Clinical Specimen Collection and Analysis

• Clinical laboratory specimen types and variables affecting clinical analyses are important

Critical Factors in Specimen Collection

• Collection
• Identification
• Processing
• Storage
• Transport

Types of Laboratory Errors

• Preanalytical errors occur during specimen collection, handling, and processing
• Analytical errors occur during the testing process
• Postanalytical errors occur in the interpretation and reporting of results

Pre-Examination/Preanalytical Errors

• Incorrect patient identification
• Improper patient preparation
• Incorrect specimen collection
• Mislabeled specimen
• Incorrect order of draw
• Tubes used incorrectly for blood collection
• Incorrect anticoagulant to blood ratio
• Improper mixing of blood and anticoagulant
• Incorrect specimen preservation
• Mishandling of specimen during transport and storage
• Incorrectly interpreted/ordered laboratory test
• Incomplete centrifugation
• Incorrect data log-in

Examination/Analytical Errors

• Incorrect sample and reagent volume
• Incorrect incubation of solution
• Equipment/instrument malfunction
• Improper calibration of equipment/calibration error

Post-Examination/Postanalytical Errors

• Unavailable or delayed laboratory results
• Long turnaround time
• Incomplete laboratory results
• Wrong transcription of patient data and laboratory results
• Missing laboratory results
• Laboratory results submitted to the wrong physician/doctors that did not request the lab test

Ten Common Errors in Specimen Collection

• Misidentification of patient
• Mislabeling of specimen
• Short draws/wrong anticoagulant/blood ratio
• Mixing problems/clots
• Wrong tubes/wrong anticoagulant
• Hemolysis/lipemia
• Hemoconcentration from prolonged tourniquet time
• Exposure to light/extreme temperatures
• Improperly timed specimens/delayed delivery to laboratory
• Processing errors like incomplete centrifugation, incorrect log-in, and improper storage

Blood Specimen

• Blood consists of 55% plasma and 45% formed elements
• Plasma consists of 90% water and 10% solutes
• Formed elements include leukocytes, platelets and erythrocytes

Plasma

• Plasma contains a buffy coat composed of WBCs and platelets
• Plasma differs from Serum because Serum = Plasma – Clotting Factors

Serum vs. Plasma

• Serum has no interference from fibrinogen, anticoagulants, and cations, while plasma is prone to them
• Serum has a lower viscosity given it lacks fibrinogen, while plasma has higher viscosity
• Serum has a lower volume yield after centrifugation in contrast to plasma with a higher yield (15-30% higher)
• Serum is prone to interferences due to post-centrifugal coagulation, while plasma has none
• Serum requires blood to clot for 20-30 minutes before centrifugation, while plasma is time-saving and can be centrifuged immediately

Types of Blood Specimens

• Arterial Blood
• Venous Blood
• Capillary Blood

Arterial Blood

• Arterial blood characteristics include oxygenated blood with uniform composition throughout the body
• Arterial blood is primarily used for measuring blood gases (oxygen and carbon dioxide levels) and pH
• Collection requires syringes due to arterial pressure with common sites including radial, brachial, and femoral arteries
• Collection is difficult and hazardous due to arterial pressure, potential hematoma formation, and difficulty stopping bleeding

Venous Blood

• Venous blood contains deoxygenated blood with variable composition based on metabolic activity
• Routine laboratory tests use venous blood
• Obtained via direct venipuncture, commonly from the antecubital vein in the arm
• Compared to arterial blood, it has lower oxygen levels and variations in chloride, glucose, pH, carbon dioxide, lactic acid, and ammonia concentrations

Capillary Blood

• Capillary blood is a mixture of arterial and venous blood along with tissue fluid
• Preferred for infants, elderly patients with fragile veins, and burn patients and frequently used in point-of-care testing
• Obtained from capillary beds in venules and arterioles
• Higher glucose concentration but lower calcium, potassium, and total protein levels compared to venous blood
• Collection sites include the ring/great finger, infant's heel, and earlobe
• Advantages include that only a very small amount of blood is needed, collection is simple and relatively painless, and collection sites can be changed to minimize scarring and pain
• Disadvantages include that not all tests can be run on capillary samples, sometimes the blood cells rupture leading to inaccurate results, and calcified nodules may develop at the site of collection, especially in infants
• Capillary puncture is appropriate for adults and older children with fragile veins or veins needed for other procedures, multiple failed venipuncture attempts, patients with clotting disorders or fear of needles, limited vein accessibility, and point-of-care testing (POCT)
• Capillary puncture is the preferred method for infants and young children because it prevents blood volume depletion and cardiac arrest due to excessive blood loss, minimizes tissue and vein damage, avoids injuries from physical restraint, and is required for specific tests

Urine Specimen

• This is the second most common specimen in clinical chemistry
• Used for creatinine clearance and urine protein analysis
• Urine sample types include Random Urine Sample, First-morning-voided, and 24-Hour Urine Collection

Cerebrospinal Fluid

• Involves a Lumbar puncture
• Used for protein, glucose, lactate, and enzyme analysis
• Required pressure before collection should measure between 90 and 180 mm Hg

Other specimen types

• Synovial fluid
• Pleural Fluid
• Pericardial Fluid
• Peritoneal Fluid
• Amniotic Fluid
• These sampling methods include Arthrocentesis, Thoracentesis, Pericardiocentesis, and Peritoneocentesis

Importance of Patient Preparation in Laboratory Testing

• Laboratory specimens should ideally be collected when the patient is in a basal state: early morning, fasting, minimal physical activity
• Basal state collection ensures consistency and comparability with normal reference values

Pre-Examination or Pre-Analytical Variables

• Factors affect patient results before laboratory analysis
• Classified into controllable (extrinsic) variables that can be modified to minimize errors and uncontrollable variables that are factors inherent to the patient that cannot be changed

Controllable (Extrinsic) Variables in Specimen Collection

• Include anticoagulant preservatives, type of specimen used, influence of collection site, prolonged tourniquet application, hemolysis, timing of blood collection, and handling, storage, and transport

Anticoagulant Preservatives

• Type of anticoagulant used affects test results
• Some analytes bind to anticoagulants, leading to false high or low values

Type of Specimen Used

• Different specimen types like serum, plasma, and whole blood yield different results for some tests

Influence of Collection Site

• Blood composition varies based on collection site

Prolonged Tourniquet Application

• Increases albumin, ammonia, calcium, cholesterol, coagulation factors, enzymes, iron, potassium, and total protein
• Decreases plasma potassium
• To prevent hemoconcentration during venipuncture, ask the patient to release the fist upon blood flow, choose an appropriate patent vein, disallow the patient to pump the fist, do not excessively massage the area in locating a vein, do not probe or redirect the needle multiple times in search of a vein, and release the tourniquet within 1 minute

Hemolysis

• Increases AST, ACP, LD, Magnisium, potassium, & phosphate
• Decreases Na, Ca, and other dominant extracellular substances
• Hemoglobin absorbs light strongly at specific wavelengths (417 nm, 540 nm, 575 nm) with most interference at 415 nm and causes an increase in the optical absorbance
• Free hemoglobin acts as a pseudoperoxidase, and causes a false low bilirubin levels because Diazonium color reaction is suppressed
• Procedural errors that can cause specimen hemolysis include: drawing blood through a hematoma or from a vein with a hematoma; failure to wipe away the first drop of capillary blood, which can contain alcohol residue; forceful aspiration of blood during a syringe draw; forcing the blood from a syringe into an evacuated tube; frothing of blood caused by improper fit of the needle on a syringe; horizontal transport of tubes, which lets the blood slosh back and forth; mixing additive tubes vigorously, shaking them, or inverting them too quickly or forcefully; partially filling a normal-draw sodium fluoride tube; pulling back the plunger too quickly during a syringe draw; rough handling during transport; squeezing the site during capillary specimen collection; syringe transfer delay in which partially clotted blood is forced into a tube; using a large-volume tube with a small-diameter butterfly needle; using a needle with a diameter that is too small for venipuncture

Timing of Blood Collection

• Biological rhythms (circadian variation) affect certain analytes like Cortisol

Handling, Storage, and Transport

• Temperature and light exposure affect certain analytes:
• Cold-sensitive tests must be kept warm during transport
• Light-sensitive tests should be protected from light to prevent degradation
• Blood gases require immediate analysis to prevent pH and oxygen level changes

Sample Identification Errors

• Mislabeled or misidentified samples are among the most critical pre-analytical errors
• Consequences include misdiagnosis or incorrect treatment due to wrong patient sample testing and test repetition, leading to delays and increased healthcare costs

Physiological Variables

• Include posture, prolonged bed rest, exercise, stress, diet, alcohol, caffeine, smoking, dehydration, circadian/diurnal variation

Posture

• When going from a supine to an upright position, blood volume decreases by 12%, while cortisol, enzymes, cholesterol, total protein, triglycerides, T4, plasma renin, serum aldosterone, and catecholamine increase
• When going from a standing to a supine position, cellular elements, proteins, compounds bound to proteins, and high molecular weight substances decrease
• Prolonged standing decreases plasma protein due to Orthostatic proteinuria and Increases potassium, creatine kinase and aldolase

Prolonged Bed Rest

• Decreases Protein, Calcium, bilirubin, potassium and reduces skeletal mass
• Increases Urinary nitrogen

Exercise

• Short-term exercise:
  • Glucose, Pyruvate, Lactate, Creatinine, FA, Lactate, UA, bili, HDL, and Hormones increase
  • AST, CK, LDH, Aldosterone, Renin, and Angiotensin (24hrs) increase
  • Cholesterol, Triglyceride, Arterial pH and pCO2 decrease
• Long-term exercise increases: -Fatty acids, Lactate, Uric acid, bilirubin, HDL, Growth hormone, and Prolactin -Plasma protein, cortisol, and catecholamines increase -Arterial pH and pCO2 decrease
• Moderate exercise increases Glucose, pyruvate, lactate, & creatinine level and decreases pH, pCO2, cholesterol, & triglyceride
• Strenuous exercise increases levels of Plasma protein, growth hormone,prolactin, and secretion of cortisol and catecholamines

Stress

• Increases in Adrenal hormones, Aldosterone, Renin, TSH, GH, Prolactin, pO2, and WBCs
• Decreases Serum iron and pCO2

Diet

• Protein Diet Increases Plasma protein, plasma urea, ammonia, and cholesterol
• High Fat Diet Increases Triglyceride, alkaline phosphatase, ALT and AST and Decreases Urea nitrogen concentration
• High Carbohydrate Diet Increases Glucose, Insulin, TAG, LDL, and Uric Acid and Decreases Serum LDL, TAG, chole, and protein
• Long Term Fasting and Starvation Results in a gradual decrease in the concentration of glucose and insulin secretion, glucagon secretion doubles including epinephrine and growth hormone, an increased breakdown of triglycerides as energy sources, fatty acids and ketoacids become the source of energy for the muscles, and Consumption of proteins
• Malnutrition increases Cortisol and Decreases Proteins, chole, TAG, Lipoproteins, BUN, Crea, Enzymes, and Thyroid Hormones

Alcohol

• Increases levels of urate, lactate, triglyceride and gamma glutamyl transferase (GGT).
• Decreases Glucose

Caffeine

• Increases Catecholamines, free cortisol, Glucose, and Lipids
• Decreases Electrolytes

Smoking

• Increases Epinephrine, norepinephrine, Glucose, GH, Chole, TAG, LDL, RBC, carboxyhemoglobin, and WBC
• Decreases Antibodies and Sperm count

Dehydration

• Increases Calcium, Coagulation factors, Enzymes, Iron, and Sodium, TP, and Albumin
• Decreases Potassium and Chloride

Circadian/Diurnal Variation

• Highest in the morning: Cortisol, aldosterone, renin, luteinizing hormone, FSH, estradiol, TSH, testosterone, bilirubin, insulin, potassium and serum iron levels
• Lowest in the morning: eosinophils, creatinine, glucose, phosphate, triglyceride

Uncontrollable Variables

• Include age, sex/gender, race, pregnancy, environmental factors, and underlying diseases

Age

• Newborns: Bilirubin, RBC, Hb, Hct, K, Uric Acid, Liver Enzymes = Increase, Glucose, Ca,Mg = Decrease
• Child To Puberty: ALP, Testosterone ,Estradiol, GH, Calcium, Phosphorus = Increase, Creatinine, BUN = Decrease
• Adult: Lipid Profile, Glucose, Uric Acid, GGT, AST, ALT (some individuals), Cr (Younger Adults) = Increase, Testosterone, Estrogen, Cr Clearance, Bone Mineral Markers = Decrease
• Elderly Adults: Lipid Profile, Glucose, Uric Acid, BUN, Cr, PTH, Cortisol = Increase, GFR, Creatinine Clearance, Na, K, Bone Mineral Markers, Testosterone, Estrogen, Progesterone = Decrease

Sex/Gender

• Males: Hb, Hct, RBC, Crea, BUA, CK, GGT, Ferritin, Iron = Increase
• FEMALES: HDL, Estrogen, Progesterone, LH, FSH, Prolactin , TSH, T4 (slightly) = Increase

Race

• Affects the levels of Hb, Hct, Creatinine, Uric Acid, Glucose, and HDL

Pregnancy

• Protein, ALP, Estradiol, and Free fatty acids, RBCs = Increase
• Iron and hematological parameters such as Erythrocyte sedimentation rate (ESR) and Clotting factors II, V, VII, IX, X = Decrease

Environmental Factors

• Altitude = Hb, Hct, RBC, pH, and Lactic Acid, GH = Increase , pCO2, Plasma Volume, Arterial Oxygen Pressure (PaO2) Oxygen Saturation (SaO2) = Decrease
• Ambient Temperature = Plasma Volume, plasma protein, and K, sweating may cause the loss of water and salt = Decrease

Underlying Diseases

• Obesity increases Glucose, Lipid Profile, BUA, and Liver enzymes and decreases HDL, Testosterone, and Free T3, T4
• Fever increases Glucose, WBC, CRP, and Liver Enzymes and decreases Na+, K+, Iron, and Albumin