White blood cell differential
A white blood cell differential is a medical laboratory test that provides information about the types and amounts of white blood cells in a person's blood. The test, which is usually ordered as part of a complete blood count, measures the amounts of the five normal white blood cell typesneutrophils, lymphocytes, monocytes, eosinophils and basophilsas well as abnormal cell types if they are present. These results are reported as percentages and absolute values, and compared against reference ranges to determine whether the values are normal, low, or high. Changes in the amounts of white blood cells can aid in the diagnosis of many health conditions, including viral, bacterial, and parasitic infections and blood disorders such as leukaemia.
White blood cell differentials may be performed by an automated analyzera machine designed to run laboratory tests – or manually, by examining blood smears under a microscope. The test was performed manually until white blood cell differential analyzers were introduced in the 1970s, making the automated differential possible. In the automated differential, a blood sample is loaded onto an analyzer, which samples a small volume of blood and measures various properties of white blood cells to produce a differential count. The manual differential, in which white blood cells are counted on a stained microscope slide, is now performed to investigate abnormal results from the automated differential, or upon request by the healthcare provider. The manual differential can identify cell types that are not counted by automated methods and detect clinically significant changes in the appearance of white blood cells.
In 1674, Antonie van Leeuwenhoek published the first microscopic observations of blood cells. Improvements in microscope technology throughout the 18th and 19th centuries allowed the three cellular components of blood to be identified and counted. In the 1870s, Paul Ehrlich invented a staining technique that could differentiate between each type of white blood cell. Dmitri Leonidovich Romanowsky later modified Ehrlich's stain to produce a wider range of colours, creating the Romanowsky stain, which is still used to stain blood smears for manual differentials.
Automation of the white blood cell differential began with the invention of the Coulter counter, the first automated hematology analyzer, in the early 1950s. This machine used electrical impedance measurements to count cells and determine their sizes, allowing white and red blood cells to be enumerated. In the 1970s, two techniques were developed for performing automated differential counts: digital image processing of microscope slides and flow cytometry techniques using light scattering and cell staining. These methods remain in use on modern hematology analyzers.
Overview
The white blood cell differential is a common blood test that is often ordered alongside a complete blood count. The test may be performed as part of a routine medical examination; to investigate certain symptoms, particularly those suggestive of infection or hematological disorders; or to monitor existing conditions, such as blood disorders and inflammatory diseases.Five types of white blood cells are normally found in blood: neutrophils, lymphocytes, monocytes, eosinophils and basophils. Marked shifts in the proportions of these cell types, as measured by the automated or manual differential, can indicate various health conditions. Additionally, cell types which do not normally occur in the blood, such as blast cells, can be identified by the manual differential. These cell types may be found in blood disorders and other pathological states. The manual differential can also identify changes in the appearance of white blood cells, such as reactive lymphocytes, or features such as toxic granulation and vacuolation in neutrophils. The results of the white blood cell differential are reported as percentages and absolute values. Absolute counts are usually reported in units of cells per microlitre or 109 cells per litre. The result are then compared against reference ranges, which are defined by individual laboratories and may vary due to different patient populations and testing methods.
CBC and differential testing is usually performed on venous or capillary blood. Capillary blood draws are generally used for infants and individuals whose veins are difficult to access. To prevent clotting, the sample is drawn into a tube containing the anticoagulant compound ethylenediaminetetraacetic acid, meaning blood that has not been centrifuged.
Types
Manual differential
In a manual differential, a stained blood smear is examined under a microscope and white blood cells are counted and classified based on their appearance. A manual differential is usually performed when the automated differential is flagged for review or when the healthcare provider requests it. If the manual differential shows findings suggestive of certain serious conditions, such as leukaemia, the blood smear is referred to a physician for confirmation.Procedure
A blood smear is prepared by placing a drop of blood on a microscope slide and using a second slide held at an angle to spread the blood and pull it across the slide, forming a "feathered edge" consisting of a single layer of cells at the end of the smear. This may be done by hand or using an automated slide maker coupled to a hematology analyzer. The slide is treated with a Romanowsky stain, commonly Wright's stain or Wright-Giemsa, and examined under the microscope. The smear is examined in a systematic pattern, scanning from side to side within the feathered edge and counting cells consecutively. The differential is typically performed at 400x or 500x magnification, but 1000x magnification may be used if abnormal cells are present. Cells are identified based on their morphologic features, such as the size and structure of their nucleus and the colour and texture of their cytoplasm. This allows abnormal cell types and changes in cellular appearance to be identified. In most cases, the microscopist counts 100 white blood cells, but 200 may be counted for better representation if the white blood cell count is high. The manual differential count produces percentages of each cell type, which can be multiplied by the total white blood cell count from the analyzer to derive the absolute values.The manual differential can be partially automated with digital microscopy software, which uses artificial intelligence to classify white blood cells from photomicrographs of the blood smear. However, this technique requires confirmation by manual review.
Limitations
Because relatively few cells are counted in the manual differential, the variability is higher than in automated techniques, especially when cells are present in low amounts. For example, in a sample containing 5 percent monocytes, the manual differential results could be between 1 and 10 percent due to sampling variation. Additionally, cell identification is subjective and the accuracy depends on the skills of the person reading the slide. Poor blood smear preparation can cause an uneven distribution of white blood cells, resulting in inaccurate counting, and improper staining can impede cell identification. Overall, manual differential counts exhibit coefficients of variation ranging from 5 to 10 percent, while automated differential counts of normal neutrophils and lymphocytes have CVs of about 3 percent.In leukemias and other hematologic malignancies, the lineage and genetic characteristics of white blood cells have important implications for treatment and prognosis, and the microscopic appearance of the cells is often insufficient for accurate classification. In these cases, other techniques such as immunophenotyping by flow cytometry or special staining can be used to definitively identify the cells.
Automated differential
Most hematology analyzers provide a five-part differential, enumerating neutrophils, lymphocytes, monocytes, eosinophils and basophils. Some instruments can also count immature granulocytes and nucleated red blood cells. If a six-part differential is provided, the IG or immature granulocyte category consists of promyelocytes, myelocytes and metamyelocytes. Hematology analyzers measure various properties of white blood cells, such as impedance, light scattering parameters, and staining reactions. This data is analyzed and plotted on a scattergram, forming distinct clusters which correspond to white blood cell types. The analyzer counts many more cells than are counted in a manual differential, resulting in improved precision. If abnormal features or cell populations that the analyzer cannot identify are present, the instrument can flag the results for manual blood smear review.Procedure
Common techniques used by hematology analyzers to identify cells include light scattering, Coulter counting, and cytochemical staining techniques. Some analyzers also use radiofrequency analysis and monoclonal antibody tagging to identify cells. Staining techniques used in differential analyzers include staining of myeloperoxidase, an enzyme found in cells of myeloid lineage, and nucleic acids, which are found in higher concentrations in immature cells.A small volume of blood is aspirated into the analyzer, where reagents are applied to lyse red blood cells and preserve white blood cells. The sample is diluted and passed into a flow cell, which uses hydrodynamic focusing to isolate single cells for accurate analysis of their properties. Various cellular parameters, such as size, complexity and staining reactions, are measured and analyzed to identify cell populations. Basophils are often quantified using a reagent that lyses the cytoplasm of other white blood cells but leaves basophils intact. Samples that have abnormal results or are suspected to contain abnormal cells are flagged by the analyzer for manual blood smear review.
To ensure that results from the automated analyzer are correct, quality control samples are run at least once per day. These are samples with known results that are most often provided by the instrument manufacturer. Laboratories compare their differential results to the known values to ensure the instrument is operating correctly. A moving average measurement may also be used, in which the average results for patient samples are measured at certain intervals. Assuming that the characteristics of the patient population remain roughly the same over time, the average should remain constant. Large shifts in the average value can indicate instrument problems.