Complete blood count
A complete blood count, also known as a full blood count, is a set of medical laboratory tests that give information about the cells in a person's blood. The CBC reports the amounts of white blood cells, red blood cells and platelets, the concentration of hemoglobin in the blood, and the hematocrit, which is the percentage of the blood that is made up of red blood cells. The red blood cell indices, which calculate the average size and hemoglobin content of red blood cells, are also reported, and a white blood cell differential, which counts the different types of white blood cells, may be included.
The CBC is often performed on healthy people as part of a medical examination and can be used to help monitor health or diagnose diseases. Anemia and thrombocytopenia can be diagnosed from abnormal results, which may indicate a need for urgent medical treatment, such as a blood transfusion, particularly if the hemoglobin concentration is very low. The red blood cell indices can provide information about the cause of a person's anemia such as iron deficiency and vitamin B12 deficiency. Complete blood count results are interpreted by comparing them to reference ranges, which vary according to gender and age and are based on the range of results obtained from healthy individuals.
A complete blood count is performed manually using basic laboratory equipment or by an automated analyzer, which is a machine designed to perform laboratory tests. The analyzer counts red and white blood cells and platelets, isolating single cells and collecting information about their size and structure. The concentration of hemoglobin is measured, and the red blood cell indices are calculated from the red blood cell count, average red cell volume, and hemoglobin. Sometimes, manual techniques are used to confirm the results of automated testing. Approximately 10–25% of complete blood count samples need a manual blood smear review. This requires the blood to be stained and viewed under a microscope to verify that the analyzer results are consistent with the appearance of the blood and to look for abnormalities in the appearance of blood cells. The hematocrit may also be performed manually if the automated results are questionable.
Karl Vierordt performed what is considered the first blood count in 1852, by spreading a specific volume of blood on a microscope slide and counting the cells. The invention of the hemocytometer in 1875 by Louis-Charles Malassez simplified the microscopic analysis of blood cells, and in the late 19th century, Paul Ehrlich and Dmitri Leonidovich Romanowsky developed methods for staining white and red blood cells that are still used to examine blood smears. Automated methods for measuring hemoglobin were developed in the 1920s, and Maxwell Wintrobe introduced the Wintrobe hematocrit method in 1929, which in turn enabled him to define the red blood cell indices. Automation of blood cell counts began with the invention of the Coulter counter by brothers Wallace and Joseph A. Coulter in the 1950s. This device used electrical impedance measurements to count blood cells and measure their sizes, a technology that remains in use in many automated analyzers. Further research in the 1970s and 1980s involved the use of light scattering measurements and fluorescent staining to count and identify cells, which enabled the automation of the white blood cell differential.
Purpose
The complete blood count provides information about the three cellular components of blood—red blood cells, white blood cells and platelets. Some medical conditions, such as anemia or thrombocytopenia, are defined by marked increases or decreases in complete blood count parameters. Changes in many organ systems may affect the blood, so CBC results are useful for investigating a wide range of conditions. Because of the amount of information it provides, the complete blood count is one of the most commonly performed medical laboratory tests.The CBC is often used to screen for diseases as part of a medical examination. It is also performed when a healthcare provider suspects a person has a disease that affects blood cells, such as anemia, bleeding disorders, infection, inflammatory conditions, and some cancers. People who have had abnormal complete blood count results in the past or who are receiving treatments that can affect blood cell counts may have a regular CBC performed to monitor their health, and the test is often performed each day on people who are hospitalized. The results may be used to determine if a person needs a blood transfusion or platelet transfusion.
The complete blood count has specific applications in many medical specialties. It is routinely performed before a person undergoes surgery to ensure that their hemoglobin and platelet levels are sufficient, as well as after surgery, so that blood loss can be monitored. Blood counts are closely monitored in people undergoing chemotherapy or radiation therapy for cancer, because these treatments suppress the production of blood cells in the bone marrow and can cause severely low levels of white blood cells, platelets and hemoglobin. Regular CBCs are also necessary for people taking some psychiatric drugs, such as clozapine and carbamazepine, which in rare cases can cause a life-threatening drop in the number of white blood cells. Because pregnancy causes many changes in the blood and is associated with serious hematologic conditions like HELLP syndrome, and because anemia during pregnancy can result in poorer outcomes for the mother and her baby, the complete blood count is a routine part of prenatal care. After the baby is born, a complete blood count and blood smear examination may be done to investigate the cause of jaundice or to count the number of immature cells in the white blood cell differential, which can be an indicator of sepsis.
In hematology, the study of blood, the complete blood count is an essential tool. The results of the CBC and blood smear examination reflect the functioning of the hematopoietic system—the organs and tissues involved in the production and development of blood cells, such as the bone marrow, lymph nodes, thymus and spleen. For example, a low count of all three cell types can indicate that blood cell production is being affected by a disease of the bone marrow, which can be checked by a bone marrow examination to further investigate the cause. Blood cancers like acute leukemia and lymphoma may present with abnormal cells on the blood smear, while an abnormally high count of neutrophils or lymphocytes, in combination with a person’s symptoms and blood smear findings, may raise suspicion for a myeloproliferative disorder or lymphoproliferative disorder. Examination of the CBC results and the blood smear can help to determine whether anemia is due to an inherited condition like sickle cell anemia or thalassemia, or an acquired cause, such as nutritional deficiency.
When the prevalence of disease in a population is low, as in when the complete blood count is performed on healthy people, abnormal results may be more likely to be false positives than to represent a real medical issue. The utility of the CBC as a screening test is therefore questionable. The US Centers for Disease Control and Prevention and US Preventive Services Task Force do not recommend using the CBC for screening in healthy adults who are not pregnant, and professional organizations in the United States, United Kingdom and Canada recommend against pre-operative CBC testing for low-risk surgeries in individuals without relevant medical conditions. Repeated blood draws for CBC testing can deplete hemoglobin and platelets, which contributes to hospital-acquired anemia and can result in unnecessary transfusions. A 2011 study in the United States found that a CBC was performed on approximately 56% of adults presenting for an annual checkup, leading to an estimated loss of $33 million USD per year.
Procedure
The sample of blood is collected by venipuncture, drawing the blood into a tube containing an anticoagulant—typically ethylenediaminetetraacetic acid —to stop its natural clotting. The blood is usually taken from a vein, but sometimes it is collected by puncturing the skin of the finger or heel for young children and people whose veins are difficult to access. The blood sample is typically tested on an automated analyzer, but manual techniques such as a blood smear examination or manual hematocrit test may be used to investigate abnormal or questionable results. Cell counts and hemoglobin measurements may be performed manually in laboratories that do not have access to automated instruments.Automated
On board the analyzer, the blood sample is agitated to evenly distribute the cells, then diluted and partitioned into at least two different channels, one of which is used to count red blood cells and platelets, the other to count white blood cells. Some instruments measure hemoglobin in a separate channel, and additional channels may be used for differential white blood cell counts and specialized measurements of platelets. A process called hydrodynamic focusing is used to isolate individual cells so that their properties can be measured. The diluted sample is injected into a stream of low-pressure fluid, which causes the cells in the sample to line up in single file through laminar flow. This method of analyzing single cells suspended in a fluid stream is known as flow cytometry.A reagent chemical is added to the sample to destroy the red cells. This releases hemoglobin from the cells, allowing its concentration to be measured, and in instruments that measure hemoglobin and white blood cell counts in the same channel, permits white blood cells to be counted more easily. Hematology analyzers measure hemoglobin using spectrophotometry and are based on the linear relationship between the absorbance of light and the amount of hemoglobin present. Chemicals are used to convert different forms of hemoglobin, such as oxyhemoglobin and carboxyhemoglobin, to one stable form, usually cyanmethemoglobin, and to create a permanent colour change. The absorbance of the resulting colour, when measured at a specific wavelength—usually 540 nanometers—corresponds with the concentration of hemoglobin in the blood.
Sensors count and identify the cells in the sample using two main principles: electrical impedance and light scattering. Impedance-based cell counting operates 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 because of their poor electrical conductivity. The height of the voltage pulse generated as a cell crosses the aperture correlates with the amount of fluid displaced by the cell, and thus the cell's volume, while the total number of peaks correlates with the number of cells in the sample. The distribution of cell volumes is plotted on a histogram, and by setting volume thresholds based on the typical sizes of each type of cell, the different cell populations can be identified and counted.
In light scattering techniques, light from a laser or a tungsten-halogen lamp is directed at the stream of cells to collect information about their size and structure. Cells scatter light at different angles as they pass through the beam, which is detected using photometers. Forward scatter, which refers to the amount of light scattered at 0 degrees from the beam's axis, is mainly caused by diffraction and correlates with cellular size. Side scatter results from reflection and refraction of light by intracellular structures and correlates with cellular complexity. Different brands of analyzers may measure light at other angles. White blood cells, red blood cells and platelets, as well as individual types of white blood cells, can be distinguished based on light scattering characteristics.
Radiofrequency-based methods can be used in combination with impedance to collect additional information. These techniques work on the same principle of measuring the interruption in current as cells pass through an aperture, but since the high-frequency RF current penetrates into the cells, the magnitude of the resulting pulse relates to factors like the relative size of the nucleus, the nucleus's structure, and the amount of granules in the cytoplasm. Small red cells and cellular debris are similar in size to platelets and may cause significant interference with the platelet count when it is very low, so some analyzers use additional techniques to measure platelets, such as fluorescent staining, multi-angle light scatter and monoclonal antibody tagging.
Red blood cell indices are reported in addition to cell counts and hemoglobin. Automated hematology analyzers measure the average size of red blood cells, which is called the mean cell volume. They use the amount of hemoglobin, the MCV, the red blood cell count and the hematocrit values to calculate the average amount of hemoglobin within each red blood cell and its concentration. Another calculation, the red blood cell distribution width, or RDW, indicates whether the red blood cells are mostly the same size, or if there is noticeable variation in their sizes.
White blood cells can be classified into three types based on measurements of cell volume by impedance or light scattering. This is called a three-part differential. More advanced analyzers use additional techniques to provide a five- to seven-part differential, such as using dyes to stain specific chemicals inside cells—for example, nucleic acids, which are found in higher concentrations in immature cells or myeloperoxidase, an enzyme found in cells of the myeloid lineage. Basophils, which are difficult to distinguish from other white blood cells using conventional methods, may be counted in a separate channel where a reagent destroys other white cells and leaves basophils intact. The data collected from these measurements is analyzed and plotted on a scattergram, where it forms clusters that correlate with each white blood cell type. Another approach to automating the differential count is the use of 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.
On most analyzers, it takes less than one minute to run all the tests in the complete blood count. Because the analyzer samples and counts many cells, the results are very precise. However, some abnormal cells in the blood may not be identified correctly, requiring manual review of the instrument's results and identification by other means of abnormal cells the instrument could not categorize.
Point-of-care testing
refers to blood tests conducted outside of the laboratory setting, such as at a person's bedside or in a clinic. This method of testing is faster and uses less blood than conventional methods, and does not require specially trained personnel, so it is useful in emergency situations and in areas with limited access to resources. Commonly used devices for point-of-care hematology testing include the HemoCue, a portable analyzer that uses spectrophotometry to measure the hemoglobin concentration of a blood sample, and the i-STAT, which derives a hemoglobin reading by estimating the concentration of red blood cells from the conductivity of the blood. Hemoglobin and hematocrit can also be measured on point-of-care devices designed for blood gas testing, but the accuracy of these readings is questionable. There are simplified versions of hematology analyzers designed for use in clinics that can provide a complete blood count and differential.Manual
The tests in the complete blood count can be performed using manual techniques when automated equipment is not available or when the analyzer results are questionable. Automated complete blood count results are flagged for manual blood smear review in 10 to 25 percent of cases, which could be because of abnormal cell populations that the analyzer cannot count properly, internal flags generated by the analyzer that suggest the results may be inaccurate, or numerical results that fall outside set thresholds. To investigate these issues, a smear is made from the blood sample, stained with a Romanowsky stain, and examined under a microscope. The appearance of the red and white blood cells and platelets is assessed, and qualitative abnormalities are reported if present. Changes in the appearance of red blood cells can have considerable diagnostic significance—for example, the presence of sickle cells is indicative of sickle cell disease, and a high number of fragmented red blood cells can suggest a serious condition called microangiopathic hemolytic anemia. The platelet count can be estimated from the blood smear, which is useful if the automated platelet results are inaccurate.A manual white blood cell differential count may be performed, in which a large number of white blood cells are counted and classified into different types. This gives the percentage of each type of white blood cell, and by multiplying these percentages by the total number of white blood cells, the absolute number of each type of white cell can be obtained. Manual counting is subject to sampling error because so few cells are counted compared with automated analysis, but it can identify abnormal cells that analyzers cannot.
Automated hematocrit measurements can be incorrect in some conditions, such as polycythemia or severe leukocytosis. In these cases, a manual hematocrit can be performed by filling a capillary tube with blood, centrifuging it, and measuring the percentage of the blood that consists of red blood cells. Outside of these conditions, the automated hematocrit is more accurate than the manual hematocrit.
Red and white blood cells and platelets can be counted under the microscope using a hemocytometer, a microscopic counting chamber that holds a specified volume of diluted blood. The cells seen in the hemocytometer's counting grid are counted and divided by the volume of blood used to determine the concentration of cells in the blood sample.This process is labour-intensive and inaccurate compared to automated methods, so it is rarely used except in under-resourced laboratories that do not have access to automated analyzers.
Hemoglobin can be measured manually using a spectrophotometer or colorimeter. To measure hemoglobin manually, the blood sample is diluted using reagents that destroy red blood cells to release the hemoglobin. Other chemicals are used to convert different types of hemoglobin to one form, allowing it to be easily measured. The solution is then placed in a measuring cuvette and the absorbance is measured at a specific wavelength, which depends on the type of reagent used. A reference standard containing a known amount of hemoglobin can be used to determine the relationship between the absorbance and the hemoglobin concentration, allowing the hemoglobin level of the sample to be determined.
In rural and economically disadvantaged areas, the tests available are limited by access to equipment and personnel. At primary care facilities in these areas, testing may be limited to examination of red cell morphology and manual measurement of hemoglobin or hematocrit, while more complex techniques like manual cell counts and differentials, and sometimes automated cell counts, are performed at district laboratories. Regional and provincial hospitals and academic centres typically have access to automated analyzers. Where laboratory facilities are not available, a rough estimate of hemoglobin concentration can be obtained by placing a drop of blood on a special absorbent paper and comparing it to a colour scale.
Quality control
Automated analyzers have to be regularly calibrated. Most manufactures supply preserved blood with defined parameters and the analyzers are adjusted if the results obtained are outside the limits allowed. To ensure that results from the automated analyzer continue to be correct, quality control samples are tested at least once per day. These are samples with known results that are usually provided by the instrument manufacturer. Laboratories compare their complete blood count results to the known values to ensure the instrument is operating correctly. For laboratories that do not have access to commercial quality control material, an Indian regulatory organization recommends running patient samples in duplicate and comparing the results. A moving average measurement, in which the average results for patient samples are measured at intervals, may be used in addition to routine quality control. 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. The MCHC values are particularly useful in this regard.Hematology laboratories may also receive external quality assessment samples from regulatory organizations, which are samples whose results are not disclosed to the laboratory. Laboratories report their results for these samples to the organization and they are compared against the known values to ensure that they are correct. External quality assessment programs have been widely adopted in North America and western Europe, and laboratories are often required to participate in these programs to maintain accreditation. However, logistical issues may make it difficult for laboratories in under-resourced areas to implement external quality assessment schemes.
Included tests
In the complete blood count, the amounts of red blood cells, white blood cells, and platelets are measured, along with the hemoglobin and hematocrit values. The red blood cell indices—MCV, MCH and MCHC—which describe the size of red blood cells and their hemoglobin content, are reported along with the red blood cell distribution width, which measures the amount of variation in the sizes of red blood cells. A white blood cell differential, which enumerates the different types of white blood cells, may be performed.Red blood cells, hemoglobin, and hematocrit
Red blood cells carry hemoglobin throughout the body—to the tissues, where it delivers oxygen, and to the lungs, where it releases carbon dioxide and is oxygenated again. The analyzer counts red blood cells, reporting the result in units of 106 cells per microlitre of blood or 1012 cells per litre, and measures their average size, which is called the mean cell volume and expressed in femtolitres or cubic millimetres. By multiplying the mean cell volume by the red blood cell count, the hematocrit or packed cell volume, a measurement of the percentage of blood that is made up of red blood cells, can be derived. Hemoglobin, measured after the red blood cells are lysed, is reported in units of grams per litre or grams per decilitre. Assuming that the red blood cells are normal, there is a constant relationship between hemoglobin and hematocrit: the hematocrit percentage is approximately three times greater than the hemoglobin value in g/dL, plus or minus three. This relationship, called the rule of three, can be used to confirm that CBC results are correct.Two other measurements are calculated from the red blood cell count, the hemoglobin concentration, and the mean cell volume: the mean cell hemoglobin and the mean cell hemoglobin concentration. These parameters describe the hemoglobin content of each red blood cell. The MCH and MCHC can be confusing; in essence the MCH is a measure of the average amount of hemoglobin per red blood cell. The MCHC gives the average proportion of the cell that is hemoglobin. The MCH does not take into account the size of the red blood cells whereas the MCHC does. Collectively, the MCV, MCH, and MCHC are referred to as the red blood cell indices. Changes in these indices are visible on the blood smear: red blood cells that are abnormally large or small can be identified by comparison to the sizes of white blood cells, and cells with a low hemoglobin concentration appear pale. Another parameter is calculated from the initial measurements of red blood cells: the red blood cell distribution width or RDW, which reflects the degree of variation in the cells' size.
An abnormally low hemoglobin, hematocrit, or red blood cell count is called anemia. Anemia is not a diagnosis on its own, but it points to an underlying condition affecting the person's red blood cells. General causes of anemia include blood loss, production of defective red blood cells, decreased production of red blood cells, and increased destruction of red blood cells. Anemia reduces the blood's ability to carry oxygen, causing symptoms like tiredness and shortness of breath. If the hemoglobin level falls below thresholds based on the person's clinical condition, a blood transfusion may be necessary.
An increased proportion of red blood cells, which usually leads to an increase in the hemoglobin and hematocrit, is called polycythemia. Dehydration or use of diuretics can cause polycythemia by decreasing the amount of blood plasma compared to red cells. Polycythemia can also occur when the body produces more red blood cells to compensate for chronically low oxygen levels in conditions like lung or heart disease, or when a person has abnormally high levels of erythropoietin, a hormone that stimulates production of red blood cells. In polycythemia vera, the bone marrow produces red cells and other blood cells at an excessively high rate. A markedly increased red blood cell count makes the blood more viscous, leading to a tendency to form clots inappropriately.
Evaluation of red blood cell indices is helpful in determining the cause of anemia. Anemia with a low MCV is referred to as microcytic anemia, and anemia with a high MCV is called macrocytic anemia. Anemia with a low MCHC is called hypochromic anemia. If anemia is present but the red blood cell indices are normal, the anemia is termed normochromic and normocytic. The term hyperchromia, referring to a high MCHC, is generally not used, as elevation of the MCHC above the upper reference value is rare, mainly occurring in conditions that cause red blood cells to be abnormally shaped, such as spherocytosis, sickle cell disease and Hemoglobin C disease. An elevated MCHC can also be a false result from conditions like red blood cell agglutination or highly elevated amounts of lipids in the blood. The MCV serves an additional purpose in laboratory quality control. It is relatively stable over time compared to other CBC parameters, so a large change in MCV may indicate that the sample was drawn from the wrong patient.
Microcytic anemia is typically associated with iron deficiency, thalassemia, and anemia of chronic disease, while macrocytic anemia is associated with alcoholism, folate and B12 deficiency, use of some drugs, and some bone marrow diseases. Acute blood loss, hemolytic anemia, bone marrow disorders and various chronic diseases can result in anemia with a normocytic blood picture. A low RDW has no clinical significance, but an elevated RDW represents increased variation in red blood cell size, a condition known as anisocytosis. Anisocytosis is common in nutritional anemias such as iron deficiency anemia and anemia due to Vitamin B12 or folate deficiency, while people with thalassemia may have a normal RDW. Based on the CBC results, further steps can be taken to investigate anemia, such as a ferritin test to confirm the presence of iron deficiency, or hemoglobin electrophoresis to diagnose a hemoglobinopathy such as thalassemia or sickle cell disease.
White blood cells
White blood cells are involved in inflammation and the immune response. A high white blood cell count, which is called leukocytosis, is frequently caused by infections, and may also occur in inflammatory disorders, states of physiologic stress, and diseases such as leukemia that cause abnormal production of blood cells. In some forms of leukemia, extremely high numbers of abnormal white blood cells can cause a medical emergency called leukostasis in which white blood cells clog blood vessels. A decreased white blood cell count, termed leukopenia, can make people more susceptible to infections, and is associated with treatments like chemotherapy and radiation therapy and many disorders that inhibit the production of blood cells. The total white blood cell count is usually reported in cells per microlitre of blood or 109 cells per litre.In the white blood cell differential, the different types of white blood cells are identified and counted. The results are reported as percentage and as an absolute number per unit volume. Five types of white blood cells—neutrophils, lymphocytes, monocytes, eosinophils, and basophils—are normally measured. White blood cell types that are not normally found in the blood, such as blast cells, are reported if present.
Differential results are useful in diagnosing and monitoring various medical conditions. For example, an elevated neutrophil count is often associated with bacterial infection and inflammation, while a decreased count may occur in individuals who are undergoing chemotherapy or taking certain drugs, or who have diseases affecting the bone marrow. People with severe neutropenia and clinical signs of infection may be treated with antibiotics and given drugs to increase their neutrophil count. An elevated lymphocyte count is associated with viral infection and may occur in lymphoproliferative disorders like chronic lymphocytic leukemia. Elevated monocyte counts are associated with chronic inflammatory states, and the eosinophil count may be high in parasitic infections and allergic conditions. The presence of some types of abnormal cells, such as blast cells and lymphoma cells, may suggest a hematologic malignancy.
Platelets
Platelets are the cellular component of the blood that is responsible for clotting. A low platelet count, called thrombocytopenia, can cause bleeding, and may occur as a consequence of chemotherapy or radiation treatment, use of some drugs, blood disorders such as acute leukemia and aplastic anemia, and autoimmune diseases. If the platelet count is extremely low, a platelet transfusion may be performed. Thrombocytosis, meaning a high platelet count, may occur in states of inflammation or trauma, and the platelet count may reach exceptionally high levels in people with essential thrombocythemia, a rare blood disease. The platelet count is usually reported in units of 103 cells per microlitre of blood or 109 cells per litre.The mean platelet volume, which is a measurement of the average size of platelets in femtolitres, may be reported. The MPV can aid in determining the cause of thrombocytopenia: an elevated MPV may occur in conditions where platelets are destroyed, such as in immune thrombocytopenic purpura, while a decreased MPV may occur in thrombocytopenia caused by malfunction of megakaryocytes. Congenital diseases that cause thrombocytopenia, like Bernard–Soulier syndrome and gray platelet syndrome, may affect the MPV. The immature platelet fraction or reticulated platelet count is reported by some analyzers and provides information about the rate of platelet production by measuring the number of immature platelets in the blood.
Other
Reticulocyte count
Reticulocytes are immature red blood cells, which, unlike the mature cells, contain RNA. A reticulocyte count is sometimes performed as part of a complete blood count, usually to investigate the cause of a person's anemia or evaluate their response to treatment. Anemia with a high reticulocyte count can indicate that the bone marrow is producing red blood cells at a higher rate to make up for blood loss or hemolysis, while anemia with a low reticulocyte count may suggest that the person has a condition that reduces the body's ability to produce red blood cells. When people with nutritional anemia are given nutrient supplementation, an increase in the reticulocyte count indicates that their body is responding to the treatment by producing more red blood cells. Hematology analyzers perform reticulocyte counts by staining red blood cells with a dye that binds to RNA and measuring the number of reticulocytes through light scattering or fluorescence analysis. The test can be performed manually by staining the blood with new methylene blue, which binds to RNA, and counting the percentage of red blood cells containing RNA under the microscope. The reticulocyte count may be expressed as an absolute number or as a percentage of red blood cells.Some instruments can measure the average amount of hemoglobin in each reticulocyte, a parameter that has been studied as an indicator of iron deficiency in people who have conditions that interfere with standard tests. The immature reticulocyte fraction is another measurement produced by some analyzers which quantifies the maturity of reticulocytes: cells that are less mature contain more RNA and thus produce a stronger fluorescent signal. This information can be useful in diagnosing anemias and evaluating red blood cell production following anemia treatment or bone marrow transplantation.
Nucleated red blood cells
During their formation in the bone marrow in adults, and the liver and spleen in the newly born, red blood cells have a cell nucleus, which is usually absent in the mature cells that circulate in the bloodstream. When detected in the blood, the presence of these cells, particularly in children and adults indicates an increased demand for red blood cells, which can be caused by bleeding, some cancers and anemia. Most analyzers can detect these cells as part of the differential cell count. High numbers of nucleated red cells can cause a falsely high white cell count, which will require adjusting.Other parameters
Hematology analyzers generate additional measurements of blood cells that are rarely used in clinical practice but have been investigated for research purposes. For example, some types of analyzers produce coordinate readings indicating the size and position of each white blood cell cluster. These parameters have been studied as potential markers for blood disorders, bacterial infections and malaria. Analyzers that use myeloperoxidase staining to produce differential counts can quantify white blood cells' expression of the enzyme, which has been found to correlate with various disorders. Other types of analyzers can detect the percentage of red blood cells that are hypochromic in addition to reporting the average MCHC value, or provide a count of fragmented red cells, which occur in some types of hemolytic anemia. Because these parameters are often specific to particular brands of analyzers, it is difficult for laboratories to interpret and compare results.Limitations
Some medical conditions or problems with the blood sample may cause analyzers to produce inaccurate complete blood count results. If the blood sample is visibly clotted, which can be caused by poor phlebotomy technique, it is unsuitable for analysis, because the platelet count will be falsely decreased and other results may be abnormal. Samples that have been stored at room temperature for extended periods may give falsely high readings for MCV, because red blood cells swell as they absorb water from the plasma. Platelet and white blood cell differential results may also be inaccurate in aged specimens, as the cells degrade over time.Blood samples drawn from individuals who have very high levels of bilirubin or lipids in their plasma may show falsely high readings for hemoglobin, because these substances change the colour and opacity of the blood sample, which interferes with hemoglobin measurement. This can be avoided by removing the lipemic or icteric plasma and replacing it with saline.
Some people produce an antibody that causes their platelets to clump when their blood is drawn into tubes containing EDTA, the anticoagulant used to collect routine complete blood count samples. Platelet clumps may be counted as single platelets by automated analyzers, leading to a falsely decreased platelet count. This can be avoided by using an alternative anticoagulant like sodium citrate or heparin. Platelet clumping can also occur if the sample has begun to clot due to a difficult blood draw.
Another antibody-mediated condition that can affect complete blood count results is red blood cell agglutination. This phenomenon causes red blood cells to clump together because of antibodies bound to the cell surface. Red blood cell aggregates are counted as single cells by the analyzer, leading to a markedly decreased red blood cell count and hematocrit, and markedly elevated MCV and MCHC. Often, these antibodies are only active at room temperature, and the agglutination can be reversed by heating the blood sample to. People with warm autoimmune hemolytic anemia may exhibit red cell agglutination that does not resolve on warming.
History
Before the invention of automated cell counters, the tests in the complete blood count were performed manually: white blood cells, red blood cells, and platelets were counted under a microscope. The earliest person to publish microscopic observations of blood cells was Antonie van Leeuwenhoek, who in 1675 viewed his blood through a microscope of his own design and described it as consisting of "small red globules, driven through a crystalline humidity of water". Throughout the 18th and 19th centuries, further improvements in microscope technology such as the invention of achromatic lenses allowed white blood cells and platelets to be counted in unstained blood samples.Physiologist Karl Vierordt is credited with performing the first blood count in 1852 at the University of Tübingen. Vierordt's technique involved aspirating a carefully measured volume of blood into a capillary tube and spreading it onto a microscope slide coated with egg white. After the blood dried, he counted every cell on the slide; this process could take more than three hours to complete. The hemocytometer, introduced in 1874 by Louis-Charles Malassez, simplified the microscopic counting of blood cells. Malassez's hemocytometer consisted of a microscope slide containing a flattened capillary tube. Diluted blood was introduced to the capillary chamber by means of a rubber tube attached to one end, and an eyepiece with a scaled grid was attached to the microscope, permitting the microscopist to count the number of cells per volume of blood. In 1877, William Gowers invented a hemocytometer with a built-in counting grid, eliminating the need to produce specially calibrated eyepieces for each microscope.
invented Romanowsky staining.
In the 1870s, Paul Ehrlich developed a staining technique using a combination of an acidic and basic dye that could distinguish between different types of white blood cells and allow red blood cell morphology to be examined. 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 became the basis for Romanowsky staining, the technique that is still used to stain blood smears for manual review.
The first techniques for measuring hemoglobin concentration, devised in the late 19th century, involved visually comparing the colour of diluted blood to a standard. Attempts to automate this process using spectrophotometry and colorimetry were limited by the fact that hemoglobin is present in the blood in many different forms, meaning that it could not be measured at a single wavelength. In 1920, a method to convert the different forms of hemoglobin to one stable form was introduced, allowing hemoglobin levels to be measured automatically. The cyanmethemoglobin method remains the reference method for hemoglobin measurement and is still used in many automated hematology analyzers.
Maxwell Wintrobe is credited with the invention of the hematocrit test. In 1929, Wintrobe undertook a PhD project at the University of Tulane to determine normal ranges for red blood cell parameters. To assist with his research, he invented a method called the Wintrobe hematocrit. Hematocrit measurements had previously been described in the literature, but Wintrobe's method differed from earlier ones in that it used a large tube that could be mass-produced to precise specifications. The Wintrobe method involved letting the blood sit upright in a glass tube for one hour, then centrifuging it and measuring the percentage of red blood cells versus the percentage of plasma to determine the hematocrit. The invention of a reproducible method for determining hematocrit values allowed Wintrobe to define the red blood cell indices.
Research into automated cell counting began in the early 20th century. A method developed in 1928 used the amount of light transmitted through a diluted blood sample, as measured by photometry, to estimate the red blood cell count, but this proved inaccurate for samples with abnormal red blood cells. Other attempts, in the 1930s and 1940s, involved photoelectric detectors attached to microscopes, which would count cells as they were scanned; these methods were unsuccessful. In the 1950s, brothers Wallace H. Coulter and Joseph A. Coulter, working together in a basement laboratory in Chicago, invented the Coulter counter. This device worked on the Coulter principle, which involves measuring the drop in current as cells pass through an aperture to count cells and measure their sizes. The Coulter counter was initially designed for counting red blood cells, but with later modifications it proved effective for counting white blood cells. Coulter counters were widely adopted by medical laboratories.
The first analyzer that could produce multiple cell counts simultaneously was the Technicon, released in 1965. The analyzer accomplished this by partitioning blood samples into two channels: one for counting red blood cells, and one for counting white blood cells and measuring hemoglobin. The instrument was unpopular because it was unreliable and difficult to maintain. In 1968, the Coulter Model S analyzer was released and found widespread use. Similarly to the Technicon instrument, it used two different reaction chambers to measure hemoglobin and the two cell types. The Model S also determined the mean cell volume using impedance measurements, which allowed the red blood cell indices and hematocrit to be derived. Automated platelet counts were introduced in 1970 with Technicon's Hemalog-8 instrument and were adopted by Coulter's S Plus series analyzers in 1980.
After basic cell counting had been automated, the white blood cell differential remained a challenge. Research directed towards 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 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. However, 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 like the Cytofluorograph and Impulscytometer 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. Other researchers refined these techniques by using fluorescent dyes, which bind to specific components of cells. This research into optical cell identification led to the development of 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 unstained 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. 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.