High levels of CRP has been associated to point mutation Cys130Arg in the  APOE  gene, coding for  apolipoprotein E , establishing a link between  lipid  values and inflammatory markers modulation. [ 62 ] [ unreliable medical source? ] [ 61 ]


The role of inflammation in cancer is not well understood. Some organs of the body show greater risk of cancer when they are chronically inflamed. [ 63 ]   While there is an association between increased levels of C-reactive protein and risk of developing cancer, there is no association between genetic polymorphisms influencing circulating levels of CRP and cancer risk. [ 64 ]


In a 2004  prospective cohort study  on colon cancer risk associated with CRP levels, people with  colon cancer  had higher average CRP concentrations than people without colon cancer. [ 65 ]  It can be noted that the average CRP levels in both groups were well within the range of CRP levels usually found in healthy people. However, these findings may suggest that low inflammation level can be associated with a lower risk of colon cancer, concurring with previous studies that indicate  anti-inflammatory drugs  could lower colon cancer risk. [ 66 ]


C-reactive protein (CRP), a marker of systemic inflammation, is also increased in  obstructive sleep apnea  (OSA).  CRP and interleukin-6 (IL-6) levels were significantly higher in patients with OSA compared to obese control subjects. [ 67 ]  Patients with OSA have higher plasma CRP concentrations that increased corresponding to the severity of their  apnea-hypopnea index  score.  Treatment of OSA with CPAP ( continuous positive airway pressure ) significantly alleviated the effect of OSA on CRP and IL-6 levels. [ 67 ]


In the context of  rheumatoid arthritis  (RA), CRP is one of the acute phase reactants, whose assessment is defined as part of the joint 2010 ACR/ EULAR  classification criteria for RA with abnormal levels accounting for a single point within the criteria.  [ 68 ]  Higher levels of CRP are associated with more severe disease and a higher likelihood of radiographic progression. Rheumatoid arthritis associated antibodies together with 14-3-3η  YWHAH  have been reported to complement CRP in predicting clinical and radiographic outcomes in patients with recent onset inflammatory polyarthritis. [ 69 ]  Elevated levels of CRP appear to be associated with common comorbidities including cardiovascular disease,  metabolic syndrome , diabetes and interstitial lung (pulmonary) disease. Mechanistically, CRP also appears to influence  osteoclast  activity leading to  bone resorption  and also stimulates  RANKL  expression in peripheral blood  monocytes . [ 70 ]


It has previously been speculated that  single-nucleotide polymorphisms  in the CRP gene may affect clinical decision-making based on CRP in rheumatoid arthritis, e.g. DAS28 (Disease Activity Score 28 joints). A recent study showed that CRP  genotype  and  haplotype  were only marginally associated with serum CRP levels and without any association to the DAS28 score. [ 71 ]  Thus, that DAS28, which is the core parameter for inflammatory activity in RA, can be used for clinical decision-making without adjustment for CRP gene variants. [ citation needed ]


Increased blood CRP levels were higher in people with  avian flu   H7N9  compared to those with  H1N1  (more common) influenza, [ 72 ]  with a review reporting that severe H1N1 influenza had elevated CRP. [ 73 ]  In 2020, people infected with  COVID-19  in  Wuhan, China , had elevated CRP. [ 74 ] [ 75 ] [ 76 ]


The  calibrated automated thrombogram  ( CAT  or  CT ) is a  thrombin generation assay  (TGA) and  global coagulation assay  (GCA) which can be used as a  coagulation test  to assess  thrombotic  risk. [ 1 ] [ 2 ]  It is the most widely used TGA. [ 3 ]  The CAT is a  semi-automated  test performed in a  96-well plate  and requires specialized  technologists  to be performed. [ 3 ]  As a result, it has seen low implementation in routine laboratories and has been more limited to research settings. [ 3 ] [ 4 ]  Lack of  standardization  with the CAT has also led to difficulties in study-to-study comparisons in research. [ 3 ] [ 5 ]  However, efforts have recently been made towards standardization of the assay. [ 5 ] [ 6 ] [ 7 ] [ 8 ]  An example of a specific commercial CAT is the Thrombinoscope by Thrombinoscope BV (now owned by Diagnostica Stago). [ 9 ]


The CAT can be used to measure  thrombogram parameters  such as the  endogenous thrombin potential  (ETP) [ 2 ] [ 10 ]  and to assess  activated protein C resistance  (APCR). [ 5 ]  The CAT ETP-based  APC resistance test  is especially sensitive to  estrogen -induced  procoagulation , such as with  combined oral contraceptives . [ 2 ] [ 5 ] [ 11 ]


In 2018, a commercial fully-automated TGA system and alternative to the CAT called the  ST Genesia  debuted. [ 3 ]  It has been said that this system should allow for more widespread adoption of TGAs in clinical laboratories. [ 3 ] [ 12 ]  The ST Genesia system also shows improved  reproducibility  compared to the CAT. [ 3 ]


Cancer Likelihood in Plasma  (CLiP) refers to a set of  ensemble learning  methods for integrating various genomic features useful for the noninvasive detection of early cancers from blood  plasma . [ 1 ]  An application of this technique for early detection of  lung cancer  (Lung-CLiP) was originally described by Chabon et al. (2020) [ 2 ]  from the labs of  Ash Alizadeh  and Max Diehn at  Stanford . [ 3 ] [ 4 ]


This method relies on several improvements to cancer personalized profiling by deep sequencing ( CAPP-Seq ) [ 5 ]  for analysis of  circulating tumor DNA  (ctDNA). The CLiP technique integrates multiple distinctive genomic features of a cancer of interest findings within a machine-learning framework for cancer detection. For example, studies have shown that the majority of somatic mutations found in cell-free DNA (cfDNA) are not tumor derived, but instead reflect  clonal hematopoeisis  (also known as CHIP). [ 2 ] [ 6 ]  Even though  CHIP  tends to target specific genes, it also involves many generally non-recurrent mutations that can be shed from  leukocytes  and detected in cfDNA, regardless of whether profiling patients with cancer and healthy adults. [ 2 ]   However, genuine tumor derived  ctDNA mutations  can be distinguished from CHIP-derived mutations. This is because unlike tumor-derived mutations, CHIP-derived mutations that are shed from leukocytes into plasma tend to occur on longer cfDNA fragments, and to lack specific  mutational signatures  such as those associated with tobacco smoking in lung cancer that are also found in tumor derived ctDNA molecules. CLiP integrates these features within hierarchical ensemble  machine learning  models that consider  somatic mutations  and  copy number alternations , among other features. [ 2 ]  While the CLiP method is unique in relying exclusively on mutations and copy number alterations, it is related to a variety of other liquid biopsy methods being commercially developed for early cancer detection using ctDNA and proteins (e.g., CancerSEEK / DETECT-A  [ 7 ] ), cfDNA fragmentation patterns (e.g., DELFI), [ 8 ] [ 9 ]  and DNA methylation (e.g., cfMeDIP-Seq, [ 10 ]  Grail [ 11 ] ).


While the CLiP method has not yet been broadly applied for population-based cancer screening, it has been shown to distinguish discriminate early-stage lung cancers from risk-matched controls across multiple cohorts of patients enrolled across the US. [ 12 ]


The  captopril challenge test  ( CCT ) is a non-invasive medical test that measures the change in  renin   plasma -levels in response to administration of  captopril , an  angiotensin converting enzyme inhibitor . It is used to assist in the diagnosis of  renal artery stenosis . It is not generally considered a useful test for children, [ 1 ]  and more suitable options are available for adult cases.


Plasma concentration of renin is measured prior to and following the administration of captopril. The CCT is considered positive if the renin levels increase substantially or the baseline renin level is abnormally high. [ 2 ]


CCT in adults is known to have high  sensitivity , but a low  specificity . [ 3 ]


Subtraction angiography  is considered a more suitable test for renal artery stenosis in adults. [ 4 ]


The  captopril suppression test  ( CST ) is a non-invasive medical test that measures  plasma  levels of  aldosterone . [ 1 ]
Aldosterone production is suppressed by captopril through the  renin–angiotensin–aldosterone system . CST results are used to assist in the diagnososis of  primary aldosteronism  (Conn syndrome).


CO 2  content  (also known as "Total  CO 2 ") is a  blood test  that usually appears on a "Chem 19" or an  electrolyte  panel. The value measures the total dissolved  Carbon dioxide  ( CO 2 ) in blood. It is determined by combining the  Bicarbonate  ( HCO − 3 ) and the  partial pressure  of  CO 2  multiplied by a factor which estimates the amount of pure  CO 2  that is dissolved in its natural form (usually 0.03). [ citation needed ]


One given reference range is 24–32 mEq/L. [ 1 ]


A  coagulation screen  is a combination of  screening laboratory tests , designed to provide rapid non-specific information, which allows an initial broad categorization of  haemostatic  problems. [ 1 ]


The basic screen consists of:


Two other tests are regularly performed at the same time:


These tests may miss mild abnormalities but they will detect major disorders. The results of these screening tests, in conjunction with the  clinical history  (especially  bleeding  history), will then direct the selection of further, more detailed and specific coagulation tests, such as:


Blood clotting tests  are the tests used for diagnostics of the  hemostasis  system.
Coagulometer is the medical laboratory analyzer used for testing of the hemostasis system. Modern coagulometers realize different methods of activation and observation of development of blood clots in blood or in blood plasma. [ citation needed ]


Substantially all coagulometers used in laboratory diagnostics are based on the methods of testing of the hemostasis system created more than fifty years ago. The majority of these methods are good to detect defects in one of the hemostasis components, without diagnosing other possible defects. Another problem of the actual hemostasis system diagnostics is the  thrombosis  prediction, i.e. sensitivity to the patient's prethrombotic state. All the diversity of clinical tests of the blood coagulation system can be divided into 2 groups: global (integral, general) tests, and «local» (specific) tests. [ citation needed ]


Global tests, also known as  global coagulation assays  (GCAs), [ 1 ]  characterize the results of work of the whole clotting cascade. They suit to diagnose the general state of the blood coagulation system and the intensity of pathologies, and to simultaneously record all attendant influences. Global methods play the key role at the first stage of diagnostics: they provide an integral picture of alterations within the coagulation system and allow predicting a tendency to hyper- or hypo-coagulation in general. [ citation needed ]


Local tests characterize the results of work of the separate components of the blood coagulation system cascade, as well as of the separate coagulation factors. They are essential for the possibility to specify the pathology localization within the accuracy of coagulation factor. [ citation needed ]


A  D-dimer  (product of thrombi degradation) test can be specified separately. The rise of D-dimers concentration in the patient's blood states the possibility of the completed thrombosis.
To obtain a complete picture of the work of hemostasis by a patient, the doctor should have a possibility to choose which test is necessary. [ citation needed ]


According to the type of the investigated object, the following complementary groups of methods can be specified: [ citation needed ]


A  combined rapid anterior pituitary evaluation panel  or  triple bolus test  or a  dynamic pituitary function test  is a medical diagnostic procedure used to assess a patient's  pituitary  function.
A triple bolus test is usually ordered and interpreted by  endocrinologists . [ citation needed ]


In rare cases, it has been associated with  pituitary apoplexy . [ 1 ]


Three  hormones [ 2 ]  (usually synthetic  analogues ) are injected as a  bolus  into the patient's vein to stimulate the  anterior pituitary  gland:


The gland's response is assessed by measuring the rise in  cortisol  and  growth hormone  (GH) in response to the hypoglycaemia caused by insulin, rises in  prolactin  and  thyroid-stimulating hormone  (TSH) caused by TRH and rises in  luteinizing hormone  (LH) and  follicle-stimulating hormone  (FSH) caused by GnRH.  Blood  glucose  levels are also monitored to ensure appropriate levels of  hypoglycemia  are achieved. [ citation needed ]


The triple bolus test was introduced in 1973 by physicians from the London  Royal Postgraduate Medical School  and  Queen Elizabeth Hospital, Birmingham . [ 3 ]  It followed earlier reports combining insulin and  vasopressin  analogues in the diagnosis of hypopituitarism. [ 4 ]


The  complement fixation test  is an  immunological   medical  test that can be used to detect the presence of either specific  antibody  or specific  antigen  in a patient's  serum , based on whether  complement fixation  occurs. It was widely used to diagnose infections, particularly with microbes that are not easily detected by culture methods, and in rheumatic diseases. However, in clinical diagnostics labs it has been largely superseded by other serological methods such as  ELISA  and by DNA-based methods of pathogen detection, particularly  PCR .


The  complement system  is a system of serum proteins that react with antigen-antibody complexes. If this reaction occurs on a cell surface, it will result in the formation of trans-membrane pores and therefore destruction of the cell.  The basic steps of a complement fixation test are as follows: [ 1 ]


If the patient's serum contains antibodies against the antigen of interest, they will bind to the antigen in step 3 to form antigen-antibody complexes.  The complement proteins will react with these complexes and be depleted.  Thus when the sRBC-antibody complexes are added in step 4, there will be no complement left in the serum.  However, if no antibodies against the antigen of interest are present, the complement will not be depleted and it will react with the sRBC-antibody complexes added in step 4, lysing the sRBCs and spilling their contents into the solution, thereby turning the solution pink. [ 1 ]


While detection of antibodies is the most common test format, it is equally possible to test for the presence of antigen. In this case, the patient's serum is supplemented with specific antibody to induce formation of complexes; addition of complement and indicator sRBC is performed as before. [ citation needed ]


The test can be made quantitative by setting up a series of dilutions of patient serum and determining the highest dilution factor that will still yield a positive CF test. This dilution factor corresponds to the  titer . [ citation needed ]


A  complete blood count  ( CBC ), also known as a  full blood count  ( FBC ), is a set of  medical laboratory  tests that provide  information about the cells  in a person's  blood . The CBC indicates the counts of  white blood cells ,  red blood cells  and  platelets , the concentration of  hemoglobin , and the  hematocrit  (the volume percentage of red blood cells). The  red blood cell indices , which indicate 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 carried out as part of a medical assessment and can be used to monitor health or diagnose diseases. The results are interpreted by comparing them to  reference ranges , which vary with sex and age. Conditions like  anemia  and  thrombocytopenia  are defined by abnormal complete blood count results. The red blood cell indices can provide information about the cause of a person's anemia such as  iron deficiency  and  vitamin B12 deficiency , and the results of the white blood cell differential can help to diagnose  viral ,  bacterial  and  parasitic infections  and  blood disorders  like  leukemia . Not all results falling outside of the reference range require medical intervention.


The CBC is usually performed by an  automated hematology analyzer , which  counts cells  and collects information on their size and structure. The concentration of hemoglobin is measured, and the red blood cell indices are calculated from measurements of red blood cells and hemoglobin. Manual tests can be used to independently confirm abnormal results. Approximately 10–25% of samples require a manual  blood smear  review, [ 5 ]  in which the blood is  stained  and viewed under a  microscope  to verify that the analyzer results are consistent with the appearance of the cells and to look for abnormalities. The hematocrit can be determined manually by  centrifuging  the sample and measuring the proportion of red blood cells, and in laboratories without access to automated instruments, blood cells are counted under the microscope using a  hemocytometer .


In 1852,  Karl Vierordt  published the first procedure for performing a blood count, which involved spreading a known volume of blood on a microscope slide and counting every cell. The invention of the hemocytometer in 1874 by  Louis-Charles Malassez  simplified the microscopic analysis of blood cells, and in the late 19th century,  Paul Ehrlich  and  Dmitri Leonidovich Romanowsky  developed techniques 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 allowed him to define the red blood cell indices. A landmark in the automation of blood cell counts was the  Coulter principle , which was patented by  Wallace H. Coulter  in 1953. The Coulter principle uses  electrical impedance  measurements to count blood cells and determine their sizes; it is a technology that remains in use in many automated analyzers. Further research in the 1970s involved the use of  optical  measurements to count and identify cells, which enabled the automation of the white blood cell differential.


Blood is composed of a fluid portion, called  plasma , and a cellular portion that contains  red blood cells ,  white blood cells  and  platelets . [ note 1 ] [ 7 ]  The complete blood count evaluates the three cellular components of blood. Some medical conditions, such as  anemia  or  thrombocytopenia , are defined by marked increases or decreases in blood cell counts. [ 8 ]  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. [ 9 ] [ 10 ] [ 11 ]


The CBC is often used to  screen  for diseases as part of a medical assessment. [ 12 ]  It is also called for when a healthcare provider suspects a person has a disease that affects blood cells, such as an  infection , a  bleeding disorder , or some  cancers . People who have been diagnosed with disorders that may cause abnormal CBC results or who are receiving treatments that can affect blood cell counts may have a regular CBC performed to monitor their health, [ 4 ] [ 12 ]  and the test is often performed each day on people who are hospitalized. [ 13 ]  The results may indicate a need for a  blood  or  platelet transfusion . [ 14 ]


The complete blood count has specific applications in many  medical specialties . It is often performed before a person undergoes  surgery  to detect anemia, ensure that platelet levels are sufficient, and screen for infection, [ 15 ] [ 16 ]  as well as after surgery, so that  blood loss  can be monitored. [ 12 ] [ 17 ]  In  emergency medicine , the CBC is used to investigate numerous symptoms, such as   fever ,  abdominal pain , and  shortness of breath , [ 18 ] [ 19 ] [ 20 ]  and to assess bleeding and  trauma . [ 21 ] [ 22 ]  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 produce severely low levels of white blood cells, platelets and  hemoglobin . [ 23 ]  Regular CBCs are 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 ( agranulocytosis ). [ 24 ] [ 25 ]  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 ; [ 26 ]   and in  newborn babies , a CBC may be needed to investigate  jaundice  or to count the number of immature cells in the  white blood cell differential , which can be an indicator of  sepsis . [ 27 ] [ 28 ]


The complete blood count is an essential tool of  hematology , which is the study of the cause, prognosis, treatment, and prevention of diseases related to blood. [ 29 ]  The results of the CBC and smear examination reflect the functioning of the  hematopoietic system —the organs and  tissues  involved in the production and development of blood cells, particularly the  bone marrow . [ 9 ] [ 30 ]  For example, a low count of all three cell types ( pancytopenia ) can indicate that blood cell production is being affected by a marrow disorder, and a  bone marrow examination  can further investigate the cause. [ 31 ]  Abnormal cells on the  blood smear  might indicate  acute leukemia  or  lymphoma , [ 30 ]  while an abnormally high count of neutrophils or lymphocytes, in combination with indicative symptoms and blood smear findings, may raise suspicion of a  myeloproliferative disorder  or  lymphoproliferative disorder . Examination of the CBC results and blood smear can help to distinguish between causes of anemia, such as  nutritional deficiencies ,  bone marrow disorders ,  acquired hemolytic anemias  and inherited conditions like  sickle cell anemia  and  thalassemia . [ 32 ] [ 33 ]


The  reference ranges  for the complete blood count represent the range of results found in 95% of apparently healthy people. [ note 2 ] [ 35 ]  By definition, 5% of results will always fall outside this range, so some abnormal results may reflect natural variation rather than signifying a medical issue. [ 36 ]  This is particularly likely if such results are only slightly outside the reference range, if they are consistent with previous results, or if there are no other related abnormalities shown by the CBC. [ 37 ]  When the test is performed on a relatively healthy population, the number of clinically insignificant abnormalities may exceed the number of results that represent disease. [ 38 ]  For this reason, 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. [ 15 ] [ 39 ] [ 40 ]  Repeated blood draws for hematology testing in hospitalized patients can contribute to  hospital-acquired anemia  and may result in unnecessary transfusions. [ 38 ]


The sample is collected by drawing blood into a tube containing an  anticoagulant —typically  EDTA —to stop its natural  clotting . [ 41 ]  The blood is usually taken from a  vein , but when this is difficult it may be collected from  capillaries  by a  fingerstick , or by a  heelprick  in babies. [ 42 ] [ 43 ]  Testing is typically performed on an automated analyzer, but manual techniques such as a blood smear examination or manual hematocrit test can be used to investigate abnormal results. [ 44 ]  Cell counts and hemoglobin measurements are performed manually in laboratories lacking access to automated instruments. [ 45 ]


On board the analyzer, the sample is agitated to evenly distribute the cells, then diluted and partitioned into at least two channels, one of which is used to count red blood cells and platelets, the other to count white blood cells and determine the hemoglobin concentration. Some instruments measure hemoglobin in a separate channel, and additional channels may be used for differential white blood cell counts,  reticulocyte  counts and specialized measurements of platelets. [ 46 ] [ 47 ] [ 48 ]  The cells are suspended in a fluid stream and their properties are measured as they flow past sensors in a technique known as  flow cytometry . [ note 3 ] [ 49 ] [ 52 ]   Hydrodynamic focusing  may be used to isolate individual cells so that more accurate results can be obtained: 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 . [ 53 ] [ 54 ]


To measure the hemoglobin concentration, a  reagent  chemical is added to the sample to destroy ( lyse ) the red cells in a channel separate from that used for red blood cell counts. On analyzers that perform white blood cell counts in the same channel as hemoglobin measurement, this permits white blood cells to be counted more easily. [ 55 ]  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  nanometres —corresponds with the concentration of hemoglobin. [ 56 ] [ 57 ]


Sensors count and identify the cells in the sample using two main principles:  electrical impedance  and  light scattering . [ 58 ]  Impedance-based cell counting operates on the  Coulter principle : cells are suspended in a fluid carrying an  electric current , and as they pass through a small opening (an aperture), they cause decreases in current because of their poor  electrical conductivity . The  amplitude  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, [ 59 ] [ 60 ]  while the total number of pulses 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. [ 61 ]


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 . [ 62 ]  Forward scatter, which refers to the amount of light scattered along the beam's axis, is mainly caused by  diffraction  of light and correlates with cellular size, while side scatter (light scattered at a 90-degree angle) is caused by  reflection  and  refraction  and provides information about cellular complexity. [ 62 ] [ 63 ]


Radiofrequency -based methods can be used in combination with impedance. 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 amplitude 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 . [ 64 ] [ 65 ]  Small red cells and cellular debris, which are similar in size to platelets, may interfere with the platelet count, and large platelets may not be counted accurately, so some analyzers use additional techniques to measure platelets, such as  fluorescent  staining, multi-angle light scatter and  monoclonal antibody  tagging. [ 48 ]


Most analyzers directly measure the average size of red blood cells, which is called the  mean cell volume  (MCV), and calculate the hematocrit by multiplying the red blood cell count by the MCV. Some measure the hematocrit by comparing the total volume of red blood cells to the volume of blood sampled, and derive the MCV from the hematocrit and red blood cell count. [ 66 ]  The hemoglobin concentration, the red blood cell count and the hematocrit are used to calculate the average amount of hemoglobin within each red blood cell, the  mean corpuscular hemoglobin  (MCH); and its concentration, the  mean corpuscular hemoglobin concentration  (MCHC). [ 67 ]  Another calculation, the  red blood cell distribution width  (RDW), is derived from the  standard deviation  of the mean cell volume and reflects variation in cellular size. [ 68 ]


After being treated with reagents, white blood cells form three distinct peaks when their volumes are plotted on a histogram. These peaks correspond roughly to populations of  granulocytes , lymphocytes, and other  mononuclear cells , allowing a three-part differential to be performed based on cell volume alone. [ 69 ] [ 70 ]  More advanced analyzers use additional techniques to provide a five- to seven-part differential, such as light scattering or radiofrequency analysis, [ 70 ]  or using dyes to stain specific chemicals inside cells—for example,  nucleic acids , which are found in higher concentrations in immature cells [ 71 ]  or  myeloperoxidase , an  enzyme  found in cells of the  myeloid lineage . [ 72 ] [ 73 ]   Basophils  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. [ 70 ] [ 72 ]  Another approach to automating the differential count is the use of digital microscopy software, [ 74 ]  which uses  artificial intelligence  to classify white blood cells from  photomicrographs  of the blood smear. The cell images are displayed to a human operator, who can manually re-classify the cells if necessary. [ 75 ]


Most analyzers take less than a minute to run all the tests in the complete blood count. [ 58 ]  Because analyzers sample and count many individual cells, the results are very precise. [ 76 ]  However, some abnormal cells 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. [ 5 ] [ 77 ]


Point-of-care testing  refers to tests conducted outside of the laboratory setting, such as at a person's bedside or in a clinic. [ 78 ] [ 79 ]  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 the sample, and the  i-STAT , which derives a hemoglobin reading by estimating the concentration of red blood cells from the conductivity of the blood. [ 79 ]   Hemoglobin and hematocrit can be measured on point-of-care devices designed for  blood gas testing , but these measurements sometimes correlate poorly with those obtained through standard methods. [ 78 ]  There are simplified versions of hematology analyzers designed for use in clinics that can provide a complete blood count and differential. [ 80 ]


The tests can be performed manually when automated equipment is not available or when the analyzer results indicate that further investigation is needed. [ 45 ]  Automated results are flagged for manual blood smear review in 10–25% of cases, which may be due to abnormal cell populations that the analyzer cannot properly count, [ 5 ]  internal flags generated by the analyzer that suggest the results could be inaccurate, [ 81 ]  or numerical results that fall outside set thresholds. [ 77 ]  To investigate these issues, blood is spread on a microscope slide, stained with a  Romanowsky stain , and examined under a  microscope . [ 82 ]  The appearance of the red and white blood cells and platelets is assessed, and qualitative abnormalities are reported if present. [ 83 ]  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 ( schistocytes ) requires urgent investigation as it can suggest a  microangiopathic hemolytic anemia . [ 84 ]  In some inflammatory conditions and in  paraprotein  disorders like  multiple myeloma , high levels of protein in the blood may cause red blood cells to appear stacked together on the smear, which is termed  rouleaux . [ 85 ]  Some  parasitic diseases , such as  malaria  and  babesiosis , can be detected by finding the causative organisms on the blood smear, [ 86 ]  and the platelet count can be estimated from the blood smear, which is useful if the automated platelet count is inaccurate. [ 77 ]


To perform a manual white blood cell differential, the microscopist counts 100 cells on the blood smear and classifies them based on their appearance; sometimes 200 cells are counted. [ 87 ]  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. [ 88 ]   Manual counting is subject to  sampling error  because so few cells are counted compared with automated analysis, [ 76 ]  but it can identify abnormal cells that analyzers cannot, [ 72 ] [ 77 ]  such as the  blast cells  seen in acute leukemia. [ 89 ]  Clinically significant features like  toxic granulation  and  vacuolation  can also be ascertained from microscopic examination of white blood cells. [ 90 ]


The hematocrit can performed manually by filling a capillary tube with blood, centrifuging it, and measuring the percentage of the blood that consists of red blood cells. [ 66 ]  This is useful in some conditions that can cause automated hematocrit results to be incorrect, such as  polycythemia  (a highly elevated red blood cell count) [ 66 ]  or severe  leukocytosis  (a highly elevated white blood cell count, which interferes with red blood cell measurements by causing white blood cells to be counted as red cells). [ 91 ]


Red and white blood cells and platelets can be counted using a  hemocytometer , a microscope slide containing a chamber that holds a specified volume of diluted blood. The hemocytometer's chamber is etched with a calibrated grid to aid in cell counting. The cells seen in the grid are counted and divided by the volume of blood examined, which is determined from the number of squares counted on the grid, to obtain the concentration of cells in the sample. [ 45 ] [ 92 ]  Manual cell counts are labour-intensive and inaccurate compared to automated methods, so they are rarely used except in laboratories that do not have access to automated analyzers. [ 45 ] [ 92 ]  To count white blood cells, the sample is diluted using a fluid containing a compound that lyses red blood cells, such as  ammonium oxalate ,  acetic acid , or  hydrochloric acid . [ 93 ]  Sometimes a stain is added to the diluent that highlights the nuclei of white blood cells, making them easier to identify. Manual platelet counts are performed in a similar manner, although some methods leave the red blood cells intact. Using a  phase-contrast microscope , rather than a  light microscope , can make platelets easier to identify. [ 94 ]  The manual red blood cell count is rarely performed, as it is inaccurate and other methods such as hemoglobinometry and the manual hematocrit are available for assessing red blood cells; but if it is necessary to do so, red blood cells can be counted in blood that has been diluted with saline. [ 95 ]


Hemoglobin can be measured manually using a  spectrophotometer  or  colorimeter . To measure hemoglobin manually, the 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 is used to determine the relationship between the absorbance and the hemoglobin concentration, allowing the hemoglobin level of the sample to be measured. [ 96 ]


In rural and economically disadvantaged areas, available testing is limited by access to equipment and personnel. At  primary care  facilities in these regions, testing may be limited to examination of red cell morphology and manual measurement of hemoglobin, 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, an estimate of hemoglobin concentration can be obtained by placing a drop of blood on a standardized type of absorbent paper and comparing it to a colour scale. [ 97 ]


Automated analyzers have to be regularly  calibrated . Most manufacturers provide preserved blood with defined parameters and the analyzers are adjusted if the results are outside defined thresholds. [ 98 ]  To ensure that results continue to be accurate, quality control samples, which are typically provided by the instrument manufacturer, are tested at least once per day. The samples are formulated to provide specific results, and laboratories compare their results against the known values to ensure the instrument is functioning properly. [ 99 ] [ 100 ]  For laboratories without access to commercial quality control material, an Indian regulatory organization recommends running patient samples in duplicate and comparing the results. [ 101 ]  A  moving average  measurement, in which the average results for patient samples are measured at set intervals, can be used as an additional quality control technique. 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. [ 99 ] [ 100 ]  The MCHC values are particularly useful in this regard. [ 102 ]


In addition to analyzing internal  quality control  samples with known results, laboratories may receive  external quality assessment  samples from regulatory organizations. While the purpose of internal quality control is to ensure that analyzer results are  reproducible  within a given laboratory, external quality assessment verifies that results from different laboratories are consistent with each other and with the target values. [ 103 ]  The expected results for external quality assessment samples are not disclosed to the laboratory. [ 104 ]  External quality assessment programs have been widely adopted in North America and western Europe, [ 99 ]  and laboratories are often required to participate in these programs to maintain  accreditation . [ 105 ]  Logistical issues may make it difficult for laboratories in under-resourced areas to implement external quality assessment schemes. [ 106 ]


The CBC measures the amounts of platelets and red and white blood cells, along with the hemoglobin and hematocrit values. 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 (RDW), 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, and a count of immature red blood cells (reticulocytes) is sometimes included. [ 4 ] [ 107 ]


Red blood cells deliver  oxygen  from the  lungs  to the tissues and on their return carry  carbon dioxide  back to the lungs where it is exhaled. These functions are mediated by the cells' hemoglobin. [ 109 ]  The analyzer counts red blood cells, reporting the result in units of 10 6  cells per microlitre of blood (× 10 6 /μL) or 10 12  cells per litre (× 10 12 /L), and measures their average size, which is called the  mean cell volume  and expressed in  femtolitres  or cubic micrometres. [ 4 ]  By multiplying the mean cell volume by the red blood cell count, the hematocrit (HCT) or packed cell volume (PCV), a measurement of the percentage of blood that is made up of red blood cells, can be derived; [ 66 ]  and when the hematocrit is performed directly, the mean cell volume may be calculated from the hematocrit and red blood cell count. [ 110 ] [ 111 ]  Hemoglobin, measured after the red blood cells are lysed, is usually reported in units of grams per litre (g/L) or grams per decilitre (g/dL). [ 112 ]  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. [ 113 ]


Two other measurements are calculated from the red blood cell count, the hemoglobin concentration, and the hematocrit: the  mean corpuscular hemoglobin  and the  mean corpuscular hemoglobin concentration . [ 114 ] [ 115 ]  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. [ 116 ]  Collectively, the MCV, MCH, and MCHC are referred to as the  red blood cell indices . [ 114 ] [ 115 ]  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. [ 117 ]  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. [ 118 ]


An abnormally low hemoglobin, hematocrit, or red blood cell count indicates anemia. [ 119 ]  Anemia is not a diagnosis on its own, but it points to an underlying condition affecting the person's red blood cells. [ 88 ]  General causes of anemia include blood loss, production of defective red blood cells (ineffective  erythropoeisis ), decreased production of red blood cells (insufficient erythropoeisis), and increased destruction of red blood cells ( hemolytic anemia ). [ 120 ]  Anemia reduces the blood's ability to carry oxygen, causing symptoms like tiredness and shortness of breath. [ 121 ]   If the hemoglobin level falls below thresholds based on the person's clinical condition, a blood transfusion may be necessary. [ 122 ]


An increased number of red blood cells, leading to an increase in the hemoglobin and hematocrit, [ note 4 ]  is called  polycythemia . [ 126 ]   Dehydration  or use of  diuretics  can cause a "relative" polycythemia by decreasing the amount of plasma compared to red cells. A true increase in the number of red blood cells, called absolute polycythemia, can 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. [ 127 ]


Evaluation of red blood cell indices is helpful in determining the cause of anemia. If the MCV is low, the anemia is termed  microcytic , while 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 considered  normochromic  and  normocytic . [ 117 ]  The term  hyperchromia , referring to a high MCHC, is generally not used. Elevation of the MCHC above the upper reference value is rare, mainly occurring in conditions such as  spherocytosis , sickle cell disease and  hemoglobin C disease . [ 115 ] [ 128 ]  An elevated MCHC can also be a false result from conditions like  red blood cell agglutination  (which causes a false decrease in the red blood cell count, elevating the MCHC) [ 129 ] [ 130 ]  or highly elevated amounts of  lipids  in the blood (which causes a false increase in the hemoglobin result). [ 128 ] [ 131 ]


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. [ 115 ] [ 132 ]  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. [ 133 ]


A low RDW has no clinical significance, but an elevated RDW represents increased variation in red blood cell size, a condition known as  anisocytosis . [ 118 ]  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. [ 118 ]  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. [ 134 ]


White blood cells defend against infections and are involved in the  inflammatory response . [ 136 ]  A high white blood cell count, which is called leukocytosis, often occurs in infections, inflammation, and states of  physiologic stress . It can also be caused by diseases that involve abnormal production of blood cells, such as  myeloproliferative  and  lymphoproliferative disorders . [ 137 ]  A decreased white blood cell count, termed  leukopenia , can lead to an increased risk of acquiring infections, [ 138 ]  and occurs in treatments like chemotherapy and radiation therapy and many conditions that inhibit the production of blood cells. [ 139 ]  Sepsis is associated with both leukocytosis and leukopenia. [ 140 ]  The total white blood cell count is usually reported in cells per microlitre of blood (/μL) or 10 9  cells per litre (× 10 9 /L). [ 4 ]


In the white blood cell differential, the different types of white blood cells are identified and counted. The results are reported as a percentage and as an absolute number per unit volume. Five types of white blood cells— neutrophils ,  lymphocytes ,  monocytes ,  eosinophils , and  basophils —are typically measured. [ 141 ]  Some instruments report the number of immature granulocytes, which is a classification consisting of precursors of neutrophils; specifically,  promyelocytes ,  myelocytes  and  metamyelocytes . [ note 5 ] [ 144 ]  Other cell types are reported if they are identified in the manual differential. [ 145 ]


Differential results are useful in diagnosing and monitoring many medical conditions. For example, an elevated neutrophil count ( neutrophilia ) is associated with bacterial infection, inflammation, and myeloproliferative disorders, [ 146 ] [ 147 ]  while a decreased count ( neutropenia ) may occur in individuals who are undergoing chemotherapy or taking certain drugs, or who have diseases affecting the bone marrow. [ 148 ] [ 149 ]  Neutropenia can also be caused by some  congenital disorders  and may occur transiently after viral or bacterial infections in children. [ 150 ]  People with severe neutropenia and clinical signs of infection are treated with antibiotics to prevent potentially life-threatening disease. [ 151 ]


An increased number of  band neutrophils —young neutrophils that lack segmented nuclei—or immature granulocytes is termed  left shift  and occurs in sepsis and some blood disorders, but is normal in pregnancy. [ 152 ] [ 153 ]  An elevated lymphocyte count ( lymphocytosis ) is associated with  viral infection [ 6 ]   and lymphoproliferative disorders like  chronic lymphocytic leukemia ; [ 154 ]  elevated monocyte counts ( monocytosis ) are associated with chronic inflammatory states; [ 155 ]  and the eosinophil count is often increased ( eosinophilia ) in parasitic infections and allergic conditions. [ 156 ]  An increased number of basophils, termed  basophilia , can occur in myeloproliferative disorders like  chronic myeloid leukemia  and polycythemia vera. [ 147 ]  The presence of some types of abnormal cells, such as blast cells or lymphocytes with  neoplastic  features, is suggestive of a  hematologic malignancy . [ 89 ] [ 157 ]


Platelets play an essential role in clotting. When the wall of a  blood vessel  is damaged, platelets adhere to the exposed surface at the site of injury and plug the gap. Simultaneous activation of the  coagulation cascade  results in the formation of  fibrin , which reinforces the platelet plug to create a stable  clot . [ 158 ]   A low platelet count, known as thrombocytopenia, may cause bleeding if severe. [ 159 ]  It can occur in individuals who are undergoing treatments that suppress the bone marrow, such as chemotherapy or radiation therapy, or taking certain drugs, such as heparin, that can induce the immune system to destroy platelets. Thrombocytopenia is a feature of many blood disorders, like acute leukemia and  aplastic anemia , as well as some  autoimmune diseases . [ 160 ] [ 161 ]  If the platelet count is extremely low, a platelet transfusion may be performed. [ 162 ]   Thrombocytosis , meaning a high platelet count, may occur in states of inflammation or trauma, [ 163 ]  as well as in iron deficiency, [ 164 ]  and the platelet count may reach exceptionally high levels in people with  essential thrombocythemia , a rare blood disease. [ 163 ]  The platelet count can be reported in units of cells per microlitre of blood (/μL), [ 165 ]  10 3  cells per microlitre  (× 10 3 /μL) , or  10 9  cells per litre  (× 10 9 /L). [ 4 ]


The mean platelet volume (MPV) measures the average size of platelets in femtolitres. It can aid in determining the cause of thrombocytopenia; an elevated MPV may occur when young platelets are released into the bloodstream to compensate for increased destruction of platelets, while decreased production of platelets due to dysfunction of the bone marrow can result in a low MPV. The MPV is also useful for differentiating between congenital diseases that cause thrombocytopenia. [ 118 ] [ 166 ]  The immature platelet fraction (IPF) 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. [ 167 ]


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 compensate for blood loss or hemolysis, [ 74 ]  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. [ 168 ]   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. [ 169 ]  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  and counting the percentage of red blood cells containing RNA under the microscope. The reticulocyte count is expressed as an absolute number [ 168 ]  or as a percentage of red blood cells. [ 170 ]


Some instruments 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. [ 171 ]  The immature reticulocyte fraction (IRF) 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 . [ 172 ]


During their formation in bone marrow, and in the  liver  and spleen in fetuses, [ 173 ]  red blood cells contain a cell nucleus, which is usually absent in the mature cells that circulate in the bloodstream. Nucleated red blood cells are normal in newborn babies, [ 174 ]  but when detected in children and adults, they indicate an increased demand for red blood cells, which can be caused by bleeding, some cancers and anemia. [ 118 ]  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. [ 175 ]


Advanced hematology analyzers generate novel measurements of blood cells which have shown diagnostic significance in research studies but have not yet found widespread clinical use. [ 171 ]  For example, some types of analyzers produce  coordinate  readings indicating the size and position of each white blood cell cluster. These parameters (termed cell population data) [ 176 ]  have been studied as potential markers for blood disorders, bacterial infections and malaria. Analyzers that use  myeloperoxidase  staining to produce differential counts can measure white blood cells' expression of the enzyme, which is altered in various disorders. [ 75 ]  Some instruments can report 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 ( schistocytes ), [ 171 ]  which occur in some types of hemolytic anemia. [ 177 ]  Because these parameters are often specific to particular brands of analyzers, it is difficult for laboratories to interpret and compare results. [ 171 ]


(4–7 years old)


(0–1 days old)


The complete blood count is interpreted by comparing the output to reference ranges, which represent the results found in 95% of apparently healthy people. [ 35 ]  Based on a statistical  normal distribution , the tested samples' ranges vary with sex and age. [ 179 ]


On average, adult females have lower hemoglobin, hematocrit, and red blood cell count values than males; the difference lessens, but is still present, after  menopause . [ 180 ]  CBC results for children and newborn babies differ from those of adults. Newborns' hemoglobin, hematocrit, and red blood cell count are extremely high to compensate for low oxygen levels in the womb and the high proportion of  fetal hemoglobin , which is less effective at delivering oxygen to tissues than mature forms of hemoglobin, inside their red blood cells. [ 181 ] [ 182 ]  The MCV is also increased, and the white blood cell count is elevated with a preponderance of neutrophils. [ 181 ] [ 183 ]  The red blood cell count and related values begin to decline shortly after birth, reaching their lowest point at about two months of age and increasing thereafter. [ 184 ] [ 185 ]  The red blood cells of older infants and children are smaller, with a lower MCH, than those of adults. In the pediatric white blood cell differential, lymphocytes often outnumber neutrophils, while in adults neutrophils predominate. [ 181 ]


Other differences between populations may affect the reference ranges: for example, people living at higher altitudes have higher hemoglobin, hematocrit, and RBC results, and people of African heritage have lower white blood cell counts on average. [ 186 ]  The type of analyzer used to run the CBC affects the reference ranges as well. Reference ranges are therefore established by individual laboratories based on their own patient populations and equipment. [ 187 ] [ 188 ]


Some medical conditions or problems with the blood sample may produce inaccurate  results. If the sample is visibly clotted, which can be caused by poor  phlebotomy  technique, it is unsuitable for testing, because the platelet count will be falsely decreased and other results may be abnormal. [ 189 ] [ 190 ]  Samples stored at room temperature for several hours may give falsely high readings for MCV ( mean corpuscular volume ), [ 191 ]  because red blood cells swell as they absorb water from the plasma; and platelet and white blood cell differential results may be inaccurate in aged specimens, as the cells degrade over time. [ 91 ]


Samples drawn from individuals with very high levels of  bilirubin  or  lipids  in their plasma (referred to as an icteric sample or a lipemic sample, respectively) [ 192 ]  may show falsely high readings for hemoglobin, because these substances change the colour and opacity of the sample, which interferes with hemoglobin measurement. [ 193 ]  This effect can be mitigated by replacing the plasma with saline. [ 91 ]


Some individuals produce an  antibody  that causes their platelets to form clumps when their blood is drawn into tubes containing EDTA, the anticoagulant typically used to collect CBC 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 such as  sodium citrate  or  heparin . [ 194 ] [ 195 ]


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. [ 196 ]  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 ( mean corpuscular hemoglobin concentration ). [ 53 ]  Often, these antibodies are only active at room temperature (in which case they are called  cold agglutinins ), and the agglutination can be reversed by heating the sample to 37 °C (99 °F). Samples from people with  warm autoimmune hemolytic anemia  may exhibit red cell agglutination that does not resolve on warming. [ 130 ]


While blast and lymphoma cells can be identified in the manual differential, microscopic examination cannot reliably determine the cells'  hematopoietic lineage . This information is often necessary for diagnosing blood cancers. After abnormal cells are identified, additional techniques such as  immunophenotyping  by flow cytometry can be used to identify  markers  that provide additional information about the cells. [ 197 ] [ 198 ]


Before automated cell counters were introduced, complete blood count tests were performed manually: white and red blood cells and platelets were counted using microscopes. [ 200 ]  The first person to publish microscopic observations of blood cells was  Antonie van Leeuwenhoek , [ 201 ]  who reported on the appearance of red cells in a 1674 letter to the  Proceedings of the Royal Society of London . [ 202 ]   Jan Swammerdam  had described red blood cells some years earlier, but did not publish 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. [ 203 ]


The physiologist  Karl Vierordt  is credited with performing the first blood count. [ 8 ] [ 204 ] [ 205 ]   His technique, published in 1852, 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. [ 206 ]  The hemocytometer, introduced in 1874 by  Louis-Charles Malassez , simplified the microscopic counting of blood cells. [ 207 ]  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. [ 208 ]


In the 1870s,  Paul Ehrlich  developed a staining technique using a combination of an acidic and basic dye that could distinguish different types of white blood cells and allow red blood cell  morphology  to be examined. [ 203 ]   Dmitri Leonidovich Romanowsky  improved on this technique in the 1890s, using a mixture of  eosin  and aged  methylene blue  to produce a wide range of hues not present when either of the stains was used alone. This became the basis for Romanowsky staining, the technique still used to stain blood smears for manual review. [ 209 ]


The first techniques for measuring hemoglobin were devised in the late 19th century, and involved visual comparisons of the colour of diluted blood against a known standard. [ 205 ]  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 (cyanmethemoglobin or hemiglobincyanide) 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. [ 57 ] [ 210 ] [ 211 ]


Maxwell Wintrobe  is credited with the invention of the hematocrit test. [ 66 ] [ 212 ]  In 1929, he undertook a PhD project at the  University of Tulane  to determine normal ranges for red blood cell parameters, and invented a method known as the Wintrobe hematocrit. Hematocrit measurements had previously been described in the literature, but Wintrobe's method differed in that it used a large tube that could be mass-produced to precise specifications, with a built-in scale. The fraction of red blood cells in the tube was measured after  centrifugation  to determine the hematocrit. The invention of a reproducible method for determining hematocrit values allowed Wintrobe to define the red blood cell indices. [ 205 ]


Research into automated cell counting began in the early 20th century. [ 211 ]  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. [ 8 ]  Other unsuccessful attempts, in the 1930s and 1940s, involved photoelectric detectors attached to microscopes, which would count cells as they were scanned. [ 211 ]  In the late 1940s,  Wallace H. Coulter , motivated by a need for better red blood cell counting methods following the  bombing of Hiroshima and Nagasaki , [ 213 ]  attempted to improve on photoelectric cell counting techniques. [ note 7 ]   His research was aided by his brother, Joseph R. Coulter, in a basement laboratory in Chicago. [ 60 ]  Their results using photoelectric methods were disappointing, and in 1948, after reading a paper relating the conductivity of blood to its red blood cell concentration, Wallace devised the Coulter principle—the theory that a cell suspended in a conductive medium generates a drop in current proportional to its size as it passes through an aperture. [ 213 ]


That October, Wallace built a counter to demonstrate the principle. Owing to financial constraints, the aperture was made by burning a hole through a piece of cellophane from a cigarette package. [ 60 ] [ 213 ]  Wallace filed a patent for the technique in 1949, and in 1951 applied to the  Office of Naval Research  to fund the development of the  Coulter counter . [ 213 ]  Wallace's patent application was granted in 1953, and after improvements to the aperture and the introduction of a mercury  manometer  to provide precise control over sample size, the brothers founded Coulter Electronics Inc. in 1958 to market their instruments. The Coulter counter was initially designed for counting red blood cells, but with later modifications it proved effective for counting white blood cells. [ 60 ]  Coulter counters were widely adopted by medical laboratories. [ 211 ]


The first analyzer able to produce multiple cell counts simultaneously was the  Technicon   SMA 4A−7A , released in 1965. It achieved this by partitioning blood samples into two channels: one for counting red and white blood cells and one for measuring hemoglobin. However, the instrument was  unreliable and difficult to maintain. In 1968, the Coulter Model S analyzer was released and gained widespread use. Similarly to the Technicon instrument, it used two different reaction chambers, one of which was used for the red cell count, and one of which was used for the white blood cell count and hemoglobin determination. 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. [ 214 ]


After basic cell counting had been automated, the white blood cell differential remained a challenge. Throughout the 1970s, researchers explored two methods for automating the differential count: 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. [ 215 ]  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 . [ 216 ]  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, [ 217 ] [ 218 ]  and by 1990, no digital image analyzers were commercially available in the United States or western Europe. [ 219 ]  These techniques enjoyed a resurgence in the 2000s with the introduction of more advanced image analysis platforms using  artificial neural networks . [ 220 ] [ 221 ] [ 222 ]


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. [ 223 ]  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. [ 224 ] [ 225 ]  Introduced in 1974, [ 226 ] [ 227 ]  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. [ 225 ] [ 228 ]  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 . [ 229 ]


The  comprehensive metabolic panel , or  chemical screen  ( CMP ;  CPT  code 80053), is a  panel  of 14  blood tests  that serves as an initial broad medical  screening  tool. The CMP provides a rough check of  kidney  function,  liver  function, diabetic and parathyroid status, and  electrolyte  and  fluid balance , but this type of screening has its limitations. Abnormal values from a CMP are often the result of  false positives  and thus the CMP may need to be repeated (or a more specific test performed), requiring a second blood drawing procedure and possibly additional expense for the patient, even though no disease is present. This test is also known as SMA12+2 test.


The CMP is an expanded version of the  basic metabolic panel  (BMP), which does not include liver tests. A CMP (or BMP) can be ordered as part of a routine  physical examination , or may be used to monitor a patient with a chronic disease, such as  diabetes mellitus  or  hypertension .


Previous names for the panel of tests have been  Chem 12 ,  Chemistry panel ,  Chemistry screen ,  SMA 12 ,  SMA 20  and  SMAC  (Sequential Multiple Analysis - Computer). [ 1 ]   The tests are performed on machines based on the  AutoAnalyzer  invented in 1957.


Typically, the patient  fasts  for ten or twelve hours before the blood is drawn for the test—this is particularly important for getting a useful blood glucose measurement. CMPs are also frequently performed on nonfasting patients, but the glucose level in those cases is not as useful. The following tests are then performed: [ 1 ]


These tests help screen for a wide variety of problems. The  glucose  test in particular helps screen for diabetes mellitus and pre-diabetes. The calcium test can indicate or monitor bone diseases or diseases of the parathyroid gland or kidneys. Calcium salts, lithium, thiazide diuretics, thyroxine, and vitamin D can all increase levels and may interfere with this test. [ 2 ]


Electrolyte  levels and the balance among them are tightly regulated by the body. Both individual values and ratios among the values are significant; abnormalities among either can indicate problems such as an  electrolyte disturbance ,  acid-base  imbalance, or kidney dysfunction.


Tests of  protein  levels in the blood help screen for both kidney and liver disorders.


The  National Institutes of Health  provides ranges considered within normal limits, though optimal levels may vary by individual. [ 3 ]


Compare also the ranges given at  Reference ranges for blood tests .


0.7–1.3 (men)


The direct and indirect  Coombs tests , also known as  antiglobulin test  ( AGT ), are  blood tests  used in  immunohematology . The direct Coombs test detects antibodies that are stuck to the surface of the red blood cells. [ 1 ]  Since these antibodies sometimes destroy red blood cells they can cause  anemia ; this test can help clarify the condition. The indirect Coombs test detects antibodies that are floating freely in the blood. [ 1 ]  These antibodies could act against certain red blood cells; the test can be carried out to diagnose reactions to a  blood transfusion . [ 1 ]


The direct Coombs test is used to test for  autoimmune hemolytic anemia , a condition where the immune system breaks down  red blood cells , leading to anemia. The direct Coombs test is used to detect antibodies or  complement proteins  attached to the surface of red blood cells. To perform the test, a blood sample is taken and the red blood cells are washed (removing the patient's plasma and unbound antibodies from the red blood cells) and then incubated with anti-human globulin ("Coombs reagent"). If the red cells then  agglutinate , the test is positive, a visual indication that antibodies or complement proteins are bound to the surface of red blood cells and may be causing destruction of those cells.


The indirect Coombs test is used in prenatal testing of pregnant women and in testing prior to a blood transfusion. The test detects antibodies against foreign red blood cells. In this case,  serum  is extracted from a blood sample taken from the patient. The serum is incubated with foreign red blood cells of known  antigenicity . Finally, anti-human globulin is added. If agglutination occurs, the indirect Coombs test is positive. [ 2 ]


The two Coombs tests are based on anti-human  antibodies  binding to human antibodies, commonly  IgG  or  IgM . These anti-human antibodies are produced by  plasma cells  of non-human animals after immunizing them with  human plasma . Additionally, these anti-human antibodies will also bind to human antibodies that may be fixed onto  antigens  on the surface of  red blood cells  (RBCs). In the appropriate test tube conditions, this can lead to  agglutination  of RBCs and allowing for visualisation of the resulting clumps of RBCs. If clumping is seen, the Coombs test is positive; if not, the Coombs test is negative. [ 3 ]


Common clinical uses of the Coombs test include the preparation of blood for  transfusion  in  cross-matching , atypical antibodies in the  blood plasma  of  pregnant  women as part of  antenatal care , and detection of antibodies for the diagnosis of immune-mediated  hemolytic anemias . [ 4 ]


Coombs tests are performed using RBCs or serum (direct or indirect, respectively) from venous whole blood samples which are taken from patients by  venipuncture . The venous blood is taken to a laboratory (or blood bank), where trained scientific technical staff do the Coombs tests.  The clinical significance of the result is assessed by the  physician  who requested the Coombs test, perhaps with assistance from a laboratory-based  hematologist . [ citation needed ]


The direct Coombs test, also referred to as the direct antiglobulin test (DAT), is used to detect if antibodies or  complement system  factors have bound to  RBCs  surface antigens. [ 5 ]  The DAT is not required for pre-transfusion testing [ 6 ]  but may be carried out by some laboratories. [ clarification needed ]  Before transfusion, an  indirect  Coombs test is often done.


The direct Coombs test is used clinically when immune-mediated  hemolytic anemia  (antibody-mediated destruction of RBCs) is suspected. A positive Coombs test indicates that an immune mechanism is attacking the patient's  RBCs . This mechanism could be  autoimmunity ,  alloimmunity  or a drug-induced immune-mediated mechanism. [ 3 ]


(A  memory device  to remember that the  DAT  tests the  RBCs  and is used to test infants for  haemolytic disease of the newborn  is:
 R h  D isease ;  R  = RBCs,  D  = DAT.)


The patient's  RBCs  are washed (removing the patient's own  serum ) and then centrifuged with  antihuman globulin  (also known as Coombs reagent).  If  immunoglobulin  or complement factors have been fixed on to the RBC surface  in-vitro , the antihuman globulin will  agglutinate  the RBCs and the direct Coombs test will be positive. (A visual representation of a positive direct Coombs test is shown in the upper half of the schematic). [ citation needed ]


The indirect Coombs test, also referred to as the indirect antiglobulin test (IAT), is used to detect  in-vitro  antibody-antigen reactions.  It is used to detect very low concentrations of antibodies present in a patient's plasma/serum prior to a blood transfusion. In antenatal care, the IAT is used to screen pregnant women for antibodies that may cause  hemolytic disease of the newborn .  The IAT can also be used for  compatibility testing , antibody identification, RBC phenotyping, and titration studies. [ citation needed ]


The indirect Coombs test is used to screen for antibodies in the preparation of  blood  for  blood transfusion .  The donor's and recipient's blood must be  ABO  and Rh D compatible.  Donor blood for transfusion is also screened for infections in separate processes. [ citation needed ]


A blood sample from the recipient and a blood sample from every unit of  donor  blood are screened for antibodies with the indirect Coombs test.  Each sample is incubated against a wide range of RBCs that together exhibit a full range of surface antigens (i.e.  blood types ).


The indirect Coombs test is used to test a sample of the recipient's serum for antibodies against a sample of the  blood donor's  RBCs.  This is sometimes called  cross-matching  blood.


The indirect Coombs test is used to screen pregnant women for  IgG   antibodies  that are likely to pass through the  placenta  into the fetal blood and cause  haemolytic disease of the newborn . [ citation needed ]


The IAT is a two-stage test. (A cross match is shown visually in the lower half of the schematic as an example of an indirect Coombs test). [ citation needed ]


Nonpatient, washed  red blood cells  (RBCs) with known antigens are incubated with patient serum containing unknown antibody content.  If the serum contains  antibodies  to antigens on the RBC surface, the antibodies will bind to the surface of the RBCs. [ citation needed ]


The RBCs are washed three or four times with isotonic saline solution and then incubated with antihuman globulin.  If antibodies have bound to RBC surface antigens in the first stage, RBCs will agglutinate when incubated with the  antihuman globulin  (also known Coombs reagent) in this stage, and the indirect Coombs test will be positive.


By diluting a serum containing antibodies the quantity of the antibody in the serum can be gauged.  This is done by performing  serial dilutions  of the serum and finding the maximum dilution of test serum that is able to produce agglutination of relevant RBCs. [ citation needed ]


Coombs reagent (also known as  Coombs antiglobulin  or  antihuman globulin ) is used in both the direct Coombs test and the indirect Coombs test.  Coombs reagent is antihuman  globulin .  It is made by injecting human globulin into animals, which produce  polyclonal antibodies  specific for human immunoglobulins and human complement system factors.  More specific Coombs reagents or  monoclonal antibodies  can be used. [ citation needed ]


Both  IgM  and  IgG   antibodies  bind strongly with their complementary antigens.  IgG  antibodies are most reactive at 37°C.  IgM antibodies are easily detected in  saline  at  room temperature  as IgM antibodies are able to bridge between RBC's owing to their large size, efficiently creating what is seen as  agglutination .  IgG antibodies are smaller and require assistance to bridge well enough to form a visual  agglutination  reaction.  Reagents used to enhance IgG detection are referred to as potentiators.  RBCs have a net negative charge called zeta potential which causes them to have a natural repulsion for one another. Potentiators reduce the zeta potential of RBC membranes. Common potentiators include low ionic strength solution (LISS),  albumin ,  polyethylene glycol  (PEG), and proteolytic  enzymes .


The Coombs test was first described in 1945 by  Cambridge   immunologists   Robin Coombs  (after whom it is named),  Arthur Mourant  and  Rob Race . [ 8 ]   Historically, it was done in  test tubes .  Today, it is commonly done using automated solid phase or gel technology.


Counterimmunoelectrophoresis  is a  laboratory technique  used to evaluate the binding of an  antibody  to its  antigen , it is similar to  immunodiffusion , but with the addition of an applied  electrical field  across the diffusion medium, usually an  agar  or  polyacrylamide  gel.  The effect is rapid migration of the antibody and antigen out of their respective wells towards one another to form a line of precipitation, or a  precipitin  line, indicating binding. [ 2 ]


The  COVID-19 Immunity Task Force  ( CITF ) is one of the  Government of Canada 's early efforts to track the 2020  coronavirus pandemic . [ 1 ]  An external, dedicated secretariat was established in order to maximize the efficiency of the CITF's work. [ 2 ]


The CITF was to use a  serology  "to survey representative samples of the population for the presence of antibodies to the virus". [ 3 ]  Trudeau's press release on 23 April 2020, on the initiation of the CCITF listed several goals it would help to achieve notably that it would: [ 2 ]


establish priorities and oversee the coordination of a series of country-wide  blood test surveys  that will tell us how widely the virus has spread in Canada and provide reliable estimates of potential immunity and vulnerabilities in Canadian populations.


A Vaccine Surveillance Reference Group (VSRG) was also established within the CITF to monitor the safety and effectiveness of  COVID-19 vaccines  made available in Canada. [ 4 ]


The CITF Board is composed of doctors, infectious disease experts, and policy makers. [ 1 ] [ 5 ]


The CCITF leadership group expanded on 2 May 2020. [ 6 ]  Its additional members as of March 2022 are:


This article about an organization in Canada is a  stub . You can help Wikipedia by  expanding it .


The  CPK-MB test  (creatine phosphokinase-MB), also known as  CK-MB test , is a  cardiac marker [ 3 ]  used to assist diagnoses of an  acute myocardial infarction ,  myocardial ischemia , or  myocarditis . It measures the blood level of CK-MB (creatine kinase myocardial band), the bound combination of two variants (isoenzymes  CKM  and  CKB ) of the enzyme  phosphocreatine kinase . [ citation needed ]


In some locations, the test has been superseded by the  troponin test . However, recently, there have been improvements to the test that involve measuring the ratio of the CK-MB1 and CK-MB2 isoforms. [ 4 ]


The newer test detects different  isoforms  of the B subunit specific to the myocardium whereas the older test detected the presence of cardiac-related  isoenzyme  dimers. [ citation needed ]


Many cases of CK-MB levels exceeding the blood level of total CK have been reported, especially in newborns with cardiac malformations, especially ventricular septal defects. This reversal of ratios is in favor of pulmonary emboli or vasculitis. An autoimmune reaction creating a complex molecule of CK and IgG should be taken into consideration. [ 5 ]


Cytometry  is the  measurement  of number and characteristics of  cells . Variables that can be measured by cytometric methods include  cell size ,  cell count , cell morphology (shape and structure),  cell cycle  phase,  DNA  content, and the existence or absence of specific  proteins  on the cell surface or in the  cytoplasm . [ 1 ]  Cytometry is used to characterize and count  blood cells  in common  blood tests  such as the  complete blood count . In a similar fashion, cytometry is also used in cell biology research and in medical diagnostics to characterize cells in a wide range of applications associated with diseases such as  cancer  and  AIDS . [ citation needed ]


Image cytometry is the oldest form of cytometry. Image cytometers operate by statically imaging a large number of
cells using  optical microscopy . Prior to analysis, cells are commonly stained to enhance contrast or
to detect specific molecules by labeling these with  fluorochromes . Traditionally,
cells are viewed within a  hemocytometer  to aid manual counting. [ citation needed ]


Since the introduction of the  digital camera , in the mid-1990s, the automation level of
image cytometers has steadily increased. This has led to the commercial availability of automated image cytometers, ranging from simple cell counters to sophisticated  high-content screening  systems.


Due to the early difficulties of automating microscopy, the  flow cytometer  has since
the mid-1950s been the dominating cytometric device. [ 2 ]
Flow cytometers operate by aligning single cells using flow techniques. The cells are characterized
optically or by the use of an  electrical impedance  method called the  Coulter principle .
To detect specific molecules when optically characterized, cells are in most cases stained with the same
type of fluorochromes that are used by image cytometers. Flow cytometers generally provide
less data than image cytometers, but have a significantly higher throughput. [ citation needed ]


Cell sorters are flow cytometers capable of sorting cells according to their characteristics.
The sorting is achieved by using technology similar to what is used in  inkjet printers .
The fluid stream is broken up into droplets by a mechanical vibration.
The droplets are then electrically charged according to the characteristics of the cell contained
within the droplet. Depending on their charge, the droplets are finally deflected by an electric field into
different containers. [ citation needed ]


A key characteristic of time-lapse cytometers is their use of non heat-generating light sources such as  light-emitting diodes .
This allows a time-lapse cytometer to be placed inside a conventional  cell culture incubator
to facilitate continuous observation of cellular processes without heat building up inside the incubator.


The early history of cytometry is closely associated with the development of the blood cell counting.
Through the work of  Karl von Vierordt ,  Louis-Charles Malassez ,  Karl Bürker  and others blood cell
concentration could by the late 19th century be accurately measured using a blood cell counting chamber,
the  hemocytometer , and an  optical microscope . [ 3 ] [ 4 ]


Until the 1950s the hemocytometer was the standard method to count blood cells. [ 5 ]
In blood cell counting applications the hemocytometer has now been replaced by  electronic cell counters .
However, the hemocytometer is still being used to count cells in cell culture laboratories.
Successively the manual task of counting, using a microscope, is taken over by small automated image cytometers. [ citation needed ]


In 1904,  Moritz von Rohr  and  August Köhler  at  Carl Zeiss  in Jena constructed the first ultraviolet microscope.
The intent of the microscope was to obtain higher optical resolution by using illumination with a shorter wavelength than visual light.
However, they experienced difficulties with  autofluorescence  when observing biological material. Fortunately,
Köhler saw the potential of fluorescence.
A filtering technique for fluorescence excitation light was developed by
 Heinrich Lehmann  at Zeiss in 1910, based on work by
 Robert Wood . However, the "Lumineszenzmikroskop" he developed was only second on the market, after the one independently developed by  Oskar Heimstädt  who worked at C Reichert, Optische Werke AG in Vienna, which today is a part of  Leica Microsystems . [ 6 ] [ 7 ] [ 8 ]


By the early 1930s various firms manufactured ultraviolet fluorescent microscopes. The stage was
set for cytometry to now go beyond the now established hemocytometer. At this time,  Torbjörn Caspersson ,
working at the Karolinska Institute in Stockholm, developed a series of progressively more sophisticated
instruments called  cytophotometers . These instruments combined a fluorescent microscope with a  spectrophotometer
to quantify cellular nucleic acids and their relation to cell growth and function. Caspersson’s early
apparatus now seems hopelessly primitive. But, even this primitive apparatus got results, and attracted
the attention of other researchers. Many of the advances in analytical cytology from the 1940s and on-wards
were made by people who made the pilgrimage to Stockholm. [ 9 ]


The first attempts to automate cell counting were made around World War II.
Gucker et al. builds a device to detect bacteria in aerosols. [ 10 ]  Lagercrantz builds an automated
cell counter based on microscopy [ 11 ]  and identifies the difficulties
in aligning cells to be individually counted using microscopy, as Moldavan had proposed in 1934. [ 12 ]
Joseph and  Wallace Coulter  circumnavigates these difficulties by inventing the principle
of using electrical impedance to count and size microscopic particles suspended in a fluid. [ 5 ] [ 13 ]  This principle is today
known as the  Coulter principle  and was used in the automated blood cell counter released by  Coulter Electronics
in 1954. The “ Coulter counter ” was the first commercial flow cytometer. [ citation needed ]


During the 1960s Dittrich, Göhde and Kamentsky improves the design pioneered by Caspersson 30 years earlier.
Dittrich and Göhde’s  pulse cytophotometer  was built around a Zeiss fluorescent microscope and commercialized
as the ICP 11 by  Partec GmbH  in 1968.
Kamentsky’s device was commercialized by Bio/Physics Systems Inc. as the Cytograph in 1970. [ 14 ] [ 15 ]
These devices were able to count cells, like the earlier Coulter counter. But more importantly, they were also capable of measuring cellular characteristics.
However, these early cytophotometers where microscopy-based. [ 16 ]


In 1953 Crosland-Taylor published an unsuccessful attempt to count red blood cells using microscopy in which he solved the problem of aligning cells by using  sheath fluid  to  hydrodynamically focus  the cells. [ 2 ]  In the late 1960s, Van Dilla at  Los Alamos National Laboratory  built the first non microscopy-based cytophotometer. He did this by combining Crosland-Taylor's breakthrough with the fluorescent dyes originally developed for microscopy and a laser-based fluorescent detection system — the flow cytometer as we know it today. [ 17 ] [ 18 ] [ 19 ]  Fulwyler, at Los Alamos as well, combines the Coulter principle with  continuous inkjet printer technology  to create the first cell sorter in 1965. [ 20 ]


In 1973 Steinkamp and the team at Los Alamos follow up with a fluorescence-based cell sorter. [ 21 ]


In 1978, at the Conference of the American Engineering Foundation in Pensacola, Florida, the name  pulse cytophotometry
was changed to  flow cytometry , a term which quickly became popular. [ 22 ]  At that point pulse cytophotometry had evolved
into the modern form of flow cytometry, pioneered by Van Dilla ten years earlier. [ citation needed ]


D-dimer  (or  D dimer ) is a  dimer  that is a  fibrin degradation product  (FDP), a small protein fragment present in the blood after a  blood clot  is degraded by  fibrinolysis . It is so named because it contains two D fragments of the  fibrin  protein joined by a  cross-link , hence forming a  protein dimer . [ 1 ]


D-dimer concentration may be determined by a  blood test  to help diagnose  thrombosis . [ 2 ]  Since its introduction in the 1990s, it has become an important test performed in people with suspected thrombotic disorders, such as  venous thromboembolism . [ 2 ] [ 3 ]  While a negative result practically rules out thrombosis, a positive result can indicate thrombosis but does not exclude other potential causes. [ 3 ]  Its main use, therefore, is to exclude  thromboembolic  disease where the probability is low. [ 1 ] [ 2 ]


D-dimer levels are used as a predictive  biomarker  for the blood disorder  disseminated intravascular coagulation  and in the coagulation disorders associated with  COVID-19 infection . [ 1 ] [ 3 ]  A four-fold increase in the protein is an indicator of poor prognosis in people hospitalized with  COVID-19 . [ 1 ] [ 3 ] [ 4 ]


Coagulation , the formation of a blood clot or  thrombus , occurs when the proteins of the  coagulation cascade  are activated, either by contact with a damaged blood vessel wall and exposure to collagen in the tissue space (intrinsic pathway) or by activation of  factor VII  by  tissue activating factors  (extrinsic pathway). Both pathways lead to the generation of  thrombin , an enzyme that turns the soluble blood protein  fibrinogen  into fibrin, which aggregates into protofibrils. Another thrombin-generated enzyme,  factor XIII , then crosslinks the fibrin protofibrils at the D fragment site, leading to the formation of an insoluble gel that serves as a scaffold for blood clot formation. [ 1 ]


The circulating enzyme  plasmin , the main enzyme of  fibrinolysis , cleaves the fibrin gel in a number of places. The resultant fragments, "high molecular weight polymers", are digested several times more by plasmin to lead to intermediate and then to small polymers ( fibrin degradation products  or FDPs). The cross-link between two D fragments remains intact, however, and these are exposed on the surface when the fibrin fragments are sufficiently digested. The structure of D-dimer is either a 180  kDa [ 6 ]  or 195 kDa [ 7 ]  molecule of two D domains, or a 340 kDa [ 7 ]  molecule of two D domains and one E domain of the original fibrinogen molecule. [ 1 ]  The  half-life  of D-dimer in blood is approximately 6 to 8 hours. [ 8 ]


D-dimers are not normally present in human blood plasma, except when the coagulation system has been activated, for instance, because of the presence of  thrombosis  or  disseminated intravascular coagulation . The D-dimer assay depends on the binding of a  monoclonal antibody  to a particular  epitope  on the D-dimer fragment. Several detection kits are commercially available; all of them rely on a different monoclonal antibody against D-dimer. For some of these, the area of the D-dimer to which the antibody binds is known. The binding of the antibody is then measured quantitatively by one of various laboratory methods. [ 1 ]


D-dimer testing is of clinical use when there is a suspicion of  deep venous thrombosis  (DVTl),  pulmonary embolism  (PE) or  disseminated intravascular coagulation  (DIC). [ 1 ] [ 3 ]


For DVT and PE, there are possible various scoring systems that are used to determine the  a priori  clinical probability of these diseases; the best-known is the  Wells score . [ 5 ]


In some hospitals, they are measured by laboratories after a form is completed showing the probability score and only if the probability score is low or intermediate. This reduces the need for unnecessary tests in those who are high-probability. [ 11 ]  Performing the D-dimer test first can avoid a significant proportion of imaging tests and is less invasive. Since the D-dimer can exclude the need for imaging,  specialty   professional organizations  recommend that physicians use D-dimer testing as an initial diagnostic. [ 12 ] [ 13 ] [ 14 ] [ 15 ]


The following are  reference ranges  for D-dimer: [ 16 ]


D-dimer increases with age. It has therefore been suggested to use a cutoff equal to patient’s age in years × 10 μg/L (or x 0.056 nmol/L) for patients aged over 50 years for the suspicion of venous thromboembolism (VTE), as it decreases the  false positive  rate without substantially increasing the  false negative  rate. [ 17 ] [ 18 ]


An alternative measurement of D-dimer is in fibrinogen equivalent units (FEU). The molecular weight of the fibrinogen molecule is about twice the size of the D-dimer molecule, and therefore 1.0 mcg/mL FEU is equivalent to 0.5 mcg/mL of d-dimer. [ 19 ]


Various kits have a 93 to 95%  sensitivity  (true positive rate). For hospitalized patients, one study found the  specificity  to be about 50% (related to false positive rate) in the diagnosis of thrombotic disease. [ 20 ]


In interpretation of the D-dimer, for patients over age 50, a value of (patient's age) × 10 μg/L may be abnormal. [ 23 ] [ 24 ]


D-dimer was originally identified, described and named in the 1970s ( Fibrinolysis, Dr P J Gaffney ) and found its diagnostic application in the 1990s. [ 1 ] [ 5 ]


Dilute Russell's viper venom time  ( dRVVT ) is a laboratory test often used for detection of  lupus anticoagulant  (LA). It is an assessment of the time for blood to clot in the presence of a diluted amount of venom from  Russell's viper  ( Daboia russelii ), a highly venomous snake native to the Indian subcontinent and named after the herpetologist  Patrick Russell . [ 1 ]


Russell's viper venom (RVV) was known to clot blood many years ago. [ 2 ]  It was widely used as a styptic to clot minor wounds when razor blades were more commonly used for shaving (e.g. "Stypven", Burroughs-Wellcome Pharma). RVV came to be useful in laboratory tests for blood clotting factors V, X, prothrombin and phospholipid. [ 3 ]


It was first used in clotting tests for lupus anticoagulant (LA) in an individual case in 1975. [ 4 ]  The "dilute Russell's viper venom time (dRVVT)" test was then applied in 1985 to detect LA in a large number of patients and it became more widely used for this purpose. This multi-step method involved adding individual solutions of dilute  phospholipid , RVV and calcium chloride to a test plasma and then measuring how long it took for the mixture to clot. [ 5 ]


In 1989, researchers at  Westmead Hospital  developed a simpler assay by combining the venom, phospholipid, and calcium into a single reagent. Its first use on LA patients was reported in 1990. [ 6 ]  It was commercialized as "LA Screen" by Gradipore Ltd, Sydney (later Life Diagnostics) and distributed widely by American Diagnostica Inc (New York) as "dVVTest".


The reagent was improved in 1992 by making it resistant to the widely used interfering anticoagulant  heparin . A new LA resistant version with increased phospholipid was also released at that time. This was introduced as "LA-Confirm" by Gradipore and "dVVConfirm" by American Diagnostica. Results with this high phospholipid reagent were not prolonged by most LA, but remained similarly affected as in the "screen" test by all other variables in test plasmas (Gradipore product information). The combination of screening and confirmatory dRVVT reagents made identification of LA more simple. [ 7 ]  Manufacture of these reagents has since passed on to the major diagnostic companies such as Diagnostica Stago, Precision Biologic, and IL/Werfen.