Pure Red Cell Aplasia and Nonregenerative Immune-Mediated Anemia: Important Causes of Nonregenerative Anemia

Pure Red Cell Aplasia and Nonregenerative Immune-Mediated Anemia: Important Causes of Nonregenerative Anemia

Class of 2010 (Kraun) and Department of Pathology (LeRoy)
College of Veterinary Medicine, The University of Georgia
Athens, GA 30602

A dog was referred to the Veterinary Teaching Hospital at the University of Georgia for the evaluation of severe anemia. The dog had a two-day history of lethargy and anorexia, and the referring veterinarian found a packed cell volume (PCV) of 14%.

On presentation, the patient's PCV was 19%, with macroscopic autoagglutination noted. A complete blood count the following day revealed a severe normocytic normochromic anemia with a hematocrit of 13.4%. A reticulocyte count was not performed. The hemogram also revealed a mild thrombocytopenia with many shift platelets, as well as a mild increase in the concentration of band neutrophils (although the total leukocyte and segmented neutrophil counts were within the reference range). A serum chemistry and urinalysis performed at the same time were largely unremarkable. Due to the severe anemia and autoagglutination, the patient was tentatively diagnosed with immune-mediated hemolytic anemia (IMHA). Immunosuppressive therapy was begun and the dog was transfused with 240mL of packed red blood cells. A subsequent reticulocyte count revealed a lack of regeneration. A bone marrow aspirate cytology indicated erythroid hyperplasia with maturation arrest and mild myeloid and megakaryocytic hyperplasia. Based on these findings, the patient was given a provisional diagnosis of pure red cell aplasia. This paper will explore how this preliminary diagnosis was re-evaluated.

Pure red cell aplasia (PRCA) is a disorder in which anemia is caused by erythroid hypoplasia or aplasia, but without abnormalities in other hematopoietic cell lines.¹ In truth, “aplasia” is probably an incorrect term, as the disease is thought to result from destruction of red blood cell precursors rather than a complete lack of erythropoiesis. Pure red cell aplasia has been reported in humans, dogs, and cats,¹ although relatively few cases are present in the veterinary literature. The objective of this paper is to increase awareness of this disease, to emphasize the importance of appropriate diagnostic testing, and to summarize the disease process, diagnostic criteria, therapy and prognosis for patients with PRCA.

Production of a mature red blood cell requires three stages of differentiation and proliferation. The first stage involves the differentiation of a pluripotent stem cell into an erythroid progenitor cell known as a BFU-E. This step in the process occurs under the influence of several non-specific cytokines such as IL-3, GM-CSF, and stem-cell factor, which are produced by the cell’s microenvironment. BFU-Es then differentiate and proliferate into a second erythroid progenitor, the CFU-E, but only in the presence of erythropoietin, a renal hormone and growth factor. The final step in erythropoiesis involves production and maturation of nucleated red blood cell precursors, and proceeds automatically unless there is an absence of crucial factors such as proteins, iron, vitamin B12, or folic acid.² In general, as an erythrocyte precursor becomes more mature, the cell size, nuclear-to-cytoplasm (N:C) ratio, and cytoplasmic basophilia all decrease.³

Pure red cell aplasia was first identified as a separate entity from aplastic anemia by Kaznelsen in 1922.^2,4 PRCA has also been know as erythroblastic hypoplasia, erythroblastopenia, erythroid hypoplasia, and red cell agenesis.⁴ Pure red cell aplasia in humans can be separated into three forms: a congenital (constitutional) form, and two acquired forms that may be either 1) acute or 2) chronic.

The congenital form, also known as Diamond-Blackfan syndrome, is thought to be caused by some sort of prenatal injury or mutation.² However, the effectiveness of steroid therapy in the disease has led to a search for an underlying immunologic cause.⁴ The acute, self-limiting form of PRCA has been most commonly associated with a viremia, and specifically with infection by the B19 parvovirus. In addition, numerous drugs and several nutritional deficiencies have been suspected of causing aplastic crises, although the link between nutritional deficiencies and aplasia is questionable. In any case, diagnosis of acute PRCA usually becomes evident from the patient’s clinical course, as recovery is usually already underway when the diagnosis is made.⁴

The chronic, acquired form of pure red cell aplasia has frequently been suspected of having an immune-mediated pathogenesis. An association between red cell aplasia and thymomas was noted in the 1930s.⁴ The mechanism was suspected to involve T lymphocyte-mediated destruction of erythroid cells,² which has since been found to play a major role in the pathogenesis of the disease.⁴ However, many patients without thymomas were also diagnosed with chronic red cell aplasia. Some have suggested that two or more forms of the disease may exist, some of which may be associated with thymomas, and some which may have no connection.⁵ It appears that thymomas were more common in PRCA a generation ago than they are today,² as a more recent case series showed only 2/37 red cell aplasias to be associated with thymomas.⁴ The chronic form of the disease has also been associated with other immune-mediated diseases such as rheumatoid arthritis, lupus erythematosus, chronic active hepatitis, hemolytic anemia, and chronic lymphatic leukemia.⁴ Chronic PRCA generally responds to treatment with blood transfusions and immunosuppressive agents, and the remission rate has been reported to be about 40% even with prednisone therapy alone.²

The complete pathogenesis of pure red cell aplasia is not completely known, but the disease can be either primary or secondary in nature. Secondary PRCA has been associated with parvovirus infection and treatment with recombinant human erythropoietin. Primary PRCA is believed to be a form of immune-mediated hemolytic anemia in which antibodies lead to destruction of erythroid precursor cells in the bone marrow.⁷ Most immune-mediated anemias are believed to be primary (idiopathic); however, this may reflect an inability to detect an underlying cause rather than true autoimmune disease.⁸

There is some overlap in the precise medical definitions of PRCA and non-regenerative immune-mediated anemia (NRIMA). Some define pure red cell aplasia as complete arrest of the erythroid cell line at any stage of maturation. However, most^6,7 agree that a patient qualifies as having pure red cell aplasia only if bone marrow samples are devoid of, or nearly completely lacking in, all red cell precursors. Those in the latter group tend to refer to immune-mediated destruction of later-stage precursors as “nonregenerative immune-mediated anemia (NRIMA),” and believe that PRCA is the most severe or end-stage expression of NRIMA.⁶ Using the most favored definition of pure red cell aplasia, the patient presented at the beginning of this paper should have been diagnosed not with PRCA, but with NRIMA.

Regardless of whether or not one separates pure red cell aplasia from nonregenerative immune-mediated anemia, it appears as though antibody-mediated immune destruction of erythrocyte precursors is a key mechanism of both manifestations of the disease. Typically, in normal animals, suppressor T lymphocytes prevent reaction of autoantibodies with host tissues. However, as is thought to be the case in humans, animals with immune-mediated anemias may have defective suppressor T cell function or hyperfunctioning immune systems. These characteristics may suggest a genetic predisposition to the development of these conditions.⁸

The exact antigen(s) to which the antibodies react in nonregenerative immune-mediated anemias is unknown, but late-stage erythroid precursors (metarubricytes and even reticulocytes) appear to be targeted in many dogs, while true PRCA is very uncommon. In some cases, the attack may be aimed at a maturation-associated antigen, which leads to destruction only of erythroid precursors. However, some antigens are common on both precursors and mature red blood cells, so it is possible that destruction of both stages may occur if a common antigen is targeted. Additionally, some patients have developed immune-mediated thrombocytopenia following successful treatment of this disease, indicating that they may have had a more generalized immune-mediated reaction.⁶ It is possible that the patient described in this paper may have been suffering from a more generalized immune attack involving both red blood cells and platelets as targets of immune destruction.

Immune-mediated anemias are much more common in dogs than in cats,⁸ although there is a report of pure red cell aplasia in 9 cats between 8 months and 3 years of age, all of whom tested seronegative for retroviruses (FeLV/FIV).⁹ Middle-aged female dogs tend to be overrepresented,^6-8 as is the case with many immune-mediated diseases. Two studies of PRCA and NRIMA have shown a significant overrepresentation of Labrador Retrievers when compared to overall hospital population, indicating that the breed may somehow be predisposed to developing this disorder. Interestingly, breeds commonly affected by immune-mediated hemolytic anemia (Cocker Spaniels, Old English Sheepdogs, Irish Setters, Poodles, English Springer Spaniels, and Collies) do not appear to be at increased risk for development of PRCA.⁶

Patients with pure red cell aplasia may present to the veterinarian with a relatively nonspecific history and very few clinical signs. This may result from the chronicity of the disease and the ability of the body to compensate for a slowly progressive anemia. Owners may report simply that their pets are lethargic and/or anorexic, with few other presenting complaints. Patients may also have a history of pallor, weakness, exercise intolerance, weight loss, and collapse.^6,7

Physical exam commonly reveals pale mucous membranes and tachypnea, and possibly hepatomegaly and splenomegaly.^6,7 Cardiovascular changes are common in severely anemic (PCV < 20%) patients, and may include tachycardia, gallop rhythm, and a grade II-III/VI systolic heart murmur. The systolic murmur heard in anemic patients is due to abnormal blood turbulence⁸ rather than a true cardiac abnormality. If a patient has a more generalized immune-mediated anemia that also affects circulating red blood cells, icterus may be seen in addition to the previously mentioned abnormalities due to marked destruction of red blood cells and increased bilirubin metabolism.

Complete Blood Count / Serum Chemistry / Urinalysis: The most readily obvious laboratory abnormality in a patient with pure red cell aplasia is a nonregenerative anemia. The anemia can be severe, and is typically normocytic and normochromic with occasional spherocytosis.¹ The patient may be positive on a Coombs’ (direct antiglobulin) test¹ depending on the antigen targeted by the immune attack. Serum chemistry and urinalysis are unremarkable in many patients. The most common biochemical abnormalities reported are increased liver enzyme activities (possibly due to hypoxia, corticosteroid administration, and cholestasis), low bicarbonate concentration (due to hypoxia and subsequent lactic acidosis), and hyperferremia (due to lysis of red blood cells).⁶ However, these findings are inconsistent between patients. Additionally, if an antigen that is common to erythroid precursors and mature RBCs is targeted, evidence of peripheral red cell lysis may rarely be seen (hemoglobinemia/hemoglobinuria, hyperbilirubinemia/bilirubinuria, etc.).

Thoracic and Abdominal Imaging: Observations may include mild hepatomegaly or splenomegaly, but radiographic and ultrasonographic exams generally detect few to no abnormalities.

Bone Marrow: It is not possible to predict the condition of the bone marrow from hematologic findings alone. Hemogram results in patients with pure red cell aplasia can appear very similar to findings in patients with erythroid hyperplasia.⁶ Therefore, it is very important to obtain bone marrow aspirates in patients with nonregenerative anemias. In one study of 13 dogs with pure red cell aplasia,⁷ the marrow of 9 dogs contained no erythroid cells, while rare rubriblasts and prorubricytes were seen in 4 dogs. Another study of PRCA and NRIMA⁶ found erythroid hypoplasia in 11/42 dogs (26%) and no erythroid precursors in 2/42 dogs (5%). Myelofibrosis has been reported to occur in dogs with immune-mediated anemias, which can make it difficult to obtain bone marrow samples. Patients with more generalized attack on erythroid cells may develop some fibrosis over time secondary to immune-mediated destruction of red cell precursors; however, in both studies cited here,^6,7 bone marrow core biopsies revealed no collagen fibrosis in patients with complete absence of erythroid precursors in the bone marrow (PRCA).

Figures 2A and 2B. Normal bone marrow (A, left) and bone marrow from the patient described in this paper (B, right). The patient’s erythroid series consists almost exclusively of high numbers of rubriblasts and prorubricytes. A few rubricytes are seen, but later stage precursors are virtually absent. These findings are consistent with a diagnosis of nonregenerative immune-mediated anemia. No bone marrow slides were available from a patient with pure red cell aplasia, and a search of medical records did not indicate that a patient with PRCA has been seen recently at the University of Georgia.

Treatment for pure red cell aplasia and nonregenerative immune-mediated anemias is multimodal, and is often similar to treatment for peripheral forms of immune-mediated hemolytic anemia. As PRCA is believed to be an immune-mediated disease, one of the major aspects of treatment involves immunosuppression. A variety of immunosuppressive drugs are now available. Corticosteroids (for example, prednisone and dexamethasone) have been the mainstay of immunosuppressive therapy for quite some time. They are relatively inexpensive, fast-acting, and fairly efficacious. However, long-term immunosuppressive doses of corticosteroids are known to produce several undesirable side effects (iatrogenic Cushings disease), so additional drugs are often utilized to allow the clinician to taper the dose of steroids over time. Two commonly used, additional immunosuppressive agents are the T cell inhibitors, azathioprine and cyclosporine. In addition to immunosuppressive therapy, patients with immune-mediated anemias require a significant amount of supportive care, such as blood transfusions.

The prognosis for patients with PRCA and nonregenerative immune-mediated anemia was once believed to be much poorer than the prognosis for patients with regenerative immune-mediated anemias. However, recent data indicates that the prognosis for NRIMA may be equivalent to or better than regenerative IMA, and the long-term prognosis for PRCA may even be better than the prognosis for other NRIMA.⁷

In one retrospective study⁷ of 13 PRCA dogs treated with prednisolone +/- cyclophosphamide, 10 dogs had complete remission of anemia, and 1 dog had partial remission. The other two dogs were euthanized within four weeks after treatment was initiated, and it is unknown whether they would have also responded if given more time. For dogs that did respond to treatment, the median initial response time (response defined as a 5% increase in hematocrit) was 38 days (range 22-87 days), and the median time for complete remission (defined as a normal Hct) was 118 days (range 58-187 days). Another study⁶ examined dogs with PRCA and NRIMA treated with various combinations of corticosteroids, cyclophosphamide, and azathioprine. In this study, 55% of dogs had a complete response to therapy, 18% had a partial response, and 27% had no response. For those who responded, initial response was seen at a median of 2 weeks (range 1-10 weeks), and remission was seen within 1-10 months after the start of treatment.

Mortality statistics obviously vary between studies, but is has been reported that in dogs with regenerative immune-mediated anemia, the mortality rate is approximately 29%, while in dogs with nonregenerative immune-mediated anemia, the mortality rate is 28%.⁷ This may result because patients with regenerative IMA do not typically die from the anemia itself.¹⁰ More commonly, they die from hemolysis-associated activation of the coagulation system, which leads to disseminated intravascular coagulopathy (DIC) or pulmonary thromboembolism (PTE).⁷ Peripheral hemolysis is not as common in patients with PRCA and NRIMA, so they do not commonly suffer from DIC or PTE. Therefore, patients with PRCA and NRIMA may actually have a better long-term prognosis than patients with regenerative IMA if given time to respond to therapy.

Pure red cell aplasia is a rare disorder in veterinary medicine in which anemia is caused by erythroid hypoplasia or aplasia, but other hematopoietic cell lines are not defective. The disease may sometimes be confused with nonregenerative immune-mediated anemia (NRIMA), but the strict definition of PRCA requires a complete (or nearly complete) absence of all erythroid precursors in the bone marrow. PRCA is believed to be the most severe, end-stage manifestation of NRIMA. The pathogenesis is not completely understood, but is thought to involve antibody-mediated destruction of red cell precursors. Antibodies may be directed against a maturation-associated antigen, or against an antigen that is common to both precursors and mature red blood cells. PRCA has been reported in humans, dogs, and cats. Patients commonly present for nonspecific signs such as lethargy and anorexia, and initial blood work shows a nonregenerative anemia. There may or may not be additional clinical signs and laboratory abnormalities. As hemogram results can be similar for PRCA and other causes of nonregenerative anemia, bone marrow sampling is essential for appropriate diagnosis. As PRCA is believed to be immune-mediated, treatment consists mainly of immunosuppression and supportive care (including blood transfusions as necessary). The prognosis in PRCA and other nonregenerative immune-mediated anemias is guarded, but may be better than the prognosis for regenerative immune-mediated anemias (such as the more classic peripheral form of IMHA) if patients are given an appropriate amount of time to respond to therapy.

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The images of the bone marrow needles, transfusion items, and the photomicrographs of the bone marrow aspirates were obtained by the primary author (MK) with permission. A big thank you goes to Dr. Tripp Almy for the erythropoiesis images, as well as the members of the Clinical Pathology and Internal Medicine services at the UGA College of Veterinary Medicine for their help with this paper and for assisting with my understanding of this disease process and case.