A disadvantage of Ivy's method is closure of puncture wound before stoppage of bleeding. [ 7 ]


With the Duke's method, the patient is pricked with a special needle or lancet, preferably on the  earlobe [ 8 ]  or  fingertip , after having been swabbed with alcohol. The prick is about 3–4 mm deep. The patient then wipes the blood every 30 seconds with a filter paper. The test ceases when bleeding ceases. The usual time is about 2–5 minutes.


This method is not recommended and cannot be standardized because it can cause a large local hematoma. [ 7 ]


Bleeding time may be affected by platelet function, certain vascular disorders and  von Willebrand Disease —not by other  coagulation  factors such as  haemophilia .  Diseases that may cause prolonged bleeding time include  thrombocytopenia ,  disseminated intravascular coagulation  (DIC),  Bernard-Soulier disease , and  Glanzmann's thrombasthenia . [ 9 ]


Aspirin  and other  cyclooxygenase  inhibitors can significantly prolong bleeding time. While  warfarin  and  heparin  have their major effects on coagulation factors, an increased bleeding time is sometimes seen with use of these medications as well.


People with  von Willebrand disease  usually experience increased bleeding time, as  von Willebrand factor  is a platelet adhesion protein, but this is not considered an effective diagnostic test for this condition. [ 9 ]


It is also prolonged in  hypofibrinogenemia . [ 10 ]


In the British comedy film  Doctor in the House  (1954), Sir Lancelot Spratt, the intimidating chief of surgery played by  James Robertson Justice  is asking instructional questions of his medical students. He asks a young student, who has been distracted by a pretty nurse, what "the bleeding time" is. The student looks at his watch and answers "Ten past ten, sir."


Blood compatibility testing  is conducted in a  medical laboratory  to identify potential incompatibilities between  blood group systems  in  blood transfusion . It is also used to diagnose and prevent some  complications of pregnancy  that can occur when the baby has a different blood group from the mother. Blood compatibility testing includes  blood typing , which detects the  antigens  on  red blood cells  that determine a person's blood type; testing for unexpected  antibodies  against blood group antigens ( antibody screening and identification ); and, in the case of blood transfusions, mixing the recipient's  plasma  with the donor's red blood cells to detect incompatibilities ( crossmatching ). Routine blood typing involves determining the  ABO  and  RhD  (Rh factor) type, [ note 1 ]  and involves both identification of ABO antigens on red blood cells (forward grouping) and identification of ABO antibodies in the plasma (reverse grouping).  Other blood group antigens  may be tested for in specific clinical situations.


Blood compatibility testing makes use of  reactions between blood group antigens and antibodies —specifically the ability of antibodies to cause red blood cells to clump together when they bind to antigens on the cell surface, a phenomenon called  agglutination . Techniques that rely on antigen-antibody reactions are termed  serologic  methods, and several such methods are available, ranging from manual testing using test tubes or slides to fully automated systems. Blood types can also be determined through  genetic testing , which is used when conditions that interfere with serologic testing are present or when a high degree of accuracy in antigen identification is required.


Several conditions can cause false or inconclusive results in blood compatibility testing. When these issues affect ABO typing, they are called  ABO discrepancies . ABO discrepancies must be investigated and resolved before the person's blood type is reported. Other sources of error include the " weak D " phenomenon, in which people who are positive for the RhD antigen show weak or negative reactions when tested for RhD, and the presence of  immunoglobulin G  antibodies on red blood cells, which can interfere with antibody screening, crossmatching, and typing for some blood group antigens.


Blood compatibility testing is routinely performed before a  blood transfusion . The full compatibility testing process involves  ABO  and  RhD  (Rh factor) typing; screening for antibodies against  other blood group systems ; and  crossmatching , which involves testing the recipient's  blood plasma  against the donor's red blood cells as a final check for incompatibility. If an unexpected blood group antibody is detected, further testing is warranted to identify the antibody [ 3 ] : 740   and ensure that the donor blood is negative for the relevant antigen. [ 1 ] : 261   Serologic crossmatching may be omitted if the recipient's antibody screen is negative, there is no history of clinically significant antibodies, and their ABO/Rh type has been confirmed against historical records or against a second blood sample; and in emergencies, blood may be transfused before any compatibility testing results are available. [ 1 ] : 262–3


Blood compatibility testing is often performed on pregnant women and on the  cord blood  from newborn babies, because incompatibility puts the baby at risk for developing hemolytic disease of the newborn. [ 4 ] [ 5 ]  It is also used before  hematopoietic stem cell transplantation , because blood group incompatibility can be responsible for some cases of acute  graft-versus-host disease . [ 6 ]


Blood types are defined according to the presence or absence of specific  antigens  on the surface of red blood cells. The most important of these in medicine are the  ABO  and  RhD antigens [ 7 ] : 585   but many other blood group systems exist and may be clinically relevant in some situations. As of 2021, 43 blood groups are officially recognized. [ 8 ]


People who lack certain blood group antigens on their red cells can form  antibodies  against these antigens. For example, a person with type A blood will produce antibodies against the B antigen. The ABO blood group antibodies are naturally occurring, meaning that they are found in people who have not been exposed to incompatible blood. [ 7 ] : 585–92   Antibodies to most other blood group antigens, including RhD, develop after people are exposed to the antigens through transfusion or pregnancy. [ 1 ] : 62   Some of these antibodies can bind to incompatible red blood cells and  cause them to be destroyed , resulting in  transfusion reactions  and other complications. [ 9 ] : 210–11


Serologic  methods for blood compatibility testing make use of these  antibody-antigen reactions . In blood typing,  reagents  containing blood group antibodies, called  antisera , [ 7 ] : 586   are added to suspensions of blood cells. If the relevant antigen is present, the antibodies in the reagent will cause the red blood cells to  agglutinate  (clump together), which can be identified visually. [ 1 ] : 65   In antibody screening, the individual's plasma is tested against a set of red blood cells with known antigen profiles; if the plasma agglutinates one of the red blood cells in the panel, this indicates that the individual has an antibody against one of the antigens present on the cells. In crossmatching, a prospective transfusion recipient's plasma is added to the donor red blood cells and observed for agglutination (or hemolysis) to detect antibodies that could cause transfusion reactions. [ 3 ] : 722–5


Blood group antibodies occur in two major forms:  immunoglobulin M  (IgM) and  immunoglobulin G  (IgG). Antibodies that are predominantly IgM, such as the ABO antibodies, typically cause immediate agglutination of red blood cells at room temperature. Therefore, a person's ABO blood type can be determined by simply adding the red blood cells to the reagent and centrifuging or mixing the sample, [ 1 ] : 122   and in crossmatching, incompatibility between ABO types can be detected immediately after centrifugation. [ 3 ] : 725   RhD typing also typically uses IgM reagents [ 10 ] : 477   although anti-RhD usually occurs as IgG in the body. [ 1 ] : 161    Antibodies that are predominantly IgG, such as those directed towards antigens of the  Duffy  and  Kidd  systems, [ 1 ] : 198–200   generally do not cause immediate agglutination because the small size of the IgG antibody prevents formation of a lattice structure. Therefore, blood typing using IgG antisera and detection of IgG antibodies requires use of the  indirect antiglobulin test  to demonstrate IgG bound to red blood cells. [ 1 ] : 104


In the indirect antiglobulin test, the mixture of antiserum or plasma and red blood cells is incubated at 37 °C (99 °F), the ideal temperature for reactivity of IgG antibodies. After incubation, the red blood cells are washed with saline to remove unbound antibodies, and anti-human globulin reagent is added. If IgG antibodies have bound to antigens on the cell surface, anti-human globulin will bind to those antibodies, causing the red blood cells to agglutinate after centrifugation. If the reaction is negative, "check cells"—reagent cells coated with IgG—are added to ensure that the test is working correctly. If the test result is indeed negative, the check cells should react with the unbound anti-human globulin and demonstrate agglutination. [ 3 ] : 716–9


In ABO and Rh typing, reagents  containing antibodies against the A, B, and RhD antigens are added to suspensions of blood cells. If the relevant antigen is present, the red blood cells will demonstrate visible  agglutination  (clumping). [ 1 ] : 65   In addition to identifying the ABO antigens, which is termed forward grouping, routine ABO blood typing also includes identification of the ABO antibodies in the person's plasma. This is called reverse grouping, [ 1 ] : 120   and it is done to confirm the ABO blood type. In reverse grouping, the person's plasma is added to type A 1  and type B red blood cells. The plasma should agglutinate the cells that express antigens that the person lacks, while failing to agglutinate cells that express the same antigens as the patient. For example, the plasma of someone with type A blood should react with type B red cells, but not with A 1  cells. If the expected results do not occur, further testing is required. [ 7 ] : 595   Agglutination is scored from 1+ to 4+ based on the strength of the reaction. In ABO typing, a score of 3+ or 4+ indicates a positive reaction, while a score of 1+ or 2+ is inconclusive and requires further investigation. [ 1 ] : 236


Prior to receiving a blood transfusion, individuals are screened for the presence of antibodies against antigens of non-ABO  blood group systems . [ 5 ]  Blood group antigens besides ABO and RhD that are significant in  transfusion medicine  include the  RhC/c and E/e  antigens and the antigens of the  Duffy ,  Kell ,  Kidd , and  MNS  systems. [ 1 ] : 158–173   If a clinically significant antibody is identified, the recipient must be transfused with blood that is negative for the corresponding antigen to prevent a transfusion reaction. This requires the donor units to be typed for the relevant antigen. [ 1 ] : 261   The recipient may also be typed for the antigen to confirm the identity of the antibody, as only individuals who are negative for a blood group antigen should produce antibodies against it. [ 7 ] : 603


In Europe, females who require blood transfusions are often typed for the Kell and extended Rh antigens to prevent sensitization to these antigens, which could put them at risk for developing  hemolytic disease of the newborn  during pregnancy. [ 11 ]  The  American Society of Hematology  recommends that people  with  sickle cell disease  have their blood typed for the RhC/c, RhE/e, Kell, Duffy, Kidd, and MNS antigens prior to transfusion, [ 12 ] : 130–1   because they often require transfusions and may become sensitized to these antigens if transfused with mismatched blood. [ 13 ]  Extended red blood cell phenotyping is also recommended for people with  beta-thalassemia . [ 14 ]  Blood group systems other than ABO and Rh have a relatively small risk of complications when blood is mixed, so in emergencies such as major  hemorrhage , the urgency of transfusion can exceed the need for compatibility testing against other blood group systems (and potentially Rh as well). [ 15 ]


Antibodies to most blood group antigens besides those of the ABO system develop after exposure to incompatible blood. [ 1 ] : 62   Such "unexpected" blood group antibodies are only found in 0.8–2% of people; however, recipients of blood transfusions must be screened for these antibodies to prevent transfusion reactions. Antibody screening is also performed as part of  prenatal care , because antibodies against RhD and other blood group antigens can cause hemolytic disease of the newborn, and because Rh-negative mothers who have developed an anti-RhD antibody are not eligible to receive  Rho(D) immune globulin  (Rhogam). [ 1 ] : 233


In the antibody screening procedure, an individual's plasma is added to a panel of two or three sets of red blood cells which have been chosen to express most clinically significant blood group antigens. Only group O cells are used in antibody screening, as otherwise the cells would react with the naturally occurring ABO blood group antibodies. The mixture of plasma and red cells is incubated at 37°C and tested via the indirect antiglobulin test. Some antibody screening and identification protocols incorporate a phase of testing after incubation at room temperature, but this is often omitted because most unexpected antibodies that react at room temperature are clinically insignificant. [ 3 ] : 722–4


Agglutination of the screening cells by the plasma, with or without the addition of anti-human globulin, indicates that an unexpected blood group antibody is present. If this occurs, further testing using more cells (usually 10–11) is necessary to identify the antibody. By examining the antigen profiles of the red blood cells the person's plasma reacts with, it is possible to determine the antibody's identity.  An "autocontrol", in which the individual's plasma is tested against their own red cells, is included to determine whether the agglutination is due to an  alloantibody  (an antibody against a foreign antigen), an  autoantibody  (an antibody against one's own antigens), or another interfering substance. [ 3 ] : 722–4


The image above shows the interpretation of an antibody panel used in  serology  to detect antibodies towards the most relevant blood group antigens. Each row represents "reference" or "control" red blood cells of donors which have known antigen compositions and are  ABO group O . The + symbol means that the antigen is present on the reference red blood cells, and 0 means it is absent; nt means "not tested". The "result" column to the right displays reactivity when mixing reference red blood cells with plasma from the patient in 3 different phases: room temperature, 37°C and AHG (with anti-human globulin, by the  indirect antiglobulin test ). [ 16 ]


In this case, the antibody panel shows that anti-Fy a  antibodies are present. This indicates that donor blood typed to be negative for the Fy a  antigen must be used. Still, if a subsequent  cross-matching  shows reactivity, additional testing should be done against previously discounted antigens (in this case potentially E, K, Kp a  and/or Lu a ). [ 16 ]


When multiple antibodies are present, or when an antibody is directed against a high-frequency antigen, the normal antibody panel procedure may not provide a conclusive identification. In these cases,  hemagglutination inhibition  can be used, wherein a  neutralizing substance  cancels out a specific antigen. [ 17 ]  Alternatively, the plasma may be incubated with cells of known antigen profiles in order to remove a specific antibody (a process termed  adsorption ); or the cells can be treated with  enzymes  such as  ficain  or  papain  which inhibit the reactivity of some blood group antibodies and enhance others. [ 3 ] : 723–9   The effect of ficain and papain on major blood group systems is as follows: [ 18 ] [ 19 ]


People who have tested positive for an unexpected blood group antibody in the past may not exhibit a positive reaction on subsequent testing; however, if the antibody is clinically significant, they must be transfused with antigen-negative blood regardless. [ 20 ]


Crossmatching, which is routinely performed before a blood transfusion, involves adding the recipient's blood plasma to a sample of the donor's red blood cells. If the blood is incompatible, the antibodies in the recipient's plasma will bind to antigens on the donor red blood cells. This antibody-antigen reaction can be detected through visible clumping or destruction of the red blood cells, or by reaction with  anti-human globulin , after centrifugation. [ 7 ] : 600–3


If the transfusion recipient has a negative antibody screen and no history of antibodies, an "immediate spin" crossmatch is often performed: the red blood cells and plasma are centrifuged immediately after mixing as a final check for incompatibility between ABO blood types. If a clinically significant antibody is detected (or was in the past), or if the immediate spin crossmatch demonstrates incompatibility, a "full" or "IgG crossmatch" is performed, which uses the indirect antiglobulin test to detect blood group incompatibility caused by IgG antibodies. The IgG crossmatching procedure is more lengthy than the immediate spin crossmatch, and in some cases may take more than two hours. [ 20 ]


Individuals who have a negative antibody screen and no history of antibodies may also undergo an "electronic crossmatch", provided that their ABO and Rh type has been determined from the current blood sample and that the results of another ABO/Rh type are on record. In this case, the recipient's blood type is simply compared against that of the donor blood, without any need for serologic testing. [ 7 ] : 600–3   In emergencies, blood may be issued before crossmatching is complete. [ 1 ] : 262–3


Blood typing can be performed using test tubes,  microplates , or blood typing slides. The tube method involves mixing a  suspension  of red blood cells with antisera (or plasma, for reverse grouping) in a test tube. The mixture is centrifuged to separate the cells from the reagent, and then resuspended by gently agitating the tube. If the antigen of interest is present, the red blood cells agglutinate, forming a solid clump in the tube. If it is absent, the red blood cells go back into suspension when mixed. [ 7 ] : 611–12  [ 9 ] : 214    The microplate method is similar to the tube method, except rather than using individual test tubes, blood typing is carried out in a plate containing dozens of wells, allowing multiple tests to be performed at the same time. The agglutination reactions are read after the plate is centrifuged. [ 21 ] : 201


Antibody screening and identification can also be carried out by the tube method. In this procedure, the plasma and red cells are mixed together in a tube containing a medium that enhances agglutination reactions, such as  low ionic strength saline  (LISS). The tubes are incubated at body temperature for a defined period of time, then centrifuged and examined for agglutination or hemolysis; first immediately following the incubation period, and then after washing and addition of anti-human globulin reagent. [ 3 ] : 722   Crossmatching, likewise, may be performed by the tube method; the reactions are read immediately after centrifugation in the immediate spin crossmatch, or after incubation and addition of AHG in the full crossmatching procedure. [ 3 ] : 725


The slide method for blood typing involves mixing a drop of blood with a drop of antisera on a slide. The slide is tilted to mix the cells and reagents together and then observed for agglutination, which indicates a positive result. This method is typically used in under-resourced areas or emergency situations; otherwise, alternative methods are preferred. [ 9 ] : 214  [ 10 ] : 476


Column agglutination techniques for blood compatibility testing (sometimes called the "gel test") use cards containing columns of  dextran - polyacrylamide gel . Cards designed for blood typing contain pre-dispensed blood typing reagents for forward grouping, and wells containing only a  buffer solution , to which reagent red blood cells and plasma are added, for reverse grouping. Antibody screening and crossmatching can also be carried out by column agglutination, in which case cards containing anti-human globulin reagent are used. The gel cards are centrifuged (sometimes after incubation, depending on the test), during which red blood cell agglutinates become trapped at the top of the column because they are too large to migrate through the gel. Cells that have not agglutinated collect on the bottom. Therefore, a line of red blood cells at the top of the column indicates a positive result. The strength of positive reactions is scored from 1+ to 4+ depending on how far the cells have travelled through the gel. The gel test has advantages over manual methods in that it eliminates the variability associated with manually re-suspending the cells and that the cards can be kept as a record of the test. [ 1 ] : 269–71   The column agglutination method is used by some  automated analyzers  to perform blood typing automatically. [ 7 ] : 590   These analyzers  pipette  red blood cells and plasma onto gel cards, centrifuge them, and scan and read the agglutination reactions to determine the blood type. [ 1 ] : 270–1


Solid-phase assays (sometimes called the "antigen capture" method) use reagent antigens or antibodies affixed to a surface (usually a microplate). [ 9 ] : 214   Microplate wells coated with anti-A, -B and -D reagents are used for forward grouping. The test sample is added and the microplate is centrifuged; in a positive reaction, the red blood cells adhere to the surface of the well. [ 7 ] : 590  [ 10 ] : 477   Some automated analyzers use solid phase assays for blood typing. [ 1 ] : 275–6


Genetic testing can be used to determine a person's blood type in certain situations where serologic testing is insufficient. For example, if a person has been transfused with large volumes of donor blood, the results of serologic testing will reflect the antigens on the donor cells and not the person's actual blood type. [ 11 ]  Individuals who produce  antibodies against their own red blood cells [ 21 ] : 202   or who are treated with certain drugs may show spurious agglutination reactions in serologic testing, so genotyping may be necessary to determine their blood type accurately. [ 1 ] : 261   Genetic testing is required for typing red blood cell antigens for which no commercial antisera are available. [ 11 ]


The  AABB  recommends RhD antigen genotyping for women with serologic  weak D  phenotypes who have the potential to bear children. This is because some people with weak D phenotypes can produce antibodies against the RhD antigen, which can cause hemolytic disease of the newborn, while others cannot. Genotyping can identify the specific type of weak D antigen, which determines the potential for the person to produce antibodies, thus avoiding unnecessary treatment with  Rho(D) immune globulin . [ 22 ] [ 23 ]  Genotyping is preferred to serologic testing for people with sickle cell disease, because it is more accurate for certain antigens and can identify antigens that cannot be detected by serologic methods. [ 13 ]


Genotyping is also used in prenatal testing for hemolytic disease of the newborn. When a pregnant woman has a blood group antibody that can cause HDN, the fetus can be typed for the relevant antigen to determine if it is at risk of developing the disease. Because it is impractical to draw blood from the fetus, the blood type is determined using  an  amniocentesis  sample or  cell-free fetal DNA  isolated from the mother's blood. [ 21 ] : 202  [ 22 ]  The father may also be genotyped to predict the risk of hemolytic disease of the newborn, because if the father is  homozygous  for the relevant antigen (meaning having two copies of the gene) the baby will be positive for the antigen and thus at risk of developing the disease. If the father is  heterozygous  (having only one copy), the baby only has a 50% chance of being positive for the antigen. [ 11 ]


In ABO typing, the results of the forward and reverse grouping should always correspond with each other. An unexpected difference between the two results is termed an ABO discrepancy, and must be resolved before the person's blood type is reported. [ 1 ] : 136


Weak reactions in the forward grouping may occur in people who belong to certain ABO subgroups—variant blood types characterized by decreased expression of the A or B antigens or changes in their structure. Weakened expression of ABO antigens may also occur in  leukemia  and  Hodgkin's lymphoma . Weak reactions in forward grouping can be strengthened by incubating the blood and reagent mixture at room temperature or 4 °C (39 °F), or by using certain  enzymes  to enhance the antigen-antibody reactions. [ 1 ] : 139


Occasionally, two populations of red blood cells are apparent after reaction with the blood typing antisera. Some of the red blood cells are agglutinated, while others are not, making it difficult to interpret the result. This is called a  mixed field reaction , and it can occur if someone has recently received a blood transfusion with a different blood type (as in a type A patient receiving type O blood), if they have received a  bone marrow  or  stem cell transplant  from someone with a different blood type, or in patients with certain ABO subgroups, such as A 3 . Investigation of the person's medical history can clarify the cause of the mixed field reaction. [ 1 ] : 138  [ 24 ] : 314


People with  cold agglutinin disease  produce antibodies against their own red blood cells that cause them to spontaneously agglutinate at room temperature, leading to false positive reactions in forward grouping. Cold agglutinins can usually be deactivated by warming the sample to 37 °C (99 °F) and washing the red blood cells with saline. If this is not effective,  dithiothreitol  can be used to destroy the antibodies. [ 1 ] : 141


Cord blood samples may be contaminated with  Wharton's jelly , a viscous substance that can cause red blood cells to stick together, mimicking agglutination. Wharton's jelly can be removed by thoroughly washing the red blood cells. [ 1 ] : 141


In a rare phenomenon known as "acquired B antigen", a patient whose true blood type is A may show a weak positive result for B in the forward grouping. This condition, which is associated with gastrointestinal diseases such as  colon cancer [ 1 ] : 139   and  intestinal obstruction , [ 24 ] : 126   results from conversion of the A antigen to a structure mimicking the B antigen by bacterial enzymes. [ 10 ] : 477   Unlike the true B antigen, acquired B antigen does not react with reagents within a certain pH range. [ 1 ] : 139


Infants under 3 to 6 months of age exhibit missing or weak reactions in reverse grouping because they produce very low levels of ABO antibodies. [ 1 ] : 136   Therefore, reverse grouping is generally not performed for this age group. [ 10 ] : 486   Elderly people may also exhibit decreased antibody production, as may people with  hypogammaglobulinemia . Weak reactions can be strengthened by allowing the plasma and red cells to incubate at room temperature for 15 to 30 minutes, and if this is not effective, they can be incubated at 4 °C (39 °F). [ 1 ] : 137–8


Approximately 20% of individuals with the blood type A or AB belong to a subgroup of A, termed A 2 , while the more common subgroup, encompassing approximately 80% of individuals, is termed A 1 . Because of small differences in the structure of the A 1  and A 2  antigens, some individuals in the A 2  subgroup can produce an antibody against A 1 . Therefore, these individuals will type as A or AB in the forward grouping, but will exhibit an unexpected positive reaction with the type A 1  red cells in the reverse grouping. The discrepancy can be resolved by testing the person's red blood cells with an anti-A 1  reagent, which will give a negative result if the patient belongs to the A 2  subgroup. Anti-A 1  antibodies are considered clinically insignificant unless they react at 37 °C (99 °F). Other subgroups of A exist, as well as subgroups of B, but they are rarely encountered. [ 1 ] : 127–32


If high levels of  protein  are present in a person's plasma, a phenomenon known as  rouleaux  may occur when their plasma is added to the reagent cells. Rouleaux causes red blood cells to stack together, which can mimic agglutination, causing a false positive result in the reverse grouping. This can be avoided by removing the plasma, replacing it with saline, and re-centrifuging the tube. Rouleaux will disappear once the plasma is replaced with saline, but true agglutination will persist. [ 1 ] : 140–1


Antibodies to blood group antigens other than A and B may react with the reagent cells used in reverse grouping. If a  cold-reacting autoantibody  is present, the false positive result can be resolved by warming the sample to 37 °C (99 °F). If the result is caused by an alloantibody, an antibody screen can be performed to identify the antibody, [ 7 ] : 141–2   and the reverse grouping can be performed using samples that lack the relevant antigen. [ 10 ] : 477


Approximately 0.2 to 1% of people have a "weak D" phenotype, [ 25 ]  meaning that they are positive for the RhD antigen, but exhibit weak or negative reactions with some anti-RhD reagents due to decreased antigen expression or atypical variants of antigen structure. If routine serologic testing for RhD results in a score of 2+ or less, the antiglobulin test can be used to demonstrate the presence of RhD. [ 1 ] : 159   Weak D testing is also performed on blood donors who initially type as RhD negative. [ 22 ]  Historically, blood donors with weak D were treated as Rh positive and patients with weak D were treated as Rh negative in order to avoid potential exposure to incompatible blood. Genotyping is increasingly used to determine the molecular basis of weak D phenotypes, as this determines whether or not individuals with weak D can produce antibodies against RhD or sensitize others to the RhD antigen. [ 25 ]


The  indirect antiglobulin test , which is used for weak D testing and typing of some red blood cell antigens, detects IgG bound to red blood cells. If IgG is bound to red blood cells  in vivo , as may occur in  autoimmune hemolytic anemia , hemolytic disease of the newborn and transfusion reactions, [ 10 ] : 260   the indirect antiglobulin test will always give a positive result, regardless of the presence of the relevant antigen. [ 10 ] : 477–8   A  direct antiglobulin test  can be performed to demonstrate that the positive reaction is due to sensitization of red cells. [ 26 ] : 289


Some groups of people have specialized transfusion requirements. Fetuses,  very low-birth-weight infants , and  immunocompromised  people are at risk for developing severe infection with  cytomegalovirus  (CMV)―an  opportunistic pathogen  for which approximately 50% of blood donors test positive―and may be transfused with CMV-negative blood to prevent infection. [ 3 ] : 749  [ 27 ]  Those who are at risk of developing  graft-versus-host disease , such as  bone marrow transplant  recipients, receive blood that has been  irradiated  to inactivate the  T lymphocytes  that are responsible for this reaction. People who have had serious  allergic reactions  to blood transfusions in the past may be transfused with blood that has been "washed" to remove plasma. [ 1 ] : 342–5   The history of the patient is also examined to see if they have previously identified antibodies and any other serological anomalies.


A direct antiglobulin test ( Coombs test ) is also performed as part of the antibody investigation. [ 28 ]


Donor blood is generally screened for  transfusion-transmitted infections  such as  HIV . As of 2018, the  World Health Organization  reported that nearly 100% of blood donations in high- and upper-middle-income countries underwent infectious disease screening, but the figures for lower-middle-income and low-income countries were 82% and 80.3% respectively. [ 29 ]


In 1901,  Karl Landsteiner  published the results of an experiment in which he mixed the serum and red blood cells of five different human donors. He observed that a person's serum never agglutinated their own red blood cells, but it could agglutinate others', and based on the agglutination reactions the red cells could be sorted into three groups: group A, group B, and group C. Group C, which consisted of red blood cells that did not react with any person's plasma, would later be known as group O. [ 30 ] : 5    A fourth group, now known as AB, was described by Landsteiner's colleagues in 1902. [ 30 ] : 7   This experiment was the first example of blood typing. [ 1 ] : 120


In 1945,  Robin Coombs , A.E. Mourant and  R.R. Race  published a description of the  antiglobulin test  (also known as the Coombs test). Previous research on blood group antibodies had documented the presence of so-called "blocking" or "incomplete" antibodies: antibodies that occupied antigen sites, preventing other antibodies from binding, but did not cause red blood cells to agglutinate. Coombs and his colleagues devised a method to easily demonstrate the presence of these antibodies. They injected human  immunoglobulins  into rabbits, which caused them to produce an  anti-human globulin  antibody. The anti-human globulin could bind to antibodies already attached to red blood cells and cause them to agglutinate. The invention of the antiglobulin test led to the discovery of many more blood group antigens. By the early 1950s, companies had begun producing commercial antisera for special antigen testing. [ 30 ] : 62–72


A  blood culture  is a  medical laboratory  test used to detect  bacteria  or  fungi  in a person's  blood . Under normal conditions, the blood does not contain  microorganisms : their presence can indicate a  bloodstream infection  such as  bacteremia  or  fungemia , which in severe cases may result in  sepsis . By  culturing  the blood, microbes can be identified and  tested for resistance to antimicrobial drugs , which allows clinicians to provide an effective treatment.


To perform the test, blood is drawn into bottles containing a liquid formula that enhances microbial growth, called a  culture medium . Usually, two containers are collected during one draw, one of which is designed for  aerobic organisms  that require oxygen, and one of which is for  anaerobic organisms , that do not. These two containers are referred to as a  set  of blood cultures. Two sets of blood cultures are sometimes collected from two different blood draw sites. If an organism only appears in one of the two sets, it is more likely to represent contamination with  skin flora  than a true bloodstream infection. False negative results can occur if the sample is collected after the person has received  antimicrobial drugs  or if the bottles are not filled with the recommended amount of blood. Some organisms do not grow well in blood cultures and require special techniques for detection.


The containers are placed in an  incubator  for several days to allow the organisms to multiply. If microbial growth is detected, a  Gram stain  is conducted from the culture bottle to confirm that organisms are present and provide preliminary information about their identity. The blood is then  subcultured , meaning it is  streaked  onto an  agar plate  to  isolate  microbial colonies for full identification and antimicrobial susceptibility testing. Because it is essential that bloodstream infections are diagnosed and treated quickly, rapid testing methods have been developed using technologies like  polymerase chain reaction  and  MALDI-TOF MS .


Procedures for culturing the blood were published as early as the mid-19th century, but these techniques were labour-intensive and bore little resemblance to contemporary methods. Detection of microbial growth involved visual examination of the culture bottles until automated blood culture systems, which monitor gases produced by microbial metabolism, were introduced in the 1970s. In developed countries, manual blood culture methods have largely been made obsolete by automated systems.


Blood is normally  sterile . [ 1 ]  The presence of  bacteria  in the blood is termed  bacteremia , and the presence of  fungi  is called  fungemia . [ 2 ]  Minor damage to the skin [ 3 ]  or  mucous membranes , which can occur in situations like  toothbrushing  or  defecation , [ 4 ] [ 5 ]  can introduce bacteria into the bloodstream, but this bacteremia is normally transient and is rarely detected in cultures because the  immune system  and  reticuloendothelial system  quickly sequester and destroy the organisms. [ 3 ] [ 6 ]  Bacteria can enter the blood from infections such as  cellulitis ,  UTIs  and  pneumonia ; [ 7 ]  and infections within the  vascular system , such as  bacterial endocarditis  or infections associated with  intravenous lines , may result in a constant bacteremia. [ 4 ]  Fungemia occurs most commonly in people with  poorly functioning immune systems . [ 2 ]  If bacteria or fungi are not cleared from the bloodstream, they can spread to other organs and tissues, [ 3 ]  or evoke an  immune response  that leads to a systemic inflammatory condition called  sepsis , which can be life-threatening. [ 8 ] [ 9 ]


When sepsis is suspected, it is necessary to draw blood cultures to identify the causative agent and provide targeted  antimicrobial therapy . [ 10 ]  People who are hospitalized and have a fever, a  low body temperature , a  high white blood cell count  or a low count of  granulocytes  (a category of  white blood cells ) commonly have cultures drawn to detect a possible bloodstream infection. [ 11 ] [ 12 ]  Blood cultures are used to detect bloodstream infections in  febrile neutropenia , a common complication of  chemotherapy  in which  fever  occurs alongside a severely low count of  neutrophils  (white blood cells that defend against bacterial and fungal pathogens). [ 13 ] [ 14 ] [ 15 ]  Bacteremia is common in some types of infections, such as  meningitis ,  septic arthritis  and  epidural abscesses , so blood cultures are indicated in these conditions. In infections less strongly associated with bacteremia, blood culture may still be indicated if the individual is at high risk of acquiring an intravascular infection or if cultures cannot be promptly obtained from the main site of infection (for example, a  urine culture  in  pyelonephritis  or a  sputum culture  in severe  community-acquired pneumonia ). [ 16 ] [ 17 ]  Blood culture can identify an underlying microbial cause in cases of  endocarditis [ 18 ]  and  fever of unknown origin . [ 11 ] [ 19 ]


The pathogens most frequently identified in blood cultures include  Staphylococcus aureus ,  Escherichia coli  and other members of the family  Enterobacteriaceae ,  Enterococcus  species,  Pseudomonas aeruginosa  and  Candida albicans . [ 20 ] [ 21 ]   Coagulase-negative staphylococci  (CNS) are also commonly encountered, although it is often unclear whether these organisms, which constitute part of the normal skin flora, [ 22 ]  are true pathogens or merely contaminants. [ 21 ]  In blood cultures taken from newborn babies and children, CNS can indicate significant infections. [ 23 ]   The  epidemiology  of bloodstream infections varies with time and place; for instance,  Gram-positive  organisms  overtook  Gram-negative  organisms as the predominant cause of bacteremia in the United States during the 1980s and 1990s, [ 24 ]  and rates of fungemia have greatly increased in association with a growing population of people receiving immunosuppressive treatments such as chemotherapy. [ 25 ]  Gram-negative sepsis is more common in Central and South America, Eastern Europe, and Asia than in North America and Western Europe; and in Africa,  Salmonella enterica  is a leading cause of bacteremia. [ 26 ]


Blood cultures are typically drawn through  venipuncture . Collecting the sample from an intravenous line is not recommended, as this is associated with higher contamination rates, although cultures may be collected from both venipuncture and an intravenous line to diagnose catheter-associated infections. [ 11 ] [ 27 ]  Prior to the blood draw, the top of each collection bottle is disinfected using an alcohol swab to prevent contamination. [ 11 ]  The skin around the puncture site is then cleaned and left to dry; some protocols recommend disinfection with an alcohol-based antiseptic followed by either  chlorhexidine  or an  iodine -based preparation, [ note 1 ] [ 27 ] [ 28 ]  while others consider using only an alcohol-containing antiseptic to be sufficient. [ 17 ] [ 29 ]  If blood must be drawn for other tests at the same time as a blood culture, the culture bottles are  drawn first  to minimize the risk of contamination. [ 30 ]  Because antimicrobial therapy can cause false negative results by inhibiting the growth of microbes, it is recommended that blood cultures are drawn before antimicrobial drugs are given, although this may be impractical in people who are critically ill. [ 10 ]


A typical blood culture collection involves drawing blood into two bottles, which together form one "culture" or "set". One bottle is designed to enhance the growth of  aerobic organisms , and the other is designed to grow  anaerobic organisms . In children, infection with anaerobic bacteria is uncommon, so a single aerobic bottle may be collected to minimize the amount of blood required. [ 31 ]  It is recommended that at least two sets are collected from two separate venipuncture locations. This helps to distinguish infection from contamination, as contaminants are less likely to appear in more than one set than true  pathogens . Additionally, the collection of larger volumes of blood increases the likelihood that microorganisms will be detected if present. [ 32 ]


Blood culture bottles contain a  growth medium , which encourages microorganisms to multiply, and an  anticoagulant  that prevents blood from  clotting . [ 33 ]  Sodium polyanethol sulfonate (SPS) is the most commonly used anticoagulant [ 33 ]  because it does not interfere with the growth of most organisms. [ 28 ]  The exact composition of the growth medium varies, but aerobic bottles use a broth that is enriched with nutrients, such as  brain-heart infusion  or  trypticase soy broth , [ 34 ]  and anaerobic bottles typically contain a  reducing agent  such as  thioglycollate . The empty space in an anaerobic bottle is filled with a gas mixture that does not contain oxygen. [ 33 ] [ 35 ]


Many commercially manufactured bottles contain a  resin  that absorbs  antibiotics  to reduce their action on the microorganisms in the sample. [ 11 ]  Bottles intended for paediatric use are designed to accommodate lower blood volumes and have additives that enhance the growth of pathogens more commonly found in children. [ 36 ]  Other specialized bottles may be used to detect fungi and  mycobacteria . [ 35 ]  In  low and middle income countries , pre-formulated culture bottles can be prohibitively expensive, and it may be necessary to prepare the bottles manually. It can be difficult to access the proper supplies and facilities, [ 37 ]  and in some regions, it may not be possible to perform blood cultures at all. [ 38 ]


It is important that the bottles are neither underfilled nor overfilled: underfilling can lead to false negative results as fewer organisms are present in the sample, while overfilling can inhibit microbial growth because the ratio of growth medium to blood is comparatively lower. A 1:10 to 1:5 ratio of blood to culture medium is suggested to optimize microbial growth. [ 28 ] [ 39 ]  For routine blood cultures in adults, the  Clinical and Laboratory Standards Institute  (CLSI) recommends the collection of two sets of bottles from two different draws, with 20–30 mL of blood drawn in each set. [ 11 ]  In children, the amount of blood to be drawn is often based on the child's age or weight. [ 36 ] [ 40 ]  If endocarditis is suspected, a total of six bottles may be collected. [ 41 ]


After the blood is collected, the bottles are  incubated  at body temperature to encourage the growth of microorganisms. Bottles are usually incubated for up to five days in automated systems, [ 43 ]  although most common bloodstream pathogens are detected within 48 hours. [ 44 ]  The incubation time may be extended further if manual blood culture methods are used or if slower-growing organisms, such as certain bacteria that cause endocarditis, are suspected. [ 43 ] [ 45 ]  In manual systems, the bottles are visually examined for indicators of microbial growth, which might include cloudiness, the production of gas,  the presence of visible microbial colonies, or a change in colour from the digestion of blood, which is called  hemolysis . Some manual blood culture systems indicate growth using a compartment that fills with fluid when gases are produced, or a miniature agar plate which is periodically inoculated by tipping the bottle. [ 46 ]  To ensure that positive blood cultures are not missed, a sample from the bottle is often inoculated onto an agar plate ( subcultured ) at the end of the incubation period regardless of whether or not indicators of growth are observed. [ 47 ]


In developed countries, manual culture methods have largely been replaced by automated systems that provide continuous computerized monitoring of the culture bottles. [ 48 ]  These systems, such as the BACTEC, BacT/ALERT and VersaTrek, consist of an incubator in which the culture bottles are continuously mixed. Growth is detected by sensors that measure the levels of gases inside the bottle—most commonly  carbon dioxide —which serve as an indicator of microbial metabolism. [ 46 ]  An alarm or a visual indicator alerts the microbiologist to the presence of a positive blood culture bottle. [ 49 ]  If the bottle remains negative at the end of the incubation period, it is generally discarded without being subcultured. [ 47 ]


A technique called the lysis-centrifugation method can be used for improved isolation of slow-growing or  fastidious  organisms, such as fungi, mycobacteria, and  Legionella . [ 50 ] [ 51 ]  Rather than incubating the blood in a bottle filled with growth medium, [ 52 ]  this method involves collecting blood into a tube containing an agent that destroys ( lyses ) red and white blood cells, then spinning the sample in a  centrifuge . This process concentrates the solid contents of the sample, including microorganisms if present, into a pellet, which is used to inoculate the subculture media. While lysis-centrifugation offers greater  sensitivity  than conventional blood culture methods, it is prone to contamination because it requires extensive manipulation of the sample. [ 53 ]


If growth is detected, a microbiologist will perform a  Gram stain  on a sample of blood from the bottle for a rapid preliminary identification of the organism. [ 54 ]  The Gram stain classifies bacteria as  Gram-positive  or  Gram-negative  and provides information about  their shape —whether they are rod-shaped (referred to as  bacilli ),  spherical (referred to as  cocci ), or spiral-shaped ( spirochetes )—as well as their arrangement. [ 55 ]   Gram-positive cocci in clusters, for example, are typical of  Staphylococcus  species. [ 56 ]   Yeast  and other fungi may also be identified from the Gram stain. [ 57 ] [ 58 ]  A Gram stain identifying microbial growth from a blood culture is considered a critical result and must immediately be reported to the clinician. [ 59 ]  The Gram stain provides information about the possible identity of the organism, which assists the clinician in the selection of a more appropriate antimicrobial treatment before the full culture and sensitivity results are complete. [ 54 ]


In traditional methods, the blood is then subcultured onto  agar plates  to  isolate  the organism for further testing. The Gram stain results inform microbiologists about what  types of agar plates  should be used and what tests might be appropriate to identify the organism. [ 60 ]  In some cases, no organisms are seen on the Gram stain despite the culture bottle showing indicators of growth or being reported as positive by automated instruments. This may represent a false positive result, but it is possible that organisms are present but cannot easily be visualized microscopically. Positive bottles with negative Gram stains are subcultured before being returned to the incubator, often using special culture media that promotes the growth of slow-growing organisms. [ 61 ]


It typically takes 24 to 48 hours for sufficient growth to occur on the subculture plates for definitive identification to be possible. [ 57 ]  At this point, the microbiologist will assess  the appearance of the bacterial or fungal colonies [ 62 ]  and carry out tests that provide information about the metabolic and biochemical features of the organism, which permit identification to the  genus  or species level. For example, the  catalase test  can distinguish  streptococci  and staphylococci (two  genera  of Gram-positive cocci) [ 63 ]  from each other, and the  coagulase test  can differentiate  Staphylococcus aureus , a common culprit of bloodstream infections, from the less pathogenic coagulase-negative staphylococci. [ 29 ] [ 64 ]


Microorganisms may also be identified using automated systems, such as instruments that perform panels of biochemical tests, [ 65 ]  or  matrix-assisted laser desorption/ionization time-of-flight mass spectrometry  (MALDI-TOF MS), in which microbial proteins are  ionized  and characterized on the basis of their  mass-to-charge ratios ; each microbial species exhibits a characteristic pattern of proteins when analyzed through  mass spectrometry . [ 66 ]


Because bloodstream infections can be life-threatening, timely diagnosis and treatment is critical, [ 67 ]  and to this end several rapid identification methods have been developed. [ 57 ]  MALDI-TOF can be used to identify organisms directly from positive blood culture bottles after separation and concentration procedures, [ 68 ]  or from preliminary growth on the agar plate within a few hours of subculturing. [ 69 ]  Genetic methods such as  polymerase chain reaction  (PCR) and  microarrays  can identify microorganisms by detection of  DNA sequences  specific to certain species in blood culture samples. Several systems designed for the identification of common blood culture pathogens are commercially available. [ 70 ]  Some biochemical and immunologic tests can be performed directly on positive blood cultures, such as the  tube coagulase  test for identification of  S. aureus [ 57 ]  or  latex agglutination  tests for  Streptococcus pneumoniae , [ 71 ]  and unlike PCR and MALDI-TOF, these methods may be practical for laboratories in low and middle income countries. [ 42 ]  It is also possible to directly inoculate microbial identification panels with blood from a positive culture bottle, although this is not as reliable as testing subcultured bacteria because additives from the growth media can interfere with the results. [ 72 ]


Even faster diagnosis could be achieved through bypassing culture entirely and detecting pathogens directly from blood samples. A few direct testing systems are commercially available as of 2018, but the technology is still in its infancy. Most panels detect only a limited number of pathogens, and the sensitivity can be poor compared to conventional blood culture methods. Culturing remains necessary in order to carry out full antimicrobial sensitivity testing. [ 73 ]


Antimicrobial treatment of bloodstream infections is initially  empiric , meaning it is based on the clinician's suspicion about the causative agent of the disease and local patterns of antimicrobial resistance. Carrying out  antibiotic susceptibility testing  (AST) on pathogens isolated from a blood culture allows clinicians to provide a more targeted treatment and to discontinue  broad-spectrum antibiotics , which can have undesirable side effects. [ 8 ] [ 10 ]  In traditional AST methods, such as the  disk diffusion test , pure colonies of the organism are selected from the subculture plate and used to inoculate a secondary medium. These methods require overnight incubation before results can be obtained. [ 74 ]  There are automated systems which use pre-formulated antibiotic panels, measure microbial growth automatically, and determine the sensitivity results using algorithms; some of these can provide results in as little as five hours, but others require overnight incubation as well. [ 65 ] [ 75 ]


Rapid administration of effective antimicrobial drugs is crucial in the treatment of sepsis, [ 8 ]  so several methods have been developed to provide faster antibiotic sensitivity results. Conventional AST methods can be carried out on young growth from the subculture plate, [ 76 ]  pellets of microorganisms obtained from concentration and purification of the positive blood culture, or directly from the culture bottle. [ 77 ] [ 78 ]  Because direct testing methods do not isolate the organisms, they do not provide accurate results if more than one microorganism is present, although this is an infrequent occurrence in blood cultures. [ 76 ]  Another source of error is the difficulty in standardizing the amount of bacteria in the sample (the  inoculum ), which has a profound effect on the test results. [ 79 ]


Genetic testing  can be used for rapid detection of certain antimicrobial resistance markers. [ 80 ]  Methods such as PCR and microarrays, which can be performed directly on positive blood culture samples, [ 70 ]  detect DNA sequences associated with genes that confer resistance, such as the  mecA  gene found in  methicillin-resistant  Staphylococcus aureus  or the  vanA  and  vanB  genes of  vancomycin-resistant enterococci . [ 68 ]  MALDI-TOF has been explored as a rapid antimicrobial sensitivity testing method; principles involve measuring microbial growth in the presence of antibiotics, identifying the breakdown of antibiotics by microbial  enzymes , and detecting protein spectra associated with bacterial strains that exhibit antibiotic resistance. [ 79 ]  Some of these methods can be performed on pellets from positive blood culture bottles. [ 81 ]  However, the lack of established methodologies for AST by MALDI-TOF limits its use in clinical practice, [ 82 ]  and direct AST by MALDI-TOF, unlike genetic testing methods, had not been approved by the  Food and Drug Administration  as of 2018. [ 81 ]


Blood cultures are subject to both false positive and false negative errors. In automated culture systems, identification of positive bottles is based on the detection of gases produced by cellular metabolism, so samples with high numbers of  white blood cells  may be reported as positive when no bacteria are present. Inspection of the growth curve produced by the instrument can help to distinguish between true and false positive cultures, but Gram staining and subculturing are still necessary for any sample that is flagged as positive. [ 61 ]


Blood cultures can become contaminated with microorganisms from the skin or the environment, which multiply inside the culture bottle, giving the false impression that those organisms are present in the blood. [ 11 ]  Contamination of blood cultures can lead to unnecessary antibiotic treatment and longer hospital stays. [ 29 ]  The frequency of contamination can be reduced by following established protocols for blood culture collection, but it cannot be eliminated; [ 83 ]  for instance, bacteria can survive in deeper layers of the skin even after meticulous disinfection of the blood draw site. [ 29 ]  The CLSI defines an acceptable contamination rate as no greater than 3% of all blood cultures. [ 11 ]  The frequency of contamination varies widely between institutions and between different departments in the same hospital; [ 83 ]  studies have found rates ranging from 0.8 to 12.5 percent. [ 29 ]


When faced with a positive blood culture result, clinicians must decide whether the finding represents contamination or genuine infection. Some organisms, such as  S. aureus  or  Streptococcus pneumoniae , are usually considered to be pathogenic when detected in a blood culture, while others are more likely to represent contamination with skin flora; but even common skin organisms such as coagulase-negative staphylococci can cause bloodstream infections under certain conditions. When such organisms are present, interpretation of the culture result involves taking into account the person's clinical condition and whether or not multiple cultures are positive for the same organism. [ 29 ]


False negatives may be caused by drawing blood cultures after the person has received antibiotics or collecting an insufficient amount of blood. The volume of blood drawn is considered the most important variable in ensuring that pathogens are detected: the more blood that is collected, the more pathogens are recovered. [ 11 ]  However, if the amount of blood collected far exceeds the recommended volume, bacterial growth may be inhibited by natural inhibitors present in the blood and an inadequate amount of growth medium in the bottle. Over-filling of blood culture bottles may also contribute to  iatrogenic anemia . [ 28 ]


Not all pathogens are easily detected by conventional blood culture methods. Particularly  fastidious organisms , such as  Brucella  and  Mycobacterium  species, may require prolonged incubation times or special culture media. Some organisms are exceedingly difficult to culture or do not grow in culture at all, so  serology testing  or molecular methods such as PCR are preferred if infection with these organisms is suspected. [ 45 ] [ 84 ]


Early blood culture methods were labour-intensive. [ 86 ]  One of the first known procedures, published in 1869, recommended that  leeches  be used to collect blood from the patient. [ 87 ]  A microbiology textbook  from 1911 noted that decontamination of the draw site and equipment could take over an hour, and that due to a lack of effective methods for preserving blood, the cultures would sometimes have to be prepared at the patient's bedside. In addition to subculturing the broth, some protocols specified that the blood be mixed with melted agar and the mixture poured into a petri dish. [ 86 ]  In 1915, a blood culture collection system consisting of glass vacuum tubes containing  glucose  broth and an anticoagulant was described.  Robert James Valentine Pulvertaft  published a seminal work on blood cultures in 1930, [ 88 ]  specifying—among other insights—an optimal blood-to-broth ratio of 1:5, which is still accepted today. [ 87 ]  The use of SPS as an anticoagulant and preservative was introduced in the 1930s and 40s and resolved some of the logistical issues with earlier methods. [ 86 ]  From the 1940s through the 1980s, a great deal of research was carried out on broth formulations and additives, with the goal of creating a growth medium that could accommodate all common bloodstream pathogens. [ 87 ]


In 1947, M.R. Castañeda invented a "biphasic" culture bottle for the identification of  Brucella  species, which contained both broth and an agar slant, allowing the agar to be easily subcultured from the broth; [ 42 ]  this was a precursor of some contemporary systems for manual blood cultures. [ 43 ]  E.G. Scott in 1951 published a protocol described as "the advent of the modern blood culture set". [ 86 ]  Scott's method involved inoculating blood into two rubber-sealed glass bottles; one for aerobes and one for anaerobes. The aerobic bottle contained trypticase soy broth and an agar slant, and the anaerobic bottle contained thioglycollate broth. The lysis-centrifugation method was introduced in 1917 by Mildred Clough, but it was rarely used in clinical practice until commercial systems were developed in the mid-1970s. [ 86 ] [ 89 ]


Automated blood culture systems first became available in the 1970s. [ 90 ]  The earliest of these—the BACTEC systems, produced by Johnston Laboratories (now  Becton Dickinson )—used culture broths containing nutrients  labelled  with  radioactive isotopes . Microbes that fed on these substrates would produce radioactive carbon dioxide, and growth could be detected by monitoring its concentration. [ 50 ] [ 91 ]  Before this technique was applied to blood cultures, it had been proposed by  NASA  as a method for detecting life on Mars. [ 86 ]   Throughout the 1970s and 80s several manufacturers attempted to detect microbial growth by measuring changes in the  electrical conductivity  of the culture medium, but none of these methods were commercially successful. [ 91 ]


A major issue with the early BACTEC systems was that they produced  radioactive waste , which required special disposal procedures, [ 50 ]  so in 1984 a new generation of BACTEC instruments was released that used  spectrophotometry  to detect CO 2 . [ 91 ]  The BacT/ALERT system, which indirectly detects production of CO 2  by measuring the decrease in the medium's pH, was approved for use in the US in 1991. Unlike the BACTEC systems available at the time, the BacT/ALERT did not require a needle to be introduced into the bottle for sampling; this reduced the frequency of contamination [ 91 ]  and made it the first system to provide truly continuous monitoring of blood cultures. [ 92 ]  This non-invasive measurement method was adopted in 1992 by the BACTEC 9000 series, which used fluorescent indicators to detect pH changes. [ 93 ]  The Difco ESP, a direct predecessor of the contemporary VersaTREK system [ 86 ]  which detects gas production by measuring pressure changes, was also first approved in 1992. [ 91 ]  By 1996, an international study found that 55% of 466 laboratories surveyed were using the BACTEC or BacT/ALERT systems, with other automated systems accounting for 10% of the total. [ 94 ]


A  blood smear ,  peripheral blood smear  or  blood film  is a thin layer of  blood  smeared on a glass  microscope slide  and then stained in such a way as to allow the various blood cells to be examined microscopically. Blood smears are examined in the investigation of  hematological  (blood) disorders and are routinely employed to look for blood  parasites , such as those of  malaria  and  filariasis .


A blood smear is made by placing a drop of blood on one end of a slide, and using a  spreader slide  to disperse the blood over the slide's length. The aim is to get a region, called a monolayer, where the cells are spaced far enough apart to be counted and differentiated. The monolayer is found in the "feathered edge" created by the spreader slide as it draws the blood forward. [ citation needed ]


The slide is left to air dry, after which the blood is  fixed  to the slide by immersing it briefly in  methanol . The fixative is essential for good staining and presentation of cellular detail. After fixation, the slide is  stained  to distinguish the cells from each other. [ citation needed ]


Routine analysis of blood in medical laboratories is usually performed on blood films stained with  Romanowsky stains  such as  Wright's stain ,  Giemsa stain , or  Diff-Quik . Wright-Giemsa combination stain is also a popular choice. These stains allow for the detection of  white blood cell ,  red blood cell , and  platelet  abnormalities.  Hematopathologists  often use other specialized stains to aid in the differential diagnosis of blood disorders. [ citation needed ]


After staining, the monolayer is viewed under a microscope using magnification up to 1000 times. Individual cells are examined and their morphology is characterized and recorded. [ 1 ] [ 2 ]


Blood smear examination is usually performed in conjunction with a  complete blood count  in order to investigate abnormal results or confirm results that the  automated analyzer  has flagged as unreliable. [ 3 ]


Microscopic examination of the shape, size, and coloration of red blood cells is useful for determining the cause of  anemia . Disorders such as  iron deficiency anemia ,  sickle cell anemia ,  megaloblastic anemia  and  microangiopathic hemolytic anemia  result in characteristic abnormalities on the blood film. [ 2 ]


The proportions of different types of  white blood cells  can be determined from the blood smear. This is known as a manual  white blood cell differential . The white blood cell differential can reveal abnormalities in the proportions of white blood cell types, such as  neutrophilia  and  eosinophilia , as well as the presence of abnormal cells such as the circulating  blast cells  seen in  acute leukemia . [ 4 ]   Qualitative abnormalities of white blood cells, like  toxic granulation , are also visible on the blood smear. Modern complete blood count analyzers can provide an automated white blood cell differential, but they have a limited ability to differentiate immature and abnormal cells, so manual examination of the blood smear is frequently indicated. [ 5 ] [ 6 ]


Blood smear examination is the preferred diagnostic method for certain parasitic infections, such as malaria and  babesiosis . [ 7 ]  Rarely, bacteria may be visible on the blood smear in patients with severe  sepsis . [ 8 ]


The preferred and most reliable diagnosis of malaria is microscopic examination of blood smears, because each of the four major parasite species has distinguishing characteristics.  Two sorts of blood smear are traditionally used. [ 9 ]


From the thick smear, an experienced microscopist can detect all parasites they encounter. Microscopic diagnosis can be difficult because the early trophozoites ("ring form") of all four species look identical and it is never possible to diagnose species on the basis of a single ring form; species identification is always based on several trophozoites. [ citation needed ]


The biggest pitfall in most laboratories in developed countries is leaving too great a delay between taking the blood sample and making the blood smears.  As blood cools to room temperature, male gametocytes will divide and release  microgametes : these are long sinuous filamentous structures that can be mistaken for organisms such as  Borrelia . If the blood is kept at warmer temperatures,  schizonts  will rupture and merozoites invading erythrocytes will mistakenly give the appearance of the accolé form of  P. falciparum . If  P. vivax  or  P. ovale  is left for several hours in EDTA, the buildup of acid in the sample will cause the parasitised erythrocytes to shrink and the parasite will roll up, simulating the appearance of  P. malariae . This problem is made worse if  anticoagulants  such as  heparin  or  citrate  are used.  The anticoagulant that causes the least problems is  EDTA . Romanowsky stain or a variant stain is usually used. Some laboratories mistakenly use the same staining pH as they do for routine haematology blood films ( pH  6.8): malaria blood films must be stained at pH 7.2, or  Schüffner's dots  and  James'  dots will not be seen. [ citation needed ]


Immunochromatographic  capture procedures (rapid diagnostic tests such as the  malaria antigen detection tests ) are nonmicroscopic diagnostic options for the laboratory that may not have appropriate microscopy expertise available. [ 11 ]


The  blood sugar level ,  blood sugar concentration ,   blood glucose level , or  glycemia  is the measure of  glucose  concentrated in the  blood . The body tightly  regulates blood glucose levels  as a part of metabolic  homeostasis . [ 2 ]


For a 70 kg (154 lb) human, approximately four grams of  dissolved  glucose (also called "blood glucose") is maintained in the  blood plasma  at all times. [ 2 ]  Glucose that is not circulating in the blood is stored in  skeletal muscle  and  liver  cells in the form of  glycogen ; [ 2 ]  in  fasting  individuals, blood glucose is maintained at a constant level by releasing just enough glucose from these glycogen stores in the liver and skeletal muscle in order to maintain homeostasis. [ 2 ]  Glucose can be transported from the intestines or liver to other tissues in the body via the bloodstream. [ 2 ]  Cellular glucose uptake is primarily regulated by  insulin , a  hormone  produced in the  pancreas . [ 2 ]  Once inside the cell, the glucose can now act as an energy source as it undergoes the process of  glycolysis .


In humans, properly maintained glucose levels are necessary for normal function in a number of tissues, including the  human brain , which consumes approximately 60% of blood glucose in fasting, sedentary individuals. [ 2 ]  A persistent elevation in blood glucose leads to glucose toxicity, which contributes to cell dysfunction and the pathology grouped together as complications of  diabetes . [ 2 ]


Glucose levels are usually lowest in the morning, before the first meal of the day, and rise after meals for an hour or two by a few  millimoles  per litre.


Abnormal persistently high glycemia is referred to as  hyperglycemia ; low levels are referred to as  hypoglycemia .  Diabetes mellitus  is characterized by persistent hyperglycemia from a variety of causes, and it is the most prominent disease related to the failure of blood sugar regulation. Diabetes mellitus is also characterized by frequent episodes of low sugar, or  hypoglycemia . There are different methods of  testing  and measuring blood sugar levels.


Drinking  alcohol  causes an initial surge in blood sugar and later tends to cause levels to fall. Also, certain drugs can increase or decrease glucose levels. [ 3 ]


There are two ways of measuring blood glucose levels: In the United Kingdom and Commonwealth countries (Australia, Canada, India, etc.) and ex-USSR countries  molar concentration , measured in  mmol/L  (millimoles per litre, or millimolar, abbreviated mM). In the United States, Germany, Japan and many other countries  mass concentration  is measured in mg/dl (milligrams per  decilitre ). [ 4 ]


Unit conversion formula from mmol/L to mg/dL [ 5 ]


m
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 =
 m
 m
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 m
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 c
 u
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 a
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 w
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 i
 g
 h
 t
 ÷
 10

 {\displaystyle mg/dL=mmol/L\times molecular\ weight\div 10}


Since the  molecular mass  of glucose C 6 H 12 O 6  is 180.156 g/mol, the factor between the two units is about 18, so 1 mmol/L of glucose is equivalent to 18 mg/dL.


m
 g

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 d
 L
 =
 m
 m
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 18

 {\displaystyle mg/dL=mmol/L\times 18}


Normal blood glucose level (tested while fasting) for non-diabetics should be 3.9–5.5 mmol/L (70–100 mg/dL). [ 6 ] [ 7 ] [ 8 ]


According to the  American Diabetes Association , the fasting blood glucose target range for diabetics, should be 3.9 - 7.2 mmol/L (70 - 130 mg/dL) and less than 10 mmol/L (180 mg/dL)  two hours after meals (as measured by a blood glucose monitor). [ 6 ] [ 7 ] [ 9 ]


Normal value ranges  may vary slightly between laboratories.  Glucose homeostasis , when operating normally, restores the blood sugar level to a narrow range of about 4.4 to 6.1 mmol/L (79 to 110 mg/dL) (as measured by a  fasting blood glucose test ). [ 10 ]


The global mean fasting plasma blood glucose level in humans is about 5.5 mmol/L (100 mg/dL); [ 11 ] [ 12 ]  however, this level fluctuates throughout the day. Blood sugar levels for those without diabetes and who are not fasting should be below 6.9 mmol/L (125 mg/dL). [ 13 ]


Despite widely variable intervals between meals or the occasional consumption of meals with a substantial  carbohydrate  load, human blood glucose levels tend to remain within the normal range. However, shortly after eating, the blood glucose level may rise, in non-diabetics, temporarily up to 7.8 mmol/L (140 mg/dL) or slightly more.


The actual amount of glucose in the blood and body fluids is very small. In a healthy adult male of 75 kg (165 lb) with a blood volume of 5 L, a blood glucose level of 5.5 mmol/L (100 mg/dL) amounts to 5 g, equivalent to about a teaspoonful of sugar. [ 14 ]  Part of the reason why this amount is so small is that, to maintain an influx of glucose into cells,  enzymes  modify glucose by adding  phosphate  or other groups to it. [ citation needed ]


In general, ranges of blood sugar in common domestic  ruminants  are lower than in many  monogastric  mammals. [ 15 ]    However this generalization does not extend to wild ruminants or  camelids .  For serum glucose in mg/dL, reference ranges of 42 to 75 for cows, 44 to 81 for sheep, and 48 to 76 for goats, but 61 to 124 for cats; 62 to 108 for dogs, 62 to 114 for horses, 66 to 116 for pigs, 75 to 155 for rabbits, and 90 to 140 for llamas have been reported. [ 16 ]   A 90 percent reference interval for serum glucose of 26 to 181 mg/dL has been reported for captured mountain goats ( Oreamnos americanus ), where no effects of the pursuit and capture on measured levels were evident. [ 17 ]   For beluga whales, the 25–75 percent range for serum glucose has been estimated to be 94 to 115 mg/dL. [ 18 ]   For the white rhinoceros, one study has indicated that the 95 percent range is 28 to 140 mg/dL. [ 19 ]   For harp seals, a serum glucose range of 4.9 to 12.1 mmol/L [i.e. 88 to 218 mg/dL] has been reported; for hooded seals, a range of 7.5 to 15.7 mmol/L [i.e. about 135 to 283 mg/dL] has been reported. [ 20 ]


The body's  homeostatic  mechanism keeps blood glucose levels within a narrow range. It is composed of several interacting systems, of which hormone regulation is the most important. [ 21 ]


There are two types of mutually antagonistic metabolic hormones affecting blood glucose levels:


These hormones are secreted from  pancreatic islets  (bundles of endocrine tissues), of which there are four types: alpha (A) cells, beta (B) cells, Delta (D) cells and F cells. Glucagon is secreted from alpha cells, while insulin is secreted by beta cells. Together they regulate the blood-glucose levels through negative feedback, a process where the end product of one reaction stimulates the beginning of another reaction. In blood-glucose levels, insulin lowers the concentration of glucose in the blood. The lower blood-glucose level (a product of the insulin secretion) triggers glucagon to be secreted, and repeats the cycle. [ 23 ]


In order for blood glucose to be kept stable, modifications to insulin, glucagon, epinephrine and cortisol are made. Each of these hormones has a different responsibility to keep blood glucose regulated; when blood sugar is too high, insulin tells muscles to take up excess glucose for storage in the form of  glycogen . Glucagon responds to too low of a blood glucose level; it informs the tissue to release some glucose from the glycogen stores. Epinephrine prepares the muscles and respiratory system for activity in the case of a "fight or flight" response. Lastly, cortisol supplies the body with fuel in times of heavy stress. [ 24 ]


If blood sugar levels remain too high the body suppresses appetite over the short term. Long-term  hyperglycemia  causes many health problems including heart disease, cancer, [ 25 ]  eye, kidney, and nerve damage. [ 26 ]


Blood sugar levels above 16.7   mmol/L (300   mg/dL) can cause fatal reactions. Ketones will be very high (a magnitude higher than when eating a very low carbohydrate diet) initiating  ketoacidosis . The ADA (American Diabetes Association) recommends seeing a doctor if blood glucose reaches 13.3 mmol/L (240 mg/dL), [ 27 ]  and it is recommended to seek emergency treatment at 15   mmol/L (270   mg/dL) blood glucose if Ketones are present. [ 28 ]  The most common cause of hyperglycemia is  diabetes . When diabetes is the cause, physicians typically recommend an  anti-diabetic medication  as treatment. From the perspective of the majority of patients, treatment with an old, well-understood diabetes drug such as  metformin  will be the safest, most effective, least expensive, and most comfortable route to managing the condition. Treatment will vary for the distinct forms of Diabetes and can differ from person to person based on how they are reacting to treatment. [ 29 ]  Diet changes and exercise implementation may also be part of a treatment plan for diabetes. [ 30 ]


Some medications may cause a rise in blood sugars of diabetics, such as steroid medications, including cortisone, hydrocortisone, prednisolone, prednisone, and dexamethasone. [ 31 ]


When the blood sugar level is below 70 mg/dL, this is referred to as having low blood sugar. Low blood sugar is very frequent among type 1 diabetics. There are several causes of low blood sugar, including, taking an excessive amount of insulin, not  consuming enough carbohydrates, drinking alcohol, spending time at a high elevation, puberty, and menstruation. [ 32 ]  If blood sugar levels drop too low, a potentially fatal condition called  hypoglycemia  develops. Symptoms may include  lethargy , impaired mental functioning;  irritability ; shaking, twitching, weakness in arm and leg muscles; pale complexion; sweating;  loss of consciousness . [ citation needed ]


Mechanisms that restore satisfactory blood glucose levels after extreme  hypoglycemia  (below 2.2 mmol/L or 40 mg/dL) must be quick and effective to prevent extremely serious consequences of insufficient glucose: confusion or unsteadiness and, in the extreme (below 0.8 mmol/L or 15 mg/dL)  loss of consciousness and seizures. Without discounting the potentially quite serious conditions and risks due to or oftentimes accompanying hyperglycemia, especially in the long-term (diabetes or pre-diabetes, obesity or overweight,  hyperlipidemia ,  hypertension , etc.), it is still generally more dangerous to have too little glucose – especially if levels are very low – in the blood than too much, at least temporarily, because glucose is so important for metabolism and nutrition and the proper functioning of the body's organs. This is especially the case for those organs that are metabolically active or that require a constant, regulated supply of blood sugar (the liver and brain are examples). Symptomatic hypoglycemia is most likely associated with  diabetes  and  liver disease  (especially overnight or postprandial), without treatment or with wrong treatment, possibly in combination with carbohydrate malabsorption, physical over-exertion or drugs. Many other less likely illnesses, like cancer, could also be a reason. Starvation, possibly due to eating disorders, like anorexia, will also eventually lead to hypoglycemia. Hypoglycemic episodes can vary greatly between persons and from time to time, both in severity and swiftness of onset. For severe cases, prompt medical assistance is essential, as damage to brain and other tissues and even death will result from sufficiently low blood-glucose levels. [ citation needed ]


In the past to measure blood glucose it was necessary to take a blood sample, as explained below, but since 2015 it has also been possible to use a  continuous glucose monitor , which involves an electrode placed under the skin. Both methods, as of 2023, cost hundreds of dollars or euros per year for supplies needed. [ citation needed ]


Glucose testing  in a fasting individual shows comparable levels of glucose in arterial, venous, and capillary blood. But following meals, capillary and arterial blood glucose levels can be significantly higher than venous levels. Although these differences vary widely, one study found that following the consumption of 50 grams of glucose, "the mean capillary blood glucose concentration is higher than the mean venous blood glucose concentration by 35%." [ 33 ] [ 34 ]


Glucose is measured in whole blood,  plasma  or  serum . Historically, blood glucose values were given in terms of whole blood, but most laboratories now measure and report plasma or serum glucose levels. Because  red blood cells  (erythrocytes) have a higher concentration of protein (e.g., hemoglobin) than serum, serum has a higher water content and consequently more dissolved glucose than does whole blood. To convert from whole-blood glucose, multiplication by 1.14 [ 35 ]  has been shown to generally give the serum/plasma level.


To prevent contamination of the sample with  intravenous fluids , particular care should be given to drawing blood samples from the arm opposite the one in which an intravenous line is inserted. Alternatively, blood can be drawn from the same arm with an IV line after the IV has been turned off for at least 5 minutes, and the arm has been elevated to drain infused fluids away from the vein. Inattention can lead to large errors, since as little as 10% contamination with a 5%  glucose  solution (D5W) will elevate glucose in a sample by 500 mg/dL or more. The actual concentration of glucose in blood is very low, even in the hyperglycemic. [ citation needed ]


Two major methods have been used to measure glucose. The first, still in use in some places, is a chemical method exploiting the nonspecific reducing property of glucose in a reaction with an indicator substance that changes color when reduced. Since other blood compounds also have reducing properties (e.g., urea, which can be abnormally high in uremic patients), this technique can produce erroneous readings in some situations (5–15 mg/dL has been reported). The more recent technique, using enzymes specific to glucose, is less susceptible to this kind of error. The two most common employed enzymes are glucose oxidase and hexokinase. [ 36 ]  Average blood glucose concentrations can also be measured. This method measures the level of  glycated hemoglobin , which is representative of the average blood glucose levels over the last, approximately, 120 days. [ 36 ]


In either case, the chemical system is commonly contained on a test strip which is inserted into a meter, and then has a blood sample applied. Test-strip shapes and their exact chemical composition vary between meter systems and cannot be interchanged. Formerly, some test strips were read (after timing and wiping away the blood sample) by visual comparison against a color chart printed on the vial label. Strips of this type are still used for urine glucose readings, but for blood glucose levels they are obsolete. Their error rates were, in any case, much higher. Errors when using test strips were often caused by the age of the strip or exposure to high temperatures or humidity. [ 37 ]  More precise blood glucose measurements are performed in a  medical laboratory , using hexokinase, glucose oxidase, or glucose dehydrogenase enzymes.


Urine glucose  readings, however taken, are much less useful. In properly functioning kidneys, glucose does not appear in urine until the  renal threshold  for glucose has been exceeded. This is substantially above any normal glucose level, and is evidence of an existing severe hyperglycemic condition. However, as urine is stored in the bladder, any glucose in it might have been produced at any time since the last time the bladder was emptied. Since metabolic conditions change rapidly, as a result of any of several factors, this is delayed news and gives no warning of a developing condition. [ 38 ]  Blood glucose monitoring is far preferable, both clinically and for home monitoring by patients. Healthy urine glucose levels were first standardized and published in 1965 [ 39 ]  by  Hans Renschler .


A noninvasive method of sampling to monitor glucose levels has emerged using an  exhaled breath condensate . However this method does need highly sensitive glucose biosensors. [ 40 ]


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The fasting blood glucose level, which is measured after a fast of 8 hours, is the most commonly used indication of overall glucose homeostasis, largely because disturbing events such as food intake are avoided. Conditions affecting glucose levels are shown in the table below. Abnormalities in these test results are due to problems in the multiple control mechanism of glucose regulation. [ citation needed ]


The metabolic response to a carbohydrate challenge is conveniently assessed by a postprandial glucose level drawn 2 hours after a meal or a glucose load. In addition, the glucose tolerance test, consisting of several timed measurements after a standardized amount of oral glucose intake, is used to aid in the diagnosis of  diabetes . [ citation needed ]


Error rates for blood glucose measurements systems vary, depending on laboratories, and on the methods used. Colorimetry techniques can be biased by color changes in test strips (from airborne or finger-borne contamination, perhaps) or interference (e.g., tinting contaminants) with light source or the light sensor. Electrical techniques are less susceptible to these errors, though not to others. In home use, the most important issue is not accuracy, but trend. Thus if a meter / test strip system is consistently wrong by 10%, there will be little consequence, as long as changes (e.g., due to exercise or medication adjustments) are properly tracked. In the US, home use blood test meters must be approved by the federal  Food and Drug Administration  before they can be sold. [ citation needed ]


Finally, there are several influences on blood glucose level aside from food intake. Infection, for instance, tends to change blood glucose levels, as does stress either physical or psychological. Exercise, especially if prolonged or long after the most recent meal, will have an effect as well. In the typical person, maintenance of blood glucose at near constant levels will nevertheless be quite effective. [ clarification needed ]


The  Bodansky unit  is an obsolete measure of  alkaline phosphatase  concentration in blood. It is defined as the quantity of alkaline phosphatase that liberates 1   mg of phosphate ion during the first hour of incubation with a buffered substrate containing  sodium β-glycerophosphate . [ 1 ]  This technique was the first test to measure blood alkaline phosphatase levels, and was developed by  Aaron Bodansky  in the early 1930s. [ 2 ]


Other units that were used in the past as a measure of blood alkaline phosphatase levels are King-Armstrong units, Kind-King units, and International units. [ 3 ]  However, the Bodansky unit and those other units have become outdated, and the current standard for measuring alkaline phosphatase is units per liter (U/L). [ 3 ]


1B09 ,  1GNH ,  1LJ7 ,  3L2Y ,  3PVN ,  3PVO


NM_000567 NM_001329057 NM_001329058 NM_001382703


NP_000558 NP_001315986 NP_001315987 NP_001369632


C-reactive protein  ( CRP ) is an annular (ring-shaped)  pentameric protein  found in  blood plasma , whose  circulating concentrations  rise in response to  inflammation . It is an  acute-phase protein  of  hepatic  origin that increases following  interleukin-6  secretion by  macrophages  and  T cells . Its physiological role is to bind to  lysophosphatidylcholine  expressed on the surface of dead or dying cells (and some types of bacteria) in order to activate the  complement system  via  C1q . [ 5 ]


CRP is synthesized by the  liver [ 6 ]  in response to factors released by  macrophages , T cells and fat cells ( adipocytes ). [ 7 ]  It is a member of the  pentraxin  family of proteins. [ 6 ]  It is not related to  C-peptide  (insulin) or  protein C  (blood coagulation). C-reactive protein was the first  pattern recognition receptor  (PRR) to be identified. [ 8 ]


Discovered by  Tillett  and Francis in 1930, [ 9 ]  it was initially thought that CRP might be a pathogenic secretion since it was elevated in a variety of illnesses, including  cancer . [ 6 ]  The later discovery of hepatic synthesis (made in the liver) demonstrated that it is a native protein. [ 10 ] [ 11 ] [ 12 ]  Initially, CRP was measured using the  quellung reaction  which gave a positive or a negative result. More precise methods nowadays use  dynamic light scattering  after reaction with CRP-specific antibodies. [ 13 ]


CRP was so named because it was first identified as a substance in the serum of patients with acute inflammation that reacted with the  cell wall   polysaccharide  (C-polysaccharide) of  pneumococcus . [ 14 ]


It is a member of the small  pentraxins  family (also known as short pentraxins). [ 15 ]  The polypeptide encoded by this gene has 224 amino acids. [ 16 ]  The full-length polypeptide is not present in the body in significant quantities due to  signal peptide , which is removed by  signal peptidase  before  translation  is completed. The complete protein, composed of five monomers, has a total mass of approximately 120,000 Da.  In serum, it assembles into stable pentameric structure with a discoid shape. [ 17 ]


CRP binds to the  phosphocholine  expressed on the surface of bacterial cells such as pneumococcus  bacteria . This activates the  complement system , promoting  phagocytosis  by macrophages, which clears  necrotic  and  apoptotic  cells and bacteria. [ 18 ] [ 13 ]  With this mechanism, CRP also binds to ischemic/hypoxic cells, which could regenerate with more time. However, the binding of CRP causes them to be disposed of prematurely. [ 19 ] [ 20 ]  CRP binds to the Fc-gamma receptor IIa, to which IgG isotype antibodies also bind. [ 21 ]  In addition, CRP activates the classical complement pathway via C1q binding. [ 22 ] [ 23 ]  CRP thus forms immune complexes in the same way as IgG antibodies.


This so-called  acute phase response  occurs as a result of increasing concentrations of  interleukin-6  (IL-6), which is produced by  macrophages [ 6 ]  as well as  adipocytes [ 7 ]  in response to a wide range of acute and chronic inflammatory conditions such as  bacterial ,  viral , or  fungal  infections;  rheumatic  and other  inflammatory diseases ;  malignancy ; and tissue injury and necrosis. These conditions cause release of IL-6 and other  cytokines  that trigger the synthesis of CRP and  fibrinogen  by the liver.


CRP binds to phosphocholine on micro-organisms. It is thought to assist in  complement  binding to foreign and damaged cells and enhances phagocytosis by macrophages ( opsonin-mediated phagocytosis ), which express a receptor for CRP. It plays a role in  innate immunity  as an early defense system against infections. [ 13 ]


Traditional CRP measurement only detected CRP in the range of 10 to 1,000 mg/L, whereas high sensitivity CRP (hs-CRP) detects CRP in the range of 0.5 to 10 mg/L. [ 25 ]  hs-CRP can detect  cardiovascular disease  risk when in excess of 3 mg/L, whereas below 1 mg/L would be low risk. [ 26 ]  Traditional CRP measurement is faster and less costly than hs-CRP, and can be adequate for some applications, such as monitoring  hemodialysis  patients. [ 27 ]  Current immunoassay methods for CRP have similar precision to hsCRP performed by nephelometry and could probably replace hsCRP for cardiovascular risk assessment, [ 28 ]  however, in the United States this would represent off-label use, making it a laboratory-developed test under FDA regulations. [ 29 ]


In healthy adults, the normal concentrations of CRP varies between 0.8 mg/L and 3.0 mg/L. However, some healthy adults show elevated CRP at 10 mg/L. CRP concentrations also increase with age, possibly due to  subclinical  conditions. There are also no  seasonal variations  of CRP concentrations.  Gene polymorphism  of  interleukin-1 family ,  interleukin 6 , and polymorphic GT repeat of the CRP gene do affect the usual CRP concentrations when a person does not have any medical illnesses. [ 6 ]


When there is a stimulus, the CRP level can increase 10,000-fold from less than 50 μg/L to more than 500 mg/L. Its concentration can increase to 5 mg/L by 6 hours and peak at 48 hours. The plasma half-life of CRP is 19 hours, and is constant in all medical conditions. [ 30 ]  Therefore, the only factor that affects the blood CRP concentration is its production rate, which increases with inflammation, infection, trauma, necrosis, malignancy, and  allergic reactions . [ citation needed ]  Other inflammatory mediators that can increase CRP are  TGF beta 1 , and  tumor necrosis factor alpha . In acute inflammation, CRP can increase as much as 50 to 100 mg/L within 4 to 6 hours in mild to moderate inflammation or an insult such as  skin infection ,  cystitis , or  bronchitis [ clarification needed ] . It can double every 8 hours and reaches its peak at 36 to 50 hours following injury or inflammation. CRP between 100 and 500 mg/L is considered highly predictive of inflammation due to bacterial infection.  Once inflammation subsides, CRP level falls quickly because of its relatively short half-life. [ 13 ]


CRP concentrations between 2 and 10 mg/L are considered as metabolic inflammation: metabolic pathways that cause  arteriosclerosis [ 31 ]  and  type II diabetes mellitus . [ 32 ]


CRP is used mainly as an inflammation marker. Apart from  liver failure , there are few known factors that interfere with CRP production. [ 6 ]   Interferon alpha  inhibits CRP production from liver cells which may explain the relatively low levels of CRP found during viral infections compared to bacterial infections  [ 33 ] [ 34 ]


Measuring and charting CRP values can prove useful in determining disease progress or the effectiveness of treatments.  ELISA  and  radial immunodiffusion  methods are available for research use, while  immunoturbidimetry  is used clinically for CRP and  nephelometry  is typically used for hsCRP. [ 35 ] [ 26 ] Cutoffs for cardiovascular risk assessment have included:


Normal levels increase with  aging . [ 36 ]  Higher levels are found in late  pregnant  women, mild  inflammation  and  viral infections  (10–40 mg/L), active inflammation, bacterial infection (40–200 mg/L), severe  bacterial infections  and  burns  (>200 mg/L). [ 37 ]


CRP cut-off levels indicating bacterial from non-bacterial illness can vary due to  co-morbidities  such as  malaria ,  HIV  and  malnutrition  and the stage of disease presentation. [ 38 ]  In patients presenting to the emergency department with suspected sepsis, a CRP/albumin ratio of less than 32 has a negative predictive value of 89% for ruling out sepsis. [ 39 ]


CRP is a more sensitive and accurate reflection of the acute phase response than the ESR [ 40 ]  ( erythrocyte sedimentation rate ). ESR may be normal while CRP is elevated. CRP returns to normal more quickly than ESR in response to therapy. [ citation needed ]


Recent research suggests that patients with elevated basal levels of CRP are at an increased risk of  diabetes , [ 41 ] [ 42 ]   hypertension  and  cardiovascular disease . A study of over 700 nurses showed that those in the highest  quartile  of  trans fat  consumption had blood levels of CRP that were 73% higher than those in the lowest quartile. [ 43 ]  Although one group of researchers indicated that CRP may be only a moderate risk factor for cardiovascular disease, [ 44 ]  this study (known as the Reykjavik Study) was found to have some problems for this type of analysis related to the characteristics of the population studied, and there was an extremely long follow-up time, which may have attenuated the association between CRP and future outcomes. [ 45 ]  Others have shown that CRP can exacerbate  ischemic   necrosis  in a  complement -dependent fashion and that CRP inhibition can be a safe and effective therapy for  myocardial  and  cerebral   infarcts ; this has been demonstrated in animal models and humans. [ 46 ] [ 47 ] [ 48 ]


It has been hypothesized that patients with high CRP levels might benefit from use of  statins . This is based on the  JUPITER trial  that found that elevated CRP levels without hyperlipidemia benefited. Statins were selected because they have been proven to reduce levels of CRP. [ 6 ] [ 49 ]  Studies comparing effect of various statins in hs-CRP revealed similar effects of different statins. [ 50 ] [ 51 ]  A subsequent trial however failed to find that CRP was useful for determining statin benefit. [ 52 ]


In a  meta-analysis  of 20 studies involving 1,466 patients with  coronary artery disease , CRP levels were found to be reduced after exercise interventions. Among those studies, higher CRP concentrations or poorer lipid profiles before beginning exercise were associated with greater reductions in CRP. [ 53 ]


To clarify whether CRP is a bystander or active participant in  atherogenesis , a 2008 study compared people with various genetic CRP variants. Those with a high CRP due to genetic variation had no increased risk of cardiovascular disease compared to those with a normal or low CRP. [ 54 ]  A study published in 2011 shows that CRP is associated with lipid responses to low-fat and high-polyunsaturated fat diets. [ 55 ]


Arterial damage results from  white blood cell  invasion and  inflammation  within the wall. CRP is a general marker for inflammation and infection, so it can be used as a very rough proxy for heart disease risk. Since many things can cause elevated CRP, this is not a very  specific  prognostic indicator. [ 56 ] [ 57 ]  Nevertheless, a level above 2.4 mg/L has been associated with a doubled risk of a coronary event compared to levels below 1 mg/L; [ 6 ]  however, the study group in this case consisted of patients who had been diagnosed with unstable angina pectoris; whether elevated CRP has any predictive value of acute coronary events in the general population of all age ranges remains unclear. Currently, C-reactive protein is not recommended as a cardiovascular disease screening test for average-risk adults without symptoms. [ 58 ]


The  American Heart Association  and U.S.  Centers for Disease Control and Prevention  have defined risk groups as follows: [ 59 ] [ 26 ]


But hs-CRP is not to be used alone and should be combined with elevated levels of  cholesterol ,  LDL-C ,  triglycerides , and glucose level.  Smoking , hypertension and diabetes also increase the risk level of cardiovascular disease.


Scleroderma ,  polymyositis , and  dermatomyositis  elicit little or no CRP response. [ citation needed ]  CRP levels also tend not to be elevated in  systemic lupus erythematosus  (SLE) unless  serositis  or  synovitis  is present. Elevations of CRP in the absence of clinically significant inflammation can occur in  kidney failure . CRP level is an independent risk factor for atherosclerotic disease. Patients with high CRP concentrations are more likely to develop  stroke ,  myocardial infarction , and severe  peripheral vascular disease . [ 60 ]  Elevated level of CRP can also be observed in  inflammatory bowel disease  (IBD), including  Crohn's disease  and  ulcerative colitis . [ 40 ] [ 61 ]