Endocrine disruptors  may have pronounced effects on step-up deiodinases, as suggested by positive correlation of SPINA-GD to combined exposure to polycyclic aromatic hydrocarbons (PAHs) [ 52 ]  and urine concentrations of  cadmium  and  phthalate  metabolites, [ 53 ] [ 54 ] [ 55 ]  negative correlation to  paraben ,  mercury  and  bisphenol A  concentration [ 56 ] [ 53 ] [ 54 ]  and a nonlinear association to the concentrations of  per- and polyfluoroalkyl substances . [ 57 ]  In a cohort of manganese-exposed workers, SPINA-GD responded to a tenfold increase in concentrations of titanium, nickel, selenium and strontium. [ 58 ]


In a longitudinal evaluation of a large sample of the general US population over 10 years, reduced SPINA-GD significantly predicted reduced overall survival. [ 59 ]


suPARnostic  is a simplified double monoclonal antibody sandwich enzyme-linked immunosorbent assay ( ELISA ) that measures the amount of soluble urokinase plasminogen activator receptor ( suPAR ) in blood. [ 1 ]  Elevated plasma suPAR levels have been observed in various  infectious ,  inflammatory  and  autoimmune diseases . [ 2 ]  suPAR concentration positively correlates to the activation level of the immune system. suPARnostic can be used as a prognostic tool [ 3 ]  to determine the severity of a disease within a patient, but is not used as a reliable diagnostic tool, as it can detect the severity of the immune response in a patient, but does not reveal the specific disease from which the patient may be suffering. Recently, increase suPAR levels were shown to be associated with increased risk of systemic inflammatory response syndrome ( SIRS )/ sepsis ,  cardiovascular disease ,  type 2 diabetes ,  infectious diseases ,  HIV , [ 4 ]   cancer [ 5 ]   tuberculosis , [ 3 ]   malaria , [ 6 ]  bacterial and viral  CNS infections , [ 7 ]   rheumatoid arthritis , [ 8 ]   multiple sclerosis [ 3 ]  and  mortality  in the general population.


Performing the suPARnostic  ELISA  requires two antibodies with high specificity for  suPAR .  The blood plasma sample from the patient that contains an unknown amount of suPAR is immobilized on the microwells on the clear  microtiter plate  and a detection antibody forms a complex with suPAR.
Between each step the plate is rinsed with a wash buffer to dispose of any proteins that do not specifically bind to any of the wells on the plate.  After the final wash step, the plate is developed by adding the  TMB  substrate to produce a visible signal, which indicates the quantity of suPAR in the sample.  The measured  absorbance  can, based on the values from the standard curve, be converted to the concentration (ng/mL) of suPAR in the sample.  This level can then suggest whether or not the patient is experiencing challenges to their immune system. [ 3 ]


The suPARnostic  ELISA  is a simplified double  monoclonal antibody  sandwich assay that measures the level of  suPAR  and suPAR II-III  in the body . The suPARnostic ELISA utilizes monoclonal mouse and rat antibodies against human  suPAR . [ 9 ]


The advantages of using  monoclonal antibodies  compared to using  polyclonal antibodies  includes: High  homogeneity , absence of nonspecific antibodies and no batch-to-batch or lot-to-lot variability.  This results in a very robust and reliable assay. [ 3 ]


A 'sandwich' is formed of solid-phase  antibody , suPAR and peroxidase-conjugated antibody. The concentration (ng/mL plasma) of suPAR in the patient sample is determined via interpolation, based on a  calibration curve  prepared from seven suPAR standards. Recombinant suPAR standards are calibrated against healthy human blood donor samples. Absorbance is measured using a  microtiter plate  reader, at 450 nm with a 650 nm reference filter. Measurement of suPAR levels from blood samples provides greater accuracy and precision than measurement from  urine  or  cerebral spinal fluid . suPAR level is not changed by transient illness such as cold. It also remains stable after a blood sample is taken despite storage. [ 10 ] [ 11 ]


suPARnostic measurements between 0.1 and 4.0 ng/mL suggest that a patient is healthy, with no challenges to their  immune system  and no signs or symptoms of an  opportunistic infection  or  inflammation ; the average level among the population is 3.4 ng/mL. However, a patient's immune system can be considered 'negatively activated' at suPAR levels above 4.0 and up to 6.0 ng/mL, indicating a potential infection or high level of inflammation. In this case, a patient's health is likely to worsen and he or she should be referred for further testing. suPARnostic measurements from 6.0 ng/mL to double digit levels can indicate a serious illness that is progressing rapidly to a critical situation. Patients in the intensive care unit average a level of 10.0 ng/mL. There is no difference in suPAR levels intrinsic to various races; however, the scale varies for male and female. [ 9 ] [ 11 ]


There are two suPARnostic tests available. The suPARnostic Standard ELISA (Code No. A001) is for research use and large trials, one batch consisting of 41 samples in doublets. The suPARnostic Flex ELISA (Code No. A002) has been developed for clinical applications consisting of 93 samples, is modular and flexible, and gives fully quantitative results in 2 hours. [ 11 ]


The suPARnostic kit has a refrigerated  shelf life  of several years and when frozen, may be kept for longer.  The kit should sit at room temperature for half an hour before use but it may be held at room temperature for as long as three to four hours. [ 12 ]  The suPARnostic Flex ELISA (Code No. A002) is able to provide fully quantitative results in 2 hours. [ 13 ]  suPARnostic is run as large, batch test with up to 41 samples in doublets for research purposes or 93 samples for clinical use at one time. [ 11 ]


Although suPARnostic currently does not have FDA approval, it is CE/ IVD  marked for distribution throughout Europe. [ 11 ]  suPAR is a prognostic test to indicate general health, and it cannot be used as a diagnostic tool to suggest a particular illness. [ 9 ]  suPAR cannot be used in the detection of brain tumors because the suPAR molecule cannot migrate through the  blood brain barrier . [ 2 ]


Syndromic testing  is a process by which a healthcare provider simultaneously tests a patient for multiple pathogens with overlapping  symptomology . This allows providers to order one test to see if patients are suffering from any one of multiple causes, rather than having to order a test for each potential underlying reason for the illness. It can be used with patients that are  immunosuppressed , in hospital environments that have limited testing assets, or patients that could be suffering from any number of or combination of reasons for a specific syndrome, such as respiratory distress,  gastroenteritis , [ 1 ]  bloodstream infections, or CNS infections. [ 2 ]  The test uses multi-panel syndromic  assays  that allow the simultaneous detection of a number of agents, increasing the accuracy of tests for microbial agents. [ 3 ]  The first multiplex panel for syndromic testing to be approved by the  FDA  received approval in 2008, [ 4 ] [ 5 ]  and since, panels for several potential pathogens have been approved. [ 6 ]


A  tetramer assay  (also known as a  tetramer stain ) is a procedure that uses  tetrameric  proteins to detect and quantify  T cells  that are specific for a given  antigen  within a blood sample. [ 1 ]  The tetramers used in the assay are made up of four  major histocompatibility complex  (MHC) molecules, which are found on the surface of most cells in the body. [ 2 ]  MHC molecules present  peptides  to T-cells as a way to communicate the presence of viruses, bacteria, cancerous mutations, or other antigens in a cell. If a T-cell's receptor matches the peptide being presented by an MHC molecule, an immune response is triggered. [ 3 ]  Thus, MHC tetramers that are bioengineered to present a specific peptide can be used to find T-cells with receptors that match that peptide.
The tetramers are labeled with a  fluorophore , allowing tetramer-bound T-cells to be analyzed with  flow cytometry . [ 4 ]  Quantification and sorting of T-cells by flow cytometry enables researchers to investigate immune response to viral infection and vaccine administration as well as functionality of antigen-specific T-cells. [ 5 ]  Generally, if a person's  immune system  has encountered a  pathogen , the individual will possess T cells with specificity toward some peptide on that pathogen. Hence, if a tetramer stain specific for a pathogenic peptide results in a positive signal, this may indicate that the person's immune system has encountered and built a response to that pathogen. [ citation needed ]


This methodology was first published in 1996 by a lab at Stanford University. [ 6 ]  Previous attempts to quantify antigen-specific T-cells involved the less accurate limiting dilution assay, which estimates numbers of T-cells at 50-500 times below their actual levels. [ 7 ] [ 8 ]  Stains using soluble MHC  monomers  were also unsuccessful due to the low binding affinity of T-cell receptors and MHC-peptide monomers. MHC tetramers can bind to more than one receptor on the target T-cell, resulting in an increased total binding strength and lower dissociation rates. [ 5 ]


Tetramer stains usually analyze  cytotoxic T lymphocyte  (CTL) populations. [ 9 ]  CTLs are also called CD8+ T-cells, because they have  CD8  co-receptors that bind to MHC class I molecules. Most cells in the body express MHC class I molecules, which are responsible for processing intracellular antigens and presenting at the cell's surface. If the peptides being presented by MHC class I molecules are foreign—for example, derived from viral proteins instead of the cell's own proteins—the CTL with a receptor that matches the peptide will destroy the cell. [ 2 ] [ 3 ]  Tetramer stains allow for the visualization, quantification, and sorting of these cells by flow cytometry, which is extremely useful in  immunology . T-cell populations can be tracked over the duration of a virus or after the application of a vaccine. Tetramer stains can also be paired with functional assays like  ELIspot , which detects the number of  cytokine  secreting cells in a sample. [ 9 ]


MHC tetramer molecules developed in a lab can mimic the antigen presenting complex on cells and bind to T-cells that recognize the antigen. Class I MHC molecules are made up of a polymorphic heavy α-chain associated with an invariant light chain  beta-2 microglobulin  (β2m).  Escherichia coli  are used to synthesize the light chain and a shortened version of the heavy chain that includes the  biotin  15 amino acid recognition tag. These MHC chains are biotinylated with the enzyme BirA and refolded with the antigenic peptide of interest. Biotin is a small molecule that forms a strong bond with another protein called streptavidin. Fluorophore tagged streptavidin is added to the bioengineered MHC monomers, and the biotin-streptavidin interaction causes four MHC monomers to bind to the streptavidin and create a tetramer. When the tetramers are mixed with a blood sample, they will bind to T-cells expressing the appropriate antigen specific receptor. Any MHC tetramers that are not bound are washed out of the sample before it is analyzed with flow cytometry. [ 9 ]


Recent advancements within recombinant MHC molecules have democratised peptide MHC complex formulation and subsequent multimerisation. Highly active formulations of a broad range of MHC class I molecules [ 10 ]  now allows non-experts users to make their own custom peptide-MHC complexes from day-to-day in any lab without special equipment. [ citation needed ]


Tetramers that bind to helper T-cells have also been developed. [ 9 ]   Helper T-cells  or CD4+ T-cells express  CD4  co-receptors. They bind to  class II MHC  molecules, which are only expressed in professional  antigen-presenting  cells like dendritic cells or  macrophages . Class II MHC molecules present extracellular antigens, allowing helper T-cells to detect bacteria, [ 11 ]  fungi, and parasites. [ 2 ]  Class II MHC tetramer use is becoming more common, but the tetramers are more difficult to create than class I tetramers and the bond between helper T-cells and MHC molecules is even weaker. [ 9 ] [ 12 ]


Natural killer T-cells  (NKT cells) can also be visualized with tetramer technology. NKT cells bind to proteins that present  lipid  or  glycolipid  antigens. [ 13 ]  The antigen presenting complex that NKT cells bind to involves  CD1  proteins, so tetramers made of CD1 can be used to stain for NKT cells. [ 9 ]


An early application of tetramer technology focused on the cell-mediated immune response to  HIV  infection. MHC tetramers were developed to present HIV antigens and used to find the percentage of CTLs specific to those HIV antigens in blood samples of infected patients. This was compared to results of cytotoxic assays and plasma  RNA  viral load to characterize the function of CTLs in HIV infection. The CTLs that bound to tetramers were sorted into ELIspot wells for analysis of cytokine secretion. [ 14 ]


Another study utilized MHC tetramer complexes to investigate the effectiveness of an influenza  vaccine  delivery method. Mice were given subcutaneous and intranasal vaccinations for influenza, and tetramer stains coupled with flow cytometry were used to quantify the CTLs specific to the antigen used in the vaccine. This allowed for comparison of the immune response (the number of T-cells that target a virus) in two different vaccine delivery methods. [ 15 ]


A  thrombin generation assay  ( TGA ) or  thrombin generation test  ( TGT ) is a  global coagulation assay  (GCA) and type of  coagulation test  which can be used to assess  coagulation  and  thrombotic  risk. [ 1 ] [ 2 ] [ 3 ]  It is based on the potential of a  plasma  to generate  thrombin  over time, following activation of coagulation via addition of  phospholipids ,  tissue factor , and  calcium . [ 4 ]  The results of the TGA can be output as a  thrombogram  or thrombin generation curve using computer software with calculation of thrombogram parameters. [ 5 ] [ 1 ]


TGAs can be performed with methods like the semi-automated  calibrated automated thrombogram  (CAT) (2003) or the fully-automated  ST Genesia  system (2018). [ 6 ] [ 1 ] [ 7 ]  TGAs were first used as manual assays in the 1950s and have since become increasingly automated. [ 1 ]


Thrombogram parameters for the TGA include: [ 1 ]


The addition of  activated protein C  (APC) to a TGA results in an inhibition of thrombin generation as measured by reduction of the endogenous thrombin potential (ETP; area under the thrombin generation curve). [ 4 ]  This can be used to assess  APC resistance  and is termed the ETP-based APC resistance test. [ 4 ]  Results may be expressed as normalized APC sensitivity ratio (nAPCsr), which corresponds to the ratio of the ETP measured in the presence and absence of APC divided by the same ratio in reference plasma. [ 4 ]  The higher the nAPCsr value, the greater the APC resistance of the person. [ 4 ]  The ETP-based APC resistance test was developed in 1997. [ 4 ]


The  thrombin time  ( TT ), also known as the  thrombin clotting time  ( TCT ), is a  blood test  that measures the time it takes for a  clot  to form in the plasma of a blood sample containing  anticoagulant , after an excess of  thrombin  has been added. [ 1 ]  It is used to diagnose blood  coagulation  disorders and to assess the effectiveness of fibrinolytic therapy.  This test is repeated with pooled plasma from normal patients. The difference in time between the test and the 'normal' indicates an abnormality in the conversion of  fibrinogen  (a soluble protein) to fibrin, an insoluble protein. [ 2 ]


The thrombin time compares the rate of clot formation to that of a sample of normal pooled plasma. Thrombin is added to the samples of plasma. If the time it takes for the plasma to clot is prolonged, a quantitative (fibrinogen deficiency) or qualitative (dysfunctional fibrinogen) defect is present. [ 3 ]  In  blood samples suspected to contain  heparin , a substance derived from snake venom called  batroxobin [ 4 ]  (formerly  reptilase ) is used for comparison to thrombin time. Batroxobin has a similar action to thrombin but unlike thrombin it is not inhibited by heparin, so  reptilase time  and thrombin time can be used concurrently to distinguish anticoagulant effect from  hypofibrinogenemia  or  dysfibrinogenemia . [ 1 ] [ 5 ]


Normal values for thrombin time may be 12 to 14 seconds, [ 6 ]  but the test has significant reagent variability. If batroxobin is used, the time should be between 15 and 20 seconds. Thrombin time can be prolonged by heparin, fibrin degradation products, and fibrinogen deficiency or abnormality.  Thrombin time is not affected by anti-Xa anticoagulants such as  rivaroxaban  or  apixaban , but is very sensitive to  direct thrombin inhibitors  including  dabigatran ,  argatroban , and  bivalirudin . [ 5 ]


After separating the plasma from the whole blood by  centrifugation , bovine  thrombin  is added to the sample of plasma. Clot formation is detected optically or mechanically by a coagulation instrument. The time between the addition of the thrombin and the clot formation is recorded as the thrombin clotting time. [ 5 ]


Whole blood is taken with either  citrate  or  oxalate  additive (if using the  vacutainer  system, this is a light blue top tube).  As with other coagulation assays, the tube must not be over- or under-filled in order to ensure the correct anticoagulant-to-blood ratio: one part anticoagulant per nine parts blood. [ 5 ]


The  reference ranges  of the thrombin clotting time is generally <22 seconds, [ 7 ]  and often from 14 to 16 seconds. [ 6 ]  Laboratories usually calculate their own ranges, based on the method used and the results obtained from healthy individuals from the local population.  Variability arises from differences in thrombin concentration, dilution of plasma, presence and/or concentration of calcium ions, as well as the influence of analyser type. [ 5 ]  TT may also be sensitive to citrate pH. Separate ranges are used for infants. [ 8 ]


Blood samples that are more than eight hours old can give inaccurate results when tested. [ 9 ]


Thrombodynamics test  is a method for blood  coagulation  monitoring and anticoagulant control. This test is based on imitation of coagulation processes occurring  in vivo , is sensitive both to pro- and anticoagulant changes in the hemostatic balance. Highly sensitive to thrombosis.


The method was developed in the Physical Biochemistry Laboratory under the direction of Prof. Fazly Ataullakhanov.


Thrombodynamics designed to investigate the  in vitro  spatial-temporal dynamics of blood coagulation initiated by localized coagulation activator under conditions similar to the conditions of the blood clotting  in vivo . Thrombodynamics takes into account the spatial heterogeneity trombodinamiki processes in blood coagulation. The test is performed without mixing in a thin layer of  plasma .


The measurement cuvette with the blood plasma sample is placed inside the water thermostat. Clotting starts when activator with immobilized  TF  is immersed into the cuvette. The clot then propagates from the activating surface into the bulk of plasma. Image of growing clot is registered via the CCD camera using a time-lapse microscopy mode in scattered light and then parameters of coagulation are calculated on the computer. Thrombodynamics analyser T-2 device also supports measurement of spatial dynamics of thrombin propagation during the process of clot growth via usage of the fluorogenic substrate for thrombin. Blood plasma sample is periodically irradiated with the excitation light and the emission of the fluorophore is registered by CCD camera.


Mathematical methods are used to restore spatio-temporal distribution of the thrombin from the fluorophore signal. This experimental model worked well in research and has demonstrated good sensitivity to various disorders of the coagulation system.


[ 1 ]
 [ 2 ]
 [ 3 ]
 [ 4 ]
 [ 5 ]
 [ 6 ]
 [ 7 ]


Thromboelastography  ( TEG ) is a method of testing the efficiency of  blood   coagulation . It is a test mainly used in  surgery  and  anesthesiology , although increasingly used in resuscitations in emergency departments, intensive care units, and labor and delivery suites. More common tests of blood coagulation include  prothrombin time  (PT) and  partial thromboplastin time  (aPTT) which measure coagulation factor function, but TEG also can assess platelet function, clot strength, and  fibrinolysis  which these other tests cannot. [ 1 ] [ 2 ]


Thromboelastometry  (TEM), previously named rotational thromboelastography (ROTEG) or rotational thromboelastometry (ROTEM), is another version of TEG in which it is the sensor shaft, rather than the cup, that rotates.


A small sample of blood is taken from the selected person and rotated gently through 4º 45', six times a minute, to imitate sluggish  venous flow  and activate coagulation. The clot forms around a thin wire probe used for measurement. The speed and strength of clot formation is measured in various ways, typically by computer. The speed at which the sample coagulates depends on the activity of the  plasma  coagulation system,  platelet  function,  fibrinolysis  and other factors which can be affected by genetics, illness, environment, and medications. The patterns of changes in strength and elasticity in the clot provide information about how well the blood can perform  hemostasis  and how well or poorly different factors are contributing to clot formation. [ 2 ]


Four values that represent clot formation are determined by this test: the reaction time (R value), the K value, the angle and the maximum amplitude (MA). The R value represents the time until the first evidence of a clot is detected. The K value is the time from the end of R until the clot reaches 20mm and this represents the speed of clot formation. The angle is the tangent of the curve made as the K is reached and offers similar information to K. The MA is a reflection of clot strength. A mathematical formula determined by the manufacturer can be used to determine a Coagulation Index (CI) (or overall assessment of coagulability) which takes into account the relative contribution of each of these 4 values into 1 equation. The G-value is a log-derivation of the MA and is meant to also represent the clot strength using dynes/sec as its units. There are some studies which suggest that an elevated G-value is associated with a hypercoagulable state and therefore increases the risk for venous thromboembolic disease. However, there are no studies dosing of prophylactic heparin products based on the G-value. TEG also measures clot lysis which is reported as both the estimated percent lysis (EPL) and the percentage of clot which has actually lysed after 30 minutes (LY 30,%). Although a normal EPL can be as high as 15% and a normal LY 30 can be as high as 8%, some studies in the trauma population suggest that a LY30 greater than 3% is associated with risk of hemorrhage. [ 2 ] [ 3 ]


Thromboelastometry  (TEM), previously named rotational thromboelastography (ROTEG) or rotational thromboelastometry (ROTEM), is another version of TEG in which it is the sensor shaft, rather than the cup, that rotates. Blood (300 μL, anticoagulated with  citrate ) is placed into the disposable cuvette using an electronic pipette. A disposable pin is attached to a shaft which is connected with a thin spring (the equivalent to Hartert's torsion wire in thrombelastography) and slowly  oscillates  back and forth. The signal of the pin suspended in the blood sample is transmitted via an optical detector system. The test is started by adding appropriate reagents. The instrument measures and graphically displays the changes in elasticity at all stages of the developing and resolving clot. The typical test temperature is 37 °C, but different temperatures can be selected, e.g. for patients with hypothermia. [ 4 ]


Parameters derived from thromboelastography are mainly: [ 5 ]


Following are examples of thromboelastography patterns and recommended treatments. [ 6 ] [ 7 ]


There are several types of assays that can be run using TEG: Standard (kaolin), RapidTEG, heparinase, Functional Fibrinogen and PlateletMapping. A standard TEG is the most commonly ordered test and includes the parameters noted above. A RapidTEG uses tissue factor in addition to kaolin thereby further speeding up the reaction. In this assay, the R-value is replaced by the TEG-ACT value which is measured in seconds rather than in minutes. The remainder of the TEG parameters do not differ between a standard and RapidTEG. A heparinase TEG is used to assess for heparin-associated anticoagulation as the cause of hemorrhage. It is used most commonly following  cardiopulmonary bypass  procedures where heparin is reversed using protamine intraoperatively. In instances where a patient develops bleeding due to recurrent coagulopathy (usually shortly after arrival to the ICU), the heparinase TEG can help quickly discern patients who can be treated with additional dosing of protamine versus those who need to be taken back to the operating room for re-exploration. In this assay, a standard TEG is run twice – once using the patient's blood only and another time using the patient's blood plus added heparinase. If the two graphs are nearly the same, the cause of bleeding is not related to heparin rebound. However, if the R-time associated with the heparinase-added specimen is significantly shorter than the R-time of the patient's blood without added heparinase, the bleeding is likely due to heparin rebound and should respond to administration of protamine. Lastly, the platelet map TEG aims to determine to what degree platelet function may be inhibited due to pharmacologic inhibition of either the arachidonic acid (AA) or adenosine diphosphate (ADP) pathways. Aspirin inhibits platelet function via the AA pathway while clopidogrel inhibits platelet function via the ADP pathway; thus, this test can be used to determine the degree to which a patient is anticoagulated due to either medication. In this assay, a standard TEG is run using patient's whole blood. Then, separate assays are run using the patient's blood with added AA or ADP. The contribution of fibrin to the MA is subtracted using a mathematical formula. This allows determination of the MA (AA) and MA (ADP), respectively. The difference between the patient's whole blood result and AA/ADP added results are used to calculate the percent inhibition.


Because the R value on the TEG represents the time it takes for clot formation to start, it is a reflection of coagulation factor activity. Coagulation factors are essentially enzymes that drive clot formation. Thus, a significantly prolonged R time could be treated with frozen plasma. The alpha angle represents the thrombin burst and conversion of fibrinogen to fibrin. Thus, a depressed alpha angle could be treated with  cryoprecipitate . 80% of the MA is derived from platelet function whereas the remaining 20% is derived from fibrin. Thus, a significantly depressed MA could be treated with platelet transfusion or medications that improve platelet function, such as DDAVP. An elevated EPL or LY30 suggests fibrinolysis and may be treated with an antifibrinolytic, such as tranexamic acid or aminocaproic acid, in the appropriate clinical setting. A single, modified TEG assay with exogenous tissue plasminogen activator (tPA) demonstrated remarkable efficiency in unmasking patients' impending risk for massive transfusion in trauma patients. [ 8 ]  The iTACTIC trial showed no difference in outcomes in trauma patients with massive hemorrhage managed with conventional coagulation tests compared to viscoelastic assays.  However, the traumatic brain injury subgroup showed slightly better survival in the TEG/ROTEM group with a “very low” quality assessment by the GRADE system. [ 9 ]


Clinical studies of thromboelastography during elective surgery (cardiac and liver surgery) and emergency resuscitation have shown improvements in clinical outcomes. [ 10 ]  In elective surgery there was a decreased need for blood products (platelets and plasma) and reduced operating room length of stay as well as duration of intensive care admission and bleeding rates; mortality was not affected. In emergency settings, mortality was reduced with an associated decrease in the need for platelets and plasma. [ 10 ]


Additional studies show thromboelastography may be used to characterize COVID-19-associated coagulopathy. TEG with platelet mapping may be used to guide use of anticoagulant and antiplatelet medications. When using a TEG-guided strategy hospital length of stay, intensive care unit length of stay, mortality, acute kidney injury, intensive care unit admissions and need for mechanical ventilation may be reduced.  [ 11 ]


Thromboelastometry  ( TEM ), previously named  rotational thromboelastography  ( ROTEG ) or  rotational thromboelastometry  ( ROTEM ), is an established  viscoelastic  method for  hemostasis  testing in  whole blood . [ 1 ]  It is a modification of traditional  thromboelastography  (TEG).


TEM investigates the interaction of  coagulation factors , their  inhibitors ,  anticoagulant drugs ,  blood cells , specifically  platelets , during clotting and subsequent  fibrinolysis . The  rheological  conditions mimic the sluggish flow of blood in  veins .


While traditional thromboelastography is a global  assay  for  blood clotting  disorders and drug effects, TEM is primarily used in combination with appropriate differential assays. They allow testing in the presence of therapeutic  heparin  concentrations and provide differential diagnostic information to support decisions in therapy. In numerous publications, the validity of the method is shown. Application of TEM at the  point of care  (POC) or in emergency laboratories is getting more and more popular. TEM detects both hypo- and hyperfunctional stages of the clotting process and is probably the only reliable rapid test for the diagnosis of  hyperfibrinolysis . In contrast to standard clotting tests, the  fibrin -stabilizing effect of  factor XIII  contributes to the result.


The rapid availability of results helps to discriminate surgical bleeding from a true haemostasis disorder and improves the therapy with  blood products , factor concentrates,  anticoagulants  and  protamine , hemostyptic and  antifibrinolytic  drugs. [ 2 ] [ 3 ]  Several reports confirm that application of TEM is cost effective by reducing the consumption of blood products. [ 4 ] [ 5 ] [ 6 ]


Blood (300 μL, anticoagulated with  citrate ) is placed into the disposable cuvette using an electronic pipette.  A disposable pin is attached to a shaft which is connected with a thin spring (the equivalent to Hartert’s torsion wire in thrombelastography) and slowly  oscillates  back and forth. The signal of the pin suspended in the blood sample is transmitted via an optical detector system. The test is started by adding appropriate reagents. The instrument measures and graphically displays the changes in elasticity at all stages of the developing and resolving clot. The typical test temperature is 37 °C, but different temperatures can be selected, e.g. for patients with hypothermia. [ 7 ]


In contrast to thrombelastography with its pendulum-like principle, the design of the TEM  viscoelastic  detection system (figure 1) makes it quite robust and insensitive against mechanical shocks or vibrations. [ citation needed ]


The primary result of TEM is a reaction curve which shows the elasticity over time when the clot forms or dissolves. This curve is also called a TEMogram. Four key parameters describe the clotting curve for clinical routine. More than 10 additional  calculated parameters, including  derivative  curves which are useful in specific research applications, e.g. in hemophilia [ 8 ]  or thrombophilia [ 9 ]  or for the effects of recombinant FVIIa [ 10 ]  are available for research purposes.


CT  (Clotting time):
The CT is the latency time from adding the start reagent to blood until the clot starts to form. Prolongation of CT may be a result of coagulation deficiencies, primarily coagulation factors, or heparin (dependent on the test used).  A potential contribution of heparin can be detected by comparing INTEM- with HEPTEM CT data (see “reagents” below). A shortening of CT indicates hypercoagulability. [ citation needed ]


CFT  (Clot formation time) and  alpha-angle :
The alpha angle is the angle of tangent between 0 mm and the curve when the clot firmness is 20 mm, while CFT is the time from CT until a clot firmness of  20 mm point has been reached. These parameters denote the speed at which a solid clot forms and are primarily influenced by  platelet  function, but to a certain extent especially  fibrinogen  and  coagulation factors  contribute. A prolonged CFT (or a lower alpha-angle) is usually caused by poor platelet function, low platelet count, fibrin polymerization disorders or fibrinogen deficiency. Apparently also FXIII seems to be involved already in this phase. [ 11 ]  Higher concentrations of  heparin  can also prolong CFT in the INTEM assay, but not in HEPTEM, EXTEM, FIBTEM or APTEM (see under “reagents”). A shortening of CFT (or a high alpha-angle) indicate hypercoagulability. [ citation needed ]


MCF  (Maximum clot firmness):
MCF is the greatest vertical  amplitude  of the trace. It reflects the absolute strength of the fibrin and platelet clot. A low MCF is indicative of decreased platelet number or function, decreased fibrinogen level or fibrin polymerization disorders, or low activity of factor XIII. A mechanically weak clot represents a severe bleeding risk and should initiate immediate therapeutic steps. High doses of heparin can lower MCF in the INTEM assay, but not in HEPTEM, EXTEM, FIBTEM or APTEM (see under “reagents”).


A5, A 10, A15 or A20 value
These values describe the clot firmness (or amplitude) obtained after 10, 15 or 20 minutes (beginning from CFT) and  provide a forecast on the expected MCF value at an earlier stage already. A recent investigation has validated this approach for the A15 value in more than 800 cases during  liver transplantation . [ 12 ]  The advantage of the A15-values is obvious: It allows for a more rapid decision about therapeutic interventions.


LI 30  (Lysis Index after 30 minutes) and  ML  (Maximum Lysis)
The LI30 value is the percentage of remaining clot stability in relation to the MCF value at 30 min after CT. A similar value can also be calculated at other time points (45 or 60 min). The ML parameter describes the percentage of lost clot stability (relative to MCF, in %) viewed at any selected time point or when the test has been stopped.  A low LI (X) value or a high ML value indicates hyperfibrinolysis. While in normal blood fibrinolysis activity is quite low, in clinical samples a more rapid loss of clot stability by hyperfibrinolysis may lead to bleeding complications which can be treated by the administration of  antifibrinolytic  drugs. [ citation needed ]


Initially,  thrombelastography  was simply performed with whole blood without adding reagents (except calcium when citrate anticoagulated blood was used). This provides a global overview about all phases of clot formation, stabilization and degradation. In the case of monocausal haemostasis disorders, the resulting reaction curves may be quite typical; however, under most clinical conditions this approach has severe limitations. In reality various effects overlap, including haemodilution or application of high doses of  parenteral  anticoagulants. High doses of heparin often prevent clot formation at all. Absence of a controlled activation step leads to inferior reproducibility and very long test times which are not acceptable for POC applications.
The assays for ROTEM analysis help to get a rapid differentiation between various potential haemostasis defects or anticoagulant drug effects and allow for a rapid differential diagnosis. They form the base for selecting a therapeutic strategy. Several diagnostic  algorithms  have been proposed and were clinically validated. [ 12 ] [ 13 ] [ 14 ]  Application of this strategy helps to minimize the exposure of patients to  allogeneic  blood products which have certain risks; and it saves costs. [ 4 ] [ 5 ] [ 6 ]  Numerous research applications have used the technique as well. [ citation needed ]


INTEM
This test mildly activates the contact phase of haemostasis. The result is influenced by coagulation factors, platelets, fibrinogen and heparin. Low molecular weight heparin is detected at higher concentrations. [ 15 ] [ 16 ]  In the absence of heparin, INTEM is a screening test for the haemostasis system. It is used for therapeutic decisions regarding the administration of fresh frozen plasma, coagulation factors, fibrinogen or platelets.


HEPTEM
This assay represents an INTEM assay performed in the presence of heparinase, a heparin (or LMWH) degrading enzyme. It allows the identification of haemostasis deficiencies even in the presence of heparin and represents an INTEM test without interference by heparin or heparin like anticoagulants. The difference between HEPTEM and INTEM CT-value comparison confirms the presence of heparin, e.g. accidental exposure. [ 17 ]


EXTEM
test mildly activates haemostasis via the physiological activator tissue factor. The result is influenced by extrinsic coagulation factors, platelets and fibrinogen. EXTEM is a screening test for the (extrinsic) haemostasis system. This assay is not influenced by heparin (heparin inhibitor included in the EXTEM reagent). It is used for therapeutic decisions regarding the administration of fresh frozen plasma, coagulation factors, fibrinogen or platelets. EXTEM is also the base activator for FIBTEM and APTEM.


FIBTEM
test is an EXTEM based assay for the fibrin part of the clot. FIBTEM eliminates the platelet contribution of clot formation by inhibiting the platelets irreversibly with  cytochalasin D , a potent  inhibitor  of  actin  polymerization which disrupts actin microfilaments, an essential part of a cytoskeleton-mediated contractibility apparatus of the platelet. The use of cytochalasin is more favorable than using  glycoprotein  IIb/ IIIa inhibitors which block platelet incompletely, especially at higher platelet counts. [ 18 ]  FIBTEM allows for the detection of fibrinogen deficiency or fibrin polymerization disorders, e.g. induced by certain plasma expanders, and may identify rapidly the need to substitute fibrinogen. FIBTEM results correlate well in many cases with the Clauss fibrinogen assay, but is additionally influenced by fibrin polymerization disorders which cannot reliably be detected with clotting tests. [ 18 ]


APTEM
test is an EXTEM based assay in which  fibrinolysis  is inhibited by  aprotinin  in the reagent. A significant improvement of the clot in APTEM compared to EXTEM allows to detect fulminant hyperfibrinolysis. Therefore, the test helps in identifying the necessity of administrating  antifibrinolytic  drugs. Furthermore, APTEM enables the estimation if an antifibrinolytic therapy alone normalizes coagulation or if additional measures have to be taken (e.g. administration of fibrinogen or platelets).


Quality control is an important requirement in laboratory and POC testing. An automatic internal electronic control procedure is implemented in the ROTEM whole blood analyzer. Furthermore, biological control materials at 2 different levels are available and should be used in order to document quality and validity of results.


TEM is successfully used in the near patient assessment of haemostasis. The method allows detection of complex haemostasis disorders (available in most coagulopathies) within a few minutes and thus enables rapid therapeutic intervention. Whole blood TEM is sensitive to haemostasis affecting agents such as plasma expanders or  acidosis  while the effects of these agents are hardly identified by plasma based laboratory tests.
TEM-guided  transfusion  of blood products or factor concentrates in  cardiac ,  hepatic  and major  orthopedic  surgery is the main application of the method. Moreover, it is successfully used in the complex situation of  (poly) trauma , or in decision making for of alternative therapy such as antifibrinolytic drug administration .
The clinical benefits of TEM have resulted in major savings [ 4 ] [ 5 ] [ 6 ]  and publications recommend that  viscoelastic  methods such as TEM should be used in surgical procedures where major blood loss followed by the need for transfusions can be expected. [ 19 ]


Like any other haemostasis evaluating method, TEM (and thrombelastography) have limitations which need to be considered when interpreting the results. The typical assays are not responsive for the effect of von Willebrand factor or platelet antagonists such as  aspirin  or thienopyridines (e.g.  clopidogrel ), and only supratherapeutic doses of  GPIIb/IIIa  antagonists may influence results. The sensitivity for coagulation factor deficiencies, including those induced by oral anticoagulation, is less pronounced as compared to clotting assays. Therefore, TEM is not meant to replace laboratory assays such as prothrombin time (PT) or factor assays. However, due to the rapid availability of differential diagnostic information, TEM has become an established method in surgical procedures where blood losses can be expected.


The  Thyroid Feedback Quantile-based Index  ( TFQI ) is a calculated parameter for thyrotropic  pituitary  function. It was defined to be more robust to distorted data than established markers including  Jostel's TSH index  (JTI) and the  thyrotroph thyroid hormone sensitivity index  (TTSI).


The TFQI can be calculated with


from  quantiles  of FT4 and TSH concentration (as determined based on  cumulative distribution functions ). [ 1 ]  Per definition the TFQI has a  mean  of 0 and a  standard deviation  of 0.37 in a reference population. [ 1 ]  This explains the reference range of –0.74 to + 0.74.


Higher values of TFQI are associated with  obesity ,  metabolic syndrome , impaired  renal  function,  diabetes , and diabetes-related  mortality . [ 1 ] [ 2 ] [ 3 ] [ 4 ] [ 5 ] [ 6 ] [ 7 ]  In a large population of community-dwelling euthyroid subjects the thyroid feedback quantile-based index predicted all-cause mortality, even after adjustment for other established risk factors and comorbidities. [ 8 ]


A cross-sectional study from Spain observed increased prevalence of type 2 diabetes, atrial fibrillation, ischemic heart disease and hypertension in persons with elevated PTFQI. [ 9 ]


Serum Concentrations of Adipocyte Fatty Acid-Binding Protein (A-FABP) are significantly correlateted to TFQI, suggesting some form of cross-talk between adipose tissue and HPT axis. [ 10 ]


TFQI results are also elevated in  takotsubo syndrome , [ 11 ]  potentially reflecting type 2  allostatic load  in the situation of  psychosocial stress . Reductions have been observed in subjects with  schizophrenia  after initiation of therapy with  oxcarbazepine [ 12 ]  and quetiapine, [ 13 ]  potentially reflecting declining allostatic load.


Despite positive association to metabolic syndrome and type 2 allostatic load a large population-based study failed to identify an association to risks of  dyslipidemia  and  non-alcoholic fatty liver disease  (NAFLD). [ 14 ]


Thyroid function tests  ( TFTs ) is a collective term for  blood tests  used to check the function of the  thyroid . [ 1 ]
TFTs may be requested if a patient is thought to suffer from  hyperthyroidism  (overactive thyroid) or  hypothyroidism  (underactive thyroid), or to monitor the effectiveness of either thyroid-suppression or hormone replacement therapy. It is also requested routinely in conditions linked to thyroid disease, such as  atrial fibrillation  and  anxiety disorder .


A TFT  panel  typically includes  thyroid hormones  such as  thyroid-stimulating hormone  (TSH, thyrotropin) and  thyroxine  (T4), and  triiodothyronine  (T3) depending on local laboratory policy.


Thyroid-stimulating hormone  (TSH, thyrotropin) is generally increased in hypothyroidism and decreased in hyperthyroidism, [ 2 ]  making it the most important test for early detection of both of these conditions. [ 3 ] [ 4 ]  The result of this  assay  is suggestive of the presence and cause of thyroid disease, since a measurement of elevated TSH generally indicates  hypothyroidism , while a measurement of low TSH generally indicates  hyperthyroidism . [ 2 ]  However, when TSH is measured by itself, it can yield misleading results, so additional thyroid function tests must be compared with the result of this test for accurate diagnosis. [ 4 ] [ 5 ] [ 6 ]


TSH is produced in the  pituitary gland . The production of TSH is controlled by  thyrotropin-releasing hormone  (TRH), which is produced in the  hypothalamus . TSH levels may be suppressed by excess free T3 (fT3) or free T4 (fT4) in the blood. [ citation needed ]


First-generation TSH assays were done by  radioimmunoassay  and were introduced in 1965. [ 3 ]  There were variations and improvements upon TSH radioimmunoassay, but their use declined as a new immunometric assay technique became available in the middle of the 1980s. [ 3 ] [ 4 ]  The new techniques were more accurate, leading to the second, third, and even fourth generations of TSH assay, with each generation possessing ten times greater functional sensitivity than the last. [ 7 ]  Third generation immunometric assay methods are typically automated. [ 3 ]  Fourth generation TSH immunometric assay has been developed for use in research. [ 4 ]


Third generation TSH assay is the requirement for modern standards of care. TSH testing in the United States is typically carried out with automated platforms using advanced forms of immunometric assay. [ 3 ]  Nonetheless, there is no international standard for measurement of thyroid-stimulating hormone. [ 4 ]


Accurate interpretation takes a variety of factors into account, such as the thyroid hormones i.e.  thyroxine  (T 4 ) and  triiodothyronine  (T 3 ), current medical status (such as pregnancy [ 3 ] ), [ 4 ]  certain medications like  propylthiouracil , [ 4 ]  temporal effects including  circadian rhythm [ 8 ]  and  hysteresis , [ 9 ]  and other past  medical history . [ 10 ]


Total thyroxine is rarely measured, having been largely superseded by free thyroxine tests. Total  thyroxine  (Total T 4 ) is generally elevated in  hyperthyroidism  and decreased in  hypothyroidism . [ 2 ]  It is usually slightly elevated in pregnancy secondary to increased levels of  thyroid binding globulin  (TBG). [ 2 ]


Total T4 is measured to see the bound and unbound levels of T4. The total T4 is less useful in cases where there could be protein abnormalities. The total T4 is less accurate due to the large amount of T4 that is bound. The total T3 is measured in clinical practice since the T3 has decreased amount that is bound as compared to T4. [ citation needed ]


Reference ranges depend on the method of analysis. Results should always be interpreted using the range from the laboratory that performed the test. Example values are:


Free  thyroxine  (fT 4  or free T4) is generally elevated in  hyperthyroidism  and decreased in  hypothyroidism . [ 2 ]


Total  triiodothyronine  (Total T 3 ) is rarely measured, having been largely superseded by free T3 tests. Total T3 is generally elevated in hyperthyroidism and decreased in hypothyroidism. [ 2 ]


Free  triiodothyronine  (fT 3  or free T3) is generally elevated in hyperthyroidism and decreased in hypothyroidism. [ 2 ]


An increased  thyroxine-binding globulin  results in an increased total thyroxine and total triiodothyronine without an actual increase in hormonal activity of thyroid hormones.


Reference ranges:


Thyroid hormone uptake (T uptake  or T 3 uptake ) is a measure of the unbound  thyroxine binding globulins  in the blood, that is, the TBG that is unsaturated with thyroid hormone. [ 2 ]  Unsaturated TBG increases with decreased levels of thyroid hormones. It is not directly related to triiodothyronine, despite the name T 3 uptake . [ 2 ]


The Free Thyroxine Index (FTI or T7) is obtained by multiplying the total T 4  with T 3  uptake. [ 2 ]  FTI is considered to be a more reliable indicator of thyroid status in the presence of abnormalities in plasma protein binding. [ 2 ]  This test is rarely used now that reliable free thyroxine and free triiodothyronine assays are routinely available.


FTI is elevated in hyperthyroidism and decreased in hypothyroidism. [ 2 ]


Derived  structure parameters  that describe constant properties of the overall  feedback control system  may add useful information for special purposes, e.g. in diagnosis of  nonthyroidal illness syndrome  or  central hypothyroidism . [ 20 ] [ 21 ] [ 22 ] [ 23 ]


Thyroid's secretory capacity  ( G T , also referred to as SPINA-GT) is the maximum stimulated amount of thyroxine the thyroid can produce in one second. [ 24 ]   G T  is elevated in hyperthyroidism and reduced in hypothyroidism. [ 25 ]


G T  is calculated with


G
 ^

 T

 =

 β

 T

 (

 D

 T

 +
 [
 T
 S
 H
 ]
 )
 (
 1
 +

 K

 41

 [
 T
 B
 G
 ]
 +

 K

 42

 [
 T
 B
 P
 A
 ]
 )
 [
 F

 T

 4

 ]

 α

 T

 [
 T
 S
 H
 ]

 {\displaystyle {\hat {G}}_{T}={{\beta _{T}(D_{T}+[TSH])(1+K_{41}[TBG]+K_{42}[TBPA])[FT_{4}]} \over {\alpha _{T}[TSH]}}}


G
 ^

 T

 =

 β

 T

 (

 D

 T

 +
 [
 T
 S
 H
 ]
 )
 [
 T

 T

 4

 ]

 α

 T

 [
 T
 S
 H
 ]

 {\displaystyle {\hat {G}}_{T}={{\beta _{T}(D_{T}+[TSH])[TT_{4}]} \over {\alpha _{T}[TSH]}}}


α

 T

 {\displaystyle \alpha _{T}}

 : Dilution factor for T4 (reciprocal of apparent volume of distribution, 0.1 l −1 )

 β

 T

 {\displaystyle \beta _{T}}

 : Clearance exponent for T4 (1.1e-6 sec −1 )
 K 41 : Dissociation constant T4-TBG (2e10 L/mol)
 K 42 : Dissociation constant T4-TBPA (2e8 L/mol)
 D T : EC 50  for TSH (2.75 mU/L) [ 24 ]


The  sum activity of peripheral deiodinases  ( G D , also referred to as SPINA-GD) is reduced in nonthyroidal illness with hypodeiodination. [ 21 ] [ 22 ] [ 26 ]


G D  is obtained with


α

 31

 {\displaystyle \alpha _{31}}

 : Dilution factor for T3 (reciprocal of apparent volume of distribution, 0.026 L −1 )

 β

 31

 {\displaystyle \beta _{31}}

 : Clearance exponent for T3 (8e-6 sec −1 )
 K M 1 : Dissociation constant of type-1-deiodinase (5e-7 mol/L)
 K 30 : Dissociation constant T3-TBG (2e9 L/mol) [ 24 ]


Jostel's TSH index  (JTI or TSHI) helps to determine thyrotropic function of anterior  pituitary  on a quantitative level. [ 27 ]  It is reduced in thyrotropic insufficiency [ 27 ]  and in certain cases of non-thyroidal illness syndrome. [ 26 ]


It is calculated with


T
 S
 H
 I
 =
 L
 N
 (
 T
 S
 H
 )
 +
 0.1345
 ∗
 F
 T
 4

 {\displaystyle TSHI=LN(TSH)+0.1345*FT4}

 .


Additionally, a standardized form of TSH index may be calculated with


s
 T
 S
 H
 I
 =
 (
 T
 S
 H
 I
 −
 2.7
 )

 /

 0.676

 {\displaystyle sTSHI=(TSHI-2.7)/0.676}

 . [ 27 ]


The  Thyrotroph Thyroid Hormone Sensitivity Index  (TTSI, also referred to as  Thyrotroph T4 Resistance Index  or TT4RI) was developed to enable fast screening for  resistance to thyroid hormone . [ 28 ] [ 29 ]  Somewhat similar to the TSH Index it is calculated from equilibrium values for TSH and FT4, however with a different equation.


The  Thyroid Feedback Quantile-based Index  (TFQI) is another parameter for thyrotropic pituitary function. It was defined to be more robust to distorted data than JTI and TTSI. It is calculated with


T
 F
 Q
 I
 =

 F

 F
 T
 4

 (
 F
 T
 4
 )
 −
 (
 1
 −

 F

 T
 S
 H

 (
 T
 S
 H
 )
 )

 {\displaystyle TFQI=F_{FT4}(FT4)-(1-F_{TSH}(TSH))}


from quantiles of FT4 and TSH concentration (as determined based on  cumulative distribution functions ). [ 30 ]  Per definition the TFQI has a  mean  of 0 and a  standard deviation  of 0.37 in a reference population. [ 30 ]  Higher values of TFQI are associated with  obesity ,  metabolic syndrome , impaired  renal  function,  diabetes , and diabetes-related  mortality . [ 30 ] [ 31 ] [ 32 ] [ 33 ] [ 34 ] [ 35 ] [ 36 ]  TFQI results are also elevated in  takotsubo syndrome , [ 37 ]  potentially reflecting type 2  allostatic load  in the situation of  psychosocial stress . Reductions have been observed in subjects with  schizophrenia  after initiation of therapy with  oxcarbazepine , potentially reflecting declining allostatic load. [ 38 ]


In healthy persons, the intra-individual variation of TSH and thyroid hormones is considerably smaller than the inter-individual variation. [ 39 ] [ 40 ] [ 41 ]  This results from a personal  set point  of thyroid homeostasis. [ 42 ]  In hypothyroidism, it is impossible to directly access the set point, [ 43 ]  but it can be reconstructed with methods of systems theory. [ 44 ] [ 45 ] [ 46 ]


A computerised algorithm, called Thyroid-SPOT, which is based on this mathematical theory, has been implemented in software applications. [ 47 ]  In patients undergoing thyroidectomy it could be demonstrated that this algorithm can be used to reconstruct the personal set point with sufficient precision. [ 48 ]


Drugs can profoundly affect thyroid function tests. Listed below is a selection of important effects.


↓: reduced serum concentration or structure parameter; ↑: increased serum concentration or structure parameter; ↔: no change; TSH: Thyroid-stimulating hormone; T 3 : Total triiodothyronine; T 4 : Total thyroxine; fT 4 : Free thyroxine; fT 3 : Free triiodothyronine; rT 3 : Reverse triiodothyronine


The  Centers for Disease Control and Prevention  has published the following laboratory procedure manuals for measuring thyroid-stimulating hormone:


Thyroid hormone binding ratio (THBR)  is a  thyroid function test  that measures the "uptake" of  T3 or T4  tracer by  thyroid-binding globulin  (TBG) in a given serum sample. This provides an indirect and  reciprocal  estimate of the available binding sites on TBG within the sample.


The results are then reported as a ratio to normal serum.


Attempts to correct for changes in thyroid binding globulin due to liver disease, protein losing states, pregnancy or various drugs [ citation needed ]


It is used to calculate  free thyroxine index  (total T4 x T3 uptake), an estimate of free T4. Free thyroxine index may be calculated with increased diagnostic accuracy using direct TBG measurement when the total hormone concentration is abnormally elevated  [ 1 ]


Invalid if other proteins or immunoglobulins compete with TBG, including  familial dysalbuminemic hyperthyroxinemia [ 2 ]


Thyroid's secretory capacity  ( G T , also referred to as  thyroid's incretory capacity ,  maximum thyroid hormone output ,  T4 output  or, if calculated from serum levels of thyrotropin and thyroxine, as  SPINA-GT [ a ] ) is the maximum stimulated amount of  thyroxine  that the  thyroid  can produce in a given time-unit (e.g. one second). [ 2 ] [ 3 ]


Experimentally,  G T  can be determined by stimulating the thyroid with a high thyrotropin concentration (e.g. by means of  rhTSH , i.e.  recombinant  human thyrotropin) and measuring its output in terms of T4 production, or by measuring the serum concentration of protein-bound  iodine-131  after administration of  radioiodine . [ 4 ]  These approaches are, however, costly and accompanied by significant exposure to radiation. [ 5 ]


In vivo ,  G T  can also be estimated from equilibrium levels of TSH and T4 or free T4. In this case it is calculated with


[ TSH ]: Serum thyrotropin concentration (in mIU/L or   μIU/mL)
[ FT4 ]: Serum free T4 concentration (in pmol/L)
[ TT4 ]: Serum total T4 concentration (in nmol/L)

 G
 ^

 T

 {\displaystyle {\hat {G}}_{T}}

 : Theoretical (apparent) secretory capacity (SPINA-GT)

 α

 T

 {\displaystyle \alpha _{T}}

 :  Dilution  factor for T4 (reciprocal of apparent volume of distribution, 0.1 L −1 )

 β

 T

 {\displaystyle \beta _{T}}

 :  Clearance  exponent for T4 (1.1e-6 sec −1 ), i. e.,  reaction rate constant  for degradation
 K 41 :  Binding constant  T4-TBG (2e10 L/mol)
 K 42 : Binding constant T4-TBPA (2e8 L/mol)
 D T :  EC 50  for TSH (2.75 mU/L) [ 2 ] [ 6 ]


The method is based on mathematical models of thyroid homeostasis. [ 2 ] [ 3 ]  Calculating the secretory capacity with one of these equations is an  inverse problem . Therefore, certain conditions (e.g. stationarity) have to be fulfilled to deliver a reliable result.


The ratio of SPINA-GT and thyroid volume  V T  (as determined e.g. by  ultrasonography )


G
 ^

 T
 S

 =

 G
 ^

 T

 V

 T

 {\displaystyle {\hat {G}}_{TS}={\frac {{\hat {G}}_{T}}{{V}_{T}}}}

 ,


i.e.


G
 ^

 T
 S

 =

 β

 T

 (

 D

 T

 +
 [
 T
 S
 H
 ]
 )
 (
 1
 +

 K

 41

 [
 T
 B
 G
 ]
 +

 K

 42

 [
 T
 B
 P
 A
 ]
 )
 [
 F

 T

 4

 ]

 α

 T

 [
 T
 S
 H
 ]

 V

 T

 {\displaystyle {\hat {G}}_{TS}={\frac {\beta _{T}(D_{T}+[TSH])(1+K_{41}[TBG]+K_{42}[TBPA])[FT_{4}]}{\alpha _{T}[TSH]{V}_{T}}}}


G
 ^

 T
 S

 =

 β

 T

 (

 D

 T

 +
 [
 T
 S
 H
 ]
 )
 [
 T

 T

 4

 ]

 α

 T

 [
 T
 S
 H
 ]

 V

 T

 {\displaystyle {\hat {G}}_{TS}={\frac {\beta _{T}(D_{T}+[TSH])[TT_{4}]}{\alpha _{T}[TSH]{V}_{T}}}}


is referred to as  specific thyroid capacity  (SPINA-GTs). [ 7 ]  It is a measure for how much one  millilitre  of thyroid tissue can produce under conditions of maximum stimulation. Thereby, SPINA-GTs is an estimate for the endocrine quality of thyroid tissue. [ citation needed ]


The equations and their parameters are calibrated for adult humans with a body mass of 70 kg and a plasma volume of ca. 2.5 L. [ 2 ]


SPINA-GT is elevated in primary  hyperthyroidism [ 8 ] [ 9 ]  and reduced in both primary  hypothyroidism [ 10 ] [ 11 ] [ 12 ] [ 9 ]  and untreated autoimmune thyroiditis. [ 13 ]  It has been observed to correlate (with positive direction) to  resting energy expenditure , [ 14 ]   resting heart rate , [ 15 ]  the colour Doppler ultrasound pattern [ 16 ]  and thyroid volume, [ 2 ] [ 7 ]  and (with negative direction) to thyroid autoantibody titres, which reflect organ destruction due to autoimmunity. [ 17 ]  Elevated SPINA-GT in  Graves' disease  is reversible with antithyroid treatment. [ 14 ]  While SPINA-GT is significantly altered in primary thyroid disorders, it is insensitive to disorders of secondary nature (e.g. pure pituitary diseases). [ 3 ]


In silico  experiments with  Monte Carlo simulations  demonstrated that both SPINA-GT and  SPINA-GD  can be estimated with sufficient reliability, even if laboratory assays have limited accuracy. [ 3 ]  This was confirmed by longitudinal  in vivo  studies that showed that GT has lower intraindividual variation (i.e. higher reliability) than  TSH ,  FT4  or  FT3 . [ 18 ]


In clinical trials SPINA-GT was significantly elevated in patients with  Graves' disease  and  toxic adenoma  compared to normal subjects. [ 2 ] [ 8 ] [ 19 ]  It is also elevated in diffuse and nodular  goiters , and reduced in untreated autoimmune thyroiditis. [ 2 ] [ 13 ]  In patients with toxic adenoma it has higher  specificity  and  positive likelihood ratio  for diagnosis of thyrotoxicosis than serum concentrations of  thyrotropin , free T4 or free T3. [ 2 ]  GT's specificity is also high in thyroid disorders of secondary or tertiary origin. [ 3 ]


Calculating SPINA-GT has proved to be useful in challenging clinical situations, e.g. for differential diagnosis of subclinical hypothyroidism and elevated TSH concentration due to type 2 allostatic load (as it is typical for obesity and certain psychiatric diseases). For this purpose, its usage has been recommended in  sociomedical assessment . [ 20 ]


In patients suffering from toxic adenoma, toxic multinodular goitre and Graves’ disease  radioiodine therapy  leads to a significant decrease of the initially elevated SPINA-GT. [ 19 ]


Correlation of SPINA-GT with  creatinine clearance  suggests a negative influence of  uremic  toxins on thyroid biology. [ 21 ] [ 22 ]  In the initial phase of major  non-thyroidal illness syndrome  (NTIS) SPINA-GT may be temporarily elevated. [ 23 ] [ 24 ]  In chronic NTIS [ 25 ]  as well as in certain non-critical chronic diseases, e.g.  chronic fatigue syndrome [ 26 ] [ 27 ]  or  asthma [ 28 ]  SPINA-GT is slightly reduced.


According to the results of a community-based study in China it was associated to sleep duration and exercise habits. [ 29 ]  With respect to iodine supply, it showed a complex U-shaped pattern, being reduced in subjects consuming iodine-rich food, but elevated in situations of iodine excess. [ 29 ]  In two other studies from China, SPINA-GT correlated with negative direction to markers of obesity including body mass index, waist circumference and waist to hip ratio. [ 30 ] [ 31 ]  This doesn't seem to be the case, however, in Western populations. [ 32 ]


In women, therapy with  Metformin  results in increased SPINA-GT, in parallel to improved  insulin sensitivity . [ 33 ] [ 34 ]  This observation was reproducible in men with hypogonadism, but not in men with normal testosterone concentrations,. [ 35 ]  In postmenopausal women this effect was only observed in subjects on oestradiol replacement therapy. [ 36 ]  Therefore, the described phenomenon seems to depend on an interaction of metformin with sex hormones. [ 35 ] [ 37 ]  In hyperthyroid [ 8 ]  men both SPINA-GT and  SPINA-GD  negatively correlate to  erectile function , intercourse satisfaction,  orgasmic function  and  sexual desire . Likewise, in women with thyrotoxicosis elevated thyroid's secretory capacity predicts depression and sexual dysfunction. [ 38 ]  Conversely, in androgen-deficient men with concomitant  autoimmune thyroiditis , substitution therapy with  testosterone  leads to a decrease in  thyroid autoantibody  titres and an increase in SPINA-GT. [ 39 ]  In a large study from mainland China, SPINA-GT was elevated in certain psychiatric diseases including bipolar disorder and schizophrenia. [ 40 ]  In bipolar disorder with manic or mixed episodes it was higher than in cases with depressive episodes. [ 40 ]


SPINA-GT is reduced in persons suffering from  hidradenitis suppurativa  compared to healthy controls with the same sex and age distribution. [ 41 ]  This phenomenon has been ascribed to  B-cell -mediated  hypothyroidism , i.e. hypothyroid  Graves' disease  due to inhibiting TSH receptor autoantibodies (iTRAb). [ 41 ]


In patients with autoimmune thyroiditis a  gluten-free diet  results in increased SPINA-GT (in parallel to sinking autoantibody titres). [ 42 ]   Statin  therapy has the same effect, but only if supply with  vitamin D  is sufficient. [ 43 ]  Accordingly, substitution therapy with  25-hydroxyvitamin D  leads to rising secretory capacity. [ 44 ] [ 45 ] [ 46 ] [ 47 ]  This effect is potentiated by substitution therapy with  myo-inositol [ 48 ]  and  selenomethionine [ 44 ] [ 45 ] [ 49 ]  or, in women, with  dehydroepiandrosterone , [ 50 ]  but impaired in males with early-onset androgenic alopecia. [ 51 ]  The effects of vitamin D and selenomethionine are attenuated in  hyperprolactinaemia , suggesting an inhibitory effect of  prolactin . [ 52 ]  Although both vitamin D supplementation and gluten-free diet result in increased SPINA-GT, there seems to be a complex interaction between both therapeutic measures, since vitamin D treatment is only able to elevate the thyroid's secretory capacity in subjects not following any dietary recommendation. [ 53 ]


On the other hand, men treated with  spironolactone  are faced with decreasing SPINA-GT (in addition to rising  thyroid antibody  titres). [ 54 ]  It has, therefore, been concluded that spironolactone may aggravate thyroid autoimmunity in men. [ 54 ]


In subjects with  type 2 diabetes , treatment with  beta blockers  resulted in decreased SPINA-GT, suggesting  sympathetic innervation  to contribute to the control of thyroid function. [ 55 ]  In diabetic women, but not in men, SPINA-GT shows a positive correlation to the β-C-terminal cross-linked telopeptides of type I collagen (β-CTX), a marker of bone resorption. [ 56 ]  In both diabetic and non-diabetic persons it correlates (negatively) with age and (positively) with the concentrations of  troponin T  and  HbA1c . [ 57 ]


SPINA-GT correlates to mechanical pain sensitivity (MPS) in  quantitative sensory testing  (QST) and to measures of respiratory arrhythmia in the analysis of  heart rate variability , indicating a potential link to both  sensorimotor  and  autonomic neuropathy . [ 58 ]


A study in  euthyroid  subjects with  structural heart disease  found that increased SPINA-GT predicts the risk of malignant  arrhythmia  including  ventricular fibrillation  and  ventricular tachycardia . [ 59 ]  This applies to both incidence and event-free survival. [ 59 ]  Likewise, SPINA-GT is elevated in a significant subgroup of patients with  takotsubo syndrome , [ 60 ]  especially in non-survivors. [ 61 ]  A stress-mediated effect on SPINA-GT is also suggested by the observation that it is increased in persons with a history of psychological trauma. [ 62 ]  On the other hand, two studies found negative correlation between SPINA-GT and markers of dispersion in cardiac repolarisation, including Tp-e interval, JT interval, Tp-e/ QT ratio and Tp-e/QTc ratio. These results suggest that reduced thyroid function may trigger cardiovascular mortality as well. [ 63 ] [ 9 ]


Among subjects with  Parkinson's disease , SPINA-GT is significantly elevated in  tremor -dominant and mixed subtypes compared to the akinetic-rigid type. [ 64 ]


Specific secretory capacity (SPINA-GTs) is reduced in  obesity [ 2 ]  and  autoimmune thyroiditis . [ 7 ] [ 65 ]


Endocrine disruptors  may affect stimulated thyroid output, as demonstrated by a positive correlation of SPINA-GT with exposure to  2-hydroxynaphthalene  (2-NAP), [ 66 ]  urinary  mercury  concentration [ 67 ]  and the excretion of certain  phthalate  metabolites, [ 68 ]  and negative correlation with combined exposure to  polycyclic aromatic hydrocarbons  (PAHs) [ 66 ]  and  nickel . [ 69 ]  Additionally, SPINA-GT was altered in young people exposed to  butylparaben . [ 70 ]


In a longitudinal evaluation of a large sample of the general US population over 10 years, reduced SPINA-GT significantly predicted all-cause mortality. [ 71 ]


The  Thyrotroph Thyroid Hormone Sensitivity Index  (abbreviated  TTSI , also referred to as  Thyrotroph T4 Resistance Index  or  TT4RI ) is a calculated structure parameter of  thyroid homeostasis . It was originally developed to deliver a method for fast  screening  for  resistance to thyroid hormone . [ 1 ] [ 2 ]  Today it is also used to get an estimate for the  set point  of thyroid homeostasis, [ 3 ]  especially to assess dynamic thyrotropic adaptation of the anterior  pituitary  gland, including  non-thyroidal illnesses . [ 4 ]


The TTSI can be calculated with


from equilibrium  serum  or  plasma  concentrations of  thyrotropin  (TSH), free  T4  (FT4) and the  assay -specific upper limit of the  reference interval  for FT4 concentration ( l u ). [ 4 ]


Some publications use a simpler form of this equation that doesn't correct for the reference range of free T4. It is calculated with


The disadvantage of this uncorrected version is that its numeric results are highly dependent on the used  assays  and their  units of measurement . [ citation needed ]


In case of resistance to thyroid hormone, the magnitude of TTSI depends on which nucleotide in the  THRB  gene is mutated, but also on the genotype of coactivators. A systematic investigation in mice demonstrated a strong association of TT4RI to the genotypes of THRB and the steroid receptor coactivator ( SRC-1 ) gene. [ 5 ]


The TTSI is used as a screening parameter for resistance to thyroid hormone due to mutations in the  THRB  gene, where it is elevated. [ 4 ]  It is also beneficial for assessing the severity of already confirmed thyroid hormone resistance, [ 6 ]  even on replacement therapy with L-T4, [ 7 ]  and for monitoring the pituitary response to substitution therapy with thyromimetics (e.g.  TRIAC ) in RTH Beta. [ 8 ]


In  autoimmune thyroiditis  the TTSI is moderately elevated. [ 9 ]


A large cohort study demonstrated TTSI to be strongly influenced by genetic factors. [ 10 ]  A variant of the TTSI that is not corrected for the upper limit of the FT4 reference range was shown to be significantly increased in offspring from long-lived siblings compared to their partners. [ 11 ]


Conversely, an elevated set point of thyroid homeostasis, as quantified by the TT4RI, is associated to higher prevalence of  metabolic syndrome [ 3 ]  and several harmonized criteria by the  International Diabetes Federation , including  triglyceride  and  HDL  concentration and  blood pressure . [ 12 ] [ 13 ]


In certain phenotypes of  non-thyroidal illness syndrome , especially in cases with concomitant  sepsis , the TTSI is reduced. [ 14 ]  This reflects a reduced set point of thyroid homeostasis, as also experimentally predicted in rodent models of  inflammation  and sepsis. [ 15 ] [ 16 ] [ 17 ]


Negative correlation of the TTSI with the urinary excretion of certain  phthalates  suggests that  endocrine disruptors  may affect the central set point of thyroid homeostasis. [ 18 ]


Total iron-binding capacity  ( TIBC ) or sometimes  transferrin iron-binding capacity  is a medical laboratory test that measures the blood's capacity to bind  iron  with  transferrin . [ 1 ]   Transferrin  can bind two atoms of  ferric iron  (Fe 3+ ) with high affinity. It means that  transferrin  has the capacity to transport approximately from 1.40 to 1.49 mg of  iron  per gram of  transferrin  present in the blood. [ 2 ]


It is performed by  drawing blood  and measuring the maximum amount of iron that it can carry, which indirectly measures  transferrin [ 3 ]  since transferrin is the most dynamic carrier. If TIBC values are known, the transferrin concentration can be estimated with the following formulas: [ 2 ]


To measure TIBC in the blood is less expensive than a direct measurement of  transferrin . [ 4 ] [ 5 ]


The TIBC should not be confused with the  unsaturated iron-binding capacity  or  UIBC  ( LOINC   2501-5 ,  22753-8  &  35216-1 ). The UIBC is calculated by subtracting the serum iron from the TIBC. [ 6 ]


Taken together with  serum iron  and  percent transferrin saturation  clinicians usually perform this test when they are concerned about  anemia ,  iron deficiency  or  iron deficiency anemia . However, because the  liver  produces transferrin, alterations in function (such as  cirrhosis ,  hepatitis , or  liver failure ) must be considered when performing this test. It can also be an indirect test of liver function, but is rarely used for this purpose. [ 7 ]


The percent transferrin saturation (i.e., the result of the formula of serum iron/TIBC x 100) can also be a useful indicator. Studies also revealed that a transferrin saturation (serum iron concentration ÷ total iron binding capacity) over 60 percent in men and over 50 percent in women identified the presence of an abnormality in iron metabolism (hereditary hemochromatosis, heterozygotes and homozygotes) with approximately 95 percent accuracy. This finding helps in the early diagnosis of hereditary hemochromatosis, especially while serum  ferritin  still remains low. The retained iron in hereditary hemochromatosis is primarily deposited in parenchymal cells, with reticuloendothelial cell accumulation occurring very late in the disease. This is in contrast to transfusional iron overload in which iron deposition occurs first in the reticuloendothelial cells and then in parenchymal cells. This explains why ferritin levels remain relative low in hereditary hemochromatosis, while transferrin saturation is high. [ 8 ] [ 9 ]


These examples demonstrate that to properly understand a value for TIBC, one also must know the serum iron, the percent transferrin saturation, and the individual clinical situation. In modern laboratory testings, serum ferritin levels are generally accepted as reliable single indicators of the presence of iron deficiency. [ citation needed ]


Laboratories often use different units of measurement and "normal ranges" may vary by population and the laboratory techniques used. Look at the individual laboratory  reference values  to interpret a specific test (for instance, your own). [ citation needed ]  Example reference ranges are:


μg/dL = micrograms per deciliter; μmol/L = micromoles per litre.


Transferrin saturation  (TS), measured as a percentage, is a  medical  laboratory value. It is the value of  serum iron  divided by the  total iron-binding capacity [ 1 ]  of the available  transferrin , the main protein that binds iron in the blood, this value tells a clinician how much serum iron is bound. For instance, a value of 15% means that 15% of iron-binding sites of transferrin are being occupied by iron. The three results are usually reported together. A low transferrin saturation is a common indicator of  iron deficiency anemia  whereas a high transferrin saturation may indicate  iron overload  or  hemochromatosis . [ 1 ] [ 2 ]
Transferrin saturation is also called transferrin saturation index (TSI) or transferrin saturation percentage (TS%)  [ 3 ]


Studies also reveal that a transferrin saturation (serum iron concentration ÷ total iron binding capacity) over 60 percent in men and over 50 percent in women identified the presence of an abnormality in iron metabolism (hereditary hemochromatosis, heterozygotes and homozygotes) with approximately 95 percent accuracy. This finding helps in the early diagnosis of hereditary hemochromatosis, especially while serum  ferritin  still remains low. The retained iron in hereditary hemochromatosis is primarily deposited in parenchymal cells, with reticuloendothelial cell accumulation occurring very late in the disease. This is in contrast to transfusional iron overload in which iron deposition occurs first in the reticuloendothelial cells and then in parenchymal cells. This explains why ferritin levels remain relative low in hereditary hemochromatosis, while transferrin saturation is high. [ 4 ] [ 5 ]


Normal  reference ranges  are: [ citation needed ]


μg/dL = micrograms per deciliter


μmol/L = micromoles per liter


Laboratories often use different units and "normal" may vary by population and the lab techniques used. To help clinicians interpret their patients' results, laboratories are generally also required to report their normal or  reference values . [ citation needed ]


Prior to the availability of sensitive  TSH assays ,  thyrotropin releasing hormone  or  TRH stimulation tests  were relied upon for confirming and assessing the degree of suppression
in suspected  hyperthyroidism . Typically, this stimulation test involves determining basal
 TSH  levels and levels 15 to 30 minutes after an intravenous  bolus  of TRH. Normally,
TSH would rise into the concentration range measurable with less sensitive TSH assays.
Third generation TSH assays do not have this
limitation and thus TRH stimulation is generally not required when third generation TSH
assays are used to assess degree of suppression.


TRH-stimulation testing however
continues to be useful for the differential diagnosis of secondary (pituitary disorder) and tertiary (hypothalamic disorder) hypothyroidism. Patients with these conditions
appear to have physiologically inactive TSH in their circulation that is recognized by
TSH assays to a degree such that they may yield misleading, " euthyroid " TSH
results. Use and Interpretation: [ citation needed ]


• Helpful in diagnosis in patients with confusing TFTs. In primary hyperthyroidism
TSH are low and TRH administration induces little or no change in TSH levels


• In hypothyroidism due to end  organ failure , administration of TRH produces a
prompt increase in TSH


• In hypothyroidism due to  pituitary disease  (secondary hypothyroidism) administration of TRH does not produce
an increase in TSH


• In hypothyroidism due to  hypothalamic disease  (tertiary hypothyroidism), administration of TRH produces a
delayed (60–120 minutes, rather than 15–30 minutes) increase in TSH


The TRH test involves administration of a small amount of TRH intravenously, [ 1 ]  following which levels of TSH will be measured at several subsequent time points using samples of blood taken from a peripheral vein. [ citation needed ]


The test is used in the differential diagnosis of secondary and tertiary hypothyroidism.  First, blood is drawn and a baseline TSH level is measured.  Then, TRH is administered via a vein.  After 30 minutes blood is drawn again and the levels of TSH are measured and compared to the baseline. Some authors recommend additional blood sampling at 15 minutes. In children, late blood sampling at 60 to 120 minutes is necessary.
An increase in the serum TSH level following TRH administration means that the cause of the hypothyroidism is in the  hypothalamus  (tertiary hypothyroidism), i.e. the hypothalamus is not producing TRH.  Therefore, when TRH is given exogenously, TSH levels increase. If the increase in serum TSH level following TRH administration is absent or very slight, then the cause of the hypothyroidism is in the  anterior pituitary gland , i.e. the pituitary is not secreting TSH.  Therefore, even when TRH is given exogenously, TSH levels do not rise as the pituitary is diseased. [ citation needed ]


TRH may cause nausea, vomiting and some patients experience an urge to urinate. [ citation needed ]


Rarely, TRH may cause blood vessel constriction leading to  hemorrhage  in patients with pre-existing  pituitary tumors . Accordingly, patients should be advised about the risks, albeit rare, of TRH testing. [ 2 ]


Typhidot  is a  medical test  consisting of a dot  ELISA  kit [ 1 ]  that detects  IgM  and  IgG   antibodies  against the  outer membrane protein  (OMP) of the  Salmonella typhi . The typhidot test becomes positive within 2–3 days of infection and separately identifies IgM and IgG antibodies. The test is based on the presence of specific IgM and IgG antibodies to a specific 50 Kd  OMP  antigen , which is impregnated on  nitrocellulose  strips. IgM shows recent infection whereas IgG signifies remote infection. The most important  limitation of this test is that it is not quantitative and result is only positive or negative hhj8b . Whereas a detailed  Widal test  can tell the titres of specific antibodies. However both tests lack sensitivity and specificity. The Widal test is losing its value as it is labor-intensive and time-consuming. It is an  immunochromatographic  test. [ citation needed ]


The  vertical auto profile  ( VAP ) test is a  cholesterol ,  lipid  and  lipoprotein  test.


The name "VAP test" uses a technology, the vertical auto profile, to measure and report risk factors for patients who are at increased risk for cardiovascular disease that are not captured by routine cholesterol testing. Its accuracy is unaffected by triglycerides and can be performed in a non-fasting state.


The VAP test has a unique ability to identify far more areas of risk to patients than the standard lipid panel, specifically because it reports 15 separate components versus four in the standard cholesterol test. Studies report that this comprehensive test is able to identify more than twice the number of patients with lipid abnormalities than the standard lipid panel (cholesterol and triglyceride test).


The VAP test directly measures and routinely reports all five lipoprotein classes and sub-classes, including LDL, HDL, intermediate-density lipoprotein (IDL), very low density lipoprotein (VLDL), and lipoprotein (a) [Lp(a)]. In routine cholesterol testing, LDL-cholesterol (LDL-C) is not directly measured, rather it is estimated using the  Friedewald equation , is generally inaccurate when patients are not fasting because of its dependence on triglycerides levels in the calculation. Also, estimated LDL-C is falsely low when directly measured LDL-C is < 100 mg/dL or when triglycerides are elevated. The VAP technology also measures and reports LDL particle concentration (LDL-P).


The test meets the American Diabetes Association and American College of Cardiology (ADA-ACC) cholesterol guidelines for people at high risk of heart attack and stroke (including people with Type 2 diabetes mellitus). The ADA-ACC consensus statement establishes measurement and treatment guidelines for Apolipoprotein B|apoB in addition to LDL and non-HDL in high-risk patients.  The VAP test was the first cholesterol profile to comply with updated National Cholesterol Education Program ATP III recommendations for LDL measurement.


The VAP technology is currently owned and operated by VAP Diagnostics Lab, an international company.


Summary of studies:


• In a Johns Hopkins study of 1.34 million patients, up to 60% of patients assessed were misclassified using the Friedewald estimated basic lipid panel, potentially leading to undertreatment of those
patients most at risk.1


• Fasting remnant lipoprotein cholesterol is independently associated with subclinical atherosclerosis: The ELSA-BRASIL Study.2


• Smaller, denser HDL3-C levels are primarily responsible for the inverse association between HDL-C and incident CHD in this diverse group of primary prevention subjects: Jackson Heart Study & Framingham
Offspring Cohort Study.3


• Remnant Lipoprotein cholesterol (RLP-C) levels are an independent predictor of the incidents of CHD in two primary prevention cohorts: Jackson Heart Study & Framingham Offspring Cohort Study.4


• Low HDL3-C, but not HDL2-C or HDL-C, independently increased the risk for long-term hard clinical events in secondary prevention patients: TRIUMPH.5


• The apo A1 remnant ratio was a significant predictor of short and intermediate-term death/myocardial infarction risk among women over 50.6


1. Friedewald-estimated versus directly measured low-density lipoprotein cholesterol and treatment implications. J Am Coll Cardiol. 2013 Aug 20;62(8):732-9.


2. Fasting remnant lipoprotein cholesterol is independently associated with subclinical atherosclerosis: The ELSA-BRASIL Study. JACC 5 April 2016 Volume 67, Issue 12.


3. Association of high-density lipoprotein subclasses and incident coronary heart disease: The Jackson Heart and Framingham Offspring Cohort Studies. Eur J Prev Cardiol. 2016 Jan;23(1):41-9.


4. Remnant lipoprotein cholesterol and incident coronary heart disease: The Jackson Heart and Framingham Offspring Cohort Studies. J Am Heart Assoc. 2016 Apr 29;5(5).


5. HDL cholesterol subclasses, myocardial infarction, and mortality in secondary prevention: the Lipoprotein Investigators Collaborative. Eur Heart J. 2015 Jan 1;36(1):22-30.


6. A new ratio for better predicting future death/myocardial infarction than standard lipid measurements in women >50 years undergoing coronary angiography: the apolipoprotein A1 remnant ratio (Apo A1/ [VLDL₃+IDL]). Lipids Health Dis. 2013 Apr 26;12:55.


A  white blood cell differential  is a  medical laboratory  test that provides information about the types and amounts of  white blood cells  in a person's blood. The test, which is usually ordered as part of a  complete blood count  (CBC), measures the amounts of the five normal white blood cell types –  neutrophils ,  lymphocytes ,  monocytes ,  eosinophils  and  basophils  – as well as abnormal cell types if they are present. These results are reported as percentages and absolute values, and compared against  reference ranges  to determine whether the values are normal, low, or high. Changes in the amounts of white blood cells can aid in the diagnosis of many health conditions, including  viral ,  bacterial , and  parasitic  infections and  blood disorders  such as  leukemia .


White blood cell differentials may be performed by an  automated analyzer  – a machine designed to run laboratory tests – or manually, by examining  blood smears  under a microscope. The test was performed manually until white blood cell differential analyzers were introduced in the 1970s, making the  automated differential  possible. In the automated differential, a blood sample is loaded onto an analyzer, which samples a small volume of blood and measures various properties of white blood cells to produce a differential count. The  manual differential , in which white blood cells are counted on a  stained  microscope slide, is now performed to investigate abnormal results from the automated differential, or upon request by the healthcare provider. The manual differential can identify cell types that are not counted by automated methods and detect clinically significant changes in the appearance of white   blood cells.


In 1674,  Antonie van Leeuwenhoek  published the first microscopic observations of blood cells. Improvements in microscope technology throughout the 18th and 19th centuries allowed the three cellular components of blood to be identified and counted. In the 1870s,  Paul Ehrlich  invented a staining technique that could differentiate between each type of white blood cell.  Dmitri Leonidovich Romanowsky  later modified Ehrlich's stain to produce a wider range of colours, creating the  Romanowsky stain , which is still used to stain blood smears for manual differentials.


Automation of the white blood cell differential began with the invention of the  Coulter counter , the first automated  hematology analyzer , in the early 1950s. This machine used electrical impedance measurements to count cells and determine their sizes, allowing white and red blood cells to be enumerated. In the 1970s, two techniques were developed for performing automated differential counts: digital image processing of microscope slides and  flow cytometry  techniques using light scattering and cell staining. These methods remain in use on modern hematology analyzers.


The white blood cell differential is a common blood test that is often ordered alongside a  complete blood count . The test may be performed as part of a routine  medical examination ; to investigate certain symptoms, particularly those suggestive of  infection  or  hematological disorders ; [ 5 ] [ 6 ]  or to monitor existing conditions, such as blood disorders and inflammatory diseases. [ 7 ]


Five types of white blood cells are normally found in blood:  neutrophils ,  lymphocytes ,  monocytes ,  eosinophils  and  basophils . [ 8 ]   Marked shifts in the proportions of these cell types, as measured by the automated or manual differential, can indicate various health conditions. [ 9 ]  Additionally, cell types which do not normally occur in the blood, such as  blast cells , can be identified by the manual differential. These cell types may be found in blood disorders and other pathological states. [ 10 ] [ 11 ]  The manual differential can also identify changes in the appearance of white blood cells, such as   reactive lymphocytes , [ 12 ]  or features such as  toxic granulation  and  vacuolation  in neutrophils. [ 13 ]  The results of the white blood cell differential are reported as percentages and absolute values. Absolute counts are usually reported in units of cells per microliter (μL) or 10 9  cells per liter (L). [ 6 ]  The result are then compared against  reference ranges , which are defined by individual laboratories and may vary due to different patient populations and testing methods. [ 14 ]


CBC and differential testing is usually performed on  venous  or  capillary  blood. Capillary blood draws are generally used for infants and individuals whose veins are difficult to access. [ 15 ]  To prevent  clotting , the sample is drawn into a tube containing the  anticoagulant  compound  ethylenediaminetetraacetic acid  (EDTA)., [ 16 ]  meaning blood that has not been  centrifuged . [ 17 ]


In a manual differential, a stained  blood smear  is examined under a microscope and white blood cells are counted and classified based on their appearance. A manual differential is usually performed when the automated differential is flagged for review or when the healthcare provider requests it. [ 1 ] [ 9 ]  If the manual differential shows findings suggestive of certain serious conditions, such as leukemia, the blood smear is referred to a physician (generally a  hematologist  or  pathologist ) for confirmation. [ 1 ]


A blood smear is prepared by placing a drop of blood on a microscope slide and using a second slide held at an angle to spread the blood and pull it across the slide, forming a "feathered edge" consisting of a single layer of cells at the end of the smear. [ 18 ]  This may be done by hand or using an automated slide maker coupled to a hematology analyzer. The slide is treated with a Romanowsky stain, commonly  Wright's stain  or Wright-Giemsa, and examined under the microscope. The smear is examined in a systematic pattern, scanning from side to side within the feathered edge and counting cells consecutively. The differential is typically performed at 400x or 500x  magnification , but 1000x magnification may be used if abnormal cells are present. [ 19 ]  Cells are identified based on their  morphologic  features, such as the size and structure of their nucleus and the colour and texture of their cytoplasm. This allows abnormal cell types and changes in cellular appearance to be identified. [ 8 ]  In most cases, the microscopist counts 100 white blood cells, but 200 may be counted for better representation if the white blood cell count is high. [ 20 ]  The manual differential count produces percentages of each cell type, which can be multiplied by the total white blood cell count from the analyzer to derive the absolute values. [ 21 ]


The manual differential can be partially automated with digital microscopy software, [ 22 ]  which uses  artificial intelligence  to classify white blood cells from  photomicrographs  of the blood smear. [ 23 ]  However, this technique requires confirmation by manual review. [ 24 ] [ 25 ]


Because relatively few cells are counted in the manual differential, the variability is higher than in automated techniques, especially when cells are present in low amounts. [ 26 ]  For example, in a sample containing 5 percent monocytes, the manual differential results could be between 1 and 10 percent due to  sampling  variation. [ 27 ]  Additionally, cell identification is subjective and the accuracy depends on the skills of the person reading the slide. [ 26 ] [ 28 ]  Poor blood smear preparation can cause an uneven distribution of white blood cells, resulting in inaccurate counting, [ 29 ]  and improper staining can impede cell identification. [ 30 ]  Overall, manual differential counts exhibit  coefficients of variation  (CVs) ranging from 5 to 10 percent, while automated differential counts of normal neutrophils and lymphocytes have CVs of about 3 percent. [ 30 ]


In  leukemias  and other hematologic  malignancies , the lineage and genetic characteristics of white blood cells have important implications for treatment and prognosis, and the microscopic appearance of the cells is often insufficient for accurate classification. [ 31 ] [ 14 ]  In these cases, other techniques such as  immunophenotyping  by flow cytometry or special staining can be used to definitively identify the cells. [ 32 ]


Most  hematology analyzers  provide a five-part differential, enumerating neutrophils, lymphocytes, monocytes, eosinophils and basophils. Some instruments can also count immature  granulocytes  and  nucleated red blood cells . [ 33 ]   If a six-part differential is provided, the IG or immature granulocyte category consists of promyelocytes, myelocytes and metamyelocytes. [ 34 ]  Hematology analyzers measure various properties of white blood cells, such as impedance,  light scattering  parameters, and staining reactions. This data is analyzed and plotted on a  scattergram , forming distinct clusters which correspond to white blood cell types. [ 35 ]  The analyzer counts many more cells than are counted in a manual differential, resulting in improved precision. [ 26 ]  If abnormal features or cell populations that the analyzer cannot identify are present, the instrument can flag the results for manual blood smear review. [ 33 ] [ 35 ]


Common techniques used by hematology analyzers to identify cells include light scattering,  Coulter counting , and cytochemical staining techniques. Some analyzers also use  radiofrequency  analysis and  monoclonal antibody  tagging to identify cells. [ 26 ] [ 36 ]  Staining techniques used in differential analyzers include staining of  myeloperoxidase , [ 30 ]  an enzyme found in cells of  myeloid  lineage, [ 37 ]  and  nucleic acids , which are found in higher concentrations in immature cells. [ 38 ]


A small volume of blood (as low as 150 microliters) is aspirated into the analyzer, where  reagents  are applied to  lyse  red blood cells and preserve white blood cells. The sample is diluted and passed into a flow cell, which uses  hydrodynamic focusing  to isolate single cells for accurate analysis of their properties. Various cellular parameters, such as size, complexity and staining reactions, are measured and analyzed to identify cell populations. Basophils are often quantified using a reagent that lyses the cytoplasm of other white blood cells but leaves basophils intact. [ 30 ]  Samples that have abnormal results [ 1 ]  or are suspected to contain abnormal cells are flagged by the analyzer for manual blood smear review. [ 35 ]


To ensure that results from the automated analyzer are correct, quality control samples are run at least once per day. These are samples with known results that are most often provided by the instrument manufacturer. Laboratories compare their differential results to the known values to ensure the instrument is operating correctly. A moving average measurement may also be used, in which the average results for patient samples are measured at certain intervals. Assuming that the characteristics of the patient population remain roughly the same over time, the average should remain constant. Large shifts in the average value can indicate instrument problems. [ 39 ] [ 40 ]


When immature or abnormal white blood cells are present, automated differential results may be incorrect, necessitating a manual blood smear review. Overall, 10 to 25 percent of CBC samples are flagged for manual review by the analyzer. [ 41 ]  Although most abnormal samples are automatically flagged, some may be missed; [ 42 ]  conversely, analyzers may generate false positive flags when no abnormal cells are present. [ 41 ]  Hematology laboratories compensate for these issues by requiring a smear review when differential or CBC results fall outside certain numerical thresholds, regardless of the presence of analyzer flags. [ 1 ]  The  sensitivity and specificity  of analyzer flagging can be determined by comparing analyzer flags to manual differential results. [ 40 ]


The automated  basophil  count is notoriously unreliable, [ 11 ] [ 30 ]  often underestimating counts in  basophilia  and producing falsely elevated results in the presence of abnormal cells. [ 43 ]  The manual differential is therefore considered the reference method for these cells. [ 11 ] [ 44 ]


Analyzers may count nucleated red blood cells,  giant  and clumped platelets,  and red blood cells containing abnormal  hemoglobins  (such as Hemoglobin S in  sickle cell disease ) as white blood cells, leading to faulty differential results. Automated differential counts on aged specimens may be incorrect due to cellular degeneration. [ 45 ]


The neutrophil count is normally higher in newborns and pregnant women than in other groups. [ 48 ]  Outside of these conditions, increased neutrophil counts ( neutrophilia ) are associated with  bacterial infection , inflammation, and various forms of physiological stress. [ 49 ]  Neutrophil counts can become extremely high in response to some infections and inflammatory states, which is termed  leukemoid reaction  because the high  white blood cell count  mimics leukemia. [ 50 ]  Neutrophilia may also occur in  myeloproliferative disorders . [ 49 ] Neutropenia , meaning a low neutrophil count, may occur as a response to drug treatment (especially chemotherapy) [ 51 ]  or in certain infections, such as  tuberculosis  and  Gram-negative   sepsis . Neutropenia also occurs in many hematologic disorders, such as leukemia and  myelodysplastic syndrome , and in a variety of autoimmune and congenital diseases. [ 52 ]  A neutrophil count below the reference interval may be normal in individuals of certain ethnicities; this is termed  benign ethnic neutropenia . [ 53 ] [ 54 ]  Very low neutrophil counts are associated with  immunosuppression . [ 55 ]


When stimulated by infection or inflammation, neutrophils may develop abnormal features in their cytoplasm, such as  toxic granulation ,  toxic vacuolation  and  Döhle bodies . These features, which are caused by the release of  cytokines , [ 56 ]  are collectively known as toxic changes. [ 13 ]


Increased lymphocyte counts ( lymphocytosis ) can be caused by viral infections [ 58 ]  and may also occur after  splenectomy . Children have higher lymphocyte counts than adults. [ 59 ]   Chronic lymphocytic leukemia  presents with an elevated lymphocyte count and abnormal lymphocyte morphology, in which the lymphocytes have extremely dense, clumped nuclei [ 60 ]  and some cells appear  smudged  on the blood smear. [ 61 ]    Low lymphocyte counts ( lymphopenia ) may be seen in infections such as  HIV/AIDS ,  influenza  and  viral hepatitis , as well as in  protein-energy malnutrition , [ 62 ]  acute illnesses and drug reactions. [ 59 ]


In response to viral infections (especially  infectious mononucleosis ), lymphocytes may increase greatly in size, developing unusually shaped nuclei and large amounts of dark blue cytoplasm. Such cells are referred to as  reactive or atypical lymphocytes [ 12 ]  and when present they are either commented on or counted separately from normal lymphocytes in the manual differential. [ 14 ]


Increased monocyte counts ( monocytosis ) are seen in chronic infection and inflammation. Extremely high monocyte counts, as well as immature forms of monocytes, occur in  chronic myelomonocytic leukemia  and acute leukemias of monocytic origin. [ 64 ]  Monocyte counts may be decreased ( monocytopenia ) in individuals who are receiving chemotherapy as well as those with  aplastic anemia , severe burns, and AIDS. [ 65 ]


Elevated eosinophil counts ( eosinophilia ) are associated with  allergic reactions ,  parasitic infections , and asthma. [ 66 ] [ 67 ]  Eosinophil counts may be decreased in pregnancy and in response to physiological stress, inflammation or treatment with certain drugs, such as  steroids  and  epinephrine . [ 67 ]


Basophilia  and eosinophilia can occur along with other white blood cell abnormalities in  chronic myeloid leukemia  and other myeloproliferative disorders. [ 68 ]  An increased basophil count may also be seen in  hypersensitivity reactions  and after splenectomy. The basophil count may decrease during  ovulation , steroid treatment, and periods of physiological stress. [ 69 ]