Evaluation of the hemogram: What do those numbers mean? (Proceedings)

The Importance of a Complete Hemogram

Of all the diagnostic tests available, blood evaluation is one of the single most valuable tools in assessing the general health of the body. Blood, and the nutrients it carries, circulates through every living cell in the body. It stands to reason that it is an incredibly valuable indicator of disease, either local or systemic. The complete blood count (CBC) is the best and most convenient mechanism to detect abnormalities in our patient's the blood. The CBC begins with the quantitative evaluation of erythrocytes, leukocytes and platelets, but it does not end there. It ends with the microscopic examination of the blood film to detect morphological abnormalities that provide valuable insight to various disease conditions.

Quantitative Analysis

Erythrocytes

The red blood cell count (RBC), the packed cell volume (PCV) and the hematocrit all assess the same parameter, red blood cell mass in the body in relation to the volume of plasma. Elevations in these parameters indicate an absolute increase in the number of erythrocytes (erythrocytosis) or a relative decrease in the volume of plasma (hemoconcentration). Another term for erythrocytosis is polycythemia. Polycythemia may be secondary to other clinical conditions such as hypoxia, renal neoplasia, hyperthyroidism or splenic contraction. However, primary polycythemia (polycythemia vera) is a myeloproliferative disease and results from increased production of red cells from the marrow independent of erythropoietin production. Measurement of endogenous erythropoietin levels can give an indication of the etiology. Reduced red blood cell mass (anemia) can either result from a red cell production problem, hemolysis, blood loss, or a combination of the above. PCV alone can provide an accurate evaluation of red blood cell mass, but only with the addition of the RBC can all of the red cell indices be calculated.

The red cell indices, mean cell volume (MCV), mean corpuscular hemoglobin (MCH) and mean cell hemoglobin concentration (MCHC), help us to classify an anemia as regenerative (blood loss or hemolysis) or nonregenerative (production problem), often giving us insight to the etiology. A nonregenerative anemia will typically have a normal MCV (normocytic) and MCHC (normochromic). This can be seen in a number of conditions resulting in decreased red cell production or in per acute blood loss of less than 3 to 5 days duration. The most common cause of a nonregenerative anemia in companion animal medicine is the anemia of chronic inflammatory disease. A regenerative anemia will have an increased MCV (macrocytic) and a decreased MCHC (hypochromic). However, because the indices used to evaluate regeneration are mean values, they will not increase until the population of immature erythrocytes (Reticulocytes) are abundant enough to push the mean values out of the reference range. Therefore, many regenerative anemias will have MCVs and MCHCs within the normal range. The most sensitive way to detect a regenerative response is by performing a reticulocyte count. A regenerative anemia may be seen in conditions resulting in blood loss or hemolysis. A particularly strong regenerative response is seen in animals with hemolytic anemia. Hemolysis results in the most dramatic changes in red cell indicies. An anemia that is the result of chronic hemorrhage and iron deficiency will have a low MCV (microcytic) and low MCHC (hypochormic). In addition, cats with FeLV infection may experience a specific type of anemia arises from disturbances in red cell maturation, resulting in a macrocytic (elevated MCV) and normochromic (normal MCHC) abnormality.

Leukocytes

The leukogram begins with the total white blood cell count (WBC). However, this number alone does not provide vital information needed to assess patient health. Many animals will have a normal WBC with significant abnormalities in the leukogram. Those hematology analyzers that can accurately perform a differential leukocyte evaluation provide superior information regarding individual leukocyte numbers to detect increased numbers of immature neutrophils, decreased numbers of mature neutrophils and increased or decreased numbers of lymphocytes, monocytes, eosinophils and basophils. These changes can occur without alterations in the total WBC, and in some cases, these changes are even more significant when the WBC is normal. Automated analyzers that utilize laser technology are better at providing accurate differential leukocyte counts than are older impedance counters because they separate cells based on their ability to cause light refraction. Impedance counters separate cells based on size alone. Some analyzers are capable of "flagging" atypical cells in circulation which could include reactive monocytes and lymphocytes or neoplastic hematopoietic blast cells.

Increased numbers of immature cells in circulation is termed a "left-shift". The type of left shift present can be of prognostic value since it serves as an indication of how well the animal's innate immune system is responding to the disease process. A left-shift may be degenerative, regenerative, or transitional, depending upon the total leukocyte count and the number of immature neutrophils in circulation.

Regenerative left shift – a left shift in which there is typically a neutrophilia and there are a higher numbers of mature cells than immature. This is a favorable response where the bone marrow has had sufficient time (3 –5 days) to respond to peripheral demands for neutrophils.

Degenerative left shift – a left shift in which there are more immature neutrophils (bands, metamyelocytes and myelocytes) than mature neutrophils (segmented). Total neutrophils counts are typically low or only slightly elevated. This indicates that the reserve of mature neutrophils in the bone marrow has been depleted, has had insufficient time to respond, or cannot meet the overwhelming demand for neutrophils. In most species this is an unfavorable prognostic indicator.

Transitional left shift – a leukogram that has a moderate to marked neutrophilia, but the immature forms still outnumber the mature forms or conversely, in situations where there is a neutropenia but mature forms still outnumber immature forms. These findings need to be interpreted in light of sequential hemograms or changes in status of the patient to determine if they indicate recovery or worsening of the disease.

Platelets

Platelet counts are difficult to obtain using any automated hematology analyzer, particularly in cats. This is because when platelets are activated they form clumps that cannot be counted. In addition, because of size similarity, impedance counters cannot distinguish large platelets from erythrocytes, a common occurrence in some dogs and most cats. Hematology analyzers that use laser technology are better at distinguishing platelets from erythrocytes because they do not identify them based on size. However, no analyzer can give an accurate assessment of platelet numbers when significant clumping has occurred. For this reason, all animals with abnormally low platelet counts should have a blood film evaluated to estimate platelet numbers from the smear and to identify the degree of platelet clumping. Two breeds of dogs (Greyhounds and King Charles Cavalier Spaniels) have platelet counts that are normally below the reference interval for other dogs.

The quantitative assessment of the hemogram is important, but the hemogram is not complete without a microscopic evaluation of the blood film.

Cytological Abnormalities in the Canine & Feline Blood Film

The evaluation of a blood smear will allow the practitioner to gain rapid, valuable information regarding the health of the patient when the evaluation is performed in a systematic fashion. The important clinical information needed for the hematologic evaluation of an animal can all be obtained by estimating cell numbers and evaluating the morphologic changes in erythrocytes, leukocytes and platelets. The value of these findings, many of which are not recognized by automated cell counters, cannot be overemphasized.

Blood Collection and Slide Preparation

Vacutainer tubes containing EDTA should be filled to the designated amount. Partial filling of vacutainer tubes with blood may cause artifactual changes in cell morphology and numerical values. Blood smears should be prepared as quickly as possible in order to minimize artifactual changes in erythrocytes and leukocytes such as red cell crenation, leukocyte vacuolation and nuclear swelling and pyknosis. In addition, prolonged exposure to EDTA may make it more difficult, or even impossible to identify infectious agents, such as Mycoplasma haemofelis (formerly Haemobartonella felis), in the blood of infected cats. The coverslip technique for making smears is preferred over the glass slide technique. This technique minimizes traumatic injury to cells during slide preparation. This technique produces a more even distribution of cells, allowing more accurate estimation of leukocyte and platelet numbers. Smears should be rapidly dried with a blow drier to eliminate artifacts of air-drying red blood cells. This is particularly important when attempting to identify red cell parasites such as haemoplasmas or evaluation of erythrocyte shape changes.

Scanning the Smear

The first step in the evaluation of a blood smear is to scan the slide using a 10× or 20× objective. With regard to the red blood cells, we should observe red cell density and presence of rouleaux or agglutination.(Rouleaux may be differentiated from agglutination by saline test where 1 drop of blood is mixed with 0.5 to 1.0 ml of physiological saline solution and observed on wet mounts, unstained. Rouleaux will disperse with saline dilution.). With regard to nucleated cells we should confirm that the mature neutrophil is the predominant cell type. The presence of any left-shifted neutrophils or large or atypical leukocytes, as well as the presence of any nucleated erythrocytes should also be recorded. Platelet clumps should also be identified at this magnification because they will affect how we interpret platelet numbers later in the evaluation. This is particularly crucial in the evaluation of the feline blood film because platelet clumping is a common occurrence in this species. Leukocyte numbers may be estimated using the following formula. Formula:# cells/µl = (Ave. # of cells per field) × (Objective power) 2. The objective used to estimate leukocyte numbers should be one where approximately 5 - 10 leukocytes are seen per field. Example: If an average of 5 cells were counted for each 50× field, the total leukocyte count would be (5) × (2,500) = 12,500 cells / µl.