Strategic Objectives



Author: Dr L.L. van der Merwe

With Input from Prof A. Leisewitz, Dr A. Goddard, and Dr F. Kettner

All of the Department of Companion Animal Clinical Studies, Faculty of Veterinary Science, Onderstepoort.

This article will primarily revolve around the management of dogs with immune mediated destruction of erythrocytes secondary to babesiosis and will discuss the ideal monitoring parameters for such a case. The other causes of secondary immune mediated haemolytic anaemia (IMHA) (drugs, bacterial infections, neoplasia, etc) or primary IMHA will not be discussed in any detail. They all show very similar presenting clinical sign, the disease will progress in a similar manner, and also develop similar complications although the long term prognosis does vary.

Immune mediated haemolytic anaemia secondary to babesiosis will be self evident due to the temporal relationship with finding the parasite on a stained bloodsmear, or a history of the patient having recently being treated for babesiosis. Other secondary causes of IMHA may be much more insidious and present a greater diagnostic challenge.


The management and monitoring of a patient with haemolytic disease is a combination of both clinical and laboratory parameters.

1. Initial Patient Evaluation


Clinical signs are attributable to:

The degree of anaemia

Clinical signs attributable to anaemia will depend on the acuteness of the erythrocyte destruction and whether compensatory mechanisms have been activated.

The type of anaemia /haemolysis

Intravascular haemolysis

Hallmarks of this form of haemolysis include haemoglobinuria, haemoglobinaemia (red serum) and splenomegaly.

Extravascular haemolysis

Haemoglobinaemia and haemoglobinuria are absent as the erythrocytes aer lysed within phagocytes, but splenomegally is usually present.

Multi-systemic signs

Multi-systemic organ disease and/or failure may develop due to excessive or imbalanced cytokine secretion, the generation of free oxygen radicals, immune system dysregulation, endothelial damage, activation of the coagulation cascade, and disturbances in oxygen supply to the tissues. These complications may occur in any haemolytic disease be it babesiosis or primary IMHA.

2. Laboratory Evaluation

  1. Full Evaluation of a blood smear

There is more to the evaluation of a blood smear than simply finding Babesia parasites:

Blood smears should always be good quality (with straight edges and well defined shoulders and a feather edge) and be properly stained for evaluation. Diagnosis may easily be missed if the quality of the smear is poor!
Red cell regenerative capacity should be assessed. Reticulocytes and normoblasts cause anisocytosis and polychromasia with an increase in red cell distribution width (RDW). The smear may appear non-regenerative in acute severe haemolysis as the bone marrow storage pool is used and a 2-3 day lag phase exists between red cell destruction and the appearance of a bone marrow response in the peripheral circulation. Acute babesiosis also seems to suppress the red cell regenerative capacity and the degree of regeneration in the presence of an active parasitaemia is usually inappropriate. Strong red cell regenerative responses should, however, be seen soon after elimination of the Babesia parasite.
Evaluate for concurrent Ehrlichia infection in both the monocytes as well as the neutrophils. It is important to note that phagocytosed erythrocytes and parasites may resemble Ehrlichia morulae.
The presence of spherocytosis (small, dark staining, round erythrocytes without central pallor) indicates an immune-mediated process in red cell destruction. When more than 40-60% of red cells are spherocytes, a diagnosis of IMHA is strongly supported (although IMHA can be present with lower percentages).
Haemolytic disease, with or without a concurrent Babesia infection is an inflammatory process. Any inflammatory process in the body may induce a neutrophila with a left shift (increased band cells). This is usually a regenerative left shift and the presence thereof does not imply bacterial infection. Babesia infection without an inflammatory leukogram may be indicative of concurrent immunosuppressive disease (such as Ehrlichia, or distemper).


  1. Packed cell volume (PCV):

The PCV is a percentage value obtained by centrifuging a blood sample in a micro-pipette tube. The haematocrit, on the other hand, is a haematology analyser calculated value based on red cell number and mean red cell volume.

Determine the initial PCV at time of presentation. Whether a blood transfusion is necessary is usually not a decision based on PCV alone. Other factors such as the clinical state of the dog, the availability of blood, the chronicity of the condition, ISA positivity with ongoing erythrocyte destruction, and financial constraints will all affect this choice. The initial PCV value is also useful to assist with the evaluation of response to therapy. Dogs that have chronic disease where the anaemia evolved slowly will be better able to cope with a low PCV than dogs that have experienced a rapid decrease in the PCV.
The buffy-coat can also be smeared to evaluate the white blood cells for the presence of Ehrlichia.
Together with the PCV the serum component should also be assessed.
If the sample is collected atraumatically, any red discolouration of the serum can be interpreted as intravascular haemolysis.
Total serum proteins (TSP) can be read by a refractometer. In young animals especially, verminosis is commonly occurs concurrently with babesiosis. A low TSP is an indication for a whole blood rather than a packed red cell transfusion, or for patient support with intravenous colloids administration.
Mild to moderate hypoalbuminaemia is common in babesiosis because of the inflammatory nature of the disease and the loss of albumin through damaged endothelium. However, the estimation of the TSP by a refractometer is a crude estimation of the albumin levels as increases in globulins (common in Babesia infection) may mask the decrease in albumin.

c) In-Saline Agglutination (ISA)/ Autoagglutination:

Auto-agglutination is the result of the spontaneous aggregation of erythrocytes due to the presence of erythrocyte-membrane bound immunoglobulins. These antibodies may be primary or acquired auto-antibodies. Autoagglutination must be differentiated from rouleaux formation, which occurs when erythrocytes stack together due to electrostatic forces caused by the presence of serum proteins (fibrinogen and acute phase globulins).
The ISA test is performed to check for an immune mediated reaction against the erythrocytes. Rouleaux will be dispersed by the addition of saline. False positives occur if insufficient saline is used to dilute the sample. The standard test procedure involves adding 1 drop of whole anti-coagulated blood to 6 drops of saline. This mixture should be well mixed. The slide must be evaluated under a microscope at 400 ´ magnification (no oil immersion) with low light intensity and the light condenser dropped low.
A negative ISA test does not, however, imply that IMHA is absent as the test is relatively crude. The interpretation can be graded according to the size of the aggregates.
It should be noted that the ISA test is useful and should be done in all cases of babesiosis at initial presentation together with the PCV. If it is not done at initial presentation, it is imperative that it be performed in all cases that respond poorly to therapy.
It should also be noted that response to immunosuppressive treatment for IMHA should be based on a rise in PCV and not by the resolution of a positive ISA reaction. Most dogs that respond well to immunosuppression (as seen by a rising PCV) will remain ISA positive for a few days despite good recovery. This occurs because the antibodies responsible for agglutination have a long half-life and will need to be cleared from circulation before the positive ISA reaction resolves. Therefore treat the PCV and not the ISA reaction.

d) Coombs’ Test

Although a threshold density of erythrocyte membrane bound antibodies is required to cause auto-agglutination, the sensitized reticuloendothelial system of the body can recognize cells that are marked (opsonised) with only very few antibodies.
The Coombs’ test is more sensitive than ISA in the diagnosis of IMHA as it involves the incubation of washed patient erythrocytes with serum containing anti-immunoglobulin antibodies which increases cross-linkages and thus potentiating agglutination at lower immunoglobulin levels. This test is indicated in patients exhibiting clinical and laboratory signs of regenerative anaemia with or without spherocytosis that do not show any evidence of blood loss. There is no need to perform this test if the patient is ISA positive.
As both the Coombs’ and ISA tests are affected by foreign erythrocytes, they must be performed PRIOR to any blood transfusion.

The initial evaluation of a patient with a haemolytic disease and laboratory monitoring is a synthesis of the abovementioned simple laboratory tests, a good clinical examination and common sense.

Always remember that TRENDS are all important and one-off examinations are often misleading.

3. Patient Monitoring

The complications associated with the haemolytic disease, be it babesiosis or primary or secondary IMHA, are generally due to a combination of anaemia and the systemic inflammatory reaction. The organs that are most frequently secondarily affected include the lungs (causing acute respiratory distress syndrome - ARDS), kidneys (causing acute renal failure - ARF), liver (causing icterus), erythrocytes (causing secondary IMHA), the brain (causing cerebral signs) and coagulation disorders (including pulmonary thromboembolism - PTE).

The assessment and monitoring must therefore include criteria that would evaluate these organ systems:

  1. Lungs:
  2. Monitor for sudden increases in respiratory rate and depth, referred breath sounds and increased effort of respiration. Always remember that very anaemic Babesia infected dogs always have profound blood gas and acid-base disturbances, which should resolve with blood transfusion. If respiratory signs worsen or improve only to suddenly deteriorate again following transfusion, consider ARDS or pulmonary thromboembolism. Increases in rate and depth are signs on an early decrease in pulmonary compliance due to fluid accumulation in the interstitium. By the time rales are auscultated the fluid accumulation is severe the alveoli are flooded, which may be a very poor prognostic sign. Radiographs, although helpful in confirming pathology do not take the place of a careful clinical evaluation as clinical changes will precede radiographic changes.

    The initial monitoring plan of a sick Babesia patient should include frequent (hourly) assessment of the respiratory rate for at least the first 6 hours. This parameter is a simple yet sensitive indicator of decreased pulmonary function.

  3. Kidneys:
  4. Monitor for urine production, especially in haemoconcentrated babesiosis cases which very frequently present with ARF. Oliguria or anuria will precede the development of azotaemia (increases in urea and creatinine). Note that haemolysis will cause an increase in urea without a linear increase in creatinine. As such urea used alone is a poor indicator of renal function in this scenario and should always be used with creatinine or creatinine should be used alone.

  5. Secondary/acquired IMHA:
  6. The majority of clinically ill babesiosis infected dogs are Coombs’ positive and it is estimated that about 20% of cases seen at Outpatients section of the Onderstepoort Veterinary Academic Hospital are ISA positive at the time of Babesia diagnosis. The decision on how to proceed with these cases is based on the PCV at the time of diagnosis, ISA positivity, the colour of the serum, and the general clinical condition of the animal Many of these patients are ISA negative within 24 – 48 hours and thus do not require a long tapering course of prednisilone as eradication of the parasite has removed the trigger or the initial test was a false positive.

    Occasionally a case can develop into a severe and clinically important secondary IMHA where auto-antibodies have developed against normal erythrocyte membrane antigens. These patients are challenging to manage and require aggressive therapy often with multiple transfusions and strong immunosuppressive therapy of several months duration.

  7. Follow-up evaluation

It is important to request a re-evaluation of the dog the day following initial therapy. Uncomplicated cases will show a distinct improvement in habitus and clinical signs, although the PCV may still be quite low or even the same as the previous day. The parasitized cells are removed from circulation and the PCV may fall between 1-7% six hours after injecting of the babesiacidal agent. This percentage will obviously be affected by the regenerative capacity of the patient and the initial parasite density.

The monitoring of a patient with haemolytic disease /true IMHA involves the following basic in-house laboratory tests:

A repeat blood smear evaluation is essential to evaluate for regeneration and spherocytosis.
Ensure that the ISA test has been performed accurately. ISA must be evaluated microscopically. If the patient is positive at a 1:6 dilution with saline, increase the ration to a 1:20. A true ISA will still be positive whereas strong rouleaux will become negative.
Determine if the PCV is stable, decreasing or increasing. This parameter must be interpreted together with the identification of regeneration on the blood smear. IMHA cases are usually highly regenerative. Good regeneration with a stable PCV indicates ongoing erythrocyte destruction. Concomitant disease or chronic inflammation may decrease the regenerative capacity.
Evaluate serum colour for haemolysis

A typical regimen for a mild, babesiosis induced IMHA is a tapering dose of 2mg/kg decreasing to 0.5 mg/kg over 7 days.

B. Primary IMHA

The diagnosis of primary IMHA is challenging as it requires the exclusion of all possible secondary causes, which are numerous.

To simplify matters an abridged version of causes follows.

Infectious: – ensure that the patient has been thoroughly examined for any chronic infections: endocarditis, osteomyelitis, discospondylitis, urinary tract infections, prostatitis, pyometra, dirofilaria, ehrlichia

Drugs: - many medications are capable inducing IMHA. Vaccinations, especially rabies are probable causes if given less than 3-4 weeks previously. Potentiated sulpha drugs are major inducers of auto-immune reactions and may cause immune mediated thrombocytopaenia, IMHA, immune mediated polyarthritis and keratoconjunctivitis sicca (KCS). Cephalosporins and penicillins are also predisposed.

Neoplastic : Neoplastic cells may express non self antigens on their cell surfaces and as such may induce a variety on immune mediated conditions e.g. Thymoma induced Myaesthenia gravis.

A thorough, logical and often costly clinical and diagnostic plan is required to eliminate secondary causes.

Primary IMHA is more resistant to therapy and will require very gradual tapering with duration of therapy up to 6 months. In large breed dogs it is often advisable to add azathioprine early in the course of therapy to allow a reduction in the prednisilone dosage and thus decrease the side effects.

These dosage regimens are not a hard and fast rule but will be modified according to regular patient and haematology re-evaluations. These parameters should be re-assessed after each dosage reduction of the immunosuppressive therapy and at regular 2-3 week intervals to ensure that the condition is resolving. Often the prednisilone is tapered too rapidly and the haemolysis starts up again. If no monitoring is performed the animal will only present when the PCV is so low that it is once again causing clinical signs.





Side effects/ comments


* Blocks macrophages

* Decreases antibody production


* 4-5 days to effect

2-4 d is immunosuppressive

(> 20kg = 3mg/day, <20kg = 4mg/kg/d … BSA effect)

taper initially by decr. dose by 50% every 2-3 weeks until 1mg/kg/day.

Maintain for 4 weeks, then decrease to 0.5mg/kgoid for 4w then 0,5mg/kg alt days for 4-8 weeks.

* Large breed dogs may develop severe muscle wasting and weakness. Advise combination with azathioprine to allow dose reduction ASAP.

* Small breeds show more typical cushingoid side effects of weight gain and pu/pd

* GIT ulceration (prevent with MisoprostilÒ )



( AzapressÒ )

* Suppresses T cell function

* 2 weeks to peak effect

Initiating dose of 1-2 mg/kg / day then maintenance at 1 -2 mg/kg every alternate day

( often alternating with prednisilone)


* Contraindicated in cats

* May cause pancreatitis and bone marrow suppression


(EndoxanÒ )

* Anti-neoplastic agent

* Antibody and lymphocyte suppression


50mg/m2 first 4 days of each week for maximum of 4-6 weeks.


Not recommended by the author

* severe bone marrow suppression

* haemorrhagic sterile cystitis


(Human Intravenous Gamma globulin)

* Saturates Macrophage Fc receptors.

* Immediate effect


Indicated for erythrophagocytic IMHA.

0.5 – 1.5 g/kg iv over 4-6 hours


* Very Expensive

* Bridging therapy



* Inhibits amplification of immune reaction


5 – 15 mg/kg / day

serum levels > 200ng/ml

* Expensive

* Bridging therapy


* Decreases RBC fragility


5mg/kg tid

* For chronic management


    1. Jacobson LS, Reyers F et al. Changes in haematocrit after treatment of uncomplicated canine babesiosis: a comparison between diminazine and trypan blue, and an evaluation of the influence of parasitaemia. Journal of the South African Veterinary Association 1996 vol. 67 (2) pp 95 – 105
    2. Grundy SA, Barton C. Influence of drug treatment on survival of dogs with immune mediated haemolytic anaemia: 88 cases (1989 – 1999) Journal of the American Veterinary Medical Assosciation. 2001 vol. 218 (4)543 – 545
    3. Kellerman DL, Bruyette DS. Intravenous Human Immunoglobulin for the Treatment of Immune-Mediated Hemolytic Anemia in 13 Dogs. Journal of Veterinary Internal Medicine 1997 vol 11 (6) pp 327 – 332
    4. Reimer ME, Troy GC, Warnick LD .Immune –Mediated Hemolytic Anemia: 70 cases (1988 – 1996). Journal of the American Animal Hospital Association 1999 Vol. 35 pp 384 – 391
    5. Slappendal R.J. Interpretation of tests for immune mediated blood diseases. In Kirks Current Veterinary Therapeutics X (Eds) Kirk and Bonagura. pg: 498 – 50.
    6. Stewart A.F., Feldman B.F. Immune mediated hemolytic anemia. Part I. Clinical Entity, Diagnosis, and Treatment Theory. Compendium on Continuing Education for the Practicing Veterinarian 1993 Vol. 15 (11) pp 1479 – 1993
    7. Stewart A.F., Feldman B.F. Immune mediated hemolytic anemia. Part II. An Overview. Compendium on Continuing Education for the Practicing Veterinarian 1993 Vol15 (3) pp 372 – 381
    8. Vaughn Scott T, Jacobson L.S. et al Systemic Inflammatory Response syndrome and multiple-organ damage /dysfunction in complicated canine babesiosis. Journal of the South African Veterinary Association 2001 vol 72 (3) pp 158 – 162