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Plasma, Serum, Urine, CSF, and Other Body Fluids.

Pancreatic cyst, synovial, pleural, chest, ascites, peritoneal, abdominal, feces, peri-nephric, peri-cardial, and PTH aspirates.

All specimens are spun down before running on the analyzer.

Green and grey = Serum, plasma, pancreatic cyst; Yellow and grey = Urine, feces lytes; Red = Stat (usually serum/plasma only); Pink and grey = Body Fluids; Brown and grey = CSF; Blue and grey = False bottom tubes; Solid grey = Samples aliquotted from the MPA.

Ion Selective Electrode, Photometric, and ECL (Electrochemiluminescent)

The ISE unit performs indirect measurement of electromotive force (EMF) in millivolts between ion-selective electrodes and the reference electrode.

It means that all samples are diluted at a 1:31 ratio.

They are converted by a calculation algorithm that uses the EMF data together with data from a 2-point calibration with two primary calibrators.

An ISE measurement before and after each routine sample measurement is used to offset the drift

Sodium, potassium, and chloride.

It is calculated from the EMF of the specific electrode.

The equation derived from the Nernst Equation.

All tests that use absorbance measurements for result calculation, including clinical chemistry and immunological tests.

Endpoint and rate assays

In endpoint assays, measurements are taken by the photometer at specific measuring points. If measurements are taken after the reactions are completed, the intensity of the colored (or turbidimetric) product is an indicator of the sample component's concentration. These are called endpoint assays.

With rate assays, measurements are taken as the reaction proceeds. In this case, the rate of the reaction is proportional to the sample component's concentration or activity being analyzed.

ECL is a process in which highly reactive species are generated from stable precursors at the surface of an electrode. These highly reactive species react with one another, producing light. The ECL processes are known to occur with numerous molecules including compounds of ruthenium, osmium, rhenium, or other elements.

The development of ECL immunology tests is based on the use of a ruthenium(II)-tris(bipyridyl) complex and tripropylamine (TPA).

The final chemiluminescent product is formed during the detection step. The chemiluminescent reactions that lead to the emission of light from the ruthenium complex are initiated electrically rather than chemically.

It is achieved by applying a voltage to the immunological complexes (including the ruthenium complex) that are attached to streptavidin-coated microbeads. The advantage of electrically initiating the chemiluminescent reaction is that the entire reaction can be precisely controlled.

The ISE module is composed of two ISE (ion selective electrode) units, measures Na (sodium), K (potassium), and Cl (chloride), and uses indirect measurement.

The c702 is the main chemistry module at UCLA used for clinical chemistry and homogeneous immunoassays, with a throughput of up to 2,000 tests per hour.

It ensures specimen integrity via serum indices, clot and liquid level detection, uses contact-free ultrasonic mixing, and has 2 sample probes and 4 reagent probes.

The c502 performs clinical chemistry and homogeneous immunoassays. At UCLA, it focuses on chemistries related to TDM (therapeutic drug monitoring) and DAU (drugs of abuse).

The c502 has a throughput of up to 600 tests per hour, ensures specimen integrity via serum indices, clot and liquid level detection, and uses contact-free ultrasonic mixing.

The e801 performs heterogeneous (separation step) immunoassays using ElectroChemiLuminescence (ECL) technology.

The e801 has a throughput of up to 300 tests per hour, uses carryover-free disposable tips, and includes clot and air bubble detection.

The purpose of calibration is to maintain assurance of accuracy and repeatability of measurements.

In order to calibrate a machine, known standards are measured and plotted against a measured (response) value, such as absorbance.

When we measure something, we convert an absorbance value to something meaningful, such as analyte concentration or enzyme activity.

Beer-Lambert Law.

Calibrations are performed when loading new lots of reagent, after major maintenance, and when troubleshooting QC.

A lot cal is a calibration curve for a specific lot of reagent.

A reagent cal is a calibration curve for a specific reagent pack.

The calibrator of the lowest analyte concentration is used for calibration, such as Calibrator 1 (for example, H₂O).

The calibration curve is adapted without changing the K-factor.

Two calibrators are used: Calibrator 1, for example H₂O, and a second calibrator, for example C.f.a.s.

The calibration curve is adapted by recalculating the K-factor.

All calibrators are used for this nonlinear calibration, meaning all calibrators defined for this test.

The calibration curve is adapted by recalculating all K-factors.

Quality Control (QC) validates whether our instrument is working properly and gives us confidence that our test results are correct.

QC is run every 24 hours, when a new reagent is loaded, after preventative maintenance, and after calibration.

Less stable tests have their QC run more frequently.

QC material is meant to mimic a patient sample, so its matrix is ideally made from serum, urine, or spinal fluid.

Each QC material can test multiple analytes.

Ideally, QC material will have a normal control and an abnormal control or span an analyte’s decision points.

It should be well mixed and at the appropriate temperature before running.

QC material is run around 20 times, once a day and by different people, to account for natural variation that occurs.

The QC range is established by calculating the mean and standard deviation.

The mean is an estimate of the analyte’s true value.

SD is a measure of precision.

Once the mean and SD are known, the QC range is calculated as mean (±) 2SD.

QC range calculation Example:
If the mean is 50 and the SD is 5, then the QC range is 40 to 60.

95% of QC runs should be within 2SD.

There are six basic rules: 1-3s, 2-2s, R-4s, 1-2s, 4-1s, and 10-x. These can be used individually or in combination (multi-rule) to evaluate the quality of analytical runs.

They reduce false rejections made when applying just the 1-2s rule for run rejection and increase error detection more than provided when applying just the 1-3s rule.

1-3s and R-4s detect random error, while 2-2s, 4-1s, and 10-x detect systematic error.

The 1-3s rule identifies unacceptable random error or possibly the beginning of a large systematic error. Any QC result outside ±3SD violates this rule. Since only 0.3% or 3 out of 1000 points will fall outside the ±3SD limit, any value outside of ±3SD is usually considered to be associated with a significant error condition.

While a value outside ±3SD may be statistically significant, it may not be biologically or medically relevant because modern laboratory instruments are often more precise than what is needed medically.

The 1-2s rule is usually a warning rule violated when a single control observation is outside the ±2SD limit. It was originally designed as a warning rule for manual application of the Westgard rules. If one control measurement within a run exceeds the mean ±2SD, evaluate other controls in the run and in previous runs before accepting the run and reporting results.

Some laboratories incorrectly consider any QC value outside ±2SD to be out of control and reject valid runs. An analytical run should not be rejected if a single QC value is outside ±2SD but within ±3SD, since about 4.5% of valid QC values naturally fall between ±2SD and ±3SD. Universally rejecting values outside ±2SD leads to rejecting good runs too frequently, and with computer-based Westgard applications, the 1-2s rule is usually unnecessary.

Using the 1-2s rule alone causes frequent rejection of valid runs because it fails to allow for valid points between 2SD and 3SD.

It may falsely reject 5% of all analytical runs when using one level of control, 10% when using two levels, and 14% when using three levels. These false rejections lead to unnecessary repeats of patient samples, wasted labor and materials, and delayed patient results.

The 2-2s rule detects systematic error only. It is violated when two consecutive QC results are greater than 2SD and on the same side of the mean.

Within a run, the rule is violated when two control values in the same run are >2SD on the same side of the mean, indicating systematic error affecting the entire analytical curve. Across runs, the rule is violated when a single level of control is >2SD from the mean in one run and again >2SD on the same side of the mean in the next run, indicating systematic error affecting only a single portion of the analytical curve.

The R-4s rule identifies random error and is applied only within the current run. It is violated when there is at least a 4SD difference between control values within a single run.

If Level 1 is +2.8SD above the mean and Level 2 is -1.3SD below the mean, the total difference is greater than 4SD (+2.8SD - (-1.3SD) = 4.1SD), indicating a violation of the R-4s rule.

These rules are violated when three or four consecutive results are greater than 1SD and on the same side of the mean.

They are applied within a control level or across control levels. Violations within a control level indicate systematic bias in a single area of the method curve, while violations across control levels indicate systematic error over a broader range of concentrations.

N-x rules are violated when there are 7, 8, 9, 10, or 12 control results on the same side of the mean.

They are applied within a control level or across control levels. Violations within a control level indicate systematic bias in a single area of the method curve, while violations across control levels indicate systematic error over a broader range of concentrations.

The 7-x control rule is far more sensitive to analytical bias than the 12-x rule because the chance of finding seven consecutive control observations on one side of the mean is much higher than finding twelve.

A Levey-Jennings Chart gives a visual representation of QC data and helps visualize systematic errors.

It graphs QC value runs, showing run-to-run or day-to-day performance.

Systematic errors can be gradual (trend) or abrupt (shift) and are built in due to an issue such as bad calibration, failing hardware, or old QC.

Random errors are due to natural variability.

The analytical measuring range is the range of an analyte that a method can measure without dilution or concentration.

The clinical measuring range is the range of an analyte expanded with dilution.

Limit of Blank (LoB), Limit of Detection (LoD), and Limit of Quantitation (LoQ) describe the smallest concentration of a measurand that can be reliably measured by an analytical procedure.

LoB is the highest apparent analyte concentration expected to be found when replicates of a blank sample containing no analyte are tested.

LoD is the lowest analyte concentration likely to be reliably distinguished from the LoB and at which detection is feasible.

LoQ is the lowest concentration at which the analyte can not only be reliably detected but at which some predefined goals for bias and imprecision are met.

Dilution is the process of decreasing the amount of analyte until it is measurable; the purpose is to decrease the analyte concentration so it is within the analytical measuring range (AMR).

Some dilutions are done by the machine, while others are performed manually.

A diluent is a substance used to dilute an analyte.

The dilution factor is the number by which you multiply the measured diluted value to obtain the original value.

A serial dilution is a series of dilutions that can be done to save sample or to dilute something with a high concentration.

It determines the relationship between the total volume, sample volume, and diluent volume in a dilution process.

C1V1 = C2V2.

Dilution factor = F, Total volume = T, Diluent volume = D, Sample volume = S.

(T / F) = S and T – S = D.

A serial dilution is performed when the concentration of an analyte is still too high (above the AMR), allowing further dilution using an already diluted sample.

It means that poor-quality specimens will produce poor or unreliable test results.

Age (stability), exposure to air (capped / uncapped), delay in centrifugation, recentrifugation, indices (HIL), pH, volume, clot, draw times, order of draw, light, IV contamination, temperature, and tourniquet time.

Indices greatly affect test results and must be checked before releasing results.

Depending on the test, indices can cause results to be either higher or lower.

Indices can cause chemical or spectral interference depending on the assay.

Hemolysis is the destruction of red blood cells, which releases intracellular components such as hemoglobin.

Biochemical, immunological, physical, or chemical causes.

Incorrect sample processing.

LDH, AST, K, and Folate.

It can be elevated in liver disease, biliary cirrhosis, alcoholism, or as a response to many drugs.

Lipemia refers to the turbidity of a sample caused by the accumulation of lipoprotein particles, with chylomicron being the largest contributor. It can be removed by airfuge.

Lipemia is usually seen if the patient was not fasting or if the patient is receiving lipids.

Other causes include diabetes mellitus, ethanol use, chronic renal failure, and pancreatitis.

Lipoproteins cause a volume depletion effect by reducing the available water in the sample volume.

Most analytes, such as sodium (Na), are dissolved in the aqueous phase of plasma or serum. Lipoproteins displace water, leading to an apparent decrease in the concentration of these analytes.

For analytes that are lipid soluble, lipemia causes an apparent increase in concentration.

Different panels provide information about the body’s chemical balance, metabolism, and body functions.

They provide an easier and more organized way of ordering multiple related tests.

Na, K, Cl, CO2, AGAP, Glu, Crea, BUN, and Ca.

Tp, Alb, t.Bili, d.Bili, ALP, AST, and ALT.

The CMP includes all tests from the BMP plus the Hepatic Panel, but d.Bili (direct bilirubin) is not included.

Electrolytes are ions capable of carrying an electric charge and are classified as anions or cations based on the type of charge they carry.

Electrolytes help control nerve and muscle function and maintain the acid-base (pH) balance and water balance of the blood.

Sodium is the major extracellular cation that maintains the distribution of water and osmotic pressure.

Potassium is the major intracellular cation that regulates neuromuscular excitability, contraction of the heart, intracellular fluid (ICF) volume, and hydrogen ion (H⁺) concentration.

Chloride is the major extracellular anion that maintains osmolality, blood volume, and electric neutrality.

Total CO₂ is important for maintaining acid-base balance.

AGAP = Na – (Cl + CO₂).

The anion gap measures the difference, or gap, between negatively charged and positively charged ions.

It takes into account the unmeasured anions and cations.

Diabetic acidosis, lactic acidosis, and renal failure.

A low or negative AGAP can be caused by low albumin or instrument issues.

An increase in ALP indicates liver and bone disease.

An increase in AST indicates liver disease.

An increase in ALT indicates liver disease.

An increase in GGT indicates liver disease.

An increase in LD indicates liver disease.

An increase in CK indicates heart or muscle disease.

An increase in Amylase indicates pancreatitis.

AST is widely distributed in human tissue, with the highest concentrations in cardiac tissue, liver, and skeletal muscle, and smaller amounts in the kidney, pancreas, and erythrocytes.

AST is mainly used to evaluate hepatocellular disorders and skeletal muscle involvement.

In AMI, AST levels begin to rise within 6 to 8 hours, peak at 24 hours, and generally return to normal within 5 days.

Because of its wide tissue distribution, AST levels are not specific enough to be useful in the diagnosis of AMI.

ALT is distributed in many tissues but has comparatively high concentrations in the liver.

ALT is considered the more liver-specific enzyme of the transferases because of its high concentration in liver tissue.

ALT assays are mainly used for evaluating hepatic disorders.

Higher elevations are found in hepatocellular disorders than in extrahepatic or intrahepatic obstructive disorders.

ALP activity is present on cell surfaces in most human tissues, with the highest concentrations in the intestine, liver, bone, spleen, placenta, and kidney.

Elevations of ALP are most significant in evaluating hepatobiliary and bone disorders.

In hepatobiliary disorders, ALP elevations are more predominant in obstructive conditions than in hepatocellular disorders.

In bone disorders, ALP elevations are observed when there is involvement of osteoblasts.

CK is widely distributed in tissue, with the highest activities found in skeletal muscle, heart muscle, and brain tissue, and much smaller quantities in the bladder, placenta, gastrointestinal tract, thyroid, uterus, kidney, lung, prostate, spleen, liver, and pancreas.

Because of the high concentrations of CK in muscle tissue, levels are frequently elevated in disorders of cardiac and skeletal muscle.

Elevated CK levels are associated with acute myocardial infarction (AMI) and muscular dystrophy, particularly the Duchenne type.

LDH is widely distributed, with high activities in the heart, liver, skeletal muscle, kidney, and erythrocytes, and lesser amounts in the lung, smooth muscle, and brain.

Because LDH is active in numerous body tissues, it can be elevated in a variety of disorders.

Increased LDH levels are found in cardiac, hepatic, skeletal muscle, and renal diseases, as well as in hematologic and neoplastic disorders.

The highest levels of total LDH are seen in pernicious anemia and hemolytic disorders.

GGT activity is found primarily in the kidney, brain, prostate, pancreas, and liver.

GGT assays are mainly used for evaluating liver and biliary system disorders.

In the liver, GGT is located in the canaliculi of the hepatic cells and in the epithelial cells lining the biliary ductules.

Because GGT is elevated in virtually all hepatobiliary disorders, it is one of the most sensitive enzyme assays for detecting these conditions.

The acinar cells of the pancreas and the salivary glands are the major tissue sources of serum amylase, with lesser concentrations found in skeletal muscle, the small intestine, and the fallopian tubes.

Serum and urine amylase measurements are primarily used in the diagnosis of acute pancreatitis.

Lipase concentration is found primarily in the pancreas, but it is also present in the stomach and small intestine.

Serum lipase measurement is used almost exclusively for the diagnosis of acute pancreatitis.

Lipase measurement is similar to amylase in diagnosing acute pancreatitis but is considered more specific for pancreatic disorders than amylase measurement.

Total bilirubin measures both conjugated and unconjugated bilirubin and is elevated in liver disease and hemolysis.

Bilirubin samples must be protected from light for accurate measurement because high levels can cause brain damage in newborns (kernicterus).

Conjugated bilirubin, or direct bilirubin, is converted in the liver so it can be removed from the body and is water soluble.

Unconjugated bilirubin, or indirect bilirubin, is water insoluble.

Jaundice is the yellowing of the skin and sclera caused by abnormally elevated levels of bilirubin in the blood.

Jaundice is categorized into three types: pre-hepatic, intra-hepatic, and post-hepatic.

Pre-hepatic and intra-hepatic jaundice are known as medical jaundice, while post-hepatic (or obstructive) jaundice is considered surgical jaundice.

In pre-hepatic jaundice, excess production of bilirubin overwhelms the liver’s ability to conjugate and excrete it into the gut, leading to predominantly unconjugated hyperbilirubinemia.

The most common cause is hemolytic anemia, which results in excess heme breakdown.

Intra-hepatic jaundice is caused by parenchymal liver disease that impairs the liver’s ability to conjugate or excrete bilirubin.

The fraction of bilirubin that is elevated varies—viral hepatitis often causes predominantly unconjugated hyperbilirubinemia, while conjugated hyperbilirubinemia occurs with cholestasis from drugs or primary biliary cholangitis.

Post-hepatic jaundice occurs when there is an impediment to the flow of bile due to a partial or complete obstruction of the extrahepatic biliary passage between the liver and duodenum.

The obstruction can occur within the biliary ducts themselves or more distally within the pancreas.

Post-hepatic jaundice is characterized by predominantly conjugated hyperbilirubinemia.

Hormones are chemical substances secreted by endocrine glands that travel through the blood and regulate the functions of tissues and organs elsewhere in the body.

The three types of hormones are steroids, amines, and proteins/peptides.

Steroid hormones are made from cholesterol and include cortisol, aldosterone, androgens (male hormones), estrogens (female hormones), and progesterone.

Amine hormones include thyroid hormones and adrenomedullary hormones.

Protein and peptide hormones include pituitary, parathyroid, and pancreatic hormones.

TSH is secreted by the pituitary gland and stimulates the production of T4 and T3.

T4 and T3 stimulate metabolic activity and increase oxygen consumption, heat production, heart rate, gastrointestinal movement, and brain activity. They also influence the growth and development of the musculoskeletal and nervous systems.

When T4 and T3 levels are low, the hypothalamus releases TRH, which stimulates TSH release. When T4 and T3 levels are high, TRH release is inhibited.

TSH regulates the production of T3 and T4 by the thyroid.

T3 and T4 regulate growth and development.

TSH is used to evaluate thyroid function.

T4 helps with diagnosis and determination of hyperthyroidism or hypothyroidism.

T3 is ordered after abnormal TSH and T4 results and helps diagnose hypothyroidism.

aTPO is used to diagnose and monitor autoimmune thyroid disease.

Tg is used as a tumor marker for thyroid cancer.

tgAB is measured because it can interfere with Tg measurement.

T-Uptake estimates protein binding of thyroid hormones

ACTH regulates cortisol and androgens.

FSH regulates and stimulates the growth and function of the ovaries and testes. It works with LH to stimulate follicle growth and maturation in women and induces spermatogonium development in men.

LH regulates and stimulates the growth and function of the ovaries and testes. In men, it stimulates testosterone production; in women, LH peaks mid-cycle to induce ovulation and the formation of the corpus luteum.

PRL regulates lactation.

TSH regulates the production of T3 and T4.

ADH regulates the reabsorption of water in the distal renal tubules.

Oxytocin regulates uterine contractions during childbirth and lactation.

T4 and T3 regulate metabolism, growth, and development.

Calcitonin inhibits calcium resorption.

The parathyroid gland produces PTH, which regulates calcium and phosphate.

The adrenal cortex produces aldosterone and cortisol.

Aldosterone regulates the reabsorption of sodium (Na) in the renal tubules.

Cortisol regulates carbohydrate, fat, and protein metabolism; maintains water and electrolyte balance; and suppresses inflammatory and allergic reactions.

The adrenal medulla produces epinephrine and norepinephrine.

Epinephrine and norepinephrine regulate the “fight or flight” response by stimulating the sympathetic nervous system.

The ovaries produce estrogen and progesterone. Estrogen regulates female reproductive organs and secondary sex characteristics, controls the menstrual cycle, and maintains pregnancy. Progesterone prepares the uterus for ovum implantation and maintains pregnancy.

The placenta produces HCG, which regulates progesterone production by the corpus luteum during early pregnancy and supports the development of fetal gonads.

The testes produce testosterone, which is responsible for the development of male reproductive organs and secondary sex characteristics.

Insulin regulates carbohydrate metabolism and increases the movement of glucose into cells for metabolism.

Glucagon regulates glycogenolysis, gluconeogenesis, and lipolysis.

Therapeutic drug monitoring is the measurement of specific drugs and/or their metabolites at timed intervals to maintain a relatively constant concentration of the medication in the blood.

The goal is to balance producing the desired therapeutic effect while avoiding adverse responses in the patient.

The therapeutic index is the ratio between the toxic dose and the therapeutic (effective) dose of a medication.

The trough is the lowest concentration of a drug in the blood and is drawn before the next dose.

The peak is the highest concentration of a drug in the blood and is drawn about one hour or more after the dose, depending on the drug.

Pharmacokinetics is the study of what the body does to the drug.

Pharmacodynamics is the study of what the drug does to the body.

Most drugs, especially water-soluble ones, are eliminated largely by the kidneys in urine.

Pharmacogenetic testing provides information about a person’s genes to help health care providers choose the medicine and dosage that are the best fit for the individual.

Before resulting, check for:
• Volume – Ensure there is enough sample.
• Clot – A clot can prevent accurate sampling and cause erroneous results, even if clot detection is missed by the machine.
• Previous history – Confirm whether a trough looks like a trough (low value) and a peak looks like a peak (high value).

Trough and peak samples may be ordered incorrectly, switched, or collected at the wrong time; if timing is uncertain, the order may need to be changed to “Random.”

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The panel includes amphetamine, barbiturates, benzodiazepine, cannabinoids, cocaine, methadone, opiates, oxycodone, ethanol, hydrocodone, and fentanyl.

DAU tests are semi-quantitative—the machine gives a numeric value that is converted to a positive or negative result based on the analyte’s cutoff.

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To assess possible contamination, look for a dilutional effect on other analytes. For example, an increase in sodium (Na) may suggest saline contamination. Checking other analytes such as creatinine and BUN for dilutional effects can support suspicion of IV saline contamination.

If results are not clearly contaminated, UCLA’s SOP states that staff should not hint at contamination but may ask the RN or MD if the value is expected. The final decision is made by the MD.

A majority of tests are auto-verified, meaning they are automatically released unless an issue causes the result to be held, in which case the CLS must address it.

Pay attention to error codes—if there’s an error code, it’s most likely not safe to release the result.

When in doubt, check the machine to see what it is doing.

Check the test’s AMR (Analytical Measuring Range) and CRR (Clinical Reportable Range), perform a dilution if necessary, and review patient history to ensure the values make sense.

Contamination can occur when drawing from a line, if blood is drawn near the source of infusion, or due to an incorrect order of draw.

Common causes include Total Parenteral Nutrition (TPN), which can increase glucose and potassium (K), or saline, which can increase sodium (Na) and chloride (Cl); EDTA contamination is also common.

Some parameters may appear diluted when contamination occurs.

At UCLA, if a sample is contaminated, all specimens drawn at the same time are considered contaminated, regardless of whether they match previous history or appear normal.