine monocytotropic ehrlichiosis, immune-mediated neutropenia due to antibodies directed at bone marrow, or an idiopathic cause of neutropenia. Plasmacytosis may point toward Ehrlichia canis infections.27

In the case of immune-mediated neutropenia, bone marrow aspiration typically reveals hypercellular bone marrow, though hypoplasia can also be seen.5 Myeloid hyperplasia with a lack of mature neutrophils in the marrow but lots of bands would suggest peripheral destruction of the neutrophils as opposed to decreased production.

DIAGNOSIS

Antineutrophil antibodies

To definitively diagnose immune-mediated neutropenia, antineutrophil antibodies must be demonstrated. However, no validated canine and feline assays for antineutrophil antibodies exist.28,29 The difficulty of developing a reliable test in veterinary medicine is related to the inherent fragility of neutrophils and their propensity to degranulate and aggregate in vitro. Many granulocyte antibodies are likely involved. In people, multiple tests are available to detect antibodies against the five different neutrophil surface antigens that are targeted in immune-mediated neutropenia.

Additionally, there are tests for people for antibodies directed against less-specific surface antigens that may be found on multiple cell types.28 The presence of antibodies against these less-specific antigens may lead to concurrent immune-mediated diseases, such as rheumatoid arthritis, immune-mediated thrombocytopenia, or immune-mediated hemolytic anemia. In veterinary medicine, IgG and complement 3 were associated with bone marrow neutrophil elements in two dogs with suspected immune-mediated neutropenia.2 More recently, IgG associated with circulating neutrophils in five of six dogs with immune-mediated neutropenia was demonstrated by using flow cytometry.30 However, it was unknown if the targets were neutrophil-specific, a shared antigen, or adsorbed surface antigen.11,30

Exclusion of other causes

Lacking tests to prove an immune-mediated cause, the diagnosis is determined by excluding other causes and by receiving a positive response to immunosuppression. Concurrent detection of other immune-mediated diseases, such as immune-mediated hemolytic anemia, immune-mediated thrombocytopenia, steroid-responsive meningitis, immune-mediated non-erosive arthritis, panniculitis, or vasculitis, also gives support to the neutropenia being immune-mediated. 3,4,8,14

TREATMENT

After a short course of antibiotic therapy, treating immune-mediated neutropenia requires immunosuppressive drug regimens.

Antibiotics

Typically, a two- to three-day course of bactericidal antibiotics (beta-lactams, aminoglycosides, fluoroquinolones, and trimethoprim-sulfonamides) is given before instituting immunosuppression. A bacteriostatic antibiotic would require a functional neutrophil population, which is lacking in these patients. A typical regimen might be ampicillin combined with an aminoglycoside. Successful treatment of a concurrent infectious process may improve clinical signs and resolve the toxic change but will not resolve the neutropenia.

Prednisone

To increase the neutrophil numbers, initiate prednisone therapy (2 to 4 mg/kg once a day). Admitting the patient to the hospital for the first few days of therapy to monitor for signs of infection (monitor rectal temperature every four to six hours) is advisable. Neutrophil counts should begin to rise within a few days of instituting immunosuppressive therapy, but it may take up to three weeks to reach acceptable counts (> 1,500/μl in dogs and > 1,000/μl in cats).5 Provided the counts remain acceptable, taper immunosuppressive therapy by reducing the dosage 25% every two to four weeks. As with other immune-mediated diseases, lifelong therapy may be required, and the lowest effective dose should be administered every other day.

Azathioprine and chlorambucil

Azathioprine at 2 mg/kg daily for one week and then at 1 mg/kg every other day may be instituted at the beginning of prednisone therapy in dogs to allow more rapid tapering of prednisone, thus minimizing prednisone-related adverse effects. Azathioprine can also be added to therapy for dogs that are not responding to prednisone alone. A CBC and hepatic serum chemistry profile should be performed one week after initiating azathioprine therapy and then monthly while the dog receives azathioprine to monitor for adverse effects.

If additional immune suppression is needed in cats, chlorambucil can be given.

Lithium carbonate

Lithium carbonate (10 mg/kg orally b.i.d.) is used in dogs for chemotherapy-associated neutropenia14 and may play a role in treating immune-mediated neutropenia. It causes a nonspecific stimulation of neutrophil production.31

Therapy in people

In people, use of recombinant granulocyte-colony stimulating factor has revolutionized therapy.32 Corticosteroids are typically contraindicated because of their affects on immunity, growth, and normal adrenal gland function in children affected with immune-mediated neutropenia. Short courses of corticosteroids, intermittent antibiotics, and intravenous IgG are core to therapy in children until disease remission occurs spontaneously. Administering human recombinant granulocyte-colony stimulating factor in dogs can lead to neutropenia because dogs may develop antibodies to it. These antibodies may then cross-react with endogenous canine granulocyte-colony stimulating factor, impairing neutrophil production in the bone marrow. Antibodies tend to develop within three weeks of initiating therapy.33 Canine recombinant granulocyte-colony stimulating factor is not commercially available, and given the induction of antineutrophil antibodies, it is contraindicated.34

Prognosis

The prognosis for remission is good, with 100% of dogs achieving normal neutrophil counts after 1 to 18 days of prednisone therapy in one retrospective study.6 In the published case reports, loss of remission once medication was discontinued was frequently, but not always, reversed with re-institution of therapy.2,11

CONCLUSION

Although not commonly recognized, immune-mediated neutropenia should be a differential diagnosis in neutropenic patients that after a diagnostic work-up have no other apparent reason for their neutropenia. These patients tend to be younger and have fairly profound neutropenias that often lack toxic change. Response to immunosuppression can be fast and dramatic and is used to confirm the diagnosis since no diagnostic test for antineutrophil antibodies is validated in animals. The validation of a canine and feline antineutrophil antibody test is needed.

Belle Marie D. Nibblett, DVM

Anthony P. Carr, Dr. med. vet., DACVIM (small animal internal medicine)

Department of Small Animal Clinical Sciences

Western College of Veterinary Medicine

University of Saskatchewan

Saskatoon, SK S7N 5B4

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