Complete blood count interpretation – cells and numbers (Proceedings)

A complete blood count (CBC) is a useful and very often-used method of screening and diagnosing patients who present for a wide variety of conditions. It may include both quantitative information about cell numbers, sizes, variability, etc. as well as descriptive information based on evaluation of a blood smear and description of any morphologic abnormalities or infectious agents present. A CBC is probably the most useful when both types of information are reviewed.

Erythrocytes

Classification of anemias

If a patient has a significant anemia, an important step is to figure out if the anemia is regenerative or non-regenerative. Being able to classify an anemia in this way is often key to determining the underlying cause, and thus, the most appropriate treatment and prognosis. Regenerative anemias are most often due to blood loss or hemolysis, while non-regenerative anemias are usually referable to a problem with effective bone marrow production of erythroid cells.

Sometimes it's relatively easy to tell whether an anemia is regenerative but other times it is not and it's very useful to have several different tools that can be used to evaluate these cases.

Classification by cell appearance

Reviewing a blood smear from an anemic patient can provide some very good subjective information, as well as occasionally revealing an underlying etiology, such as Mycoplasma spp. Regenerative anemias will (except in horses) show polychromasia and anisocytosis. Polychromatophilic (bluish) cells are immature erythrocytes released prematurely from the bone marrow, and these are usually also macrocytic cells. There are, however, some causes of larger-than-normal, or macrocytic cells that are not part of a regenerative response. These include FeLV infection in cats, bone marrow disorders (myelodysplasia), folate deficiency, and poodle macrocytosis.

Nucleated red blood cells, also known as metarubricytes, can also be seen as part of a regenerative response as they are erythrocytes that are even less mature than polychromatophilic cells. These cells may also be seen in other conditions, however, including lead toxicity, hemangiosarcoma or other splenic disorder, corticosteroid influence, and bone marrow disorders so they are not specific indicators of regeneration.

Similarly, basophilic stippling may be seen occasionally in a very regenerative anemia, especially in ruminants and sometimes cats and dogs, but is does not confirm regeneration, as it also may be seen in acute lead poisoning.

Reticulocytes

Although reviewing a blood smear may give a very good subjective view of the regenerative response (or lack thereof), in some cases, a more quantitative and accurate way of assessing regeneration is to perform a reticulocyte count. This may be easily done manually by mixing equal parts of fresh blood and new methylene blue stain. The blood and stain mixture is allowed to incubate for 10-15 minutes and then a blood smear made in the usual manner and air-dried. Some of the newer in-house hematology analyzers will provide a reticulocyte count as part of a CBC.

Usually, the percentage of reticulocytes out of 1000 red blood cells is counted. In dogs, this is relatively straightforward, but in cats, the distinction is made between aggregate and punctuate reticulocytes. Aggregate reticulocytes are those that have clumped or "stringy" dark blue reticulum, while punctuate reticulocytes have dots of reticulum. The punctuate reticulates are older as they can circulate for up to two weeks. Thus, they don't really reflect the current regenerative ability of the bone marrow and are not typically counted in a reticulocyte count.

The percentage of reticulocytes can be misleading as it doesn't allow and correct for the degree of anemia. There are a couple of ways that this can be handled. One is by calculating an absolute number, rather than percentage of reticulocytes. This can only be done if the RBC number is known. As normal, non-anemic animals can have up to 60,000 reticulocytes/ml, an animal with a regenerative anemia should have > 60,000 reticulocytes/ml with markedly regenerative anemias associated with > 200,000 reticulocytes/ml. If the absolute RBC count is not done, the correction for degree of anemia can be done by using the patient's PCV divided by the "normal" PCV for the species (e.g. 45% for dogs) as a correction factor. This factor is multiplied by the reticulocyte percentage obtained to come up with a corrected reticulocyte percentage.

In reviewing reticulocyte counts or percentages to determine whether regeneration is occurring, it's important to keep in mind that, if there is a sudden demand for erythrocytes (i.e. acute onset of anemia), it will take 2-3 days before any reticulocytes will be seen in the peripheral blood and they will not peak until 4-7 days in dogs. Some people use the term "pre-regenerative" to describe the situation when reticulocytes and other indicators of regeneration are not seen in the blood, but there has not been sufficient time for them to appear.

Other clues to anemia found on blood smears

In addition to the evidence of immature RBC's that may be seen on peripheral blood smears, other erythrocyte abnormalities can contribute to an assessment of anemia. Small erythrocytes, or microcytes, are usually associated with non-regenerative anemias, with iron-deficiency the most common underlying cause. In iron deficiency, the cells also appear hypochromic, or pale, with increased central pallor. Other causes of microcytosis that are not associated with hypochromasia are portosystemic shunts, and Akitas and Shiba Inu dogs that have normally have smaller-than-normal RBCs.

Spherocytes are another clue to the cause of anemia that may be seen on blood smears. These are small, dark, dense cells that have lost membrane due to immune-mediated anemia, so they are very significant when present in significant numbers. Although not as commonly observed, agglutatination of RBCs on a blood smear is also evidence of immune-mediated hemolytic anemia.

Erythrocytes with Heinz bodies may be seen on regular Wright's-stained slides or on slides stained with new methylene blue. Although a small percentage may be seen in normal cat blood, these are otherwise indicative of oxidative damage of cells, which can lead to extravascular or intravascular hemolysis.

Howell-Jolly bodies, or small nuclear remnants in RBC's are not specific to any particular condition. Although they may be seen in increased numbers in regenerative anemia, they are also seen in non-anemic animals.

Erythrocytes parasites that are associated with anemia include Mycoplasma hemofelis, M. hemominutum and Cytauxzoon felis in cats, M. hemocanis and Babesia spp. in dogs, Anaplasma spp. and other hemotropic mycoplasmas in large animals. Most of these are readily observed on well-stained blood smears made shortly after the blood is drawn.

Classification by rbc indices

The other way to classify anemias is to use red blood cell indices. These are generated by essentially all hematology analyzers and are commonly provided by commercial laboratories. The most valuable of these indices are the mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC ).

Anemias can be microcytic, normocytic, or macrocytic, depending on whether the RBC MCV is less than, within, or greater than the reference values. Anemias can be hypochromic or normochromic, depending on whether the MCHC is less than, or within the reference values. Hyperchromia doesn't really occur, but the MCHC may be higher than the reference range as a result of artifact (hemolysis or administration of cell-free hemoglobin-based blood substitute).

As a general rule, regenerative anemias due to hemorrhage or hemolysis will be macrocytic, hypochromic anemias. The stronger the regenerative response, the higher the MCV will typically be. Anemias that are microcytic and hypochromic are most often due to iron deficiency.

Another index that is provided by automated hematology analyzers is the red cell distribution width (RDW). This is basically a quantitative (rather than qualitative) measurement of anisocytosis. It is a coefficient of variation of the erythrocyte volume. It is expected to be higher in cases of regenerative anemia, where younger (and therefore larger) cells are present. It may give a better idea of the variation in cell size than the MCV, which is an average cell volume. The RDW may also be increased in iron-deficiency anemia where there are smaller than normal cells, and when there are lots of fragmented cells. It is usually normal in non-regenerative anemias.

Using the total protein and pcv together

Determining a plasma or serum protein is potentially useful in categorizing anemia, and this should always be done even if the CBC doesn't include it. The combination of a decreased PCV and a decreased total protein suggests ongoing or recent blood loss, though fluid therapy that's too vigorous can also cause this combination. A low PCV with normal total protein is seen in a larger variety of conditions, such as chronic blood loss, immune-mediated anemia, or decreased RBC production. If the PCV is low and total protein is high, anemia of chronic disease might be considered.

Other useful tests to further characterize anemia

Once it's been determined that an anemia is regenerative or non-regenerative, additional tests are often indicated to refine the diagnosis. These may include:

• Examination of plasma/serum for hemolysis

• Urine dipstick for hemoglobinuria-typical of intravascular hemolysis

• Coomb's test for immune-mediated hemolytic anemia, if there is a regenerative anemia and spherocytes and/or autoagglutination

• Bone marrow aspirate for non-regenerative anemias

• Assay for serum iron, iron saturation and serum ferritin

• Assay for serum cobalamin and folate (rarely done, but useful in non-regenerative, macrocytic anemias)

• Serum chemistries to determine whether renal disease, endocrinopathies, etc. may be contributing to anemia

• PCR for infectious agents if not apparent on a blood smear

• Serology for diagnosis of other diseases that are associated with anemia (e.g. FeLV, ehrlichiosis)

Leukocytes

Interpretation of leukocytes is based on the total WBC count, the absolute numbers of each type of leukocyte present and leukocyte morphology as seen on a blood smear. Although it is uncommon to make a definitive diagnosis based on the leukogram, there are several patterns that suggest underlying conditions.

Stress leukogram

This pattern may be seen in response to endogenous corticosteroid release such as that involved with illness or endocrine disease, as well as when exogenous corticosteroids have been administered. The classic pattern is that of mature neutrophilia, lymphopenia, eosinopenia and (in dogs) monocytosis

Inflammatory leukogram

Acute inflammation is most often associated with leukocytosis and neutrophilia that may be accompanied by a left shift to immature neutrophils. As inflammation becomes less acute, the left shift may resolve and in very chronic inflammation, there may also be no peripheral leukocytosis. Toxic neutrophils may be present in severe inflammation. These are neutrophils with some abnormal morphologic features that are due to the influence of inflammatory mediators on the bone marrow.

In very acute, severe inflammation, a leukopenia may be present. In more long-standing severe inflammation, a marked leukocytosis known as a leukemoid reaction (> 50,000 WBCs/ul) may be present.

Epinephrine/excitement leukogram

Physiologic leukocytosis may result from epinephrine release in animals that are being handled for examination or blood draw-especially young cats and horses. This is characterized by a mature neutrophilia with no left shift and a lymphocytosis. It results from leukocytes being shifted from the marginated pool to the circulation and typically resolves in 30-60 minutes.

Neutropenia

Neutropenia most often is due to excessive consumption of neutrophils at a site of severe inflammation in the tissues. A left shift may also be present and this leukogram pattern is sometimes known as a degenerative left shift, reflecting the associated poor prognosis. Less common causes of neutropenia are immune-mediated and neutropenia that occurs when the bone marrow is not normally producing mature neutrophils. Causes of the latter include viral disease, ehrlichial disease, toxins, drugs, and neoplasia.

Leukocyte morphologic abnormalities

Examination of a blood smear may reveal several different types of leukocyte abnormalities including:

• Infectious agents, such as Hepatozoon, and Ehrlichia spp.

• Genetic diseases: such as Chediak Higashi Syndrome and lysosomal storage diseases

• Abnormal nuclear morphology: such as Pelger Huet Anomaly

• Neoplasia