Immune Mediated Hemolytic Anemia

WHAT IS YOUR DIAGNOSIS? A 1.5 year, male neuter, domestic shorthair cat was presented to the R(D)SVS Internal Medicine Service with a three month history of pica (ingestion of cat litter and licking concrete) and a two-week history of marked weight loss and progressive lethargy. Pallor, tachycardia and anaemia (PCV 6.7%) were detected by the primary veterinarian, leading to emergency referral on the same day. On clinical examination, the cat was quiet but alert with a body condition score of 2/9. Mucous membranes were slightly tacky with marked pallor. The heart rate was 220 bpm with a grade II/VI systolic murmur but no arrhythmia or pulse deficits. The respiratory rate was 52 breaths/minute, with no adventitious lung sounds. Splenomegaly was noted on abdominal palpation. Peripheral lymph nodes were unremarkable and the rectal temperature was 39.0°C. .A blood sample was taken for routine haematology and serum biochemistry (results below). Haematology Parameter Result Reference Range RBC (x 1012/l) 0.91* 5.5-10 PCV (%) 8.0* 24-45 Hb (g/dl) 2.5* 8-14 MCV (fl) 58* 39-55 MCHC (%) 31.0 30-36 TWBC (x 109/l) 9.4 7-20 Neutrophils (x 109/l) 1.97* 2.5-12.8 Lymphocytes (x 109/l) 7.05* 1.5-7.0 Monocytes (x 109/l) 0.28 0.07-0.85 Eosinophils (x 109/l) 0.1 0-1.0 Basophils (x 109/l) 0 0-0.2 Platelets (x 109/l) 44* 300-600 page 1 of 6 Serum biochemistry Parameter Result Reference Range Total protein (g/l) 72 69-79 Albumin (g/l) 31.0 28-39 Globulin (g/l) 51* 23-50 Bile acids (μmol/l) 6.9 0-7 Total bilirubin (μmol/l) 14.1* 0-6.8 Urea (mmol/l) 7.6 2.8-9.8 Creatinine (μmol/l) 95.0 40-177 Glucose (mmol/l) 6.9* 3-5 AP (iu/l) 26 10-100 ALT (iu/l) 399* 6-83 Sodium (mmol/l) 152 145-156 Chloride (mmol/l) 120 117-140 Potassium (mmol/l) 4.4 4-5 Inorganic phosphate (mmol/l) 1.57 1.4-2.5 Total calcium (mmol/l) 2.26 2.1-2.9 1) What additional information about the anaemia would be clinically helpful? 2) How do the other haematology and biochemistry parameters help you in establishing the underlying cause of the anaemia? 3) What other tests would you like to perform? page 2 of 6 1) Establishing whether the anaemia is regenerative is likely to be clinically informative in this case. This is achieved by determining the reticulocyte count. In the cat there are two types of reticulocytes, aggregate and punctate. Aggregate reticulocytes are similar in appearance to canine reticulocytes which are short lived and rise sharply after the development of acute or severe anaemia and mature into punctate reticulocytes. In cats, blood normally contains only very small numbers of aggregate reticulocytes (0-0.9%) and up to 10% punctate reticulocytes. The punctate count can remain elevated for up to 4 weeks following a single episode of blood loss so are less useful to assess the bone marrow response. In mild anaemia, the punctate count can be useful as an indicator of the regenerative response, since in this scenario the bone marrow tends to hold onto the aggregate reticulocytes until they mature into the punctate form. It is the absolute reticulocyte count which is the most useful indicator in cats of a regenerative response and is calculated as follows: Absolute reticulocyte count(x109/l)=observed % retics x RBC count (x1012/l) x 10 Values >50 indicate a regenerative response (>60 in dogs) Values >50 would prompt investigation into causes of Haemolysis or Haemorrhage. Values <50 would prompt investigation into causes of Lack of Bone Production (but can due to acute haemolysis or haemorrhage and the bone marrow has not had time to respond i.e. first 4-5 days). For this patient the absolute reticulocyte count was 8.19 x 109/l indicating a nonregenerative anaemia. 2) The haematology results, before the reticulocyte count became available, showed a profound anaemia. The raised MCV would suggest the presence of larger cells (reticulocytes) or red cell aggregates (e.g. IMHA). However, the decrease in other cell lines (neutropenia and thrombocytopenia) was suggestive of bone marrow disease. The elevated bilirubin, together with normal albumin levels, would be consistent with haemolysis. However, the bilirubin may be elevated due to liver disease. The ALT was elevated which is commonly seen in cats with severe anaemia and is considered to be secondary to hypoxia. 3) A blood smear is always mandatory in the investigation of anaemia. In this case, the smear revealed numerous ghost cells and red cell aggregates, suggestive of immune mediated haemolysis. This was supported by a positive Coombs test. In cats with haemolysis it is important to rule out haemoplasma infection (Mycoplasma haemofelis, Mycoplasma haemominutum, Mycoplasma turicensis). The PCR was negative for all species in this cat. FeLV and FIV are implicated as cause of anaemia in cats and may affected prognosis. An in-house ELISA snap test was negative for both viruses. page 3 of 6 The cat had a severe anaemia that required a blood transfusion. Therefore, blood typing was performed and the patient was blood group B. Exact guidelines which define at what level of anaemia require transfusion with chronic anaemias are not possible because cats become quite well adapted to the anaemic over time. However, if there are signs of compromise (tachycardia, tachypnoea) such as in this case, transfusion is indicated. Most anaemias with a PCV <10% will require transfusion. Despite good evidence of an immune-mediated haemolytic chronic anaemia, there was no evidence of regeneration. Therefore, a bone marrow aspirate and core samples were taken for the right femur. Results showed marked myelofibrosis and hypocellulariy with no evidence of myeloproliferative i.e. neoplastic disease (see Figures 1 and 2). Figure 1. Bone marrow core histopathology, H&E. Note low cellularity with marrow replaced with amorphous eosinophilic material (disorganised fibrosis) page 4 of 6 Figure 2. Bone marrow core histopathology, Masson’s Trichrome. Bone marrow replaced by widespread fibrosis (green-blue). Management A matched whole blood transfusion was given which increased the PCV to 12% but this reduced to 7.5% by day four, likely due to ongoing haemolysis. Transfusions of bovine haemoglobin (Oxyglobin®) 6ml/kg were given on day 4 and 5 of treatment. By day seven the PCV had increased to 13% and the patient was discharged. Immunosuppressive treatment was commenced at diagnosis with prednisolone 2mg/kg PO q12hrs and also doxycycline 10mg/kg PO q24hrs which was discontinued once haemoplasma results were received. On re-examination on day 14, demeanour was brighter and PCV had increased to 17%. By day 24 the anaemia and heart murmur had resolved, there was weight gain to 3.1kg and progressive improvement in exercise tolerance. Prednisolone therapy was tapered off by the referring veterinarian over the following six weeks and the cat was clinically normal at followup one year later. page 5 of 6 Discussion The presence of hyperbilirubinaemia, ghost cells, autoagglutination, positive Coombs’ test and positive response to immunosuppressive therapy are consistent with a diagnosis of IMHA. Since no triggering cause could be identified, a diagnosis of primary IMHA was made. IMHA is typically thought to be cause a regenerative, which stimulated investigation into causes of an inadequate regenerative response. However, in a study of cats with primary IMHA, 58% had a non-regenerative anaemia at initial presentation. They postulated that may be due to the production of antibodies against erythrocyte precursors in the bone marrow, although bone marrow cytology was not performed. On examination of bone marrow of cats with non-regenerative immune-mediated haemolytic anaemia (NRIMHA), the pathogenesis was thought to be a combination of immune-mediated destruction and alterations in the bone marrow environment (including myelofibrosis, necrosis, inflammation and oedema) leading to ineffective haemopoiesis. A secondary dysmyelopoiesis affecting other cell lines is recognised in IMHA in cats. Myelofibrosis can occur as a primary condition or can be secondary to various conditions including IMHA, myelodysplastic syndromes, chronic renal failure, infectious diseases, lymphoproliferative and myeloproliferative disease. The majority of secondary causes are associated with a normocellular or hypercellular bone marrow, so it is unusual that the bone marrow was hypocellular in this case. However, it has been noted as an uncommon finding in dogs with NRIMHA. www.ed.ac.uk/vet/hfsa-int-med page 6 of 6