The manual differential blood test is a specialized laboratory procedure where a skilled professional manually examines a patient’s blood under a microscope. This examination is performed on a thin smear of peripheral blood, using light microscopy to visually identify and count different types of white blood cells (WBCs), also called leukocytes. The test is frequently ordered as a follow-up or confirmation to an automated Complete Blood Count (CBC) when the machine detects irregularities or flags the sample as abnormal. It provides a detailed, cell-by-cell analysis that automated instruments cannot fully replicate.
Why a Manual Differential is Necessary
While automated hematology analyzers are fast and highly accurate for counting the total number of blood cells, they have limitations when assessing cellular details. These machines rely on measuring properties like cell size, granularity, and light scatter, which can lead to misidentification if cells are unusually shaped or sized. The automated analyzer will apply a “flag” to the result when it detects an abnormality, such as a possible presence of immature or atypical cells, signaling the need for human intervention.
A primary reason for performing a manual differential is the ability to assess cell morphology, which relates to the cell’s shape, size, and internal features. A laboratory scientist can identify subtle changes in the appearance of white blood cells, red blood cells, and platelets that may indicate a specific underlying condition. The manual process is necessary to detect rare or abnormal cells, such as blast cells (highly immature white blood cells not normally found in peripheral blood) or parasitic inclusions within blood cells.
The Procedure for Performing the Differential
The manual differential begins with the creation of a peripheral blood smear, which involves placing a small drop of blood near one end of a glass slide. A second “spreader” slide is then used to drag the drop across the first slide at an angle, typically around 25 degrees, to create a thin film of cells that air-dries quickly. The goal is to produce a thin, smooth smear where the blood cells are evenly distributed in a single layer without excessive clumping or overlap.
Once dry, the slide is treated with a specialized multi-component stain, often a Romanowsky-type stain such as Wright-Giemsa, which chemically colors different cellular components. This staining process selectively highlights the nuclei, cytoplasm, and granules of the various blood cells, allowing for clear visual differentiation under the microscope.
The laboratory professional systematically scans a specific area of the smear known as the “monolayer” or “feathered edge,” where the cells are well-separated and undistorted. A common counting method, such as the “battlement” pattern, is used to ensure different areas of the slide are sampled for a representative count. The scientist identifies and counts a minimum of 100 white blood cells, categorizing each one by its type using a specialized counter. The final result is reported as a percentage of each of the five types of white blood cells present in the sample.
Identification of the Five White Blood Cell Types
The manual differential provides a breakdown of the five primary types of leukocytes, each distinguishable by its unique microscopic appearance and function.
Neutrophils
Neutrophils are the most numerous, characterized by a multi-lobed nucleus and pale pink or light purple granules in the cytoplasm; these cells are primarily involved in fighting bacterial infections. An earlier form, the band neutrophil, is also counted, identifiable by its horseshoe-shaped, non-segmented nucleus, and its presence can indicate a rapid response to infection.
Lymphocytes
Lymphocytes are the second most common type, recognized by a large, round, dark-staining nucleus that occupies most of the cell, surrounded by a small rim of pale blue cytoplasm. They are the central players in the body’s immune response, responsible for generating antibodies and directly attacking foreign invaders.
Monocytes
Monocytes are the largest of the white blood cells, featuring an abundant, gray-blue cytoplasm and a large, often kidney-bean or horseshoe-shaped nucleus. These cells circulate in the blood before migrating into tissues, where they mature into macrophages that consume cellular debris and pathogens.
Eosinophils
Eosinophils are distinct because of their large, bright red or orange granules that fill the cytoplasm and a typically bi-lobed nucleus. Their primary role is in defending against parasitic infections and moderating allergic reactions by releasing specific chemical mediators.
Basophils
Basophils are the least common type, easily identified by large, dark purple or black granules that are so dense they often obscure the nucleus. Basophils contain histamine and other substances that are released during allergic responses and inflammation.