Sensitivity Information and Courses from MediaLab, Inc.

The three most commonly used methods in antibody detection and identification are tube, gel and solid phase.Increased sensitivity in detection of antibodies is seen when using the tube method with PEG or the gel method (especially for mixed field reactions). Solid phase testing has a better sensitivity than just using the test tube method with LISS.

C-reactive protein (CRP) is an acute-phase protein produced by the liver in response to injury or tissue damage. It has been assayed for many years as a non-specific marker of acute inflammatory diseases, infections, neoplastic diseases, and other conditions where inflammation occurs. It is still assayed in this manner as a marker of inflammation by immunoassay methods that are sensitive to concentrations of 5-20 mg/L. Atherosclerosis is a subclinical chronic inflammatory condition. Highly sensitive measurements of CRP have been developed to detect this protein in lower levels that are sensitive to 0.5-10.0 mg/L. This assay is referred to as high sensitivity C-reactive protein (hs-CRP).

Dipsticks measure pH using methyl red and bromthymol blue indicator dyes. The color change that occurs in this test area correlates with the urine's pH. Sensitivity to pH ranges from 5.0 (acid pH) to 9.0 (alkaline pH) on a urine reagent strip.

False negative results may occur with some methods when the concentration of ascorbic acid is greater than 5 mg/dL. The sensitivity of the blood portion of the test strip is decreased in specimens with a high specific gravity and increased protein. High levels of nitrites may delay the reaction, causing a false negative to be reported. If the pH of a urine sample is below 5, hemolysis of red cells as part of the test reaction is inhibited which results in a false negative reaction. An improperly mixed specimen may test negative if the red blood cells are in the sediment.

Most clinical laboratories will use a 1:1 mix when performing mixing studies; however, some will use various dilutions of patient plasma and pooled normal plasma for their protocols.In addition, the analysis of the mixing study involves interpreting the pre- and post-mix results. This can be performed using various methods including: the Rosner Index, the <70% correction formula, or a laboratories own calculation and cut-off value. Finally, it is suggested that each laboratory test the sensitivity of their PT and aPTT reagents before running mixing studies. The sensitivity of the reagent system can be tested by running dilutions of the pooled normal plasma controls with specific factor deficient plasma. This ensures that the system will detect a normal result, even if the factor level is as low as 40%.

Molecular diagnostic techniques utilize CE extensively. Automation, microvolume sample, increased sensitivity, immediate detection, and the computerization provided by CE enhance the analysis of nucleic acids. A multiple fluorescence detection system available with CE is also valuable.CE analysis of short tandem repeat polymorphisms is used in forensics, parentage testing, bone marrow engraftment analysis and other identification assays. Other testing for diagnosis of genetic diseases, oncology studies and DNA sequencing frequently utilize CE. DNA sequencing uses CE for separation of nucleotides labeled with multiple colored fluorescence dyes; CE and these markers enable computerized determination of the nucleotide sequence of DNA segments.

The traditional CRP test uses immunoassay methods that are sensitive to concentrations of 5-20 mg/L. The hs-CRP test, with its increased sensitivity, is able to detect C-reactive protein in lower levels, 0.5-10.0 mg/L. As with most risk markers, the results of hs-CRP testing are generally interpreted on a relative scale; the higher the value, the higher the risk of a future cardiovascular event.The American Heart Association and Centers for Disease Control and Prevention has defined risk groups with hs-CRP as follows: Low risk: < 1.0 mg/L Average risk: 1.0 to 3.0 mg/L High risk: > 3.0 mg/L It is important to note that hs-CRP assays are measuring the same protein as traditional CRP assays. Thus, in patients with active inflammation (such as chronic, active arthritis; lupus; infection; etc.) hs-CRP values would be expected to be high and would not necessarily implicate cardiovascular risk. If values greater than 10 mg/L are seen in repeated measurements, a non-cardiovascular cause should be considered. Taking anti-inflammatory drugs (NSAIDs, aspirin, etc.) or the statin-class of cholesterol-lowering drugs may reduce CRP levels in patients. This is not an artifact, but is thought to be an effect of treating the underlying inflammatory process.

The INR component of the laboratory result is a calculated value that is used by the clinician to monitor anticoagulant therapy and adjust dosage as dictated by clinical status. An INR of 2.0 - 3.0 is often desired as the therapeutic range. The following formula is used by the clinical laboratory to derive an INR value. The INR must be adjusted for every new lot of PT reagent. INR= (PT of patient/PT of geometric mean of the normal population)ISI The International Sensitivity Index, or ISI value, is provided by the reagent manufacturer as the relative sensitivity of the reagent itself. The INR is used to standardize PT results, and in turn, anticoagulant therapy, across laboratory instrumentation, methodologies, and locale. Be sure to frequently check that ISI values match those of the lot currently in use as erroneous results may otherwise occur .

A molecular method may be the test method of choice for a variety of reasons, including the following: Organisms of interest, especially in microbiology or virology, may be extremely slow growing, fastidious or extremely limited in quantity. Other technologies lack the necessary sensitivity or specificity Patient population requirements

Serum ferritin (SF) level reflects the amount of storage iron in tissues. An elevated SF combined with elevated TS implies primary iron overload. Patients with hereditary hemochromatosis (HH) generally show increases in SF as adults, but a normal SF does not rule out the diagnosis of the disease. Children and premenopausal females with HFE mutations may have had inadequate time to develop iron overload, but may do so later in life.SF alone is inadequate as the sole screening test for HH because it lacks the necessary sensitivity and specificity. SF is frequently elevated in persons with inflammation, cancer, or infection. SF is often ordered along with the serum iron and TIBC when iron overload is suspected. SF is also important is assessing the efficacy of treatment of HH.Upper limits of reference intervals for SF are 200 ng/mL for premenopausal women and 300 ng/mL for men and postmenopausal women. 40 ng/mL is a typical lower limit for the reference interval.SF is measured in serum using immunochemical methods such as enzyme-linked immunosorbent assay (ELISA), immunoradiometric assay, immunochemiluminescent assay, and immunofluorometry. SF tests are available as automated assays and in kit form.(2)

Cervical cytology has been quite successful in the detection of cervical cancer. Since the carcinogenesis of cervical cancer is usually very slow, taking many years to decades to develop, cervical cytology can detect HPV-related cervical cancerous or precancerous states in most cases. The Papanicolaou-stained smear (Pap smear), is used most often to screen for pre-cancerous and cancerous processes by looking for changes to normal cervical cells. Abnormal cytology findings are investigated with colposcopy and biopsy. Colposcopy utilizes a powerful light source and lenses to locate and identify lesions that led to abnormal cells on Pap smear slides. If lesions are found, they too can be biopsied during colposcopy. For a Pap test, cervical cells are collected, fixed on a glass slide or placed in a liquid medium, fixed on slides, stained, and microscopically examined for abnormal cells. Sensitivity of a Pap smear is only 55%-80%; however its specificity is greater than 90%. Further information regarding cell types and terminology associated with the Pap test/cervical cytology testing are provided in the PDF resource that can be accessed from this page.

Eyestrain is a common problem associated with the prolonged use of computers or microscopes. Symptoms of eyestrain include: Blurred or double vision Itchy, dry, sore, burning, or tired eyes Headaches Color fringes / after images Sensitivity to light

Surveillance is a critical component of any program for controlling multi-drug resistant organisms. Many institutions are using active surveillance cultures to identify patients who are colonized with a targeted MDRO. With respect to MRSA, an increasing number of hospitals are screening patients upon admission and on a periodic basis (usually weekly). The anterior nares is the primary site that is swabbed for screening.There are several selective and/or differential media that can be used for this purpose.Baird Parker Agar is a selective medium for the isolation of S. aureus; on this medium S. aureus produces black colonies with a clear halo.Mannitol Salt Agar is also a selective medium; S. aureus produces yellow colonies which contrast with the red color of the medium.Chromogenic agars have been developed for the isolation and presumptive identification of different species of bacteria and yeast. The media are formulated so that as different organisms utilize various substrates in the media, the organism of interest produce colonies with a unique color. Chromogenic agars specifically designed for the detection of MRSA are commercially available.In addition to culture methods, there are now commercially available, FDA approved methodologies for screening for MRSA by PCR. Although equipment and cost factors may not make these a viable option for every laboratory, they may offer greater sensitivity and improved turnaround times.

Stool culture is very effective in detecting C. difficile. Unfortunately, non-toxigenic strains will also grow, requiring strains to be tested for toxin production. The greatest disadvantage to culture is the length of time that is needed before results are available, which may be up to four days. However, antibiotic sensitivity testing following culture is useful for strain-typing that would provide necessary epidemiological information during nosocomial outbreaks.Colonies of C. difficile will appear white, flat, and spreading on blood agar (see top image on the right). Cycloserin- cefoxitin-fructose agar(CCFA) is a selective media that is used for isolation of C. difficile. There is however, no distinction between pathogenic and commensal strains, which all produce yellow colonies with a characteristic "ground glass" appearance. as shown in the bottom image on the right. The characteristic odor of “horse manure” aids in identification of C. difficile. Stool samples are directly inoculated onto CCFA and incubated in an anaerobic atmosphere at 37ºC for 48 hours. Large, thin, gram-positive bacilli with spores will be observed on a Gram stain of a typical colony, as shown below.

Some literature in recent years has suggested that assays for GDH (glutamate dehydrogenase - an enzyme produced by C. difficile) could provide a more sensitive means of screening for C. difficile.Published studies have indicated that toxin immunoassays, by themselves, may not provide adequate sensitivity of detection. Several investigators have examined the utilization of a two step algorithm. This first step is an enzmye immunoassay for the GDH antigen.A negative result for GDH has been associated with a high value for prediction of a true negative result; however, a positive result is not necessarily associated with a toxin producing strain. A second assay on positive samples for detection of toxin production is required in these algorithms.

The genes involved in warfarin metabolism are CYP2C9 and vitamin K epoxide reductase complex subunit 1 (VKOR). Warfarin owes its anticoagulant action to its inhibition of VKOR. This enzyme recycles vitamin K, a critical element for the clotting factors II, VII, IX, and X, as well as for proteins C, S, and Z. There are six CYP2C9 alleles that are known to cause prolonged metabolism of warfarin: CYP2C9 *2, *3, *4, *5, *6, and *11. (Polymorphisms in CYP450 genes are denoted with asterisks.)One-third of the patients that receive warfarin metabolize it differently than expected and experience a higher risk of bleeding.Genetic testing for the two most common polymorphisms (CYP2C9*2 and *3) as well as for VKOR may be able to reduce the variability associated with warfarin dosing response. Labs performing PGx testing can provide general warfarin dosing recommendations based on the patient's genotype analysis. The lab report will indicate whether a patient has a normal, mild, moderate, high, or very high sensitivity to warfarin. For example, a patient who has one CYP2C9 normal wild-type allele (CYP2C9 *1), one polymorphism (CYP2C9*3), and also a VKOR polymorphism is predicted to have a moderate sensitivity to warfarin. This patient should have frequent INR monitoring and possible warfarin dose reduction. It is important to recognize that knowing a genotype does not necessarily guarantee accurate dose prediction; other drugs and/or environmental or disease factors can also alter CYP2C9 activity. Therefore, monitoring the INR is still very important.

Sensitivity is the ability of a test to correctly identify individuals who do have a particular disease or disorder. To determine sensitivity, we use the following equation: True Positives (TP) Divided by True Positives (TP) plus False Negatives (FN) Times 100 or (TP ÷ (TP + FN)) x 100 The result will be a percentage.

The tried-and-true method also had 600 true positives but had 50 false negatives. Once again inserting these numbers into our equation, (600 ÷ (600 + 50)) x 100, we find that the tried-and-true method has a sensitivity of 92%.

The sensitivity of our experimental method is 96% and our tried-and-true method is 92%. Sensitivity, reflects the ability of a test to categorize those individuals with the condition as positive. A highly sensitive test will be positive in most individuals having the condition. Conversely, those individuals who test negatively with a highly sensitive test are unlikely to have the condition.

Gram stain is used primarily as a differential stain for bacteria, although it will also stain most fungi (especially yeasts) and some parasites, including Strongyloides and Trichomonas. The Gram stain procedure is commonly performed on direct smears of clinical specimens and on smears from cultures. This course will focus on Gram-stained direct smears. A direct smear from a clinical specimen can be used to: Judge the quality of the specimen. Provide the clinician with same-day information regarding possible pathogenic organisms, pending results of culture and sensitivity. Contribute to selection of culture media, especially with mixed flora. Provide internal quality control when direct smear results are compared to culture results.

Gram stain is used primarily as a differential stain for bacteria, although it will also stain most fungi (especially yeasts) and some parasites, including Strongyloides and Trichomonas. The Gram stain procedure is commonly performed on direct smears of clinical samples and on smears from cultures. This course will focus on Gram stained direct smears. A direct smear is made from a clinical specimen. It can be used to: Guide the physician on initial choice of antibiotic, pending results of culture and sensitivity. Judge specimen quality. Contribute to selection of culture media, especially with mixed flora. Provide internal quality control when direct smear results are compared to culture results.

The pH of normal fresh semen is 7.0 or greater. Acid conditions can lead to reduced sperm motility and viability.Secretions of the different glands of the male reproductive tract contain fluids of different pH. The portion of semen that originates in the seminal vesicles is basic, that of the prostate gland is acidic. If the pH is low it could mean that there is an obstruction in the ejaculatory duct below the level of the seminal vesicles or it could signal absence of the vas deferens. Low pH is often associated with low volume since both basic pH and much of semen volume are products of the seminal vesicles. Measuring pH can thus help a clinician determine the cause of azoospermia (absence of sperm in semen).The pH is measured using pH testing strips. A drop of semen is placed on a pH strip and allowed to develop full color. Final color is compared to a standard. For the most accurate results, use test strips with a sensitivity in the basic range, for example from 6.0 to 10.0.

A variety of tests are available for the detection of influenza A viruses, including the 2009 H1N1 strain. These tests include: rapid antigen tests, direct fluorescent antibody tests to detect the presence of virus in patient specimens, shell vial cell cultures, classical tube cell cultures, and reverse transcriptase PCR (RT-PCR), which detects influenza-specific viral genes. These tests differ in sensitivity, specificity, availability, and the ability to distinguish between different influenza strains and subtypes, such as influenza A 2009 H1N1.The rapid tests, such as the direct rapid antigen tests or immunofluorescence assays, have lower sensitivity and specificity compared to cell culture and the RT-PCR based tests. Rapid tests vary in their ability to detect the 2009 H1N1 virus. The range of sensitivity is 10% to 70% and none of the rapid tests that are currently available are specific for H1N1. However, results of rapid tests are available within 30 minutes to one hour so that a positive test will provide further information toward a diagnosis when it is coupled with a patient's symptoms. A few FDA-cleared RT-PCR kits are available for the detection of influenza A viruses. For the subtyping of influenza A viruses, such as Influenza A seasonal H3N2, and 2009 H1N1, the FDA has given the status of "Emergency Use Authorization" (EUA) to a few of the RT-PCR kits; currently available kits under this emergency status category include those made by the CDC, ELITech, Prodesse, Focus Diagnostics, and Roche. (http://www.fda.gov/MedicalDevices/Safety/EmergencySituations/ucm161496.htm)State Departments of Health have been provided with RT-PCR kits from the CDC for the subtyping of influenza A viruses. This testing has also been FDA-reviewed and given the status of EUA. State and local health department guidelines determine which specimens should be submitted to public health laboratories for RT-PCR testing. In addition, several commercial reference laboratories, academic labs, and hospital labs have been able to perform influenza A subtyping for 2009 H1N1 under the same EUA status. Any laboratory that performs an EUA method would be required to perform an internal validation process.