Lab
د. حسين محمد جمعه
اختصاصي الامراض الباطنة
البورد العربي
كلية طب الموصل
2011
In vitro diagnostic (IVD) tests are medical devices intended to perform diagnoses from assays in a test tube, or more generally in a controlled environment outside a living organism. In vitro means in glass in Latin.
In microbiology, colony-forming unit (CFU or cfu) is a measure of viable bacterial or fungal numbers. Unlike direct microscopic counts where all cells, dead and living, are counted, CFU measures viable cells. For convenience the results are given as CFU/mL (colony-forming units per milliliter) for liquids, and CFU/g (colony-forming units per gram) for solids. A measure of viable cells in which a colony represents an aggregate of cells derived from a single progenitor cell.
Supplement
CFU is used to determine the number of viable bacterial cells in a sample per mL. Hence, it tells the degree of contamination in samples of water, vegetables, soil or fruits, or the magnitude of the infection in humans and animals.The term efficacy generally refers to the ability of a product to achieve its desired effect under ideal conditions, such as a controlled clinical trial in which the product is consistently administered as prescribed.
The effectiveness of a product is its ability to achieve the desired effect under real-world conditions. The charge to the committee uses both terms, and in many situations the terms are used interchangeably, with the context showing whether the effects were observed under laboratory-controlled or real-world conditions.
Polymerase Chain Reaction vs. Blood Culture in Sepsis
Correlation with sepsis severity parameters was better for PCR than for blood culture; in patients with sepsis, concordance between the two methods was modest.In patients with sepsis, rapid initiation of adequate antibiotic therapy is a key component of therapy. Empirical therapy with broad-spectrum antibiotics is used until the causative pathogen is identified. (PCR)-based detection systems could markedly shorten the time to pathogen identification, thus hastening the start of targeted antibiotic therapy.But how well do such PCR-based systems perform?
To find out, researchers in Germany — sponsored by industry — compared results of a whole-blood bacterial and fungal DNA multiplex PCR system with conventional blood culture (BC) in 142 surgical patients with severe sepsis or septic shock and 63 surgical patients with no signs of infection (controls). Blood samples were obtained for 3 days following study inclusion (patients with sepsis) or admission (controls).Among controls, 1.6% of BCs and 4.8% of PCRs were positive at baseline, but none of these positives were confirmed by a positive result using the alternative technique. Among patients with sepsis, these rates were 17.8% and 33.1%; the sensitivity and specificity of PCR to detect culture-positive bacteremia were 0.80 and 0.77, respectively. PCR detected 70.3% of microorganisms identified by BC, whereas BC detected 21.4% of those identified by PCR. Overall, PCR-suspected infections were microbiologically confirmed from blood (in 23%) or a focus of infection (in 38%); BC-positive cases were confirmed in 42% from the site of infection. Positive PCR results correlated significantly with elevated procalcitonin and interleukin (IL)-6 levels and with greater organ dysfunction scores; positive BCs were significantly associated only with higher IL-6 levels.
Comment: For patients with sepsis, PCR appears to provide more information on causative pathogens than conventional BCs, possibly by avoiding false-negative results if antibiotics have been initiated before specimens are obtained. Nevertheless, PCR results must be interpreted with caution. Furthermore, PCR has a significant shortcoming: It yields no information on susceptibility results — yet
Drugs that can increase BUN measurements include:
AllopurinolAminoglycosides
Amphotericin B
Bacitracin
Carbamazepine
Cephalosporins
Chloral hydrate
Cisplatin
Colistin
Furosemide
Gentamicin
Guanethidine
High-dose aspirin
Indomethacin
Drugs that can decrease BUN measurements
Chloramphenicol
Streptomycin
Methicillin
Methotrexate
Methyldopa
Neomycin
Penicillamine
Polymyxin B
Probenecid
Propranolol
Rifampin
Spironolactone
Tetracyclines
Thiazide diuretics
Triamterene
Vancomycin
Anti-transglutaminase antibodies (ATA) are autoantibodies against the transglutaminase protein.
High levels (titers) of ATA are found in coeliac disease.
ATA can be classified according to 2 different schemes. Transglutaminase isoform and the reactivity of immunoglobulin subclass (IgA, IgG) toward transglutaminases.
Transglutaminase isoform reactivity
Anti-tissue transglutaminase
found in patients with several conditions, including coeliac disease, juvenile diabetes[2], inflammatory bowel disease[3], and various forms of arthritis[4].
In Coeliac Disease, ATA are involved in the destruction of the villous extracellular matrix and target the destruction of intestinal villous epithelial cells by killer cells. Deposits of anti-tTG in the intestinal epithelium predict coeliac disease.[
Anti-endomysial reactivity
The endomysium is a layer of connective tissue that ensheaths a muscle fiber. The endomysium contains a form of transglutaminase called "tissue transglutaminase" or "tTG" for short, and antibodies that bind to this form of transglutaminase are called anti-endomysial antibodies or EMA for short,[6] therefore targeting those cells for apoptosis. The antiendomysial antibody test is a histological assay for patient serum binding to esophageal tissue from primate. EMA are present in celiac disease. They do not cause any direct symptoms to muscles, but detection of EMA is useful in the diagnosis of the disease.Anti-epidermal transglutaminase
Antibodies to epidermal transglutaminase (eGT, also Keratinocyte transglutaminase) are the autoantibodies believed to cause dermatitis herpetiformis.Immunoglobin subclass
ATA IgA are more frequently found in CD; however, ATA IgG are found in CD and at higher levels when affected individual had the IgA-less phenotype. The IgA-less phenotype is more common in CD than the normal population; however, one haplotype, DQ2.5 is found in most CD, has genetic linkage to the IgA-less gene location.Associated Conditions
Coeliac Disease.Inflammatory bowel disease. A study published in Nature in 2001 found high levels of anti-transglutaminase antibodies in Inflammatory Bowel Disease - specifically in Crohn's Disease and Ulcerative rectocolitis.[3]
Arthritis. Studies of patients with various forms of arthritis showed highly increased frequencies of antibodies against guinea pig transglutaminase, human recombinant transglutaminase and peptidylarginine deiminase type 4 (PAD4). This suggests a potential for crossreactive antibodies between anti-tTG and anti-PAD4.[10]
Juvenile diabetes and anti-tTG. Childhood (male) Type 1 diabetes (T1D) increases the risk for CD and vice versa[11] and the early signs of CD may precede T1D in many cases.[12] A search for CD in juvenile diabates patients revealed that GF diet resulted in some improvements.[13] A elevated number of diabetes patients have ATA[14] along with increased numbers of gluten-specific T-cells.
Mechanism of Autoimmunity
The antibodies to tissue transglutaminase follow a complex pathway of generation. For most antigens, T-cells specific to those antigens develop, for autoimmunity autoreactive T-cells are not suppressed or antigens escape the protective process. T-cells are stimulated by antigen, presented by MHC molecules (HLA in humans) and surface IgM on antigen reactive B-cells. These T-helper cells then stimulate B-cells to multiply and mature into plasma cells that make IgG to that protein.In the case of Coeliac Disease, the current understanding is that tTG autoimmunity arises when T-cells are generated against wheat gliadin and similar gluten proteins of the trib Triticeae.
The T-cells are defined by the ability to react to HLA-DQ8 and DQ2.5 restricted antigens and gliadin is one of the antigens. Gliadin is a favored dietary substrate for transglutaminase because of many enzyme reaction sites on gliadin.
In disease, transglutaminase reacts with gliadin forming a linkage[20]. In forming this bond transglutaminase becomes linked to T-cell epitopes on gliadin. B-cells with surface IgM that react to transglutaminase can present it with bound gliadin peptides to T-cells which stimulate B-cell maturation and proliferation to plasma cells making IgA or IgM.
ATA changes the behavior of tTG. Some studies have revealed that antibodies increase the activity of tTG, instead of inhibiting activity as is commonly encountered with function alterning antibodies. A recent study has shown that ATA also modify and increase replication in intestinal epithileal cells, by apparently interacting with cell-surface transglutaminase.[
Blood test :A tissue transglutaminase
IgA and/or IgG testused as part of an evaluation for certain autoimmune conditions, most notably celiac disease.
An autoimmune disease can occur when the body's immune system mistakenly perceives a nonthreatening substance — in the case of people with celiac disease, gluten, a protein found in wheat, barley, rye, and oats — as a foreign invader and attacks it. This immune system response in celiac disease also involves the production of antibodies directed against an enzyme normally present in the intestines called tissue transglutaminase (tTG).
In celiac disease, the body produces two types of antibodies that attack tTG: immunoglobulin A (IgA) and immunoglobulin G (IgG).
Measuring the IgA form of tTG antibody in the blood is more useful in detecting celiac disease because it's made in the small intestine, where gluten causes inflammation and irritation in sensitive people.
Levels of the IgG form of tTG antibody, on the other hand, are less specific to celiac disease, but may still be useful in diagnosing the condition in people who are unable to make normal amounts of IgA antibodies.
Preparation
For accurate results, your child must currently be eating a diet that includes gluten-containing foods, such as breads, pastas, and baked goods. The body will only produce antibodies when it's exposed to gluten. If gluten has been removed from the diet, there won't be an antibody response to measure.Getting the Results
The blood sample will be processed by a machine. Results are commonly available within a day or two. If results suggest celiac disease, the doctor will likely perform further tests, including a biopsy of the small intestine to look for evidence of gluten-induced inflammation.What are the appropriate screening tests for celiac disease?
The tests of choice are antibody measurements in the blood, ideally performed before the patient has removed gluten from the diet. However, patients and physicians must remember that no screening test is perfect, and that the keys to confirming the diagnosis of CELIAC DISEASE remain a small intestinal biopsy combined with the patient’s subsequent clinical response to a gluten-free diet. Thus, a patient (especially a young child) with symptoms of CELIAC DISEASE should have a small bowel biopsy, even if the antibodies are not highly suggestive.
The blood tests can be divided into 2 different types of antibodies: those which are “anti-gluten”, and those that “anti-self”. The “anti-gluten” antibodies are the anti-gliadin IgG and IgA.
The “anti-self” antibodies are anti-endomysial IgA and anti-tissue transglutaminase IgA. Each antibody test varies widely in its sensitivity and specificity for predicting whether the disease is present in any individual.
There are also several conditions which may yield false negative antibody results. One of the conditions that may give a false negative result is Immunoglobulin A or IgA deficiency. If a patient has a low total IgA level, the antibodies may be falsely low. This is why I always recommend that a patient have a total IgA level drawn at the same time the antibody testing is done. Young children may not make the some of the “anti-self” antibodies, as it takes a somewhat mature immune system to make them. So in a young child, antiendomysial antibody, or the TTG antibody, can have false negative results. A gluten-free diet will make the antibody tests negative.
Antigliadin antibodies
The antigliadin antibodies IgG and IgA recognize a small piece of the gluten protein called gliadin. These antibodies became available during the late 1970′s and were the first step towards recognizing CELIAC DISEASE as an autoimmune disorder. Antigliadin IgG has good sensitivity, while antigliadin IgA has good specificity, and therefore their combined use provided the first reliable screening test for CELIAC DISEASE.Unfortunately, many normal individuals without CELIAC DISEASE will have an elevated antigliadin IgG, causing much confusion among physicians. The antigliadin IgG is useful in screening individuals who are IgA deficient, as the other antibodies used for routine screening are usually of the IgA class. It is thought that 0.2-0.4% of the general population has selective IgA deficiency, while 2 to 3% or more of celiacs are IgA deficient.
If a patient’s celiac panel is only positive for antigliadin IgG, this is not highly suggestive for CELIAC DISEASE if the patient has a normal total IgA level, corrected for age. Younger children make less IgA than older children and adults. A markedly elevated antigliadin IgG, such as greater than three to four times the upper limit of normal for that lab, is highly suggestive of a condition where the gut is leakier to gluten. This can happen in food allergies, cystic fibrosis, parasitic infections, Crohn’s disease, and other types of autoimmune GI diseases. These antibodies may also be slightly elevated in individuals with no obvious disease.
A strength of the antigliadin antibodies is that they are ELISA tests. This is a rapid immunochemical test that involves an enzyme, which a protein that causes a biochemical reaction. An ELISA test also involves an antibody or antigen. ELISA tests are utilized to detect substances that have antigenic properties, primarily proteins, such as gliadin. The importance of an ELISA test is that is it rapid, inexpensive, and run by a machine. Thus the results are independent of observer variability. The TTG test is also an ELISA test. This is in contrast to the antiendomysial IgA, where a slide has to be made, and a person has to look at it through a microscope. These are more prone to human error
Antiendomysium antibodies
excellent screening test for CELIAC DISEASE, with both a high sensitivity and specificity. It is considered the gold standard of antibodies. However, the subjective nature of this test may lead to false negative values and unacceptable variability between laboratories. This antibody was discovered in the early 1980′s, and rapidly gained use as part of a screening “celiac panel” by commercial labs in combination with antigliadin IgG and IgA. Its major drawbacks are that it may be falsely negative in young children, in patients with IgA deficiency and a lesser degree of villous atrophy, and in the hands of an inexperienced laboratory.Tissue transglutaminase antibodies or TTG
Since tTG had been first described as the autoantigen of celiac disease in 1997, it has been utilized to develop innovative diagnostic tools. The tTG IgA ELISA test is highly sensitive and specific. The tTG assay correlates well with EMA-IgA and biopsy. However, it represents an improvement over the antiendomysial antibody assay because it inexpensive, rapid, is not a subjective test, and can be performed on a single drop of blood using a dot-blot technique. One negative aspect of the TTG antibody is that it can be falsely positive in a patient who has another autoimmune condition. TTG false positivity has been described in patients with both type I diabetes and autoimmune hepatitis.
