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.

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 November 2014 only 17,970 POLs, representing only 14.8 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.

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 8 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 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.

Fluid composition

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

Other common chemical elements that may be tested for with cholesterol and other 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

While Kalorama expects modest growth from an otherwise mature global clinical chemistry 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.

As mentioned in the hematology section, with only 14.8 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, other 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.

Immunology

• concepts in immunology

• instruments and test kits

• CLIA-waived tests

Microbiology

• common pathogens

• topics in microbiology

• instruments and test kits

• CLIA-waived tests

Toxicology

• topics in toxicology

• instruments and test kits

• CLIA-waived tests