Quantum dots  are sometimes used in place of traditional fluorophores because of their narrower emission peaks. [ citation needed ]


Mass cytometry overcomes the fluorescent labeling limit by utilizing  lanthanide  isotopes attached to antibodies. This method could theoretically allow the use of 40 to 60 distinguishable labels and has been demonstrated for 30 labels. [ 39 ]  Mass cytometry is fundamentally different from flow cytometry: cells are introduced into a  plasma , ionized, and associated isotopes are quantified via  time-of-flight mass spectrometry . Although this method permits the use of a large number of labels, it currently has lower throughput capacity than flow cytometry. It also destroys the analysed cells, precluding their recovery by sorting. [ 39 ]


In addition to the ability to label and identify individual cells via fluorescent antibodies, cellular products such as cytokines, proteins, and other factors may be measured as well. Similar to  ELISA  sandwich assays, cytometric bead array ( CBA ) assays use multiple bead populations typically differentiated by size and different levels of fluorescence intensity to distinguish multiple analytes in a single assay. The amount of the analyte captured is detected via a biotinylated antibody against a secondary epitope of the protein, followed by a streptavidin-R-phycoerythrin treatment. The fluorescent intensity of R-phycoerythrin on the beads is quantified on a flow cytometer equipped with a 488 nm excitation source. Concentrations of a protein of interest in the samples can be obtained by comparing the fluorescent signals to those of a standard curve generated from a serial dilution of a known concentration of the analyte. Commonly also referred to as cytokine bead array (CBA).


Impedance -based single cell analysis systems are commonly known as  Coulter counters . They represent a well-established method for counting and sizing virtually any kind of cells and particles. The label-free technology has recently been enhanced by a " lab-on-a-chip " based approach and by applying high frequency  alternating current  (AC) in the radio frequency range (from 100 kHz to 30 MHz) instead of a static  direct current  (DC) or low frequency AC field. [ 40 ] [ 41 ]  This patented technology allows a highly accurate cell analysis and provides additional information like membrane  capacitance  and  viability . The relatively small size and robustness allow battery powered on-site use in the field.


Measurable factors in cellular analysis and flow cytometry include a diverse set of traits and indicators that provide important insights into cell biology and function. Flow cytometry techniques can quantify and evaluate these factors, allowing researchers to investigate and analyze various aspects of cells. Here are some important quantifiable parameters that are frequently investigated:


The technology has applications in a number of fields, including  molecular biology ,  pathology ,  immunology , virology, [ 44 ]   plant biology  and  marine biology . [ 45 ]  It has broad application in  medicine  especially in transplantation, hematology, tumor immunology and chemotherapy, prenatal diagnosis, genetics and  sperm sorting  for  sex preselection . Flow cytometry is widely applied to detect sperm cells abnormality associated with  DNA fragmentation [ 46 ]  in  male fertility  assays. [ 47 ]  Also, it is extensively used in research for the detection of  DNA damage , [ 48 ] [ 49 ]  caspase cleavage and  apoptosis . [ 50 ]   Photoacoustic flow cytometry  is used in the study of multi-drug-resistant bacteria (most commonly MRSA) to detect, differentiate, and quantify bacteria in the blood marked with dyed bacteriophages. [ 51 ]  In  neuroscience , co-expression of cell surface and intracellular antigens can also be analyzed. [ 52 ]  In microbiology, it can be used to screen and sort transposon mutant libraries constructed with a GFP-encoding transposon (TnMHA), [ 53 ]  or to assess viability. [ 54 ]  In protein engineering, flow cytometry is used in conjunction with  yeast display  and  bacterial display  to identify cell surface-displayed protein variants with desired properties.
The main advantages of flow cytometry over histology and IHC is the possibility to precisely measure the quantities of antigens and the possibility to stain each cell with multiple antibodies-fluorophores, in current laboratories around 10 antibodies can be bound to each cell. This is much less than mass cytometer where up to 40 can be currently measured, but at a higher price and a slower pace.


In aquatic systems, flow cytometry is used for the analysis of autofluorescing cells or cells that are fluorescently-labeled with added stains. This research started in 1981 when  Clarice Yentsch  used flow cytometry to measure the fluorescence in a red tide producing dinoflagellate. [ 55 ]  The next year researchers published flow cytometric measurements of multiple algal species which could be distinguished based on their fluorescence characteristics. [ 56 ]  By 1983, marine researchers were assembling their own flow cytometers [ 57 ]  or using commercially available flow cytometers on seawater samples collected off Bermuda to demonstrate that phytoplankton cells could be distinguished from non-living material and that cyanobacteria could be sorted from a mixed community and subsequently cultured in the lab. [ 58 ]  Flow cytometry also allowed marine researchers to distinguish between dimly-fluorescing  Prochlorococcus  and heterotrophic microorganisms, a distinction that is difficult with microscopy-based assessments. [ 59 ]  Advances in technology now allow aquatic scientists to use flow cytometers continuously during research cruises [ 60 ]  and flow cytometers are used to provide images of individual phytoplankton cells. [ 61 ] [ 62 ]  Marine scientists use the sorting ability of flow cytometers to make discrete measurements of cellular activity and diversity, [ 63 ] [ 64 ]  to conduct investigations into the mutualistic relationships between microorganisms that live in close proximity, [ 65 ]  and to measure biogeochemical rates of multiple processes in the ocean. [ 66 ]


Cell proliferation is the major function in the immune system. Often it is required to analyse the proliferative nature of the cells in order to make some conclusions. One such assay to determine the cell proliferation is the tracking dye carboxyfluorescein diacetate succinimidyl ester (CFSE). It helps to monitor proliferative cells. This assay gives quantitative as well as qualitative data during time-series experiments. [ 67 ]  This dye binds covalently with the long-lived molecules present inside the cell. When the cells divide, the molecules divide too and, the daughter cells possess half the dye than the parent population. This decrease in the intensity can be visualized by flow cytometry. [ 68 ]  In literature, this powerful technique of flow cytometry and CFSE has been used to find the efficiency of T-cells in killing the target cells in cancer such as leukemia. In order to visualize the target cell death, both rapid and slow, scientists have used CFSE labelling with antibody staining of certain kinds of cells and fluorescently labelled microbeads. This also gave information regarding the proliferation of the target cells upon the treatment of certain cytokines. [ 69 ]


Flow cytometry has been used to measure  genome sizes , or more precisely: the amount of  DNA  in a  cell  or  nucleus . Although genomes can be analyzed with more precision by  genome sequencing , this is often difficult due to a high fraction of  micro-chromosomes  or  repetitive sequences  which may be missed by sequencing (or which get filtered out during the analysis step when they cannot be assigned to  chromosomes ). However, flow cytometry is not perfect either. The resulting genome sizes may differ based on the dye used. An analysis of fish genomes resulted in significantly different genome sizes when  propidium iodide  (PI) and  DAPI  were used, respectively. For instance, the genome of  Anguilla japonica  was found to contain 1.09 pg of DNA with PI vs. 1.25 pg with DAPI. Similarly, the genome of  Myxocyprinus asiaticus  was found to contain 2.75 pg of DNA (PI) vs. 3.08 pg (DAPI). That is, the differences were on the order of 12–14%. [ 70 ]


Fluorescein-labeled proaerolysin  ( FLAER ) is used in a flow cytometric assay to diagnose  paroxysmal nocturnal hemoglobinuria  (PNH). [ 1 ] [ 2 ]   The assay takes advantage of the action of proaerolysin, a prototoxin of  aerolysin , a virulence factor  of the bacterium   Aeromonas hydrophila . Proaerolysin binds to the  glycophosphatidylinositol (GPI) anchor in the  plasma membrane  of cells.  Cells affected by PNH lack GPI anchoring proteins, and thus are not bound by proaerolysin.  Of note, the FLAER-based assay is not suitable for evaluation of  erythrocytes  and  platelets  in PNH but flow cytometry assays based on  CD55 ,  CD59  and others are suitable. [ 1 ]


Fructosamines  are compounds that result from  glycation  reactions between glucose and a  primary amine , followed by  isomerization  via the  Amadori rearrangement . Biologically, fructosamines are recognized by  fructosamine-3-kinase , which may trigger the degradation of  advanced glycation end-products  (though the true clinical significance of this pathway is unclear). Fructosamine can also refer to the specific compound 1-amino-1-deoxy-D-fructose ( isoglucosamine ), first synthesized by Nobel laureate  Hermann Emil Fischer  in 1886. [ citation needed ]


Most commonly, fructosamine refers to a laboratory test for  diabetes management  that is rarely used in human clinical practice (simple  blood glucose monitoring  or  hemoglobin A1c  testing are preferred). In small animal veterinary practice however it is part of the diabetic cat or dog diagnosis and monitoring [ 1 ]  giving an indication of blood glucose levels over the previous week. [ 2 ]  Many  direct-to-consumer  lab testing companies sell fructosamine tests.


In  diabetes , maintaining a normal  blood glucose  is essential to preventing many medical complications, including  heart attacks ,  diabetic nephropathy ,  diabetic neuropathy , and  diabetic retinopathy  eventually leading to  blindness . Most commonly, blood sugars are measured by either  blood glucose monitoring  which measures the current blood glucose level, or by  glycated hemoglobin  (HbA1c) which measures average glucose levels over approximately 3 months. In a similar way to hemoglobin A1c testing (which measures the  glycation  of hemoglobin), fructosamine testing determines the fraction of total  serum  proteins that have undergone glycation (the  glycated serum proteins ). Since albumin is the most abundant protein in blood, fructosamine levels typically reflect albumin glycation. (Some fructosamine tests specifically quantify the glycation of albumin, or  glycated serum albumin  instead of all proteins.). Because albumin has a  half-life  of approximately 20 days, the plasma fructosamine concentration reflects relatively recent (1–2 week) changes in blood glucose. [ citation needed ]


In patients with diseases that reduce red blood cell lifespan, such as  hemolytic anaemia  or  hemoglobinopathies  such as  sickle-cell disease , a hemoglobin-based A1c test can be misleadingly low. A1c results may also be falsely high or low in  hemoglobinopathies  because abnormal hemoglobin variants can interfere in the analysis. In these cases, fructosamine measurement can be used as a marker of  blood sugar  levels, as its measurements are based on albumin instead of hemoglobin. However, any condition that changes serum albumin (such as the  nephrotic syndrome ) will affect the fructosamine result. [ citation needed ]


In practice, fructosamine is rarely measured clinically (even in individuals with hemoglobinopathies or other red cell disorders) due to a number of pragmatic concerns. First, diabetes care is rarely changed in short (1–4 week) intervals, since diabetes medications can take months to reach a  steady state . An exception to this is pregnancy, where medication needs can change more rapidly and fructosamine may help provide closer short-term monitoring. Second, fructosamine has higher variability than A1c tests. Third, the overwhelming majority of studies in diabetes care are based on A1c measurements, which can make fructosamine results difficult to interpret. Fourth, the A1c test is very well standardized [ 3 ]  and trusted due to its nearly universal use. A variety of more advanced forms of the A1c test (e.g. some types of  HPLC ,  immunoassay , and  capillary electrophoresis ) can more accurately assay A1c levels during complex hemoglobinopathies and other conditions. [ 4 ]  However this does not overcome the effect of shortened red blood cell lifespan on A1c results. [ citation needed ]


Because glycated albumin (GA) has a shorter half-life than glycated hemoglobin (HbA1c), glycated albumin reflects recent glycemic control more accurately and usefully for monitoring patients with diabetic  end-stage renal disease  (ESRD) ( hemodialysis  and  peritoneal dialysis  patients). It can be used less often than blood sugar testing. An average blood glucose level of 155–160 mg/dL could be matched to a GA value of 18–19% in patients with ESRD. The ratio of GA/HbA1c is 3.0 approximately. [ 5 ]


There is no standard reference range available for this test. The reference values depend upon the factors of patient age, gender, sample population, and test method. Hence, each laboratory report will include the patient's specific reference range for the test. An increase in fructosamine in lab testing results usually means an increase in glucose in the blood.


On average, each change of 3.3 mmol (60 mg/dL) in average blood sugar levels will give rise to changes of 2% HbA1c and 75 μmol fructosamine values. [ 6 ]  However, this overemphasizes the upper limit of many laboratories' reference ranges of 285 μmol/L as equivalent to HbA1c 7.5% rather than 6.5%. A comparative study, [ 7 ]  which has been used in official advice for  Quality and Outcomes Framework  guidance in the UK [ 8 ]  and summarized by the United States'  National Quality Measures Clearinghouse , [ 9 ]  gives the following formula and resulting values:


Protein electrophoresis  is a method for analysing the proteins in a fluid or an extract. The  electrophoresis  may be performed with a small volume of sample in a number of alternative ways with or without a supporting medium, namely  agarose  or  polyacrylamide . Variants of  gel electrophoresis  include  SDS-PAGE ,  free-flow electrophoresis ,  electrofocusing ,  isotachophoresis ,  affinity electrophoresis ,  immunoelectrophoresis ,  counterelectrophoresis , and  capillary electrophoresis . Each variant has many subtypes with individual advantages and limitations. Gel electrophoresis is often performed in combination with  electroblotting  or  immunoblotting  to give additional information about a specific protein. [ 1 ]


SDS-PAGE,  sodium dodecyl sulfate  polyacrylamide gel electrophoresis, describes a collection of related techniques to separate  proteins  according to their  electrophoretic mobility  (a function of the molecular weight of a polypeptide chain) while in the  denatured  (unfolded) state. In most proteins, the binding of SDS to the polypeptide chain imparts an even distribution of charge per unit mass, thereby resulting in a fractionation by approximate size during electrophoresis. [ 2 ]


SDS is a strong detergent agent used to denature native proteins to unfolded, individual  polypeptides . When a protein mixture is heated to 100 °C in presence of SDS, the  detergent  wraps around the polypeptide backbone. In this process, the intrinsic charges of polypeptides becomes negligible when compared to the negative charges contributed by SDS. Thus polypeptides after treatment become rod-like structures possessing a uniform charge density, that is same net negative charge per unit length. The electrophoretic mobilities of these proteins will be a linear function of the  logarithms  of their molecular weights. [ 3 ]


Native gels, also known as non-denaturing gels, analyze proteins that are still in their folded state. Thus, the electrophoretic mobility depends not only on the charge-to-mass ratio, but also on the physical shape and size of the protein. [ 4 ]


BN-PAGE is a native  PAGE  technique, where the  Coomassie brilliant blue  dye provides the necessary  charges  to the protein complexes for the electrophoretic separation. [ 5 ] [ 6 ]  The disadvantage of Coomassie is that in binding to proteins it can act like a  detergent  causing complexes to  dissociate . Another drawback is the potential  quenching  of  chemoluminescence  (e.g. in subsequent  western blot  detection or activity assays) or  fluorescence  of proteins with  prosthetic groups  (e.g.  heme  or  chlorophyll ) or labelled with fluorescent dyes. [ citation needed ]


CN-PAGE (commonly referred to as Native PAGE) separates acidic water-soluble and membrane  proteins  in a  polyacrylamide  gradient gel. It uses no charged dye so the electrophoretic mobility of proteins in CN-PAGE (in contrast to the charge shift technique BN-PAGE) is related to the intrinsic charge of the proteins. [ 7 ]  The migration distance depends on the protein charge, its size and the pore size of the gel. In many cases this method has lower resolution than BN-PAGE, but CN-PAGE offers advantages whenever  Coomassie  dye would interfere with further analytical techniques, for example it has been described as a very efficient microscale separation technique for  FRET  analyses. [ 8 ]  Additionally, as CN-PAGE does not require the harsh conditions of BN-PAGE, it can retain the  supramolecular assemblies  of  membrane protein  complexes that would be  dissociated  in BN-PAGE. [ 7 ]


The  folded   protein complexes  of interest separate cleanly and predictably without the risk of denaturation due to the specific  properties  of the polyacrylamide gel, electrophoresis buffer solution, electrophoretic equipment and standardized parameters used. The separated proteins are continuously  eluted  into a physiological eluent and transported to a fraction collector. In four to five PAGE fractions each the different metal cofactors can be identified and absolutely quantified by high-resolution  ICP-MS . The associated structures of the isolated  metalloproteins  in these fractions can be specifically determined by solution  NMR  spectroscopy. [ 9 ]


Most protein separations are performed using a "discontinuous" (or DISC)  buffer  system that significantly enhances the sharpness of the bands within the gel. During electrophoresis in a discontinuous gel system, an ion gradient is formed in the early stage of electrophoresis that causes all of the proteins to focus into a single sharp band. The formation of the ion gradient is achieved by choosing a pH value at which the ions of the buffer are only moderately charged compared to the SDS-coated proteins. These conditions provide an environment in which  Kohlrausch's  reactions determine the  molar conductivity . As a result, SDS-coated proteins are concentrated to several fold in a thin zone of the order of 19 μm within a few minutes. At this stage all proteins migrate at the same migration speed by  isotachophoresis . This occurs in a region of the gel that has larger pores so that the gel matrix does not retard the migration during the focusing or "stacking" event. [ 10 ] [ 11 ]  Separation of the proteins by size is achieved in the lower, "resolving" region of the gel. The resolving gel typically has a much smaller pore size, which leads to a sieving effect that now determines the electrophoretic mobility of the proteins. At the same time, the separating part of the gel also has a pH value in which the buffer ions on average carry a greater charge, causing them to "outrun" the SDS-covered proteins and eliminate the ion gradient and thereby the stacking effect. [ citation needed ]


A very widespread discontinuous buffer system is the tris-glycine or " Laemmli " system that stacks at a  pH  of 6.8 and resolves at a  pH  of ~8.3-9.0. A drawback of this system is that these pH values may promote  disulfide  bond formation between  cysteine  residues in the proteins because the  pKa  of cysteine ranges from 8-9 and because reducing agent present in the loading buffer doesn't co-migrate with the proteins. Recent advances in buffering technology alleviate this problem by resolving the proteins at a pH well below the pKa of cysteine (e.g.,  bis-tris , pH 6.5) and include reducing agents (e.g. sodium bisulfite) that move into the gel ahead of the proteins to maintain a reducing environment. An additional benefit of using buffers with lower pH values is that the acrylamide gel is more stable at lower pH values, so the gels can be stored for long periods of time before use. [ 12 ] [ 13 ]


As voltage is applied, the anions (and negatively charged sample molecules) migrate toward the positive electrode (anode) in the lower chamber, the leading ion is  Cl −  ( high mobility and high concentration); glycinate is the trailing ion (low mobility and low concentration). SDS-protein particles do not migrate freely at the border between the Cl −  of the gel buffer and the  Gly −  of the cathode buffer.  Friedrich Kohlrausch  found that  Ohm's law  also applies to dissolved  electrolytes . Because of the voltage drop between the Cl −  and Glycine-buffers,  proteins are compressed (stacked) into micrometer thin layers. [ 14 ]  The boundary moves through a pore gradient and the protein stack gradually disperses due to a frictional resistance increase of the gel matrix. Stacking and unstacking occurs continuously in the gradient gel, for every protein at a different position. For a complete protein unstacking the polyacrylamide-gel concentration must exceed 16% T. The two-gel system of "Laemmli" is a simple gradient gel. The pH discontinuity of the buffers is of no significance for the separation quality, and a "stacking-gel" with a different pH is not needed. [ 15 ]


The most popular protein stain is  Coomassie brilliant blue . It is an anionic dye, which non-specifically binds to proteins. Proteins in the gel are fixed by acetic acid and simultaneously stained. The excess dye incorporated into the gel can be removed by destaining with the same solution without the dye. The proteins are detected as blue bands on a clear background. [ 16 ] [ 17 ]


When more sensitive method than staining by Coomassie is needed, silver staining is usually used. Silver staining is a sensitive procedure to detect trace amounts of proteins in gels, but can also visualize nucleic acid or polysaccharides. [ 17 ]


Visualization methods without using a dye such as Coomassie and silver are available on the market. [ 18 ]  For example  Bio-Rad Laboratories  markets ”stain-free” gels for SDS-PAGE gel electrophoresis. Alternatively, reversible fluorescent dyes, such as those from  Azure Biosystems  such as AzureRed or Azure TotalStain Q can be used. [ 17 ] [ 18 ] [ 19 ]


Similarly as in nucleic acid gel electrophoresis,  tracking dye  is often used. Anionic dyes of a known electrophoretic mobility are usually included in the sample buffer. A very common tracking dye is  Bromophenol blue . This dye is coloured at alkali and neutral pH and is a small negatively charged molecule that moves towards the anode. Being a highly mobile molecule it moves ahead of most proteins. [ 20 ]


In  medicine ,  protein electrophoresis  is a method of analysing the  proteins  mainly in  blood serum .  Before the widespread use of  gel electrophoresis , protein electrophoresis was performed as free-flow electrophoresis (on paper) or as immunoelectrophoresis. [ citation needed ]


Traditionally, two classes of  blood proteins  are considered:  serum albumin  and  globulin . They are generally equal in proportion, but  albumin  as a molecule is much smaller and lightly, negatively-charged, leading to an accumulation of albumin on the electrophoretic gel. A small band before albumin represents  transthyretin  (also named prealbumin). Some forms of medication or body chemicals can cause their own band, but it usually is small. Abnormal bands (spikes) are seen in  monoclonal gammopathy of undetermined significance  and  multiple myeloma , and are useful in the diagnosis of these conditions. [ citation needed ]


The globulins are classified by their banding pattern (with their main representatives): [ citation needed ]


Blood glucose monitoring  is the use of a  glucose meter  for testing the concentration of  glucose  in the blood ( glycemia ). Particularly important in  diabetes management , a blood glucose test is typically performed by piercing the  skin  (typically, via  fingerstick ) to draw blood, then applying the blood to a chemically active disposable 'test-strip'. The other main option is  continuous glucose monitoring  (CGM). Different manufacturers use different technology, but most systems measure an electrical characteristic and use this to determine the glucose level in the blood. Skin-prick methods measure capillary blood glucose (i.e., the level found in capillary blood), whereas CGM correlates interstitial fluid glucose level to blood glucose level. Measurements may occur after  fasting  or at random nonfasting intervals ( random glucose tests ), each of which informs diagnosis or monitoring in different ways.


Healthcare professionals advise patients with  diabetes mellitus  on the appropriate monitoring regimen for their condition. Most people with  type 2 diabetes  test at least once per day. The  Mayo Clinic  generally recommends that diabetics who use insulin (all  type 1 diabetics  and many  type 2 diabetics ) test their blood sugar more often (4–8 times per day for type 1 diabetics, 2 or more times per day for type 2 diabetics), [ 1 ]  both to assess the effectiveness of their prior insulin dose and to help determine their next insulin dose.


Blood glucose monitoring reveals individual patterns of blood glucose changes, and helps in the planning of meals, activities, and at what time of day to take medications. [ 2 ]


Also, testing allows for a quick response to high blood sugar ( hyperglycemia ) or low blood sugar ( hypoglycemia ). This might include diet adjustments, exercise, and insulin (as instructed by the health care provider). [ 2 ]


A  blood glucose meter  is an electronic device for measuring the blood glucose level. A relatively small drop of blood is placed on a disposable test strip which interfaces with a digital meter. Within several seconds, the level of blood glucose will be shown on the digital display. Needing only a small drop of blood for the meter means that the time and effort required for testing are reduced and the compliance of diabetic people to their testing regimens is improved significantly. Blood glucose meters provide results in various units such as eAG (mg/dL) and eAG (mmol/L), and may also estimate A1C levels. These measurements can aid in classifying blood glucose levels as normal, prediabetic, or diabetic, facilitating effective diabetes management for users. While some models offer interpretative features that indicate the health status based on these results, not all meters provide this functionality, focusing instead on providing raw glucose measurements. Users of blood glucose meters without interpretative features can utilize online calculators to determine their blood glucose status based on measured values.  [ 3 ]  The cost of using blood glucose meters is believed to be a cost-benefit relative to the avoided medical costs of the  complications of diabetes . [ 4 ]


Recent advances include: [ 5 ]


A  continuous glucose monitor  determines glucose levels on a continuous basis (every few minutes). [ 6 ]  A typical system consists of:


Continuous glucose monitors measure the concentration of glucose in a sample of  interstitial fluid .  Shortcomings of CGM systems due to this fact are:


Patients, therefore, require traditional fingerstick measurements for calibration (typically twice per day) and are often advised to use fingerstick measurements to confirm hypo- or hyperglycemia before taking corrective action.


The lag time discussed above has been reported to be about 5 minutes. [ 8 ] [ 9 ] [ 10 ]   Anecdotally, some users of the various systems report lag times of up to 10–15 minutes. This lag time is insignificant when blood sugar levels are relatively consistent. However, blood sugar levels, when changing rapidly, may read in the normal range on a CGM system while in reality the patient is already experiencing symptoms of an out-of-range blood glucose value and may require treatment. Patients using CGM are therefore advised to consider both the absolute value of the blood glucose level given by the system as well as any trend in the blood glucose levels. For example, a patient using CGM with a blood glucose of 100 mg/dl on their CGM system might take no action if their blood glucose has been consistent for several readings, while a patient with the same blood glucose level but whose blood glucose has been dropping steeply in a short period of time might be advised to perform a fingerstick test to check for hypoglycemia. [ citation needed ]


Continuous monitoring allows examination of how the blood glucose level reacts to insulin, exercise, food, and other factors. The additional data can be useful for setting correct  insulin  dosing ratios for food intake and correction of hyperglycemia. Monitoring during periods when blood glucose levels are not typically checked (e.g. overnight) can help to identify problems in insulin dosing (such as basal levels for insulin pump users or long-acting insulin levels for patients taking injections). Monitors may also be equipped with alarms to alert patients of hyperglycemia or hypoglycemia so that a patient can take corrective action(s) (after fingerstick testing, if necessary) even in cases where they do not feel symptoms of either condition. While the technology has its limitations, studies have demonstrated that patients with continuous sensors experience a smaller number of hyperglycemic and hypoglycemic events, a reduction in their  glycated hemoglobin  levels and a decrease in glycemic variability. [ 11 ] [ 12 ] [ 13 ] [ 14 ] [ 15 ]  Compared to intermittent testing, it is likely to help reduce hypertensive complications during pregnancy. [ 16 ]  In a recent systematic review with meta-analysis  about glycaemia monitoring in critical patients [ 17 ]  who are haemodynamically unstable and require intensive monitoring of glycaemia it concluded that should be undertaken using arterial blood samples and POC blood gas analysers, as this is more reliable and is not affected by the variability of different confusion factors. Determining glycaemia in capillary blood using glucometry may be suitable in stable patients or when close monitoring of glycaemia is not required.


Continuous blood glucose monitoring is not automatically covered by  health insurance  in the United States in the same way that most other diabetic supplies are covered (e.g. standard glucose testing supplies,  insulin , and  insulin pumps ). However, an increasing number of insurance companies do cover continuous glucose monitoring supplies (both the receiver and disposable sensors) on a case-by-case basis if the patient and doctor show a specific need. The lack of insurance coverage is exacerbated by the fact that disposable sensors must be frequently replaced. Some sensors have been U.S.  Food and Drug Administration  (FDA) approved for 7- and 3-day use, (although some patients wear sensors for longer than the recommended period) and the receiving meters likewise have finite lifetimes (less than 2 years and as little as 6 months). This is one factor in the slow uptake in the use of sensors that have been marketed in the United States. [ citation needed ]


The principles, history and recent developments of operation of electrochemical glucose biosensors are discussed in a chemical review by  Joseph Wang . [ 18 ]


Investigations on the use of test strips have shown that the required self-injury acts as a psychological barrier restraining the patients from sufficient glucose control. [ 19 ]  As a result,  secondary diseases  are caused by excessive glucose levels. A significant improvement of diabetes therapy might be achieved with an implantable sensor that would continuously monitor blood sugar levels within the body and transmit the measured data outside. The burden of regular blood testing would be taken from the patient, who would instead follow the course of their glucose levels on an intelligent device like a laptop or a smartphone. [ citation needed ]


Glucose concentrations do not necessarily have to be measured in blood vessels, but may also be determined in the  interstitial fluid , where the same levels prevail – with a time lag of a few minutes – due to its connection with the  capillary system . However, the enzymatic glucose detection scheme used in single-use test strips is not directly suitable for  implants . One main problem is caused by the varying supply of oxygen, by which glucose is converted to  glucono lactone  and H 2 O 2  by  glucose oxidase . Since the implantation of a sensor into the body is accompanied by growth of encapsulation tissue, [ 20 ]  the diffusion of oxygen to the reaction zone is continuously diminished. This decreasing oxygen availability causes the sensor reading to drift, requiring frequent re-calibration using finger-sticks and test strips.


One approach to achieving long-term glucose sensing is to measure and compensate for the changing local oxygen concentration. [ 21 ]  Other approaches replace the troublesome glucose oxidase reaction with a reversible sensing reaction, known as an  affinity assay . This scheme was originally put forward by Schultz & Sims in 1978. [ 22 ] [ 23 ]  A number of different affinity assays have been investigated, [ 24 ] [ 25 ] [ 26 ]  with  fluorescent assays  proving most common. [ 27 ] [ 28 ] [ 29 ]   MEMS  technology has recently allowed for smaller and more convenient alternatives to fluorescent detection, via measurement of  viscosity . [ 30 ]  Investigation of affinity-based sensors has shown that encapsulation by body tissue does not cause a drift of the sensor signal, but only a time lag of the signal compared to the direct measurement in blood. [ 31 ]  A new implantable continuous glucose monitor based on affinity principles and fluorescence detection is the Eversense device manufactured by Senseonics Inc. This device has been approved by the FDA for 90 day implantation. [ 32 ] [ 33 ]


Some new technologies to monitor blood glucose levels will not require access to blood to read the glucose level.  Non-invasive  technologies include microwave/RF sensing, [ 34 ] [ 35 ]   near IR  detection, [ 36 ]   ultrasound [ 37 ]  and  dielectric spectroscopy . [ 38 ]  These may free the person with diabetes from finger sticks to supply the drop of blood for blood glucose analysis. [ citation needed ]


Most [ citation needed ]  of the non-invasive methods under development are continuous glucose monitoring methods and offer the advantage of providing additional information to the subject between the conventional finger stick, blood glucose measurements, and overtime periods where no finger stick measurements are available (i.e. while the subject is sleeping).


For patients with  diabetes mellitus type 2 , the importance of monitoring and the optimal frequency of monitoring are not clear. A 2011 study found no evidence that blood glucose monitoring leads to better patient outcomes in actual practice. [ 39 ]  Randomized controlled trials found that self-monitoring of blood glucose did not improve  glycated hemoglobin  (HbA1c) among "reasonably well controlled non-insulin treated patients with type 2 diabetes" [ 40 ]  or lead to significant changes in quality of life. [ 41 ]   However a recent meta-analysis of 47 randomized controlled trials encompassing 7677 patients showed that self-care management intervention improves glycemic control in diabetics, with an estimated 0.36% (95% CI, 0.21–0.51) reduction in their glycated hemoglobin values. [ 42 ]  Furthermore, a recent study showed that patients described as being "Uncontrolled Diabetics" (defined in this study by HbA1C levels >8%) showed a statistically significant decrease in the HbA1C levels after a 90-day period of seven-point self-monitoring of blood glucose (SMBG) with a relative risk reduction (RRR) of 0.18% (95% CI, 0.86–2.64%, p<.001). [ 43 ]  Regardless of lab values or other numerical parameters, the purpose of the clinician is to improve quality of life and patient outcomes in diabetic patients. A recent study included 12 randomized controlled trials and evaluated outcomes in 3259 patients. The authors concluded through a qualitative analysis that SMBG on quality of life showed no effect on patient satisfaction or the patients' health-related quality of life.  Furthermore, the same study identified that patients with type 2 diabetes mellitus diagnosed greater than one year prior to initiation of SMBG, who were not on insulin, experienced a statistically significant reduction in their HbA1C of 0.3% (95% CI, -0.4 –  -0.1) at six months follow up, but a statistically insignificant reduction of 0.1% (95% CI, -0.3 – 0.04) at twelve months follow up.  Conversely, newly diagnosed patients experienced a statistically significant reduction of 0.5% (95% CI, -0.9 – -0.1) at 12 months follow up. [ 44 ]  A recent study found that a treatment strategy of intensively lowering blood sugar levels (below 6%) in patients with additional  cardiovascular disease  risk factors poses more harm than benefit. [ 45 ]   For type 2 diabetics who are not on insulin, exercise and diet are the best tools. [ citation needed ]  Blood glucose monitoring is, in that case, simply a tool to evaluate the success of diet and exercise. Insulin-dependent type 2 diabetics do not need to monitor their blood sugar as frequently as type 1 diabetics. [ 46 ]  In a recent systematic review with meta-analysis, about glycaemia monitoring in critical patients who are haemodynamically unstable and require intensive monitoring of glycaemia it concluded that should be undertaken using arterial blood samples and POC blood gas analysers, as this is more reliable and is not affected by the variability of different confusion factors. Determining glycaemia in capillary blood using glucometry may be suitable in stable patients or when close monitoring of glycaemia is not required.


The  National Institute for Health and Clinical Excellence  (NICE), UK released updated diabetes recommendations on 30 May 2008, which recommend that self-monitoring of plasma glucose levels for people with newly diagnosed type 2 diabetes must be integrated into a structured self-management education process. [ 47 ]  The recommendations have been updated in August 2015 for children and young adults with type 1 diabetes. [ 48 ]


The  American Diabetes Association (ADA) , which produces guidelines for diabetes care and  clinical practice recommendations , recently updated its "Standards of Medical Care" in January 2019 to acknowledge that routine self-monitoring of blood glucose in people who are not using insulin is of limited additional clinical benefit. [ 49 ]  A randomized controlled trial evaluated once-daily self-monitoring that included tailored patient messaging and did not show that this strategy led to significant changes in A1C after a year. [ 41 ]


Many types of  glucose tests  exist and they can be used to estimate  blood sugar levels  at a given time or, over a longer period of time, to obtain average levels or to see how fast the body is able to normalize changed  glucose  levels. Eating food for example leads to elevated blood sugar levels. In healthy people, these levels quickly return to normal via increased  cellular  glucose uptake which is primarily mediated by increase in blood  insulin  levels. [ citation needed ]


Glucose tests can reveal temporary/long-term  hyperglycemia  or  hypoglycemia . These conditions may not have obvious symptoms and can damage organs in the long-term. Abnormally high/low levels, slow return to normal levels from either of these conditions and/or inability to normalize blood sugar levels means that the person being tested probably has some kind of medical condition like  type 2 diabetes  which is caused by cellular insensitivity to insulin. Glucose tests are thus often used to diagnose such conditions.


Tests that can be performed at home are used in  blood glucose monitoring  for illnesses that have already been diagnosed medically so that these illnesses can be maintained via medication and meal timing. Some of the home testing methods include [ citation needed ]


Laboratory tests are often used to diagnose illnesses and such methods include


Some laboratory tests don't measure glucose levels directly from body fluids or tissues but still indicate elevated blood sugar levels. Such tests measure the levels of  glycated hemoglobin , other  glycated  proteins,  1,5-anhydroglucitol  etc. from blood. [ 1 ]


Glucose testing can be used to diagnose or indicate certain medical conditions. [ citation needed ]


High blood sugar  may indicate


Low blood sugar  may indicate


Fasting  prior to glucose testing may be required with some test types. Fasting blood sugar test, for example, requires 10–16 hour-long period of not eating before the test. [ 1 ]


Blood sugar levels can be affected by some drugs and prior to some glucose tests these medications should be temporarily given up or their dosages should be decreased. Such drugs may include  salicylates  (Aspirin),  birth control pills ,  corticosteroids ,  tricyclic antidepressants ,  lithium ,  diuretics  and  phenytoin . [ medical citation needed ]


Some foods contain  caffeine  ( coffee ,  tea ,  colas ,  energy drinks  etc.). Blood sugar levels of healthy people are generally not significantly changed by caffeine, but in diabetics caffeine intake may elevate these levels via its ability to stimulate the  adrenergic nervous system . [ 4 ]


A level below 5.6 mmol/L (100 mg/dL) 10–16 hours without eating is normal. 5.6–6 mmol/L (100–109 mg/dL) may indicate  prediabetes  and  oral glucose tolerance test  (OGTT) should be offered to high-risk individuals (old people, those with  high blood pressure  etc.). 6.1–6.9 mmol/L (110–125 mg/dL) means OGTT should be offered even if other indicators of diabetes are not present. 7 mmol/L (126 mg/dL) and above indicates diabetes and the fasting test should be repeated. [ 5 ]


The  glucose tolerance test  (GTT, not to be confused with  GGT test ) is a  medical test  in which  glucose  is given and  blood  samples taken afterward to determine how quickly it is cleared from the blood. [ 1 ]  The test is usually used to test for  diabetes ,  insulin resistance , impaired  beta cell function , [ 2 ]  and sometimes  reactive hypoglycemia  and  acromegaly , or rarer disorders of  carbohydrate metabolism . In the most commonly performed version of the test, an  oral glucose tolerance test  (OGTT), a standard dose of glucose is ingested by mouth and blood levels are checked two hours later. [ 3 ]  Many variations of the GTT have been devised over the years for various purposes, with different standard doses of glucose, different routes of administration, different intervals and durations of sampling, and various substances measured in addition to blood glucose.


The glucose tolerance test was first described in 1923 by  Jerome W. Conn . [ 4 ]


The test was based on the previous work in 1913 by A. T. B. Jacobson in determining that carbohydrate ingestion results in blood glucose fluctuations, [ 5 ]  and the premise (named the  Staub-Traugott Phenomenon  after its first observers H. Staub in 1921 and K. Traugott in 1922) that a normal patient fed glucose will rapidly return to normal levels of blood glucose after an initial spike, and will see improved reaction to subsequent glucose feedings. [ 6 ] [ 7 ]


Since the 1970s, the  World Health Organization  and other organizations interested in diabetes agreed on a standard dose and duration. [ 8 ]


The patient is instructed not to restrict  carbohydrate  intake in the days or weeks before the test. [ 9 ]  The test should not be done during an illness, as results may not reflect the patient's glucose metabolism when healthy. A full adult dose should not be given to a person weighing less than 42.6 kg (94 lb), or the excessive glucose may produce a  false positive  result. Usually the OGTT is performed in the morning as glucose tolerance can exhibit a diurnal rhythm with a significant decrease in the afternoon. The patient is instructed to  fast  (water is allowed) for 8–12 hours prior to the tests. Medication such as large doses of  salicylates ,  diuretics ,  anticonvulsants , and  oral contraceptives  affect the glucose tolerance test. [ 10 ]


If  renal glycosuria  (sugar excreted in the urine despite normal levels in the blood) is suspected, urine samples may also be collected for testing along with the fasting and 2 hour blood tests. [ 15 ]


For  gestational diabetes , the  American College of Obstetricians and Gynecologists  (ACOG) recommends a two-step procedure, wherein the first step is a 50 g glucose dose. [ 12 ] [ 16 ]  If after 1 hour the blood glucose level is more than 7.8 mmol/L (140 mg/dL), [ 12 ]  it is followed by a 100 g glucose dose. [ 12 ]  The diagnosis of gestational diabetes is then defined by a blood glucose level meeting or exceeding the cutoff values on at least two intervals, [ 12 ]  with cutoffs as follows: [ 12 ]


The diagnosis criteria stated above by the World Health Organization (WHO) are for venous samples only (a blood sample taken from a vein in the arm). An increasingly popular method for measuring blood glucose is to sample capillary or finger-prick blood, which is less invasive, more convenient for the patient and requires minimal training to conduct. Though fasting blood glucose levels have been shown to be similar in both capillary and venous samples, postprandial blood glucose levels (those measured after a meal) can vary. [ 17 ]  The diagnosis criteria issued by the WHO are only suitable for venous blood samples. Given the increasing popularity of capillary testing, the WHO has recommended that a conversion factor between the two sample types be calculated, but as of 2017 [update]  no conversion factor had been issued by the WHO, despite some medical professionals adopting their own. [ citation needed ]  A 2020 study on pregnant women for gestational diabetes mellitus (GDM) found that 0-hour venous and capillary levels were similar, but that 2-hour samples were different. The authors compared their study with others, and concluded that capillary samples could be used for diagnosis of GDM during pregnancy using corrected cutoffs with acceptable accuracy in an antenatal care setting. [ 17 ]


A standard two-hour GTT (glucose tolerance test) is sufficient to diagnose or exclude all forms of diabetes mellitus at all but the earliest stages of development.


Longer tests have been used for a variety of other purposes, such as detecting reactive hypoglycemia or defining subsets of  hypothalamic obesity . Insulin levels are sometimes measured to detect insulin resistance or deficiency. [ 18 ]


The GTT (glucose tolerance test) is of limited value in the diagnosis of reactive hypoglycemia, since normal levels do not preclude the diagnosis, abnormal levels do not prove that the patient's other symptoms are related to a demonstrated atypical OGTT, and many people without symptoms of reactive hypoglycemia may have the late low glucose. [ 19 ]


The oral glucose challenge test (OGCT) is a short version of the OGTT, used to check pregnant women for signs of  gestational diabetes . [ 3 ] [ 20 ]  It can be done at any time of day, not on an empty stomach. [ 3 ]  The test involves 50 g of glucose, with a reading after one hour. [ 3 ]


The OGTT does not distinguish between insulin resistance in peripheral tissues and reduced capacity of the pancreas  beta-cells  to produce insulin. The OGTT is less accurate than the  hyperinsulinemic-euglycemic clamp technique  (the "gold standard" for measuring insulin resistance), or the  insulin tolerance test , but is technically less difficult. Neither of the two technically demanding tests can be easily applied in a clinical setting or used in epidemiological studies. HOMA-IR ( homeostatic model assessment ) is a convenient way of measuring insulin resistance in normal subjects, which can be used in epidemiological studies, but can give erroneous results for diabetic patients. [ 21 ] [ 22 ]


Glycated hemoglobin  (also called  glycohemoglobin ) is a form of  hemoglobin  (Hb) that is chemically linked to a sugar. ("Glycosylated haemoglobin" is a misnomer because  glycation  and  glycosylation  are different processes, of which only the former is relevant in this case.)


Most  monosaccharides , including  glucose ,  galactose , and  fructose , spontaneously (that is,  non-enzymatically ) bond with hemoglobin when they are present in the bloodstream. However, glucose is only 21% as likely to do so as galactose and 13% as likely to do so as fructose, which may explain why glucose is used as the primary metabolic fuel in humans. [ 1 ] [ 2 ]


The formation of excess sugar-hemoglobin linkages indicates the presence of excessive sugar in the bloodstream and is an indicator of  diabetes  or other hormone diseases in high concentration (HbA1c >6.4%). [ 3 ]  A1c is of particular interest because it is easy to detect. The process by which sugars attach to hemoglobin is called  glycation  and the reference system is based on HbA1c, defined as beta-N-1-deoxy fructosyl hemoglobin as component. [ 4 ]


There are several ways to measure glycated hemoglobin, of which  HbA1c  (or simply  A1c ) is a standard single test. [ 5 ]  HbA1c is measured primarily to determine the three-month average  blood sugar level  and is used as a standard diagnostic test for evaluating the risk of complications of  diabetes  and as an assessment of  glycemic control . [ 5 ] [ 6 ]  The test is considered a three-month average because the average lifespan of a red blood cell is three to four months. Normal levels of glucose produce a normal amount of glycated hemoglobin. As the average amount of plasma glucose increases, the fraction of glycated hemoglobin increases in a predictable way. In diabetes, higher amounts of glycated hemoglobin, indicating higher of blood glucose levels, have been associated with  cardiovascular disease ,  nephropathy ,  neuropathy , and  retinopathy . [ 7 ]


Glycated  hemoglobin is preferred over  glycosylated  hemoglobin to reflect the correct (non-enzymatic) process. Early literature often used  glycosylated  as it was unclear which process was involved until further research was performed. The terms are still sometimes used interchangeably in English-language literature. [ 8 ]


The naming of HbA1c derives from hemoglobin type A being separated on  cation exchange chromatography . The first fraction to separate, probably considered to be pure hemoglobin A, was designated HbA0, and the following fractions were designated HbA1a, HbA1b, and HbA1c, in their order of  elution . Improved separation techniques have subsequently led to the isolation of more  subfractions . [ 9 ]


Hemoglobin A1c was first separated from other forms of hemoglobin by Huisman and Meyering in 1958 using a  chromatographic column . [ 10 ]  It was first characterized as a  glycoprotein  by Bookchin and Gallop in 1968. [ 11 ]  Its increase in diabetes was first described in 1969 by  Samuel Rahbar   et al . [ 12 ]  The reactions leading to its formation were characterized by Bunn and his coworkers in 1975. [ 13 ]


The use of hemoglobin A1c for monitoring the degree of control of glucose metabolism in diabetic patients was proposed in 1976 by  Anthony Cerami , Ronald Koenig, and coworkers. [ 14 ]


Glycated hemoglobin causes an increase of highly reactive  free radicals  inside blood cells, altering the properties of their  cell membranes . This leads to  blood cell aggregation  and increased blood  viscosity , which results in impaired blood flow. [ 15 ]


Another way glycated hemoglobin causes damage is via  inflammation , which results in  atherosclerotic  plaque ( atheroma ) formation. Free-radical build-up promotes the  excitation  of  Fe 2+ -hemoglobin through  Fe 3+ -Hb  into abnormal  ferryl  hemoglobin (Fe 4+ -Hb). Fe 4+  is unstable and reacts with specific  amino acids  in hemoglobin to regain its Fe 3+   oxidation state . Hemoglobin molecules clump together via  cross-linking reactions , and these hemoglobin clumps (multimers) promote cell damage and the release of Fe 4+ -hemoglobin into the  matrix  of innermost layers ( subendothelium ) of arteries and veins. This results in increased permeability of interior surface ( endothelium ) of blood vessels and production of pro-inflammatory  monocyte   adhesion  proteins, which promote  macrophage  accumulation in blood vessel surfaces, ultimately leading to harmful plaques in these vessels. [ 15 ]


Highly glycated Hb- AGEs  go through  vascular smooth muscle  layer and inactivate  acetylcholine -induced endothelium-dependent relaxation, possibly through binding to  nitric oxide  (NO), preventing its normal function. NO is a potent  vasodilator  and also inhibits formation of plaque-promoting  LDLs  (sometimes called "bad cholesterol")  oxidized  form. [ 15 ]


This overall degradation of blood cells also releases  heme  from them. Loose heme can cause oxidation of endothelial and LDL proteins, which results in plaques. [ 15 ]


Glycation of proteins is a frequent occurrence, but in the case of hemoglobin, a nonenzymatic condensation reaction occurs between glucose and the N-end of the  beta chain . This reaction produces a  Schiff base  ( R−N=CHR' , R=beta chain, CHR'=glucose-derived), which is itself converted to 1-deoxyfructose. This second conversion is an example of an  Amadori rearrangement . [ citation needed ]


When blood glucose levels are high,  glucose  molecules attach to the hemoglobin in  red blood cells . The longer hyperglycemia occurs in blood, the more glucose binds to hemoglobin in the red blood cells and the higher the glycated hemoglobin. [ 17 ]


Once a hemoglobin molecule is glycated, it remains that way. A buildup of glycated hemoglobin within the red cell, therefore, reflects the average level of glucose to which the cell has been exposed during its  life-cycle . Measuring glycated hemoglobin assesses the effectiveness of therapy by monitoring long-term serum glucose regulation.


A1c is a weighted average of blood glucose levels during the life of the red blood cells (117 days for men and 106 days in women [ 18 ] ). Therefore, glucose levels on days nearer to the test contribute substantially more to the level of A1c than the levels in days further from the test. [ 19 ]


This is also supported by data from clinical practice showing that HbA1c levels improved significantly after 20 days from start or intensification of glucose-lowering treatment. [ 20 ]


Several techniques are used to measure hemoglobin A1c. Laboratories may use  high-performance liquid chromatography ,  immunoassay ,  enzymatic  assay,  capillary electrophoresis , or  boronate affinity chromatography .  Point of care  (e.g., doctor's office) devices use immunoassay boronate affinity chromatography. [ 17 ]


In the United States, HbA 1c  testing laboratories are certified by the National Glycohemoglobin Standardization Program to standardize them against the results of the 1993  Diabetes Control and Complications Trial  (DCCT). [ 21 ]  An additional percentage scale, Mono S has previously been in use by Sweden and KO500 is in use in Japan. [ 22 ] [ 23 ]


The American Diabetes Association,  European Association for the Study of Diabetes , and  International Diabetes Federation  have agreed that, in the future, HbA 1c  is to be reported in the  International Federation of Clinical Chemistry and Laboratory Medicine  (IFCC) units. [ 24 ]  IFCC reporting was introduced in Europe except for the UK in 2003; [ 25 ]  the UK carried out dual reporting from 1 June 2009  [ 26 ]  until 1 October 2011.


Conversion between DCCT and IFCC is by the following equation: [ 27 ]


Laboratory results may differ depending on the analytical technique, the age of the subject, and biological variation among individuals. Higher levels of HbA 1c  are found in people with persistently elevated blood sugar, as in  diabetes mellitus . While diabetic patient treatment goals vary, many include a target range of HbA 1c  values. A diabetic person with good glucose control has an HbA 1c  level that is close to or within the reference range. [ citation needed ]


The International Diabetes Federation and the American College of Endocrinology recommend HbA 1c  values below 48 mmol/mol (6.5 DCCT %), while the  American Diabetes Association  recommends HbA 1c  be below 53 mmol/mol (7.0 DCCT %) for most patients. [ 28 ]  Results from large trials in  2008–09  suggested that a target below 53 mmol/mol (7.0 DCCT %) for older adults with type 2 diabetes may be excessive: Below 53 mmol/mol, the health benefits of reduced A1c become smaller, and the intensive glycemic control required to reach this level leads to an increased rate of dangerous hypoglycemic episodes. [ 29 ]


A retrospective study of 47,970 type 2 diabetes patients, aged 50 years and older, found that patients with an HbA 1c  more than 48 mmol/mol (6.5 DCCT %) had an increased mortality rate, [ 30 ]  but a later international study contradicted these findings. [ 31 ] [ 32 ] [ 33 ]


A review of the  UKPDS , Action to Control Cardiovascular Risk in Diabetes (ACCORD), Advance and Veterans Affairs Diabetes Trials (VADT) estimated that the risks of the main complications of diabetes ( diabetic retinopathy ,  diabetic nephropathy ,  diabetic neuropathy , and  macrovascular disease ) decreased by about 3% for every 1 mmol/mol decrease in HbA 1c . [ 34 ]


However, a trial by ACCORD designed specifically to determine whether reducing HbA 1c  below 42 mmol/mol (6.0 DCCT %) using increased amounts of medication would reduce the rate of cardiovascular events found higher mortality with this intensive therapy, so much so that the trial was terminated 17 months early. [ 35 ]


Practitioners must consider patients' health, their risk of hypoglycemia, and their specific health risks when setting a target HbA 1c  level. Because patients are responsible for averting or responding to their own hypoglycemic episodes, their input and the doctors' assessments of the patients'  self-care  skills are also important. [ citation needed ]


Persistent elevations in blood sugar (and, therefore, HbA 1c ) increase the risk of long-term vascular complications of diabetes, such as  coronary disease ,  heart attack ,  stroke ,  heart failure ,  kidney failure ,  blindness ,  erectile dysfunction ,  neuropathy  (loss of sensation, especially in the feet),  gangrene , and  gastroparesis  (slowed emptying of the stomach). Poor blood glucose control also increases the risk of short-term complications of surgery such as poor  wound healing . [ citation needed ]


All-cause mortality  is higher above 64 mmol/mol (8.0 DCCT%) HbA1c as well as below 42 mmol/mol (6.0 DCCT %) in diabetic patients, and above 42 mmol/mol (6.0 DCCT %) as well as below 31 mmol/mol (5.0 DCCT %) in non-diabetic persons, indicating the risks of  hyperglycemia  and  hypoglycemia , respectively. [ 7 ]  Similar risk results are seen for  cardiovascular disease . [ 7 ]


The 2022 ADA guidelines reaffirmed the recommendation that HbA1c should be maintained below 7.0% for most patients. Higher target values are appropriate for children and adolescents, patients with extensive co-morbid illness and those with a history of severe hypoglycemia.  More stringent targets (<6.0%) are preferred for pregnant patients if this can be achieved without significant hypoglycemia. [ 36 ]


Lower-than-expected levels of HbA 1c  can be seen in people with shortened red blood cell lifespans, such as with  glucose-6-phosphate dehydrogenase deficiency ,  sickle-cell disease , or any other condition causing premature red blood cell death. For these patients, alternate assessment with  fructosamine  or glycated  albumin  is recommended; these methods reflect glycemic control over the preceding 2-3 weeks. [ 37 ]  Blood donation will result in rapid replacement of lost RBCs with newly formed red blood cells. Since these new RBCs will have only existed for a short period of time, their presence will lead HbA 1c  to underestimate the actual average levels. There may also be distortions resulting from  blood donation  during the preceding two months, due to an abnormal synchronization of the age of the RBCs, resulting in an older than normal average blood cell life (resulting in an overestimate of actual average blood glucose levels). Conversely, higher-than-expected levels can be seen in people with a longer red blood cell lifespan, such as with iron deficiency. [ 38 ]


Results can be unreliable in many circumstances, for example after blood loss, after surgery, blood transfusions, anemia, or high erythrocyte turnover; in the presence of chronic renal or liver disease; after administration of high-dose vitamin C; or  erythropoetin  treatment. [ 39 ]   Hypothyroidism  can artificially raise the A1c. [ 40 ] [ 41 ] [ 42 ]  In general, the  reference range  (that found in healthy young persons), is about 30–33 mmol/mol (4.9–5.2 DCCT %). [ 43 ]  The mean HbA 1c  for diabetics type 1 in Sweden in 2014 was 63 mmol/mol (7.9 DCCT%) and for type 2, 61 mmol/mol (7.7 DCCT%). [ 44 ]  HbA1c levels show a small, but statistically significant, progressive uptick with age; the clinical importance of this increase is unclear. [ 37 ]


The approximate mapping between HbA 1c  values given in DCCT percentage (%) and eAG (estimated average glucose) measurements is given by the following equation: [ 39 ]


The 2010  American Diabetes Association  Standards of Medical Care in Diabetes added the HbA 1c  ≥ 48 mmol/mol (≥6.5 DCCT %) as another criterion for the diagnosis of diabetes. [ 46 ]


Glycated hemoglobin testing is recommended for both checking the blood sugar control in people who might be prediabetic and monitoring blood sugar control in patients with more elevated levels, termed diabetes mellitus. For a single blood sample, it provides far more revealing information on glycemic behavior than a fasting blood sugar value. However, fasting blood sugar tests are crucial in making treatment decisions. The American Diabetes Association guidelines are similar to others in advising that the glycated hemoglobin test be performed at least twice a year in patients with diabetes who are meeting treatment goals (and who have stable glycemic control) and quarterly in patients with diabetes whose therapy has changed or who are not meeting glycemic goals. [ 48 ] [ 36 ]


Glycated hemoglobin measurement is not appropriate where a change in diet or treatment has been made within six weeks. Likewise, the test assumes a normal red blood cell aging process and mix of hemoglobin subtypes (predominantly HbA in normal adults). Hence, people with recent blood loss,  hemolytic anemia , or genetic differences in the hemoglobin molecule ( hemoglobinopathy ) such as  sickle-cell disease  and other conditions, as well as those who have donated blood recently, are not suitable for this test. [ 49 ]


Due to glycated hemoglobin's variability (as shown in the table above), additional measures should be checked in patients at or near recommended goals. People with HbA 1c  values at 64 mmol/mol or less should be provided additional testing to determine whether the HbA 1c  values are due to averaging out high blood glucose ( hyperglycemia ) with low blood glucose ( hypoglycemia ) or the HbA 1c  is more reflective of an elevated blood glucose that does not vary much throughout the day. Devices such as continuous  blood glucose monitoring  allow people with diabetes to determine their blood glucose levels on a continuous basis, testing every few minutes. Continuous use of blood glucose monitors is becoming more common, and the devices are covered by many health insurance plans, including  Medicare  in the United States. The supplies tend to be expensive, since the sensors must be changed at least every 2 weeks. Another useful test in determining if HbA 1c  values are due to wide variations of blood glucose throughout the day is  1,5-anhydroglucitol , also known as  GlycoMark . GlycoMark reflects only the times that the person experiences hyperglycemia above 180 mg/dL over a two-week period. [ citation needed ]


Concentrations of hemoglobin A1 (HbA1) are increased, both in diabetic patients and in patients with  kidney failure , when measured by  ion-exchange chromatography . The thiobarbituric acid method (a chemical method specific for the detection of glycation) shows that patients with kidney failure have values for glycated hemoglobin similar to those observed in normal subjects, suggesting that the high values in these patients are a result of binding of something other than glucose to hemoglobin. [ 50 ]


In  autoimmune hemolytic anemia , concentrations of HbA1 is undetectable. Administration of  prednisolone  will allow the HbA1 to be detected. [ 51 ]  The alternative  fructosamine  test may be used in these circumstances and it also reflects an average of blood glucose levels over the preceding 2 to 3 weeks. [ 52 ]


All the major institutions such as the International Expert Committee Report, drawn from the International Diabetes Federation, the European Association for the Study of Diabetes, and the American Diabetes Association, suggest the HbA 1c  level of 48 mmol/mol (6.5 DCCT %) as a diagnostic level. [ 53 ]  The Committee Report further states that, when HbA 1c  testing cannot be done, the fasting and glucose-tolerance tests be done. Screening for diabetes during pregnancy continues to require fasting and glucose-tolerance measurements for  gestational diabetes  at 24 to 28 weeks gestation, although glycated hemoglobin may be used for screening at the first prenatal visit. [ 37 ]


Meta-analysis  has shown  probiotics  to cause a statistically significant reduction in glycated hemoglobin in  type-2 diabetics . [ 54 ]  Trials with multiple strains of probiotics had statistically significant reductions in glycated hemoglobin, whereas trials with single strains did not. [ 54 ]


Most clinical studies recommend the use of HbA1c assays that are traceable to the DCCT assay. [ 55 ]  The National Glycohemoglobin Standardization Program (NGSP) and IFCC have improved assay standardization. [ 37 ]  For initial diagnosis of diabetes, only HbA1c methods that are NGSP-certified should be used, not  point-of-care testing  devices. [ 36 ]  Analytical performance has been a problem with earlier  point-of-care  devices for HbA1c testing, specifically large standard deviations and negative bias. [ 37 ]


HbA1c testing has not been found useful in the monitoring during the treatment of cats and dogs with diabetes, and is not generally used; monitoring of  fructosamine  levels is favoured instead. [ 56 ]


Glycogen phosphorylase isoenzyme BB  (abbreviation:  GPBB ) is an  isoenzyme  of  glycogen phosphorylase . This isoform of the enzyme exists in cardiac (heart) and brain tissue.


The enzyme is one of the "new  cardiac markers " which are discussed to improve early diagnosis in  acute coronary syndrome . A rapid rise in blood levels can be seen in  myocardial infarction  and  unstable angina .


Other enzymes related to glycogen phosphorylase are abbreviated as  GPLL  (liver) and  GPMM  (muscle).


The  T-Lymphocyte Helper/Suppressor Profile  (Helper/Suppressor ratio, T4:T8 ratio, CD4:CD8 ratio) is a basic laboratory test in which the percentage of  CD3 -positive  lymphocytes  in the blood positive for  CD4  ( T helper cells ) and  CD8  (a class of  regulatory T cells ) are counted and compared.  Normal values (95% confidence intervals) are approximately 30-60% CD4 and 10-30% CD8 depending on age (ratio 0.9 to 3.7 in adults). [ 1 ]   One reason for abnormal results is the loss of CD4-positive cells to the  human immunodeficiency virus  (HIV) infection. The loss of CD4-positive cells to HIV infection can result in various distortions in the ratio, as in the initial period, production of HIV specific CD8 positive cells will cause a large fall in the ratio, but subsequent immunosuppression over time may lead to overall non production of immune cells and inversion of the ratio. It has been shown that the degree of inversion of this ratio in individuals on antiretroviral therapy is indicative of the age of the infection and independently predictive of mortality associated with non HIV events. [ 2 ] [ 3 ]


The  hematocrit  ( / h ɪ ˈ m æ t ə k r ɪ t / ) ( Ht  or  HCT ), also known by  several other names , is the  volume percentage  (vol%) of  red blood cells  (RBCs) in  blood , [ 1 ] [ 2 ]  measured as part of a  blood test . [ 3 ]  The measurement depends on the number and size of red blood cells. [ 3 ]  It is normally 40.7–50.3% for males and 36.1–44.3% for females. [ 3 ]  It is a part of a person's  complete blood count  results, [ 4 ]  along with  hemoglobin  concentration,  white blood cell  count and  platelet  count.


Because the purpose of red blood cells is to transfer oxygen from the lungs to body tissues, a blood sample's hematocrit—the red blood cell volume percentage—can become a point of reference of its capability of delivering oxygen. Hematocrit levels that are too high or too low can indicate a blood disorder,  dehydration , or other medical conditions. [ 4 ]  An abnormally low hematocrit may suggest  anemia , a decrease in the total amount of red blood cells, while an abnormally high hematocrit is called  polycythemia . [ 5 ]  Both are potentially life-threatening disorders.


There are other names for the hematocrit, such as  packed cell volume  (PCV),  volume of packed red cells  (VPRC), or  erythrocyte volume fraction  (EVF). The term  hematocrit  (or  haematocrit  in  British English ) comes from the  Ancient Greek  words  haima  ( αἷμα , "blood") and  kritēs  ( κριτής , "judge"), and hematocrit means "to separate blood". [ 6 ] [ 7 ]  It was coined in 1891 by Swedish physiologist  Magnus Blix  as  haematokrit , [ 8 ] [ 9 ] [ 10 ]  modeled after  lactokrit .


With modern lab equipment, the hematocrit can be calculated by an  automated analyzer  or directly measured, depending on the analyzer manufacturer. Calculated hematocrit is determined by multiplying the red cell count by the  mean cell volume . The hematocrit is slightly more accurate, as the PCV includes small amounts of  blood plasma  trapped between the red cells. An estimated hematocrit as a percentage may be derived by tripling the  hemoglobin  concentration in  g / dL  and dropping the units. [ 11 ]


The packed cell volume (PCV) can be determined by  centrifuging   EDTA -treated or  heparinized  blood in a  capillary tube  (also known as a microhematocrit tube) at 10,000  RPM  for five minutes. [ 12 ]  This separates the blood into layers. The volume of packed red blood cells divided by the total volume of the blood sample gives the PCV. Since a tube is used, this can be calculated by measuring the lengths of the layers. [ citation needed ]


Another way of measuring hematocrit levels is by optical methods such as  spectrophotometry . [ 13 ]  Through differential spectrophotometry, the differences in optical densities of a blood sample flowing through small-bore glass tubes at  isosbestic  wavelengths for deoxyhemoglobin and oxyhemoglobin and the product of the luminal diameter and hematocrit create a linear relationship that is used to measure hematocrit levels. [ 14 ]


Other than potential bruising at the puncture site, and/or dizziness, there are no complications associated with this test. [ 12 ]


While known hematocrit levels are used in detecting conditions, it may fail at times due to hematocrit being the measure of concentration of red blood cells through volume in a blood sample. It does not account for the mass of the red blood cells, and thus the changes in mass can alter a hematocrit level or go undetected while affecting a subject's condition. [ 15 ]  Additionally, there have been cases in which the blood for testing was inadvertently drawn proximal to an intravenous line that was infusing packed red cells or fluids. In these situations, the hemoglobin level in the blood sample will not be the true level for the patient because the sample will contain a large amount of the infused material rather than what is diluted into the circulating whole blood. That is, if packed red cells are being supplied, the sample will contain a large amount of those cells and the hematocrit will be artificially very high. [ citation needed ]


Hematocrit can vary from the determining factors of the number of red blood cells. These factors can be from the age and sex of the subject. [ clarification needed ] [ 16 ]  The normal hematocrit level is around 40% for adult women and about 45% for adult men. In newborns, it is approximately 55% and drops to around 35% by 2 months of age. After that, it gradually increases during development, reaching adult levels at puberty. [ 17 ]  Following this, the hematocrit level gradually decreases with aging. [ 18 ]  Typically, a higher hematocrit level signifies the blood sample's ability to transport oxygen, [ 19 ]  which has led to reports that an "optimal hematocrit level" may exist. Optimal hematocrit levels have been studied through combinations of assays on blood sample's hematocrit itself, viscosity, and hemoglobin level. [ 19 ]


Hematocrit levels also serve as an indicator of health conditions. Thus, tests on hematocrit levels are often carried out in the process of diagnosis of such conditions, [ 3 ]  and may be conducted prior to surgery. [ 6 ]  Additionally, the health conditions associated with certain hematocrit levels are the same as ones associated with certain hemoglobin levels. As blood flows from the arterioles into the capillaries, a change in pressure occurs. In order to maintain pressure, the capillaries branch off to a web of vessels that carry blood into the venules. Through this process blood undergoes micro-circulation. In micro-circulation, the  Fåhræus effect  will take place, resulting in a large change in hematocrit. As blood flows through the arterioles, red cells will act a feed hematocrit (Hf), while in the capillaries, a tube hematocrit (Ht) occurs. In tube hematocrit, plasma fills most of the vessel while the red cells travel through in somewhat of a single file line. From this stage, blood will enter the venules increasing in hematocrit, in other words the discharge hematocrit (Hd).In large vessels with low hematocrit, viscosity dramatically drops and red cells take in a lot of energy. [ citation needed ]


Relationships between hematocrit, viscosity, and shear rate are important factors to put into consideration. Since blood is non-Newtonian, the viscosity of the blood is in relation to the hematocrit, and as a function of shear rate. This is important when it comes to determining shear force, since a lower hematocrit level indicates that there is a need for more force to push the red blood cells through the system. This is because shear rate is defined as the rate to which adjacent layers of fluid move in respect to each other. [ 20 ]  Plasma is a more viscous material than typically red blood cells, since they are able to adjust their size to the radius of a tube; the shear rate is purely dependent on the amount of red blood cells being forced in a vessel. [ 21 ]


Generally at both sea levels and high altitudes, hematocrit levels rise as children mature. [ 22 ]  These health-related causes and impacts of elevated hematocrit levels have been reported:


Hematocrit levels were also reported to be correlated with social factors that influence subjects. In the 1966–80 Health Examination Survey, there was a small rise in mean hematocrit levels in female and male adolescents that reflected a rise in annual family income. Additionally, a higher education in a parent has been put into account for a rise in mean hematocrit levels of the child. [ 33 ]


Lowered hematocrit levels also pose health impacts. These causes and impacts have been reported:


This template's  initial visibility currently defaults to  autocollapse , meaning that if there is another collapsible item on the page (a  navbox, sidebar , or  table with the collapsible attribute ), it is hidden apart from its title bar; if not, it is fully visible.


To change this template's initial visibility, the  |state=   parameter  may be used:


Hemoglobin electrophoresis  is a  blood test  that can detect different types of  hemoglobin . The test can detect hemoglobin S, the form associated with  sickle cell disease , as well as other abnormal types of hemoglobin, such as  hemoglobin C . It can also be used to investigate  thalassemias , which are disorders caused by defective hemoglobin production.


The test uses the principles of  gel electrophoresis  to separate out the various types of hemoglobin and is a type of  native gel electrophoresis . After the sample has been treated to release the hemoglobin from the red cells, it is introduced into a porous gel (usually made of  agarose  or  cellulose acetate ) and subjected to an  electrical field , most commonly in an  alkaline  medium. Different hemoglobins have different charges, and according to those charges, they move at different speeds in the gel and eventually form discrete bands (see  electrophoretic migration patterns ). A  quality control  sample containing hemoglobins A, F, S, and C is run along with the patient sample to aid in identifying the different bands. [ 1 ] [ 2 ]  The relative amounts of each type of hemoglobin can be estimated by measuring the  optical density  of the bands, though this method is not reliable for hemoglobins that are present in low quantities. [ 2 ]


Because hemoglobins exhibit different migration patterns depending on the  pH  level, testing the same sample at both an acid and an alkaline pH can help to identify some abnormal hemoglobins that would otherwise be impossible to distinguish from others. [ 3 ] : 437


Adult human blood normally contains three types of hemoglobin:  hemoglobin A , which makes up approximately 95% of the total;  hemoglobin A 2 , which accounts for less than 3.5%; and a minute amount of  hemoglobin F . [ 3 ] : 140   If abnormal hemoglobin variants such as hemoglobin S (which occurs in  sickle cell disease ),  C  or  E  are present, they will appear as unexpected bands on electrophoresis (provided they do not migrate to the same place as other hemoglobins). [ 1 ]


Hemoglobin electrophoresis can also be used to investigate  thalassemias , which are caused by decreased production of subunits of the hemoglobin molecule. Hemoglobin A 2  levels are typically elevated in  beta-thalassemia minor  and hemoglobin F may be slightly increased. In  beta-thalassemia major , hemoglobin A is decreased (or in some cases absent) and hemoglobin F is markedly elevated; A 2  levels are variable. [ 3 ] : 461–2   In  hemoglobin H disease , a form of  alpha-thalassemia , an abnormal band of hemoglobin H can be detected, and sometimes a band of  Hemoglobin Barts ; but in the milder  alpha-thalassemia trait , electrophoresis results are effectively normal. [ 4 ]


Linus Pauling  is credited with the invention of hemoglobin electrophoresis in 1949. Newer alternatives to conventional hemoglobin electrophoresis include  isoelectric focusing ,  capillary zone electrophoresis , and  high-performance liquid chromatography . [ 2 ]


Hemoximetry  is the monitoring of  hemoglobin  and  oxygen saturation , especially during procedures such as  cardiac catheterization . [ 1 ]


Hemoximetry presents hemoglobin oxygen saturation, dyshemoglobines, and total hemoglobin concentration data. The functional oxygen saturation measured by this procedure is the basis for calibrating  pulse oximeters . Pulse oximeters cannot be calibrated using physical procedures, but only by directly comparing the reported measurements and the parallel arterial oxygen saturation measured by hemoximetry in a group of healthy subjects. [ citation needed ]


INR self-monitoring  is a medical kit that is used by patients both on long-term and on lifetime anti-coagulation therapy to measure their INR ( International Normalized Ratio ) levels themselves at your premises without going to a clinic. People who self-monitor their INR levels use a portable INR monitor as in a clinic. It requires the patient to test a drop of blood, drawn from a finger at scheduled times, and record the INR level measured by a monitor.


People on anti-coagulation therapy who are self-testing provide the INR upi antem76102824@axl reading they obtain from their monitor to their healthcare professionals at an agreed time, generally by telephone. The healthcare professional decides if any change to the  warfarin  dose is required and lets the person know what action is needed. A PT/INR meter can be obtained by contacting an Independent Diagnostic Testing Facility (IDTF). [ 1 ]  They are able to provide patients with all necessary testing equipment and bill the insurance for test results reported. [ citation needed ]


People who  self-manage  adjust their warfarin doses themselves, following training with their healthcare professional. This means that if the INR reading obtained from their monitor is out of the normal  therapeutic range , they are able to make an adjustment to their own warfarin dose by themselves. [ citation needed ]


International normalized ratio (INR) which is a derivative of prothrombin time is a measurement of blood coagulation in the circulatory system. Both are used to determine the  clotting  rate of blood, which can be affected by anticoagulant usage, liver damage and Vitamin K levels. The preferred range of INR levels for a patient on anticoagulation therapy is usually between 2 and 3, but it tends to vary depending on the patient's requirements. [ 2 ]


Patients who self-monitor tend to choose this route for the greater control they feel it gives them over their lives and their condition. This helps to reduce the number of visits being made to their anticoagulation clinic for routine appointments to measure their INR levels. This is a lengthy process in comparison to self-testing and management. Results of clinical studies, which have been recognized by the National Patients Safety Authority (NPSA), show that people who self-monitor keep more frequently within their  therapeutic range  and have fewer complications including clots and bleeding, compared with people who have their INR levels tested only at their anticoagulation clinic. [ citation needed ]


Patient self-care is a key initiative in the  NHS Plan  for a patient-centered health service and an important component in supporting people with long-term conditions. [ 3 ]  It is seen to provide:


Self-monitoring may be a suitable option for a variety of people who are on long term anticoagulation therapy and want the convenience of being able to monitor their own INR levels at a time and place of their own choosing (e.g. if travelling abroad either with work or on vacation). They do have continuous support from their healthcare professionals and there are no upper age limits for self-monitoring and parents can take charge for their children. Self-monitoring is ideal if: [ citation needed ]


In the past 5 years, there have been a number of clinical trials to highlight the advantages of self-monitoring, whether self-testing or self-managing. It also gives an indication of what improvements the self-testing and self-management can do to INR levels. [ citation needed ]


" Quality of life changed in a positive way. Independence and better organisation of vacation and spare time were most frequently mentioned advantages ."


Extract from a study into the impact of self-monitoring on the quality of life of patients under anticoagulation therapy. [ 6 ]


" Patient self-testing . . . is an effective method of monitoring oral anticoagulation therapy, providing outcomes at least as good as, and possibly better than, those achieved with an anti-coagulation clinic. "


From international consensus guidelines prepared by the International Self-Monitoring Association for Oral Anti-coagulation. [ 7 ]


" Self-monitoring can improve the quality of oral anti-coagulation therapy, with patients more frequently in the therapeutic range, while improving benefits and decreasing harm ."


From the conclusions of a review of studies of self-monitoring in oral anti-coagulation therapy. [ 8 ] [ 9 ]


The results of the study  Effect of Home Testing of International Normalized Ratio on Clinical Events  (2010), comparing whether weekly home-testing of the INR-level offers any advantage over monthly testing in a clinic, reduces the risk of a major hemorrhage event (i.e.  stroke , major bleeding, or death), indicated that the time to the first primary-event (stroke, major bleeding, or death) was not significantly longer in the self-testing group of patients than in the clinic-testing group of patients (hazard ratio, 0.88; 95% confidence interval, 0.75 to 1.04; p=0.14). The study was a prospective, randomized, non-blinded trial for which the patients were randomized into two groups, (i) weekly INR self-testing and (ii) monthly INR clinic-testing, using a stratified method of adaptive-allocation that was determined according to the duration of anti-coagulation and the indication for  Warfarin . Although the study was non-blinded, blinding is less critical because the objective outcomes of the study: stroke, major bleeding episode, and death. [ 10 ]


Patients who are self-monitoring have to use a monitor in order to measure their INR level. There are a range of INR testing monitors on the market, such as the CoaguChek XS, [ 11 ] [ 12 ]  MicroINR., [ 13 ]  INRatio2, [ 14 ]  and  QLabs [ 15 ]


An  insulin tolerance test  ( ITT ) is a medical diagnostic procedure during which  insulin  is injected into a patient's vein, after which blood glucose is measured at regular intervals. This procedure is performed to assess  pituitary  function,  adrenal  function,  insulin sensitivity , [ 1 ] [ 2 ]  and sometimes for other purposes. An ITT is usually ordered and interpreted by an  endocrinologist .


When used to assess insulin sensitivity, a standard dose of insulin is administered, and blood glucose is monitored with frequent sampling. The plasma glucose disappearance rate (KITT) indicates the degree of whole-body insulin sensitivity, and correlates well with the gold-standard  glucose clamp technique . [ 1 ]


When used for assessing the integrity of the  hypothalamic–pituitary–adrenal axis  (HPA), insulin injections are continued to the point of inducing extreme  hypoglycemia  below 2.2 mmol/L (40 mg/dL).  Patient must have symptomatic neuroglycopenia to trigger counter-regulatory cascade. Glucose levels below 2.2 mmol/L are insufficient absent symptoms. The brain must register low glucose levels.  In response,  adrenocorticotropic hormone  (ACTH) and  growth hormone  (GH) are released as a part of the stress mechanism. ACTH elevation causes the  adrenal cortex  to release  cortisol . Normally, both cortisol and GH serve as  counterregulatory hormones , opposing the action of insulin, i.e. acting against the hypoglycemia. [ 3 ]


ITT to the point of extreme hypoglycemia is considered to be the  gold standard  for assessing the integrity of the HPA. Sometimes ITT is performed to assess the adrenal function, e.g. before  surgery . It is assumed that the ability to respond to insulin-induced hypoglycemia translates into appropriate cortisol rise in the stressful event of acute illness or major surgery. [ 4 ]  The extreme hypoglycemic version of the ITT is potentially very dangerous and must be undertaken with great care, because it can  iatrogenically  induce the equivalent of a  diabetic coma . A health professional must attend it at all times. Other provocation tests which cause much less release of growth hormone include the use of  glucagon ,  arginine  and  clonidine .


Side effects include sweating, palpitations, loss of consciousness and rarely convulsions due to severe hypoglycemia which may cause coma. If extreme symptoms are present, glucose should be given  intravenously . In subjects with no adrenal reserve an  Addisonian crisis  may occur.  For cortisol stimulation, the  ACTH stimulation test  has much less risk. [ citation needed ]


Insulin tolerance test contraindications are: [ citation needed ]


The test cannot be interpreted unless hypoglycaemia (< 2.2 mmol/L (or) < 40 mg/dL) is achieved. [ citation needed ]


An adequate cortisol response is defined as a rise to greater than 550 nmol/L. Patients with impaired cortisol responses (less than 550 but greater than 400 nmol/L) may only need steroid cover for major illnesses or stresses. An adequate GH response occurs with an absolute response exceeding 20 mU/L. [ citation needed ]


There will be a rise of less than 170 nmol/L above the fluctuations of basal levels of cortisol. [ citation needed ]


In medicine,  intravascular volume status  refers to the volume of  blood  in a patient's  circulatory system , and is essentially the  blood plasma  component of the overall  volume status  of the body, which otherwise includes both  intracellular fluid  and  extracellular fluid . Still, the intravascular component is usually of primary interest, and  volume status  is sometimes used synonymously with  intravascular volume status .


It is related to the patient's state of hydration, but is not identical to it. For instance, intravascular volume depletion can exist in an adequately hydrated person if there is loss of water into  interstitial tissue  (e.g. due to  hyponatremia  or  liver failure ).


Volume contraction  of intravascular fluid ( blood plasma ) is termed  hypovolemia , [ 1 ] [ 2 ]  and its signs include, in order of severity:


Signs of  intravascular volume overload  (high blood volume) include:


For the clinical assessment of intravascular blood volume, the BVA-100, a semi-automated blood volume analyzer device that has FDA approval, determines the status of a patient’s blood volume based on the Ideal Height and Weight Method. [ 3 ]
Using a patient’s ideal weight and actual weight, the percent deviation from the desirable weight is found using the following equation:


±
 %

  Desirable Weight

 =

 Actual Weight

 −

 Desirable Weight

 Desirable Weight

 ×
 100

 {\displaystyle \pm \%{\text{ Desirable Weight}}={\frac {{\text{Actual Weight}}-{\text{Desirable Weight}}}{\text{Desirable Weight}}}\times 100}

Using the deviation from desirable weight, the BV ratio (ml/kg), i.e. Ideal Blood Volume, can be determined.
The machine was tested in clinical studies for the treatment of a broad range of medical conditions related to Intravascular Volume Status, such as anemia, [ 4 ]  congestive heart failure, [ 5 ]  sepsis, [ 6 ]  CFS, [ 7 ]  Hyponatremia, [ 8 ]  Syncope [ 9 ]  and more. This tool for measuring blood volume may foster improved patient care [ 10 ]  as both a stand-alone and complementary diagnostic tool as there has been a statistically significant increase in patient survival. [ 6 ]


The most common cause of  hypovolemia  is diarrhea or vomiting. The other causes are usually divided into  renal  and  extrarenal  causes. Renal causes include overuse of diuretics, or trauma or disease of the kidney.  Extrarenal causes include bleeding, burns, and any causes of  edema  (e.g. congestive heart failure, liver failure). [ citation needed ]


Intravascular volume depletion is divided into three types based on the blood sodium level:


Intravascular volume overload  can occur during surgery, if water rather than isotonic saline is used to wash the incision. It can also occur if there is inadequate urination, e.g. with certain kidney diseases. [ citation needed ]


Iron tests  are groups of  clinical chemistry  laboratory  blood tests  that are used to evaluate  body iron stores  or the  iron  level in  blood serum .


Other terms used for the same tests are  iron panel ,  iron profile ,  iron indices ,  iron status  or  iron studies .


*  = or normal.


Jostel's TSH index  ( TSHI  or  JTI ), also referred to as  Jostel's thyrotropin index  or  Thyroid Function index  ( TFI ), is a method for estimating the thyrotropic (i.e. thyroid stimulating) function of the anterior  pituitary  lobe in a quantitative way. [ 1 ] [ 2 ]  The equation has been derived from the logarithmic standard model of  thyroid homeostasis . [ 3 ] [ 4 ] [ 5 ] [ 6 ]  In a paper from 2014 further study was suggested to show if it is useful, [ 7 ]  but the 2018 guideline by the European Thyroid Association for the diagnosis of uncertain cases of central hypothyroidism regarded it as beneficial. [ 2 ]  It is also recommended for purposes of differential diagnosis in the  sociomedical expert assessment . [ 8 ]


Jostel's TSH index can be calculated with


from equilibrium serum concentrations of  thyrotropin  (TSH), free  T4  (FT4) and a correction coefficient derived from the logarithmic standard model (β = 0.1345).


An alternative standardised form ( standardised TSH index  or  sTSHI ) is calculated with. [ 1 ]


as a  z-transformed  value incorporating  mean  (2.7) and  standard deviation  (0.676) of TSHI in a reference population [ 5 ]


The TSH index is reduced in patients with  secondary hypothyroidism  resulting from thyrotropic insufficiency. [ 1 ] [ 9 ] [ 10 ] [ 11 ]  For this indication, it has, however, up to now only been validated in adults. [ 12 ]  JTI was also found reduced in cases of  TACITUS syndrome (non-thyroidal illness syndrome)  as an example of type 1 thyroid  allostasis . [ 13 ] [ 14 ]  Conversely, an elevated thyroid function index may serve as a biomarker for type 2 allostasis and contextual stress. [ 15 ] [ 16 ]


Jostel's TSH index may decrease under therapy with the antidiabetic drug  metformin , [ 17 ]  especially in women under oral  contraceptives . [ 18 ]


In two large population-based cohorts included in the  Study of Health in Pomerania  differentially correlated to some markers of  body composition . Correlation was positive to  body mass index  (BMI),  waist circumference  and fat mass, but negative to body cell mass. [ 19 ]  With the exception of fat mass all correlations were age-dependent. [ 19 ]  Very similar observations have been made earlier in the  NHANES  dataset. [ 20 ]


In  Parkinson's disease , JTI is significantly elevated in early sub-types of the disease compared to an advanced group. [ 21 ]


A longitudinal study in  euthyroid  subjects with  structural heart disease  found that JTI predicts the risk of malignant  arrhythmia  including  ventricular fibrillation  and  ventricular tachycardia . [ 22 ]  This applies to both incidence and event-free survival. [ 22 ]  A second study in a different population undergoing coronary angiography arrived at similar results. [ 23 ]  It was therefore concluded that an elevated set point of thyroid homeostasis may contribute to cardiovascular risk. A positive correlation of JTI to SIQALS 2, [ 16 ]  a score for  allostatic load , suggests that thyroid hormones are among the mediators linking stress to major cardiovascular endpoints. [ 24 ]


Jostel's TSH index and the  thyroid feedback quantile-based index , another biomarker for the central thyrotropic function, were observed to be elevated in certain psychiatric diseases including schizophrenia. [ 25 ]


Another study demonstrated the TSH index to inversely correlate to  thyroid's secretory capacity  and thyroid volume. [ 26 ]  It is unclear if this finding reflects shortcomings of the index (i.e. low specificity in the setting of subclinical hypothyroidism) or plastic responses of the pituitary gland to beginning hypothyroidism. [ citation needed ]


In subjects with  type 2 diabetes , treatment with  beta blockers  resulted in increased TSH index, but the mechanism is unclear. [ 27 ]


Negative correlation of Jostel's TSH index to the urinary excretion of certain  phthalates  suggests that  endocrine disruptors  may affect the central set point of thyroid homeostasis. [ 28 ]


Drugs that reduce the TSH index, probably via effects on the central set point of the feedback loop, include mirtazapine [ 29 ]  and oxcarbazepine. [ 30 ]


A reduction of Jostel's TSH index may predict the development of  hypophysitis  due to therapy with  immune checkpoint inhibitors , e. g.  ipilimumab . [ 31 ]


Kaolin clotting time  ( KCT ) is a  sensitive  test to detect  lupus anticoagulants . [ 2 ]  There is evidence that suggests it is the most sensitive test for detecting lupus anticoagulants. [ 3 ]  It can also detect  factor VIII  inhibitors but is sensitive to  unfractionated heparin  as well. [ 4 ]


The KCT on whole blood is known as the "Activated Clotting Time" (ACT) and is widely used in various instruments during surgery such as cardiac bypass to monitor heparin. [ 5 ]


KCT was first described by Dr. Joel Margolis in 1958. [ 6 ]  Later on, it was found to be very sensitive to lupus anticoagulants but was only reliable when test plasmas were mixed with normal plasma in various proportions. [ 3 ]  It became the preferred method for lupus anticoagulant testing after Dr. Wilhelm Lubbe showed it to be a good marker for recurrent fetal loss. [ 7 ]


KCT is similar to the  activated partial thromboplastin time  test, except it does not use exogenous  phospholipid . [ 2 ]  Thus, a confirmatory test that uses excess phospholipid is needed to validate the presence of lupus anticoagulants. [ 2 ]  Otherwise, diluting the test plasma in normal plasma before testing provides characteristic mixing patterns. [ 8 ]


Kaolin is the surface activator, and the test also requires small amounts of cell fragments and plasma lipids to provide the phospholipid surface required for coagulation. [ 2 ] [ 4 ]  Therefore, the sample quality is important for the validity of the screening test. [ 2 ]


The test combines a test  plasma  with  kaolin , and after a brief pre-incubation and the addition of calcium chloride, the time to clot (in seconds) is measured. [ 6 ]  Mixes of patient plasma with normal plasma are recommended for testing. [ 9 ]


The KCT test/control ratio of greater than or equal to 1.2 indicates that a defect is present. [ 4 ]  If the test/control ratio is between 1.1 and 1.2, the test is equivocal. [ 4 ]


A good way of expressing the result using mixes is to calculate the Rosner index. [ 10 ]  If A is the KCT of normal plasma, B is that of the 1:1 mix and C is that of the patient plasma, then the Rosner index is 100x(B-A)/C. Values above 15 indicate a positive result but in most cases labs set their own cutoff values. [ 9 ]