Clinical Chemistry - 03 Specimen Collection and Handling

• CLINICAL CHEMISTRY SPECIMEN COLLECTION AND HANDLING OUTLINE • Introduction o Patient Identification • Blood Collection o Blood Specimens o Blood o Arterial Puncture o Capillary Puncture o Venipuncture o Phlebotomy Needles o Tourniquet Application o Blood Collection Tubes Additives o Centrifugation • Specimen Variables o Pre-analytical Variables • Other Body Fluids o Urine o Cerebrospinal Fluid (CSF) o Serous Fluids o Synovial Fluid o Amniotic Fluid • • INTRODUCTION PATIENT IDENTIFICATION • • • First step in sample collection; most critical step A prime factor for accuracy of laboratory reports o Correct patient identification allows correct production of laboratory report Should match accurately with the details in the requisition form o Ask patient to spell their name for discrepancy BLOOD • • • PROCEDURE 1. 2. 3. 4. • Conscious Inpatients o Verbally ask their full name and verify with the ID bracelet placed on the arm of the patient Sleeping Patients o Gently awaken the patient and verbally ask their full name comparing it with the ID bracelet Unconscious, Mentally-incompetent patients, Infants and Children o Ask the attending nurse or the relative verifying with the ID bracelet Outpatient/Ambulatory Patient o Verbally ask their full name, address, and birthdate, and countercheck with a valid ID 3-WAY ID Method - to avoid misidentification o Patient’s verbal identification statement o Verify with a ID bracelet o Compare labeled specimen with the patient’s ID bracelet before leaving the bedside BLOOD COLLECTION Venous whole Blood o Whole blood from vein o Deoxygenated blood o Dark red in color o For hematology, immunohematology o Only used in Chemistry for HbA1c determination ▪ Aka glycosylated Hgb/glycated Hgb ▪ Reflects the average blood glucose level over the previous three months ▪ HbA1c - largest subfraction of Hgb A Average Human Body = 1.73L (5 quarts) of whole blood o Adult Male - 5-6L of blood o Adult Female - 4-5 L of blood Composition of Whole Blood o Plasma (55-60%) - 2.84 L (3 quarts) ▪ Liquid part in circulation o Blood cells (40-45%) - 1.89 L (2 quarts) Method of Collection: o Arterial Puncture o Capillary Puncture o Venipuncture ARTERIAL PUNCTURE • • • BLOOD SPECIMENS • Serum o Liquid part of a clotted whole blood o Centrifuged clotted blood o Clearer than plasma o Absence of majority of clotting factors o Most common sample in clinical chemistry section o Chemistry and Immunology/serology Plasma o Liquid part of the anticoagulated blood o Clotting factors are all present causing haziness o For hematology ▪ Citrated plasma for coagulation studies o Used in chemistry, only is serum is not available ▪ Utilizes heparinized plasma (STAT cases) Arterial Blood o For chemistry o Used for blood gas analysis and pH measurement • • • • Process of obtaining blood from patient’s artery Blood collected is arterial blood o Oxygenated blood with bright red color (oxyhemoglobin) o Used for Chmistry: blood gas analysis and pH measurement Sites o Redial artery - most common arterial puncture site for adults (45° angle) o Brachial artery (45° angle) o Femoral artery, scalp artery, umbilical artery Tourniquet is not needed during collection Anticoagulant used - heparin o Heparin - already in syringe for collection Modified Allen test - used to determine whether the ulnar artery can provide collateral circulation to the hand after the radial artery puncture. o Ensure the compensation of lost arterial blood Performed by respiratory therapists and physicians • CAPILLARY PUNCTURE • • • • • • • Also known as dermal puncture/skin puncture. Method of collection for pediatrics and sometimes geriatrics. Rarely used in chemistry. o Due to concentration difference Povidone-Iodine: not used as antiseptic for capillary puncture. May interfere in analyte measurement Blood collected is Capillary Blood o Composed of arterial blood, venous blood, and interstitial fluids o Concentration differences (compared to venous blood): ▪ Glucose: higher than venous blood ▪ K+, total protein, Ca2+: lower than venous blood Sites: o Heel (Lateral Plantar Area) – used on infants o Finger ▪ Central palmar area of 3rd or 4th digit of nondominant hand ▪ used on adults or children (>1 y.o) Order of Draw for microcollection tubes o Important as platelets has tendency to accumulate at the puncture site. Order of Draw Capillary Blood Gas Additives Heparin Blood Smears (Glass Slide) Lavender top Heparinized Plasma Separator Tubes Oxalate/Fluoride Tubes Serum tube with additives Serum tube without additive Newborn Screening N/A Inversion/mixing Rotate between palms N/A EDTA Lithium heparin Lithium heparin with gel separator Sodium fluoride with Potassium oxalate Clot activator 10 10 10 N/A 0 Use spot/filter cards 0 VENIPUNCTURE METHODS • 10 5 VENIPUNCTURE • • • • • Most common type of blood collection Collection of blood from the vein through puncture methods to draw blood. Performed by phlebotomist Blood collected is venous blood o Deoxygenated blood with dark red color (deoxyhemoglobin). Preferred site for Venipuncture: o Antecubital Fossa (elbow pit) ▪ Collectively termed as antecubital veins ▪ Median cubital vein • preferred vein • Largest and the best anchored vein ▪ Cephalic vein – 2nd preferred vein • Away from the body ▪ Basilic vein – last choice • Painful puncture • Close proximity to artery o Other arm and hand veins – only used if antecubital area are not accessible. Sites to be avoided (venipuncture): o Arm with IV lines ▪ First, collect from the opposite arm ▪ Alternatively, collect from the lower extremities with the permission of the physician ▪ Collect below the IV site ▪ Stop IV infusion for 2 minutes and then collect blood sample. Discard the first 5mL (contaminated with IV fluid) ▪ IV fluid contamination • Increased: Glucose, Cl-, K+, Na+ o 10% contamination with 5% dextrose = increased blood glucose by ≥500 mg/dL o May lead to diabetic coma • Decreased: Urea, Creatinine o Hematoma o Burned or scarred areas o Mastectomy side ▪ Collect from the arm opposite to the mastectomy site. ▪ Bilateral mastectomy: collect from veins at the back of the hand without tourniquet; capillary puncture may be alternative o Casts on arm o AV Shunt/Fistula • Syringe Method o Used as an alternative method when drawing blood o Preferred method when drawing blood from patients with small or fragile veins. o Suction pressure on the vein can be controlled o Routinely used: 2mL, 5mL and 10mL o Composed of: ▪ Syringe needle – with plastic hub attached designed to fit onto the barrel. • Hub - indicates presence of blood • Some have protection devices – hypodermic needle ▪ Syringe – divided into graduated barrel and plunger • Barrel – graduated into mL or cc • Plunger – fits tightly within the barrel creating a vacuum when retracted Evacuated Tube System o Closed collection system o The most efficient and the most frequently used method for venipuncture o Blood is collected directly into the evacuated tube; no need for transferring of blood o It enables multiple tubes to be collected in a single venipuncture o Composed of: ▪ Multi-sample needle – allows collection of multiple tubes during venipuncture • Double-pointed needle • With rubber sleeve - inserted to the tube • Bare needle - inserted to the patient ▪ • Tube holders - plastic cylinder with a small opening for a needle at one end and a large opening for tubes ▪ Evacuated tubes – collection tubes with premeasured vacuum that automatically draws the volume of blood indicated in the tube label.\ Butterfly System o Winged infusion set o Used for IV fluid infusion and venipuncture for very small and fragile vein (pediatrics and geriatrics) o Has short needle with a plastic part resembling butterfly wings ▪ Length: ½ - ¾ inches o With long tubing with: ▪ Luer fitting – syringe ▪ Luer adapter – ETS PHLEBOTOMY NEEDLES • • • • Color coded based on gauge Needle gauge is inversely proportional to the needle size. o Lower gauge number - larger bore o Higher gauge number - smaller bore Length: o 1-1.5 inches o 1⁄2 to 3⁄4 inch – butterfly needle Gauge: 16-23 o 21g – standard for venipuncture o 23g – used for children o 23g or 25g – used for butterfly system o 25g – used by trained personnel to collect blood from scalp or other tiny veins. o Using needle smaller than 23g may increase chance of hemolysis. • • BLOOD COLLECTION TUBES ADDITIVES • • • • • Additive Sodium citrate None Red (plastic) Red/light gray (plastic) Red/black (tiger) Gold Red/gold Green/gray Light green Green Clot activator Nonadditive Lavender Pink Gray Orange Gray/yellow Royal blue Tan (glass tube) Tan (plastic) Yellow Yellow Department(s) Coagulation Chemistry, blood bank, serology/ immunology Chemistry NA (Discard tube only) Clot activator and gel separator Chemistry Lithium heparin and gel separator Lithium heparin Sodium heparin EDTA Chemistry Sodium fluoride and potassium oxalate Sodium fluoride and EDTA Sodium fluoride Thrombin None (red label) EDTA (lavender label) Sodium heparin (green label) Sodium heparin EDTA Sodium polyanethol sulfonate (SPS) Acid citrate dextrose (ACD) Chemistry Hematology Blood bank Chemistry Chemistry Chemistry Chemistry Microbiology Blood bank/ Immunohematology ORDER OF DRAW A special sequence of tube collection that reduces the risk of specimen contamination by microorganisms and additive carry-over. ORDER OF DRAW 1. Blood culture tubes (sterile tubes) 2. Coagulation tubes 3. Serum tube with no additives 4. Serum tube with additives, SST 5. Plasma Separator Tubes Heparin 6. EDTA tubes Plasma Preparator Tubes Oxalate/Fluoride tubes TOURNIQUET APPLICATION Should be 3 to 4 inches above the puncture site Constricts blood flow to palpate veins easily Shall not be applied for more than one minute. o > 1 minute: hemoconcentration ▪ Increased number of formed elements due to decrease in plasma volume. ▪ Trapped formed elements May contain none to more than one additives depending on the test ordered. Forms of additives: Liquid, Spray-dried, or Powdered Color coding for evacuated tubes are universal Evacuated tubes are available in glass and plastic Have thick color-coded rubber stopper Stopper Color Light blue Red (glass) • • • • Disposable (reusing may transmit bacteria such as MRSA) o MRSA - Methicillin-resistant Staphylococcus aureus Alternative: o Blood pressure cuff (60mmHg) o For latex-sensitive patients • • TUBE STOPPER COLOR Yellow (SPS) Light Blue Top Red (Glass) Red/Black, Gold, Red/Gold Light Green, Green/Gray Green Lavender/Purple, Pink Pearl Top Gray top SPS - inhibits phagocytosis, prevents complement activation, neutralizes some antibiotics o An anticoagulant Light blue top - 3.2% sodium citrate o 9:1 Blood-to-Anticoagulant Ratio CENTRIFUGATION • • • The process of applying centrifugal force to separate solid materials from a liquid mixture. In blood sample, it separates the formed elements from the liquid part (serum or plasma). Serum/Plasma should be separated from blood cells within an hour. o To prevent glycolysis. ▪ 2 mg NaF/mL of blood – prevents glycolysis for up to 48-72 hrs o To minimize deterioration of unstable analytes. ▪ Unstable (after 30 mins): K+, Phosphorus, glucose ▪ Unstable (after 6 hrs): albumin, bicarbonate, Cl-, C-peptide, HDL, Iron, LDL, Total Protein o To prevent electrolyte shift. ▪ Falsely Increased: Potassium ▪ Falsely Decreased: Sodium o To prevent hemolysis. • • • • SPECIMEN VARIABLES PRE-ANALYTICAL VARIABLES • • • • • Factors affecting analyte concentration prior to collection Exercise o ↑ lactate, fatty acid, ammonia, creatinine kinase, aspartate aminotransferase, lactate dehydrogenase, aldolase, prolactin, growth hormone. o ↓ FSH, LH, Estrogen and testosterone o Vigorous hand exercise (fist clenching): ↑ potassium, lactate and phosphate. Fasting o Required fasting hours: 8-12 hours ▪ No food intake; sips of water is allowed o Analytes requiring fasting specimen: FBS, OGTT, Lipids, Lipoproteins, Gastrin and Insulin. o Basal State Collection – collection of blood sample in the morning before rising, 12 hrs after ingestion of food. ▪ For FBS, Lipids, Lipoproteins and Inorganic Phosphorus o Overfasting: ▪ 48 hours – ↑ serum bilirubin ▪ 72 hours • Males: ↑ triglyceride • Females: ↓ glucose to 45 mg/dL. Diet o High Protein Diet: ↑ urea and uric acid o Atkins Diet (low carb-high protein diet): ↑ urea and urine ketones o High caffeine intake: ↑ glucose (due to catecholamine) ▪ Inhibits insulin Posture o Preferred position during phlebotomy: upright or supine o Recommendation: patient should be seated/supine for 15-20 minutes before blood collection to prevent hemodilution or hemoconcentration. o Abrupt change in posture may affect analytes o Supine to sitting: ↑ albumin, enzymes, calcium o Sitting to supine: ↑ proteins, lipids, BUN, iron and calcium o Standing to supine: ↓ Lipids and lipoproteins • • Tobacco Smoking o ↑ ammonia, glucose, GH, lipids, urea, lactate, insulin, catecholamines, cortisol ▪ 200x increase in ammonia (first-hand & secondhand) = 100-200 mg increase ▪ Ammonia determination - should not have exposure to tobacco or nicotine ▪ Last consume/exposure to tobacco should be 8 hours before collection o ↓ Vitamin B12 Alcohol Ingestion o ↑ Gamma glutamyl transferase, urate, lactate, triglyceride o ↓ Glucose Stress o ↑ catecholamines, cortisol, ACTH, prolactin, albumin, glucose, cholesterol, lactate, Tourniquet Application o Recommended application: 1 minute o > 1 minute application = hemoconcentration and anaerobiosis o ↑ potassium, protein, enzymes, lactate, cholesterol, ammonia ▪ Potassium - 1st protein to increase ▪ Protein - 2nd protein to increase ▪ Tourniquet application increases pressure which causes analytes to leak from tissues to the blood. ▪ Lactate determination: tourniquet should not be applied and fist shall not be clenched during phlebotomy. ▪ Prolongs tourniquet application + fist exercise: ↑ potassium by 1 mmol/L (drastic increase) • Normal potassium - 3.5 and 5.0 mmol/L o ↓ venous pO2, pH Hemolysis o Pinkish/reddish discoloration of the liquid part (serum, plasma) of whole blood caused by extravagation of hemoglobin from red blood cells due to bursting. o It interferes with color reactions. o Decreased (due to dilution effect): ▪ Creatinine, Glucose o Increased: ▪ Electrolytes: Potassium, phosphorus, magnesium ▪ Enzymes: Lactate dehydrogenase, acid phosphatase, alanine aminotransferase, aspartate aminotransferase ▪ Total Protein, Albumin ▪ Cholesterol ▪ Iron ▪ Bilirubin Drugs o Therapeutic Drug Monitoring (TDM) should be scheduled according to the time of the last dose. ▪ Specimen used is serum in red top only o Hepatotoxic drugs: ↑ liver function enzymes o Diuretics: ↓ Na+, K+ o Opiates: ↑ liver and pancreatic enzymes o Blood samples for TDM shall not be placed on SST. ▪ Thixotropic gels absorb drugs ▪ Phenytoin, phenobarbital, lidocaine, quinidine, and carbamazepine. • Blood Specimen Rejection o Hemolyzed sample o Lipemic sample – if non-fasting o Clots (anticoagulated tubes) o Non-fasting specimen – if fasting is required o Short draws - insufficient volume o Improper transport o Error between labelling and requisition form o Unlabeled/Mislabeled specimen o Contaminated specimen OTHER BODY FLUIDS o • • URINE • • Ultrafiltrate of plasma o Blood is filtered in the kidneys o The filtrates are excreted through the urine Most analyzed non-blood specimen • Inhibiting water source prevent adulteration of urine specimen to prevent false negative in testing o Collection may be “observed” (by ASC-authorized specimen collection) or “unobserved” o ASC - not necessarily a medical technologist; ensures proper collection and prevent adulteration of urine Catheterized – Urine collection from a sterile catheter inserted through the urethra into the bladder o For bacterial culture o Urine will not be exposed to genitals which may contaminate the urine Suprapubic Aspiration – Collection through aspiration by inserting needle directly into the bladder. o For anaerobic bacterial culture and cell cytology Pediatric – A soft, clear plastic bag with adhesive is attached to the genitalia of the patient (wee bag) o Checked every 15 minutes until required volume is collected CEREBROSPINAL FLUID (CSF) TYPES OF URINE SPECIMEN • • • • • Random – collected any time of the day o For routine and qualitative urinalysis First Morning/8-hour Urine – First voided urine after waking up from 8 hours of sleep o Ideal Specimen for routine urinalysis & pregnancy test o Most concentrated (↑HcG levels for pregnancy test) ▪ Due to 8-hour gap between voiding ▪ Accumulation of substances during 8-hour period Fasting/Second Morning – second voided urine after fasting (important for glucose determination) o Example ▪ Fasting @ 10 pm ▪ 1st urine @ 6 am - formed before fasting, may have false increased glucose, discarde ▪ 2nd urine - accepted Urine for Drug testing – random urine specimen collected in a special urine container (white opaque urine container with tight fitting lid) Timed Specimen – collected urine or pooling of multiple urine at specific time period o Glucose Tolerance Urine – fasting (30 minutes, 1 hours). Optional for glucose tolerance test (blood) o 2-hour Post Prandial Urine – two hours after meal (for diabetes screening/monitoring) o 24-hours Urine – collection and pooling of all urine voided within 24 hours. o Fractional Specimen (Double-voided) – series of urine samples (at least 2) collected at specific time intervals for comparison • • • • ORDER OF COLLECTION Tube number (in order of collection) First tube • • • Regular Voided – patient voids or urinates into a clean container (no specific timing) Midstream – first and last drop of uninterrupted urination are discarded. Midstream is collected. Midstream Clean-Catch – cleaning of genitals prior to midstream collection. o Cleaning minimizes contamination of urine specimen Drug Testing – patient voids random urine inside a restroom with no water source (toilet bowl, sinks) located inside testing area Tests Notes Storage Least affected by Frozen bacteria or blood introduced as a result of collection Second tube Microbiology Least likely to be Room contaminated with temperature bacteria introduced only in spinal tap Third tube Cell count Least likely to Refrigerated (Hematology) contain cells introduced in the spinal tap *Fourth tube Microbiology, For better exclude Frozen (Im) Immunology skin contamination URINE COLLECTION METHOD • 3rd major body fluid o 3rd most commonly analyzed body fluid o 1st - urine; 2nd - stool Clear and colorless fluid that surrounds the brain and spinal cord Specimen: o Method of Collection: ▪ Lumbar puncture/spinal tap between 3rd, 4th or 5th vertebra ▪ Collected by trained physicians o Collection tube: collected in 3 to 4 sterile tube ▪ If one tube only is filled (insufficient sample): Microbiology → Cell Count → Chemistry/Serology Diagnostic value: o diagnosis of meningitis o brain abscess o CNS cancer o multiple sclerosis Chemistry, Serology SEROUS FLUIDS • • Ultrafiltrate of plasma found in between serous cavities. Lubricates the membranes of serous cavities to prevent friction as a result of movement of enclosed organs (heart, lungs, peritoneum) • • • Specimen: o Pleural fluid (lungs) – collected through thoracentesis o Pericardial fluid (heart) – collected through pericardiocentesis o Peritoneal fluid (peritoneum cavity in abdominal area) – collected through paracentesis o Collection tube: ▪ EDTA – cell counts and smears ▪ Heparin/Sodium fluoride – chemistry ▪ Sterile – culture and sensitivity ▪ Non-additive tube – biochemical tests o Should be delivered in the laboratory within 2 hrs. If delay is expected, store in refrigerator (4C) except for microbial culture (sterile tube). Diagnostic Value: o Detect causes of Effusion (increased serous fluid volume due to inflammation, infection, or hypoproteinemia) Chemical Examination Glucose Decreased in tuberculosis and malignancy Lactate Malignancy dehydrogenase Amylase Pancreatitis, GIT perforation, intestinal necrosis Alkaline phosphatase Intestinal perforation Lactate Increased in bacterial peritonitis, malignancy and tuberculosis BUN & Creatinine Ruptured Bladder Ammonia Perforated peptic ulcer, ruptured appendix, bower strangulation, ruptured bladder Serum ascitesTransudates: >1.1 albumin gradient Exudates: <1.1 CEA and CA125 Tumor markers SYNOVIAL FLUID • • • • • Joint fluid viscous fluid circulating in diarthroses (movable joints) Specimen: o Collection: Arthrocentesis – aspiration collection by inserting needle in between joints o Specimen Container: ▪ Liquid EDTA/Heparin – cell counts, crystal ID, and smear preparation • Avoid powdered to prevent crystal formation ▪ Sterile Container – Culture and Sensitivity ▪ Sodium fluoride – glucose analysis ▪ Non-additive tubes – macroscopic appearance, chemistry, immunology, and clot formation observation Diagnostic value: o Arthritis o Gout o Inflammatory conditions Chemical Examination Glucose Reference value: 0-10 mg/dL lower than serum glucose. Synovial fluid-to-serum glucose ratio: 0.9:1 Decreased in septic arthritis (arthritis due to infection) Protein Lactate Uric Acid Lactate dehydrogenase • • • • • Reference value: <3 g/dL Increased: Inflammation, hemorrhagic disorders Reference value: <25 mg/dL Septic arthritis: >250 - 1000 mg/dL Reference value: 6 mg/dL Increased in gouty arthritis Used if crystal examination is not available Increased: rheumatoid arthritis, infectious arthritis, gout Increased: rheumatoid arthritis, infectious arthritis, gout Rheumatoid arthritis: increased synovial LDH but normal serum LDH AMNIOTIC FLUID product of fetal metabolism provides cushion for fetus, allow fetal movement, and stabilize temperature for the fetus. Collection: o Amniocentesis – needle aspiration technique o First 2-3 mL are discarded – contaminated with maternal blood, tissue fluid and cells. o Container: Sterile container o Storage: ▪ Fetal Lung Maturity: refrigerated ▪ Cytogenetic analysis: room to body temp ▪ Hemolytic disease of the Newborn (HDN): protect from light. Diagnostic Value: o Checks fetal development (esp. fetal lung maturity) o Detects any genetic disorders o Verify gestational age o Diagnosis of Hemolytic disease of the newborn Chemical Examination Alpha-fetoprotein Neural tube <2.0 MoM Acetylcholinesterase defects ABO-Rh HDN OD at 365 nm compatibility Bilirubin scan OD at 450>0.025 LecithinFetal Lung ≥ 2.0 Sphingomyelin ratio Maturity Foam stability Index ≥ 47 Fluorescence 55 mg/g Polarization surfactant/albumin content Lamellar body count ≥ 32,000/mL o OD - optical density