The dRVVT assay relies on the venom of the  Russelli viper . The venom contains enzymes (RVV-X) that directly activates clotting  factor X  (bypassing  intrinsic  and  extrinsic  cascades). [ 8 ]  In the presence of calcium and phospholipids, these factors convert  prothrombin  into  thrombin , leading to  fibrin  clot formation. The assay uses low concentrations of venom and phospholipids, resulting in a standard  clotting time  of 35 to 37 seconds. The test has three phases: In screening phase a lower phospholipid quantity is used (35–37 seconds clotting time) to enhance sensitivity to lupus anticoagulant. [ 9 ]  In confirmatory phase full phospholipid dose (30–35 seconds clotting time) helps validate results. In a  mixing study , the patient plasma is mixed with normal pooled plasma (NPP) in a 1:1 ratio to assess clotting factor deficiency. If clotting time prolongs during screening, lupus antibodies may be present. Mixing study differentiates between lupus antibodies and factor deficiency. Excess phospholipids in the confirmatory phase shorten clotting time if lupus antibodies are present. [ 10 ]


Through the three phases, following ratios are calculated which help interpret the diagnosis:


By using these ratios and analyzing the possible result combinations, the test's interpretation and reporting are conducted. [ 11 ]


Additional calculations are made using percentage correction of dRVVT and normalized percentage correction as under:


Normalized percentage correction of >10% is taken as positive and suggests the presence of lupus anticoagulant. [ 12 ]


dRVVT tests are strongly affected by the new direct oral anticoagulants (DOACs) and false positive LA results are obtained particularly with  rivaroxaban . [ 13 ]  It is now possible to specifically remove DOACs from test plasmas with activated carbon and enable the correct diagnosis of LA with the dRVVT system despite their initial presence. [ 14 ]


The dRVVT is one component of a workup of a suspected  antiphospholipid antibody , the other component being the  serological  testing for  anticardiolipin antibodies  and  anti-β2 glycoprotein-I antibodies  using  ELISA  technology. The Sapporo criteria require at least one of the above laboratory tests to be positive and the patient to have at least one clinical manifestation of  antiphospholipid syndrome , such as vascular thrombosis or fetal mortality/morbidity, in order to diagnose the antiphospholipid syndrome. [ 15 ]  Positive laboratory test results should be seen on two occasions at least 12 weeks apart in order for diagnosis.  Antiphospholipid antibody syndrome is an important marker for recurrent thrombosis, and often warrants indefinite  anticoagulant  (blood thinner) therapy. Warfarin appears to be preferable to DOACs as the latter have recently been found less effective than expected. [ 16 ]


The criteria were defined in 1999, and revised in 2006. [ 17 ]


A  direct agglutination test  ( DAT ) is any test that uses whole organisms as a means of looking for  serum   antibodies .  The abbreviation, DAT, is most frequently used for the  serological  test for  visceral leishmaniasis . [ 1 ]


Dried blood spot testing  (DBS) is a form of  biosampling  where blood samples are blotted and dried on filter paper. The dried samples can easily be shipped to an  analytical laboratory  and analysed using various methods such as  DNA amplification  or  high-performance liquid chromatography . [ citation needed ]


Ivar Bang first described the DBS as an unusual sampling method in 1913. [ 1 ]  The concept that capillary blood, obtained from pricking the heel or finger and blotted onto filter paper, could be used to screen for metabolic diseases in large populations of neonates was introduced in Scotland by  Robert Guthrie  in 1963. Neonatal screening for  phenylketonuria  became nationwide in 1969–70. Since then,  Guthrie  card samples have been collected routinely from infants in over 20 countries to screen for phenylketonuria and more recently for  congenital hypothyroidism ,  sickle cell disorders  and HIV infection. The limitations of sensitivity and specificity when screening such small volumes of blood restricted the use of dried blood spots for many years. However, recent advances such as the production of  monoclonal antibodies , expression of synthetic proteins, and the introduction of the polymerase chain reaction have overcome many of these problems. [ 2 ]


This type of blood testing is now available for use at home by consumers in the U.S.  Available blood tests include vitamin D, estrogen, testosterone, cortisol, TSH and lipids.  New York is the only state that prohibits home blood spot testing. [ citation needed ]


By 2001, over 175 analytes had been measured using DBS, ranging from acylcarnitines and C-reactive protein to cyclosporine A, cytokines, hepatitis B virus, glucose, and antibodies for over 30 viruses and microorganisms. Other analytes included gentamicin, lipoproteins, prolactin, selenium, trace elements, vitamin A, and zinc protoporphyrin were also measured. [ 3 ] [ 4 ]


In the 20th century, the use of blood and serum collected and dried on a filter paper for serologic testing for syphilis was already reported. Both field and home sample collections were described. [ 3 ]


The first report of blood absorbed onto filter paper for enzyme measurements was published in 1953. [ 3 ] [ 5 ]


In 1962, Berry explored the use of filter paper urine samples for population-based screening programs. [ 3 ] [ 6 ]


In 1980, an immunochemical test for colorectal cancer screening using fecal occult blood smears on specially treated filter paper was introduced. [ 3 ] [ 7 ]


In 1987, successful extraction of DNA from blood collected on “blotter” paper and dried was first reported by McCabe. [ 3 ] [ 8 ]


Dried blood spot specimens are collected by applying a few drops of blood, drawn by lancet from the finger, heel or toe, onto specially manufactured absorbent filter paper. The blood is allowed to thoroughly saturate the paper. It is air dried for several hours. [ 9 ]  Specimens are stored in low gas-permeability plastic bags with  desiccant  added to reduce humidity, and may be kept at ambient temperature, even in tropical climates. [ citation needed ]


Once in the laboratory, technicians separate a small disc of saturated paper from the sheet using an automated or manual hole punch, dropping the disc into a flat bottomed microtitre plate.  The blood is eluted out in phosphate buffered saline containing 0.05%  Tween 80  and 0.005%  sodium azide , overnight at 4 °C. The resultant plate containing the eluates forms the "master" from which dilutions can be made for subsequent testing. [ 2 ]


As an alternative to punching out a paper disc, recent automation solutions extract the sample by flushing an eluent through the filter without punching it out. [ 10 ] [ 11 ]  An automation including the application of an internal standard prior extraction was developed by the Swiss company CAMAG. [ 12 ]


The technology holds promise for expanding diagnostic services to HIV-infected infants in resource-poor settings due to the samples' longer lifespan with reduced need for refrigeration and the less invasive nature of the test compared with other methods.  Unlike  ELISA  testing for HIV-antibodies in the blood, which may be transmitted to infants in pregnancy independently of the virus itself, dried blood spot testing can be used to detect the genetic material of the actual virus, thereby avoiding the likelihood of a  false positive  result.
Dried blood spot testing for HIV is not considered sensitive enough for diagnostic testing, but may be useful in estimating the prevalence of HIV through surveillance.
DBS specimens also pose less of a biohazard risk to handlers, and are easier to transport or store than liquid blood specimens. [ 13 ]


The reason for the stability of DNA, RNA or protein could be attributed to the fact that the biological material binds to the matrix of the filter paper and the process of drying excludes water which is an important factor necessary for protease or nuclease to act. Binding of the biological material also binds several inhibitors which may interfere with various nucleic acid amplification methods. [ citation needed ]


DBS has important characteristics that make it suitable for current and future applications.  It presents minimal potential risk of bacterial contamination and/or hemolysis. It is an easy, non-invasive and economical collection method. DBS samples can be stored for extended periods with almost no deterioration of the analytes, they require less blood compared to conventional venipuncture. [ 14 ]


However, there are some concerns associated with DBS biosampling. These include challenges related to sample volume, analyte recovery, the hematocrit effect, sample homogeneity, and the characteristics of the filter paper used. [ 3 ]


Ecarin clotting time  ( ECT ) is a laboratory test used to monitor anticoagulation during treatment with  hirudin , an  anticoagulant   medication  which was originally isolated from  leech  saliva. Ecarin, the primary reagent in this assay, is derived from the venom of the saw-scaled viper,  Echis carinatus . [ citation needed ]


In the clinical assay, a known quantity of  ecarin  is added to the plasma of a patient treated with hirudin. Ecarin activates  prothrombin  through a specific proteolytic cleavage, which produces meizothrombin, a prothrombin-thrombin intermediate which retains the full molecular weight of prothrombin, but possesses a low level of procoagulant enzymatic activity. Crucially, this activity is inhibited by hirudin and other direct thrombin inhibitors, but not by  heparin . The ECT is also unaffected by prior treatment with  warfarin  or the presence of phospholipid-dependent anticoagulants, such as  lupus anticoagulant . Thus, the ECT is prolonged in a specific and linear fashion with increasing concentrations of hirudin. An enhancement of the ECT is the ecarin chromogenic assay (ECA) in which diluted sample is mixed with an excess of purified prothrombin and the generated meizothrombin is measured with a specific chromogenic substrate. This assay shows no interference from prothrombin or fibrinogen in the sample and is suitable for the measurement of all direct thrombin inhibitors. [ 1 ] [ 2 ] [ 3 ] [ 4 ]


Epitope Detection in Monocytes (EDIM)  is a technology that uses the  innate immune system 's mechanisms to detect  biomarkers  or  antigens  in  immune cells . It is a non-invasive form of liquid  biopsy , i.e. biopsy from blood, which analyzes activated  macrophages  ( CD14+ / CD16+ ) for disease-specific epitopes, such as tumor cell components. [ 1 ] [ 2 ]


Macrophages are part of the human immune system. They are involved in the detection, phagocytosis and destruction of organisms which are deemed harmful. [ 2 ] [ 3 ]


In case of cancerous tumors,  macrophages  ingest tumor cells and dissolve them with the help of enzymes, storing tumor proteins intracellularly, even when little tumor mass is present. With the help of EDIM technologie, activated macrophages containing intracellular tumor epitopes can be detected using CD14 and CD16 specific antibodies. [ 4 ] [ 5 ] [ 6 ]


Currently, EDIM technology is used for the blood test PanTum Detect. Here, the method is applied to examine which individuals would benefit from further cancer detection examinations with imaging procedures ( MRI ,  PET/CT ) to clarify a possible tumor disease. The two biomarkers used for PanTum Detect are  TKTL1  and  DNaseX .


The PanTum Detect blood test exploits the EDIM technology utilizing the fact that activated monocytes/macrophages phagocytose tumor cells and contain tumor proteins intracellularly. [ 7 ] [ 8 ]


This  biotechnology  article is a  stub . You can help Wikipedia by  expanding it .


An  electrolyte  is a substance that conducts  electricity  through the movement of  ions , but not through the movement of  electrons . [ 1 ] [ 2 ] [ 3 ]  This includes most soluble  salts ,  acids , and  bases , dissolved in a  polar solvent  like water. Upon dissolving, the substance separates into  cations  and  anions , which disperse uniformly throughout the solvent. [ 4 ]   Solid-state electrolytes  also exist. In medicine and sometimes in chemistry, the term electrolyte refers to the substance that is dissolved. [ 5 ] [ 6 ]


Electrically, such a solution is neutral. If an  electric potential  is applied to such a solution, the cations of the solution are drawn to the  electrode  that has an abundance of  electrons , while the anions are drawn to the electrode that has a deficit of electrons. The movement of anions and cations in opposite directions within the solution amounts to a current. Some gases, such as  hydrogen chloride  (HCL), under conditions of high temperature or low pressure can also function as electrolytes. [ clarification needed ]  Electrolyte solutions can also result from the dissolution of some biological (e.g.,  DNA ,  polypeptides ) or  synthetic polymers  (e.g.,  polystyrene sulfonate ), termed " polyelectrolytes ", which contain charged  functional groups . A substance that dissociates into ions in solution or in the melt acquires the capacity to conduct electricity.  Sodium ,  potassium ,  chloride ,  calcium ,  magnesium , and  phosphate  in a liquid phase are examples of electrolytes.


In medicine,  electrolyte replacement  is needed when a person has prolonged  vomiting  or  diarrhea , and as a response to sweating due to strenuous athletic activity. Commercial electrolyte solutions are available, particularly for sick children (such as  oral rehydration  solution,  Suero Oral , or  Pedialyte ) and athletes ( sports drinks ). Electrolyte monitoring is important in the treatment of  anorexia  and  bulimia .


In science, electrolytes are one of the main components of  electrochemical cells . [ 2 ]


In clinical  medicine , mentions of electrolytes usually refer  metonymically  to the ions, and (especially) to their  concentrations  (in blood, serum, urine, or other fluids). Thus, mentions of electrolyte levels usually refer to the various ion concentrations, not to the fluid volumes.


The word  electrolyte  derives from  Ancient Greek  ήλεκτρο- ( ēlectro -), prefix originally meaning  amber  but in modern contexts related to electricity, and λυτός ( lytos ), meaning "able to be untied or loosened". [ citation needed ]


In his 1884 dissertation,  Svante Arrhenius  put forth his explanation of solid crystalline salts disassociating into paired charged particles when dissolved, for which he won the 1903  Nobel Prize  in Chemistry. [ 7 ] [ 8 ] [ 9 ] [ 10 ]  Arrhenius's explanation was that in forming a solution, the salt dissociates into charged particles, to which  Michael Faraday  (1791-1867) had given the name " ions " many years earlier. Faraday's belief had been that ions were produced in the process of  electrolysis . Arrhenius proposed that, even in the absence of an electric current, solutions of salts contained ions. He thus proposed that chemical reactions in solution were reactions between ions. [ 8 ] [ 9 ] [ 10 ]


Shortly after Arrhenius's hypothesis of ions,  Franz Hofmeister  and Siegmund Lewith [ 11 ] [ 12 ] [ 13 ]  found that different ion types displayed different effects on such things as the solubility of proteins. A consistent ordering of these different ions on the magnitude of their effect arises consistently in many other systems as well. This has since become known as the  Hofmeister series .


While the origins of these effects are not abundantly clear and have been debated throughout the past century, it has been suggested that the charge density of these ions is important [ 14 ]  and might actually have explanations originating from the work of  Charles-Augustin de Coulomb  over 200 years ago.


Electrolyte solutions are normally formed when salt is placed into a  solvent  such as water and the individual components dissociate due to the  thermodynamic  interactions between solvent and solute molecules, in a process called " solvation ". For example, when table salt ( sodium chloride ), NaCl, is placed in water, the salt (a solid) dissolves into its component ions, according to the dissociation reaction: [ citation needed ]


It is also possible for substances to react with water, producing ions.  For example,  carbon dioxide  gas dissolves in water to produce a solution that contains  hydronium ,  carbonate , and  hydrogen carbonate  ions. [ citation needed ]


Molten salts  can also be electrolytes as, for example, when sodium chloride is molten, the liquid conducts electricity. In particular, ionic liquids, which are molten salts with melting points below 100 °C, [ 15 ]  are a type of highly conductive non-aqueous electrolytes and thus have found more and more applications in fuel cells and batteries. [ 16 ]


An electrolyte in a solution may be described as "concentrated" if it has a high concentration of ions, or "dilute" if it has a low concentration. If a high proportion of the solute dissociates to form free ions, the electrolyte is strong; if most of the solute does not dissociate, the electrolyte is weak. The properties of electrolytes may be exploited using electrolysis to extract constituent elements and compounds contained within the solution. [ citation needed ]


Alkaline earth metals form hydroxides that are strong electrolytes with limited solubility in water, due to the strong attraction between their constituent ions. This limits their application to situations where high solubility is required. [ 17 ]


In 2021, researchers have found that electrolyte can "substantially facilitate electrochemical corrosion studies in less conductive media". [ 18 ]


In  physiology , the primary ions of electrolytes are  sodium  (Na + ),  potassium  (K + ),  calcium  (Ca 2+ ),  magnesium  (Mg 2+ ),  chloride  (Cl − ),  hydrogen phosphate  (HPO 4 2− ), and  hydrogen carbonate  (HCO 3 − ). [ 19 ] [ failed verification ]  The electric charge symbols of plus (+) and minus (−)  indicate that the substance is ionic in nature and has an imbalanced distribution of electrons, the result of  chemical dissociation . Sodium is the main electrolyte found in extracellular fluid and potassium is the main intracellular electrolyte; [ 20 ]  both are involved in fluid balance and  blood pressure  control. [ 21 ]


All known multicellular lifeforms require a subtle and complex electrolyte balance between the  intracellular  and  extracellular  environments. [ 19 ]  In particular, the maintenance of precise  osmotic   gradients  of electrolytes is important. Such gradients affect and regulate the  hydration  of the body as well as  blood pH , and are critical for  nerve  and  muscle  function. Various mechanisms exist in living species that keep the concentrations of different electrolytes under tight control. [ 22 ]


Both muscle tissue and  neurons  are considered electric tissues of the body. Muscles and neurons are activated by electrolyte activity between the  extracellular fluid  or  interstitial fluid , and  intracellular fluid . Electrolytes may enter or leave the cell membrane through specialized protein structures embedded in the  plasma membrane  called " ion channels ". For example,  muscle contraction  is dependent upon the presence of calcium (Ca 2+ ), sodium (Na + ), and potassium (K + ). Without sufficient levels of these key electrolytes, muscle weakness or severe muscle contractions may occur. [ citation needed ] [ 23 ]


Electrolyte balance is maintained by oral, or in emergencies, intravenous (IV) intake of electrolyte-containing substances, and is regulated by  hormones , in general with the  kidneys  flushing out excess levels. In humans, electrolyte  homeostasis  is regulated by hormones such as  antidiuretic hormones ,  aldosterone  and  parathyroid hormones . Serious  electrolyte disturbances , such as  dehydration  and  overhydration , may lead to cardiac and neurological complications and, unless they are rapidly resolved, will result in a  medical emergency .


Measurement of electrolytes is a commonly performed diagnostic procedure, performed via  blood testing   with  ion-selective electrodes  or  urinalysis  by  medical technologists . The interpretation of these values is somewhat meaningless without analysis of the  clinical history  and is often impossible without parallel measurements of  renal function . The electrolytes measured most often are sodium and potassium. Chloride levels are rarely measured except for  arterial blood gas  interpretations since they are inherently linked to sodium levels. One important test conducted on urine is the  specific gravity  test to determine the occurrence of an  electrolyte imbalance . [ citation needed ]


According to a study paid for by the  Gatorade Sports Science Institute , electrolyte drinks containing sodium and potassium salts replenish the body's water and electrolyte concentrations after dehydration caused by  exercise ,  excessive alcohol consumption ,  diaphoresis  (heavy sweating), diarrhea, vomiting,  intoxication  or starvation; the study says that athletes exercising in extreme conditions (for three or more hours continuously, e.g. a  marathon  or  triathlon ) who do not consume electrolytes risk  dehydration  (or  hyponatremia ). [ 24 ] [ needs independent confirmation ]


A home-made electrolyte drink can be made by using water, sugar and salt  in precise proportions . [ 25 ]  It is important to include  glucose  (sugar) to utilise the co-transport mechanism of sodium and glucose. Commercial preparations are also available [ 26 ]  for both human and veterinary use.


Electrolytes are commonly found in  fruit juices , sports drinks, milk, nuts, and many fruits and vegetables (whole or in juice form) (e.g., potatoes,  avocados ).


When  electrodes  are placed in an electrolyte and a  voltage  is applied, the electrolyte will conduct electricity. Lone  electrons  normally cannot pass through the electrolyte; instead, a chemical reaction occurs at the  cathode , providing electrons to the electrolyte.   Another reaction occurs at the  anode , consuming electrons from the electrolyte. As a result, a negative charge cloud develops in the electrolyte around the cathode, and a positive charge develops around the anode. The ions in the electrolyte neutralize these charges, enabling the electrons to keep flowing and the reactions to continue. [ citation needed ]


For example, in a solution of ordinary table salt (sodium chloride, NaCl) in water, the cathode reaction will be


and  hydrogen  gas will bubble up; the anode reaction is


and  chlorine  gas will be liberated into solution where it reacts with the sodium and hydroxyl ions to produce  sodium hypochlorite  - household  bleach . The positively charged sodium ions Na +  will react toward the cathode, neutralizing the negative charge of OH −  there, and the negatively charged hydroxide ions OH −  will react toward the anode, neutralizing the positive charge of Na +  there. Without the ions from the electrolyte, the charges around the electrode would slow down continued electron flow;  diffusion  of H +  and OH −  through water to the other electrode takes longer than movement of the much more prevalent salt ions.
Electrolytes dissociate in water because water molecules are dipoles and the dipoles orient in an energetically favorable manner to  solvate  the ions.


In other systems, the electrode reactions can involve the metals of the electrodes as well as the ions of the electrolyte.


Electrolytic conductors are used in electronic devices where the chemical reaction at a metal-electrolyte interface yields useful effects.


Solid electrolytes can be mostly divided into four groups described below.


Gel electrolytes – closely resemble liquid electrolytes. In essence, they are liquids in a flexible  lattice framework . Various  additives  are often applied to increase the  conductivity  of such systems. [ 27 ] [ 29 ]


Solid ceramic electrolytes –  ions  migrate through the ceramic phase by means of vacancies or  interstitials  within the  lattice . There are also  glassy-ceramic  electrolytes.


Dry polymer electrolytes differ from liquid and gel electrolytes in that salt is dissolved directly into the solid medium. Usually it is a relatively high- dielectric constant   polymer  ( PEO ,  PMMA ,  PAN ,  polyphosphazenes ,  siloxanes , etc.) and a salt with low  lattice energy . In order to increase the  mechanical strength  and conductivity of such electrolytes, very often  composites  are made, and inert ceramic phase is introduced. There are two major classes of such electrolytes: polymer-in-ceramic, and ceramic-in-polymer. [ 30 ] [ 31 ] [ 32 ]


Organic ionic plastic crystals – are a type  organic salts  exhibiting  mesophases  (i.e. a  state of matter  intermediate between liquid and solid), in which mobile ions are orientationally or rotationally disordered while their centers are located at the ordered sites in the crystal structure. [ 28 ]  They have various forms of disorder due to one or more solid–solid  phase transitions  below the  melting point  and have therefore  plastic  properties and good mechanical flexibility as well as an improved electrode-electrolyte interfacial contact. In particular, protic organic ionic plastic crystals (POIPCs), [ 28 ]  which are solid  protic  organic salts formed by  proton  transfer from a  Brønsted acid  to a Brønsted base and in essence are protic  ionic liquids  in the  molten state , have found to be promising solid-state  proton conductors  for  fuel cells . Examples include  1,2,4-triazolium   perfluorobutanesulfonate [ 28 ]  and  imidazolium   methanesulfonate . [ 33 ]


Enzyme multiplied immunoassay technique  ( EMIT ) is a common method for qualitative and quantitative determination of therapeutic and recreational drugs and certain proteins in  serum  and  urine . [ 1 ]


It is an  immunoassay  in which a drug or metabolite in the sample competes with a drug/metabolite labelled with an enzyme, to bind to an antibody. The more drug there is in the sample, the more free enzyme there will be, and the increased enzyme activity causes a change in color. [ 2 ] : 70


Determination of drug levels in serum is particularly important when the difference in the concentrations needed to produce a therapeutic effect and adverse side reactions (the  therapeutic window ) is small. EMIT therapeutic drug monitoring tests provide accurate information about the concentration of such drugs such as  immunosuppressant  drugs and some antibiotics. [ 3 ] [ 4 ]


EMIT urine assays for drugs such as cannabinoids, morphine, and amphetamine are designed to detect the drug itself or a metabolite of the drug present in a concentration above a pre-specified minimum detection cutoff limit. [ 5 ]   In the U.S., the cutoff limits must be set in accordance with Mandatory Guidelines for Federal Workplace Drug Testing Programs that were developed by SAMHSA (The Substance Abuse and Mental Health Services Administration is a branch of the U.S. Department of Health and Human Services).  The setting of reasonable cutoff limits help reduce false positive results that occur from assay limitations. Because of the social and legal consequences, a positive test result must be confirmed by an alternative method, usually Gas Chromatography/Mass spectrometry. As an example the SAMHSA cutoffs for cannabinoids are 50 ng/ml for the immunoassay and 15 ng/ml as confirmed by GC/MS. [ 6 ]  Immunoassays that do not conform with SAMHSA, featuring a cutoff of 20 ng/ml, have been shown to produce false positives from passive inhalation of marijuana smoke. [ 7 ]


Erythrocyte fragility  refers to the propensity of  erythrocytes  (red blood cells, RBC) to hemolyse (rupture) under stress. It can be thought of as the degree or proportion of  hemolysis  that occurs when a sample of  red blood cells  are subjected to stress (typically physical stress, and most commonly osmotic and/or mechanical stress). Depending on the application as well as the kind of fragility involved, the amount of stress applied and/or the significance of the resultant hemolysis may vary. [ citation needed ]


When multiple levels of stress are applied to a given population/sample of cells, a  fragility profile  can be obtained by measuring the relative or absolute extent of hemolysis existing at each such level, [ 1 ]  in addition to finding one or more single-number  indexes [ 2 ]  (either measured directly or interpolated) associated with particular respective levels of hemolysis and/or corresponding stress. Fragility testing can be useful to assess cells' ability (or lack thereof) to withstand sustained or repeated stress. Moreover, it can be used to assess how fragility itself varies under different or changing environmental or stress conditions, during or prior to the inducement of the hemolysis. Low fragility is often termed " stability ," though technically stability refers to cells' resistance to  both  stress-induced lysis  and  spontaneous auto-lysis. [ citation needed ]


Osmotic fragility  (OF) refers to the degree or proportion of  hemolysis  that occurs when a sample of  red blood cells  are subjected to osmotic stress by being placed in a  hypotonic  solution. Osmotic fragility is affected by various factors, including membrane composition and integrity as well as the cells' sizes or surface-area-to-volume ratios. [ 3 ] [ 4 ] [ 5 ]


The osmotic fragility test is common in  hematology , and is often performed to aid with diagnosis of diseases associated with RBC membrane abnormalities. Some diseases linked to  increased  OF include  hereditary spherocytosis  and  hypernatremia , while some linked to  decreased  OF include chronic liver disease, iron deficiency anemia, thalassemia, hyponatremia, polycythemia vera, hereditary xerocytosis, [ 6 ]  and sickle cell anemia after splenectomy. [ 7 ]


New approaches to testing OF are under development to better facilitate its use in disease diagnosis and screening, such as by utilizing microfluidic devices along with cell counting. [ 8 ]


Mechanical fragility  (MF) refers to the degree or proportion of  hemolysis  that occurs when a sample of  red blood cells  are subjected to mechanical stress, such as (typically) some kind of  shear stress . Yet unlike with osmotic fragility, no single approach for testing mechanical fragility has yet gained sufficient acceptance to enable standardization. [ 9 ]   This has led to some insurance companies not currently covering the test. [ 10 ]


Uses of erythrocyte mechanical fragility can include diagnostic testing, [ 11 ]  calibrations to aid comparisons of hemolysis caused by blood-handling devices, [ 9 ]  or assessment of sublethal (i.e., non-hemolysing) damage caused to cells from devices that manipulate blood (such as for dialysis [ 12 ]  or intraoperative autotransfusion [ 13 ] ). It can also help in assessing damage of stored RBC product [ 14 ]  (so-called "storage lesion"), leading to applications in  blood transfusion  and  blood banking .


It's also notable that there can be a  qualitative  difference between a mechanical fragility test involving a comparatively lower energy stress, such as by agitating one or more beads in the presence of the sample (a common approach [ 15 ] ), versus a higher energy stress, such as by applying ultrasound to the sample. [ 16 ]  The difference is that the lower-energy category of stress can more prominently reflect cell membrane properties, whereas the higher-energy category largely reflects other properties like hemoglobin viscosity and cell size. Viscous or fluidic-mechanical stresses can be of either sort. [ citation needed ]


Mechanical fragility is increased  in the cases of sickle cell anemia, thalassemia, hereditary spherocytosis etc. .


Susceptibility to hemolysis from causes  other than  osmotic or mechanical forces are not as common, but may sometimes be referred to in terms of fragility or stability.  For example, photons or radicals can induce hemolysis.Erythrocytes/RBC may also be tested for related membrane properties aside from fragility, including  erythrocyte deformability  and cell morphology. Morphology can be measured by indexes which characterize shape changes of differences among cells. Deformability testing involves measuring the degree or ease of cells' contortion or shape change under a given level of applied force - or some indirect inference of the like. Other related red blood cell properties can include adhesion and aggregation, which along with deformability are often classed as RBC "flow properties." [ citation needed ]


Erythrocyte rosetting  or  E-rosetting  is a phenomenon seen through a  microscope  where  red blood cells   (erythrocytes)  are arranged around a central cell to form a cluster that looks like a flower.  The red blood cells surrounding the cell form the  petal,  while the central cell forms the  stigma  of the flower shape.  This formation occurs due to an  immunological  reaction between an epitope on the central cell's surface and a receptor or antibody on a red cell. [ 1 ]   The presence of E-rosetting can be used as a test for  T cells  although more modern tests such as  immunohistochemistry  are available. Rosetting is caused by parasites in the genus  Plasmodium  and is a cause of some malaria-associated symptoms. [ 2 ] [ citation needed ]


Three types of rosette techniques have been developed and used experimentally. [ citation needed ]


The  Rosette test  is performed on postpartum maternal blood to estimate the volume of  fetal-maternal hemorrhage  in case of an  Rh negative  mother and an Rh positive child. This estimate, in turn, also estimates the required amount of  Rho(D) immune globulin  to administer. [ 3 ]  In this test, a sample of maternal blood is incubated with Rho(D) immune globulin, which will bind to any fetal Rh positive  red blood cells , if present. [ 4 ]  Upon addition of enzyme-treated cDE indicator cells, the presence of Rh positive fetal blood causes rosetting, which can be seen by  light microscopy . [ 4 ]  The test is recommended for Rh negative mothers within 72 hours of giving birth to an Rh-positive infant. [ 3 ]  In a positive test, it is recommended that a  Kleihauer–Betke test  should be performed to confirm and quantify any positive rosette tests. [ 3 ]


E-rosetting is used in the identification of T cells where a T cells  CD2  surface protein is bound to a sugar based  LFA-3  homologue on the surface of a sheep red blood cell. [ 1 ]   Because the LFA-3 homologue is only present on the surface of sheep red blood cells other species red blood cells can not be used in this type of rosetting. [ citation needed ]


Erythrocyte antibody rosetting (EA-rosetting), occurs when an antibody molecule that is specific for an epitope on another cell is embedded in the membrane of a red blood cell and then reacted against a cell carrying the epitope that the antibody is specific for. [ citation needed ]


Erythrocyte antibody complement rosetting (EAC-rosetting), occurs when antibody in the presence of complement is bound to the surface of a red blood cell.  The complement binds to the tail region (Fc region) of the antibody.  Finally T-cells with a complement receptor are added and the T-cells bind to the complement on the antibody completing the rosette. [ citation needed ]


The  erythrocyte sedimentation rate  ( ESR  or  sed rate ) is the rate at which  red blood cells  in anticoagulated  whole blood  descend in a standardized tube over a period of one hour. It is a common  hematology  test, and is a non-specific measure of  inflammation .
To perform the test,  anticoagulated  blood is traditionally placed in an upright tube, known as a Westergren tube, and the distance which the red blood cells fall is measured and reported in  millimetres  at the end of one hour. [ 3 ]


Since the introduction of  automated analyzers  into the clinical laboratory, the ESR test has been automatically performed.


The ESR is influenced by the aggregation of red blood cells: blood plasma proteins, mainly  fibrinogen , promote the formation of red cell clusters called  rouleaux  or larger structures (interconnected rouleaux, irregular clusters). As according to  Stokes' law  the sedimentation velocity varies like the square of the object's diameter, larger aggregates settle faster. While aggregation already takes place at normal physiological fibrinogen levels, these tend to increase when an inflammatory process is present, leading to increased ESR.


The ESR is increased in inflammation,  pregnancy ,  anemia ,  autoimmune disorders  (such as  rheumatoid arthritis  and  lupus ), infections, some kidney diseases and some cancers (such as  lymphoma  and  multiple myeloma ). The ESR is decreased in  polycythemia ,  hyperviscosity ,  sickle cell anemia ,  leukemia ,  chronic fatigue syndrome , [ 4 ]  low  plasma protein  (due to liver or kidney disease) and  congestive heart failure . Although increases in immunoglobulins usually increase the ESR, very high levels can reduce it again due to hyperviscosity of the plasma. [ 5 ]  This is especially likely with  IgM -class paraproteins, and to a lesser extent,  IgA -class. The basal ESR is slightly higher in females. [ 6 ]


Erythrocyte sedimentation rate (ESR) is the measure of ability of erythrocytes ( red blood cell ) to fall through the  blood plasma  and accumulate together at the base of container in one hour. [ 7 ]


There are three stages in erythrocyte sedimentation: [ 8 ]


In normal conditions, the red blood cells are negatively charged and therefore repel each other rather than stacking. ESR is also reduced by high blood  viscosity , which slows the rate of fall. [ 7 ]


The rate of erythrocyte sedimentation is affected by both  inflammatory  and non-inflammatory conditions.


In inflammatory conditions,  fibrinogen , other clotting proteins, and  alpha globulin  are positively charged, thus increasing the ESR. [ 9 ]  ESR begins to rise at 24 to 48 hours after the onset of acute self-limited inflammation, decreases slowly as inflammation resolves, and can take weeks to months to return to normal levels. For ESR values more than 100 mm/hour, there is a 90% probability that an underlying cause would be found upon investigation. [ 9 ]


In non-inflammatory conditions, plasma albumin concentration, size, shape, and number of red blood cells, and the concentration of  immunoglobulin  can affect the ESR. Non-inflammatory conditions that can cause raised ESR include  anemia ,  kidney failure ,  obesity , ageing, and female sex. [ 7 ]  ESR is also higher in women during  menstruation  and  pregnancy . [ 9 ]  The value of ESR does not change whether dialysis is performed or not. Therefore, ESR is not a reliable measure of inflammation in those with kidney injuries as the ESR value is already elevated. [ 10 ]


An increased number of red blood cells (polycythemia) causes reduced ESR as blood viscosity increases.  Hemoglobinopathy  such as  sickle-cell disease  can have low ESR due to an improper shape of red blood cells that impairs stacking. [ citation needed ]


ESR can sometimes be useful in diagnosing diseases, such as multiple myeloma,  temporal arteritis ,  polymyalgia rheumatica , various autoimmune diseases,  systemic lupus erythematosus ,  rheumatoid arthritis , inflammatory bowel disease [ 11 ]  and chronic kidney diseases.  In many of these cases, the ESR may exceed 100 mm/hour. [ 12 ]


It is commonly used for a differential diagnosis for  Kawasaki's disease  (from  Takayasu's arteritis ; which would have a markedly elevated ESR) and it may be increased in some chronic infective conditions like  tuberculosis  and infective  endocarditis . It is also elevated in subacute  thyroiditis  also known as DeQuervain's.


In markedly increased ESR of over 100 mm/h, infection is the most common cause (33% of cases in an American study), followed by cancer (17%), kidney disease (17%) and noninfectious inflammatory disorders (14%). [ 13 ]  Yet, in  pneumonia  the ESR stays under 100. [ 14 ]


The usefulness of the ESR in current practice has been questioned by some, as it is a relatively imprecise and non-specific test compared to other available diagnostic tests. [ 15 ]  Current literature suggests that and ESR should be "obtained on all patients over the age of 50" who have an intense headache. [ 16 ]


It is a component of the  PCDAI  (pediatric Crohn's disease activity index), an index for assessment of the severity of inflammatory bowel disease in children. [ citation needed ]


The clinical usefulness of ESR is limited to monitoring the response to therapy in certain inflammatory diseases such as temporal arteritis, polymyalgia rheumatica and rheumatoid arthritis. It can also be used as a crude measure of response in  Hodgkin's lymphoma . Additionally, ESR levels are used to define one of the several possible adverse prognostic factors in the staging of Hodgkin's lymphoma. [ citation needed ]


Note: mm/h. = millimeters per hour.


Westergren's original normal values (men 3 mm/h and women 7 mm/h) [ 17 ]  made no allowance for a person's age. Later studies from 1967 confirmed that ESR values tend to rise with age and to be generally higher in women. [ 18 ]
Values of the ESR also appear to be slightly higher in normal populations of African-Americans than Caucasians of both genders. [ 19 ]  Values also appear to be higher in anemic individuals than non-anemic individuals. [ 20 ]


The widely used [ 21 ]  rule calculating normal maximum ESR values in adults (98% confidence limit) is given by a formula devised in 1983 from a study of ≈1000 individuals over the age of 20: [ 22 ]  The normal values of ESR in men is age (in years) divided by 2; for women, the normal value is age (in years) plus 10, divided by 2. [ 9 ]


Other studies confirm a dependence of ESR on age and gender, as seen in the following:


ESR reference ranges from a large 1996 study of 3,910 healthy adults (NB. these use 95% confidence intervals rather than the 98% intervals used in the study used to derive the formula above, and because of the skewness of the data, these values appear to be less than expected from the above formula): [ 23 ]


Normal values of ESR have been quoted as 1 [ 24 ]  to 2 [ 25 ]  mm/h at birth, rising to 4 mm/h 8 days after delivery, [ 25 ]  and then to 17 mm/h by day 14. [ 24 ]


Typical normal ranges quoted are: [ 6 ]


C-reactive protein  (CRP) is an  acute phase protein . Therefore, it is a better marker for acute phase reaction than ESR. While ESR and CRP generally together correlate with the degree of inflammation, this is not always the case and results may be discordant [ 9 ]  in 12.5% of the cases. [ 7 ]  Cases with raised CRP but normal ESR may demonstrate a combination of infection and some other tissue damage such as  myocardial infarction , and  venous thromboembolism . Such inflammation may not be enough to raise the level of ESR. Those with high ESR usually do not have demonstrable inflammation. However, in cases of low grade bacterial infections of bone and joints such as coagulase negative  staphylococcus  (CoNS), and systemic lupus erythematosus (SLE), ESR can be a good marker for the inflammatory process. This may be due to the production of  Interferon type I  that inhibits the CRP production in liver cells during SLE. [ 27 ]  CRP is a better marker for other autoimmune diseases such as polymyalgia rheumatica,  giant cell arteritis , [ 7 ]  post-operative sepsis, and  neonatal sepsis . ESR may be reduced in those who are taking  statins  and  non-steroidal anti-inflammatory drugs  (NSAIDs). [ 9 ]


Bone and joint infections


Ischemic stroke


Waldenstrom's macroglobulinemia


Multiple myeloma


IgG4 related disease


Chronic kidney disease


Low serum albumin


Myocardial infarction


Venous thromboembolic disease


Rheumatoid arthritis


The test was invented in 1897 by the Polish pathologist  Edmund Biernacki . [ 28 ] [ 29 ]  In some parts of the world the test continues to be referred to as Biernacki's Reaction ( Polish :  odczyn Biernackiego , OB). [ 30 ]  In 1918, Dr Robert Fåhræus noted that ESR differed only during pregnancy. Therefore, he suggested that ESR could be used as an indicator of pregnancy. In 1921, Dr Alf Vilhelm Albertsson Westergren used ESR to measure the disease outcome of  tuberculosis . He defined the measurement standards of ESR which is still being used today. [ 7 ]  Robert Fåhræus and Alf Vilhelm Albertsson Westergren are eponymously remembered for the 'Fahraeus-Westergren test' (abbreviated as FW test; in the UK, usually termed Westergren test), [ 30 ]  which uses  sodium citrate-anti-coagulated specimens. [ 31 ]


According to a study released in 2015, a  stop gain mutation  in  HBB  gene (p. Gln40stop) was shown to be associated with ESR values in  Sardinian population . The red blood cell count, whose values are inversely related to ESR, is affected in carriers of this  SNP . This mutation is almost exclusive of the inhabitants of Sardinia and is a common cause of  beta thalassemia . [ 32 ]


According to a 2010 study, there is a reverse correlation between ESR and  general intelligence  (IQ) in Swedish males aged 18–20. [ 33 ]


The  euglobulin lysis time  ( ELT ) is a test that measures overall  fibrinolysis . The test is performed by mixing citrated platelet-poor  plasma  with acid in a glass test tube. This acidification causes the precipitation of certain  clotting factors  in a complex called the  euglobulin fraction . The euglobulin fraction contains the important  fibrinolytic  factors  fibrinogen ,  PAI-1 ,  tissue plasminogen activator  (tPA),  plasminogen , and to a lesser extent  α 2 -antiplasmin . The euglobulin fraction also contains  factor VIII . [ citation needed ]


After precipitation, the euglobulin fraction is resuspended in a borate solution. Clotting is then activated by the addition of calcium chloride at 37 °C. Historically, subsequent amount of fibrinolysis was determined by eye, by observing the clot within the test tube at ten-minute intervals until complete lysis had occurred. [ 1 ]  Newer automated methods have also been developed. These methods use the same principle as the older technique, but use a  spectrophotometer  to track clot lysis as a function of  optical density . [ 2 ]


Fecal occult blood  ( FOB ) refers to  blood  in the  feces  that is not visibly apparent (unlike other types of  blood in stool  such as  melena  or  hematochezia ). A fecal occult blood test (FOBT) checks for hidden (occult) blood in the stool (feces). [ 1 ]


The American College of Gastroenterology has recommended the abandoning of gFOBT testing as a colorectal cancer screening tool, in favor of the  fecal immunochemical test  (FIT). [ 2 ]   The newer and recommended tests look for  globin ,  DNA , or other blood factors including  transferrin , while conventional  stool guaiac tests  look for  heme .


Fecal occult blood testing (FOBT), as its name implies, aims to detect subtle blood loss in the  gastrointestinal tract , anywhere from the  mouth  to the  colon . Positive tests ("positive stool") may result from either  upper gastrointestinal bleeding  or  lower gastrointestinal bleeding  and warrant further investigation for  peptic ulcers  or a  malignancy  (such as  colorectal cancer  or  gastric cancer ). The test does not directly detect  colon cancer  but is often used in clinical  screening  for that disease. It can also be used to look for active occult blood loss in  anemia [ 3 ]  or when there are gastrointestinal symptoms. [ 4 ]


An estimated 1–5% of large tested populations have a positive fecal occult blood test. [ citation needed ]  Of those, about 2–10% have cancer, while 20–30% have  adenomas .  Screening  methods for colon cancer depend on detecting either precancerous changes such as certain kinds of polyps or on finding early and thus more treatable cancer. The extent to which screening procedures reduce the risk of gastrointestinal cancer or deaths depends on the rate of precancerous and cancerous disease in that population. gFOBT (guaiac fecal occult blood test) and flexible sigmoidoscopy screening have each shown benefit. Other colon cancer screening tools such as iFOBT (immunochemical fecal occult blood test) or colonoscopy are also included in guidelines. [ 5 ]


In 2009, the  American College of Gastroenterology  (ACG) suggested that colon cancer screening modalities that are also directly preventive by removing precursor lesions should be given precedence, and prefer a  colonoscopy  every ten years in average-risk individuals, beginning at age 50. [ 2 ]  The ACG suggests that cancer detection tests such as any type of FOB are an alternative that is less preferred, and if a colonoscopy is declined, the FIT (fecal immunochemical test, or iFOBT) should be offered instead. The 2017 US Multi-Society Task Force (MSTF)'s recommended first-tier tests are a colonoscopy every 10 years or annual FIT test. [ 6 ]  If FIT is utilized, proper steps must be taken to ensure appropriate use and follow-up of abnormal FIT results. [ 7 ]  FIT tests however are not that useful in picking up adenomas, even when advanced. [ 8 ]


The  United States Preventive Services Task Force  (USPSTF)'s 2016 recommendation, instead of emphasizing specific screening approaches, has instead chosen to highlight that there is convincing evidence that colorectal cancer screening substantially reduces deaths from the disease among adults aged 50 to 75 years and that not enough adults are using this effective preventive intervention. [ 9 ]  The ACG and MSTF also included CT colonography every five years, and fecal DNA testing as considerations. All three recommendation panels recommended replacing any older low-sensitivity, guaiac-based fecal occult blood testing (gFOBT) with either newer high-sensitivity guaiac-based fecal occult blood testing (hs gFOBT) or fecal immunochemical testing (FIT). MSTF looked at six studies that compared high-sensitivity gFOBT (Hemoccult SENSA) to FIT, and concluded that there was no clear difference in overall performance between these methods.


The English  National Health Service  (NHS) introduced a Bowel Cancer Screening Program in 2006. [ 10 ]  It is now offered to patients aged 60–74 years. In 2019 FIT was introduced as the primary screening test in England and Wales, replacing gFOBt.  [ 11 ] [ 12 ]  However, research carried out in the UK has suggested that the FIT threshold for further investigation is set at a point that may miss more than half of bowel cancer cases and only identifies one in four high-risk polyps. [ 13 ] [ 14 ]


The American College of Gastroenterology has recommended the abandoning of gFOBT testing as a colorectal cancer screening tool, in favor of the fecal immunochemical test. [ 2 ]  Though the FIT test is preferred, even the guaiac FOB testing of average risk populations may have been sufficient to reduce the mortality associated with  colon cancer  by about 25%. [ 15 ]  With this lower efficacy, it was not always cost-effective to screen a large population with gFOBT. [ 16 ] [ 17 ] [ 18 ] [ 19 ]


If colon cancer is suspected in an individual (such as in someone with an unexplained  anemia ), fecal occult blood tests may not be clinically helpful. If a doctor suspects colon cancer, more rigorous investigation is necessary, whether or not the test is positive. [ citation needed ]


In 2006, the Australian Government introduced the National Bowel Cancer Program which has been updated several times since; targeted screening will be done of all Australians aged from 50 to 74 by 2020. Cancer Council Australia recommended that FOBT should be done every two years. People over 50 not yet eligible for the national program can arrange with their doctor for an FOBT. [ 20 ]
The  Canadian Cancer Society  recommends that men and women aged 50 and over have an FOBT at least every two years. [ 21 ]
In colon cancer screening, using only one sample of feces collected by a doctor performing a  digital rectal examination  is discouraged. [ 22 ]


The use of the  M2-PK Test  is encouraged over gFOBT for routine screening, as it may pick up tumors whether or not they are bleeding. [ 23 ]  It is able to detect 80 percent of colorectal cancers and 44 percent for adenoma > 1 centimeter, while gFOBT picks up 13 to 50 percent of colorectal cancers. [ 23 ]


Gastrointestinal bleeding  has many potential sources, and positive results usually result in further testing for the bleeding site, usually looking for  lower gastrointestinal bleeding  before  upper gastrointestinal bleeding  causes unless there are other clues. [ 24 ]  Colonoscopy is usually preferred to computerized tomographic colonography. [ 25 ]


A positive test can result from  upper gastrointestinal bleeding  or  lower gastrointestinal bleeding . The common causes are:


In the event of a positive fecal occult blood test, the next step in the workup is a form of visualization of the gastrointestinal tract by one of several means:


The use of an FOBT for bleeding from the mouth, nose, esophagus, lungs, stomach and the initial portion of the small intestine, while the same as fecal testing, is discouraged, due to technical considerations including poorly characterized test performance characteristics such as sensitivity, specificity, and analytical interference. [ 29 ]  However, chemical confirmation that coloration is due to blood rather than coffee, beets, medications, or food additives can be of significant clinical assistance.


Gastrointestinal (GI) complaints and low-intensity GI bleeding frequently occur in marathon runners. [ 30 ]  Strenuous exercise, particularly in elite athlete runners and less frequently in other exercise activities, can cause acute incapacitating gastrointestinal symptoms including heartburn, nausea, vomiting, abdominal pain, diarrhea and gastrointestinal bleeding. [ 31 ]  Approximately one third of endurance runners experience transient but exercise-limiting symptoms, and repetitive gastrointestinal bleeding occasionally causes iron deficiency and anaemia. [ 32 ] [ 33 ]  Runners can sometimes experience significant symptoms including  hematemesis . [ 34 ]  Exercise is associated with extensive changes in gastrointestinal (GI) tract physiology, including diversion of blood flow from the GI tract to muscles and lungs, decreased GI absorption and small intestinal motility, increased colonic transit, neuroimmunoendocrine changes in hormones and peptides such as  vasoactive intestinal peptide ,  secretin  and peptide-histidine-methionine. [ 35 ]  Substantial changes occur in stress hormones including cortisol, in circulating concentrations and metabolic behavior of various  leucocytes , and in  immunoglobulin  levels and  major histocompatibility complex  expression. [ 36 ]  Symptoms can be exacerbated by dehydration or by pre-exercise ingestion of certain foods and  hypertonic  liquids, and lessened by adequate training. [ 35 ]


Ingestion of 800   mg of  cimetidine  two hours before running a marathon did not significantly affect the frequency of gastrointestinal symptoms or occult gastrointestinal bleeding. [ 37 ]  Conversely, 800   mg of cimetidine 1 hr before the start and again at 50 miles of a 100-mile running race substantially decreased GI symptoms and post-race guaiac test positivity but did not affect race performance. [ 38 ]


There are four methods in clinical use to test for occult blood in feces. These look at different properties, such as  antibodies ,  heme ,  globin , or  porphyrins  in blood, or at  DNA  from cellular material such as from lesions of the intestinal mucosa.


Additional methods of looking for occult blood are being explored, including  transferrin  dipstick [ 48 ]  and stool  cytology . [ 49 ]


The estimates for test performance characteristics are based on comparison with a variety of  reference methods  including 51-chromium studies, [ citation needed ]  analytical recovery studies in spiked stool samples, analytical recovery after ingestion of autologous blood, rarer studies of carefully quantified blood instilled at bowel surgery  [ citation needed ] , as well as other research approaches. [ citation needed ]  Additionally, clinical studies look at a variety of additional factors.


In healthy people about 0.5 to 1.5   ml of blood escapes blood vessels into the stool each day. [ 50 ] [ 51 ] [ 52 ]  Significant amounts of blood can be lost without producing visible blood in the stool, estimated as 200   ml in the stomach, [ 53 ]  100   ml in the duodenum, and lesser amounts in the lower intestine. Tests for occult blood identify lesser blood loss.


Fecal immunochemical testing (FIT) can identify as little as 0.3   ml of daily blood in the stool; yet this test threshold does not cause undue false positives from normal upper intestinal blood leakage because it does not detect occult blood from the stomach and upper small intestine. Thus, the FIT test is much more specific for bleeding from the colon or lower gastrointestinal tract than alternatives. [ 54 ]  The detection rate of the test decreases if the time from sample collection to laboratory processing is delayed; processing the sample in under five days from collection is recommended. [ 55 ]  It does not appear to be affected by aspirin, anticoagulants, or nonsteroidal anti-inflammatory drugs. [ 56 ]


Stool guaiac test  for fecal occult blood (gFOBT) sensitivity varies depending on the site of bleeding. Moderately sensitive gFOBT can pick up a daily blood loss of about 10   ml (about two teaspoonfuls), and higher sensitivity gFOBT can pick up lesser amounts, requires at least 2   ml to become positive. The sensitivity of a single-stool guaiac test to pick up bleeding has been quoted at 10 to 30%, but if a standard three tests are done as recommended the sensitivity rises to 92%. [ 57 ]  Reduced patient compliance with the collection of three samples hampers the usefulness of this test. Further discussion of sensitivity and specificity issues that relate particularly to the guaiac method is found in the  stool guaiac test  article.


Fecal  porphyrin  quantification by HemoQuant can yield a  false positive  result due to exogenous blood and various porphyrins. HemoQuant is the most sensitive test for upper gastrointestinal bleeding and therefore may be most appropriate fecal occult blood test to use in the evaluation of iron deficiency. [ 58 ]  It is advisable to stop ingesting red meat and aspirin for three days prior to specimen collection. [ 59 ]  False positives can occur with myoglobin, catalase, or protohemes [ 60 ]  and in certain types of porphyria. [ citation needed ]


Fecal DNA tests as of 2008 had not been studied enough to support widespread use. [ 61 ]


Safety regulations from US accreditor  the Joint Commission  may have unintentionally decreased digital rectal examination and FOBT in hospital settings such as Emergency Departments. [ 62 ] [ 63 ]


Ferritin  is a universal intracellular and extracellular  protein  that stores  iron  and releases it in a controlled fashion. The protein is produced by almost all living organisms, including  archaea ,  bacteria ,  algae ,  higher plants , and  animals . It is the primary  intracellular iron-storage protein  in both  prokaryotes  and  eukaryotes , keeping iron in a soluble and non-toxic form. In humans, it acts as a buffer against  iron deficiency  and  iron overload . [ 3 ]


Ferritin is found in most tissues as a  cytosolic  protein, but small amounts are secreted into the  serum  where it functions as an iron carrier. Plasma ferritin is also an indirect  marker  of the total amount of iron stored in the body; hence, serum ferritin is used as a  diagnostic test  for  iron-deficiency anemia  and  iron overload . [ 4 ]  Aggregated ferritin transforms into a water insoluble, crystalline and amorphous form of storage iron called  hemosiderin . [ 5 ]


Ferritin is a  globular protein  complex consisting of 24  protein subunits  forming a hollow spherical nanocage with multiple metal–protein interactions. [ 6 ]  Ferritin with iron removed is called  apoferritin . [ 7 ] : e10


Ferritin genes are highly  conserved  between species. All vertebrate ferritin genes have three  introns  and four  exons . [ 8 ]  In human ferritin, introns are present between  amino acid   residues  14 and 15, 34 and 35, and 82 and 83; in addition, there are one to two hundred  untranslated bases  at either end of the combined exons. [ 9 ]  The  tyrosine  residue at amino acid position 27 is thought to be associated with  biomineralization . [ 10 ]


Ferritin is a hollow  globular protein  of mass 474  kDa  and comprising 24 subunits. Typically it has internal and external diameters of about 8 and 12 nm, respectively. [ 11 ]  The nature of these subunits varies by class of organism:


All the aforementioned ferritins are similar, in terms of their primary sequence, with the vertebrate H-type. [ 12 ]  In  E. coli , a 20% similarity to human H-ferritin is observed. [ 12 ]  Some ferritin complexes in vertebrates are  hetero-oligomers  of two highly related  gene  products with slightly different  physiological  properties. The ratio of the two  homologous  proteins in the complex depends on the relative expression levels of the two genes.


Cytosolic ferritin  shell (apoferritin) is a heteropolymer of 24 subunits of heavy (H) and light (L) peptides that form a hollow spherical nanocage that covers an iron core composed of  crystallites  together with  phosphate  and  hydroxide  ions. The resulting particle is similar to  ferrihydrite  (5Fe 2 O 3 ·9H 2 O). Each ferritin complex can store about 4500 iron (Fe 3+ ) ions. [ 9 ] [ 12 ]  The proportion of H to L subunits varies in ferritin from different tissues, explaining its heterogeneity on isoelectric focusing. L-rich ferritins (from spleen and liver) are more basic than H-rich ferritins (from heart and red blood cells).


Serum ferritin , which is typically iron-poor, consists almost exclusively of L subunits. Serum ferritin is heterogeneous due to glycosylation. The glycosylation and direct relationship of serum ferritin concentration to iron storage in macrophages suggest it is secreted by macrophages in response to changing iron levels.


Human  mitochondrial ferritin , MtF, was found to express as a  pro-protein . [ 14 ]  When a mitochondrion takes it up, it processes it into a mature protein similar to the ferritins found in the  cytoplasm , which it assembles to form functional ferritin shells. Unlike other human ferritins, it is a homopolymer of H type ferritin  and appears to have no introns (intronless) in its genetic code. The mitochondrial ferritin's  Ramachandran plot [ 15 ]  shows its structure to be mainly  alpha helical  with a low prevalence of  beta sheets . It accumulates in large amounts in the erythroblasts of subjects with impaired heme synthesis.


Ferritin is present in every cell type. [ 9 ]  It serves to store iron in a non-toxic form, to deposit it in a safe form, and to transport it to areas where it is required. [ 16 ]  The function and structure of the expressed ferritin protein varies in different cell types. This is controlled primarily by the amount and stability of  messenger RNA  (mRNA), but also by changes in how the mRNA is stored and how efficiently it is transcribed. [ 9 ]  One major trigger for the production of many ferritins is the mere presence of iron; [ 9 ]  an exception is the yolk ferritin of  Lymnaea sp. , which lacks an iron-responsive unit. [ 12 ]


Free iron is  toxic  to  cells  as it acts as a  catalyst  in the formation of  free radicals  from  reactive oxygen species  via the  Fenton reaction . [ 17 ]  Hence vertebrates have an elaborate set of protective mechanisms to bind iron in various  tissue  compartments [ discuss ] . Within cells, iron is stored in a protein complex as ferritin or the related complex  hemosiderin . Apoferritin binds to free ferrous iron and stores it in the ferric state. As ferritin accumulates within cells of the  reticuloendothelial system , protein aggregates are formed as hemosiderin. Iron in ferritin or hemosiderin can be extracted for release by the RE cells, although hemosiderin is less readily available. Under  steady-state  conditions, the level of ferritin in the  blood serum  correlates with total body stores of iron; thus, the serum ferritin FR5Rl is the most convenient laboratory test to estimate iron stores. [ citation needed ]


Because iron is an important mineral in mineralization, ferritin is employed in the shells of organisms such as  molluscs  to control the concentration and distribution of iron, thus sculpting shell morphology and colouration. [ 18 ] [ 19 ]  It also plays a role in the  haemolymph  of the  polyplacophora , where it serves to rapidly transport iron to the mineralizing  radula . [ 20 ]


Iron is released from ferritin for use by ferritin degradation, which is performed mainly by  lysosomes . [ 21 ]


Vertebrate ferritin consists of two or three subunits which are named based on their molecular weight: L "light", H "heavy", and M "middle" subunits. The M subunit has only been reported in bullfrogs. In bacteria and archaea, ferritin consists of one subunit type. [ 22 ]  H and M subunits of eukaryotic ferritin and all subunits of bacterial and archaeal ferritin are H-type and have ferroxidase activity, which means they are able to convert iron from the ferrous (Fe 2+ ) to ferric (Fe 3+ ) forms. This limits the deleterious reaction which occurs between ferrous iron and  hydrogen peroxide  known as the Fenton reaction which produces the highly damaging  hydroxyl radical . The ferroxidase activity occurs at a diiron binding site in the middle of each H-type subunits. [ 22 ] [ 23 ]  After oxidation of Fe(II), the Fe(III) product stays metastably in the ferroxidase center and is displaced by Fe(II), [ 23 ] [ 24 ]  a mechanism that appears to be common among ferritins of all three domains of life. [ 22 ]  The light chain of ferritin has no ferroxidase activity but may be responsible for the electron transfer across the protein cage. [ 25 ]


Ferritin concentrations increase drastically in the presence of an infection or cancer.  Endotoxins  are an up-regulator of the gene coding for ferritin, thus causing the concentration of ferritin to rise. By contrast, organisms such as  Pseudomonas , although possessing endotoxin, cause plasma ferritin levels to drop significantly within the first 48 hours of infection. Thus, the iron stores of the infected body are denied to the infective agent, impeding its metabolism. [ 26 ]


The concentration of ferritin has been shown to increase in response to stresses such as  anoxia , [ 27 ]  which implies that it is an  acute phase protein . [ 28 ]


Mitochondrial ferritin  has many roles pertaining to molecular function. It participates in ferroxidase activity, binding, iron ion binding, oxidoreductase activity, ferric iron binding, metal ion binding as well as transition metal binding. Within the realm of biological processes it participates in oxidation-reduction, iron ion transport across membranes and cellular iron ion homeostasis. [ citation needed ]


In some snails, the protein component of the egg yolk is primarily ferritin. [ 29 ]  This is a different ferritin, with a different genetic sequence, from the somatic ferritin.  It is produced in the midgut glands and secreted into the haemolymph, whence it is transported to the eggs. [ 29 ]


In vertebrates, ferritin is usually found within cells, although it is also present in smaller quantities in the plasma. [ 26 ]


Serum  ferritin levels are measured in  medical laboratories  as part of the iron studies workup for  iron-deficiency anemia . [ 6 ]  They are measured in nanograms per milliliter (ng/mL) or micrograms per liter (μg/L); the two units are equivalent.


The ferritin levels measured usually have a direct correlation with the total amount of iron stored in the body. However, ferritin levels may be artificially high in cases of  anemia of chronic disease , where ferritin is elevated in its capacity as an inflammatory  acute phase protein  and not as a marker for iron overload. [ citation needed ]


A normal ferritin blood level, referred to as the  reference interval  is determined by many  testing laboratories . The ranges for ferritin can vary between laboratories but typical ranges would be between 40 and 300 ng/mL (=μg/L) for males, and 20–200 ng/mL (=μg/L) for females. [ 30 ]


According to a 2014 review in the  New England Journal of Medicine  stated that a ferritin level below 30 ng/mL indicates  iron deficiency , while a level below 10 ng/mL indicates iron-deficiency anemia. [ 30 ]  A 2020 World Health Organization guideline states that ferritin indicates iron deficiency below 12 ng/mL in apparently-healthy children under 5 and 15 ng/mL in apparently-healthy individuals of 5 and over. [ 31 ]


Some studies suggest that women with  fatigue  and ferritin below 50 ng/mL see reduced fatigue after iron supplementation. [ 32 ] [ 33 ]


In the setting of anemia, low serum ferritin is the most specific lab finding for  iron-deficiency anemia . [ 34 ]  However it is less sensitive, since its levels are increased in the blood by infection or any type of chronic inflammation, [ 35 ]  and these conditions may convert what would otherwise be a low level of ferritin from lack of iron, into a value in the normal range. For this reason, low ferritin levels carry more information than those in the normal range. A  falsely low  blood ferritin (equivalent to a  false positive  test) is very uncommon, [ 35 ]  but can result from a  hook effect  of the measuring tools in extreme cases. [ 36 ]


Low ferritin may also indicate  hypothyroidism , [ 37 ]   vitamin C deficiency  or  celiac disease . [ citation needed ]


Low serum ferritin levels are seen in some patients with  restless legs syndrome , not necessarily related to anemia, but perhaps due to low iron stores short of anemia. [ 38 ] [ 39 ]


Vegetarianism  is not a cause of low serum ferritin levels, according to the American Dietetic Association's position in 2009: "Incidence of iron-deficiency anemia among vegetarians is similar to that of non-vegetarians. Although vegetarian adults have lower iron stores than non-vegetarians, their serum ferritin levels are usually within the normal range." [ 40 ]


If  ferritin is high, there is iron in excess or else there is an acute inflammatory reaction in which ferritin is mobilized without iron excess. For example, ferritins may be high in infection without signaling body iron overload.


Ferritin is also used as a  marker  for  iron overload disorders , such as  hemochromatosis  or  hemosiderosis .  Adult-onset Still's disease , some  porphyrias , and  hemophagocytic lymphohistiocytosis / macrophage activation syndrome  are diseases in which the ferritin level may be abnormally raised.


As ferritin is also an  acute-phase reactant , it is often elevated in the course of disease. A normal  C-reactive protein  can be used to exclude elevated ferritin caused by acute phase reactions.  [ citation needed ]


Ferritin has been shown to be elevated in some cases of  COVID-19  and may correlate with worse clinical outcome. [ 41 ] [ 42 ]  Ferritin and  IL-6  are considered to be possible immunological biomarkers for severe and fatal cases of COVID-19. Ferritin and C-reactive protein may be possible screening tools for early diagnosis of  systemic inflammatory response syndrome  in cases of COVID-19. [ 43 ] [ 44 ]


According to a study of  anorexia nervosa  patients, ferritin can be elevated during periods of acute  malnourishment , perhaps due to iron going into storage as intravascular volume and thus the number of red blood cells falls. [ 45 ]


Another study suggests that due to the catabolic nature of anorexia nervosa, isoferritins may be released. Furthermore, ferritin has significant non-storage roles within the body, such as protection from  oxidative damage . The rise of these isoferritins may contribute to an overall increase in ferritin concentration. The measurement of ferritin through  immunoassay  or immunoturbidimeteric methods may also be picking up these isoferritins thus not a true reflection of iron storage status. [ 46 ]


Studies reveal that a transferrin saturation (serum iron concentration ÷ total iron binding capacity) over 60 percent in men and over 50 percent in women identified the presence of an abnormality in iron metabolism ( hereditary hemochromatosis , heterozygotes, and homozygotes) with approximately 95 percent accuracy. This finding helps in the early diagnosis of hereditary hemochromatosis, especially while serum ferritin still remains low. The retained iron in hereditary hemochromatosis is primarily deposited in parenchymal cells, with reticuloendothelial cell accumulation occurring very late in the disease. This is in contrast to transfusional iron overload in which iron deposition occurs first in the reticuloendothelial cells and then in parenchymal cells. This explains why ferritin levels remain relative low in hereditary hemochromatosis, while transferrin saturation is high. [ 47 ] [ 48 ]


Hematological abnormalities often associate with chronic liver diseases. Both iron overload and iron deficient anemia have been reported in patients with liver cirrhosis. [ 49 ] [ 50 ]  The former is mainly due to reduced  hepcidin  level caused by the decreased synthetic capacity of the liver, while the latter is due to acute and chronic bleeding caused by  portal hypertension . Inflammation is also present in patients with advanced chronic liver disease. As a consequence, elevated hepatic and serum ferritin levels are consistently reported in chronic liver diseases. [ 51 ] [ 52 ] [ 53 ]


Studies showed association between high serum ferritin levels and increased risk of short-term mortality in cirrhotic patients with acute decompensation [ 54 ]  and acute-on-chronic liver failure. [ 55 ]  An other study found association between high serum ferritin levels and increased risk of long-term mortality in compensated and stable decompensated cirrhotic patients. [ 56 ]  The same study demonstrated that increased serum ferritin levels could predict the development of bacterial infection in stable decompensated cirrhotic patients, while in compensated cirrhotic patients the appearance of the very first acute decompensation episode showed higher incidence in patients with low serum ferritin levels. This latter finding was explained by the association between chronic bleeding and increased portal pressure. [ 56 ]


Ferritin was discovered in 1937 by the  Czechoslovakian  scientist  Vilém Laufberger  [ cs ] . [ 57 ] [ 7 ] : e9    Sam Granick  and  Leonor Michaelis  produced apoferritin in 1942 [ 7 ] : e10


Ferritin is used in  materials science  as a precursor in making iron  nanoparticles  (NP) for  carbon nanotube  growth by  chemical vapor deposition .  It has also been shown to effectively store electrons for hours and  [ 58 ]  to facilitate electron tunneling under ambient conditions, [ 59 ]  properties that may be involved in biological processes. [ 60 ]


Cavities formed by ferritin and mini-ferritins ( Dps ) proteins have been successfully used as the reaction chamber for the fabrication of metal nanoparticles. [ 61 ] [ 62 ] [ 63 ] [ 64 ]   Protein  shells served as a template to restrain particle growth and as a coating to prevent coagulation/aggregation between NPs. Using various sizes of protein shells, various sizes of NPs can be easily synthesized for chemical, physical and bio-medical applications. [ 6 ] [ 65 ]


Experimental COVID-19 vaccines have been produced that display the  spike protein 's receptor binding domain on the surface of ferritin nanoparticles. [ 66 ]


The primary peptide sequence of human ferritin is: [ 67 ]


The  fibrinopeptides ,  fibrinopeptide A  ( FpA ) and  fibrinopeptide B  ( FpB ), are  peptides  which are located in the central region of the  fibrous   glycoprotein   fibrinogen  (factor I) and are  cleaved  by the  enzyme   thrombin  (factor IIa) to convert fibrinogen into  covalently-linked   fibrin  (factor IA)  monomers . [ 1 ] [ 2 ]  The  N-terminal  FpA is cleaved from the  Aα chains  of fibrinogen and FpB from the  Bβ chains  of fibrinogen, with FpA released before FpB. [ 3 ] [ 4 ]  Subsequent to their formation, fibrin monomers are converted to cross-linked fibrin  polymers  by the action of thrombin-activated  factor XIII  (fibrin stabilizing factor), and these fibrin polymers form the backbone of a  thrombus  (blood clot). [ 2 ]  Hence, the fibrinopeptides are sensitive markers of  fibrinogenesis  (fibrin generation),  thrombin  activity, and  coagulation . [ 5 ] [ 6 ] [ 7 ] [ 8 ]


FpA is a 16- amino acid  peptide. [ 8 ]  The  half-life  of FpA is very short at approximately 3 to 5 minutes. [ 5 ] [ 8 ]  Hence, FpA levels provide a relatively transient measure of coagulation activation. [ 8 ]


Levels of FpA increase with age. [ 5 ]  FpA levels also gradually increase throughout  pregnancy . [ 9 ] [ 10 ]  Likewise, FpA levels have been reported to increase with  estrogen  therapy, including with  combined birth control pills  and  menopausal hormone therapy , although research on FpA levels with these therapies appears to be relatively limited. [ 11 ] [ 12 ] [ 13 ] [ 7 ]


FibroTest , known as FibroSure in the US, is a  biomarker  test that uses the results of six  blood serum  tests to generate a score that is correlated with the degree of  liver  damage in people with a variety of liver diseases. FibroTest has the same prognostic value as a liver biopsy.  FibroSure uses quantitative results of five serum biochemical markers, α2-macroglobulin, haptoglobin, apolipoprotein A1, bilirubin, gamma glutamyl transpeptidase (GGT),  with a patient’s age and gender to generate a measure of fibrosis and necroinflammatory activity in the liver.


FibroTest has been evaluated in relation to  liver biopsy  (the current reference standard in liver disease assessment) in people with  hepatitis C ,  hepatitis B , [ 1 ]   alcoholic liver disease , [ 2 ]  and  non-alcoholic fatty liver disease . [ 3 ]  They are most useful for  cirrhosis  and less useful for other stages of liver disease. [ 4 ]


By 2008 it had been used in over 350,000 patients. [ 5 ]  In 2006, the French National Authority for Health recommended the use of FibroTest as one of a number of first-line assessment tool for fibrosis with untreated chronic hepatitis C. [ 6 ]


The FibroTest score is calculated from the results of a six-parameter blood test, combining six serum markers with the age and gender of the patient:  Alpha-2-macroglobulin ,  Haptoglobin ,  Apolipoprotein A1 ,  Gamma-glutamyl transpeptidase  (GGT), Total  bilirubin , and  Alanine transaminase  (ALT). ALT is used in a second assessment called ActiTest that is part of FibroTest. [ citation needed ]


The equation for calculating the FibroTest score regression coefficient ( logistic regression ) is: [ 7 ]


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 {\displaystyle z=4.467\times \log _{10}[Alpha2M(g/L)]-1.357\times \log _{10}[Haptoglobulin(g/L)]+1.017\times \log _{10}[GGT(IU/L)]+0.0281\times [Age]}


where B=1 for male and B=0 for female. The score (between 0 and 1) is then


F
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 {\displaystyle F=1/(1+e^{-z})}


Due to variability of components assays and analyzers, FibroTest assays can only be performed in validated laboratories. [ 8 ]  FibroTest cannot be used without algorithms that detects false positives and false negatives; the equation alone is not a diagnosis tool. [ citation needed ]


The laboratory or physician connects to the BioPredictive website [ 9 ]  for calculation of the test results and prints the results sheet, which is available immediately and is accompanied by an interpretation aid and precautions for use.


Over 95% of tests are interpretable and allow a diagnosis of fibrosis and liver activity. In less than 5% of cases, likely false positives or false negatives are highlighted. FibroTest has been validated for chronic  hepatitis C , [ 10 ]  chronic  hepatitis B , [ 5 ]  chronic hepatitis C or B with HIV co-infection, [ 11 ]  alcoholic liver diseases (steatosis and steatohepatitis), [ 2 ]  and non-alcoholic steatohepatitis (diabetes, overweight, hypertriglyceridemia, hypercholesterolemia, hypertension). [ 3 ]


FibroTest is independent of ethnic origin, sex, genotype, viral load, transaminases or the presence of comorbidities. The test has been validated in those over the age of 65 years, [ 12 ]  children, [ 13 ]  people with  chronic kidney disease  or  kidney transplantation ,  hemophiliacs , patients with chronic inflammatory disease, and the general population.


The tests are not applicable in 1 to 5% of cases. These cases can be detected by laboratory safety algorithms and when detected they are indicated on the results sheet: [ 5 ]


The conversion of FibroTest score into stages according to the three most used histological classifications (METAVIR, Knodell and Ishak) for  liver biopsies  is:


Liver biopsy  is an imperfect tool; due to sampling errors, biopsy size (5 to 30 mm) and intra- and interobservor variability, it is now agreed that biopsy is an "imperfect  Gold Standard
" (citation required).  Biopsy continues to present inconveniences: 30% of patients complain of pain, up to 3% have been noted to have complications severe enough to require hospitalization [ 14 ] [ 15 ]  and a 0.01-0.3% rate of deaths has been reported. [ 16 ] [ 17 ] [ 18 ]  There is a mean discordance of 25% between FibroTest and biopsy. Half of these discordances are attributable to an error of the biopsy, often too small, and the other half to FibroTest. [ 19 ]  The inventors report that FibroTest has comparable diagnostic and prognostic value as a 25 mm biopsy, while being noninvasive and easily repeatable. [ 10 ] [ 2 ] [ 20 ]


In  medicine , some  blood tests  are conducted on capillary blood obtained by  fingerstick  (or  fingerprick ) (or, for  neonates , by an analogous  heelprick ). The site, free of surface  arterial flow , where the blood is to be collected is sterilized with a  topical   germicide , and the skin pierced with a sterile  lancet . [ 1 ]  After a droplet has formed,  capillary blood  is captured in a  capillary tube  (usually relying on  surface tension ). Blood cells drawn from fingersticks have a tendency to undergo  hemolysis , especially if the finger is "milked" to obtain more blood. [ 2 ]


Tests commonly conducted on the  capillary blood  collected are:


Fingersticks are routine for hardy [ citation needed ]  adults, but are generally performed on children and the elderly only if a small amount of blood suffices for needed tests.  Neonates  are given heelpricks instead, as this is less likely to cause permanent damage. [ citation needed ]


Flow cytometry  ( FC ) is a technique used to detect and measure the physical and chemical characteristics of a population of  cells  or particles. [ 1 ] [ 2 ] [ 3 ] [ 4 ]


In this process, a sample containing cells or particles is suspended in a fluid and injected into the flow  cytometer  instrument. The sample is focused to ideally flow one cell at a time through a laser beam, where the light scattered is characteristic to the cells and their components. Cells are often labeled with fluorescent markers so light is absorbed and then emitted in a band of wavelengths. Tens of thousands of cells can be quickly examined and the data gathered are processed by a computer. [ 5 ]


Flow cytometry is routinely used in basic research, clinical practice, and  clinical trials . Uses for flow cytometry include:


A flow cytometry analyzer is an instrument that provides quantifiable data from a sample. Other instruments using flow cytometry include cell sorters which physically separate and thereby purify cells of interest based on their optical properties.


The first  impedance -based flow cytometry device, using the  Coulter principle , was disclosed in U.S. Patent 2,656,508, issued in 1953, to  Wallace H. Coulter . Mack Fulwyler was the inventor of the forerunner to today's flow cytometers – particularly the cell sorter. [ 6 ]  Fulwyler developed this in 1965 with his publication in  Science . [ 7 ]  The first fluorescence-based flow cytometry device (ICP 11) was developed in 1968 by Wolfgang Göhde from the  University of Münster , filed for patent on 18 December 1968 [ 8 ]  and first commercialized in 1968/69 by German developer and manufacturer Partec through Phywe AG in  Göttingen . At that time,  absorption  methods were still widely favored by other scientists over  fluorescence  methods. [ 9 ]  Soon after, flow cytometry instruments were developed, including the Cytofluorograph (1971) from Bio/Physics Systems Inc. (later: Ortho Diagnostics), the PAS 8000 (1973) from Partec, the first FACS (fluorescence-activated cell sorting) instrument from  Becton Dickinson  (1974), the ICP 22 (1975) from Partec/Phywe and the Epics from  Coulter  (1977/78). The first label-free high-frequency  impedance flow cytometer  based on a patented microfluidic "lab-on-chip", Ampha Z30, was introduced by Amphasys (2012). [ citation needed ]


The original name of the fluorescence-based flow cytometry technology was "pulse cytophotometry" ( German :  Impulszytophotometrie ), based on the first patent application on fluorescence-based flow cytometry. At the 5th American Engineering Foundation Conference on Automated Cytology in Pensacola (Florida) in 1976 – eight years after the introduction of the first fluorescence-based flow cytometer (1968) – it was agreed to commonly use the name "flow cytometry", a term that quickly became popular. [ 10 ]


Modern flow cytometers are able to analyze many thousands of particles per second, in "real time" and, if configured as cell sorters, can actively separate and isolate particles with specified optical properties at similar rates. A flow cytometer is similar to a  microscope , except that, instead of producing an image of the cell, flow cytometry offers high-throughput, automated  quantification  of specified optical parameters on a cell-by-cell basis. To analyze solid  tissues , a single-cell suspension must first be prepared. [ citation needed ]


A flow cytometer has five main components: a flow cell, a measuring system, a detector, an amplification system, and a computer for analysis of the signals. The flow cell has a liquid stream (sheath fluid), which carries and aligns the cells so that they pass single file through the light beam for sensing. The measuring system commonly uses measurement of impedance (or conductivity) and optical systems – lamps ( mercury ,  xenon ); high-power water-cooled lasers ( argon ,  krypton , dye laser); low-power air-cooled lasers (argon (488 nm), red-HeNe (633 nm), green-HeNe, HeCd (UV));  diode lasers  (blue, green, red, violet) resulting in light signals. The detector and analog-to-digital conversion (ADC) system converts analog measurements of forward-scattered light (FSC) and side-scattered light (SSC) as well as dye-specific fluorescence signals into digital signals that can be processed by a computer. The amplification system can be  linear  or  logarithmic . [ citation needed ]


The process of collecting data from samples using the flow cytometer is termed "acquisition". Acquisition is mediated by a computer physically connected to the flow cytometer, and the software which handles the digital interface with the cytometer. The software is capable of adjusting parameters (e.g., voltage, compensation) for the sample being tested, and also assists in displaying initial sample information while acquiring sample data to ensure that parameters are set correctly. Early flow cytometers were, in general, experimental devices, but technological advances have enabled widespread applications for use in a variety of both clinical and research purposes. Due to these developments, a considerable market for instrumentation, analysis software, as well as the reagents used in acquisition such as  fluorescently labeled   antibodies  have been developed.


Modern instruments usually have multiple lasers and fluorescence detectors. The current record for a commercial instrument is ten lasers [ 11 ]  and 30 fluorescence detectors. [ 12 ]  Increasing the number of lasers and detectors allows for multiple antibody labeling, and can more precisely identify a target population by their  phenotypic  markers. Certain instruments can even take digital images of individual cells, allowing for the analysis of fluorescent signal location within or on the surface of cells. [ citation needed ]


Cells must pass uniformly through the center of focused laser beams to accurately measure optical properties of cells in any flow cytometer. [ 13 ] [ 14 ] [ 15 ]  The purpose of the fluidic system is to move the cells one by one through the lasers beam and throughout the instrument. Fluidics in a flow cytometer with cell sorting capabilities also use the stream to carry sorted cells into collection tubes or wells. [ 13 ]


For precise positioning of cells in a liquid jet, hydrodynamic focusing is used in most cytometers. [ 13 ] [ 14 ]  The cells in suspension enter into the instrument enclosed by an outer sheath fluid. The sample core is maintained in the center of the sheath fluid. The sample input rate or how fast the cells flow through to the laser interrogation can be controlled by the pressure of the sheath fluid on the sample core. Under optimal conditions, the central fluid stream and sheath fluid do not mix. [ citation needed ]


Acoustic focusing technology is used in some flow cytometers to support hydrodynamic focusing. [ 13 ] [ 15 ]  Acoustic waves (>2 MHz) pre-focus the sample before introduction to sheath fluid. The pre-focused sample is then injected into the hydrodynamic core and flowed through the instrument. This may help with increasing data accuracy under high sample input rates.


Light emitted from  fluorophores  are in a spectrum of wavelengths, so combining multiple fluorophores may cause overlap. To add specificity,  optical filters  and  dichroic mirrors  are used to filter and move light to the detectors such as  photomultiplier tubes  (PMTs) or  avalanche photodiodes  (APD). [ 13 ]  Optical filters are designed as band pass (BP), long pass (LP), or short pass (SP) filters. Most flow cytometers uses dichroic mirrors and band pass filters to select specific bands of the optical spectrum.


Spectral flow cytometry uses  prisms  or  diffraction gratings  to disperse the emitted light of a marker across a detector array. [ 13 ] [ 16 ]  This allows for the full spectra from each particle to be measured. The measured spectra from single cells are subsequently unmixed by using reference spectra of all used dyes and the autofluorescence spectrum. This may allow for a wider panel design and the application of new biological markers. [ citation needed ]


Imaging flow cytometry (IFC) captures multichannel images of cells. [ 13 ] [ 17 ]  Detectors used in imaging platforms can be equipped with  charge-coupled device  (CCD) or complementary metal–oxide–semiconductor (CMOS) to capture images of individual cells.


Each fluorochrome has a broad fluorescence spectrum. When more than one fluorochrome is used, an overlap between fluorochromes can occur. This situation is called spectrum overlap, and must be corrected. For example, the emission spectrum for FITC and PE is one in which the light emitted by the fluorescein overlaps the same wavelength as it passes through the filter used for PE. This spectral overlap is corrected by removing a portion of the FITC signal from the PE signals or vice versa. This process is called color compensation, which calculates a fluorochrome as a percentage to measure itself. [ 18 ]


Compensation is the mathematical process by which spectral overlap of multiparameter flow cytometric data is corrected. Since fluorochromes can have wide-ranging spectrum, they can overlap, causing the undesirable result of confusion during the analysis of data. This overlap, known as spillover and quantified in the spillover coefficient, is usually caused by detectors for a certain fluorochrome measuring a significant peak in wavelength from a different fluorochrome. Linear algebra is most often used to make this correction. [ 18 ]


In general, when graphs of one or more parameters are displayed, it is to show that the other parameters do not contribute to the distribution shown. Especially when using the parameters which are more than double, this problem is more severe. Currently, no tools have been discovered to efficiently display multidimensional parameters. Compensation is very important to see the distinction between cells.


[ citation needed ]


The data generated by flow cytometers can be plotted in a single  dimension  to produce a  histogram , or in two-dimensional dot plots, or even in three dimensions. The regions on these plots can be sequentially separated, based on fluorescence  intensity , by creating a series of subset extractions, termed "gates." Specific gating  protocols  exist for diagnostic and clinical purposes, especially in relation to  hematology . Individual single cells are often distinguished from cell doublets or higher aggregates by their "time-of-flight" (denoted also as a "pulse-width") through the narrowly focused laser beam [ 19 ]


The plots are often made on logarithmic scales. Because different fluorescent dyes' emission spectra overlap, [ 20 ] [ 21 ]  signals at the detectors have to be compensated electronically as well as computationally. Data accumulated using the flow cytometer can be analyzed using software. Once the data is collected, there is no need to stay connected to the flow cytometer and analysis is most often performed on a separate computer. [ citation needed ]  This is especially necessary in core facilities where usage of these machines is in high demand. [ citation needed ]


Recent progress on automated population identification using computational methods has offered an alternative to traditional gating strategies. Automated identification systems could potentially help findings of rare and hidden populations. Representative automated methods include FLOCK [ 22 ]  in Immunology Database and Analysis Portal (ImmPort), [ 23 ]  SamSPECTRAL [ 24 ]  and flowClust [ 25 ] [ 26 ] [ 27 ]  in  Bioconductor , and FLAME [ 28 ]  in  GenePattern . T-Distributed Stochastic Neighbor Embedding (tSNE) is an algorithm designed to perform  dimensionality reduction , to allow visualization of complex multi-dimensional data in a two-dimensional "map". [ 29 ]  Collaborative efforts have resulted in an open project called FlowCAP (Flow Cytometry: Critical Assessment of Population Identification Methods, [ 30 ] ) to provide an objective way to compare and evaluate the flow cytometry data clustering methods, and also to establish guidance about appropriate use and application of these methods.


Fluorescence minus one (FMO) controls are important for data interpretation when building multi-color panels – in which a cell is stained with multiple fluorochromes simultaneously. FMO controls provide a measure of fluorescence spillover in a given channel and allow for compensation. To generate a FMO control, a sample is stained with all the fluorochromes except the one that is being tested – meaning if you are using 4 different fluorochromes your FMO control must contain only 3 of them (example: fluorochromes – A, B, C, D; FMOs – ABC_, AB_D, A_CD, _BCD). [ citation needed ]


Cell sorting  is a method to purify cell populations based on the presence or absence of specific physical characteristics. [ 13 ] [ 15 ] [ 31 ]  In flow cytometers with sorting capabilities, the instrument detects cells using parameters including cell size, morphology, and protein expression, and then droplet technology to sort cells and recover the subsets for post-experimental use. [ 13 ] [ 15 ]


The first prototype sorter was built at the  Los Alamos National Laboratory  (LANL) in 1965 by physicist Mack J. Fulwyler by joining a Coulter volume sensor with the newly invented ink jet printer. [ 32 ]  Live cell cell sorter or fluorescence-activated cell sorter (FACS) [ a ]  was generated by  Len Herzenberg , who subsequently won the  Kyoto Prize  in 2006 for his seminal work. [ 34 ]


Flow cytometry cell sorters have a collection system unlike flow cytometry analyzers. The collection process starts when a sample is injected into a stream of sheath fluid that passes through the flow cell and laser intercepts. [ 35 ]  The stream then carries the cell through a vibrating nozzle which generates droplets with most containing either one cell or no cells. An electrical charging ring is placed just at the point where the stream breaks into droplets and a  charge  is placed on the ring based immediately prior to fluorescence intensity being measured; the opposite charge is trapped on the droplet as it breaks from the stream and the droplets are therefore charged. The charged droplets then fall through an  electrostatic deflection  system that diverts droplets into containers based on their charge. In some systems, the charge is applied directly to the stream, and the droplet breaking off retains charge of the same sign as the stream. The stream is then returned to neutral after the droplet breaks off. After collecting, these cells can be further cultured, manipulated, and studied. [ citation needed ]


Flow cytometry uses the light properties scattered from cells or particles for identification or quantitative measurement of physical properties. Labels, dyes, and stains can be used for multi-parametric analysis (understand more properties about a cell).  Immunophenotyping  is the analysis of heterogeneous populations of cells using labeled  antibodies [ 36 ]  and other fluorophore containing reagents such as dyes and stains.


A wide range of fluorophores can be used as labels in flow cytometry. [ 20 ]  Fluorophores, or simply "fluors", [ citation needed ]  are typically attached to an antibody that recognizes a target feature on or in the cell; they may also be attached to a chemical entity with affinity for the  cell membrane  or another cellular structure. Each fluorophore has a characteristic peak  excitation  and  emission  wavelength, and the emission spectra often overlap. Consequently, the combination of labels which can be used depends on the wavelength of the lamp(s) or laser(s) used to excite the fluorochromes and on the detectors available. [ 37 ]  Flow cytometry uses fluorescence as a quantitative tool; the utmost sensitivity of flow cytometry is unmatched by other fluorescent detection platforms such as  confocal microscopy . Absolute fluorescence sensitivity is generally lower in  confocal microscopy  because out-of-focus signals are rejected by the confocal optical system and because the image is built up serially from individual measurements at every location across the cell, reducing the amount of time available to collect signal. [ 38 ]