LII:The Comprehensive Guide to Physician Office Laboratory Setup and Operation/Primary laboratory testing domains in the POL

A wide variety of testing may be done in the physician office laboratory (POL), though much of it may be CLIA-waived testing. This chapter talks about the primary testing domains encountered in POLs. These testing domains seem to be relatively common in a POL and offer CLIA-waived tests.

This third chapter on the topic of primary testing domains has six sections.

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3. Primary Laboratory Testing Domains in the POL

Urinalysis

In June 2014, healthcare market research company Kalorama Information estimated that by volume, dipstick urinalysis easily was the most common test type in the POL. Urinalysis' popularity in the POL is in part due to advanced technology, low cost, and high market competition, which have in turn helped make many such in vitro diagnostic (IVD) tests eligible for CLIA's waived category. In fact, more than 1,200 CLIA waivers have been granted for dipstick urinalysis kits alone since 2004. Advances in CLIA-waived benchtop and portable dipstick readers have further improved dipstick interpretation accuracy and increased urinalysis test volumes in the POL.

Another perhaps more important reason for dipstick urinalysis' popularity in the POL is the sheer usefulness of the IVD tool. The College of American Pathologists (CAP) have referred to the urine dipstick as "a remarkable 'lab-on-a-strip'" for its ability to "help detect infection, bleeding, diabetes, and other problems." For example, the pH, specific gravity, and protein prevalence as well as the presence of constituents such as bilirubin, glucose, and nitrites all can aid a physician in ultimately making a solid diagnosis. Even pregnancy can be determined using urine dipsticks specifically designed to detect the hormone human chorionic gonadotropin (hCG).

Basic concepts and collection procedures

The human urinary system is responsible for several tasks, including filtering blood, excreting waste, regulating pH, regulating blood pressure, and creating the hormone erythropoietin, which stimulates red blood cell production in the body. The system is composed of kidneys, ureters, the bladder, and the urethra, all of which play an important role in forming and excreting urine from the body. In short, blood passes through the kidney, and through capillary action much of the liquid of the blood is filtered out and collected, eventually passing through the ureters to the bladder and out the urethra.

When collected properly using quality assurance methods, the expelled urine then has clinical significance when analyzed using physical, chemical, and even microscopic procedures. However, maintaining quality standards is vital; otherwise, specimens could become contaminated or reach a state not ideal for testing. Clinical collection procedures encourage mid-stream collection of urine into a sterile, well-labeled container. The contents should be tested within an hour of collection or otherwise rapidly refrigerated. The implementation of standardized terminology, equipment calibration techniques, quality control procedures, and proper reagent storage techniques also help ensure test results and interpretation are as accurate as possible. And of course proper hand, eye, and body protection should be correctly used when handling urine.

Urine composition

Urine is composed predominately of water (95 to 96 percent), with the rest being made up of dissolved organic and inorganic solid waste. When collected over a 24-hour period, the average healthy adult's urine contains approximately 40 to 50 grams of dissolved solid waste. The primary constituents of that waste are, in order of prevalence (assuming a 1,400 mL sample):

• urea, the molecule CO(NH₂)₂ which acts as a primary carrier of waste nitrogen from protein breakdown in the body: 25.0 grams
• chloride, the anion Cl^- which is a vital electrolyte in the blood: 6.3 grams
• sodium, the cation Na⁺ which is vital to osmotic equilibrium in the body: 3.0 grams
• potassium, the cation K⁺ which is vital to proper neuron function and osmotic equilibrium: 1.7 grams
• creatinine, an endogenous byproduct of muscle creatine metabolism: 1.5 grams
• sulfate, the anion SO₄²⁻ which is a byproduct of protein turnover and the metabolism of several sulfur-containing compounds in food and water: 1.4 grams
• dihydrogen phosphate, the anion H₂PO^4- (as a titratable acid) excreted as a byproduct of renal acid-base regulation: 1.2 grams
• ammonium, the cation NH⁴⁺ which is excreted as a byproduct of renal acid-base regulation: 0.8 grams
• amino acids, organic compounds composed of amine (-NH2) and carboxylic acid (-COOH) that are building blocks of proteins: 0.8 grams
• uric acid, the compound C₅H₄N₄O₃ which is a byproduct of the breaking down of substances called purines in the body: 0.7 grams
• calcium, the cation Ca²⁺ which is vital to many functions of human biology: 0.2 grams
• magnesium, the cation Mg²⁺ which is vital to the nucleic acid chemistry of most life: 0.15 grams

Clinical laboratories set reference ranges for these constituents, and when a sample shows one or more of them outside of that reference range, the abnormality is often a sign of an ailment or problem in the body or with a diet. For example, the U.S. National Library of Medicine indicates the normal reference range for urea is 12 to 20 grams (though they also note "normal value ranges may vary slightly among different laboratories"), and numbers outside that range could indicate anything from kidney problems and malnutrition to too much protein in the diet.

Other constituents that normally aren't found in urine in significant quantities (or not at all) can make their way there, and their measurable presence are strong indicators of an underlying illness. Those constituents include:

• bilirubin, a bile pigment resulting from red blood cell breakdown that potentially indicates bile duct blockage, hepatitis, or cirrhosis
• casts, cylindrical particles formed from kidney cell proteins, appear in minute amounts as hyaline in normal samples; the presence of cellular casts containing red and white blood cells indicate a kidney disorder
• crystals, formed from solutes in urine under specific conditions; crystals formed from solutes typical to the healthy individual appear, but the presence of crystals formed from non-typical solutes such as cystine, tyrosine, and leucine potentially indicate illness such as liver disease
• epithelial cells, which line the cavities and surfaces of bodily structures, are normal in tiny amounts; the heavy presence of certain types of these cells in the urine indicate a urinary tract condition such as an infection
• erythrocytes, otherwise known as red blood cells, are a primary oxygen delivery vehicle, and their presence in urine potentially indicates a urinary tract infection or hemorrhage, prostate issues, or cancer of the bladder or kidney
• glucose, a sugar that is typically reabsorbed into blood in the kidney during filtering; its presence could indicate a type of diabetes or alimentary glycosuria
• hemoglobin, a protein found in red blood cells that when found in urine potentially indicate anything from kidney infection to tuberculosis
• ketones, byproducts of fat metabolism that potentially indicate diabetes mellitus or a dietary issue
• leukocytes, otherwise known as white blood cells, protect the body from infectious disease, and their presence (indicated by leukocyte esterase) in urine potentially indicates bladder or kidney infection
• nitrite, the anion NO₂⁻ which appears in urine typically due to endogenous nitrates being converted to nitrites by bacteria, indicating potential bacterial infection
• protein, a building block of life, that when found in large quantities in urine could indicate heart and blood pressure problems, urinary tract problems, or dehydration
• urobilinogen, created from the breakdown of bilirubin, is also potentially indicative of bile duct blockage, hepatitis, or cirrhosis

Testing

The presence of the above constituents — as well as the pH, specific gravity, and clarity of the urine — is determined through visual, chemical, and microscopic laboratory testing. Visual inspections of a urine sample aren't meant to be precise, but the designations of clear, hazy, and cloudy provide a starting point for determining the type and number of constituents in a sample. Later, chemical and microscopic analyses provide more concrete information.

Some constituents like glucose, ketones, nitrites, and proteins are most easily detected through chemical analysis. Others like casts, crystals, epithelial cells, and red and white blood cells are better detected using microscopy. As such, the constituents (or properties) being tested for largely drive the instruments and IVD test kits used. The specific gravity test, for example, can be measured using either a reagent strip or refractometer. The choice of which device to use is most often determined by the CLIA status of a laboratory; a dipstick analysis is largely considered waived testing whereas a refractometer test is non-waived. A similar consideration is made when testing for blood in urine: a chemical-based dipstick test for hemoglobin or a microscopic analysis for the presence of red blood cells can be performed. The dipstick test will likely be CLIA-waived, whereas the microscopic examination is certainly not CLIA-waived. As such, if the highest level of testing done at a POL will be urine dipstick testing, only a CLIA certificate of waiver is needed for the lab; if microscopic urinalysis is to be done, the lab must acquire the next highest level of certification, a provider-performed microscopy (PPM) certificate.

For POLs with only a CLIA certificate of waiver, the primary tools used for urinary analysis will be CLIA-waived urine reagent strips and automated urine analyzers. Express Diagnostics' UrinCheck HealthScreen-10 reagent strips, for example, are CLIA-waived and test for eight of the above mentioned constituents as well as pH and specific gravity. The results are then compared visually with a color-coded guide on the bottle. To avoid color-coded visual inspection, a CLIA-waived urine analyzer like Roche Diagnostics' Urisys 1100 allows associated test reagent strips to be inserted and electronically analyzed, printing out the results.

For POLs with a PPM certificate, CLIA-waived urine reagent strips and analyzers can still be used. However, certified physicians and mid-level practitioners at the lab may also turn to the bright-field or phase contrast microscope to further evaluate urine for constituents that would indicate disease or injury. Bright-field microscopes are one of the simplest, transmitting white light that gets partially absorbed by the denser parts of the sample, creating contrast. The phase contrast microscope is more complicated, separating the transmitted background light from the light scattered by the specimen to make phase changes more visible. Specimens have to be minimally processed and labile to ensure the accuracy of the test. Microscopic examination is often done reflexively upon confirming abnormal visual and/or chemical results, as suggested by The Clinical and Laboratory Standards Institute. The chemical-based urine reagent strips in some cases can only provide a preliminary diagnosis, requiring microscopy to verify amounts of bacteria or types of crystals in the urine, for example.

You can find an extensive listing of CLIA-waived POL instrument and test kit vendors (including for the constituents mentioned above) in the Additional Resources section of this guide.

Hematology and blood collection

In July 2014, healthcare market research company Kalorama Information estimated that by volume, the hematological complete blood count (CBC) test was second only to the dipstick urinalysis in U.S. POLs. Kalorama estimated that while 75 percent of CBC tests in the country were being performed in hospitals and commercial reference labs, the other 25 percent of tests — nearly 100 million — were being realized by POLs and "near-patient clinics." What's not directly mentioned by Kalorama, however, is the fact that CBC testing falls in the domain of at minimum CLIA moderate complexity testing; no CLIA-waived CBC test devices yet exist. This means that as of July 2015 only 17,737 POLs, representing only 14.5 percent of all registered POLs, were capable of offering CBC testing. So where does that leave the other 85 percent of CLIA-certified labs with PPM and waiver certificates? What hematological testing is available to them? That will be addressed later in the testing section.

Kalorama also noted that another hematological test is third most popular in U.S. POLs: prothrombin time (PT) and the related international normalized ratio (INR), referred to collectively as PT/INR. The market research company estimated that 20 million such tests are conducted in U.S. POLs every year. PT/INR is a coagulation test used to measure the tendency for blood to clot and is a vital test for patients taking the anticoagulant Warfarin or some other type of oral anticoagulant therapy. Several dedicated handheld analyzers are available to CLIA-waived physician office laboratories wishing to conduct PT/INR testing. (See the testing section for more.)

Basic concepts and collection procedures

Blood is responsible for moving oxygen and nutrients to all the necessary cells of the body while also removing metabolic waste products that are formed from bodily processes. Blood is pumped through the body by the heart through the various blood vessels of the circulatory system. Arteries carry the blood away from the heart to the various parts of the bodies that contain capillaries. These tiny vessels allow water, oxygen, and chemicals to pass from the blood to the tissues and back. Veins, a third type of vessel, usher the blood from the capillaries back toward the heart. In all but the pulmonaries, arteries carry highly oxygenated blood while veins carry oxygen-depleted blood.

When blood must be collected for analysis, it will typically come from either specific veins in the antecubital area of the arm or the capillary beds of the fingers or heel, depending on the volume needed for testing. In rare cases such as respiratory emergencies, an arterial blood draw may be necessary, in which case the radial or brachial artery is used. The procedures used to draw and collect blood from these areas differ, and careful attention must be paid to handling of the blood upon collection, especially with arterial draws. Likewise, collection equipment may vary slightly depending on method and area of collection. Butterfly needles, for example, may be used for patients with small veins or for pediatric patients, while special heparinized syringes are used for arterial draws. However, safety equipment like gloves and sharps containers will always need to be used regardless of area and method, as required by the Occupational Safety and Health Administration (OSHA).

Blood composition

Blood is roughly 52 to 62 percent plasma and 48 to 38 percent formed elements such as red blood cells (RBC), white blood cells (WBC), and platelets. The plasma portion of blood is 92 percent water and eight percent constituents such as albumin (a protein that helps move small molecules through blood), fibrinogen (a protein that helps with clotting), and globulins (a protein that includes antibodies). In times of illness or poor health, the formed elements and proteins of blood can be used as important indicators to help diagnose diseases and other ailments.

The study of blood and its constituents is called hematology, practiced in laboratories all around the world. The complete blood count (CBC) represents one of the most common hematology tests used, analyzing RBC, WBC and platelet counts; hemoglobin concentration; hematocrit; WBC differential; and three RBC indices. Suggested reference ranges for these are:

• WBC count: 4,300 to 10,800/mm³
• RBC count: 4.2–5.9 x 10⁶/mm³ for adult females; 4.6–6.2 x 10⁶/mm³ for adult males
• platelet count: 150–450 x 10³/mm³
• hemoglobin concentration: 12–16 g/dL for adult females; 13–18 g/dL for adult males
• hematocrit: 37–48 percent for adult females; 45–52 percent for adult males
• red blood cell indices: MCV of 80–100 femtoliters; MCH of 27–31 picograms/cell; MCHC of 32–36 g/dL
• WBC differential: neutrophils 54–65 percent; lymphocyte 25–110 percent; monocyte 2–8 percent; eosinophil l–4 percent; basophil 0–l percent

Plasma constituents such as albumin, fibrinogen, and globulins can also be measured with hematological testing. Abnormal results could indicate disseminated intravascular coagulation, kidney or liver disease, an inflammatory disease, an infection, or dietary issues among other things. Their reference range are:

• albumin: 3.4–5.4 g/dL
• fibrinogen: 200-400 mg/dL
• globulins: serum globulin 2.0–3.5 g/dL; IgM 75–300 mg/dL; IgG 650–1850 mg/dL; IgA 90–350 mg/dL

Testing

For POLs with compliance and accreditation certificates (meaning they can conduct moderate- and/or high-complexity tests), CLIA-waived point-of-care (POC) hematology analyzers can of course be used. Additionally, moderate-complexity benchtop hematology analyzers capable of CBC and white blood cell differential testing give added flexibility to the physician's offerings. The caveat, though, for many such labs: while better, rapid, more convenient patient care is desirable through POC technology, the need to perform enough tests and receive sufficient reimbursement for CBC testing (or alternatively make up for any loss associated with CBC testing elsewhere in practice operations) typically must still justify the added expense. And as mentioned in Chapter 1, upcoming changes to the Clinical Laboratory Fee Schedule will likely drive reimbursement lower for CBC and other hematology tests, making such offerings less attractive to the POL.

For POLs with only a CLIA certificate of waiver, the inability to perform CBC testing may be frustrating: as of June 2015, the CBC test hasn't yet been integrated into a CLIA-waived device. In June 2012, researchers at the Point-of-Care Testing Center for Teaching and Research described the challenges underlying creating a multiplex POC hematology device that can handle CBC testing:

Currently the CLIA-waived hematology and coagulation POC devices only test for hemoglobin (Hb), hematocrit (Hct), and prothrombin time/international normalized ratio (PT/INR). The problem with these devices is the lack of multiplexing. POC coagulation and hematology devices face challenges for obtaining a waiver. These challenges include the lack of clinical needs assessment, miniaturized assays that correct for interfering substances, and assays simple enough to be combined in a multiplex platform.

Later in October 2013, Kalorama contributed its own insight into the problem of making CBC and blood differential (white blood cell count) testing waived, saying that "[w]aiving differentials under CLIA has proven problematic as even the most sophisticated analyzer commonly requires verification of automated parameters due to instrument error in classifying nucleated cells and variant leukocytes."

However, CLIA-waived handheld hemoglobin, hematocrit, and PT/INR (coagulation) analyzers give POLs at least some hematology analysis technology if they desire it. Most if not all benefit from requiring only a finger stick for blood collection rather than a tube of venous blood. The following are examples of CLIA-waived POC hematology devices that may prove useful to the POL:

CLIA-waived POC hematology devices Device Test(s) HemoCue Hb 201+ System hemoglobin StabBio STAT-Site MHgb Hemoglobin Photometer hemoglobin Clarity HbCheck Hemoglobin Testing System hemoglobin, hematocrit StanBio HemoPoint H2 System hemoglobin, hematocrit Alere INRatio 2 PT/INR Monitor PT/INR Coag-Sense PT/INR Monitoring System PT/INR Roche CoaguChek XS System PT/INR

Clinical chemistry

In July 2014, healthcare market research company Kalorama Information estimated that by volume, "clinical chemistry panels and parameters represent the fourth-most commonly performed test in U.S. POLs." These panels and parameters are used to make qualitative and quantitative assessments of the level of chemical elements dissolved in body fluids such as serum, plasma, and urine as well as cerebrospinal, synovial, pleural, pericardial, and peritoneal fluids. Clinical chemistry tests are useful for checking glucose, cholesterol, electrolytes, thyroid-stimulating hormone, or creatinine in blood plasma, serum, or urine. The presence of these analytes and others (listed in the section of fluid composition) in quantities outside a lab's standard reference ranges could indicate disease processes like diabetes, hyperlipidemia, and hypothyroidism as well as kidney, liver, and heart dysfunction.

Basic concepts and collection procedures

As most testing in clinical chemistry is done using blood and urine, the collection information presented in the urinalysis and hematology sections is also relevant to clinical chemistry testing. When collecting blood for testing, the type of blood collected will depend on the testing device and the type of test performed. Most modern blood glucose test devices are handheld and require only capillary blood which can be obtained using a lancet, a tiny scalpel-like device used to prick the skin of a fingertip or the heel of a foot. This blood is collected either after the patient has fasted or has eaten a full meal. The process is typically the same for some of the handheld lipid testing devices. However, more advanced metabolic, electrolyte, and lipid panels will usually require a larger sample, necessitating venous blood collected from the antecubital area of the arm. While whole blood may be used for a few clinical chemistry tests, plasma or, to a lesser extent, serum that has been separated by centrifugation is most frequently used for testing. If a whole blood sample isn't sufficiently centrifuged, "it may result in an incomplete gel barrier, and the content of serum and plasma separators may produce significant analytical interference." Other preanalytical errors such as hemolysis (the damaging of red blood cells during or after collection), lipemia (an excess of lipoproteins often caused by not fasting appropriately), and incorrect tube use or collection timing can also cause a sample to become unsuitable for testing.

Fluid composition

Like urinalysis and hematological testing, clinical chemistry looks at body fluids and determines qualitatively and/or quantitatively their constituents. Clinical laboratories set reference ranges for the chemical elements found in those body fluids, and when a sample shows one or more of them outside of that reference range, the abnormality is often a sign of an ailment or problem in the body or with a diet.

Diabetes mellitus (DM) is such a disorder, one in which glucose is not properly metabolized due to inadequate production or use of insulin. It can also occur when the body is unable to produce enough effective insulin for the movement of glucose into body tissues. The two tests recommended by the American Diabetes Association for identifying diabetes or prediabetes is the fasting plasma glucose (FPG) and the oral glucose tolerance test (OGTT). FPG samples are usually taken in the morning after a patient has been fasting for 10 to 14 hours, while the OGTT is performed after the FBG, requiring a patient be given either a large meal or a high glucose drink and then rested for two hours. In both cases, glucose levels are tested.

Suggested reference ranges for glucose levels include:

• Normal: fasting plasma glucose of 70–100 mg/dL, with a two-hour postprandial glucose of less than 140 mg/dL
• Prediabetes: fasting plasma glucose of 101–125 mg/dL, with a two-hour postprandial glucose of 141–199 mg/dL
• Diabetes: fasting plasma glucose of 126 mg/dL or greater, with a two-hour postprandial glucose of 200 mg/dL or greater

Of course, glucose isn't the only chemical element examined in clinical chemistry. Other common chemical elements may be tested for with cholesterol and chemistry analyzers, and their suggested reference ranges include:

• albumin (Alb), a plasma constituent made by the liver: 3.4–5.4 g/dL
• alkaline phosphatase (ALP), a phosphate removing enzyme: 42–136 U/L
• alanine aminotransferase (ALT), a biomarker for measuring liver health: 10–35 U/L
• aspartate aminotransferase (AST), a biomarker for measuring liver health: 0–35 U/L
• bilirubin, total (TBili), a product of heme catabolism: 0.3–1 mg/dL
• blood urea nitrogen (BUN), an indicator for renal health: 10–20 mg/dL
• brain natriuretic peptide (BNP), a polypeptide secreted by the ventricles of the heart: 0–100 ng/L
• calcium (Ca), vital to many functions of human biology: 8.2–10.5 mg/dL
• carbon dioxide (CO₂), an end product of cellular respiration: 22–30 mEq/L
• chloride (Cl), which helps maintain proper body water distribution and extracellular osmotic pressure: 96–106 mEq/L
• cholesterol, total (Chol), an essential structural component of animal cells: less than 200 mg/dL
• creatinine (Creat), an endogenous byproduct of muscle creatine metabolism: 0.6–1.2 mg/dL
• creatine kinase (CK), a marker of damage to muscular and renal functions: 55–170 U/L
• high-density lipoprotein (HDL), which can remove and transport fat molecules around the body: greater than 50 mg/dL
• lactate dehydrogenase (LDH), a common enzyme that acts as a marker of common injuries and disease: 100–190 U/L
• low-density lipoprotein (LDL), which can transport and deposit fat molecules around the body: less than 100 mg/dL
• myoglobin, a protein that acts as a marker of muscle damage: less than 90 µU/L
• potassium (K), vital to proper neuron function and osmotic equilibrium: 3.5–5.0 mEq/L
• sodium (Na), vital to osmotic equilibrium in the body: 136–145 mEq/L
• thyroid-stimulating hormone (TSH), which prompts the thyroid gland to produce thyroxine: 0.4–4.2 µU/mL
• thyroxine (T4), which helps regulate metabolism: 4.5–11.2 µg/dL
• triglyceride (Trig), which enables adipose fat and blood glucose to transfer to and from the liver: less than 150 mg/dL
• triiodothyronine (T3), which affects numerous physiological processes: 75–220 ng/dL

Testing

As mentioned in the hematology section, with only 14.5 percent of POLs having CLIA compliance certificates, most POLs wanting to perform in-house clinical chemistry tests will be limited to a few CLIA-waived options. Glucose testing is the most common of such tests, relying on one of more than 100 easy-to-use handheld CLIA-waived monitors that can analyze a tiny amount of capillary blood for glucose or glycosylated hemoglobin (Hb A1c). Cholesterol and lipid testing is another common type of CLIA-waived testing that can be performed in the POL, testing basic cholesterol levels or running complete lipid profiles. To a much lesser degree, CLIA-waived clinical chemistry tests like metabolic, electrolyte, and liver panels can be performed on a handful of analyzers like the Piccolo Xpress from Abaxis, the i-STAT from Abbott Point of Care, and the SPOTCHEM EZ from Arkray. For POL labs with moderate- and high-complexity CLIA test certification, larger, more advanced clinical chemistry analyzers like the ACE Alera from Alfa Wassermann and the ABX Pentra 400 and Pentra C200 from HORIBA provide more complex test panels and greater batch testing of patient specimens.

While Kalorama expects modest growth from an otherwise mature global clinical chemistry test market, market growth in the U.S. POL market is expected to be minimal through 2020. The research company found "[p]rohibitive factors to the expansion of the U.S. POL clinical chemistry market include the lack of growth in the number of CLIA compliance (moderate- and high-complexity) POLs, centralization of core lab testing, and declining reimbursement for heavily automated tests." As such, it seems likely only the busiest of POLs — depending on high in-house test volumes — may choose to or continue to invest in low-volume and handheld clinical chemistry analyzers.

You can find an extensive listing of CLIA-waived POL instrument and test kit vendors (including for the elements mentioned above) in the Additional Resources section of this guide.

Immunology

In December 2014, healthcare market research company Kalorama Information estimated that by volume, several immunological tests ranked in the top 10 tests conducted in POLs. Kalorama ranked the rapid antigen detection test for group A streptococcus and the human Chorionic Gonadotropin (hCG) urine pregnancy test fifth and eighth respectively in physician office use. The popularity of these two immunological tests, among others, in the POL is likely due to their availability as inexpensive qualitative CLIA-waived tests. This is exemplified by CLIA-waived over-the-counter products like Proctor and Gamble's Clearblue PLUS Pregnancy Test, which can be bought for less than $10 U.S. and provide a qualitative result in three minutes. And CLIA-waived Strep A test kits that provide a result in five minutes can by bought with multiple tests per box, working out in some cases to less than $2 U.S. per test. In addition to these types of tests, a handful of other CLIA-waived immunological tests are available to POLs (detailed in the testing section).

Basic concepts and collection procedures

The human body's immune system is largely responsible for defending against a wide variety of foreign entities including microorganisms and allergens among others. When these enter or appear in the body, the immune system must determine what belongs and what doesn't belong. The foreign substance, often referred to as an antigen, is met with a wave of antibodies designed to attach to and destroy it. Additionally, the antibodies are tasked with "remembering" its encounter with the antigen so the antibody can be more agile the next time it is confronted with the foreign body. Usually this process works as intended, but in some cases the body's ability to identify properly goes wrong, and the immune system rebels against useful constituents of the body. As laboratory science has progressed, we've created new and novel ways to search for antigens, antibodies, and attacks against the "self" by applying various in vivo and in vitro tests to various body fluids.

The most common body fluids used in immunological testing are blood, serum, urine, and oral fluids, though occasionally body tissues may need to be examined. As such, the collection procedures used for these fluids will be the same as in urinalysis, hematology, and clinical chemistry. In complex cases — likely not conducted in a POL — body tissues such as placenta, mucosa-associated lymphoid, and even bone marrow may need to be collected for immunological analysis, requiring special surgical techniques.

Specimen composition and testing

Several testing methodologies are used in immunological testing; however, for CLIA-waived testing the most common methodology is lateral flow immunoassay. Practically speaking, this means a specimen is absorbed by the testing device, flowing laterally over the testing materials to reach the antigen or antibody contained within the testing materials. Color changes can be matched to a key to indicate a positive or negative qualitative change. Lateral flow testing has many advantages as well as a few disadvantages. The technology is relatively cheap, easy-to-use, quick, versatile, easy to commercialize, and testing devices typically maintain a long shelf life. However, they also tend to only give qualitative results, manufactured lots can vary in quality (with reagents used), and test accuracy may suffer from cross-reactivity.

In the opening section, we mentioned pregnancy and group A strep tests as examples of immunology-based tests that are often CLIA-waived. These and other examples, by disease or condition, include:

• eye disorders: Either 1. testing for adenoviral conjunctivitis (Ad-CS, an affliction commonly known as pink eye) via examination of eye fluid or 2. or the inflammatory marker MMP-9 (associated with dry eye disease) found in tear fluid
• fertility: Detection of luteinizing hormone (LH) in urine
• gastric disorders: Either 1. testing for the H. pylori bacterium (found in the stomach) via examination of whole blood (or in some cases serum or plasma) or 2. gastric occult blood in gastric aspirates or vomitus by directing antibodies against human hemoglobin that might be present
• hepatitis C: Testing for the presence of the hepatitis C virus antibody via fingerstick or whole blood
• HIV-1 and -2 infection: Testing for the presence of the human immunodeficiency virus (HIV) in blood
• influenza: Detection of the influenza type A and type B antigens via nasal or nasopharyngeal swabs
• intestinal disorders: Testing for fecal occult blood in the stool by directing antibodies against human hemoglobin that might be in the stool
• menopause: Detection of the follicle-stimulating hormone (FSH) in urine
• mononucleosis: Testing for the presence of infectious mono heterophile antibodies in whole blood (or in some cases serum or plasma)
• pregnancy: Detection of the hormone human chorionic gonadotropin (hCG) in urine
• respiratory syncytial virus: Testing for the presence of the respiratory syncytial virus (RSV) via nasal secretions
• strep throat: Detection of the group A streptococcal antigen, typically from a throat swab
• syphilis: Detection of Treponema pallidum (syphilis) antibodies in fingerstick blood (or in some cases whole blood, serum, or plasma)
• trichomoniasis: Testing for the presence of Trichomonas vaginalis antigens via vaginal swabs
• urinary disorders: Detection of the bladder tumor associated antigen (BTA) in urine

CLIA-waived instruments that can perform multiple qualitative immunological tests are somewhat limited. Alere's i System is currently CLIA-waived for influenza A and B as well as group A strep, while Becton, Dickson and Company offers their Veritor System that is CLIA-waived for influenza A and B, group A strep, and respiratory syncytial virus (RSV).

You can find an extensive listing of CLIA-waived POL instrument and test kit vendors (including for the immunological analytes mentioned above) in the Additional Resources section of this guide.

Toxicology and pain management

Toxicology is the study of the effects of chemicals on living organisms. In the CLIA-waived POL, this testing is essentially limited to drugs of abuse and the chemical element lead. Drug screenings in particular are becoming more common, mandated not only by employers but also in some cases by the federal government. While this sort of testing holds importance inside and outside the POL, it also has been abused. As previously discussed in the first section of this guide, the number of POL-based drug screenings paid for by Medicare skyrocketed between 2000 and 2009 as physician offices realized they could make good money on easy testing. As such, CMS amended their rules in 2010 to prevent this type of unintended billing behavior, decreasing the financial viability of such tests for the POL. Regardless, drug screening remains an important part of POL operations today.

Tangentially related is the practice of pain management, the study of how best to improve the quality of life of those living with pain. Chronic opioid therapy is one such related treatment method, though it doesn't come without risk of abuse. Physician-owned pain management clinics or private practices offering pain management services are increasingly regulated, though those regulations neither explicitly state that urine drug tests or some other form of adherence monitoring are required, nor do they currently prohibit opioid and other drug-based therapies on patients abusing controlled substances. Despite this, "compliance monitoring has been shown to be crucial in delivering proper opioid therapy and preserving this therapy for the future," and urine drug testing is one of the major tools used in this monitoring.

Testing

As drugs are metabolized, they produce metabolites which show up in urine and saliva. As such, both urine- and saliva-based tests have been manufactured to make qualitative and quantitative determinations of a metabolite's presence relatively easy and accurate. Ethanol is a common legal drug in the U.S. and constitutes one of the drugs tested for in CLIA-waived tests. Note, however, that ethanol testing is not typically part of a qualitative immunoassay-based urine test; the associated metabolites ethyl glucuronide (EtG) and ethyl sulfate (EtS) may appear in urine for reasons other than direct alcohol consumption, including the ingestion of partially fermented juices and absorption through the skin of alcohol-based lotions. Since ethanol equilibrates rapidly between saliva and blood, and a saliva test is rapid and easy to administer, most CLIA-waived tests for ethanol — usually quantitative — will be saliva-based. Qualitative testing for amphetamines, cannabinoids, morphine, and many other constituents of drugs classified as drugs of abuse is typically done with relatively simple CLIA-waived urine test cups and cards. However, proper care in specimen labeling, storage, technique (including timing recommendations), and interpretation are still vital to ensure the most accurate results.

In the particular case of pain management, a simple qualitative immunoassay-based urine drug screen isn't always appropriate or accurate. Differences in metabolites and problems with accuracy come with those simplified tests. More accurate measurements may be necessary in the pain management laboratory, necessitating chromatographic tools and techniques that fall under CLIA moderate to advanced status. Given the previously mentioned fact that only 14.5 percent of all registered POLs would be CLIA certified for such testing, this sort of advanced testing would be limited to pain management POLs willing to go through the financial and regulatory hoops involved.

For testing levels of lead in blood, the LeadCare II lead testing system — partially funded by the Centers for Disease Control and Prevention (CDC) — remains the only CLIA-waived option.

Molecular diagnostics

We would be remiss for not at least mentioning the field of molecular diagnostics and its application in the POL. We initially addressed the topic in the CLIA market and industry trends section of this guide, mentioning molecular in vitro tests for both moderate CLIA complexity labs (Cepheid's Flu Xpert) and CLIA-waived labs (Alere i System for influenza A & B and strep A). Molecular testing is largely nucleic acid-based testing, which the FDA describes as "tests [that] analyze variations in the sequence, structure, or expression of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in order to diagnose disease or medical conditions, infection with an identifiable pathogen, and determine genetic carrier status." The FDA maintains a list of approved nucleic acid-based human genetic, microbial, and companion diagnostic tests, most of which are classified as CLIA moderate complexity. As such, with the exception of the Alere i System, molecular diagnostic testing in the POL is limited to the previously mentioned 14.5 percent that maintain a CLIA compliance certificate.

It is clear, however, that many experts in the industry believe that CLIA-waived molecular testing will gradually become more prevalent. Alberto Gutierrez, FDA director of the Office of In Vitro Diagnostics and Radiological Health, emphasized this earlier this year with the first CLIA-waived approval of the Alere i System, saying "We expect many other simple and accurate tests using nucleic acid-based technology to be developed in the near future." This expectation will largely be met through test devices that have the greatest practical clinical use, according to Ihor Boszko, vice president of business development for molecular diagnostics company Xagenic Inc. "[W]idespread adoption of POC molecular diagnostics will be driven by tests that have the greatest clinical utility and that provide demonstrable efficiencies to the healthcare system that are unattainable through traditional central laboratory methods."

One potential barrier to wider adoption of molecular testing in the POL appears to be how public payers will cover and reimburse such tests. In an April 2015 piece for the American Association for Clinical Chemistry's (AACC's) Clinical Laboratory News, AACC managing editor Bill Malone and Quorum Consulting director of strategic product planning Genevieve Tang highlighted some of the concerns and considerations of reimbursement in the U.S. Tang argued one of the most important ways for manufacturers to have private payers cover their new molecular tests is to tie patient outcomes to clinical decision making. "Securing payer coverage is increasingly going to require clinical utility studies that clearly demonstrate how use of the test improves patient outcomes," Tang said, elaborating with the following:

Payers look for three levels of evidence when they decide whether or not to cover a test, Tang explained. “The first is analytical validity, which refers to the accuracy, precision, and reproducibility of the test results. The second is clinical validity, which is the correlation of the test results with the clinical outcomes of interest,” Tang said. “Third is clinical utility, which may seem an abstract concept, but essentially refers to how use of the test influences clinical decision-making and/or improving patient outcomes. This third level of evidence is what payers are really looking for.”