If the KCT is less than 60 seconds, this suggests that the test plasma is contaminated with platelet fragments; therefore, the test is not valid. [ 4 ]


The  Kleihauer–Betke ("KB") test ,  Kleihauer–Betke ("KB") stain ,  Kleihauer test  or  acid elution test  is a  blood test  used to measure the amount of  fetal hemoglobin  transferred from a  fetus  to a mother's  bloodstream . [ 1 ]  It is usually performed on  Rh-negative  mothers to determine the required dose of  Rho(D) immune globulin  (RhIg) to inhibit formation of Rh  antibodies  in the mother and prevent  Rh disease  in future Rh-positive children. [ 2 ]  It is named after Enno Kleihauer and Klaus Betke who described it in 1957.


The KB test is the standard method of quantitating  fetal–maternal hemorrhage  (FMH). It takes advantage of the differential resistance of fetal hemoglobin to acid. A standard  blood smear  is prepared from the mother's  blood  and exposed to an acid bath. This removes adult  hemoglobin , but not fetal hemoglobin, from the red blood cells. Subsequent staining, using Shepard's method, [ 3 ]  makes fetal cells (containing fetal hemoglobin) appear rose-pink in color, while adult red blood cells are only seen as "ghosts". 2,000 cells are counted under the microscope and a percentage of fetal to maternal cells is calculated. [ 2 ]


In those with positive tests, follow up testing at a  postpartum  check should be done to rule out the possibility of a  false positive . This could be caused by a  hemoglobinopathy  in the mother which causes persistent elevation of fetal hemoglobin, e.g.  sickle cell trait . [ 2 ]


Comparison with other more expensive or technologically advanced methods such as  flow cytometry  has shown that the KB stain, like the more advanced methods, is sensitive in its detection of FMH. [ 1 ]  However, publications have shown that in comparison with flow cytometry methods, the KB Test overestimates FMH due to false positive results when F-cells are present in the maternal blood sample. Background counting errors can result in estimates of as much as 5 mL fetal  blood loss  when there actually is no such blood loss, but standard methods available in most laboratories admit an extremely low probability of the return of a false positive when more severe FMH has taken place. [ 2 ]


Thin smears are prepared from capillary blood or venous blood collected into anticoagulants such as  heparin ,  oxalate ,  citrate , or  EDTA . Smears are air dried between 10 and 60 minutes, fixed in 80 vol% ethanol for 5 min at 20-22 °C, rinsed with tap water, and air dried. Films are then immersed in the citrate-phosphate buffer for 5 minutes at 37 °C and gently agitated for about 3 minutes. Slides are rinsed with tap water, dried, and stained with Ehrlich's acid hematoxylin for 3 min, rinsed with water, and dried again. They are counterstained with erythrosine for 3 min. After a final rinse, films are dried and examined under light microscopy. [ 4 ]


Hemoglobin F cells are densely stained with erythrosine, Hemoglobin A cells appear as ghost cells, while intermediate cells are stained more or less pink. Reticulocytes containing Hemoglobin A may appear as intermediate cells and/or may show intracellular granulation.  Inclusion bodies  (Heinz bodies, precipitated α-chains or β-chains) are visible in eluted cells as compact inclusions of different size. Hemoglobin A is eluted regardless of whether it is oxyhemoglobin, methemoglobin, cyanmethemoglobin, reduced hemoglobin, or carboxyhemoglobin.


Methods developed by Schneider and Ludwig [ 5 ]  and Bartsch' are recommended. For determination of the intracellular distribution of Hemoglobin F, the semi-quantitative method of Shepard, Weatherall, and Conley' may be employed.


Normal values for Hemoglobin F cells in adults as published originally by Kleihauer were below 0.01%; in full-term newborns they are above 90%.


To determine if a positive test for FMH indicates the likely cause of fetal death, the percent of total fetal blood volume lost should be calculated, making appropriate adjustments based on the following known relationships:


These  constraints  can then be applied to yield the formula


where


An estimate of the required number of Rho(D) immune globulin vials may assume the following equations: [ 6 ]


Combining those two equations results in: [ 6 ]


This is approximately equal to:


Practically, if the number to the right of the decimal point is ≥5, it is rounded up to add one vial. [ 6 ]


Suppose that a KB stain is performed and

 T
 C
 =
 5000

 {\displaystyle TC=5000}

  total red blood cells are observed,

 F
 C
 =
 200

 {\displaystyle FC=200}

  of which are found to be fetal red blood cells.  Suppose further that the stillbirth weight of the fetus under consideration is

 F
 W
 =
 2.0
 k
 g

 {\displaystyle FW=2.0kg}

 .  Then we would conclude that the total percentage of fetal blood lost is approximately:


to five  significant digits .  We would hence conclude that the fetus under consideration lost 66.667% (two-thirds) of its blood via FMH.  Generally, stillbirth is highly probable for any value of

 P
 F
 B
 ≥
 20

 {\displaystyle PFB\geq 20}

 , particularly if the fetus abruptly loses this much blood; in this example, we would hence be likely to suspect FMH as the cause of the stillbirth.  It is important to note, however, that such a  diagnosis  is still not completely conclusive; fetuses losing large quantities of blood over long periods of time are able to compensate for this slower blood loss; since the KB stain tells us nothing with regard to the level of acuity of FMH.  This means that it is not possible to entirely correlate a positive KB stain and high

 P
 F
 B

 {\displaystyle PFB}

  with a stillbirth, though in many cases, given other information, such as known hereditary complications of pregnancy, extremely high positive  correlation coefficients

 r
 ≈
 +
 1.000

 {\displaystyle r\approx +1.000}

  between FMH and stillbirth have been observed. [ citation needed ]


Since fetal and maternal  blood cells  have the same life expectancy in the maternal bloodstream, it is possible to obtain informative results from a KB stain for a fair period of time after a stillbirth. However, if the mother and fetus are  ABO  incompatible, it is more crucial to quickly perform the KB stain following a stillbirth, as the fetal red blood cells will be eliminated from the maternal bloodstream quickly, causing the KB stain to underestimate the degree of FMH, if any. Much concern has been raised in the literature concerning false positives when sampling is done after delivery. In general this is not a problem. Delivery does result in higher frequency of detection of micro-hemorrhages but this should not confound interpretation of FMH as a possible cause of stillbirth. It is not necessary to draw the sample before induction, onset of labor, delivery, placental delivery etc. despite what some published literature purports. However, if Caesarean section is to be used, failure to draw the sample prior to that will result in a 2% false positive rate. [ citation needed ]


Finally, anything which causes persistence of fetal hemoglobin in maternal blood cells will make interpretation much trickier. Certain  hemoglobinopathies , the most common of which is sickle cell trait, do this. Overall, somewhere around 1–3% of the time this could result in false interpretation.


An article published in 2004 concluded that a Kleihauer-Betke (KB) test is necessary in all cases of maternal trauma, as clinical evaluation is not sensitive enough for determination of risk of pre-term labour. It accurately predicts the risk of preterm labor after maternal trauma whereas the article concluded that clinical assessment does not. With a negative KB test, posttrauma electronic fetal monitoring duration may be limited safely. With a positive KB test, the significant risk of pre-term labour mandates detailed monitoring. KB testing has important advantages to all maternal trauma victims, regardless of Rh status. [ 7 ]


A  latex fixation test , also called a  latex agglutination assay  or  test  ( LA assay  or  test ), is an  assay  used clinically in the identification and  typing  of many important  microorganisms . These tests use the patient's  antigen - antibody  immune response. This response occurs when the body detects a  pathogen  and forms an antibody specific to an identified antigen (a protein configuration) present on the surface of the pathogen. [ citation needed ]


Agglutination tests, specific to a variety of pathogens, can be designed and manufactured for clinicians by coating  microbeads  of  latex  with pathogen-specific antigens or antibodies.  In performing a test, laboratory clinicians will mix a patient's  cerebrospinal fluid ,  serum  or  urine  with the coated latex particles in  serial dilutions  with  normal saline  (important to avoid the  prozone effect ) and observe for  agglutination  (clumping). Agglutination of the beads in any of the dilutions is considered a positive result, confirming either that the patient's body has produced the pathogen-specific antibody (if the test supplied the antigen) or that the specimen contains the pathogen's antigen (if the test supplied the antibody). Instances of  cross-reactivity  (where the antibody sticks to another antigen besides the antigen of interest) can lead to confusing results.


Agglutination techniques are used to detect antibodies produced in response to a variety of  viruses  and  bacteria , as well as  autoantibodies , which are produced against the self in  autoimmune diseases . For example, assays exist for  rubella virus ,  rotavirus , and  rheumatoid factor , and an excellent LA test is available for  cryptococcus . [ 1 ]  Agglutination techniques are also used in definitive diagnosis of  group A streptococcal infection .


A  lipid profile  or  lipid panel  is a  panel  of  blood tests  used to find abnormalities in  blood lipid  ( such as  cholesterol  and  triglycerides ) concentrations. [ not verified in body ]  The results of this test can identify certain  genetic diseases  and can determine approximate risks for  cardiovascular disease , certain forms of  pancreatitis , and other diseases.


Lipid panels are usually ordered as part of a  physical exam , along with other panels such as the  complete blood count  (CBC) and  basic metabolic panel  (BMP). [ not verified in body ]


A lipid profile report typically includes: [ 1 ]


LDL is not usually actually measured, but calculated from the other three using the  Friedewald equation . [ 1 ]  A laboratory can optionally calculate the two extra values from the report:


Recommendations for cholesterol testing come from the Adult Treatment Panel (ATP) III guidelines, and are based on many large clinical studies, such as the  Framingham Heart Study . [ citation needed ]


For healthy adults with no cardiovascular risk factors, the ATP III guidelines recommend screening once every five years. [ 2 ]  A lipid profile may also be ordered at regular intervals to evaluate the success of lipid-lowering drugs such as  statins . [ citation needed ]


In the pediatric and adolescent population, lipid testing is not routinely performed. However, the  American Academy of Pediatrics  and the  National Heart, Lung, and Blood Institute  (NHLBI) recommend that children aged 9–11 be screened once for severe cholesterol abnormalities. [ 3 ]  This screening can be valuable to detect  genetic diseases  such as  familial hypercholesterolemia  that can be lethal if not treated early. [ citation needed ]


Traditionally, most laboratories have required patients to fast for 9–12 hours before screening. However, studies have questioned the utility of  fasting  before lipid panels, and some diagnostic labs routinely accept non-fasting samples. [ 4 ] [ 5 ] [ 6 ] [ 7 ]


Typically the laboratory measures only three quantities: total cholesterol; HDL; Triglycerides. A typical procedure used by  NHANES  2004 uses the following measurement methods: [ 8 ]


From these three data LDL may be calculated. According to Friedewald's equation: [ 9 ]


Other calculations of LDL from those same three data have been proposed which yield some significantly different results. [ 10 ]


VLDL can be defined as the total cholesterol that is neither HDL nor LDL. With that definition, Friedewald's equation [ 9 ]  yields:


The alternative calculations mentioned above may yield significantly different values for VLDL.


The Friedewald method is reasonably reliable for the majority of patients, but is notably inaccurate in patients with  hypertriglyceridemia  (> 400 mg/dL or 4.5 mmol/L). It also underestimates LDL-C in patients with low LDL-C (< 25 mg/dL or 0.6 mmol/L). It does not take into account  intermediate-density lipoprotein . [ 1 ]


A "Martin/Hopkins" variation that takes into how triglycerides-to-VLDL ratio tends to vary with other parameters appears more reliable and accurate. [ 11 ] [ 12 ] [ 13 ]


Every part of the lipid panel can be measured directly using ultracentrifugation, which is the  gold standard . [ 1 ]   This type of measurement involves no errors from estimation and can also measure  IDL -C and  Lp(a) -C levels. Fully direct measurement is more costly, however. [ 14 ]


Laboratories may also use proprietrary tests for "direct chemical LDL-C" which require no prior separation by centrifugation. These tests are not yet standardized in US and Europe and lack validation. [ 13 ]  A specific version of the test seems popular in Japan, however. [ 15 ]  A number of other LDL-C determination methods have been used in the past or have been proposed for future use. [ 16 ]


This test is used to identify  dyslipidemia  (various disturbances of cholesterol and triglyceride levels), many forms of which are recognized risk factors for  cardiovascular disease  and rarely  pancreatitis .


A total cholesterol reading can be used to assess an individual's risk for heart disease; however, it should not be relied upon as the only indicator. The individual components that make up total cholesterol reading— LDL ,  HDL , and  VLDL —are also important in measuring risk. [ citation needed ]


For instance, someone's total cholesterol may be high, but this may be due to very high HDL ("good cholesterol") cholesterol levels,—which can help prevent heart disease (the test is mainly concerned with high LDL, or "bad cholesterol" levels). So, while a high total cholesterol level may help give an indication that there is a problem with cholesterol levels, the components that make up total cholesterol should also be measured. [ citation needed ]


Live blood analysis  ( LBA ),  live cell analysis ,  Hemaview  or  nutritional blood analysis  is the use of high-resolution  dark field microscopy  to observe live blood cells. Live blood analysis is promoted by some  alternative medicine  practitioners, who assert that it can diagnose a range of diseases. There is no  scientific evidence  that live blood analysis is reliable or effective, and it has been described as a fraudulent means of convincing people that they are ill and should purchase  dietary supplements . [ 1 ] [ 2 ] [ 3 ]


Live blood analysis is not accepted in laboratory practice and its validity as a laboratory test has not been established. [ 4 ]  There is no scientific evidence for the validity of live blood analysis, [ 4 ]  it has been described as a pseudoscientific, bogus and fraudulent medical test, [ 5 ] [ 6 ]  and its practice has been dismissed by the medical profession as  quackery . [ 7 ]  The field of live blood microscopy is  unregulated , there is no training requirement for practitioners and no recognised qualification, no recognised medical validity to the results, and proponents have made false claims about both medical  blood pathology testing  and their own services, which some have refused to amend when instructed by the  Advertising Standards Authority . [ 8 ]


It has its origins in the now-discarded theories of  pleomorphism  promoted by  Günther Enderlein , notably in his 1925 book  Bakterien-Cyklogenie .


In January 2014 prominent live blood proponent and teacher  Robert O. Young  was arrested and charged for practising medicine without a license, [ 9 ]  and in March 2014 Errol Denton, a former student of his, a UK live blood practitioner, was convicted on nine counts in a rare prosecution under the  Cancer Act 1939 , [ 7 ]  followed in May 2014 by another former student, Stephen Ferguson. [ citation needed ]


Proponents claim that live blood analysis provides information "about the state of the immune system, possible vitamin deficiencies, amount of toxicity, pH and mineral imbalance, areas of concern and weaknesses, fungus and yeast." Some even claim it can "spot cancer and other degenerative immune system diseases up to two years before they would otherwise be detectable" or say they can diagnose "lack of oxygen in the blood, low trace minerals, lack of exercise, too much alcohol or yeast, weak kidneys, bladder or spleen." [ 1 ]  Practitioners include alternative medicine providers such as nutritionists, herbologists, naturopaths, and chiropractors. [ 4 ]


Dark field microscopy  is useful to enhance contrast in unstained samples, but live blood analysis is not proven to be useful for any of its claimed indications. Two journal articles published in the alternative medical literature found that darkfield microscopy seemed unable to detect cancer, and that live blood analysis lacked reliability,  reproducibility , and  sensitivity and specificity . [ 10 ] [ 11 ]   Edzard Ernst , professor of  complementary medicine  at the  University of Exeter  and  University of Plymouth , notes: "No credible scientific studies have demonstrated the reliability of LBA for detecting any of the above conditions." Ernst describes live blood analysis as a "fraudulent" means of convincing patients to buy  dietary supplements . [ 1 ]
 Quackwatch  has been critical of live blood analysis, noting dishonesty in the claims brought forward by its proponents. [ 12 ]  The alternative medicine popularizer  Andrew Weil  dismissed live blood analysis as "completely bogus", writing: "Dark-field microscopy combined with live blood analysis may sound like cutting-edge science, but it's old-fashioned hokum. Don't buy into it." [ 3 ]


There are several common diagnoses by the LBA practitioners that are actually based on observation of artifacts normally found in microscopy, and ignorance of basic biological science: [ 13 ] [ 14 ]


Acid in the blood:  When the  red blood cells  stack on top of one another and appear like stacks of coins, it is called ' rouleaux ' formation.   By observation of the rouleaux, the LBA practitioners diagnose 'acid in the blood', while other practitioners suggest a weak pancreas.  Rouleaux of red blood cells under the microscope is an artifact which occurs when the blood sample at the edge of the coverslip [ 15 ]  starts to dry out; where a large number of red blood cells clump together; or when the blood starts to clot when contacted with the glass.  These artifacts are observed in only small, selected areas on the slide, while near the center of the slide the red blood cells are free floating.  Blood  acidosis  is a severe illness and can not be diagnosed by observation of blood, nor treated by dietary supplements. [ 13 ] [ 14 ]


In 1996, the Pennsylvania Department of Laboratories informed three Pennsylvania chiropractors that Infinity2's "Nutritional Blood Analysis" could not be used for diagnostic purposes unless they maintain a laboratory that has both state and federal certification for complex testing. [ 16 ]


In 2001, the  Health and Human Services  Office of the Inspector General issued a report on regulation of "unestablished laboratory tests" that focused on live blood cell analysis and the difficulty of regulating unestablished tests and laboratories. [ 4 ]


In 2002, an Australian  naturopath  was convicted and fined for  falsely claiming  that he could diagnose illness using live blood analysis [ 17 ]  after the death of a patient. He was acquitted of  manslaughter . He subsequently changed his name and was later banned from practice for life. [ 18 ]


In 2005, the  Rhode Island  Department of Health ordered a  chiropractor  to stop performing live blood analysis. An attorney for the State Board of Examiners in Chiropractic Medicine described the test as "useless" and a "money-making scheme... The point of it all is apparently to sell nutritional supplements." A state medical board official said that live blood analysis has no discernible value, and that the public "should be very suspicious of any practitioner who offers this test." [ 2 ]


In 2011, the UK  General Medical Council  suspended a doctor's licence to practise after he used live blood analysis to diagnose patients with  Lyme disease . The doctor accepted he had been practising "bad medicine". [ 19 ]


In 2013, following several  Advertising Standards Authority  adjudications  [ 20 ]  against claims made by LBA practitioners, the  Committee of Advertising Practice  added new guidelines to their  AdviceOnline  database advising what LBA marketers may claim in their advertising material. These state that "CAP is yet to see any evidence for the efficacy of this therapy which, without rigorous evidence to support it, should be advertised on an availability-only platform." [ 21 ]


One of these practitioners, Errol Denton, who practised out of a serviced office in Harley Street, was prosecuted in December 2013 under the  Cancer Act 1939 , and chose to use a  Freeman on the Land  defence. [ 22 ]  On March 20, 2014, he was convicted on nine counts under the  Cancer Act 1939  and fined £9,000 plus around £10,000 in costs. [ 7 ] [ 23 ]  In April 2018, Denton was further convicted of two counts of "engaging in unfair commercial practice" and one of "selling food not of the quality demanded", for selling a bottle of  colloidal silver  drink to an undercover  trading standards  officer in February 2016, after examining a drop of her blood and from it claiming that she had dislocated her shoulder. [ 24 ] [ 25 ]  He was made the subject of a  Criminal Behaviour Order , fined £2,250, and ordered pay £15,000 in costs. [ 26 ]


Liver function tests  ( LFTs  or  LFs ), also referred to as a hepatic panel or liver panel, are groups of blood tests that provide information about the state of a patient's  liver . [ 1 ]  These tests include  prothrombin time  (PT/INR),  activated partial thromboplastin time  (aPTT),  albumin ,  bilirubin  (direct and indirect), and others. The liver transaminases  aspartate transaminase (AST or SGOT)  and  alanine transaminase (ALT or SGPT)  are useful biomarkers of liver injury in a patient with some degree of intact liver function. [ 2 ] [ 3 ] [ 4 ]


Most  liver diseases  cause only mild symptoms initially, but these diseases must be detected early. Hepatic (liver) involvement in some diseases can be of crucial importance.  This testing is performed on a patient's blood sample. Some tests are associated with functionality (e.g., albumin), some with cellular integrity (e.g.,  transaminase ), and some with conditions linked to the biliary tract ( gamma-glutamyl transferase  and  alkaline phosphatase ). Because some of these tests do not measure function, it is more accurate to call these liver chemistries or liver tests rather than liver function tests. [ 5 ]


Several biochemical tests are useful in the evaluation and management of patients with hepatic dysfunction. These tests can be used to detect the presence of liver disease. They can help distinguish among different types of liver disorders, gauge the extent of known liver damage, and monitor the response to treatment. Some or all of these measurements are also carried out (usually about twice a year for routine cases) on individuals taking certain medications, such as anticonvulsants, to ensure that these medications are not adversely impacting the person's liver. [ citation needed ]


Standard liver tests for assessing liver damage include alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP). Bilirubin may be used to estimate the excretory function of the liver and coagulation tests and albumin can be used to evaluate the metabolic activity of the liver. [ 6 ]


Although example reference ranges are given, these will vary depending on method of analysis used at the administering laboratory, as well as age, gender, ethnicity, and potentially unrelated health factors. Individual results should always be interpreted using the reference range provided by the laboratory that performed the test. [ 7 ]


Measurement of total  bilirubin  includes both unconjugated (indirect) and conjugated (direct) bilirubin. Unconjugated bilirubin is a breakdown product of  heme  (a part of  hemoglobin  in red blood cells). The liver is responsible for clearing the blood of unconjugated bilirubin, by 'conjugating' it (modified to make it water-soluble) through an enzyme named  UDP-glucuronyl-transferase . When the total bilirubin level exceeds 17 μmol/L, it indicates liver disease. When total bilirubin levels exceed 40 μmol/L, bilirubin deposition at the sclera, skin, and mucous membranes will give these areas a yellow colour, thus it is called  jaundice . [ 6 ]


The increase in predominantly unconjugated bilirubin is due to overproduction, reduced hepatic uptake of the unconjugated bilirubin and reduced conjugation of bilirubin. Overproduction can be due to the reabsorption of a  haematoma  and ineffective  erythropoiesis  leading to increased red blood cell destruction.  Gilbert's syndrome  and  Crigler–Najjar syndrome  have defects in the  UDP-glucuronyl-transferase  enzyme, affecting bilirubin conjugation. [ 6 ]


The degree of rise in conjugated bilirubin is directly proportional to the degree of hepatocyte injury. Viral  hepatitis  can also cause the rise in conjugated bilirubin. In parenchymal liver disease and incomplete extrahepatic obstruction, the rise in conjugated bilirubin is less than the complete  common bile duct  obstruction due to malignant causes. In  Dubin–Johnson syndrome , a mutation in  multiple drug-resistance protein 2 (MRP2)  causes a rise in conjugated bilirubin. [ 6 ]


In  acute appendicitis , total bilirubin can rise from 20.52 μmol/L to 143 μmol/L. In pregnant women, the total bilirubin level is low in all three trimesters. [ 6 ]


The measurement of bilirubin levels in the newborns is done through the use of bilimeter or transcutanoeus bilirubinometer instead of performing LFTs. When the total serum bilirubin increases over 95th percentile for age during the first week of life for high risk babies, it is known as hyperbilirubinemia of the newborn ( neonatal jaundice ) and requires  light therapy  to reduce the amount of bilirubin in the blood. Pathological jaundice in newborns should be suspected when the serum bilirubin level rises by more than 5 mg/dL per day, serum bilirubin more than the physiological range, clinical jaundice more than 2 weeks, and conjugated bilirubin (dark urine staining clothes).  Haemolytic jaundice  is the commonest cause of pathological jaundice. Those babies with Rh hemolytic disease, ABO incompatibility with the mother,  Glucose-6-phosphate dehydrogenase  (G-6-PD) deficiency and minor blood group incompatibility are at increased risk of getting haemolytic jaundice. [ 9 ]


Apart from being found in high concentrations in the liver, ALT is found in the kidneys, heart, and muscles. It catalyses the  transamination  reaction, and only exists in a cytoplasmic form. Any kind of liver injury can cause a rise in ALT. A rise of up to 300 IU/L is not specific to the liver, but can be due to the damage of other organs such as the kidneys or muscles. When ALT rises to more than 500 IU/L, causes are usually from the liver. It can be due to hepatitis, ischemic liver injury, and toxins that causes liver damage. The ALT levels in  hepatitis C  rises more than in hepatitis A and B. Persistent ALT elevation more than 6 months is known as  chronic hepatitis .  Alcoholic liver disease ,  non-alcoholic fatty liver disease  (NAFLD), fat accumulation in liver during childhood obesity,  steatohepatitis  (inflammation of fatty liver disease) are associated with a rise in ALT. Rise in ALT is also associated with reduced insulin response, reduced glucose tolerance, and increased free  fatty acids  and  triglycerides . Bright liver syndrome (bright liver on ultrasound suggestive of fatty liver) with raised ALT is suggestive of  metabolic syndrome . [ 6 ]


In pregnancy, ALT levels would rise during the second trimester. In one of the studies, measured ALT levels in pregnancy-related conditions such as  hyperemesis gravidarum  was 103.5 IU/L,  pre-eclampsia  was 115,  HELLP syndrome  was 149. ALT levels would reduce by greater than 50% in three days after child delivery. Another study also shows that  caffeine  consumption can reduce the risk of ALT elevation in those who consume alcohol, overweight people, impaired glucose metabolism, and viral hepatitis. [ 6 ]


AST exists in two  isoenzymes  namely mitochondrial form and cytoplasmic form. It is found in highest concentration in the liver, followed by heart, muscle, kidney, brain, pancreas, and lungs. [ 10 ]  This wide range of AST containing organs makes it a relatively less specific indicator of liver damage compared to ALT. An increase of mitochondrial AST in bloods is highly suggestive of tissue  necrosis  in  myocardial infarction  and chronic liver disease. More than 80% of the liver AST activity are contributed by mitochondrial form of the isoenzymes, while the circulating AST in blood are contributed by cytoplasmic form of AST. AST is especially markedly raised in those with  liver cirrhosis . [ 6 ]  AST can be released from a variety of other tissues and if the elevation is less than two times the normal AST, no further workup needs to be performed if a patient is proceeding to surgery. [ citation needed ]


In certain pregnancy related conditions such as hyperemesis gravidarum, AST can reach as high as 73 IU/L, 66 IU/L in pre-eclampsia, and 81 IU/L in HELLP syndrome. [ 6 ]


The AST/ALT ratio increases in liver functional impairment. In alcoholic liver disease, the mean ratio is 1.45, and mean ratio is 1.33 in post necrotic liver cirrhosis. Ratio is greater than 1.17 in viral cirrhosis, greater than 2.0 in alcoholic hepatitis, and 0.9 in non-alcoholic hepatitis. Ratio is greater than 4.5 in  Wilson disease  or  hyperthyroidism . [ 6 ]


Alkaline phosphatase  (ALP) is an enzyme in the cells lining the  biliary ducts  of the liver.  It can also be found on the mucosal epithelium of the small intestine,  proximal convoluted tubule  of the kidneys, bone, liver, and placenta. It plays an important role in lipid transposition in small intestines and calcification of bones. 50% of all the serum ALP activities in blood are contributed by bone. Acute viral hepatitis usually has normal or increased ALP. For example, hepatitis A has increased ALP due to  cholestasis  (impaired bile formation or bile flow obstruction) and would have the feature of prolonged itching. Other causes include: infiltrative liver diseases, granulomatous liver disease, abscess,  amyloidosis  of the liver and  peripheral arterial disease . Mild elevation of ALP can be seen in liver cirrhosis, hepatitis, and  congestive cardiac failure . Transient  hyperphosphataemia  is a benign condition in  infants , and can reach normal level in 4 months. In contrast, low levels of ALP is found in  hypothyroidism ,  pernicious anemia ,  zinc deficiency , and  hypophosphatasia . [ 6 ]


ALP activity is significantly increased in the third trimester of  pregnancy . [ 11 ]  This is due to increased synthesis from the  placenta  as well as increased synthesis in the  liver  induced by large amounts of estrogens. [ 11 ] [ 12 ] [ 13 ]  Levels in the third trimester can be as much as 2-fold greater than in non-pregnant women. [ 11 ]  As a result, ALP is not a reliable marker of hepatic function in pregnant women. [ 11 ]  In contrast to ALP, levels of ALT, AST, GGT, and  lactate dehydrogenase  are only slightly changed or largely unchanged during pregnancy. [ 11 ]   Bilirubin  levels are significantly decreased in pregnancy. [ 11 ]


In pregnancy conditions such as hyperemesis gravdirum, ALP levels can reach 215 IU/L, meanwhile, in pre-eclampsia, ALP can reach 14 IU/L, and in HELLP syndrome ALP levels can reach 15 IU/L. [ 6 ]


GGT is a microsomal enzyme found in hepatocytes, biliary epithelial cells, renal tubules, pancreas, and intestines. It helps in  glutathione  metabolism by transporting peptides across the cell membrane. Much like ALP, GGT measurements are usually elevated if cholestasis is present. [ 10 ]  In acute viral hepatitis, the GGT levels can peak at 2nd and 3rd week of illness, and remained elevated at 6 weeks of illness. GGT is also elevated in 30% of the hepatitis C patients. GGT can increase by 10 times in alcoholism. GGT can increase by 2 to 3 times in 50% of the patients with non-alcoholic liver disease. When GGT levels is elevated, the  triglyceride  level is elevated also. With insulin treatment, the GGT level can reduce. Other causes of elevated GGT are: diabetes mellitus,  acute pancreatitis , myocardial infarction,  anorexia nervosa ,  Guillain–Barré syndrome , hyperthyroidism, obesity and  myotonic dystrophy . [ 6 ]


In pregnancy conditions GGT activity is reduced in 2nd and 3rd trimesters. In hyperemesis gravidarum, GGT level value can reach 45 IU/L, 17 IU/L in pre-eclampsia, and 35 IU/L in HELPP syndrome. [ 6 ]


Albumin  is a protein made specifically by the liver, and can be measured cheaply and easily. It is the main constituent of total protein (the remaining constituents are primarily  globulins ). Albumin levels are decreased in chronic liver disease, such as  cirrhosis . It is also decreased in  nephrotic syndrome , where it is lost through the urine.  The consequence of low albumin can be edema since the intravascular  oncotic pressure  becomes lower than the extravascular space. An alternative to albumin measurement is prealbumin, which is better at detecting acute changes (half-life of albumin and prealbumin is about 2 weeks and about 2 days, respectively). [ 14 ] [ 15 ]


Other tests are requested alongside LFT to rule out specific causes.


5'  Nucleotidase  (5NT) is a  glycoprotein  found throughout the body, in the cytoplasmic membrane, catalyzing the conversion to inorganic phosphates from nucleoside-5-phosphate. Its level is raised in conditions such as obstructive jaundice, parenchymal liver disease, liver metastases, and bone disease. [ 6 ]


Serum NT levels are higher during 2nd and 3rd trimesters in pregnancy. [ 6 ]


Ceruloplasmin is an  acute phase protein  synthesized in the liver. It is the carrier of the copper ion. Its level is increased in infections,  rheumatoid arthritis , pregnancy, non-Wilson liver disease and obstructive jaundice. In Wilson disease, the ceruloplasmin level is depressed which lead to copper accumulation in body tissues. [ 6 ]


Alpha-fetoprotein (AFP) is significantly expressed in foetal liver. However, the mechanism that led to the suppression of AFP synthesis in adults is not fully known. Exposure of the liver to cancer-causing agents and arrest of liver maturation in childhood can lead to the rise in AFP. AFP can reach until 400–500 μg/L in  hepatocellular carcinoma . AFP concentration of more than 400 μg/L is associated with greater tumour size, involvement of both lobes of liver, portal vein invasion and a lower median survival rate. [ 6 ]


The liver is responsible for the production of the vast majority of  coagulation  factors. In patients with liver disease,  international normalized ratio  (INR) can be used as a marker of liver synthetic function as it includes  factor VII , which has the shortest half life (2–6 hours) of all coagulation factors measured in INR. An elevated INR in patients with liver disease, however, does not necessarily mean the patient has a tendency to bleed, as it only measures procoagulants and not anticoagulants. In liver disease the synthesis of both are decreased and some patients are even found to be hypercoagulable (increased tendency to clot) despite an elevated INR. In liver patients, coagulation is better determined by more modern tests such as  thromboelastogram  (TEG) or thomboelastrometry (ROTEM). [ citation needed ]


Prothrombin time (PT) and its derived measures of prothrombin ratio (PR) and INR are measures of the  extrinsic pathway  of  coagulation . This test is also called "ProTime INR" and "INR PT". They are used to determine the clotting tendency of blood, in the measure of  warfarin  dosage, liver damage, and  vitamin K  status. [ 16 ]


The serum glucose test, abbreviated as "BG" or "Glu", measures the liver's ability to produce glucose ( gluconeogenesis ); it is usually the last function to be lost in the setting of  fulminant liver failure . [ 17 ]


Lactate dehydrogenase  (LDH) is found in many body tissues, including the liver. Elevated levels of LDH may indicate liver damage. [ 18 ]  LDH isotype-1 (or cardiac) is used for estimating damage to cardiac tissue, although troponin and creatine kinase tests are preferred. [ 19 ]


Macro-creatine kinase (macro-CK)  is a macroenzyme, an enzyme of high  molecular weight  and prolonged  half-life  found in human  serum . [ 1 ]  It is one of the most common macroenzymes. [ 1 ]  Macro-CK type 1 is a  complex  formed by one of the  creatine kinase   isoenzyme  types, typically CK-BB, and  antibodies ; typically  IgG , sometimes  IgA , rarely  IgM . Macro-CK type 2 is formed from  mitochondrial  CK polymer. [ 2 ]


Macro-CK type 1 has been associated with autoimmune and other chronic conditions. [ 1 ]  Macro-CK type 2 has been associated with  malignancy . [ 1 ]


Macro-CK has been implicated as a source of interference in interpretation of medical labs. [ 3 ]


Magnetic immunoassay  ( MIA ) is a type of diagnostic  immunoassay  using magnetic beads as labels in lieu of conventional enzymes ( ELISA ), radioisotopes ( RIA ) or fluorescent moieties ( fluorescent immunoassays ) [ 1 ]  to detect a specified  analyte . MIA involves the specific binding of an antibody to its antigen, where a magnetic label is conjugated to one element of the pair. The presence of magnetic beads is then detected by a magnetic reader ( magnetometer ) which measures the magnetic field change induced by the beads. The signal measured by the magnetometer is proportional to the analyte (virus, toxin, bacteria, cardiac marker, etc.) concentration in the initial sample.


Magnetic beads are made of nanometric-sized iron oxide particles encapsulated or glued together with polymers. These magnetic beads range from 35 nm up to 4.5 μm. The component  magnetic nanoparticles  range from 5 to 50 nm and exhibit a unique quality referred to as  superparamagnetism  in the presence of an externally applied magnetic field. [ 2 ]  First discovered by Frenchman  Louis Néel , Nobel Physics Prize winner in 1970, this superparamagnetic quality has already been used for medical application in Magnetic Resonance Imaging (MRI) and in biological separations, but not yet for labeling in commercial diagnostic applications.
Magnetic labels exhibit several features very well adapted for such applications: [ citation needed ]


Magnetic Immunoassay (MIA) is able to detect select molecules or pathogens through the use of a magnetically tagged antibody. Functioning in a way similar to that of an ELISA or Western Blot, a two-antibody binding process is used to determine concentrations of analytes. MIA uses antibodies that are coating a magnetic bead. These anti-bodies directly bind to the desired pathogen or molecule and the magnetic signal given off the bound beads is read using a magnetometer. The largest benefit this technology provides for immunostaining is that it can be conducted in a liquid medium, where methods such as ELISA or Western Blotting require a stationary medium for the desired target to bind to before the secondary antibody (such as HRP [Horse Radish Peroxidase]) is able to be applied. Since MIA can be conducted in a liquid medium a more accurate measurement of desired molecules can be performed in the model system. Since no isolation must occur to achieve quantifiable results users can monitor activity within a system. Getting a better idea of the behavior of their target.  [ citation needed ]


The manners in which this detection can occur are very numerous. The most basic form of detection is to run a sample through a gravity column that contains a polyethylene matrix with the secondary anti-body. The target compound binds to the antibody contained in the matrix, and any residual substances are washed out using a chosen buffer. The magnetic antibodies are then passed through the same column and after an incubation period, any unbound antibodies are washed out using the same method as before. The reading obtained from the magnetic beads bound to the target which is captured by the antibodies on the membrane is used to quantify the target compound in solution. [ citation needed ]


Also, because it is so similar in methodology to ELISA or Western Blot the experiments for MIA can be adapted to use the same detection if the researcher wants to quantify their data in a similar manner.


A simple instrument can detect the presence and measure the total magnetic signal of a sample, however, the challenge of developing an effective MIA is to separate naturally occurring magnetic background (noise) from the weak magnetically labeled target (signal). Various approaches and devices have been employed to achieve a meaningful signal-to-noise ratio (SNR) for bio-sensing applications: [ citation needed ]


But improving SNR often requires a complex instrument to provide repeated scanning and extrapolation through data processing, or precise alignment of target and sensor of miniature and matching size. Beyond this requirement, MIA that exploits the non-linear magnetic properties of magnetic labels [ citation needed ]  can effectively use the intrinsic ability of a magnetic field to pass through plastic, water,  nitrocellulose , and other materials, thus allowing for true volumetric measurements in various immunoassay formats. Unlike conventional methods that measure the susceptibility of superparamagnetic materials, a MIA-based on non-linear magnetization eliminates the impact of linear dia- or paramagnetic materials such as sample matrix, consumable plastics and/or nitrocellulose. Although the intrinsic magnetism of these materials is very weak, with typical susceptibility values of –10 −5  (dia) or +10 −3  (para), when one is investigating very small quantities of superparamagnetic materials, such as nanograms per test, the background signal generated by ancillary materials cannot be ignored. In MIA based on non-linear magnetic properties of magnetic labels the beads are exposed to an alternating magnetic field at two frequencies, f1 and f2. In the presence of non-linear materials such as superparamagnetic labels, a signal can be recorded at combinatorial frequencies, for example, at f = f1 ± 2×f2. This signal is exactly proportional to the amount of magnetic material inside the reading coil.


This technology makes magnetic immunoassay possible in a variety of formats such as:


It was also described for in vivo applications [ 4 ]  and for multiparametric testing.


MIA is a versatile technique that can be used for a wide variety of practices.


Currently it has been used to detect viruses in plants to catch pathogens that would normally devastate crops such as  Grapevine fanleaf virus , [ 5 ] [ full citation needed ]  and  Potato virus X . Its adaptations now include portable devices that allow the user to gather sensitive data in the field. [ 6 ] [ full citation needed ]


MIA can also be used to monitor therapeutic drugs. A case report of a 53-year-old [ 7 ] [ full citation needed ]  kidney transplant patient details how the doctors were able to alter the quantities of the therapeutic drug.


Malaria antigen detection tests  are a group of commercially available  rapid diagnostic tests  of the  rapid antigen test  type that allow quick  diagnosis  of  malaria  by people who are not otherwise skilled in traditional laboratory techniques for diagnosing malaria or in situations where such equipment is not available. There are currently over 20 such tests commercially available (WHO product testing 2008). The first malaria  antigen  suitable as target for such a test was a soluble  glycolytic   enzyme   Glutamate dehydrogenase . [ 1 ] [ 2 ] [ 3 ]
None of the rapid tests are currently as sensitive as a thick  blood film , nor as cheap. A major drawback in the use of all current  dipstick  methods is that the result is essentially qualitative. In many endemic areas of  tropical Africa , however, the quantitative assessment of parasitaemia is important, as a large percentage of the population will test positive in any qualitative assay.


Malaria is a curable  disease  if the patients have access to early diagnosis and prompt  treatment . Antigen-based  rapid diagnostic tests   (RDTs)  have an important role at the periphery of health services capability because many rural  clinics  do not have the ability to diagnose malaria on-site due to a lack of microscopes and trained  technicians  to evaluate blood films. Furthermore, in regions where the disease is not  endemic , laboratory technologists have very limited experience in detecting and identifying malaria parasites. An ever increasing numbers of travelers from temperate areas each year visit  tropical countries  and many of them return with a malaria infection. The RDT tests are still regarded as complements to conventional  microscopy  but with some improvements it may well replace the  microscope . The tests are simple and the procedure can be performed on the spot in field conditions. These tests use finger-stick or venous  blood , the completed test takes a total of 15–20 minutes, and a  laboratory  is not needed.  The threshold of detection by these rapid diagnostic tests is in the range of 100 parasites/μL of blood compared to 5 by thick film microscopy. [ 4 ] [ 5 ] [ 6 ]


An accurate diagnosis is becoming more and more important, in view of the increasing resistance of  Plasmodium falciparum  and the high price of alternatives to  chloroquine . The enzyme pGluDH does not occur in the host  red blood cell  and was recommended as a marker enzyme for  Plasmodium  species by Picard-Maureau et al. in 1975. [ 7 ]  The malaria marker enzyme test is suitable for routine work and is now a standard test in most departments dealing with malaria. Presence of pGluDH is known to represent parasite viability [ 2 ]  and a rapid diagnostic test using pGluDH as antigen would have the ability to differentiate live from dead organisms. A complete RDT with pGluDH as antigen has been developed in  China  and is now undergoing  clinical trials . [ 3 ]
GluDHs are ubiquitous enzymes that occupy an important branch-point between carbon and nitrogen metabolism. Both  nicotinamide adenine dinucleotide  (NAD)  [EC  1.4.1.2] and  nicotinamide adenine dinucleotide phosphate  (NADP) dependent GluDH [EC 1.4.1.4] enzymes are present in  Plasmodia ; the NAD-dependent GluDH is relatively unstable and not useful for diagnostic purposes. Glutamate dehydrogenase provides an oxidizable carbon source used for the production of energy as well as a reduced electron carrier, NADH.  Glutamate  is a principal amino donor to other  amino acids  in subsequent transamination reactions. The multiple roles of glutamate in nitrogen balance make it a gateway between free ammonia and the amino groups of most amino acids. Its  crystal structure  is published. [ 8 ]  The GluDH activity in  P.vivax ,  P.ovale  and  P. malariae  has never been tested, but given the importance of GluDH as a branch point enzyme, every cell must have a high concentration of GluDH. It is well known that enzymes with a high molecular weight (like GluDH) have many  isozymes , which allows strain differentiations (given the right  monoclonal antibody ). The host produces antibodies against the parasitic enzyme indicating a low sequence identity. [ 1 ]


The histidine-rich protein II (HRP II) is a  histidine - and  alanine -rich, water-soluble protein, which is localized in several cell compartments including the parasite cytoplasm. The antigen is expressed only by  P. falciparum   trophozoites . [ 9 ] [ 10 ]  HRP II from  P. falciparum  has been implicated in the  biocrystallization  of  hemozoin , an inert, crystalline form of ferriprotoporphyrin IX (Fe(3+)-PPIX) produced by the parasite.  A substantial amount of the HRP II is secreted by the parasite into the host bloodstream and the antigen can be detected in  erythrocytes ,  serum ,  plasma ,  cerebrospinal fluid  and even  urine  as a secreted water-soluble protein. [ 11 ]  These antigens persist in the circulating blood after the parasitaemia has cleared or has been greatly reduced. It generally takes around two weeks after successful treatment for HRP2-based tests to turn negative, but may take as long as one month, which compromises their value in the detection of active infection. [ 12 ]  False positive dipstick results were reported in patients with rheumatoid-factor-positive  rheumatoid arthritis . [ 9 ]  Since HRP-2 is expressed only by  P. falciparum , these tests will give negative results with samples containing only  P. vivax ,  P. ovale , or  P. malariae ; many cases of non-falciparum malaria may therefore be misdiagnosed as malaria negative (some  P.falciparum  strains also don't have HRP II). The variability in the results of pHRP2-based RDTs is related to the variability in the target antigen. [ 13 ]


P. falciparum   lactate dehydrogenase  (PfLDH) is a 33 kDa  oxidoreductase  [EC 1.1.1.27]. [ 14 ]  It is the last enzyme of the glycolytic pathway, essential for  ATP  generation and one of the most abundant enzymes expressed by  P. falciparum . [ 15 ]   Plasmodium  LDH (pLDH) from  P. vivax ,  P. malariae , and  P. ovale ) exhibit 90-92% identity to PfLDH from  P. falciparum . [ 16 ]  pLDH levels have been seen to reduce in the blood sooner after treatment than HRP2. [ 17 ]  In this respect, pLDH is similar to pGluDH.  Nevertheless, the kinetic properties and sensitivities to inhibitors targeted to the cofactor binding site differ significantly and are identifiable by measuring dissociation constants for inhibitors which, differ by up to 21-fold. [ 18 ]


Fructose-bisphosphate aldolase  [EC 4.1.2.13] catalyzes a key reaction in  glycolysis  and energy production and is produced by all four species. [ 19 ]  The  P.falciparum  aldolase is a 41 kDa protein and has 61-68% sequence similarity to known  eukaryotic  aldolases. [ 20 ]  Its crystal structure has been published. [ 21 ]  The presence of antibodies against p41 in the sera of human adults partially immune to malaria suggest that p41 is implicated in protective immune response against the parasite. [ 22 ]


mChip  is a  portable blood test device  which is capable of  diagnosing  an  infection  of  HIV  or  Syphilis [ 1 ] [ 2 ]  within 15 minutes [ 3 ]  and could be used effectively against  HIV/AIDS  in developing countries. [ 1 ] [ 3 ] [ 4 ]  The mChip costs about  US$  1 [ 3 ]  and the entire diagnostic kit costs about  US$  100. [ 3 ] [ 5 ]  mChip was developed so that people in regions with poor health facilities can access portable diagnosis for HIV/AIDS rather than travelling long distances to go to clinics for diagnosis. [ 4 ]


Lateral flow test  is one of the blood testing methods used, in which a  blood sample  or  oral fluid  is placed on a strip of paper. In this method, a colored band indicates  infection . [ citation needed ]


People in lesser developed regions like the  Sub-Saharan Africa  are adversely affected by  HIV/AIDS  and have very limited access to  clinical labs  or  hospitals . There have been estimates which have indicated that there are about 22.5 million people with HIV/AIDS in such regions and hence there is a high demand for  portable blood test devices . [ 6 ]  Hence devices like mChip will be able to diagnose HIV/AIDS in such regions [ citation needed ]


mChip was developed by scientists at  Columbia University [ 7 ]  in  New York City . Initial testing of this device was undertaken in a village in  Rwanda , [ 2 ] [ 8 ]  where, according to the  World Health Organization , approximately 3 percent of the population has  HIV/AIDS . [ 1 ]  Of the 400 volunteers who turned up for testing, 399 were correctly diagnosed with an accuracy of nearly 100 percent. [ 7 ] [ 9 ] [ 10 ] [ 11 ]  mChip was also tested for its effectiveness in diagnosing  syphilis , where, out of the 67 volunteers who turned up for testing, 63 were correctly diagnosed with an accuracy of nearly 94 percent. [ 1 ] [ 3 ]  The appearance of mChip resembles a  credit card . [ 1 ] [ 5 ]  and is estimated to cost just  US$  1. [ 3 ]


The operation of mChip is similar to that of  ELISA  (Enzyme-Linked Immunosorbent Assay). [ 2 ]  The ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample. It is a useful tool for determining  serum  antibody concentrations such as with the  HIV test . [ 12 ]  The mChip contains 10 zones [ 5 ]  which detect the passage of a small amount (about 1μl) of blood. [ 2 ]  The results can be obtained in a color-coded format [ 4 ]  in about 15 minutes. [ 5 ]


The  mean corpuscular hemoglobin , or "mean cell hemoglobin" ( MCH ), is the average mass of  hemoglobin  (Hb) per  red blood cell  (RBC) in a sample of blood. It is reported as part of a standard  complete blood count . MCH value is diminished in  hypochromic anemias . [ 1 ]  RBCs are either  normochromic  or hypochromic. They are never "hyperchromic". If more than the normal amount of hemoglobin is made, the cells get larger—they do not become darker. [ 2 ]


It is calculated by dividing the total mass of hemoglobin by the number of red blood cells in a volume of blood.


MCH=(Hb*10)/RBC (in millions) [ 3 ]


A normal MCH value in humans is 27 to 33  picograms  (pg)/cell. The amount of hemoglobin per RBC depends on hemoglobin synthesis and the size of the RBC. [ 4 ]


The mass of the red cell is determined by the iron (as part of the hemoglobin molecule), thus MCH in picograms is roughly the mass of one red cell. In  iron deficiency anemia  the cell mass becomes lighter, thus a MCH below 27 pg is an indication of iron deficiency.


The MCH decreases when Hb synthesis is reduced, or when RBCs are smaller than normal, such as in cases of iron-deficiency anemia. [ 1 ]  Conversion to SI-units: 1 pg of hemoglobin = 0.06207  femtomole  (fmol). [ 5 ]  Normal value converted to  SI-units : 1.68 – 1.92 fmol/cell.


The  mean corpuscular hemoglobin concentration  ( MCHC ) is a measure of the concentration of  hemoglobin  in a given volume of packed red blood cell.


It is calculated by dividing the hemoglobin by the  hematocrit .  Reference ranges for blood tests  are 32 to 36 g/dL (320 to 360g/L), [ 1 ]  or between 4.81 and 5.58 mmol/L. It is thus a mass or  molar concentration . Still, many instances measure MCHC in  percentage  (%), as if it were a  mass fraction  (m Hb  / m RBC ). [ 2 ] [ 3 ]  Numerically, however, the MCHC in g/dL and the mass fraction of hemoglobin in red blood cells in % are identical, assuming an RBC  density  of 1g/mL and negligible hemoglobin in plasma. [ citation needed ]


A low MCHC can be interpreted as identifying decreased production of hemoglobin. MCHC can be normal even when hemoglobin production is decreased (such as in iron deficiency) due to a calculation artifact. MCHC can be elevated ("hyperchromic") in  hereditary spherocytosis ,  sickle cell disease  and  homozygous hemoglobin C disease , depending upon the hemocytometer. [ 4 ] [ 5 ]  MCHC can be elevated in some megaloblastic anemias. MCHC can be falsely elevated when there is agglutination of red cells (falsely lowering the measured RBC count) or when there is opacification of the plasma (falsely increasing the measured hemoglobin). Causes of plasma opacification that can falsely increase the MCHC include hyperbilirubinemia, hypertriglyceridemia, and free hemoglobin in the plasma (due to hemolysis). [ citation needed ]


Because of the way  automated analysers  count blood cells, a very high MCHC (greater than about 370 g/L) may indicate the  blood  is from someone with a  cold agglutinin , or there may be some other problem resulting in one or more artifactual results affecting the MCHC. [ citation needed ]


For example, for some patients with cold agglutinins, when their blood gets colder than 37 °C, the red cells will clump together. As a result, the analyzer may incorrectly report a low number of very dense red blood cells. This will result in an impossibly high number when the analyzer calculates the MCHC. This problem is usually picked up by the laboratory before the result is reported. The blood can be warmed until the cells separate from each other, and quickly put through the machine while still warm. [ citation needed ]


There are four steps to perform when a suspect increased MCHC (>370 g/L or >37.0 g/dL) is received from the analyzer: [ citation needed ]


The  mean corpuscular volume , or  mean cell volume  ( MCV ), is a measure of the average volume of a red blood corpuscle (or  red blood cell ). The measure is obtained by multiplying a volume of blood by the proportion of blood that is cellular (the  hematocrit ), and dividing that product by the number of  erythrocytes  (red blood cells) in that volume. The mean corpuscular volume is a part of a standard  complete blood count .


In patients with  anemia , it is the MCV measurement that allows classification as either a  microcytic anemia  (MCV below normal range),  normocytic anemia  (MCV within normal range) or  macrocytic anemia  (MCV above normal range). Normocytic anemia is usually deemed so because the bone marrow has not yet responded with a change in cell volume. It occurs occasionally in acute conditions, namely blood loss and hemolysis.


If the MCV was determined by automated equipment, the result can be compared to RBC morphology on a  peripheral blood smear , where a normal RBC is about the size of a normal lymphocyte nucleus. Any deviation would usually be indicative of either faulty equipment or technician error, although there are some conditions that present with high MCV without megaloblastic cells.


For further specification, it can be used to calculate  red blood cell distribution width  (RDW).  The RDW is a statistical calculation made by automated analyzers that reflects the variability in size and shape of the RBCs.


To calculate MCV, the  hematocrit  (Hct) is divided by the concentration of RBCs ([RBC]) [ 1 ]


MCV

 =

 Hct

 [

 RBC

 ]

 {\displaystyle {\textit {MCV}}={\frac {\textit {Hct}}{[{\text{RBC}}]}}}


Normally, MCV is expressed in femtoliters ( fL , or 10 −15  L), and [RBC] in millions per microliter (10 6  / μL).  The normal range for MCV is 80–100 fL.


If the hematocrit is expressed as a percentage, the red blood cell concentration as millions per microliter, and the MCV in femtoliters, the formula becomes


MCV

 /

 L

 =

 H
 c
 t
 %

 /

 100

 [

 RBCmmL

 ]
 ×
 (

 10

 6

 /

 10

 −
 6

 )

 /

 L

 −
 1

 {\displaystyle {\textit {MCV}}/\mathrm {L} ={\frac {{\mathit {Hct\%}}/100}{[{\text{RBCmmL}}]\times (10^{6}/10^{-6})/\mathrm {L} ^{-1}}}}


MCV

 /

 f
 L

 =

 MCV

 /

 (

 10

 −
 15

 L

 )
 =

 10

 15

 H
 c
 t
 %

 /

 100

 [

 RBCmmL

 ]
 ×

 10

 12

 =

 H
 c
 t
 %

 ×
 10

 [

 RBCmmL

 ]

 {\displaystyle {\textit {MCV}}/\mathrm {fL} ={\textit {MCV}}/(10^{-15}\,\mathrm {L} )=10^{15}{\frac {{\mathit {Hct\%}}/100}{[{\text{RBCmmL}}]\times 10^{12}}}={\frac {{\mathit {Hct\%}}\times 10}{[{\text{RBCmmL}}]}}}


For example, if the Hct = 42.5% and [RBC] = 4.58 million per microliter (4,580,000/μL), then


MCV

 =

 0.425

 4.58
 ⋅

 10

 6

 /

 (

 10

 −
 6

 L

 )

 =
 92.8
 ⋅

 10

 −
 15

 L

 =
 92.8

 f
 L

 {\displaystyle {\textit {MCV}}={\frac {0.425}{4.58\cdot 10^{6}/(10^{-6}\,\mathrm {L} )}}=92.8\cdot 10^{-15}\,\mathrm {L} =92.8\,\mathrm {fL} }


Using implied units,


MCV

 /

 fL

 =

 42.5
 ×
 10

 4.58

 =
 92.8

 {\displaystyle {\textit {MCV}}/{\textrm {fL}}={\frac {42.5\times 10}{4.58}}=92.8}


The MCV can be determined in a number of ways by automatic analyzers. In volume-sensitive automated blood cell counters, such as the  Coulter counter , the red cells pass one-by-one through a small aperture and generate a signal directly proportional to their volume.
Other automated counters measure red blood cell volume by means of techniques that measure refracted, diffracted, or scattered light. [ 2 ]


The normal  reference range  is typically 80-100  fL . [ 3 ]


In  pernicious anemia  (macrocytic), MCV can range up to 150  femtolitres . [ 4 ]  (as are an elevated  GGT  and an  AST/ALT ratio  of 2:1).  Vitamin B12  and/or  folic acid  deficiency has also been associated with  macrocytic anemia  (high MCV numbers).


The most common causes of  microcytic anemia  are  iron deficiency  (due to inadequate  dietary  intake,  gastrointestinal blood loss , or  menstrual blood loss ),  thalassemia ,  sideroblastic anemia  or  chronic disease . In  iron deficiency anemia  (microcytic anemia), it can be as low as 60 to 70 femtolitres. In some cases of  thalassemia , the MCV may be low even though the patient is not iron deficient. [ citation needed ]


The MCV can be conceptualized as the total volume of a group of cells divided by the number of cells.  For a real world sized example, imagine you had 10 small jellybeans with a combined volume of 10 μL. The mean volume of a jellybean in this group would be 10 μL / 10 jellybeans = 1 μL / jellybean.  A similar calculation works for MCV. [ citation needed ]


1.  Measure the RBC index in cells/μL. Take the reciprocal (1/RBC index) to convert it to μL/cell.


2.  The 1 μL is only made of a proportion of red cells (e.g. 40%) with the rest of the volume composed of plasma. Multiply by the hematocrit (a unitless quantity) to take this into account.


3.  Finally, convert the units of μL to fL by multiplying by

 10

 9

 {\displaystyle 10^{9}}

 .  The result would look like this:


Note: the shortcut proposed above just makes the units work out:

 10
 ×
 40
 ÷
 5
 =
 80

 {\displaystyle 10\times 40\div 5=80}


Mean platelet volume  ( MPV ) is a machine-calculated measurement of the average size of  platelets  found in blood and is typically included in  blood tests  as part of the  CBC . Since the average platelet size is larger when the body is producing increased numbers of platelets, the MPV test results can be used to make inferences about platelet production in  bone marrow  or platelet destruction problems. [ 1 ]


An increased mean platelet volume (MPV) increases the risk to suffer a heart disease  [ 2 ]


MPV may be higher when there is destruction of platelets. This may be seen in  immune thrombocytopenic purpura  (ITP),  myeloproliferative diseases  and  Bernard–Soulier syndrome . It may also be related to  pre-eclampsia  and recovery from transient  hypoplasia . [ 3 ]


Abnormally low MPV values may correlate with  thrombocytopenia  when it is due to impaired production of  megakaryocytes  in the  bone marrow , such as in  aplastic anemia . A low MPV may indicate  inflammatory bowel disease  (IBD), such as  Crohn's disease  and  ulcerative colitis . [ 4 ]  A high MPV is also a bad prognostic marker in patients with sepsis or septic shock. [ 5 ] [ 6 ]  In addition, low MPV may correlate with abnormally small platelet size, sometimes a symptom of a spectrum referred to as  Wiskott–Aldrich syndrome  (WAS), [ 7 ]  caused by a genetic mutation of the WAS gene.


Sample for MPV testing is obtained in a Lavender-Top  EDTA tube. A typical range of platelet volumes is 7.2 - 11.7 fL [ 8 ]  ( femtolitre ), equivalent to spheres 2.65 to 2.9  μm  in diameter.


MELISA  ( Memory Lymphocyte Immunostimulation Assay ) is a blood test that detects  type IV hypersensitivity  to metals, chemicals, environmental toxins and molds. Type IV hypersensitivity reactions, particularly to nickel, are well established and may affect 20% of the population. [ 1 ]


The MELISA test measures type-IV delayed hypersensitivity reaction. Type-IV reactions are mediated by  T-lymphocytes  (or memory lymphocytes) that have had prior contact with a given allergen. [ 2 ]  This is in contrast to a type-I allergy, which is mediated by IgE  antibodies  and is often tested using an  ELISA  test. In genetically predisposed individuals, an ongoing exposure to allergens can induce type-IV hypersensitivity. [ citation needed ]


The MELISA  assay  is a cell culture and requires live memory lymphocytes. Lymphocytes are isolated from a blood sample and cultured in an incubator for five days. A portion of the blood is kept intact (unexposed to allergens) to serve as a negative control. A second portion is exposed to a universal allergen,  Pokeweed , to serve as a positive control. Finally, the blood is exposed to the suspected allergen/s in several different concentrations, to ensure that the conditions in vitro are as similar as possible to those in vivo. The lymphocyte reaction to each allergen is measured by two separate technologies: one based on the uptake of a radioactive  isotope ,  3 H-thymidine, by dividing lymphocytes (proliferation); the other by cell staining and microscopy evaluation. The level of reactivity is measured as a Stimulation Index (SI), against the naïve lymphocytes from the unexposed sample (negative control). Viability and reactivity are determined by cell count as well as reaction to the positive control. [ 3 ]


MELISA is an optimised, standardised version of the lymphocyte transformation test (LTT) which was developed in the early 1960s to help identify allergies to drugs, metabolites and metals. The LTT for beryllium is now accepted as the gold standard for diagnosing  berylliosis . [ 4 ]


MELISA differs from standard LTTs in several ways:


MELISA was further developed to help to assess the impact of hypersensitivity to metals used in dentistry. Hypersensitivity to dental metals may be associated with local oral reactions including oral lichen planus, stomatitis and ulceration. [ 6 ] [ 7 ] [ 8 ] [ 9 ]


The frequency of metal-induced lymphocyte responses was examined in 3,162 dental patients in three European laboratories using the MELISA test. [ 10 ]  The patients suffered from local and systemic symptoms attributed to their dental restorations. The effect of dental metal removal was studied in 111 patients with metal hypersensitivity and symptoms resembling  chronic fatigue syndrome  ( CFS ). After consultation with a dentist, a subgroup of 111 patients who showed allergy to their dental metals replaced their restorations with non-metallic materials. Nickel was the most common sensitizer, followed by inorganic mercury, gold, phenylmercury, cadmium and palladium. As compared to lymphocyte responses in healthy subjects, the CFS group had significantly increased responses to several metals, especially to inorganic mercury, phenylmercury and gold.  [ citation needed ]


Following dental metal removal:


Type IV hypersensitivity to metals is common, particularly to nickel, however hypersensitivity related complications associated with metal implants are less frequently reported. Potential hypersensitivity complications include skin rashes, chronic joint pain, swelling, aseptic loosening, and joint failure.  [ 11 ] [ 12 ]


Many authors conclude that LTT-based blood tests like MELISA may be a better option for detecting systemic allergies from implants, while patch testing is better suited to detecting dermal hypersensitivity. They add that LTT based testing may also be a good option in cases of indeterminate hypersensitivity or in patients with joint failure of an unknown cause since it has higher sensitivity than patch testing  [ 13 ] [ 14 ]


Some surgeons and researchers suggest LTT testing prior to surgery in patients with suspected/self-reported metal allergy  [ 15 ] [ 16 ] [ 17 ] [ 18 ]


Titanium and its main alloy (Ti6Al4V) are generally seen as hypoallergenic options for arthroplasty and for dental implants. Although uncommon, with a prevalence estimated at between 0.6-6.3%, [ 19 ] [ 20 ]  titanium hypersensitivity has been reported post-implantation with symptoms including impaired fracture healing, local eczema, pain, swelling, systemic dermatitis, implant loosening, and failure, all of which have been reported to resolve with implant removal and replacement with a non-titanium implant  [ 21 ] [ 22 ] [ 23 ] [ 24 ]


In spinal surgery, microscopic titanium particles are present in the tissues surrounding the implant. [ 25 ]  These particles activate macrophages that increase bone absorption and inflammatory reactions. [ 26 ]  Released nanoparticles will circulate in the body fluids, eventually accumulating in remote organs. [ 27 ]


Titanium has been shown to induce clinically relevant hypersensitivity which can be detected with MELISA testing. [ 28 ]  The accuracy of patch testing for titanium allergy, in particular, seems to be variable; the Mayo clinic failed to find any positive reactions to titanium in over a decade, [ 29 ]  despite several published cases of titanium allergy. [ 22 ] [ 30 ]


The test is also used to determine whether metal allergy is a contributing factor in the development of chronic diseases such as  CFS  (Sterzl, et al., 1999) and  multiple sclerosis . The authors hypothesize that if the immune system is constantly displaying an allergic reaction to a metal present in the body, this will alert the  HPA axis  inducing fatigue-like symptoms. A study (Stejskal, et al., 1999)  of 930 patients with CFS-like symptoms showed 62% testing MELISA-positive to metal allergy. Of those who removed the offending metals, 76% improved, but there was no placebo control. Metal hypersensitivity is not widely recognized by researchers as an accepted cause of  CFS  or  multiple sclerosis . [ citation needed ]


The MELISA test is used in  occupational medicine  and environmental health. It has been used to screen workers exposed to metals, chemicals or other allergens in their workplace. This is what the test was developed for originally, at Astra’s (now Astra-Zeneca) laboratories in Södertälje, Sweden. In the U.S., a similar technique (BeLT) is routinely used to screen for  beryllium  allergy in asymptomatic workers exposed to beryllium dust (Mroz, et al., 1991) (Newman LS, 1996).


Whilst two articles have concluded that the MELISA test may give false positive (Cederbrant, et al., 1999) (Cederbrant, et al., 1997) reactions, a subsequent study concluded the MELISA test is "reproducible, sensitive, specific, and reliable for detecting metal sensitivity in metal-sensitive patients." (Valentine-Thon & Schiawara, 2003)


The developers of the MELISA test argue that the critical articles calculated the sensitivity and specificity of in vitro lymphocyte proliferation tests using patch testing as its reference. It is well-known that patch testing can cause irritative local reactions.
It is generally agreed that LTT based tests like MELISA are better suited for diagnosing implant-related metals sensitivity than patch testing as the relationship between skin hypersensitivity and systemic hypersensitivity (Ständer, et al., 2017) is ill-defined. Performing the LTT under optimised stimulating conditions might be a useful additional tool for the diagnosis of hypersensitivity (Ständer, et al., 2017) (FDA, 2019).
To test whether patients with symptoms attributed to dental amalgam differed from healthy controls, a study compared 23 amalgam patients, 30 subjects who considered themselves healthy with amalgams and 10 subjects without amalgam using MELISA and other tests. The researchers found that a high frequency of positive results was obtained among healthy subjects with or without dental amalgam, and concluded that the test cannot be used as an objective test for mercury allergy (Cederbrant, et al., 1999).


Another study by the same critical author used 34 patients to test the sensitivity and specificity if the MELISA test, and concluded that it is not useful for diagnosis of contact allergy to the metals gold, palladium and nickel, since many false-positive results will be obtained (Cederbrant, et al., 1997).
The clinical relevance of the test has been shown by the decrease of patient-reported metal-specific responses following the removal of the allergy-causing metals (however the trial did not have a placebo control). (Stejskal, et al., 2006) (Valentine-Thon, et al., 2006)


The  Mentzer index,  described in 1973 by William C. Mentzer, [ 1 ]    is the MCV divided by the RBC count. It is said to be helpful in differentiating  iron deficiency anemia  from  beta thalassemia trait . [ 2 ] [ 3 ]


The index is calculated from the results of a  complete blood count . If the  quotient  of the  mean corpuscular volume  (MCV, in fL) divided by the  red blood cell  count (RBC, in million per microliter) is less than 13, β-thalassemia trait is said to be more likely. If the result is greater than 13, then iron-deficiency anemia is said to be more likely. [ 4 ] [ 3 ]


The principle involved is as follows:  In iron deficiency, the marrow cannot produce as many RBCs and they are small (microcytic), so the RBC count and the MCV will both be low, and as a result, the index will be greater than 13. Conversely, in thalassemia, which is a disorder of globin synthesis, the number of RBCs produced is normal, but the cells are smaller and more fragile. Therefore, the RBC count is normal, but the MCV is low, so the index will be less than 13. [ 5 ]


Missouri v. McNeely , 569 U.S. 141 (2013), was a case decided by  United States Supreme Court , on appeal from the  Supreme Court of Missouri , regarding exceptions to the  Fourth Amendment to the United States Constitution  under  exigent circumstances . [ 1 ] [ 2 ]  The United States Supreme Court ruled that police must generally obtain a warrant before subjecting a drunken-driving suspect to a blood test, and that the natural metabolism of blood alcohol does not establish a  per se  exigency that would justify a blood draw without consent.


At approximately 2:08 a.m. on October 3, 2010, Tyler McNeely was stopped after a highway patrol officer observed him exceed the posted speed limit, and cross over the centerline. The officer reportedly noticed signs of intoxication from McNeely, including bloodshot eyes, slurred speech, and the smell of alcohol on his breath. McNeely failed field-sobriety tests administered by the officer. After refusing to blow into a handheld breathalyzer, and stating that he would refuse a breathalyzer at the police station, the officer drove McNeely directly to a medical center instead of the station. The officer did not seek a warrant to conduct the blood test, but asked McNeely for his consent. McNeely was warned by the officer that by refusing a chemical test, his license would be revoked for one year. McNeely continued to refuse, and at 2:35 a.m., the officer proceeded to instruct the lab technician to draw a specimen of blood from McNeely. The results of the blood test showed a BAC of 0.154 percent, which was above the state's legal limit of 0.08 percent. McNeely was charged with driving while intoxicated, and later moved to suppress the results of his blood test, as he argued that it was done unconstitutionally as an unreasonable search and seizure. [ 3 ]


A trial judge sided with McNeely, ruling in their favor by suppressing the results of the blood test. The judge emphasized that conducting a blood test without a warrant constituted a breach of the suspect's  Fourth Amendment  protection against unreasonable searches and seizures.


Later, state prosecutors argued that justifying the administration of the test without a warrant was valid because blood alcohol would metabolize with time, and a delay in obtaining a warrant would amount to  destruction of evidence , citing the exigent circumstances exception in the 1966 United States Supreme Court decision  Schmerber v. California . On appeal, the  state appeals court  stated an intention to reverse, but transferred the case directly to the  Missouri Supreme Court . The Missouri Supreme Court affirmed the trial court's decision that the officer had violated McNeely's Fourth Amendment rights. The United States Supreme Court granted a petition for  writ of certiorari  on 25 September 2012. [ 4 ]


A 5-4 Supreme Court affirmed the Missouri Supreme Court, agreeing that an involuntary blood draw is a "search" as that term is used in the Fourth Amendment. [ 5 ]  As such, a warrant is generally required. In its majority opinion, the Court found that because McNeely's "case was unquestionably a routine  DWI  case" in which no factors other than the natural dissipation of blood-alcohol suggested that there was an emergency, the court held that the nonconsensual warrantless blood draw violated McNeely's Fourth Amendment right to be free from unreasonable searches of his person. However, the Court left open the possibility that the "exigent circumstances" exception to that general requirement might apply in some drunk-driving cases. [ 6 ]


Mitchell v. Wisconsin , 588 U.S. ___ (2019), is a  United States Supreme Court  case in which the Court held that "when a driver is unconscious and cannot be given a  breath test , the  exigent-circumstances doctrine  generally permits a  blood test  without a  warrant ." [ 1 ]


In May 2013, Gerald Mitchell crashed his car near a lake in  Sheboygan, Wisconsin . When police arrived, they used a breathalyzer to test his  blood alcohol content . Mitchell registered a 0.24% BAC and was subsequently arrested for OWI. As police were driving him to the police station, he fell unconscious, so the officers changed plans and drove him to a local hospital to have his blood drawn intravenously. This test registered his BAC at 0.22%, and prosecutors formally charged Mitchell with violating several Wisconsin drunk driving laws. [ 2 ]


At the trial court, Mitchell made a motion to suppress the results of the hospital blood draw on the grounds that it was a warrantless search and thus unconstitutional under the  Fourth Amendment . The prosecutor argued that Wisconsin's state laws constitute implied consent to blood draws once someone begins driving a vehicle. [ 2 ]  Sheboygan County Judge Terence Bourke sided with the prosecutor, denying Mitchell's motion to suppress. A jury then convicted Mitchell of all charges. [ 3 ]


Mitchell appealed his conviction to the state appellate court on the basis that the evidence gained from his blood draw should have been suppressed. The appellate court declined to hear the case, and instead certified two questions to the  Wisconsin Supreme Court  – whether the "implied consent" rule was constitutional, and whether a warrantless blood draw from an unconscious person was a violation of the Fourth Amendment. [ 3 ]


In a 5–2 decision written by Chief Justice  Roggensack , the  Wisconsin Supreme Court  upheld Mitchell's conviction, answering that the "implied consent" rule was constitutional, and thus the blood draw was permissible under the Fourth Amendment. Justice  Kelly  wrote a concurring opinion that was joined by  Justice Rebecca Bradley . In it, he argued that the "implied consent" rule is unconstitutional, but that the  exigent circumstances doctrine , along with United States Supreme Court precedent, allow for a warrantless blood draw from an unconscious driver who is suspected of being intoxicated. Justice  Ann Walsh Bradley  wrote a dissent joined by Justice  Abrahamson , which argued that "implied consent" is not the same as actual consent, and that a blood draw is such an invasive type of search that exigent circumstances do not apply. Thus, nothing the officers did was constitutional, and the blood draw should have been thrown out as evidence. [ 4 ]


Mitchell applied for certiorari before the United States Supreme Court, which accepted the case to decide "[w]hether a statute authorizing a blood draw from an unconscious motorist provides an exception to the Fourth Amendment warrant requirement."


Oral argument was held on April 23, 2019. [ 5 ]


On June 27, 2019, the Court announced its decision. In a plurality opinion written by Justice  Samuel Alito  and joined by Chief Justice  Roberts  and Justices  Breyer  and  Kavanaugh , the United States Supreme Court reversed the judgement of the  Wisconsin Supreme Court . Justice  Thomas  wrote an opinion concurring in the judgement. In opposition, Justice  Sotomayor  wrote a dissenting opinion that was joined by Justices  Ginsburg  and  Kagan . Justice  Gorsuch  wrote a lone one-paragraph dissenting opinion, arguing that the Court did not properly decide the question presented. He said that he would have dismissed the case as  improvidently granted . [ 2 ]


Mixing studies  are tests performed on  blood plasma  of patients or test subjects to distinguish factor deficiencies from  factor inhibitors , such as  lupus anticoagulant , or specific factor inhibitors, such as  antibodies  directed against  factor VIII . [ 1 ]  Mixing studies are screening tests widely performed in coagulation laboratories. The basic purpose of these tests is to determine the cause of prolongation of  Prothrombin Time  (PT),  Partial Thromboplastin Time , or sometimes of  thrombin time  (TT). Mixing studies take advantage of the fact that factor levels that are 50 percent of normal should give a normal  Prothrombin time  (PT) or  Partial thromboplastin time  (PTT) result. [ 2 ]


Fresh normal plasma has all the blood coagulation factors with normal levels.


If the problem is a simple factor deficiency, mixing the patient plasma 1:1 with plasma that contains 100% of the normal factor level results in a level ≥50% in the mixture (say the patient has an activity of 0%; the average of 100% + 0% = 50%). [ 3 ]  The PT or PTT will be normal (the mixing study shows correction).  Correction with mixing indicates factor deficiency.  Failure to correct with mixing indicates an inhibitor.  Performing a thrombin time on the test plasma can provide useful additional information for the interpretation of mixing tests, such as by demonstrating the presence of anticoagulants, hypofibrinogenemia or dysfibrinogenemia. [ 4 ]


Factor deficient plasmas (Adsorbed Plasma and Aged Plasma, etc.) have been used historically in mixing studies. Plasma with known factor deficiencies are commercially available but are very expensive, so they have been prepared in the laboratory and used for mixing tests. Adsorbed plasma or plasma from patients on oral anticoagulants ( Warfarin  etc.) for a week or more is deficient in Factor II, Factor VII, Factor IX, and Factor X. Plasma from patients on oral anticoagulants ( Warfarin  etc.) for 48–72 hours is deficient in  Factor VII .  Aged plasma is deficient in  Factor V  &  Factor VIII C. Serum is deficient in factors I, V & VIIIC.


Prothrombin time  (PT) may be corrected as follows: [ 5 ] [ 6 ]


Partial thromboplastin time  (PTT) may be corrected as follows: [ 5 ]


Some inhibitors are time dependent. In other words, it takes time for the antibody to react with and inactivate the added clotting factor. The clotting test performed immediately after the specimens are mixed may show correction because the antibody has not had time to inactivate its target factor. A test performed after the mixture is incubated for 1 to 2 hours at 37°C will show significant prolongation over the clotting time obtained after immediate mixing. Nonspecific inhibitors like the lupus anticoagulant usually are not time dependent; the immediate mixture will show prolongation. Many specific factor inhibitors are time dependent, and the inhibitor will not be detected unless the test is repeated after incubation (factor VIII inhibitors are notorious for this). [ 7 ]


A common problem is an unexplained increase in the PT and/or PTT. If this is observed, the test should be repeated with a fresh sample. Another consideration is  heparin . It is possible that the blood sample was mistakenly drawn though a running line. Interference by heparin can be detected by absorbing the heparin with a resin (“Heparsorb”) or by using an  enzyme  to digest the heparin (“Hepzyme [ 8 ] ”). Also, the patient's  history  should be checked, especially  with regard to anticoagulant use or  liver disease . Provided that the abnormal result is reproduced on a fresh specimen and there is no obvious explanation from the history, a mixing study should be performed. If the mixing study shows correction and no prolongation with incubation, factor deficiency should be looked for, starting with VIII and IX.  Vitamin K -dependent and nonvitamin K–dependent factors should be considered to rule out vitamin K deficiency, or accidental or surreptitious  warfarin  ingestion.


If the mixing study fails to correct, then an inhibitor should be suspected. [ 9 ] [ 10 ]  The most common inhibitor is a nonspecific inhibitor such as a  lupus anticoagulant . [ 9 ]  Perform a test to demonstrate a phospholipid-dependent antibody, such as a  platelet  neutralization procedure. Spontaneous specific inhibitors against clotting factors occur (i.e. not in  hemophiliacs ), most often against factor VIII. [ 11 ]  This can occur in patients with  systemic lupus erythematosus ,  monoclonal gammopathies , other  malignancies , during  pregnancy  and for no apparent reason ( idiopathic ). These patients can have devastating bleeding. The thing to do is identify the specific factor involved and find out how high the titer is. If the patient has a low titer inhibitor, try to overwhelm it with high doses of the factor. If the patient has a high titer antibody against  factor VIII , try  porcine factor VIII , activated prothrombin complex concentrate  FEIBA  (Factor Eight Inhibitor Bypassing Agent), [ 12 ]  or  NovoSeven [ 13 ]  to stop the bleeding.  Prednisone  will often lower the titer over time.  Intravenous immunoglobulin  has been reported to also help but it does not seem to work for hemophiliacs with an inhibitor.   Rituximab ,  cyclophosphamide  or other immunosuppressive therapy may be required. [ 14 ]


In order to provide specific cutoffs to distinguish an inhibitor defect from a factor deficiency, the "Rosner index" (index of circulating anticoagulant) [ 15 ]  and/or the "Chang percentage" (percent correction method) can be used: [ 16 ]


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 {\displaystyle Rosner\;index={\frac {(aPTT\;of\;1:1\;mix)-(aPTT\;of\;normal\;pooled\;plasma)}{aPTT\;of\;nonmixed\;patient\;plasma}}x100}


Results are: ≤10 is classified as correction, ≥15 indicates presence of an inhibitor, and 11-15 is classified as "indeterminate". [ 17 ]


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 {\displaystyle Chang\;percentage={\frac {(aPTT\;of\;nonmixed\;patient\;plasma)-(aPTT\;of\;1:1\;mix)}{(aPTT\;of\;nonmixed\;patient\;plasma)-(aPTT\;of\;normal\;pooled\;plasma)}}x100}


Results are classified as follows: <58% as inhibitor and >70% as correction. [ 18 ] >


Alternatively, correction into the reference range can be used to define complete correction. [ 19 ]


A fourth method is known as Estimated Factor Correction (EFC). This method involves four steps.  First, determine the most likely factor suspected to be deficient, based on PT, aPTT, and clinical history.  Next, choose the appropriate curve — single factor deficiency, vitamin K-dependent factor deficient, or all factor-deficient. Use this curve to estimate the factor level in the patient sample.  Then, predict the factor level and PT or aPTT that will occur after 1:1 mix in case of deficiency.  Finally, compare the actual mix results with the predicted results for deficiency. [ 19 ]


Monocyte distribution width  ( MDW ) is a cytometry-based parameter that measures the range of variation of  monocytes . If the parameter is available, it is reported as part of the standard  complete blood count  (CBC) with differential. [ 1 ]


The parameter was FDA cleared as an early sepsis indicator for ER patients in 2019 for  Beckman Coulter . [ 2 ] [ 3 ]


MDW serves as an indicator for early screening of sepsis in conjunction with CRP and PCT and for differentiating false positive blood cultures. [ 4 ] [ 5 ]


The  monocyte monolayer assay  (MMA) is used to determine the clinical significance of alloantibodies produced by blood transfusion recipients. [ 1 ]  The assay is used to assess the potential for  intravascular hemolysis  when incompatible cellular blood products are transfused to the anemic patient. When donor cells possess substances that are not produced by the recipient, the recipient's immune system produces antibodies against the substance; these are called alloantibodies. Specific white blood cells, called monocytes, are tasked with ingesting foreign material and become activated during certain inflammatory events. [ 2 ] [ 3 ]  These activated  monocytes  come in contact with antibody-sensitized red blood cells (RBC) and may or may not exhibit  phagocytosis  (ingestion) and destroy the donor red blood cells. If monocytes destroy the RBC, the antibody attached to those RBC is considered clinically significant. [ 1 ]


Blood banking  is a concentration within a clinical laboratory that analyzes specimens from potential transfusion recipients and provides compatible blood products to the healthcare team in charge of that patient's care. Several routine tests are performed including blood typing (determination of  ABO / Rh  status), antibody screening, serologic  cross-matching , direct antiglobulin testing, and antibody identification. Beyond the presence of naturally occurring antibodies (isohemagglutinins) to the ABO and Rh(D) blood group antigens, additional immune-stimulated antibodies are considered unexpected alloantibodies. [ 4 ]


The identification of unexpected antibodies is a labor-intensive process, and sometimes requires the addition of special laboratory techniques to aid in the proper identification of the antibody. Among these techniques are  elutions , adsorptions, and enzyme treatment. [ 4 ] [ 5 ]  Some patients produce antibodies to high frequency antigens. That is, the red cell antigens are present in a significant portion of the human population. It may be questionable and very difficult to assess if the antibody is considered clinically significant or not. [ 6 ]  A clinically significant antibody is an antibody that is capable of causing in vitro hemolysis or a decreased survival of transfused donor red blood cells. [ 7 ]  Antibodies to high frequency antigens can be assessed for clinical significance using the monocyte monolayer assay.  [ 8 ]


There are many different varieties of abnormal reactions to blood transfusion. Among these, a potentially life-threatening reaction is known as a  hemolytic transfusion reaction . This is an immune mediated reaction where recipient antibodies attack donor red blood cell antigen(s), causing hemolysis of donor cells. The reaction may occur during, immediately after, or up to 28 days later. An acute reaction is observed within the first 24 hours, whereas a delayed reaction will be observed between 24 hours and 28 days after transfusion. [ 4 ]


When talking about the ABO blood group system, Landsteiner's Law states that if an individual possesses the A and/or B antigen, they will not form antibodies to these antigens. [ 9 ]  However, if an individual does not have either A or B antigens, they will naturally produce anti-A and anti-B antibodies. [ 10 ]  According to the  International Society of Blood Transfusion  (ISBT), 43 blood group systems containing hundreds of different red blood cell antigens have been described.  [ 11 ]  With some exceptions, many non-ABO blood group system antigens require a sensitizing event to stimulate antibody production. In other words, the immune system must be exposed to the antigen in order to illicit antibody production. Exposure to antigens can occur through blood transfusion, stem cell/bone marrow transplant, and pregnancy. [ 4 ]


The clinical significance of an alloantibody depends on its ability to cause a decrease in donor red blood cell survival. [ 12 ]  Characteristics of clinically significant alloantibodies include:  immunoglobulin G  antibody subclass, reactivity at body temperature, and ability to cause red blood cell agglutination in the presence of anti-human globulin (AHG) in an indirect antiglobulin test. [ 4 ]  Sometimes, clinical significance of an antibody can be difficult to determine. [ 6 ]  Antibodies to high prevalence red cell antigens can sometimes mask the detection of clinically significant alloantibodies because the corresponding antigen is present on most, if not all, of the screening red blood cells used to detect these antibodies possess the antigen. This is where the monocyte monolayer assay may be useful.


The MMA is a very labor-intensive, manual laboratory testing method. The following steps are performed in this assay: [ 13 ]


If the positive or negative controls fail, than the entire testing procedure is invalid and must be repeated. Criteria for a positive MMA will vary by laboratory, though the originally established threshold set by Sandra Nance et al., is >20% phagocytic activity observed. [ 13 ]


Clinically, a positive MMA would indicate that the patient's serum used in the assay contains clinically significant antibodies that are capable of causing  antibody-mediated phagocytosis .


Nightscout  is a  free and open-source  project, and associated social movement, that enables accessing and working with  continuous glucose monitor  (CGM) data. [ 1 ] [ 2 ]
Nightscout software aims to give users access to their real time  blood sugar  data by putting this data in the  cloud . In addition to browser-based data visualization, Nightscout can also be used to review data from a phone or  smartwatch , or to remotely monitor CGM data for individuals with  type 1 diabetes . Associated with Nightscout software is a broader "CGM in the Cloud" social movement, supporting individuals seeking to access and use realtime CGM data through commercial and  DIY  ("do it yourself") approaches. [ 3 ]


The Nightscout Project traces its origin to February 2013, when the parents of a 4-year-old boy newly diagnosed with type 1 diabetes began using a continuous glucose monitoring system.
 [ 1 ] [ 2 ]
This data was inaccessible to the parents when the child was at school – there was no commercially available way to access the data in real time. The boy's father, John Costik, a software engineer, developed software to access and transfer CGM data to cloud computing infrastructure. Costik shared his accomplishment on  Twitter  and, after others expressed interest, privately shared the source code. [ citation needed ]


Costik's uploader was expanded by Lane Desborough and Ross Naylor, [ 4 ]  to develop "Nightscout": adding a blood glucose chart display that could be viewed throughout a home. Further development to make the software accessible to the general public occurred within a private community of developers, including Ben West, [ 5 ] [ 6 ]  Ross Naylor, Kevin Lee, [ 7 ]  Jason Calabrese, Jason Adams, and Toby Canning.  Because this software was, in effect, an unlicensed medical device, the community delayed releasing the code as open source to explore and address legal concerns. Once this was done, the combined code was released in 2014 as the Nightscout Project. [ 8 ]


A website,  Facebook  group, and  Gitter channel  were also created to support new users. [ 3 ] [ 9 ]  The primary Facebook group for the movement is "CGM in the Cloud", broadly supporting individuals seeking to use realtime CGM data, via commercial and/or  DIY  methods. As of May 2017, this group has over 23,000 members. In addition, the related  501(c)(3) organization   Nightscout Foundation  was formed in 2014 to encourage and support open source technology projects for individuals with type 1 diabetes. [ 10 ]


The #WeAreNotWaiting hashtag used by the group was initially coined by Lane Desborough [ 11 ]  and Howard Look, in reference to a growing call for a "diabetes data exchange" hosted by  Tidepool  and  DiabetesMine  in November 2013. [ 1 ]


A survey of the Nightscout community indicated that the software was most highly used by parents of young children with type 1 diabetes (less than 12 years of age), but is also used by adolescents and adults.
 [ 12 ]


Because Nightscout software displays information relevant to medical care, there are safety concerns regarding reliability, liability, and regulation. The United States  Food and Drug Administration  (FDA) expressed concern that a singular entity was needed to review code, monitor safety, and respond to issues. [ 2 ]  As of 2016, no such entity had arisen, beyond the unstructured approach of community communication channels (e.g. Facebook group and Gitter channel). [ 3 ] [ 13 ]


As a non-commercial open source project, Nightscout software has not been regulated by the FDA. [ 2 ] [ 14 ]  The software is released with a strong disclaimer:


Highly experimental. Not intended for therapy. Use at your own risk. Intended only as an investigational and educational tool to learn about this technology.


On November 20, 2020,  Medical Data Networks LLC  (Ben West, CEO) met with and formally petitioned the FDA for clearance of the service product “ T1Pal .”    T1Pal  is a hosted Nightscout platform that runs copies of the latest Nightscout software on its servers for the benefit of individual subscribers.


Nightscout, when operated as a hosted service, is expected to enable many more people to use and benefit from Nightscout.    T1Pal.com  charges a monthly fee to do this ($12.00/mo.), which includes technical support to set up and use the platform.
 Medical Data Networks LLC  has asserted with the FDA that its implementation of  T1Pal  meets all of the stated FDA requirements for code review, safety monitoring, and responding to issues.  To support this view, a complete copy of the “Quality Management System” currently used by  Medical Data Networks LLC  was provided to the FDA.