chemotherapy) or in certain infections, such as tuberculosis and gram-negative sepsis. neutropenia also occurs in many hematologic disorders, such as leukemia and myelodysplastic syndrome, and in a variety of autoimmune and congenital diseases. a neutrophil count below the reference interval may be normal in individuals of certain ethnicities; this is termed benign ethnic neutropenia. very low neutrophil counts are associated with immunosuppression. when stimulated by infection or inflammation, neutrophils may develop abnormal features in their cytoplasm, such as toxic granulation, toxic vacuolation and döhle bodies. these features, which are caused by the release of cytokines, are collectively known as toxic changes. lymphocytelymphocytes, 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. increased lymphocyte counts (lymphocytosis) can be caused by viral infections and may also occur after splenectomy. children have higher lymphocyte counts than adults. chronic lymphocytic leukemia presents with an elevated lymphocyte count and abnormal lymphocyte morphology, in which the lymphocytes have extremely dense, clumped nuclei and some cells appear smudged on the blood smear. low lymphocyte counts (lymphopenia) may be seen in infections such as hiv/aids, influenza and viral hepatitis, as well as in protein-energy malnutrition, acute illnesses and drug reactions. 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 and when present they are either commented on or counted separately from normal lymphocytes in the manual differential. monocytemonocytes are large cells with a curved or folded nucleus and finely granulated, grey-blue cytoplasm that often contains vacuoles. monocytes are the third most common white blood cell after neutrophils and lymphocytes. 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. monocyte counts may be decreased (monocytopenia) in individuals who are receiving chemotherapy as well as those with aplastic anemia, severe burns, and aids. eosinophileosinophils have large orange granules in their cytoplasm and bi-lobed nuclei. they are found in low amounts in normal blood. elevated eosinophil counts (eosinophilia) are associated with allergic reactions, parasitic infections, and asthma. eosinophil counts may be decreased in pregnancy and in response to physiological stress, inflammation or treatment with certain drugs, such as steroids and epinephrine. basophilbasophils exhibit large, dark purple granules that often cover the cell's nucleus. they are the rarest of the five normal cell types. basophilia and eosinophilia can occur along with other white blood cell abnormalities in chronic myeloid leukemia and other myeloproliferative disorders. 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. band neutrophilband neutrophils are young forms of neutrophils which lack segmentation of the nucleus. these cells, which are identified by manual counting, are found in low numbers in normal adult blood. a left shift, meaning an increase in band neutrophils or immature granulocytes, can indicate infection, inflammation or bone marrow disorders, although it can also be a normal finding in pregnancy. some laboratories do not separate bands from mature neutrophils in the differential count because the classification is highly subjective and unreliable. immature granulocyteimmature 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. igs may also be increased in steroid use and pregnancy. chronic myeloid leukemia often presents with a high number of immature granulocytes in the peripheral blood. abnormal promyelocytes with multiple auer rods, called faggot cells, occur in acute promyelocytic leukemia. blast cellblast cells are very immature cells that are normally found in the bone marrow, where they develop into mature cells (hematopoiesis) before being released into the blood. they can be identified by their large overall size, deep blue cytoplasm, and large nucleus with fine chromatin and prominent nucleoli. when seen on the blood smear, blast cells 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. other morphologic features can provide information about the lineage of blast cells: for example, myeloblasts tend to be large with distinct nucleoli, while lymphoblasts can be smaller with a denser chromatin pattern. however, these features are not diagnostic, and flow cytometry or special staining is generally used to confirm the lineage. other cellsvarious 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, and in mast cell leukemia, mast cells, which are normally confined to tissue, circulate in the blood. there is a very rare phenomenon called carcinocythemia in which tumour cells are seen on the peripheral blood smear. history before automated cell counters were introduced, cell counts were performed manually; white and red blood cells, and platelets were counted using microscopes. the first person to publish microscopic observations of blood cells was antonie van leeuwenhoek, who reported on the appearance of red cells in a 1674 letter to the proceedings of the royal society of london; jan swammerdam had described red blood cells some years earlier, but had not published his findings at the time. throughout the 18th and 19th centuries, improvements in microscope technology such as achromatic lenses allowed white blood cells and platelets to be counted in unstained samples. in the 1870s, paul ehrlich 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. dmitri leonidovich romanowsky improved on this technique in the 1890s by using a mixture of eosin and aged methylene blue, which produced a wide range of hues that was not present when either of the stains was 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. by the early years of the 20th century, the white blood cell differential had become a common practice in the united states, but difficulties in interpreting the results cast doubt on the test's utility. in 1906, charles langdon gibson introduced the gibson chart, which compared the total white blood cell count against the neutrophil count to distinguish between "pyogenic" and "non-pyogenic" conditions and to predict the severity of infections. around the same time, josef arneth proposed a system of classifying neutrophils by their number of nuclear lobes – termed the "lobe index" or arneth count – and established a set of reference ranges for neutrophil lobularity. arneth's analysis of neutrophil segmentation was later found to have limited clinical significance, but the association of hypersegmented neutrophils with vitamin b12 and folate deficiency remains accepted. viktor schilling in 1912 proposed a different classification of neutrophils, separating them into "myelozyten, jugendliche, stabkernige and segmentkernige" – that is, myelocytes, "juveniles" (metamyelocytes), band neutrophils (sometimes called "stabs"), and mature, fully segmented neutrophils – and remarked on the clinical significance of the neutrophilic left shift in conjunction with the white blood cell count and the presence of toxic changes. schilling's monograph, das blutbild und seine klinische verwertung (the blood picture and its clinical significance), was translated into english in 1926, and his neutrophil classification system quickly found acceptance in american laboratories. the first automated hematology analyzer, the coulter counter, was invented in the early 1950s by wallace h. coulter. the analyzer worked on the coulter principle, which states that when cells are suspended in a fluid carrying an electric current and passed through an aperture, they cause decreases in current proportional to their volume because of their poor electrical conductivity. the number and magnitude of these decreases can be used to count blood cells and calculate their sizes. the coulter counter was initially designed for counting red blood cells, but it proved effective for counting white blood cells as well. the model a coulter counter, the first commercial hematology analyzer after basic cell counting had been automated, the white blood cell differential remained a challenge. research into automating the differential count began in the 1970s and took two main approaches: digital image processing and flow cytometry. using technology developed in the 1950s and 60s to automate the reading of pap smears, several models of image processing analyzers were produced. these instruments would scan a stained blood smear to find cell nuclei, then take a higher resolution snapshot of the cell to analyze it through densitometry. they were expensive, slow, and did little to reduce workload in the laboratory because they still required blood smears to be prepared and stained, so flow cytometry-based systems became more popular, and by 1990, no digital image analyzers were commercially available in the united states or western europe. these techniques enjoyed a resurgence in the 2000s with the introduction of more advanced image analysis platforms using artificial neural networks. early flow cytometry devices shot beams of light at cells in specific wavelengths and measured the resulting absorbance, fluorescence or light scatter, collecting information about the cells' features and allowing cellular contents such as dna to be quantified. one such instrument—the rapid cell spectrophotometer, developed by louis kamentsky in 1965 to automate cervical cytology—could generate blood cell scattergrams using cytochemical staining techniques. leonard ornstein, who had helped to develop the staining system on the rapid cell spectrophotometer, and his colleagues later created the first commercial flow cytometric white blood cell differential analyzer, the hemalog d. introduced in 1974, this analyzer used light scattering, absorbance and cell staining to identify the five normal white blood cell types in addition to "large unidentified cells", a classification that usually consisted of atypical lymphocytes or blast cells. the hemalog d could count 10,000 cells in one run, a marked improvement over the manual differential. by 1977 it was estimated that "at least 200" automated differential analyzers were in use throughout the world. in 1981, technicon combined the hemalog d with the hemalog-8 analyzer to produce the technicon h6000, the first combined complete blood count and differential analyzer. this analyzer was unpopular with hematology laboratories because it was labour-intensive to operate, but in the late 1980s to early 1990s similar systems were widely produced by other manufacturers such as sysmex, abbott, roche and beckman coulter.