Parts and functions of a compound microscope
(A) Arm: Used to carry the microscope.
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
- Specimen slide (will need tofocus under 10× power)
- Compound microscope.
- Mount the specimen slide onthe stage and focus under 10×.
- Close the iris diaphragm completely.
- If the ball of light is not in the center, use the condenser centering screws to move it so that it is centered.
- 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).
- Open the iris diaphragm until the entire field is illuminated.
- During regular microscope maintenance
- After the microscope is moved/transported
- Whenever you suspect objects do not appear as sharp as they could be.
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
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 (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 (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 (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 (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.
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 (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.
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.
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.
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 (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, 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.
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.
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.
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.
- "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
How will samples required for PCR, ELISA, or other immunoassay applications affect which pipette should be used?