The following report describes an unusual case of poor performance. The findings of clinical examination were unremarkable in a 16 year-old gelding, however neutropaenia and anaemia were identified on haematological examination. A bone marrow disorder was suspected and ultimately confirmed. The investigation of anaemia in horses is discussed with reference to this unusual case.
A 16-year-old Arab gelding was presented for investigation of poor performance. The horse was being brought back into training for endurance competition having been turned out for the winter and was not attaining previous levels of performance. In addition, recovery from exercise was protracted and heart rate was slow to return to normal. The owner felt the horse was in poor body condition compared to previous years, despite regular administration of anthelmintics and regular dental treatment.
CLINICAL EXAMINATION AND INITIAL LABORATORY FINDINGS
The horse was in moderate body condition (body condition score 4/9) and had a normal demeanour. Heart and respiratory rates were increased, at 48 beats and 30 breaths per minute respectively; rectal temperature was within normal limits (37.6°C).
The findings of clinical examination were otherwise unremarkable and blood was collected for haematological and biochemical examination.
Haematology revealed neutropaenia, with a resultant leucopaenia, and anaemia (Table 1). Mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) were highly suggestive of a regenerative response.
Table 1. Haematology and clinical chemistry results in an Arab gelding diagnosed with disseminated lymphoma (bold type indicates abnormal values)
3.7 × 109/l
5.50–12.50 × 109/l
1.9 × 109/l
2.50–8.00 × 109/l
1.7 × 109/l
1.50–5.50 × 109/l
0.1 × 109/l
0.00–0.900 × 109/l
0.0 × 109/l
0.00–0.800 × 109/l
0.00–0.300 × 109/l
2.74 × 1012/l
6.50–12.5 × 1012/l
130 × 109/l
100–500 × 109/l
Gamma glutamyl transferase
On blood biochemistry, globulin and fibrinogen were high and albumin low, consistent with a chronic inflammatory response.
Abdominocentesis was performed in an attempt to identify whether a focus of inflammation might be present within the abdominal cavity. Peritoneal fluid had a normal gross appearance and normal total protein concentration. Cytological examination revealed normal numbers of cells with normal morphology.
Transcutaneous and transrectal ultrasonographic examinations of the abdominal cavity did not reveal any abnormalities and similarly no abnormalities were detected on transcutaneous ultrasonographic examination of the thoracic cavity.
An inflammatory response resulting in consumption of neutrophils with concurrent anaemia of chronic disease was considered as an explanation for the changes in the haemogram. However, with chronic inflammation a neutrophilia would be expected rather than a neutropaenia and, in the face of a process resulting in marked consumption of neutrophils, an increase in the release of band forms from the bone marrow would also be expected. Furthermore, the degree of anaemia in this case was profound for anaemia of chronic disease. A bone marrow disorder was considered a more likely explanation.
The horse was sedated with detomidine and butorphanol and the skin overlying the sternum was aseptically prepared in preparation for the collection of a bone marrow aspirate and core biopsy.
Bone marrow aspirate
Following ultrasonographic examination to identify the site of the sternebrae, 5 ml of 2% lignocaine was injected beneath the skin and into the subcutis, muscle and periosteum in the midline. A small stab incision was made through the skin using a 15 blade and a 15 cm, 18 G spinal needle, inserted until it made contact with the bone. With application of steady pressure the needle was inserted through the cortical bone for 2 cm before the stylet was withdrawn. There was no palpable change in resistance as the needle passed from the cortical bone to the marrow cavity. The stylet was removed and a 20 ml syringe containing a small amount of heparin was attached (prior to commencing the procedure heparin was drawn into the syringe and then injected out leaving the syringe heparin-lined).
Short, rapid and forceful draws were applied to the syringe until a small bead of red material was drawn into the hub of the syringe (Fig. 1). The needle was then withdrawn and the material injected onto a series of glass slides (Fig. 2). The slides were placed together and drawn over one another to create smears. The sample contained small gritty spicules of bone marrow that could be felt when the slides were drawn across one other and globules of fat could be visualized. Both of these findings were suggestive of the sample being marrow rather than frank blood. The slides were allowed to air dry and were submitted for cytological examination.
In order to obtain a core of bone marrow for histopathology a Jamshidi needle was inserted through the same hole in the skin and, as it made contact with the sternum, the screw cap and stylet were removed. With steady pressure and rotation the Jamshidi needle was passed through cortical bone to the marrow cavity. A finger was placed on the needle 3 cm from its tip from the outset and when this finger reached the skin it was estimated that the desired depth had been reached. The needle was then rotated and gently rocked back and forth to ensure the core had detached, after which the needle was withdrawn. The core was removed from the needle into 10% formal saline by passing the stylet through the barrel of the needle. The core that was collected comprised dark red marrow at one end and cortical bone at the other (Fig. 3).
Examination of both smears and sections of the core biopsy revealed cells from erythroid and myeloid cell lines, intermixed with megakaryocytes. There was evidence of progressive and normal maturation of erythroid forms, however, given the degree of anaemia, there were subjectively low numbers of immature erythroid forms. The myeloid:erythroid ratio was calculated to be 2:1.
In some areas there was a predominance of small- to medium-sized lymphocytes, some of which were nucleated, effacing the normal bone-marrow architecture. The abnormal appearance and infiltrative nature of the lymphoid aggregates was strongly suggestive of lymphoid neoplasia. In view of these findings the horse was euthanased.
Post mortem examination
At examination post mortem the humerus, ribs and sternum were sectioned. In all of these bones there were only small areas of red bone marrow representative of regions of haematopoiesis (Fig. 4). On histopathological examination, the grossly red bone marrow was moderately cellular, but scattered throughout the areas of haematopoiesis were numerous areas of dense lymphoid infiltration. It was estimated that 30–50% of the bone marrow from the bones examined was effaced by aggregations of small- to medium-sized lymphocytes.
The cranial thoracic, mesenteric and splenic lymph nodes were enlarged and some were an abnormal dark-red colour (Fig. 5). In addition, there was a large amount of lymphoid tissue around the trachea. On histopathological examination, the architecture of all of these lymph nodes was effaced by a mixed infiltrate of cells, with a predominance of medium-sized lymphocytes with round to rarely cleaved nuclei that occasionally contained nucleoli. The mitotic rate varied from 3–8 per high-power field.
Infiltrates of abnormal lymphocytes were also identified within sections taken from the spleen, kidneys, liver and large colon. These findings were consistent with a diagnosis of disseminated lymphoma with infiltration of the bone marrow.
The presence of a peripheral neutropaenia and anaemia as seen in this case would be expected to stimulate a regenerative response for both cell lines in the bone marrow. Subjectively, however, there was insufficient evidence of regenerative bone marrow in the current case. Reported myeloid-to-erythroid ratios in normal horses are wide (0.5–2.4) and interpretation in individual cases is therefore made in conjunction with examination of peripheral blood. The relatively high myeloid:erythroid ratio, whilst still within the normal range, was not consistent with an adequate erythroid regenerative response given the degree of anaemia. Small lymphocytes are normally present in low numbers in bone marrow, however, in the current case, there were large numbers and in areas of the bone marrow they were the dominant cell type.
This abnormal and focal proliferation of a single cell line was consistent with a neoplastic process and hence a diagnosis of lymphoid leukaemia. The results of histopathology of peripheral lymphoid tissues were also indicative of lymphoid leukaemia. Immunophenotyping using monoclonal antibodies to specific cell-surface antigens could have been performed to further classify the neoplastic cell line.
Analysis of bone marrow is a useful diagnostic tool in horses because equine erythrocytes are generally not released from the bone marrow until they are fully mature. An increase in MCV, as in this case, may be suggestive of regenerative anaemia, however the normal MCV of horses is variable and, in the absence of reference values for the individual horse, increases in MCV may be missed. Increased red-cell distribution width indicative of variability in red blood cell size is not seen commonly in horses, further hampering the diagnosis of regenerative anaemia in this species. Therefore the only means of reliably assessing the regenerative response is to obtain a sample of bone marrow.
Bone marrow neoplasia may be classified as lymphoproliferative or myeloproliferative and may or may not be associated with the release of abnormal cells into the peripheral blood. In the majority of cases, absolute numbers of each cell type in peripheral blood remain within normal limits. In the present case, lymphocytes of abnormal appearance were seen on a blood smear but the changes were insufficient to make a definitive diagnosis of lymphoid neoplasia on haematology alone.
Abnormal lymphocytes have been estimated to be present in 30–50% of cases of lymphoma with bone marrow involvement. The abnormal circulating leukocyte and erythrocyte numbers in this case were due to the secondary effects of destruction of normal bone marrow by the neoplastic process rather than the release of neoplastic cells. In this case it is likely that the bone marrow became affected secondary to a primary lymphoid neoplasm at a distant site. Typically, with primary lymphocytic leukaemia, there is diffuse involvement of the bone marrow and aggregations of neoplastic lymphocytes tend not to be seen at sites other than the bone marrow. By contrast, secondary lymphocytic leukaemia is generally associated with extensive involvement of other tissues and more focal aggregates of neoplastic cells within the marrow. Secondary disease is also far more common.
Lymphoma is the most common form of haemopoietic neoplasia in horses and has been reported to affect a wide range of tissue types, although infiltration of the bone marrow is uncommon. Four anatomic forms of lymphoma are described traditionally: generalised, intestinal, mediastinal and cutaneous. However, this classification is overly simplistic and in the majority of cases dissemination is widespread before clinical signs develop. Clinical signs depend upon the tissues most severely affected, but the most common signs are weight loss and subcutaneous oedema with peripheral lymphadenopathy being an inconsistent finding. Haematological and blood biochemical changes in horses with lymphoma may be similarly diverse.
Neoplastic infiltration often results in evidence of chronic inflammation including neutrophilia, leukocytosis, anaemia, hyperfibrinogenaemia, hypergammaglobulinaemia and high serum amyloid A concentrations. Paraneoplastic syndromes may also be recognised. The peripheral lymphocyte count is rarely increased and, as in this case, tends to be normal or decreased.
In conclusion, this case highlights the benefits of analysis of bone marrow aspirates and biopsies in cases of unexplained anaemia and cases in which abnormal haematopoietic cells are identified in a peripheral blood smear. The presence of concurrent neutropaenia in this case was further evidence of bone marrow involvement. The bicytopaenia was ultimately determined to be due to infiltration of bone marrow by metastatic spread of lymphoid neoplasia (lymphoma). Although this is an uncommon presentation for lymphoma it highlights the diverse nature of this neoplasm in horses.
CONTINUING PROFESSIONAL DEVELOPMENT
In order to test your understanding of this article, answer these multiple choice questions, or if you are a subscriber, go online at http://www.ukvet.co.uk, and find many more multiple choice questions to test your understanding.
1Which of the following is the most reliable means of identifying regenerative anaemia in the horse:a. Reticulocyte countb. Red-cell distribution widthc. Bone marrow myeloid:erythroid ratiod. Mean corpuscular volume
2Which of the following clinical biochemical findings would not be expected in association with chronic inflammation:a. Increased serum amyloid A concentrationb. Increased globulin concentrationc. Increased albumin concentrationd. Increased fibrinogen concentration
3Which of the following sites is not a recognised site for collection of bone marrow in the horse:a. Dorsal ribb. Tuber ischiumc. Sternumd. Distal tibia