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PARTS AND FUNCTIONS OF A COMPOUND MICROSCOPE

Published in Microbiology
Tuesday, 25 July 2017 10:42
The microscope is the most important piece of equipment in the clinic laboratory. The microscope is used to review fecal, urine, blood, and cytology samples on a daily basis (see Figure). Understanding how the microscope functions, how it operates, and how to care for it will improve the reliability of your results and prolong the life of this valuable piece of equipment.

Parts and functions of a compound microscope

Compound Microscope(A) Arm: Used to carry the microscope.
 
(B) Base: Supports the microscope and houses the light source.
 
(C) Oculars (or eyepieces): The lens of the microscope you look through. The ocular also magnifies the image. The total magnification can be calculated by multiplying the objective power by the ocular power. Oculars come in different magnifications, but 10× magnification is common.
 
(D) Diopter adjustment: The purpose of the diopter adjustment is to correct the differences in vision an individual may have between their left and right eyes.
 
(E) Interpupillary adjustment: This allows the oculars to move closer or further away from one another to match the width of an individual’s eyes. When looking through the microscope, one should see only a single field of view. When viewing a sample, always use both eyes. Using one eye can cause eye strain over a period of time.
 
(F) Nosepiece: The nosepiece holds the objective lenses. The objectives are mounted on a rotating turret so they can be moved into place as needed. Most nosepieces can hold up to five objectives.
 
(G) Objective lenses: The objective lens is the lens closest to the object being viewed, and its function is to magnify it. Objective lenses are available in many powers, but 4×, 10×, 40×, and 100× are standard. 4× objective is used mainly for scanning. 10× objective is considered “low power,” 40× is “high power” and 100× objective is referred to as “oil immersion.” Once magnified by the objective lens, the image is viewed through the oculars, which magnify it further. Total magnification can be calculated by multiplying the objective power by the ocular lens power.
 
For example: 100× objective lens with 10× oculars = 1000× total magnification.
 
(H) Stage: The platform on which the slide or object is placed for viewing.
 
(I) Stage brackets: Spring-loaded brackets, or clips, hold the slide or specimen in place on the stage.
 
(J) Stage control knobs: Located just below the stage are the stage control knobs. These knobs move the slide or specimen either horizontally (x-axis) or vertically (y-axis) when it is being viewed.
 
(K) Condenser: The condenser is located under the stage. As light travels from the illuminator, it passes through the condenser, where it is focused and directed at the specimen.
 
(L) Condenser control knob: Allows the condenser to be raised or lowered.
 
(M) Condenser centering screws: These crews center the condenser, and therefore the beam of light. Generally, they do not need much adjustment unless the microscope is moved or transported frequently.
 
(N) Iris diaphragm: This structure controls the amount of light that reaches the specimen. Opening and closing the iris diaphragm adjusts the diameter of the light beam.
 
(O) Coarse and fine focus adjustment knobs: These knobs bring the object into focus by raising and lowering the stage. Care should be taken when adjusting the stage height. When a higher power objective is in place (100× objective for example), there is a risk of raising the stage and slide and hitting the objective lens. This can break the slide and scratch the lens surface. Coarse adjustment is used for finding focus under low power and adjusting the stage height. Fine adjustment is used for more delicate, high power adjustment that would require fine tuning.
 
(P) Illuminator: The illuminator is the light source for the microscope, usually situated in the base. The brightness of the light from the illuminator can be adjusted to suit your preference and the object you are viewing.

KOHLER ILLUMINATION

Published in Microbiology
Tuesday, 25 July 2017 08:43
What is Kohler illumination?

Kohler illumination is a method of adjusting a microscope in order to provide optimal illumination by focusing the light on the specimen. When a microscope is in Kohler, specimens will appear clearer, and in more detail.

Process of setting Kohler
 
Materials required
 
  • Specimen slide (will need tofocus under 10× power)
  • Compound microscope.
 
Kohler illumination
 
  1. Mount the specimen slide onthe stage and focus under 10×.
  2. Close the iris diaphragm completely.
  3. If the ball of light is not in the center, use the condenser centering screws to move it so that it is centered.
  4. Using the condenser adjustment knobs, raise or lower the condenser until the edges of the field becomes sharp (see Figure 797.1 and Figure 797.2).
  5. Open the iris diaphragm until the entire field is illuminated.
 
Note the blurry edges of the unfocused light
Figure 797.1 Note the blurry edges of the unfocused light
 
Adjusting the condenser height sharpens the edges of the ball of light
Figure 797.2 Adjusting the condenser height sharpens the edges of the “ball of light.”
 
When should you set/check Kohler?
 
  • During regular microscope maintenance
  • After the microscope is moved/transported
  • Whenever you suspect objects do not appear as sharp as they could be.
 
Further Reading:
 

Wallach’s Interpretation of Diagnostic Tests, 9th Edition (Interpretation of Diagnostric Tests)

Published in Downloads
Monday, 10 April 2017 19:42

Description: Wallach’s Interpretation of Diagnostic Tests, now in its Ninth Edition, has been completely revised and updated by a new author team from the Department of Hospital Laboratories, UMass Memorial Medical Center faculty, who are carrying on the tradition of Jacques Wallach’s teachings. This text serves as a practical guide to the use of laboratory tests which aids physicians in using tests more effectively and efficiently by offering test outcomes, possible meanings, differential diagnosis, and summaries of tests available.

The book has been reorganized into 2 sections. The first section is devoted to an alphabetical listing of laboratory tests while stressing the integration of the clinical laboratory in the clinical decision making process. Test sensitivity, specificity, and positive and negative infectious disease probabilities are included whenever appropriate. Microbiology tests are listed in a separate chapter. The second section is devoted to disease states. Where appropriate, a patient’s chief complaint and/or physical findings are initially presented with subsequent discussions focused on discrete disease states as they relate to a patient’s chief complaint. Current molecular diagnostic testing, cytogenetics, common pitfalls, test limitations, and identification of appropriate tests for specific clinical presentations are also addressed.

Ninth Edition highlights include:
Detailed listing and description of routine and esoteric tests listed alphabetically, with information on when to order and how to interpret the test results based on evidence-based laboratory medicine.
Information on how to work up patients with specific symptoms and the appropriate lab tests to order
Up-to-date test procedures including molecular diagnostic tests
Detailed microbiology chapter of infectious diseases

Urine strip test — Understanding its limitations

Published in Biochemistry
Monday, 21 November 2016 20:46

Routine urinalysis is a cost-effective, non-invasive test used as an indicator of health or disease for metabolic and renal disorders, infection, drug abuse, pregnancy, and nutrition. Urine chemistry can be completed in a number of different ways, ranging from manual reading of a visual urine test strip to the use of semi-automated analyzers to loading the sample on a fully automated urine chemistry analyzer. There is one thing that all methods have in common: a urine chemistry reagent strip.

While urinalysis remains a routinely ordered laboratory test, today most of the emphasis focuses on automating urine microscopy to reduce manual, subjective microscopic work. Urine chemistry analysis is viewed by many as a screening tool that can help aid in the diagnosis of some common conditions such as urinary tract infections (UTIs), kidney or liver diseases, or diabetes, among others. It is important to remain focused on urine chemistry and better understand common test interferences.

Urine chemistry reagent strips comes in many different configurations, depending on their use. The most common tests include bilirubin, urobilinogen, glucose, ketones, protein, blood, nitrite, leukocyte esterase, and pH. In addition, some manufacturers include urine chemistry reagent pads for specific gravity, ascorbic acid, microalbumin, creatinine, and color. While urine chemistry testing is common, it is important to understand the test and its limitations to ensure accuracy of the test and recognize the factors that can cause incorrect results. Manufacturers have improved urine chemistry analysis by including additional tests to easily identify common interferences.

Bilirubin

Bilirubin (BIL) is a waste product of red blood cell (RBC) destruction. The primary source of bilirubin is the daily release of hemoglobin from the breakdown of RBCs in the reticuloendothelial system. In addition, RBC breakdown can occur in the bone marrow or other heme-containing proteins. The liver normally breaks down most of the bilirubin.

Healthy individuals exhibit a “negative” reading; very small amounts (0.02 mg/dL) can be found in urine but are undetected by routine testing techniques. The presence of bilirubin can indicate liver dysfunction such as jaundice, hemolytic disease, or obstruction of the bile duct or biliary system. A high amount of bilirubin, especially, affects the brain of newborns.

False positives can be caused by drugs that color the urine red, such as phenazopyridine, or large quantities of chlorpromazine metabolites. False negatives can be caused by the presence of ascorbic acid, increased nitrite concentrations, or improper sample storage.

Urobilinogen

Urobilinogen (URO) is a breakdown product of bilirubin. When high concentrations form in the body, the liver may not be able to break down all of the bilirubin present. Urobilinogen is produced in the intestines as bacteria metabolizes bilirubin. Small amounts (≤1 mg/dL or ≈1 Ehrlich unit) may be found in normal urine. However, the presence of urobilinogen is found with liver dysfunction, excessive destruction of RBC (hemolytic anemia, pernicious anemia and malaria), hepatitis, portal cirrhosis, and congestive heart failure.

Interferences for urobilinogen include formalin, high concentrations of nitrites, and drugs or substances that color the urine. If samples don’t equilibrate to room temperature before testing, that can produce an incorrect result.

Ketones

Ketones (KET) are normally not found in urine, but can be present when the body breaks down fat for energy. The body normally obtains energy from carbohydrates. If the carbohydrate supply is reduced, not absorbed properly, or not broken down metabolically, the body will use fat for energy. Ketones are associated with uncontrolled diabetes, vomiting, starvation, fasting, frequent strenuous exercise, and when the body uses fat instead of glucose for energy, which often occurs in people on a high-protein diet.

Agents containing free sulfhydryl groups can cause interference with ketone detection. Highly pigmented urine can result in false positive results, and improper sample or test strip storage may provide false negative results.

Glucose

Glucose (GLU) supplies the body with energy. In healthy individuals, glucose is reabsorbed by the kidney tubules and not present in the urine. However, if the concentration of blood glucose becomes too high (160-180 mg/dl), then the tubules can no longer reabsorb glucose and it will pass into the urine. This presence of glucose in the urine is called glycosuria. It is often associated with endocrine disorders such as diabetes, kidney impairment, central nervous system damage, and pancreatic disease. Other conditions associated with glycosuria include burns, infections, and fractures. Glycosuria is also associated with pregnancy.

High concentrations of ketones, decreased urine sample temperature, and increased specific gravity affect the sensitivity of the glucose pad. Increased ascorbic acid can also pose an interference. Bacterial glycolysis can occur with improper storage and can provide a false negative result.

Protein

The presence of protein (PRO) in the urine, otherwise known as proteinuria, is often the first indicator of kidney disease. It can also be indicative of other diseases such as nephrotic syndrome, glomerulonephritis, multiple myeloma, and pre-eclampsia. Exposure to cold, strenuous exercise, high fever, and dehydration can also cause the presence of protein in the urine.

The protein pad is most sensitive to albumin as opposed to other proteins. False positive results can be found with extremely alkaline samples. In addition to protein urine chemistry pads, there are also urine chemistry strips that test for microalbumin and creatinine for further assessment.

Blood

Blood (BLD) is not normally present in the urine and may not be visually present. The abnormal presence of RBCs in the urine is called hematuria, and the presence of hemoglobin in the urine is called hemoglobinuria. Blood in the urine is associated with kidney or urinary tract diseases, severe burns, infections, trauma, exposure to toxic chemicals or drugs, pyelonephritis, glomerulonephritis, renal or genital disorders, tumors, transfusion reactions, intravascular hemolysis, and hemolytic anemia. Strenuous exercise and menstruation can also cause the presence of blood in the urine. A positive result should be followed up with a microscopic correlation to assess for the present of RBCs and casts.

Urine specimens must be well mixed to ensure that RBCs have not settled out. Ascorbic acid should be considered an interferent when RBCs are present during a microscopic exam but the blood urine chemistry test is negative.

Nitrites

Nitrates (NIT) are consumed in the diet as green vegetables and are normally excreted without nitrite formation. The presence of bacteria in the urinary tract (e.g., bladder, kidney, etc.), can lead to the production of nitrites. Nitrite and leukocyte esterase screening help identify the presence of an infection. This screen should not replace further microscopic examination for bacteria or a culture to identify and quantify the bacteria present. It is used to quickly identify nitrate-reducing bacteria at a low cost.

Proper nitrite screening should be performed on a urine sample collected in the morning or after it has been retained in the bladder for at least four hours. High concentrations of ascorbic acid and improper storage can provide false results.

Leukocytes

Normal urine may contain a small number of white blood cells (WBCs) or leukocytes (LEUs). An increase in the presence of leukocyte esterase, an enzyme found in leukocytes, indicates inflammation in the urinary system. A WBC increase can be present with or without bacteriuria. If leukocytes are present without bacteria, there is usually a kidney or urinary tract infection (UTI) involving trichomonas, yeast, chlamydia, mycoplasmas, viruses, or tuberculosis. A positive nitrite and leukocyte esterase is a good indication for the performance of further microscopic examination.

High glucose, protein, and specific gravity can interfere with the leukocyte-esterase reaction, causing inaccurate results. In addition, specific antibiotics, drugs, and food (beets) can affect the chemical reaction.

pH

The kidneys play a major role in maintaining proper pH balance. Urine pH can affect the stability of formed particles in the body. Acidic urine (i.e., 4.5-6.9) is associated with, but not limited to, high-protein diets or the ingestion of cranberries, starvation, severe diarrhea, chronic lung disease, and UTIs with acid-producing bacteria (Escherichia coli) as well as certain medications. Alkaline urine (i.e., 7.0-7.9) is associated with, but not limited to, vegetarian or low-carbohydrate diets, vomiting, hyperventilation, UTIs with urease-producing bacteria, and certain medications. pHs that are below 4.5 should be suspected of adulteration, and pHs that are above 8 are often tied to improperly stored urine specimens.

Specific gravity

Specific gravity (SG) is a measure of the density of a urine. The more particles (i.e., salts, glucose, protein, etc.) in a urine, the higher the specific gravity. High specific gravity is caused by dehydration, diarrhea, heart failure, and glucose in the urine (i.e., diabetes). Low specific gravity is caused by kidney failure, diabetes insipidus, renal tubular necrosis, and the intake of too much fluids.

Urine test strips used for visual analysis often have a pH reagent pad. A limitation of the reagent pad is that it only measures the ionic solutions and can be susceptible to pH readings. Fully automated urine chemistry analyzers often use an onboard refractometer to obtain a specific gravity reading. A refractometer can be affected by particle size, temperature, and concentration of the solution as well as light wavelength. Some manufacturers have a specific gravity correction factor for high protein and glucose concentrations.

Ascorbic acid

Ascorbic acid, otherwise known as vitamin C, can be found in various foods and supplements. It is also a common interferent with urine chemistry reagent pads. When a urine sample has high levels of ascorbic acid, the reagent pads for blood, glucose, nitrite, and bilirubin may not react properly. This especially interferes with blood measurements at low levels. Clinicians should consider asking whether the patient is taking vitamin C when collecting a urine sample. We see more people taking vitamin C or vitamin C-like substances during the winter months or when traveling by plane, in an effort to boost their immune system.

Not all strip manufacturers have an ascorbic acid detection pad, as ascorbic acid is not commonly reported out. When the sample tests positive for ascorbic acid, the laboratorian may append a note with the results identifying potential interferences to the physician.

Color

Normal urine ranges from yellow/amber in color to clear or transparent and has a characteristic odor. A change in color, clarity, or odor is not necessarily a sign that something is incorrect. Urine changes color based on the body’s chemistry, food, medication intake, and state of hydration. Below is a list of colors, other than shades of yellow, found during urinalysis testing, along with their associated causes:

  • Orange: dehydration; certain medications; liver or bile duct issues
  • Blue/green: dyes in food or for kidney and bladder tests; medications such as amitriptyline, indomethacin (Indocin) and propofol (Diprivan); familial benign hypercalcemia, also known as blue diaper syndrome; UTIs caused by pseudomonas bacteria
  • Red/pink: UTIs; enlarged prostate; tumors; kidney cysts; long-distance running; kidney or bladder stones; the use of medications such as rifampin (Rifadin, Rimactane) or phenazopyridine (Pyridium); the use of some laxatives; the use of chemotherapy drugs. In addition, eating beets, blackberries, or rhubarb may cause the urine to turn red or pink
  • Brown: liver and kidney disorders; UTIs; extreme exercise; ingesting large amounts of certain foods (e.g., fava beans, rhubarb, or aloe); medications such as the antimalarial drugs chloroquine and primaquine, antibiotics metronidazole (Flagyl) and nitrofurantoin
  • Cloud/murky: urinary tract infection (UTI)

Urine color can interfere with some of the aforementioned tests during the color reaction process that takes place on the pad. For this reason, some manufacturers have a “blank” or color compensation pad on the dipstick. This color compensation pad will identify the color of the urine, and the analyzer will “subtract out” the color from other readings to provide a more accurate result.

The lab’s perspective

As noted above, specimen storage is a concern for a number of tests. Most manufacturers require testing within one to two hours of collection. If this is not feasible, samples are often refrigerated or stored in a preservative tube for testing at a later date. It’s important to note that few manufacturers have validated the use of preservative tubes for analysis on their urine analyzers, so lab leaders should assess their needs before purchasing a system.

In summary, urinalysis remains an informative laboratory test. It is important to understand what is being tested and what can interfere with the test, since certain medications and vitamins interfere with urinalysis testing. For example, during the winter months, more and more people are taking vitamin C in an effort to “starve a cold,” and we see ascorbic acid as an interference in bilirubin, glucose, blood, and nitrite testing. It is also important to understand the patient’s diet and exercise level, since they can impact results as well. Laboratorians should become very familiar with the manufacturer’s instructions for use to know what the limitations of the analyte are in order to ensure accurate reporting.

iDu Optics LabCam Microscope Adapter for iPhone

Published in Technology
Saturday, 22 October 2016 20:06

iPhones take great pictures. This adapter makes it super easy to take images and make videos with your iPhone through your microscope. You can even use your iPhone to live project/stream your view. The iDu adapter fits iPhone6/6s. It's fitted with a 10x magnifying lens and comes with two adapters to fit a 30 mm or 23 mm eyepiece slot (it should fit all Nikon, Olympus, Zeiss, Leica and other common brand microscopes). Compatible with any compound, dissection, or fluorescent microscope. Simply remove the microscope eyepiece and insert the iDu. Easy as pie.

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Status of laboratory testing for HIV

Published in Immunology
Monday, 17 October 2016 10:41

Two years have passed since the CDC finally published guidelines addressing HIV laboratory testing and officially endorsed the “new” HIV laboratory testing algorithm. Although many had become aware of the algorithm in the four years prior, and had adopted it to various degrees, this was the final word on this long-awaited guidance. The algorithm gained visibility prior to the official endorsement mainly because it had been heavily referenced in CDC publications and numerous scientific articles.

Advantages of the new algorithm

Why is the new algorithm superior to the old algorithm? First, the new algorithm emphasizes the use of an antigen/antibody (Ag/Ab) combination assay to screen for HIV infection, as the first step. The use of this more advanced technology (fourth generation) provides improved detection of acute HIV-1 infection because antigen/antibody combination assays not only detect established infection in those who have seroconverted, but can also diagnose HIV infection prior to seroconversion by detecting p24 antigen. Fourth generation assays detect acute HIV infections, on average, five to seven days earlier than the third generation, antibody-only assays.

Second, substituting the HIV-1/HIV-2 differentiation assay for the Western blot in the second step allows for correct identification of HIV-2 infection and earlier detection of HIV-1 infection, compared to the Western blot.

Third, the official addition of nucleic acid testing (NAT) is used to rule out acute HIV-1 infection, which is necessary because although HIV-1/HIV-2 differentiation assays can detect HIV infection on average a few days earlier than the Western blot, none of these can detect HIV infection prior to seroconversion.

There is ample evidence that the new algorithm has increased detection of acute HIV-1 infections, due to the use of Ag/Ab combination assays. This is important both for the patient, who can receive prompt treatment that improves health outcome, and also from a public health perspective, because it reduces disease transmission. Many laboratories now have access to a fourth generation assay, since they are offered by multiple vendors on a variety of automated platforms.

The data are not yet in as to whether the new algorithm has resulted in a significant increase in yield of HIV-2 diagnoses; this would provide critical information regarding prevalence and transmission of HIV-2 infections in the United States.

Challenges of the new algorithm

The new algorithm, however, has presented some real challenges for the laboratory. The biggest adjustment to adopting the new algorithm has been replacing the Western blot with an HIV-1/HIV-2 differentiation assay. The only assay with this capability until recently was the Multispot (Bio-Rad). However, the Multispot is no longer available and will be replaced with Bio-Rad’s Geenius. Although the Geenius is also a single use test (FDA-cleared) for confirming reactive HIV screen results and differentiating between HIV-1 and HIV-2 antibodies, it differs from the Multispot in a number of important aspects. The test uses either recombinant or synthetic peptides corresponding to four HIV-1 antigens, gp160, gp41, p31 and p24, and two corresponding to HIV-2 antigens, gp140 and gp36. There are eight possible interpretations based on the pattern observed. Performance characteristics are comparable to Multispot. Sensitivity is 100 percent for both assays, and specificity values are 99.1 percent and 96.3 percent for the Multispot and Geenius, respectively. The results can be read within 30 minutes and are interpreted using an automated cassette reader, therefore eliminating inter-observer subjectivity. The cassette system also allows for placement of a bar code label on each specimen, improving sample tracking. Additionally, because software is necessary for interpretation, the results are digitally captured, automatically recorded, and stored.

However, because the new HIV-1/HIV-2 differentiation assay requires an additional investment in the reader/software component, beyond the cost of the reagents, there is some concern that some small hospital laboratories will revert to sending out supplemental HIV testing to a reference laboratory. It should also be noted that, although adoption of the new algorithm has grown significantly, there is still substantial demand for Western blot testing. Importantly, when a third or fourth generation assay was used for screening, an indeterminate or negative Western blot should also be followed up with NAAT.

There is also much confusion regarding appropriate use of the fourth generation rapid HIV test. Although at first glance it would appear that this assay can be used in lieu of the laboratory based Ag/Ab combination assay and serve as the entry point into the algorithm, that is not the current CDC recommendation. Citing insufficient evidence for such an approach, the CDC suggests that a preliminary positive result obtained with any rapid test, including an antigen/antibody combination rapid test, must be followed up with a laboratory-based antigen/antibody combination assay.

Fifth generation testing

The horizon appears even more complicated now that the “fifth generation” HIV testing is available. This technology is currently offered only by one vendor, but it has the ability to differentiate between antigen, HIV-1 and HIV-2 antibody-positive specimens. While this simplifies the answer with regard to HIV infection status for the patient, there are no guidelines as to how to proceed with follow-up testing. For example, if the sample is positive for antigen only, then the logical follow-up would be to send out for NAT testing, as there is no reason to test with the supplemental HIV-1/HIV-2 differentiation assay that only detects antibodies. If the sample is positive for HIV-2 only, is it appropriate to follow up with the HIV-1/HIV-2 differentiation assay, because the fifth generation test is FDA-approved as a screen only and a supplemental test is needed? Fifth generation technology presents further complications to the algorithm and more complexity for the laboratory in terms of appropriate follow-up and interpretation for clinicians.

Last, one unintended consequence of the new algorithm is the effect on HIV surveillance programs. Ideally for the purpose of HIV surveillance, public health departments would like to have the final answer as to whether a patient has HIV-1, HIV-2, or acute HIV-1 infection, once the HIV testing algorithm is complete. The problem is that this is almost impossible because testing is almost always fragmented and different steps of the algorithm are performed in different laboratories. Often primary institution laboratories have the ability to perform the screening, even with a fourth generation Ag/Ab combination assay, but cannot complete the remainder of the algorithm. The sample is then sent to the reference laboratory, and that laboratory has to determine how to interpret the results without having the screen results. How to report a partial result and make it clear to the clinician that additional testing is needed and also satisfy public reporting needs is much more difficult in the context of the new algorithm, for both the primary and reference laboratory.

In summary, many technological advances have been made that importantly improve detection of HIV-2 and acute HIV-1 infections. These advances are beneficial for both the patient and society. Although most clinicians and laboratories are now familiar with and support the implementation of the algorithm, laboratories are challenged more than ever to provide appropriate test result interpretation and utilization as well as adequate public health reporting for HIV.

References

  1. "Laboratory Testing for the Diagnosis of HIV Infection: Updated Recommendations". BioScience.pk Digital Library Database. Centers for Disease Control and Prevention (CDC). Published June 27, 2014.

    About the author: Patricia Slev, PhD, DABCC, is Associate Professor of Pathology (Clinical), University of Utah and Medical Director of the Serologic Hepatitis and Retrovirus Laboratory, Core Immunology Laboratory and Co-Director Microbial Immunology Laboratory,  at ARUP. Board certified by the American Board of Clinical Chemistry, Dr. Slev’s research interests are immunogenetics and pathogen interactions, particularly HIV and viral hepatitis.

Source: Medical Laboratory Observer: The status of laboratory testing for the diagnosis of HIV infection

Contamination of Laboratory Equipment with Bloodborne Pathogens

Published in Pathology
Sunday, 09 October 2016 11:08
Clinical laboratory workers encounter a variety of occupational hazards, including exposure to infectious agents. The routes of pathogen exposure associated with laboratory work include ingestion, inhalation, direct inoculation, and contamination of skin and mucous membranes. The accidental inoculation of infectious materials (i.e., via contaminated needles, broken glass, or other sharps) is the leading cause of laboratory-associated infections.
 
In the 1980s, the emergence of the HIV epidemic created an appreciation for biosafety and laboratory-acquired infections, ultimately leading to both practice guidelines and legislation to reduce the risk of exposure of laboratory workers to bloodborne pathogens; this was implemented by the adoption of Universal Precautions in 1987, and later, in 1996, these became a component of Standard Precautions. The actual incidence and risk of laboratory-acquired infections is very difficult to quantify in the absence of standardized reporting systems, as well as the challenge of attributing a specific exposure to acquisition of infection. The most comprehensive studies to date attempting to estimate the incidence and epidemiology of laboratory-acquired infections were completed prior to 1980, and it is difficult to generalize them to today's laboratory environment, for a variety of reasons—standards for personal protective equipment have changed, the availability of vaccines, the epidemiology of bloodborne infections has changed, and the way laboratory testing is performed has changed (with a shift away from primarily manual methods to more automated methods). That said, since 1999, only 1 confirmed case of laboratory-acquired HIV infection has been reported in the US; this case was secondary to a needle stick injury sustained in an individual working with a live HIV culture.
 
The fear associated with the recent Ebola virus (EV) epidemic triggered a renewed interest in occupationally acquired infections in healthcare workers in the US, including the safety of laboratory workers in handling samples from persons under investigation (PUIs) for EV disease (EVD). Individuals at risk for EVD are also at risk for several other infectious diseases with overlapping symptom profiles (such as malaria, influenza, and bacteremia) thus obligating a number of diagnostic laboratory tests. In addition, the clinical management of patients with EVD requires ongoing laboratory testing to optimize care (such as complete blood count, coagulation testing, electrolyte analysis, etc.). Laboratory testing for suspect or confirmed EVD patients is unfamiliar to most healthcare workers in the US, and thus determining the safest approach to this testing generated anxiety and controversy.
 
The CDC recommended that laboratories perform a risk assessment to minimize the risk to laboratory staff, and determine what equipment would be available or needs to be acquired for providing critically important testing. The infectious dose for EV is estimated to be 10 viral particles, and patients with EVD may have EV viral loads of ≥108 plaque-forming units/mL. Thus, in the absence of data regarding the potential risk of laboratory testing using automated analyzers, many laboratories have opted to utilize point-of-care testing devices or conduct testing for PIUs in a separate lab, outside of the routine laboratory workflow. Even among designated US Ebola Treatment Centers, the approach to laboratory testing is highly variable, with some centers performing testing in a Biosafety Level-3 laboratory, some within a patient isolation unit, and some within the clinical laboratory of the hospital.
 
In this issue of Clinical Chemistry, to gather data on the frequency of contamination of laboratory equipment with blood-borne pathogens, Bryan et al. conducted an important evaluation of total laboratory automation (TLA) used in the routine clinical laboratory. Quantitative PCR assays were used to evaluate the extent of TLA contamination after blood samples were processed through routine workflow. Because it was not practical to test EV directly, contamination with hepatitis B virus (HBV) and hepatitis C virus (HCV) were used as surrogates to measure contamination events. To establish a baseline level of instrument contamination, the authors evaluated numerous components of the TLA at multiple time points. Out of 79 baseline swab samples, 10 (12.6%) and 8 (10.1%) were positive for HBV and HCV, respectively. The contaminated sites were associated with visible flecks of dried blood, and generally located near the decapper portion of the instrument. The authors then tested TLA surfaces and clean glass slides placed throughout the TLA after sample processing of high titer HCV samples (mean of 5.8 × 107 IU/mL). The purpose of the clean glass slides was to directly assess contamination events specifically attributed to the high titer HCV samples, thereby eliminating detection of baseline contamination events. The authors detected HCV contamination on 1 glass slide out of the 54 that were tested. Of note, although HBV and HCV were detected in this study, it is unknown if the viral particles were still viable/infectious. No contamination events were identified when sites away from the TLA were sampled, indicating that cross-contamination from the TLA to other sites did not occur in the sites examined. Although the authors examined only HBV and HCV, we would anticipate similar contamination patterns with other bloodborne pathogens, such as EV.
 
The results from this important study reveal that background contamination of TLA with HBV and HCV occurs during routine clinical use. This is an important finding, especially considering HBV viability in dried blood has been demonstrated for up to 7 days at room temperature and HBV can be present in very high concentrations in clinical samples (up to 109 IU/mL). Although HBV was the most commonly reported laboratory-acquired infection, the incidence of such infections has decreased drastically in the era of standard precautions and HBV vaccination. Additionally, although HCV seropositivity is slightly higher in healthcare workers compared to the general public, laboratory-acquired HCV infection appears to be rare, with only single case reports.
 
The evolving guidance from the CDC on the testing of PUIs for EVD stresses the importance of performing a risk assessment to identify potential exposure sources and to mitigate those events. It should be emphasized that infection with EV, similar to HIV, HBV, and HCV, requires direct exposure to EV-contaminated blood or bodily fluids, so proper personal protective equipment is essential when handling clinical samples. Additionally, the CDC recommendations state that laboratories should minimize processes that would generate aerosols that would expose laboratory staff to EV, by the use of engineering controls and safety equipment when available. There are several reports of hospitalized patients in the Netherlands, the US, and South Africa with initially undiagnosed EV or the related Marburg virus infections that had extensive contact with healthcare workers, including laboratory staff working with clinical samples from these patients. In these reports, none of the healthcare workers contracted EV or Marburg virus from these patients, which could be attributed to adherence to proper use of personal protective equipment and standard precautions.
 
It is incumbent on healthcare facilities to provide the tools and training to be able to properly evaluate PUIs for EVD, as well as other infections that could mimic EVD (i.e., meningitis, malaria). These other infections are potent and can be deadly if they are not treated promptly and properly. To this point, a recent report describes 3 instances between 2014–2015, in which malaria testing was significantly delayed in PUIs for EVD. One patient was initially empirically treated for malaria without having had malaria testing (which is counter to CDC recommendations), and in another patient the level of parasitemia was not evaluated, which is an important factor in determining which antimalarial treatment should be administered.
 
Importantly, the investigation by Bryan et al. illustrates that contamination events do occur with pathogens (i.e., HBV and HCV) that are frequently encountered, likely on a daily basis. Although contamination of laboratory equipment with bloodborne pathogens may be common, laboratory-acquired infection with these agents is not. This underscores the importance of adherence to standard precautions when handling all patient samples, and treating every clinical sample as though it may contain an infectious agent.
 
Important questions for clinical laboratories that are not addressed by this investigation include what methods are needed to adequately decontaminate laboratory equipment, the frequency with which these methods could or should be deployed, and the risk that these methods may pose to laboratory workers. Laboratories should work closely with device manufacturers to understand best practices for instrument decontamination.
 
EV and other emerging pathogens will continue to be encountered in the clinical laboratory. It is the joint responsibility of laboratorians and laboratory leadership to create a culture of safety and adherence to safety protocols, which are essential to reduce the risk of laboratory-acquired infections.
 

What to Know Before Buying Pipettes

Published in Others
Friday, 07 October 2016 19:14
Why is it important to know the viscosity of the samples that will be aspirated / dispensed?
 
The viscosity of the sample will have a direct influence on which pipette is required. For aqueous samples which are low viscosity, an air displacement pipette is ideal. These pipettes are driven by a piston in an airtight sleeve which generates a vacuum. For more viscous or heavy liquids, a positive displacement pipette should be used. These pipettes are driven by a disposable piston which comes into direct contact with the sample.

How will samples required for PCR, ELISA, or other immunoassay applications affect which pipette should be used?
 
PCR, ELISA, and many other immunoassay methods utilize microwell plates. Microplates come in many configurations: 6, 24, 96, 384, and 1536 well plates arranged in a 2x3 matrix are typically used. In order to accommodate faster throughput with such methods, many pipette manufacturers offer multichannel pipettes. These allow for faster pipetting of multiple samples: instead of having to fill 96 wells individually, an 8 channel pipette can be used, reducing the number of aspirations and dispenses to 12.
 
How does the volume of samples being worked with influence which pipette is the best fit?
 
Pipettes come in a variety of different sizes to suit whichever volume needs are required. If you know you will always be pipetting the same volume of liquid, then a fixed volume pipette will be best. If the amount to be pipetted is changing from sample to sample, then a variable pipette will be ideal.
 

Handbook of HPLC, 2011

Published in Downloads
Monday, 25 April 2016 20:01
Description: High performance liquid chromatography (HPLC) is one of the most widespread analytical and preparative scale separation techniques used for both scientific investigations and industrial and biomedical analysis. Now in its second edition, this revised and updated version of the Handbook of HPLC examines the new advances made in this field since the publication of the benchmark first edition twelve years ago. It reports detailed information on fundamental and practical aspects of HPLC related to conventional format and sophisticated novel approaches which have been developed to address a variety of separation problems in different fields.

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