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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 white blood cell types in normal human blood – neutrophils, lymphocytes, monocytes, eosinophils and basophils – as well as abnormal cell types if they are present. These results are reported as percentages and absolute values. 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 leukemia.

White blood cell differentials may be performed by an automated analyzer or manually, by microscopic examination of blood smears. In the 1950s and 1960s, the development of methods for counting, sizing, and identifying cells based on electrical impedance made the automated differential possible. Further refinements added light scattering measurements and fluorescent staining to the repertoire of automated hematology methods. Hematology analyzers now use various combinations of these methods to provide fast and accurate differential results.

The manual differential, in which a medical laboratory technologist counts white blood cells on a stained microscope slide, is performed to investigate abnormal or potentially unreliable 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.

• 1Automated differential
• 2Manual differential
• 4Interpretation

Automated differential[edit]

As of 2019, 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.^[4]:795 The analyzers measure various properties of white blood cells, such as impedance, light scattering parameters, and staining reactions, and the data is analyzed and plotted on a scattergram, where the points form distinct clusters which correlate with white blood cell types. The relative size of each cluster describes the amount of cells present, and the shape of the clusters can indicate qualitative abnormalities.^[5]:51 Several thousand cells are counted, resulting in improved accuracy over the manual differential.^[6]:5 If abnormal features or cell populations that the analyzer cannot identify are present, the instrument can flag the results for further review (i.e. manual examination of the blood smear).^{[4]:795[5]:51}

History[edit]

The first automated hematology analyzer, the Coulter counter, was invented in the early 1950s by Wallace H. Coulter and Joseph A. Coulter.^[4]:794[7] The analyzer worked on the Coulter principle, in which cells are suspended in a fluid carrying an electrical current and passed through a small aperture. As cells are poor conductors, they cause decreases in current proportional to their volume as they pass through the aperture. The number and magnitude of these decreases are then used to count blood cells and calculate their sizes. The Coulter counter was initially designed for counting red blood cells, but it also proved effective for counting white blood cells.^[7]

In 1967, M. J. Fulwyler used a modified version of the Coulter counter to sort white blood cells by size.^[8] This cell sorting method was based on a technology invented by R. G. Sweet for use in inkjet printers, in which ink droplets are deflected based on electrostatic charge.^[9][10] Fulwyler's method used Coulter counting to determine the volume of the white blood cells, then partitioned cells into fluid droplets and applied charges based on the cells' sizes, allowing them to be sorted into collection vessels by volume. The collected cells could then undergo further analysis.^[11][12]

Using this method, Fulwyler found that human white blood cells formed three distinct peaks when their volumes were plotted on a histogram, and by microscopically examining cells from each peak, he determined that the smallest-volume peak predominantly contained lymphocytes, the intermediate-volume peak contained neutrophils, and the largest-volume peak contained a mixture of granulocytes and monocytes.^[8] Fulwyler's technique was applied by other researchers to generate automated neutrophil counts, although counts for other cell types were not reliable due to the low number of cells counted.^[13] In 1983, the Coulter S Plus system, a commercial analyzer that produced a three-part differential of neutrophils, monocytes and lymphocytes based on cell size histograms, was introduced.^[14] The principle of identifying white blood cells by their sizes is still used in many hematology analyzers, although other techniques are used to improve discrimination between white blood cell types.^[15]

In the late 1960s and early 1970s, researchers began to combine Coulter counting techniques with analysis of the optical properties of cells.^[12] These early flow cytometry devices, such as Kamentsky's Cytofluorograph^[16] and Dittrich & Goehde's Impulscytometer,^[17] shot beams of light at cells in specific wavelengths and measured the resulting absorbance or fluorescence, allowing for quantification of cellular contents such as DNA.^[10] Julius and colleagues improved upon these techniques with the use of fluorescent dyes (fluorophores), which bind to specific components of cells to permit discrimination of cell types.^[18] The combination of fluorescent labelling and electrostatic cell sorting came to be known as fluorescence activated cell sorting.^[10]

Meanwhile, other researchers such as George & Groner^[12] and Ansley & Ornstein^[10] focused on using light scattering patterns to classify cells. By directing a laser at individual cells and measuring the light scattered at different angles, various features of white blood cells can be ascertained. The light scattered within 1 to 10 degrees of the beam's axis (forward scatter) describes cellular size, while light scattered at a 90 degree angle (side scatter) describes granularity.^[6]:22–3 Ansley & Ornstein's work, along with later improvements by Mansfield,^[10] resulted in the first commercial flow cytometric differential analyzer, the Hemalog D.^[19][20] Introduced in 1974,^[19] this analyzer used light scattering and three different staining channels to identify the five normal white blood cell types, plus what were referred to as large unstained cells – usually atypical lymphocytes or blast cells. It could count 10,000 cells in one run, a massive improvement over the 100-cell manual differential, and it significantly decreased workload in the hematology laboratory as samples that were identified as normal did not require manual smear review.^[21]

Procedure[edit]

Most hematology analyzers use some combination of light scattering, Coulter counting, and cytochemical staining techniques. Some analyzers also use radiofrequency analysis to determine cellular structure.^[4] Staining techniques used in differential analyzers include staining of myeloperoxidase,^[6]:5 an enzyme found in cells of myeloid lineage,^[22] and nucleic acids, which are found in higher concentrations in immature cells.^[23]

Analysis is usually performed on blood samples anticoagulated with ethylenediaminetetraacetic acid (EDTA), although alternative anticoagulants may be used for individuals in whom EDTA causes platelet clumping.^[6]:1 A small volume of blood (35 to 150 microliters) is aspirated into the analyzer, where reagents are applied to lyse red blood cells and preserve the structure of 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 are measured and analyzed to identify cell populations. Basophils, which are difficult to differentiate from other white blood cells by routine methods, are usually quantified using a reagent that lyses the cytoplasm of other white blood cells but leaves basophils intact.^[6]:3–5

Samples that have abnormal numerical results^[1] or are suspected to contain abnormal cells are flagged by the analyzer for manual blood smear review.^[5]:43

Limitations[edit]

When immature or abnormal white blood cells are present, automated differential results may be incorrect, necessitating a manual blood smear review.^[6]:5–7 While the majority of abnormal samples are flagged for review by the analyzer, some are occasionally missed.^[5]:43-4 Hematology laboratories compensate for this issue by requiring smear review when differential or complete blood count results are above or below certain numerical thresholds, regardless of the presence of flags.^[1]

The automated basophil count is notoriously unreliable,^[15] often underestimating counts in basophilia and producing falsely elevated results in the presence of abnormal cells.^[24] The manual differential is therefore considered the reference method for these cells.^[15][25]

Nucleated red blood cells, giant platelets, and red blood cells containing abnormal hemoglobins (such as Hemoglobin S in sickle cell disease), may be counted as white blood cells by some analyzers, leading to faulty differential results. Automated differential counts on aged specimens may be incorrect due to cellular degeneration.^[26]:191

Manual differential[edit]

A manual differential involves examination of a stained blood smear by a medical laboratory technologist,^[27] who counts and classifies white blood cells based on their microscopic appearance. The manual differential is usually performed when the automated differential is flagged for review due to abnormal or potentially unreliable results or when requested by the healthcare provider.^[1][28]

History[edit]

The first microscopic observations of blood cells were published by Antonie van Leeuwenhoek in 1675. Using a microscope of his own design, van Leeuwenhoek discovered that blood consisted of 'small red globules, driven through a crystalline humidity of water'.^[14] Throughout the 18th and 19th centuries, further improvements in microscope technology such as the invention of achromatic lenses permitted the discrimination and counting of white blood cells and platelets in unstained blood samples. It was Paul Ehrlich who, in the 1870s, first developed a staining technique that could differentiate between the five white blood cell types. Ehrlich's stain used a combination of an acidic and basic dye to stain white and red blood cells simultaneously.^[14] The Russian physician Dmitri Leonidovich Romanowsky improved upon this technique in the 1890s by using a mixture of eosin and aged methylene blue, which produced a wide range of hues that were not present when either of the stains were used alone. This was termed the Romanowsky effect and became the basis for Romanowsky staining, the technique that is still used to stain blood smears for manual differentials.^[29]

Procedure[edit]

A blood smear is prepared by placing a drop of blood on a microscope slide and using a second slide held at a 30 to 45 degree angle to spread the blood, forming a 'feathered edge' consisting of a single layer of cells.^{[6]:7[30]:346–8} This may be done by hand or using an automated slide maker coupled to a hematology analyzer. The slide is stained with a Romanowsky stain, commonly Wright's stain or Wright-Giemsa, and then examined by a technologist. The smear is examined in a systematic pattern, with the technologist scanning from edge to edge within the monolayer 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.^[31]:10–1 Cells are identified based on their morphologic features, such as the size and structure of the nucleus and the colour and texture of the cytoplasm. This permits the differentiation of abnormal cell types and detection of qualitative changes in morphology.^[30]:303 In most cases, the technologist counts 100 white blood cells; 200 may be counted for better representation if the white blood cell count is high.^[31]:10 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.^[30]:329

The manual differential can be partially automated with digital microscopy software,^[30]:318 which uses artificial intelligence algorithms to classify white blood cells from photomicrographs of the blood smear.^[5]:44–5 However, this technique has limitations similar to those of the automated differential and still requires review of the results by a technologist.^[32][33]

Limitations[edit]

The number of individual cells counted in the manual differential is low compared to automated techniques, which leads to variability in the results, especially when cells are present at low percentages.^[6]:5 For example, in a sample containing 5 percent monocytes, the manual differential results could be between 1 to 10 percent due to sampling variation.^[34] The manual differential is subject to further variation because cell identification is subjective^[35] and its accuracy depends upon the skills of the technologist reading the slide.^[6]:5 Additionally, poor blood smear preparation can cause an uneven distribution of white blood cells, resulting in inaccurate counting,^[5]:30–1 and improper staining can impede cell identification.^[6]:7–9 Overall, manual differential results exhibit coefficients of variation (CVs) ranging from 6.5 percent to 15 percent, while automated differential results have CVs of 1 to 6 percent.^[36]:102

The microscopic appearance of white blood cells is sometimes insufficient for classification,^[4]:336 such as in leukemias and other hematologic malignancies where the lineage and genetic characteristics of the cells have important implications for treatment and prognosis.^[37] In these cases, other techniques such as flow cytometry with cell marker labels or special staining can be used for definitive identification.^[31]:9

Medical uses[edit]

The white blood cell differential is a common blood test^[38] that is often ordered alongside a complete blood count. The test may be performed as part of a routine medical examination or to investigate certain symptoms, particularly those suggestive of infection or hematological disorders.^[27] Marked increases or decreases in the proportions of various cell types, as measured by the automated or manual differential, can indicate pathological conditions. The manual differential can also detect cell populations that are not normally found in peripheral blood and reveal clinically significant changes in the appearance of white blood cells.^[28]

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 microliter (µL) or 10⁹ cells per liter (L).^[27]

Interpretation[edit]

Neutrophils[edit]

Neutrophils are the most common white blood cells in normal adult blood. When stained with a Romanowsky stain, they exhibit a multi-lobed nucleus and pink cytoplasm that contains small purple granules.^{[4]:306[5]:88–93}

The neutrophil count is normally higher in newborns and pregnant women than in other groups.^[39]:252 Outside of these conditions, increased neutrophil counts (neutrophilia) are associated with bacterial infection, inflammation, and various forms of physiological stress.^[30]:306 Neutrophil counts can become extremely high in some infections and inflammatory states, which is termed leukemoid reaction because the high white blood cell count mimics leukemia.^[39]:253 Neutrophilia may also occur in myeloproliferative disorders.^[30]:306

Neutropenia, meaning a low neutrophil count, may occur as a response to drug treatment (especially chemotherapy)^[40] or to certain infections, such as tuberculosis and Gram-negativesepticemia. Neutropenia also occurs in many hematologic disorders, such as leukemia and myelodysplastic syndrome, and in a variety of autoimmune and congenital diseases.^[39]:247-52 A decreased neutrophil count may be normal in individuals of certain ethnicities; this is termed benign ethnic neutropenia.^[39]:8[41] Very low neutrophil counts are associated with immunosuppression.^[4]:308–11

When stimulated by infection or inflammation, the appearance of neutrophils can change. They may develop abnormal features in their cytoplasm, like toxic granulation, toxic vacuolation and Döhle bodies. These features are collectively known as toxic changes,^[42]:153 and they are caused by the release of cytokines.^[43]:4

Lymphocytes[edit]

Lymphocytes, which are the second most common type of white blood cell in adults, are typically small cells with a round, dark nucleus and a thin strip of pale blue cytoplasm. Some lymphocytes are larger and contain a few blue granules.^[30]:309 However, the appearance of lymphocytes is highly variable. In response to viral infections (especially infectious mononucleosis), lymphocytes may increase greatly in size, developing unusually shaped nuclei and large amounts of dark blue cytoplasm. Such cells are referred to as reactive or atypical lymphocytes^[5]:9–97 and when present they are either commented on or counted separately from normal lymphocytes in the manual differential.^[37]

Increased lymphocyte counts (lymphocytosis) can be caused by various viral infections^[30]:309 and may also occur after splenectomy.^[39]:258-259 Children have higher lymphocyte counts than adults.^[39]:258-259Chronic lymphocytic leukemia presents with an elevated lymphocyte count and abnormal lymphocyte morphology, in which the lymphocytes have extremely dense, clumped nuclei^[44] and some cells appear as smudges on the blood smear.^[45] Low lymphocyte counts (lymphopenia) may be seen in infections such as HIV/AIDS, influenza and viral hepatitis, as well as in protein-energy malnutrition,^[46] acute illnesses and drug reactions.^[39]:258-259

Monocytes[edit]

Cap Color Atlas Of Hematology

Monocytes are large cells with a curved or folded nucleus and finely granulated, grey-blue cytoplasm that often contains vacuoles (holes). Monocytes are the third most common white blood cell after neutrophils and lymphocytes.^[30]:307 Increased monocyte counts (monocytosis) are seen in chronic infection and inflammation. Extremely high monocyte counts, as well as immature forms of monocytes, occur in chronic myelomonocytic leukemia and acute leukemias of monocytic origin.^[5]:95 Monocyte counts may be decreased (monocytopenia) in individuals who are receiving chemotherapy as well as those with aplastic anemia, severe burns, and AIDS.^[39]:258

Eosinophils[edit]

Eosinophils have large orange granules in their cytoplasm and nuclei with two lobes. They are found in low amounts in normal blood.^[30]:307 Elevated counts (eosinophilia) are associated with allergic reactions, parasitic infections, and asthma.^{[5]:94[39]:256} Eosinophil counts may be decreased in pregnancy and in response to physiological stress, inflammation or treatment with certain drugs, such as steroids and epinephrine.^[39]:256

Basophils[edit]

Basophils exhibit large, dark purple granules that often cover the cell's nucleus. They are the rarest of the five normal cell types, making up less than one percent of the white blood cell count under normal conditions. Basophilia and eosinophilia can occur along with other white blood cell abnormalities in chronic myeloid leukemia and other myeloproliferative disorders.^[5]:94–5 An increased basophil count may also be seen in hypersensitivity reactions and after splenectomy. The basophil count may decrease during ovulation, steroid treatment, and periods of physiological stress.^[39]:257

Band neutrophils[edit]

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Band neutrophils are young forms of neutrophils that lack segmentation of the nucleus. These cells, which are identified by manual counting, are found in low numbers in normal adult blood. The term left shift refers to an increase in bands or immature granulocytes, and it can indicate infection, inflammation or bone marrow disorders, although it can also be a normal finding in pregnancy.^{[5]:93[42]:153–4} Some laboratories do not separate bands from mature neutrophils in the differential count because the classification is highly subjective and unreliable.^[37]

Immature granulocytes[edit]

Immature granulocytes are immature forms of neutrophils and other granulocytes (eosinophils and basophils). This classification consists of metamyelocytes, myelocytes and promyelocytes, which may be enumerated separately in the manual differential or reported together as immature granulocytes (IG) by automated methods. Immature granulocytes are normally found in the bone marrow, but not in peripheral blood. When present in significant quantities in the blood, immature granulocytes can indicate infection and inflammation, as well as myeloproliferative disease, leukemia and other conditions affecting the marrow.^[47]Chronic myeloid leukemia often presents with a high number of immature granulocytes in the peripheral blood.^[48] Abnormal promyelocytes with multiple Auer rods, called faggot cells, occur in acute promyelocytic leukemia.^[49]

Blasts[edit]

Blast cells are very immature cells that are normally found in the bone marrow, where they develop into mature cells before being released into the blood. They can be identified by their large overall size, deep blue cytoplasm, and large nucleus with fine, lace-like chromatin and prominent nucleoli.^[50] When seen on the blood smear, blasts are an abnormal finding and may be indicative of acute leukemia or other serious blood disorders. Rarely, they may be seen in severe cases of left shift. The presence of Auer rods inside blast cells indicates that they are of myeloid origin, which has important implications for leukemia treatment.^{[50][51]:6–7}

Others[edit]

Various other abnormal cells may be present in the blood in certain conditions. For example, lymphoma cells may be found on the manual differential in some cases of lymphoma,^[51]:228 and in mast cell leukemia, mast cells, which are normally confined to tissue, circulate in the blood.^[51]:328 There is a very rare phenomenon called carcinocythemia in which tumour cells are seen on the peripheral blood smear.^[52]:185

Reference ranges[edit]

Reference ranges for the white blood cell differential are defined by individual laboratories and can vary based on the patient population and the equipment in use.^[37] A general overview of differential reference ranges is presented below.

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53. ^References at Reference ranges for blood tests#White blood cells 2