A  left shift , meaning an increase in band neutrophils or immature granulocytes, can indicate infection,  inflammation  or bone marrow disorders, although it can also be a normal finding in  pregnancy . [ 70 ] [ 71 ]  Some laboratories do not separate bands from mature neutrophils in the differential count because the classification is highly subjective and unreliable. [ 14 ]


When present in significant quantities in the blood, immature granulocytes can indicate infection and inflammation, [ 11 ]   as well as  myeloproliferative disease ,  leukemia  and other conditions affecting the marrow. [ 73 ]  IGs may also be increased in steroid use and pregnancy. [ 11 ]   Chronic myeloid leukemia  often presents with a high number of immature granulocytes in the peripheral blood. [ 74 ]  Abnormal promyelocytes with multiple  Auer rods , called  faggot cells , occur in  acute promyelocytic leukemia . [ 75 ]


When seen on the blood smear, blast cells are an abnormal finding and may be indicative of  acute leukemia  or other serious blood disorders. Rarely, they may be seen in severe cases of left shift. The presence of Auer rods inside blast cells indicates that they are of  myeloid  origin, which has important implications for leukemia treatment. [ 10 ] [ 76 ]  Other morphologic features can provide information about the lineage of blast cells: for example,  myeloblasts  tend to be large with distinct nucleoli, while  lymphoblasts  can be smaller with a denser chromatin pattern. However, these features are not diagnostic, and flow cytometry or special staining is generally used to confirm the lineage. [ 77 ]


Before automated cell counters were introduced, cell counts were performed manually; white and red blood cells, and platelets were counted using microscopes. [ 81 ]  The first person to publish microscopic observations of blood cells was  Antonie van Leeuwenhoek , [ 82 ]  who reported on the appearance of red cells in a 1674 letter to the  Proceedings of the Royal Society of London ; [ 83 ]   Jan Swammerdam  had described red blood cells some years earlier, but had not published his findings at the time. Throughout the 18th and 19th centuries, improvements in microscope technology such as  achromatic lenses  allowed white blood cells and  platelets  to be counted in unstained samples. In the 1870s,  Paul Ehrlich  developed a staining technique that could differentiate between the five white blood cell types. Ehrlich's stain used a combination of an acidic and basic dye to stain white and red blood cells simultaneously. [ 84 ]   Dmitri Leonidovich Romanowsky  improved on this technique in the 1890s by using a mixture of  eosin  and aged  methylene blue , which produced a wide range of hues that was not present when either of the stains was used alone. This was termed the Romanowsky effect and became the basis for  Romanowsky staining , the technique that is still used to stain blood smears for manual differentials. [ 85 ]


By the early years of the 20th century, the white blood cell differential had become a common practice in the United States, but difficulties in interpreting the results cast doubt on the test's utility. [ 86 ]  In 1906, [ 87 ]  Charles Langdon Gibson introduced the Gibson chart, which compared the total white blood cell count against the neutrophil count to distinguish between " pyogenic " and "non-pyogenic" conditions and to predict the severity of infections. Around the same time,  Josef Arneth  proposed a system of classifying neutrophils by their number of nuclear lobes – termed the "lobe index" or  Arneth count  – and established a set of reference ranges for neutrophil lobularity. Arneth's analysis of neutrophil segmentation was later found to have limited clinical significance, but the association of  hypersegmented neutrophils  with  vitamin B12  and  folate deficiency  remains accepted. [ 88 ] [ 89 ] [ 90 ]   Viktor Schilling  [ de ]  in 1912 proposed a different classification of neutrophils, separating them into " myelozyten ,  jugendliche ,  stabkernige  and  segmentkernige " – that is, myelocytes, "juveniles" (metamyelocytes), band neutrophils (sometimes called "stabs"), and mature, fully segmented neutrophils – and remarked on the clinical significance of the neutrophilic left shift in conjunction with the white blood cell count and the presence of toxic changes. Schilling's monograph,  Das Blutbild und seine klinische Verwertung  ( The Blood Picture and its Clinical Significance ), was translated into English in 1926, and his neutrophil classification system quickly found acceptance in American laboratories. [ 91 ] [ 92 ]


The first automated hematology analyzer, the  Coulter counter , was invented in the early 1950s by  Wallace H. Coulter . [ 93 ] [ 94 ]  The analyzer worked on the Coulter principle, which states that when cells are suspended in a fluid carrying an  electric current  and passed through an aperture, they cause decreases in current proportional to their volume because of their poor  electrical conductivity . The number and magnitude of these decreases can be used to count blood cells and calculate their sizes. The Coulter counter was initially designed for counting  red blood cells , but it proved effective for counting white blood cells as well. [ 94 ]


After basic cell counting had been automated, the white blood cell differential remained a challenge. Research into automating the differential count began in the 1970s and took two main approaches: digital image processing and flow cytometry. Using technology developed in the 1950s and 60s to automate the reading of  Pap smears , several models of image processing analyzers were produced. [ 95 ]  These instruments would scan a stained blood smear to find cell nuclei, then take a higher resolution snapshot of the cell to analyze it through  densitometry . [ 96 ]  They were expensive, slow, and did little to reduce workload in the laboratory because they still required blood smears to be prepared and stained, so flow cytometry-based systems became more popular, [ 97 ] [ 98 ]  and by 1990, no digital image analyzers were commercially available in the United States or western Europe. [ 99 ]  These techniques enjoyed a resurgence in the 2000s with the introduction of more advanced image analysis platforms using  artificial neural networks . [ 24 ] [ 25 ]


Early flow cytometry devices shot beams of light at cells in specific wavelengths and measured the resulting absorbance, fluorescence or light scatter, collecting information about the cells' features and allowing cellular contents such as  DNA  to be quantified. [ 100 ]  One such instrument—the Rapid Cell Spectrophotometer, developed by Louis Kamentsky in 1965 to automate cervical cytology—could generate blood cell scattergrams using cytochemical staining techniques. Leonard Ornstein, who had helped to develop the staining system on the Rapid Cell Spectrophotometer, and his colleagues later created the  first commercial flow cytometric white blood cell differential analyzer, the Hemalog D. [ 101 ] [ 102 ]  Introduced in 1974, [ 103 ] [ 104 ]  this analyzer used light scattering, absorbance and cell staining to identify the five normal white blood cell types in addition to "large unidentified cells", a classification that usually consisted of  atypical lymphocytes  or blast cells. The Hemalog D could count 10,000 cells in one run, a marked improvement over the manual differential. [ 102 ] [ 105 ]
By 1977 it was estimated that "at least 200" automated differential analyzers were in use throughout the world. [ 106 ]  In 1981, Technicon combined the Hemalog D with the Hemalog-8 analyzer to produce the Technicon H6000, the first combined complete blood count and differential analyzer. This analyzer was unpopular with hematology laboratories because it was labour-intensive to operate, but in the late 1980s to early 1990s similar systems were widely produced by other manufacturers such as  Sysmex ,  Abbott ,  Roche  and  Beckman Coulter . [ 107 ]


The  whole blood clotting test  is a blood test used to check the coagulation mechanism in the blood following a  snake bite . [ 1 ]  If the test is positive after a bite in South East Asia it indicates the snake was a  viper  rather than an  elapid . [ 1 ]
It can also be used to assess the effectiveness of antivenin therapy. [ 2 ]


This test indirectly measures the severity of  defibrinogenation  in  envenomed  samples. [ 3 ]  The test is done by collecting 2 ml  of venous blood in a dry and clean glass tube. The clot and stability of the formed clot is checked after 20 minutes. [ 4 ]


The  Widal test , developed in 1896 and named after its inventor,  Georges-Fernand Widal , is an indirect  agglutination  test for  enteric fever  or  undulant fever  whereby bacteria causing typhoid fever are mixed with a serum containing specific  antibodies  obtained from an infected individual. In cases of  Salmonella  infection, the test assesses for host antibodies to the O soma antigen and the H flagellar antigen of the bacteria. [ 1 ]   False positive and false negative results may occur. Test results need to be interpreted carefully to account for any history of enteric fever,  typhoid  vaccination, and the general level of antibodies in the populations in endemic areas of the world. As with all serological tests, the rise in antibody levels needed to perform the diagnosis takes 7–14 days, which limits its applicability in early diagnosis. Other means of diagnosing  Salmonella typhi  (and  paratyphi ) include cultures of blood, urine and  faeces . These organisms produce H 2 S from thiosulfate and can be identified easily on differential media such as  bismuth sulfite agar . [ 2 ] [ 3 ] [ 4 ]   Typhidot  is the other test used to ascertain the diagnosis of  typhoid fever . A new serological test called the Tubex test is neither superior nor better performing than the Widal test. Therefore, Tubex test is not recommended for diagnosis of typhoid fever. [ 5 ]


2-mercaptoethanol  is often added to the Widal test. This agent more easily denatures the  IgM  class of  antibodies , so if a decrease in the titer is seen after using this agent, it means that the contribution of IgM has been removed leaving the  IgG  component. This differentiation of  antibody  classes is important as it allows for the distinction of a recent (IgM) from an old infection (IgG).


The Widal test is positive if TO antigen  titer  is more than 1:160 in an active infection, or if TH antigen titer is more than 1:160 in past infection or in immunized persons. A single Widal test is of little clinical relevance especially in endemic areas such as Indian subcontinent, Africa and South-east Asia. This is due to recurrent exposure to the typhoid causing bacteria, immunization and high chances of cross-reaction from infections, such as malaria and non typhoidal salmonella.  [ 6 ]


If no other tests (either bacteriologic culture or more specific serology) are available, a fourfold increase in the titer (e.g., from 1:40 to 1:640) in the course of the infection, or a conversion from an IgM reaction to an IgG reaction of at least the same titer, would be consistent with a typhoid infection. The normal widal ranges are 1:20 and 1:80, these are in the normal range anything more is a concern and you should seek medical consulation.


D-xylose absorption test  is a medical test performed to diagnose conditions that present with  malabsorption  of the proximal  small intestine [ 1 ]  due to defects in the integrity of the gastrointestinal mucosa. [ 2 ]   D-xylose  is a  monosaccharide , or simple sugar, that does not require enzymes for digestion prior to absorption. Its absorption requires an intact mucosa only. In contrast,  polysaccharides  require enzymes, such as  amylase , to break them down so that they can eventually be absorbed as monosaccharides. This test was previously in use but has been made redundant by  antibody tests . [ citation needed ]


In normal individuals, a 25 g oral dose of D-xylose will be absorbed and excreted in the urine at approximately 4.5 g in 5 hours. A decreased urinary excretion of D-xylose is seen in conditions involving the gastrointestinal mucosa, such as  small intestinal bacterial overgrowth  and  Whipple's disease . In cases of bacterial overgrowth, the values of D-xylose absorption return to normal after treatment with  antibiotics . In contrast, if the D-xylose urinary excretion is not normal after a course of antibiotics, then the problem must be due to a non-infectious cause of malabsorption (i.e.,  celiac disease ). [ citation needed ]


Carbaminohemoglobin  ( carbaminohaemoglobin   BrE ) ( CO 2 Hb , also known as  carbhemoglobin  and  carbohemoglobin ) is a  compound  of  hemoglobin  and  carbon dioxide , and is one of the forms in which carbon dioxide exists in the  blood . [ 1 ]   Twenty-three percent of carbon dioxide is carried in  blood  this way (70% is converted into   bicarbonate  by  carbonic anhydrase  and then carried in plasma, 7% carried as free CO 2 , dissolved in plasma). [ 2 ]


The structure of carbaminohemoglobin can be described as the binding of carbon dioxide to the amino groups of the globin chains of hemoglobin. This occurs at the N-terminals of the globin chains and at the amino sidebranches of  arginine  and  lysine  residues. [ 3 ]  The process of carbon dioxide binding to hemoglobin is generally known as carbamino formation. This is the source from where the protein gets its name, as it is a combination of carbamino and hemoglobin. [ 4 ]


One of the primary functions of carbaminohemoglobin is to enable the transport of carbon dioxide in the bloodstream. When carbon dioxide is produced as a waste product of cellular metabolism in tissues, the compound is diffused into the bloodstream and it works to react with hemoglobin. [ 5 ]


When the binding of molecules occurs to form carbaminohemoglobin, it allows for the transport of carbon dioxide from the tissues to the lungs. Once it is in the lungs, carbon dioxide is released from carbaminohemoglobin and can be let out from the body during the exhalation process. This complete process is very important for maintaining the balance of gases in the blood and to ensure that gas exchange is being transported between tissues and organs. [ 6 ]


Carbaminohemoglobin interacts with carbon dioxide in a process known as respiratory gas exchange. The interaction involves the binding of carbon dioxide to hemoglobin. Carbon dioxide binds to the protein chains of hemoglobin. The ability of hemoglobin to bind to both oxygen and carbon dioxide molecules is what makes it an important protein to the respiratory system in respiratory gas exchange.


The interactions between carbon dioxide and hemoglobin helps in the transport of carbon dioxide from the tissues to the lungs for eliminations. When carbon dioxide is transported from the tissues, it is produced as a waste product of a set of reactions known as cellular metabolism. Most importantly, the binding of carbon dioxide to hemoglobin plays a part in the buffering of blood pH by preventing the drop of pH due to the production of carbonic acid. [ 6 ]


Although, the carbaminohemoglobin protein interacts with another protein (like hemoglobin) found in red blood cells, this interaction only takes place in the bloodstream and its products can be expelled. Carbaminohemoglobin does not interact with DNA since DNA is a molecule that is found in cell nucleus and its function is to carry genetic information. [ 7 ]


The formation and dissociation of the protein carbaminohemoglobin are controlled by many factors to guarantee the transport of carbon dioxide to the blood stream. A list of regulatory factors are listed below:


When the tissues release carbon dioxide into the bloodstream, around 10% is dissolved into the plasma. The rest of the carbon dioxide is carried either directly or indirectly by  hemoglobin . Approximately 10% of the carbon dioxide carried by hemoglobin is in the form of carbaminohemoglobin. This carbaminohemoglobin is formed by the reaction between carbon dioxide and an amino (-NH 2 ) residue from the globin molecule, resulting in the formation of a carbamino residue (-NH.COO − ). The rest of the carbon dioxide is transported in the plasma as bicarbonate anions. [ 12 ]


When carbon dioxide binds to hemoglobin, carbaminohemoglobin is formed, lowering hemoglobin's affinity for  oxygen  via the  Bohr effect . The reaction is formed between a carbon dioxide molecule and an amino residue. [ 12 ]  In the absence of oxygen, unbound hemoglobin molecules have a greater chance of becoming carbaminohemoglobin. The  Haldane effect  relates to the increased affinity of de-oxygenated hemoglobin for  H + : offloading of oxygen to the tissues thus results in increased affinity of the hemoglobin for  carbon dioxide ,  and  H + , which the body needs to get rid of, which can then be transported to the lung for removal. Because the formation of this compound generates hydrogen ions, haemoglobin is needed to buffer it. [ 12 ]


Hemoglobin can bind to four molecules of carbon dioxide. The carbon dioxide molecules form a  carbamate  with the four terminal-amine groups of the four protein chains in the deoxy form of the molecule. Thus, one hemoglobin molecule can transport four carbon dioxide molecules back to the lungs, where they are released when the molecule changes back to the oxyhemoglobin form.  [ 6 ]


When carbon dioxide diffuses as a dissolved gas from the tissue capillaries, it binds to the α-amino terminus of the globulin chain, forming Carbaminohemoglobin. Carbaminohemoglobin is able to directly stabilise the T conformation as part of the carbon dioxide  Bohr effect . Deoxyhemoglobin in turn subsequently increases the uptake of carbon dioxide in the form of favouring the formation of Bicarbonate as well as Carbaminohemoglobin through the  Haldane effect . [ 13 ]


Dysfunctional or altered levels of carbaminohemoglobin do not generally cause disease or disorders. Carbaminohemoglobin is a part of the carbon dioxide transport process in the body. The levels of this protein can decrease and increase based on factors that regulate the protein in the body. [ 14 ]


A way that carbaminohemoglobin can be associated with disease is when there is a change in its level caused by a pre-existing condition or imbalance in the respiratory and metabolic systems of the human body.


Some of these existing medical conditions can be the following:


The protein carbaminohemoglobin plays an important role in the transport of carbon dioxide in the blood, and its biologically important in many functions:


Carboxyhemoglobin  ( carboxyhaemoglobin   BrE ) (symbol  COHb  or  HbCO ) is a stable  complex  of  carbon monoxide  and  hemoglobin  (Hb) that forms in  red blood cells  upon contact with carbon monoxide. Carboxyhemoglobin is often mistaken for the compound formed by the combination of carbon dioxide ( carboxyl ) and hemoglobin, which is actually  carbaminohemoglobin . Carboxyhemoglobin terminology emerged when carbon monoxide was known by its historic name, "carbonic oxide", and evolved through Germanic and British English etymological influences; the preferred  IUPAC  nomenclature is  carbonylhemoglobin . [ 2 ] [ 3 ] [ 4 ]


The average non-smoker maintains a systemic carboxyhemoglobin level under 3% COHb whereas smokers approach 10% COHb. [ 4 ]  The biological threshold for carboxyhemoglobin tolerance is 15% COHb, meaning toxicity is consistently observed at levels in excess of this concentration. [ 5 ]  The  FDA  has previously set a threshold of 14% COHb in certain  clinical trials  evaluating the therapeutic potential of carbon monoxide. [ 6 ]


The average  red blood cell  contains 250 million  hemoglobin  molecules. [ 7 ]  Hemoglobin contains a  globin  protein unit with four  prosthetic   heme  groups (hence the name  heme   -o-   globin ); each heme is capable of reversibly binding with one gaseous molecule (oxygen, carbon monoxide, cyanide, etc.), [ 8 ]  therefore a typical red blood cell may carry up to one billion gas molecules. As the binding of carbon monoxide with hemoglobin is reversible, certain models have estimated that 20% of the carbon monoxide carried as carboxyhemoglobin may dissociate in remote tissues. [ 7 ]


In biology, carbon monoxide is naturally produced through many enzymatic and non-enzymatic pathways. [ 7 ]  The most extensively studied pathway is the metabolism of  heme  by  heme oxygenase  which occurs throughout the body with significant activity in the spleen to facilitate  hemoglobin  breakdown during  erythrocyte  recycling. Therefore heme can both carry carbon monoxide in the case of carboxyhemoglobin, or, undergo enzymatic catabolism to generate carbon monoxide.


Carbon monoxide was characterized as a neurotransmitter in 1993 and has since been subcategorized as a  gasotransmitter . [ 4 ]


Most endogenously produced carbon monoxide is stored as carboxyhemoglobin. The gas primarily undergoes  pulmonary  excretion, however trace amounts may be oxidized to  carbon dioxide  by certain  cytochromes , metabolized by resident  microbiota , or excreted by transdermal diffusion. [ 4 ] [ 7 ]


Compared to oxygen, carbon monoxide binds with approximately 240 times greater affinity, [ 9 ] [ 4 ]  however the affinity of carbon monoxide for hemoglobin varies both across species and within a species. In the 1950s,  Esther Killick  was among the first to recognize a difference in carbon monoxide affinity between adult and  foetal  blood, and a difference between humans and sheep. [ 4 ] [ 10 ] [ 11 ]  In humans, the Hb-Kirklareli mutation has a relative 80,000 times greater affinity for carbon monoxide than oxygen resulting in systemic carboxyhemoglobin reaching a sustained level of 16% COHb. [ 5 ]  Other human mutations have been described (see also:  hemoglobin variants ). [ 12 ] [ 13 ]  Structural variations and mutations across other  hemoproteins  likewise affect carbon monoxide's interaction with the heme prosthetic group as exemplified by  Cytochrome P450  where certain forms of the  CYP3A   family  is relatively less affected by the inhibitory effects of carbon monoxide. [ 4 ]


Murinae  species have a COHb half-life of 20 minutes compared to 300 minutes for a typical human (see  § Toxicokinetics ). [ 4 ]  As a result, the metabolic kinetics, blood saturation point, and tolerance for carbon monoxide exposure vary across species, potentially leading to data inconsistencies pertaining to the  toxicology  of  carbon monoxide poisoning  and  pharmacology  of low-dose therapeutic protocols. [ 4 ]


Some deep-diving marine mammal species are known to contain concentrations of carbon monoxide in their blood that resembles levels seen in chronic cigarette smokers, which may provide benefits against hypoxia. [ 14 ]  Similarly, the elevated levels in smokers has been suggested to be a basis for the  smoker's paradox . [ 4 ]  Prolonged exposure to carbon monoxide and elevated carboxyhemoglobin, such as in smoking, results in  erythremia . [ 4 ]  Furthermore, humans can  acclimate  to toxic levels of carbon monoxide based on findings reported by  Esther Killick . [ 4 ]


A bright red skin complexion is commonly associated with elevated carboxyhemoglobin levels. Trace evidence for an endogenous presence of carbon monoxide dates back to  Marcellus Donato  circa 1570 who noted an unusually red complexion upon conducting an autopsy of victims who died from charcoal fumes in  Mantua . [ 4 ]  Similar findings pertaining to red complexion later emerged as documented by  Johann Jakob Wepfer  in the 1600s, and  M.   Antoine Portal  in the late 1700s. [ 4 ]


Phlogiston theory  is a trace origin for the first chemical explanations of endogenous carboxyhemoglobin exemplified by the work of  Joseph Priestley  in the eighteenth century who suspected phlogiston to be a cellular waste product carried by the blood of animals which was subsequently exhaled. [ 4 ]


Thomas Beddoes ,  James Watt ,  Humphry Davy ,  James Lind , and many others investigated the therapeutic potential of inhaling  factitious airs  in the late eighteenth century (see also:  Pneumatic Institution ). Among the gases experimented with,  hydrocarbonate  had received significant attention. Hydrocarbonate is  water gas  generated by passing steam over  coke , the process of which generates carbon monoxide and hydrogen, and some considered it contain  phlogiston . Beddoes and Watt recognized hydrocarbonate brightened venous blood in 1793. Watt suggested coal fumes could act as an  antidote  to the oxygen in blood, and Beddoes and Watt likewise speculated hydrocarbonate has a greater affinity for animal fiber than oxygen in 1796. [ 4 ]


After the discovery of carbon monoxide by  William Cruickshank  in 1800,  Johann Dömling  (1803) and  John Bostock  (1804) developed hypotheses suggesting blood returned to the heart loaded with carbon monoxide to subsequently be oxidized to carbon dioxide in the lung prior to exhalation. [ 4 ]  Later in 1854,  Adrien Chenot  similarly suggested carbon monoxide could remove oxygen from blood and be oxidized within the body to carbon dioxide. [ 4 ]  The mechanism for carbon monoxide poisoning in the context of carboxyhemoglobin formation is widely credited to  Claude Bernard  whose memoirs beginning in 1846 and published in 1857 notably phrased, "prevents arterials blood from becoming venous". [ 4 ]   Felix Hoppe-Seyler  independently published similar conclusions in the following year.


The first analytical method to detect carboxyhemoglobin emerged in 1858 with a colorimetric method developed by  Felix Hoppe-Seyler , and the first quantitative analysis method emerged in 1880 with  Josef von Fodor . [ 4 ]


Carbon  is derived from the Latin term  carbo , meaning coal, via the French  charbone , which first appeared in print in 1786. [ 15 ]  The  etymology of oxygen  is generally accepted mean 'acid' based on Lavoisier's system, which also recognized carbon as a nonmetallic element capable of oxidation, although the original degrees of oxides were based on diamond, graphite, coal and  carbonic acid (CO 2 )  as the most oxidized form; [ 15 ]  Lavoisier's system was superseded by other obsolete oxide nomenclature systems. [ 16 ]


Upon discovering carbon monoxide through a series of experiments originating from  coke  (short for coal-cake [ 15 ] ),  Cruickshank  named the new molecule "gaseous oxide of carbon" which evolved to "carbonic oxide" and was translated into German as "kohlenoxyd". Kohlen is the German word for coal. [ 4 ] [ 17 ]  As carbonic acid (CO 2 ) was considered to be the most highly oxidized form in Lavoisier's system, the name carbonic oxide implied an intermediate oxidized species between coal and carbonic acid (i.e. use of the word acid indicated maximum oxidation).


Haem is derived from Greek  meaning blood, [ 18 ] [ 19 ]  and globin is Latin derived from  globus  typically accepted to mean glob/spherical/round object; the terms are conjoined with an  -o-  . Regarding haem,  the use of "ae / æ" remains prevalent in British English  in modern day [ 20 ]  whereas the American English spelling evolved to heme from hema. [ 19 ]


Felix Hoppe-Seyler  coined the name "hämoglobin" in 1864. [ 21 ]  In German, an  umlaut  such as  ä  is synonymous with spelling as "ae", therefore hämoglobin is commonly spelled as haemoglobin throughout German literature, hence haemoglobin is the term adopted by English literature.


Hoppe-Seyler likewise coined the name Kohlenoxydhämoglobin [ 22 ]  which may have similarly been directly translated back into English as "carbonic oxide hæmoglobin". [ 23 ]  The term carboxyhæmoglobin appeared as early as 1895 in works by  John Haldane  while the name for CO was still widely regarded as carbonic oxide. [ 24 ]


The term "carbon monoxide" was formally introduced in 1879, but the name would not become mainstream for several decades. [ 4 ]  Variations of COHb terminology, such as carbonmonoxyhemoglobin, [ 25 ] [ 11 ]  followed and eventually evolved and simplified back into "carboxyhemoglobin".


As carboxy is now firmly associated with the CO 2   carboxyl  group, and carbon monoxide is generally regarded as a  carbonyl , IUPAC has recommended "carbonylhemoglobin" as the preferred COHb nomenclature. [ 4 ]  Despite the IUPAC guidance, carboxyhemoglobin remains the most widely used term (akin to the  survival  of  bicarbonate  nomenclature).


Historically, carboxyhemoglobin detection has been achieved by  colorimetric analysis , chemical reactivity,  spectrophotometry , gasometric and  thermoelectric  detection methods. [ 4 ]   Gas chromatography  analysis emerged in 1961 and remains a commonly used method. [ 4 ]


Modern methods include  pulse oximetry  with a  CO-oximeter , and a variety of other analytical techniques. [ 26 ] [ 27 ]  Most methods require laboratory equipment, skilled technicians, or expensive electronics therefore rapid and economical detection technologies remain in development.


Breath carbon monoxide  is another detection method that may correlate with carboxyhemoglobin levels. [ 28 ]


Carbon monoxide poisoning, also known as carboxyhemoglobinemia, [ 29 ] [ 30 ]  has plagued humankind since primitive ancestors first harnessed fire. In modern times, carboxyhemoglobin data assist physicians in making a poisoning diagnosis. However, carboxyhemoglobin levels do not necessarily correlate with the symptoms of carbon monoxide poisoning. [ 31 ]  In general, 30% COHb is considered severe carbon monoxide poisoning. [ 4 ]  The highest reported non-fatal carboxyhemoglobin level was 73% COHb. [ 4 ]


Gas exchange  is an essential process for many organisms to maintain homeostasis. Oxygen accounts for about 20% of Earth's atmospheric  air . While inhaling air is critical to supply cells with oxygen for  aerobic respiration  via the  Bohr effect  and  Haldane effect  (and perhaps local low oxygen partial pressure e.g. active muscles), [ 32 ]  exhaling the cellular waste product  carbon dioxide  is arguably the more critical aspect of respiration. Whereas the body can tolerate brief periods of  hypoxia  (as commonly occurs in  anaerobic exercise , although the brain, heart, liver and kidney are significantly less tolerant than skeletal muscle), failure to expel carbon dioxide may cause  respiratory acidosis  (meaning bodily fluids and blood become too acidic thereby affecting homeostasis). [ 33 ]  In absence of oxygen, cells switch to  anaerobic respiration  which if prolonged may significantly increase  lactic acid  leading to  metabolic acidosis . [ 34 ]


To provide a simplified synopsis of the molecular mechanism of systemic gas exchange, upon inhalation of air it was widely thought oxygen binding to any of the heme sites triggers a  conformational change  in the protein unit of hemoglobin which then enables the binding of additional oxygen to each of the other heme sites. Upon arrival to the cellular region, oxygen is released at the tissue due to a conformational change in hemoglobin as caused by ionization of hemoglobin's surface due to the "acidification" of the tissue's local pH (meaning a relatively higher concentration of 'acidic' protons / hydrogen  ions  annotated as H + ; an acidic pH is commonly referenced to as either low pH based on the acidity of pH 1-7 having a  low  number, or, referred to as a high pH due to the  high  concentration of H +  ions as the scale approaches pH 1); the local acidity is caused by an increase in the biotransformation of carbon dioxide waste into  carbonic acid  via  carbonic anhydrase . In other words, oxygenated arterial blood arrives to cells in the " hemoglobin R-state " which has deprotonated/unionized amino acid  residues  (regarding hemoglobin's  amines  transitioning between the deprotonated/unionized Hb-NH2 to the protonated/ionized Hb-NH3 +  state) based on the less-acidic pH (arterial blood averages pH 7.407 whereas venous blood is slightly more acidic at pH 7.371 [ 35 ] ). The "T-state" of hemoglobin is deoxygenated in venous blood partially due to protonation/ionization as caused by the acidic environment hence causing a conformation unsuited for oxygen-binding [ 36 ]  (i.e. oxygen is 'ejected' upon arrival at the cell due to H +  ions bombarding the hemoglobin surface residues to convert Hb from "R-state" to "T-state"). Furthermore, the mechanism for formation of  carbaminohemoglobin  generates additional H +  ions that may further stabilize the protonated/ionized deoxygenated hemoglobin. Upon return of venous blood into the lung and subsequent exhalation of carbon dioxide, the blood is "de-acidified" (see also:  hyperventilation ) for the deprotonation/unionization of hemoglobin to re-enable oxygen binding as part of the transition to arterial blood (note this process is complex due to involvement of  chemoreceptors , pH buffers and other physiochemical functionalities). Carbon monoxide poisoning disturbs this physiological process hence the venous blood of poisoning patients is bright red akin to arterial blood since the carbonyl/carbon monoxide is retained, whereas deoxygenated hemoglobin is dark red and carbaminohemoglobin has a blue hue. [ 13 ]


At toxic concentrations, carbon monoxide as carboxyhemoglobin significantly interferes with respiration and gas exchange by simultaneously inhibiting acquisition and delivery of oxygen to cells, and preventing formation of  carbaminohemoglobin  which accounts for approximately 30% of carbon dioxide exportation. [ 37 ]  Therefore a patient suffering from carbon monoxide poisoning may experience severe  hypoxia  and  acidosis  in addition to the toxicities of excess carbon monoxide binding to numerous hemoproteins, metallic and non-metallic targets which affect cellular machinery (such as inhibition of  cytochrome c oxidase ). [ 7 ] [ 38 ]


In common air under normal atmospheric conditions, a typical patient's carboxyhemoglobin has a half-life around 300 minutes. [ 4 ]  This time can be reduced to 90 minutes upon administration of high-flow pure oxygen, and the time is further reduced when oxygen is administered with 5% carbon dioxide as first identified by  Esther Killick . [ 4 ]  Additionally, treatment in a  hyperbaric chamber  is a more effective manner of reducing the half-life of carboxyhemoglobin to 30 minutes [ 4 ]  and allows oxygen to dissolve in biological fluids for delivery to tissues. [ citation needed ]


Supplemental oxygen takes advantage of  Le Chatelier's principle  to quicken the decomposition of carboxyhemoglobin back to hemoglobin: [ 39 ]


As carbon monoxide is now understood to have a therapeutic potential, pharmaceutical efforts have focused on development of  carbon monoxide-releasing molecules  and selective  heme oxygenase  inducers. [ 40 ]


An alternative method for drug delivery consists of carbon monoxide immobilized on  polyethylene glycol  (PEG)-lyated  bovine  carboxyhemoglobin which is currently in late clinical development. Similarly,  maleimide  PEG conjugated human carboxyhemoglobin had previously been the subject of pharmaceutical development. [ 41 ]


The human embryonic haemoglobins were discovered in 1961. [ 1 ] [ 2 ]  These include Hb-Gower 1, consisting of 2 zeta chains and 2 epsilon chains, and Hb-Gower 2, which consists of 2 αlpha-chains and 2 epsilon-chains, the zeta and epsilon chains being the embryonic haemoglobin chains.


Embryonic hemoglobin  is a  tetramer  produced in the  blood islands  in the embryonic  yolk sac  during the  mesoblastic stage  (from 3rd week of pregnancy until 3 months). The protein is commonly referred to as  hemoglobin ε .


Chromosomal abnormalities  can lead to a delay in switching from embryonic hemoglobin. [ 3 ]


Hemoglobin Gower 1  (also referred to as  ζ 2 ε 2  or  HbE Gower-1 ) is a form of hemoglobin existing only during embryonic life, and is the primary embryonic hemoglobin.  It is composed of two  zeta chains  and two  epsilon chains , and is relatively unstable, breaking down easily. [ 4 ]


Hemoglobin Gower 2  (also referred to as  α 2 ε 2  or  HbE Gower-2 ) is a form of hemoglobin existing at low levels during embryonic and fetal life.  It is composed of two alpha chains and two epsilon chains, and is somewhat unstable, though not as much as hemoglobin Gower 1. [ 4 ]   Due to its relative stability compared to hemoglobin Gower 1 and  hemoglobin S , it has been proposed as a subject for reactivation in the adult in cases of severe  β thalassemia  and  hemoglobinopathies  in subjects for which the reactivation of  hemoglobin F  is contraindicated due to toxicity concerns. [ 4 ]


Hemoglobin Portland I  (also referred to as  ζ 2 γ 2  or  HbE Portland-1 ) is a form of hemoglobin existing at low levels during embryonic and fetal life, composed of two zeta chains and two gamma chains. [ 4 ]


Hemoglobin Portland II  (also referred to as  ζ 2 β 2  or  HbE Portland-2 ) is a form of hemoglobin existing at low levels during embryonic and fetal life, composed of two zeta chains and two beta chains.  It is quite unstable, more so than even hemoglobin Gower 1, and breaks down very rapidly under stress. [ 4 ]   Despite this, it has been proposed as a candidate for reactivation in cases of severe  α thalassemia  or hemoglobinopathies afflicting the alpha chain. [ 4 ] [ 5 ]


Fetal hemoglobin , or  foetal haemoglobin  (also  hemoglobin F ,  HbF , or  α 2 γ 2 ) is the main  oxygen   carrier protein  in the human  fetus . Hemoglobin   F is found in fetal  red blood cells , and is involved in transporting oxygen from the mother's  bloodstream  to organs and tissues in the fetus. It is produced at around 6   weeks of pregnancy  [ 1 ]  and the levels remain high after birth until the baby is roughly 2–4   months old. [ 2 ]  Hemoglobin   F has a different composition than adult forms of  hemoglobin , allowing it to bind (or attach to) oxygen more strongly; this in turn enables the developing fetus to retrieve oxygen from the mother's bloodstream, which occurs through the  placenta  found in the mother's  uterus . [ 3 ]


In the newborn, levels of hemoglobin F gradually decrease and reach adult levels (less than 1% of total hemoglobin) usually within the first year, as adult forms of hemoglobin begin to be produced. [ 4 ]  Diseases such as  beta thalassemias , which affect components of the  adult hemoglobin , can delay this process, and cause hemoglobin F levels to be higher than normal. [ 5 ]  In  sickle cell anemia , increasing the production of hemoglobin F has been used as a treatment to relieve some of the symptoms. [ 6 ]


Hemoglobin F, like adult hemoglobin ( hemoglobin A  and  hemoglobin A2 ), has four  subunits  or chains. Each subunit contains a  heme  group with an iron element which is key in allowing the binding and unbinding of oxygen. As such, hemoglobin F can adopt two states:  oxyhemoglobin  (bound to oxygen) and  deoxyhemoglobin  (without oxygen). As hemoglobin F has 4 heme groups, it can bind to up to four oxygen molecules. [ 7 ]  It is composed of  two α (alpha)  subunits and  two γ (gamma)  subunits, whereas hemoglobin A (97% of total hemoglobin in adults) is composed of two α and two β (beta) subunits.


In humans, the α subunit is encoded on  chromosome 16  and the γ subunit is encoded on  chromosome 11 . There are two very similar  genes  that code for the α subunit,  HBA1  and  HBA2 . The protein that they produce is identical, but they differ in gene regulatory regions that determine when or how much of the protein is produced. This leads to HBA1 and HBA2 contributing 40% and 60%, respectively, of the total α subunits produced. As a consequence, mutations on the  HBA2  gene are expected to have a stronger effect than mutations on the  HBA1  gene. [ 8 ]
There are also two similar copies of the gene coding for the γ subunit,  HBG1  and  HBG2 , but the protein produced is slightly different, just in one  protein unit :  HBG1  codes for the protein form with an  alanine  at position 136, whereas  HBG2  codes for a  glycine [ 9 ]
BCL11A and ZBTB7A are major repressor proteins of hemoglobin F production, by binding to the gene coding for the γ subunit at their promoter region. [ 10 ]  This happens naturally as the newborn baby starts to switch from producing hemoglobin F to producing hemoglobin A.
Some genetic diseases can take place due to mutations to genes coding for components of hemoglobin F. Mutations to  HBA1  and  HBA2  genes can cause  alpha-thalassemia [ 11 ]  and mutations to the promoter regions of  HBG1  and  HBG2  can cause hemoglobin F to still be produced after the switch to hemoglobin A should have occurred, which is called  hereditary persistence of fetal hemoglobin . [ 10 ]


During the first 3 months of pregnancy, the main form of hemoglobin in the embryo/fetus is  embryonic hemoglobin , which has 3 variants depending on the types of subunits it contains. The production of hemoglobin F starts from week 6, but it's only from 3 months onwards that it becomes the main type found in fetal red blood cells. [ 4 ]
The switch to produce adult forms of hemoglobin (essentially hemoglobin A) starts at around 40 weeks of gestation, which is close to the expected time of birth. [ 1 ]  At birth, hemoglobin F accounts for 50-95% of the infant's hemoglobin and at around 6 months after birth, hemoglobin A becomes the predominant type. By the time the baby is one year old, the proportions of different types of hemoglobin are expected to approximate the adult levels, with hemoglobin F reduced to very low levels. [ 4 ]  The small proportion of red blood cells containing hemoglobin F are called F-cells, which also contain other types of hemoglobin.


In healthy adults, the composition of hemoglobin is hemoglobin A (~97%), hemoglobin A2 (2.2 - 3.5%) and hemoglobin F (<1%). [ 12 ]


Certain genetic abnormalities can cause the switch to adult hemoglobin synthesis to fail, resulting in a condition known as  hereditary persistence of fetal hemoglobin .


The four hemes, which are the oxygen-binding parts of hemoglobin, are similar between hemoglobin F and other types of hemoglobin, including hemoglobin A. Thus, the key feature that allows hemoglobin F to bind more strongly to oxygen is by having γ subunits (instead of β, for example). In fact, some naturally existing molecules in our body can bind to hemoglobin and change its binding affinity for oxygen. One of the molecules is  2,3-bisphosphoglycerate (2,3-BPG)  and it enhances hemoglobin's ability to release oxygen. [ 13 ]  2,3-BPG interacts much more with hemoglobin A than hemoglobin F. This is because the adult β subunit has more positive charges than the fetal γ subunit, which attract the negative charges from 2,3-BPG. Due to the preference of 2,3-BPG for hemoglobin A, hemoglobin F binds to oxygen with more affinity, in average. [ 14 ]


Hemoglobin Barts  is an abnormal form of hemoglobin produced in hemoglobin Barts syndrome or alpha-thalassemia major, the most severe form of  alpha-thalassemia . Alpha-thalassemia is a genetic blood disorder and one of the most common hemoglobin-related diseases, affecting the production of α subunits from hemoglobin. [ 15 ]  Depending on how many genes coding for the α subunit are impacted (between one and four), patients with this disease can have reduced to no production of the α subunit of the hemoglobin. As a consequence, less hemoglobin is available and this affects oxygen supply to the tissues.
Hemoglobin Barts syndrome manifests when all four genes coding for α subunit are deleted. This is often fatal for the fetus carrying the disorder, as in the absence of α subunits, a form of hemoglobin with four γ subunits, hemoglobin Barts, is produced. This form of hemoglobin isn't fit for oxygen exchange precisely due to its very high affinity for oxygen.  While hemoglobin Barts is very efficient at binding oxygen, it doesn't release oxygen to the organs and tissues. [ 16 ]  The disease is fatal for the fetus or newborn unless early diagnosis and intervention is carried out during pregnancy, and the child will be dependent on lifelong blood transfusions.


To quantify how strongly a certain type of hemoglobin binds to oxygen (or its affinity for oxygen), a parameter called P50 is often used. In a given situation, P50 can be understood as the partial pressure of oxygen at which Hb is 50% saturated. [ 17 ]  For example, Hemoglobin F has a lower P50 than hemoglobin A. This means that if we have the same amount of hemoglobin F and hemoglobin A in the blood and add oxygen to it, half of hemoglobin F will bind to oxygen before half of hemoglobin A manages to do so. Therefore, a lower P50 means stronger binding or higher affinity for oxygen.


For reference, the P50 of fetal hemoglobin is roughly 19 mmHg (a measure of pressure), whereas adult hemoglobin is approximately 26.8 mmHg (see  Blood gas tension ). [ 18 ]


During pregnancy, the mother's circulatory system delivers oxygen and nutrients to the fetus and carries away nutrient-depleted blood enriched with carbon dioxide. The maternal and fetal blood circulations are separate and the exchange of molecules occurs through the placenta, in a region called  intervillous space  which is located in between maternal and fetal blood vessels. [ 3 ]


Focusing on oxygen exchange, there are three important aspects that allow oxygen to pass from the maternal circulation into the fetal circulation. Firstly, the presence of hemoglobin F in the fetus allows a stronger binding to oxygen than maternal hemoglobin (see  Factors affecting oxygen affinity ). Secondly, the mother's bloodstream is richer in oxygen than that of the fetus, so oxygen naturally flows towards the fetal circulation by diffusion. [ 19 ]  The final factor is related to the effects of pH on maternal and fetal hemoglobin. As the maternal blood acquires more carbon dioxide, it becomes more acidic and this favors the release of oxygen by the maternal hemoglobin. At the same time, the decrease in carbon dioxide in fetal blood makes it more alkaline and favors the uptake of oxygen. This is called the Bohr effect or  Haldane effect , which also happens in the air exchange in the lungs. [ 20 ]  All of these three factors are present simultaneously and cooperate to improve the fetus’ access to oxygen from the mother.


F-cells are the subpopulation of red blood cells that contain hemoglobin F, in amongst other types of hemoglobin. While common in fetuses, in normal adults, only around 3-7% of red blood cells contain hemoglobin F. [ 21 ]  The low percentage of F-cells in adults owes to two factors: very low levels of hemoglobin F being present and its tendency to be produced only in a subset of cells rather than evenly distributed amongst all red blood cells. In fact, there is a positive correlation between the levels of hemoglobin F and number of F-cells, with patients with higher percentages of hemoglobin F also having a higher proportion of F-cells. [ 22 ]   Despite the correlations between hemoglobin F levels and F-cell numbers, usually they are determined by direct measurements. While the amount of hemoglobin F is calculated using cell lysates, which are fluids with contents of cells that were broken open, F-cell numbers are done by counting intact red blood cells. [ 21 ]


Due to the correlation between the amount of hemoglobin F and F-cells, F-cell numbers are higher in some inherited hemoglobin disorders, including  beta-thalassemia ,  sickle cell anemia  and  hereditary persistence of fetal hemoglobin . Additionally, some acquired conditions can also have higher F-cell numbers, such as acute erythropoietic stress (response to poor oxygenation which includes very rapid synthesis of new red blood cells) [ 23 ]  and pregnancy. [ 21 ]
F-cells have similar mass of haemoglobin per cell compared to red blood cells without haemoglobin F, which is measured  mean cell haemoglobin  values (MCH). [ 24 ]


There is a significant increase in hemoglobin F levels during early pregnancy. However, it's not clear whether these levels are stable or decrease as the pregnancy goes on, as different sources reported different results. [ 25 ] [ 26 ]
The increase in hemoglobin F then induces a 3 to 7 fold increase in the number of F-cells in pregnant women, which was observed between the 23rd to 31st week of gestation. [ 27 ]
However, as to the reason of the increase in hemoglobin F levels in pregnant women, there doesn't seem to be conclusive evidence. While an early study suggested that maternal red blood cells switch on hemoglobin F production during pregnancy, [ 27 ]  more recent literature suggested that the increase in haemoglobin F could be, at least in part, due to fetal red blood cells being transferred to the maternal circulation. [ 28 ] [ 21 ]


Presence of high levels of hemoglobin F in pregnant women can impact the growth of the fetus, as fetal red blood cells struggle to compete for the oxygen from the mother's circulation. This is because instead of competing with hemoglobin A, which has a weaker association to oxygen than hemoglobin F, it becomes a competition between fetal and maternal hemoglobin F which have similar affinities for oxygen. As a result, women with hemoglobin F as >70% of total hemoglobin are much more likely to have fetuses that are small for their gestational age compared women with <70% hemoglobin F (at a rate of 100% compared to 8%, respectively). [ 29 ]


This is a rare benign genetic disease where production of hemoglobin F persists after twelve months of life and into the adulthood. As a result, hemoglobin F is present in a higher number of adult red blood cells than normal. [ 30 ]
It doesn't present symptoms and is usually discovered when screening for other blood-related diseases.
In this condition, the genes coding for the γ subunit (HBG1 and HBG2) are not suppressed shortly before birth. This can happen when a mutation occurs in the promoter region of HBG1 and HBG2, preventing the binding of BCL11A and ZBTB7A proteins. These proteins would normally bind and suppress the production of γ subunits and as they can't bind due to the mutation, γ subunits continue to be produced. [ 10 ]  There are two types of patients with HPFH: either with one normal copy of the gene and one disease form or with two disease copies. Whereas normal adults have less than 1% of hemoglobin F, patients with only one disease gene have 5-30%. Patients with two disease copies can have hemoglobin F in up to 100% of red blood cells. [ 31 ]
As other diseases such as sickle cell disease could also cause a higher level of hemoglobin F to be present, it can sometimes be misdiagnosed. [ 32 ]


Delta beta-thalassemia is a rare genetic blood disorder in which the production of both δ and β subunits are reduced or absent. In these cases, the production of the γ subunit increases to compensate for the loss of δ and β subunits, resulting in a higher amount of hemoglobin F present in the blood. Normally,  people have two sets of genes for producing δ and β subunits. People with only one set of working genes don't get any symptoms and in the rarely reported cases where both sets of genes are affected, the patients only experienced mild symptoms. [ 33 ]


The discovery that hemoglobin F alleviated the symptoms of sickle cell disease occurred in 1948. Janet Watson observed that red blood cells from infants with the disease took longer to sickle and did not deform as much compared to their mother's cells, which carried the disease trait. Later, it was noted that patients with sickle cell trait as well as hereditary persistence of hemoglobin F (HPFH) didn't have symptoms. [ 34 ]  Additionally, in sickle cell patients, F-cells were found to be more long living than non-F cells as they contain hemoglobin F.


When fetal hemoglobin production is switched off after birth, normal children begin producing adult hemoglobin (HbA). Children with  sickle-cell disease  begin producing a defective form of hemoglobin called  hemoglobin S  instead, which form chains that cause  red blood cells  to change their shape from round to  sickle -shaped. [ 35 ]
These defective red blood cells have a much shorter life span than normal red blood cells (10–20 days compared to up to 120 days). [ 36 ]  They also have a greater tendency to clump together and block small  blood vessels , preventing blood supply to tissues and organs. This leads to the so-called  vaso-occlusive crisis , which is a hallmark of the disease. [ 37 ]
If fetal hemoglobin remains relatively high after birth, the number of painful episodes decreases in patients with sickle-cell disease and they have a better prognosis. [ 38 ]
Fetal hemoglobin's role in reducing disease severity comes from its ability to disrupt the formation of hemoglobin S chains within red blood cells. [ 39 ]
Interestingly, while higher levels of hemoglobin F were associated with improvement of some symptoms, including the frequency of painful episodes, leg ulcers and the general severity of the disease, it had no correlation to others. A few examples are  priapism , stroke and systemic blood pressure. [ 34 ]   As hemoglobin F are only produced by some red blood cells, in different quantities, only a subpopulation of cells are protected against sickling. It could be that the symptoms that high hemoglobin F doesn't prevent are quite sensitive to the rupture of the sickled non-F cells. [ 34 ]


Hydroxyurea  is a chemical that promotes the production of fetal hemoglobin and reduces the premature rupturing of red blood cells. [ 6 ] [ 40 ]  Combination therapy with hydroxyurea and recombinant  erythropoietin  — rather than treatment with hydroxyurea alone — has been shown to further elevate hemoglobin F levels and to promote the development of HbF-containing F-cells. [ 41 ]


There have been some studies evaluating the possibility of using hemoglobin F as an indicator of the prognosis for cancer. It has been suggested that elevated concentrations of haemoglobin F can be found in main kinds of solid tumours and blood cancers. [ 42 ]
Examples include acute lymphoblastic leukemia and myeloid leukemia in children, where higher concentrations of hemoglobin F were associated with a worse outcome, including a higher risk of relapse or death. [ 43 ]
Other cancer types where higher hemoglobin F levels have been observed are transitional cell cancer, [ 44 ]  colorectal carcinoma [ 45 ]
and various types of blastomas. [ 46 ]  In fact, in several types of blastomas, including neuroblastoma and retinoblastoma (affecting the nerve cells and the eyes, respectively), F-cells were found in newly formed blood vessels and spaces in between tumour cells. Clusters of F-cells were also present in the  bone marrow  of some of these patients. [ 46 ]
Interestingly, hemoglobin F is not directly produced by tumour cells, but seems to be induced by the biological environment of the cancer in nearby blood cells. A reason suggested for this increase in hemoglobin F is that it may favor cancer growth by providing better oxygen supply to the developing cancerous cells. [ 44 ]  In adults, increased hemoglobin F production is thought to be caused by factors leading to the activation of the gene coding for the γ subunit, such as  DNA demethylation  (which can activate normally silent genes and is a hallmark of cancer). [ 47 ]


1SHR ,  1SI4


Hemoglobin subunit delta  is a  protein  that in  humans  is encoded by the  HBD   gene . [ 3 ]


The delta (HBD) and beta (HBB) genes are normally expressed in the adult: two alpha chains plus two beta chains constitute  HbA , which in normal adult life comprises about 97% of the total hemoglobin. Two alpha chains plus two delta chains constitute  HbA2 , which with  HbF  comprises the remaining 3% of adult hemoglobin. Five beta-like globin genes are found within a 45 kb cluster on chromosome 11 in the following order: 5' -  epsilon  –  gamma-G  –  gamma-A  –  delta  –  beta  - 3'. [ 4 ]


Mutations in the delta-globin gene are associated with  Delta-thalassemia . [ 4 ]


NP_005321 NP_005321.1


Hemoglobin subunit epsilon  is a  protein  that in humans is encoded by the  HBE1   gene . [ 5 ]


The epsilon globin gene (HBE) is normally expressed in the embryonic yolk sac: two epsilon chains together with two  zeta chains  (an alpha-like globin) constitute the embryonic hemoglobin  Hb Gower I ; two epsilon chains together with two alpha chains form the embryonic  Hb Gower II . Both of these embryonic hemoglobins are normally supplanted by fetal, and later, adult hemoglobin. The five beta-like globin genes are found within a 45 kb cluster on chromosome 11 in the following order:  5' -  epsilon  –  gamma-G  –  gamma-A  –  delta  –  beta  - 3'. [ 6 ]


This article incorporates text from the  United States National Library of Medicine , which is in the  public domain .


1I3D ,  1I3E


Hemoglobin subunit gamma-1  is a  protein  that in humans is encoded by the  HBG1   gene . [ 3 ]


The gamma globin genes (HBG1 and HBG2) are normally expressed in the fetal liver, spleen and bone marrow. Two gamma chains together with two alpha chains constitute fetal hemoglobin (HbF) which is normally replaced by adult hemoglobin (HbA) in the year following birth. In the non-pathological condition known as hereditary persistence of fetal hemoglobin (HPFH), gamma globin expression is continued into adulthood. Also, in cases of beta-thalassemia and related conditions, gamma chain production may be maintained, possibly as a mechanism to compensate for the mutated beta-globin. The two types of gamma chains differ at residue 136 where glycine is found in the G-gamma product (HBG2) and alanine is found in the A-gamma product (HBG1). The former is predominant at birth. The order of the genes in the beta-globin cluster is: 5' -  epsilon  –  gamma-G  –  gamma-A  –  delta  –  beta  - 3'. [ 4 ]


1FDH ,  4MQJ ,  4MQK


Hemoglobin subunit gamma-2  is a  protein  that in humans is encoded by the  HBG2   gene . [ 5 ]


The gamma globin genes ( HBG1  and HBG2, this gene) are normally expressed in the fetal liver, spleen and bone marrow. Two gamma chains together with two alpha chains constitute fetal hemoglobin (HbF) which is normally replaced by adult hemoglobin (HbA) at birth. In some  beta-thalassemias  and related conditions, gamma chain production continues into adulthood. The two types of gamma chains differ at residue 136 where  glycine  is found in the G-gamma product (HBG2) and  alanine  is found in the A-gamma product (HBG1). The former is predominant at birth. The order of the genes in the beta-globin cluster is: 5' -  epsilon  –  gamma-G  –  gamma-A  –  delta  –  beta  - 3'. [ 6 ]


Hemoglobin subunit theta-1  is a  protein  that in humans is encoded by the  HBQ1   gene . [ 5 ] [ 6 ]


Theta-globin mRNA is found in human  fetal  erythroid tissue but not in adult  erythroid  or other nonerythroid tissue. The theta-1 gene may be expressed very early in embryonic life, perhaps sometime before 5 weeks. Theta-1 is a member of the human alpha-globin gene cluster that involves five functional genes and two pseudogenes. The order of genes is: 5' -  zeta  - pseudozeta -  mu  - pseudoalpha-1 -  alpha-2  -  alpha-1  -  theta-1  - 3'. Research supports a transcriptionally active role for the gene and a functional role for the peptide in specific cells, possibly those of early erythroid tissue. [ 6 ]


This article on a  gene  on human  chromosome 16  is a  stub . You can help Wikipedia by  expanding it .


Hemoglobin  ( haemoglobin , [ a ]   Hb  or  Hgb ) is a  protein  containing  iron  that facilitates the transportation of  oxygen  in  red blood cells . Almost all  vertebrates  contain hemoglobin, [ 3 ]  with the sole exception of the fish family  Channichthyidae . [ 4 ]  Hemoglobin in the  blood  carries oxygen from the respiratory organs ( lungs  or  gills ) to the other tissues of the body, where it releases the oxygen to enable  aerobic respiration  which powers an animal's  metabolism . A healthy human has 12   to 20   grams of hemoglobin in every 100   mL of blood. Hemoglobin is a  metalloprotein , a  chromoprotein , and  globulin .


In  mammals , hemoglobin makes up about 96% of a red blood cell's  dry weight  (excluding water), and around 35% of the total weight (including water). [ 5 ]  Hemoglobin has an oxygen-binding capacity of 1.34   mL of O 2  per gram, [ 6 ]  which increases the total  blood oxygen capacity  seventy-fold compared to dissolved oxygen in blood plasma alone. [ 7 ]  The mammalian hemoglobin molecule can bind and transport up to four oxygen molecules. [ 8 ]


Hemoglobin also transports other gases. It carries off some of the body's respiratory  carbon dioxide  (about 20–25% of the total) [ 9 ]  as  carbaminohemoglobin , in which CO 2  binds to the  heme protein . The molecule also carries the important regulatory molecule  nitric oxide  bound to a  thiol  group in the globin protein, releasing it at the same time as oxygen. [ 10 ]


Hemoglobin is also found in other cells, including in the  A9 dopaminergic neurons  of the  substantia nigra ,  macrophages ,  alveolar cells , lungs, retinal pigment epithelium, hepatocytes,  mesangial cells  of the kidney, endometrial cells, cervical cells, and vaginal epithelial cells. [ 11 ]  In these tissues, hemoglobin absorbs unneeded oxygen as an  antioxidant , and regulates  iron metabolism . [ 12 ]  Excessive glucose in the blood can attach to hemoglobin and raise the level of hemoglobin A1c. [ 13 ]


Hemoglobin and hemoglobin-like molecules are also found in many invertebrates, fungi, and plants. [ 14 ]  In these organisms, hemoglobins may carry oxygen, or they may transport and regulate other small molecules and ions such as carbon dioxide, nitric oxide, hydrogen sulfide and sulfide. A variant called  leghemoglobin  serves to scavenge oxygen away from  anaerobic  systems such as the nitrogen-fixing nodules of  leguminous  plants, preventing oxygen poisoning.


The medical condition  hemoglobinemia , a form of  anemia , is caused by  intravascular hemolysis , in which hemoglobin leaks from red blood cells into the  blood plasma .


In 1825, Johann Friedrich Engelhart discovered that the ratio of iron to protein is identical in the hemoglobins of several species. [ 16 ] [ 17 ]  From the known atomic mass of iron, he calculated the molecular mass of hemoglobin to  n  × 16000 ( n =number of iron atoms per hemoglobin molecule, now known to be 4), the first determination of a protein's molecular mass. This "hasty conclusion" drew ridicule from colleagues who could not believe that any molecule could be so large. However,  Gilbert Smithson Adair  confirmed Engelhart's results in 1925 by measuring the osmotic pressure of hemoglobin solutions. [ 18 ]


Although blood had been known to carry oxygen since at least 1794, [ 19 ] [ 20 ]  the oxygen-carrying property of hemoglobin was described by Hünefeld in 1840. [ 21 ]  In 1851, German physiologist  Otto Funke  published a series of articles in which he described growing hemoglobin crystals by successively diluting red blood cells with a solvent such as pure water, alcohol or ether, followed by slow evaporation of the solvent from the resulting protein solution. [ 22 ] [ 23 ]  Hemoglobin's reversible oxygenation was described a few years later by  Felix Hoppe-Seyler . [ 24 ]


With the development of  X-ray crystallography , it became possible to sequence protein structures. [ 25 ]  In 1959,  Max Perutz  determined the molecular structure of hemoglobin. [ 26 ] [ 27 ]  For this work he shared the 1962  Nobel Prize in Chemistry  with  John Kendrew , who sequenced the globular protein  myoglobin . [ 25 ] [ 28 ]


The role of hemoglobin in the blood was elucidated by French  physiologist   Claude Bernard .


The name  hemoglobin  (or  haemoglobin ) is derived from the words  heme  (or  haem ) and  globin , reflecting the fact that each  subunit  of hemoglobin is a  globular protein  with an embedded  heme  group. Each heme group contains one iron atom, that can bind one oxygen molecule through ion-induced dipole forces. The most common type of hemoglobin in mammals contains four such subunits. [ 29 ]


Hemoglobin consists of  protein subunits  ( globin  molecules), which are  polypeptides , long folded chains of specific  amino acids  which determine the protein's chemical properties and function. The amino acid sequence of any polypeptide is  translated  from a segment of DNA, the corresponding  gene .


There is more than one hemoglobin gene. In humans,  hemoglobin A  (the main form of hemoglobin in adults) is coded by genes  HBA1 ,  HBA2 , and  HBB . [ 30 ]  Alpha 1 and alpha 2 subunits are respectively coded by genes  HBA1  and  HBA2  close together on chromosome 16, while the beta subunit is coded by gene  HBB  on chromosome 11. The amino acid sequences of the globin subunits usually differ between species, with the difference growing with evolutionary distance. For example, the most common hemoglobin sequences in humans, bonobos and chimpanzees are completely identical, with exactly the same alpha and beta globin protein chains. [ 31 ] [ 32 ] [ 33 ]  Human and gorilla hemoglobin differ in one amino acid in both alpha and beta chains, and these differences grow larger between less closely related species. [ citation needed ]


Mutations  in the genes for hemoglobin can result in  variants of hemoglobin  within a single species, although one sequence is usually "most common" in each species. [ 34 ] [ 35 ]  Many of these mutations cause no disease, but some cause a group of  hereditary diseases  called  hemoglobinopathies . The best known hemoglobinopathy is  sickle-cell disease , which was the first human disease whose  mechanism  was understood at the molecular level. A mostly separate set of diseases called  thalassemias  involves underproduction of normal and sometimes abnormal hemoglobins, through problems and mutations in globin  gene regulation . All these diseases produce  anemia . [ 36 ]


Variations in hemoglobin sequences, as with other proteins, may be adaptive. For example, hemoglobin has been found to adapt in different ways to the thin air at high altitudes, where lower partial pressure of oxygen diminishes its binding to hemoglobin compared to the higher pressures at sea level. Recent studies of deer mice found mutations in four genes that can account for differences between high- and low-elevation populations. It was found that the genes of the two breeds are "virtually identical—except for those that govern the oxygen-carrying capacity of their hemoglobin. . . . The genetic difference enables highland mice to make more efficient use of their oxygen." [ 37 ]   Mammoth  hemoglobin featured mutations that allowed for oxygen delivery at lower temperatures, thus enabling mammoths to migrate to higher latitudes during the  Pleistocene . [ 38 ]  This was also found in hummingbirds that inhabit the Andes. Hummingbirds already expend a lot of energy and thus have high oxygen demands and yet Andean hummingbirds have been found to thrive in high altitudes. Non-synonymous mutations in the hemoglobin gene of multiple species living at high elevations ( Oreotrochilus, A. castelnaudii, C. violifer, P. gigas,  and  A. viridicuada ) have caused the protein to have less of an affinity for  inositol hexaphosphate  (IHP), a molecule found in birds that has a similar role as 2,3-BPG in humans; this results in the ability to bind oxygen in lower partial pressures. [ 39 ]


Birds' unique  circulatory lungs  also promote efficient use of oxygen at low partial pressures of O 2 . These two adaptations reinforce each other and account for birds' remarkable high-altitude performance. [ citation needed ]


Hemoglobin adaptation extends to humans, as well. There is a higher offspring survival rate among Tibetan women with high oxygen saturation genotypes residing at 4,000 m. [ 40 ]  Natural selection seems to be the main force working on this gene because the mortality rate of offspring is significantly lower for women with higher hemoglobin-oxygen affinity when compared to the mortality rate of offspring from women with low hemoglobin-oxygen affinity. While the exact genotype and mechanism by which this occurs is not yet clear, selection is acting on these women's ability to bind oxygen in low partial pressures, which overall allows them to better sustain crucial metabolic processes. [ citation needed ]


Hemoglobin (Hb) is synthesized in a complex series of steps. The heme part is synthesized in a series of steps in the  mitochondria  and the  cytosol  of immature red blood cells, while the  globin  protein parts are synthesized by  ribosomes  in the cytosol. [ 41 ]  Production of Hb continues in the cell throughout its early development from the  proerythroblast  to the  reticulocyte  in the  bone marrow . At this point, the  nucleus  is lost in mammalian red blood cells, but not in  birds  and many other species. Even after the loss of the nucleus in mammals, residual  ribosomal RNA  allows further synthesis of Hb until the reticulocyte loses its RNA soon after entering the  vasculature  (this hemoglobin-synthetic RNA in fact gives the reticulocyte its reticulated appearance and name). [ 42 ]


Hemoglobin has a  quaternary structure  characteristic of many multi-subunit globular proteins. [ 43 ]  Most of the amino acids in hemoglobin form  alpha helices , and these helices are connected by short non-helical segments. Hydrogen bonds stabilize the helical sections inside this protein, causing attractions within the molecule, which then causes each polypeptide chain to fold into a specific shape. [ 44 ]  Hemoglobin's quaternary structure comes from its four subunits in roughly a tetrahedral arrangement. [ 43 ]


In most vertebrates, the hemoglobin  molecule  is an assembly of four  globular protein  subunits. Each subunit is composed of a protein chain tightly associated with a non-protein  prosthetic   heme  group. Each protein chain arranges into a set of  alpha-helix  structural segments connected together in a  globin fold  arrangement. Such a name is given because this arrangement is the same folding motif used in other heme/globin proteins such as  myoglobin . [ 45 ] [ 46 ]  This folding pattern contains a pocket that strongly binds the heme group. [ citation needed ]


A heme group consists of an iron (Fe)  ion  held in a  heterocyclic  ring, known as a  porphyrin . This porphyrin ring consists of four  pyrrole  molecules cyclically linked together (by  methine  bridges) with the iron ion bound in the center. [ 47 ]  The iron ion, which is the site of oxygen binding, coordinates with the four  nitrogen  atoms in the center of the ring, which all lie in one plane. The heme is bound strongly (covalently) to the globular protein via the N atoms of the  imidazole  ring of F8  histidine  residue (also known as the proximal histidine) below the porphyrin ring. A sixth position can reversibly bind oxygen by a  coordinate covalent bond , [ 48 ]  completing the octahedral group of six ligands. This reversible bonding with oxygen is why hemoglobin is so useful for transporting oxygen around the body. [ 49 ]  Oxygen binds in an "end-on bent" geometry where one oxygen atom binds to Fe and the other protrudes at an angle. When oxygen is not bound, a very weakly bonded water molecule fills the site, forming a distorted  octahedron .


Even though carbon dioxide is carried by hemoglobin, it does not compete with oxygen for the iron-binding positions but is bound to the amine groups of the protein chains attached to the heme groups.


The iron ion may be either in the  ferrous Fe 2+  or in the  ferric Fe 3+  state, but ferrihemoglobin ( methemoglobin ) (Fe 3+ ) cannot bind oxygen. [ 50 ]  In binding, oxygen temporarily and reversibly oxidizes (Fe 2+ ) to (Fe 3+ ) while oxygen temporarily turns into the  superoxide  ion, thus iron must exist in the +2 oxidation state to bind oxygen. If superoxide ion associated to Fe 3+  is protonated, the hemoglobin iron will remain oxidized and incapable of binding oxygen. In such cases, the enzyme  methemoglobin reductase  will be able to eventually reactivate methemoglobin by reducing the iron center.


In adult humans, the most common hemoglobin type is a  tetramer  (which contains four subunit proteins) called  hemoglobin A , consisting of two α and two β subunits non-covalently bound, each made of 141 and 146 amino acid residues, respectively. This is denoted as α 2 β 2 . The subunits are structurally similar and about the same size. Each subunit has a molecular weight of about 16,000  daltons , [ 51 ]  for a total  molecular weight  of the tetramer of about 64,000 daltons (64,458 g/mol). [ 52 ]  Thus, 1 g/dL=0.1551 mmol/L. Hemoglobin A is the most intensively studied of the hemoglobin molecules. [ citation needed ]


In human infants, the  fetal hemoglobin  molecule is made up of 2 α chains and 2 γ chains. The γ chains are gradually replaced by β chains as the infant grows. [ 53 ]


The four  polypeptide chains  are bound to each other by  salt bridges ,  hydrogen bonds , and the  hydrophobic effect .


In general, hemoglobin can be saturated with oxygen molecules (oxyhemoglobin), or desaturated with oxygen molecules (deoxyhemoglobin). [ 54 ]


Oxyhemoglobin  is formed during  physiological respiration  when oxygen binds to the heme component of the protein hemoglobin in red blood cells. This process occurs in the  pulmonary capillaries  adjacent to the  alveoli  of the lungs. The oxygen then travels through the blood stream to be dropped off at cells where it is utilized as a terminal electron acceptor in the production of  ATP  by the process of  oxidative phosphorylation . It does not, however, help to counteract a decrease in blood pH.  Ventilation , or breathing, may reverse this condition by removal of  carbon dioxide , thus causing a shift up in pH. [ 55 ]


Hemoglobin exists in two forms, a  taut (tense) form  (T) and a  relaxed form  (R). Various factors such as low pH, high CO 2  and high  2,3 BPG  at the level of the tissues favor the taut form, which has low oxygen affinity and releases oxygen in the tissues. Conversely, a high pH, low CO 2 , or low 2,3 BPG favors the relaxed form, which can better bind oxygen. [ 56 ]  The partial pressure of the system also affects O 2  affinity where, at high partial pressures of oxygen (such as those present in the alveoli), the relaxed (high affinity, R) state is favoured. Inversely, at low partial pressures (such as those present in respiring tissues), the (low affinity, T) tense state is favoured. [ 57 ]  Additionally, the binding of oxygen to the iron(II) heme pulls the iron into the plane of the porphyrin ring, causing a slight conformational shift. The shift encourages oxygen to bind to the three remaining heme units within hemoglobin (thus, oxygen binding is cooperative). [ citation needed ]


Classically, the iron in oxyhemoglobin is seen as existing in the iron(II) oxidation state. However, the complex of oxygen with heme iron is  diamagnetic , whereas both oxygen and high-spin iron(II) are  paramagnetic . Experimental evidence strongly suggests heme iron is in the iron(III) oxidation state in oxyhemoglobin, with the oxygen existing as  superoxide anion  (O 2 •− ) or in a covalent charge-transfer complex. [ 58 ]


Deoxygenated hemoglobin (deoxyhemoglobin) is the form of hemoglobin without the bound oxygen. The  absorption spectra  of oxyhemoglobin and deoxyhemoglobin differ. The oxyhemoglobin has significantly lower absorption of the 660 nm  wavelength  than deoxyhemoglobin, while at 940 nm its absorption is slightly higher. This difference is used for the measurement of the amount of oxygen in a patient's blood by an instrument called a  pulse oximeter . This difference also accounts for the presentation of  cyanosis , the blue to purplish color that tissues develop during  hypoxia . [ 59 ]


Deoxygenated hemoglobin is  paramagnetic ; it is weakly attracted to  magnetic fields . [ 60 ] [ 61 ]  In contrast, oxygenated hemoglobin exhibits  diamagnetism , a weak repulsion from a magnetic field. [ 61 ]


Scientists agree that the event that separated myoglobin from hemoglobin occurred after  lampreys  diverged from  jawed vertebrates . [ 62 ]  This separation of myoglobin and hemoglobin allowed for the different functions of the two molecules to arise and develop: myoglobin has more to do with oxygen storage while hemoglobin is tasked with oxygen transport. [ 63 ]  The α- and β-like globin genes encode the individual subunits of the protein. [ 30 ]  The predecessors of these genes arose through another duplication event also after the gnathosome common ancestor derived from jawless fish, approximately 450–500 million years ago. [ 62 ]  Ancestral reconstruction studies suggest that the preduplication ancestor of the α and β genes was a dimer made up of identical globin subunits, which then evolved to assemble into a tetrameric architecture after the duplication. [ 64 ]  The development of α and β genes created the potential for hemoglobin to be composed of multiple distinct subunits, a physical composition central to hemoglobin's ability to transport oxygen. Having multiple subunits contributes to hemoglobin's ability to bind oxygen cooperatively as well as be regulated allosterically. [ 63 ] [ 64 ]  Subsequently, the α gene also underwent a duplication event to form the  HBA1  and  HBA2  genes. [ 65 ]  These further duplications and divergences have created a diverse range of α- and β-like globin genes that are regulated so that certain forms occur at different stages of development. [ 63 ]


Most ice fish of the family  Channichthyidae  have lost their hemoglobin genes as an adaptation to cold water. [ 4 ]


When oxygen binds to the iron complex, it causes the iron atom to move back toward the center of the plane of the  porphyrin  ring (see moving diagram). At the same time, the  imidazole  side-chain of the histidine residue interacting at the other pole of the iron is pulled toward the porphyrin ring. This interaction forces the plane of the ring sideways toward the outside of the tetramer, and also induces a strain in the protein helix containing the histidine as it moves nearer to the iron atom. This strain is transmitted to the remaining three monomers in the tetramer, where it induces a similar conformational change in the other heme sites such that binding of oxygen to these sites becomes easier.


As oxygen binds to one monomer of hemoglobin, the tetramer's conformation shifts from the T (tense) state to the R (relaxed) state. This shift promotes the binding of oxygen to the remaining three monomers' heme groups, thus saturating the hemoglobin molecule with oxygen. [ 66 ]


In the tetrameric form of normal adult hemoglobin, the binding of oxygen is, thus, a  cooperative process . The binding affinity of hemoglobin for oxygen is increased by the oxygen saturation of the molecule, with the first molecules of oxygen bound influencing the shape of the binding sites for the next ones, in a way favorable for binding. This positive cooperative binding is achieved through  steric  conformational changes of the hemoglobin protein complex as discussed above; i.e., when one subunit protein in hemoglobin becomes oxygenated, a conformational or structural change in the whole complex is initiated, causing the other subunits to gain an increased affinity for oxygen. As a consequence, the oxygen binding curve of hemoglobin is  sigmoidal , or  S -shaped, as opposed to the normal  hyperbolic  curve associated with noncooperative binding.


The dynamic mechanism of the cooperativity in hemoglobin and its relation with low-frequency  resonance  has been discussed. [ 67 ]


Besides the oxygen  ligand , which binds to hemoglobin in a cooperative manner, hemoglobin ligands also include  competitive inhibitors  such as  carbon monoxide  (CO) and  allosteric ligands  such as carbon dioxide (CO 2 ) and  nitric oxide  (NO). The carbon dioxide is bound to amino groups of the globin proteins to form  carbaminohemoglobin ; this mechanism is thought to account for about 10% of carbon dioxide transport in mammals.  Nitric oxide  can also be transported by hemoglobin; it is bound to specific  thiol  groups in the globin protein to form an S-nitrosothiol, which dissociates into free nitric oxide and thiol again, as the hemoglobin releases oxygen from its heme site. This nitric oxide transport to peripheral tissues is hypothesized to assist oxygen transport in tissues, by releasing  vasodilatory  nitric oxide to tissues in which oxygen levels are low. [ 68 ]


The binding of oxygen is affected by molecules such as carbon monoxide (for example, from  tobacco smoking ,  exhaust gas , and incomplete combustion in furnaces). CO competes with oxygen at the heme binding site. Hemoglobin's binding affinity for CO is 250 times greater than its affinity for oxygen, [ 69 ] [ 70 ]  Since carbon monoxide is a colorless, odorless and tasteless gas, and poses a potentially fatal threat,  carbon monoxide detectors  have become commercially available to warn of dangerous levels in residences. When hemoglobin combines with CO, it forms a very bright red compound called  carboxyhemoglobin , which may cause the skin of  CO poisoning  victims to appear pink in death, instead of white or blue. When inspired air contains CO levels as low as 0.02%,  headache  and  nausea  occur; if the CO concentration is increased to 0.1%, unconsciousness will follow. In heavy smokers, up to 20% of the oxygen-active sites can be blocked by CO.


In similar fashion, hemoglobin also has competitive binding affinity for  cyanide  (CN − ),  sulfur monoxide  (SO), and  sulfide  (S 2− ), including  hydrogen sulfide  (H 2 S). All of these bind to iron in heme without changing its oxidation state, but they nevertheless inhibit oxygen-binding, causing grave toxicity.


The iron atom in the heme group must initially be in the  ferrous  (Fe 2+ ) oxidation state to support oxygen and other gases' binding and transport (it temporarily switches to ferric during the time oxygen is bound, as explained above). Initial oxidation to the  ferric  (Fe 3+ ) state without oxygen converts hemoglobin into "hem i globin" or  methemoglobin , which cannot bind oxygen. Hemoglobin in normal red blood cells is protected by a reduction system to keep this from happening. Nitric oxide is capable of converting a small fraction of hemoglobin to methemoglobin in red blood cells. The latter reaction is a remnant activity of the more ancient  nitric oxide dioxygenase  function of globins.


Carbon  di oxide occupies a different binding site on the hemoglobin. At tissues, where carbon dioxide concentration is higher, carbon dioxide binds to allosteric site of hemoglobin, facilitating unloading of oxygen from hemoglobin and ultimately its removal from the body after the oxygen has been released to tissues undergoing metabolism. This increased affinity for carbon dioxide by the venous blood is known as the  Bohr effect . Through the enzyme  carbonic anhydrase , carbon dioxide reacts with water to give  carbonic acid , which decomposes into  bicarbonate  and  protons :


Hence, blood with high carbon dioxide levels is also lower in  pH  (more  acidic ). Hemoglobin can bind protons and carbon dioxide, which causes a conformational change in the protein and facilitates the release of oxygen. Protons bind at various places on the protein, while carbon dioxide binds at the α-amino group. [ 71 ]  Carbon dioxide binds to hemoglobin and forms  carbaminohemoglobin . [ 72 ]  This decrease in hemoglobin's affinity for oxygen by the binding of carbon dioxide and acid is known as the  Bohr effect . The Bohr effect favors the T state rather than the R state. (shifts the O 2 -saturation curve to the  right ). Conversely, when the carbon dioxide levels in the blood decrease (i.e., in the lung capillaries), carbon dioxide and protons are released from hemoglobin, increasing the oxygen affinity of the protein. A reduction in the total binding capacity of hemoglobin to oxygen (i.e. shifting the curve down, not just to the right) due to reduced pH is called the  root effect . This is seen in bony fish.


It is necessary for hemoglobin to release the oxygen that it binds; if not, there is no point in binding it. The sigmoidal curve of hemoglobin makes it efficient in binding (taking up O 2  in lungs), and efficient in unloading (unloading O 2  in tissues). [ 73 ]


In people acclimated to high altitudes, the concentration of  2,3-Bisphosphoglycerate  (2,3-BPG) in the blood is increased, which allows these individuals to deliver a larger amount of oxygen to tissues under conditions of lower  oxygen tension . This phenomenon, where molecule Y affects the binding of molecule X to a transport molecule Z, is called a  heterotropic  allosteric effect. Hemoglobin in organisms at high altitudes has also adapted such that it has less of an affinity for 2,3-BPG and so the protein will be shifted more towards its R state. In its R state, hemoglobin will bind oxygen more readily, thus allowing organisms to perform the necessary metabolic processes when oxygen is present at low partial pressures. [ 74 ]


Animals other than humans use different molecules to bind to hemoglobin and change its O 2  affinity under unfavorable conditions. Fish use both  ATP  and  GTP . These bind to a phosphate "pocket" on the fish hemoglobin molecule, which stabilizes the tense state and therefore decreases oxygen affinity. [ 75 ]  GTP reduces hemoglobin oxygen affinity much more than ATP, which is thought to be due to an extra  hydrogen bond  formed that further stabilizes the tense state. [ 76 ]  Under hypoxic conditions, the concentration of both ATP and GTP is reduced in fish red blood cells to increase oxygen affinity. [ 77 ]


A variant hemoglobin, called  fetal hemoglobin  (HbF, α 2 γ 2 ), is found in the developing  fetus , and binds oxygen with greater affinity than adult hemoglobin. This means that the oxygen binding curve for fetal hemoglobin is left-shifted (i.e., a higher percentage of hemoglobin has oxygen bound to it at lower oxygen tension), in comparison to that of adult hemoglobin. As a result, fetal blood in the  placenta  is able to take oxygen from maternal blood.


Hemoglobin also carries  nitric oxide  (NO) in the globin part of the molecule. This improves oxygen delivery in the periphery and contributes to the control of respiration. NO binds reversibly to a specific cysteine residue in globin; the binding depends on the state (R or T) of the hemoglobin. The resulting S-nitrosylated hemoglobin influences various NO-related activities such as the control of vascular resistance, blood pressure and respiration. NO is not released in the cytoplasm of red blood cells but transported out of them by an anion exchanger called  AE1 . [ 78 ]


Hemoglobin variants  are a part of the normal  embryonic  and  fetal  development. They may also be pathologic mutant forms of hemoglobin in a  population , caused by variations in genetics. Some well-known hemoglobin variants, such as  sickle-cell anemia , are responsible for diseases and are considered  hemoglobinopathies . Other variants cause no detectable  pathology , and are thus considered non-pathological variants. [ 34 ] [ 79 ]


In  embryos :


In fetuses:


In  neonates  (newborns inmmediately after birth):


Abnormal forms that occur in diseases:


When  red blood cells  reach the end of their life due to aging or defects, they are removed from the circulation by the phagocytic activity of macrophages in the spleen or the liver or hemolyze within the circulation.  Free hemoglobin  is then cleared from the circulation via the hemoglobin transporter  CD163 , which is exclusively expressed on monocytes or macrophages. Within these cells the hemoglobin molecule is broken up, and the iron gets recycled. This process also produces one molecule of carbon monoxide for every molecule of heme degraded. [ 80 ]  Heme degradation is the only natural source of carbon monoxide in the human body, and is responsible for the normal blood levels of carbon monoxide in people breathing normal air. [ 81 ]


The other major final product of heme degradation is  bilirubin . Increased levels of this chemical are detected in the blood if red blood cells are being destroyed more rapidly than usual. Improperly degraded hemoglobin protein or hemoglobin that has been released from the blood cells too rapidly can clog small blood vessels, especially the delicate blood filtering vessels of the  kidneys , causing kidney damage. Iron is removed from heme and salvaged for later use, it is stored as hemosiderin or  ferritin  in tissues and transported in plasma by beta globulins as  transferrins . When the porphyrin ring is broken up, the fragments are normally secreted as a yellow pigment called bilirubin, which is secreted into the intestines as bile. Intestines metabolize bilirubin into urobilinogen. Urobilinogen leaves the body in faeces, in a pigment called stercobilin. Globulin is metabolized into amino acids that are then released into circulation.


Hemoglobin deficiency can be caused either by a decreased amount of hemoglobin molecules, as in  anemia , or by decreased ability of each molecule to bind oxygen at the same partial pressure of oxygen.  Hemoglobinopathies  (genetic defects resulting in abnormal structure of the hemoglobin molecule) [ 82 ]  may cause both. In any case, hemoglobin deficiency decreases  blood oxygen-carrying capacity . Hemoglobin deficiency is, in general, strictly distinguished from  hypoxemia , defined as decreased  partial pressure  of oxygen in blood, [ 83 ] [ 84 ] [ 85 ] [ 86 ]  although both are causes of  hypoxia  (insufficient oxygen supply to tissues).


Other common causes of low hemoglobin include loss of blood, nutritional deficiency, bone marrow problems, chemotherapy, kidney failure, or abnormal hemoglobin (such as that of sickle-cell disease).


The ability of each hemoglobin molecule to carry oxygen is normally modified by altered blood pH or CO 2 , causing an altered  oxygen–hemoglobin dissociation curve . However, it can also be pathologically altered in, e.g.,  carbon monoxide poisoning .


Decrease of hemoglobin, with or without an absolute decrease of red blood cells, leads to symptoms of anemia. Anemia has many different causes, although  iron deficiency  and its resultant  iron deficiency anemia  are the most common causes in the Western world. As absence of iron decreases heme synthesis, red blood cells in iron deficiency anemia are  hypochromic  (lacking the red hemoglobin pigment) and  microcytic  (smaller than normal). Other anemias are rarer. In  hemolysis  (accelerated breakdown of red blood cells), associated  jaundice  is caused by the hemoglobin metabolite bilirubin, and the circulating hemoglobin can cause  kidney failure .


Some mutations in the globin chain are associated with the  hemoglobinopathies , such as sickle-cell disease and  thalassemia . Other mutations, as discussed at the beginning of the article, are benign and are referred to merely as  hemoglobin variants .


There is a group of genetic disorders, known as the  porphyrias  that are characterized by errors in metabolic pathways of heme synthesis. King  George III of the United Kingdom  was probably the most famous porphyria sufferer.


To a small extent, hemoglobin A slowly combines with  glucose  at the terminal valine (an alpha aminoacid) of each β chain. The resulting molecule is often referred to as  Hb A 1c , a  glycated hemoglobin . The binding of glucose to amino acids in the hemoglobin takes place spontaneously (without the help of an enzyme) in many proteins, and is not known to serve a useful purpose. However, as the concentration of glucose in the blood increases, the percentage of Hb A that turns into Hb A 1c  increases. In  diabetics  whose glucose usually runs high, the percent Hb A 1c  also runs high. Because of the slow rate of Hb A combination with glucose, the Hb A 1c  percentage reflects a weighted average of blood glucose levels over the lifetime of red cells, which is approximately 120 days. [ 87 ]  The levels of glycated hemoglobin are therefore measured in order to monitor the long-term control of the chronic disease of type 2 diabetes mellitus (T2DM). Poor control of T2DM results in high levels of glycated hemoglobin in the red blood cells. The normal reference range is approximately 4.0–5.9%. Though difficult to obtain, values less than 7% are recommended for people with T2DM. Levels greater than 9% are associated with poor control of the glycated hemoglobin, and levels greater than 12% are associated with very poor control. Diabetics who keep their glycated hemoglobin levels close to 7% have a much better chance of avoiding the complications that may accompany diabetes (than those whose levels are 8% or higher). [ 88 ]  In addition, increased glycated of hemoglobin increases its affinity for oxygen, therefore preventing its release at the tissue and inducing a level of hypoxia in extreme cases. [ 89 ]


Elevated levels of hemoglobin are associated with increased numbers or sizes of red blood cells, called  polycythemia . This elevation may be caused by  congenital heart disease ,  cor pulmonale ,  pulmonary fibrosis , too much  erythropoietin , or  polycythemia vera . [ 90 ]  High hemoglobin levels may also be caused by exposure to high altitudes, smoking, dehydration (artificially by concentrating Hb), advanced lung disease and certain tumors. [ 53 ]


Hemoglobin concentration measurement is among the most commonly performed  blood tests , usually as part of a  complete blood count . For example, it is typically tested before or after  blood donation . Results are reported in  g / L , g/ dL  or  mol /L. 1 g/dL equals about 0.6206 mmol/L, although the latter units are not used as often due to uncertainty regarding the polymeric state of the molecule. [ 91 ]  This conversion factor, using the single globin unit molecular weight of 16,000  Da , is more common for hemoglobin concentration in blood. For MCHC (mean corpuscular hemoglobin concentration) the conversion factor 0.155, which uses the tetramer weight of 64,500 Da, is more common. [ 92 ]  Normal levels are:


Normal values of hemoglobin in the 1st and 3rd trimesters of pregnant women must be at least 11 g/dL and at least 10.5 g/dL during the 2nd trimester. [ 95 ]


Dehydration or hyperhydration can greatly influence measured hemoglobin levels. Albumin can indicate hydration status.


If the concentration is below normal, this is called anemia. Anemias are classified by the size of red blood cells, the cells that contain hemoglobin in vertebrates. The anemia is called "microcytic" if red cells are small, "macrocytic" if they are large, and "normocytic" otherwise.


Hematocrit , the proportion of blood volume occupied by red blood cells, is typically about three times the hemoglobin concentration measured in g/dL. For example, if the hemoglobin is measured at 17 g/dL, that compares with a hematocrit of 51%. [ 96 ]


Laboratory hemoglobin test methods require a blood sample (arterial, venous, or capillary) and analysis on hematology analyzer and CO-oximeter. Additionally, a new noninvasive hemoglobin (SpHb) test method called Pulse CO-Oximetry is also available with comparable accuracy to invasive methods. [ 97 ]


Concentrations of oxy- and deoxyhemoglobin can be measured continuously, regionally and noninvasively using  NIRS . [ 98 ] [ 99 ] [ 100 ] [ 101 ] [ 102 ]  NIRS can be used both on the head and on muscles. This technique is often used for research in e.g. elite sports training, ergonomics, rehabilitation, patient monitoring, neonatal research, functional brain monitoring,  brain–computer interface , urology (bladder contraction), neurology (Neurovascular coupling) and more.


Hemoglobin mass can be measured in humans using the non-radioactive, carbon monoxide (CO) rebreathing technique that has been used for more than 100 years. With this technique, a small volume of pure CO gas is inhaled and rebreathed for a few minutes. During rebreathing, CO binds to hemoglobin present in red blood cells. Based on the increase in blood CO after the rebreathing period, the hemoglobin mass can be determined through the dilution principle. [ 103 ]


Long-term control of  blood sugar  concentration can be measured by the concentration of Hb A 1c . Measuring it directly would require many samples because blood sugar levels vary widely through the day. Hb A 1c  is the product of the  irreversible reaction  of hemoglobin A with glucose. A higher glucose  concentration  results in more Hb A 1c . Because the reaction is slow, the Hb A 1c  proportion represents glucose level in blood averaged over the half-life of red blood cells, is typically ~120 days. An Hb A 1c  proportion of 6.0% or less show good long-term glucose control, while values above 7.0% are elevated. This test is especially useful for diabetics. [ c ]


The  functional magnetic resonance imaging  (fMRI) machine uses the signal from deoxyhemoglobin, which is sensitive to magnetic fields since it is paramagnetic. Combined measurement with  NIRS  shows good correlation with both the oxy- and deoxyhemoglobin signal compared to the  BOLD signal . [ 104 ]


Hemoglobin can be tracked noninvasively, to build an individual data set tracking the hemoconcentration and hemodilution effects of daily activities for better understanding of sports performance and training. Athletes are often concerned about endurance and intensity of exercise. The sensor uses light-emitting diodes that emit red and infrared light through the tissue to a light detector, which then sends a signal to a processor to calculate the absorption of light by the hemoglobin protein. [ 105 ]  This sensor is similar to a  pulse oximeter , which consists of a small sensing device that clips to the finger.


A variety of oxygen-transport and -binding proteins exist in organisms throughout the animal and plant kingdoms. Organisms including  bacteria ,  protozoans , and  fungi  all have hemoglobin-like proteins whose known and predicted roles include the reversible binding of gaseous  ligands . Since many of these proteins contain  globins  and the heme  moiety  (iron in a flat porphyrin support), they are often called hemoglobins, even if their overall tertiary structure is very different from that of vertebrate hemoglobin. In particular, the distinction of "myoglobin" and hemoglobin in lower animals is often impossible, because some of these organisms do not contain  muscles . Or, they may have a recognizable separate  circulatory system  but not one that deals with oxygen transport (for example, many  insects  and other  arthropods ). In all these groups, heme/globin-containing molecules (even monomeric globin ones) that deal with gas-binding are referred to as oxyhemoglobins. In addition to dealing with transport and sensing of oxygen, they may also deal with NO, CO 2 , sulfide compounds, and even O 2  scavenging in environments that must be anaerobic. [ 106 ]  They may even deal with detoxification of chlorinated materials in a way analogous to heme-containing P450 enzymes and peroxidases.