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 Specimen/ samples – analyzed  Substances – measured SUBSTANCES MEASURED IN SERUM 1. Substances normally present w/ function: - glucose, TP, albumin, individual proteins, electrolytes, total albumin globulin (TAG), cholesterol, hormones, vit 2. Metabolites – waste-products - urea, creatinine, uric acid, NH3, Bb SAMPLE VARIABLES: 1. Physiological consideration 2. Proper Px preparation 3. Problems w/ collection 4. transport 5. processing 6. storage IMPORTANT CONSIDERATIONS 1. Physical activity a. Transient effects – inc, dec (FFA, alanine, lactate, K+) b. Long term effects – inc alanine, aldolase, ALT, LDH c. 6 months of physical training – inc: plasma testosterone: 21% androstenedione: 25% LH: 25% 2. Fasting (prolonged)—FBS: 12hrs a. after 48 hrs: serum Bb inc: 240% b. after 72 hrs: glucose dec to 45mg/dl inc plasma trig, glycerol & FFA cholesterol: no change 3. Posture - supine upright (inc albumin, drug-bound CHON, cholesterol, enzymes, total CHON, triG, Bb, Ca) 4. Ethanol ingestion - inc plasma lactate, urate, trig - ROH abusers – inc GGT (γ-glutamyl transferase), urate, MCV 5. Tobacco smoking - inc blood carboxy Hb levels a. heavy smokers: inc 8% b. non-smokers - <1% c. acute tobacco smoking – inc plasma catecholamines, serum cortisol GENERAL RULES FOR BLOOD SPECIMEN COLLECTION: 1. Px ID 2. approaching Px 3. Collection site 4. Tourniquet app 5. checking Px 6. specimen handling & transport 7. storing specimen prior to transport  serum pH: 8.5 – ACP destroyed  Glycolysis – dec serum glucose  Changes in RBC permeability – inc K, P, Mg  Freezing, subseq thawing, refreezing, rethawing: CHON denaturation  Frozen specimen, thawed at RT before analysis – inc ALP  Exposure to light – dec Bb VENIPUNCTURE - deoxygenated blood; contains subs that come from metabolic activities of diff organs - blood chem. & immunologic studies - more easily collected than arterial blood Sites:

 antecubital fossa veins – median (most preferred), cephalic, basilica  wrist, hand & ankle veins Complications (immediate/ delayed & local/ systemic):  hematoma (missed vein)  collapsed small veins (excessive pull of plunger)  syncope (loss of consciousness)  excessive bleeding  thrombosis of vein  blood-borne infection: Hep B & AIDS Methods: a. Syringe method (most conventional) - single sample needles – specifically for syringes - barrel, plunger, needle, needle holder (5&10), hub, bevel b. Evacuated tube system - multiple needles should be used if more than 1 tube of blood is to be collected (prevent leakage during tube changes - gel: barrier bet serum & cells - marked w/ expiration date (lost vacuum/ ineffective additive) - correct blood-AC ratio - needle: 2 way longer for tube; other for vein c. Butterfly infusion set - stainless steel beveled needle w/ attached plastic wing for phlebo to grasp during needle insertion - plastic tubing connects needle to adaptor- screws into a tube holder = modified evacuated system - for pedia/ babies & geriatrics *gauge: bigger number = smaller bore (21:ideal; 26: blood donation) VENIPUNCTURE PROCEDURE 1. assembling the patient 2. positioning & ID the patient 3. preparing needle & evacuated tubes or syringe 4. applying tourniquet to select the vein & removal of tourniquet - 3 mins application increases CHON, Fe, AST, Bb & total lipids - Repeated fist clinching increases K+ 1-2nm 5. applying the antiseptic (wet then dry) - circular motion (inside to out) 6. reapplying tourniquet 7. inserting the needle 8. withdrawing blood - IV avoided - Turn off IV for 2-5 mins, discard 1st 5ml of draw 9. releasing tourniquet 10. withdrawing needle 11. removing needle from syringe/ removing evacuated tube from needle 12. transferring blood to an appropriate container 13. checking patient’s wound - Betadine – falsely high P, uric acid, K+ - Isopropyl (70%) alcohol should not be used for medical or legal ethanol levels 14. labeling specimen ADVANTAGES OF EVACUATED TUBE SYSTEM OVER SYRINGE METHOD:  multiple blood collection  no prior preparation  wider range of tube sizes & AC  safer method of blood collection

ADVANTAGES OF VENIPUNCTURE OVER SKIN PUNCTURE:  Large amount of blood can be obtained for a variety of tests  Additional & repeated test can be done  Fastest method of collecting samples from a number of patients  Blood can be transported to lab and stored for future use  blood chemistry determination DISADVANTAGES OF VENIPUNCTURE:  Harm on infants, children & obese individuals  more time & skill (operator)  complications COMPLICATIONS IN VENIPUNCTURE: 1. local immediate complications - hemoconcentration - failure of blood to enter syringe - circulatory failure - fainting or syncope 2. local delayed complications - hematoma - thrombosis of vein - thrombophlebitis 3. general delayed complications - serum hepatitis, AIDS  SST – clot activator – reduce clotting time  PST – green/ gray - Li Heparin w/ gel - Electrolytes, routine chem. (invert 505x)  Trace element tube: heparin, EDTA or none  SPS: Soidum/ Polyanithol sulfonate (micro)  ACD – acid citrate dextrose (HLA typing) PARTS: 1. needles - adults: 20, 21 - pediatrics/ geriatrics: 22; veterinary: 18 2. needle holder - regular – 13, 16mm - pediatric – 10mm 3. tubes/ evacuated tubes - regular – 75mm; 100mm ARTERIAL PUNCTURE - oxygenated blood; uniform composition throughout body - measure O2 , CO2 tension & blood pH - BGA: critical to patients w/ pulmonary problems, O2 therapy, cardiovascular problems & those undergoing major operations - Sites: radial, brachial & femoral arteries - Radial & brachial: preferred sites - Newborns: umbilical artery catheter - Brachial: 18-20 gauge, 45-60 - Radial: 23-25, 90 - AC: heparin Complications:  Hematoma (increases pressure in artery)  Arterial spasm (restriction of BF due to reflex constriction)  Temporary discomfort (aching, throbbing, tenderness, sharp sensation & cramping)  Thrombosis, hemorrhage & infection Considerations:  Intense care must be administered

 Not be selected: irritated, edematous, near wound or area of AV shunt or fistula  Ice water/ coolant (1-5C): minimize WBC O2 consumption  Capillary blood: substitute for arterial blood determination of pH & pCO2 (warmed site: increases BF through capillaries & arterioles arterial-rich blood) SKIN PUNCTURE/ CAPILLARY PUNCTURE - small quantity blood needed - pediatrics, obese w/ thrombotic tendencies & severe burns - geriatric patients: thinner & less elasticity of skin Sites:  earlobe – free edges, not on sides  palmar surface of finger  plantar surface of heel & big toe Infants:  digits of 2nd, 3rd or 4th fingers  lateral plantar heel surface  median plantar heel surface Children  plantar surface of big toe  lateral side of finger, adjacent to nose Blood obtained:  capillary: BG (prewarmed)  peripheral (most common term used)  arteriolar Sites to avoid:  inflamed or palmar surface  congested or edematous site  cold & cyanotic areas (concentrated blood)  scarred & heavily calloused area Advantages using earlobe:  less painful (few nerve endings)  free flow of blood (thinner skin)  less tissue contamination of blood  searching Abnormal cells (histiocytes in bacterial endocarditis) Advantages using finger:  accessible to phleb  easy to manipulate  ideal for peripheral blood smears  less intimidating Disadvantages of skin puncture:  less amt of blood obtained  add & repeated tests can’t be done  blood hemolyzes easily  painful (finger) Procedure: 1. ID patient 2. reassure 3. assemble equipment 4. prepare finger 5. puncture finger 6. eliminate 1st drop 7. produce large rounded drop 8. withdraw/collect blood 9. stop bleeding 10. thank patient 11. transport specimen to lab Things to remember in doing skin puncture:  depth: 2.5-3mm

avoid pressure & squeezing first drop should be discarded (alcohol & tissue juice) RBC, Hct, Hb & platelets: low WBC: higher than in venous Capillary blood from skin puncture is an admixture of venous, arterial & may contain tissue juice Microcapillary tubes used in hema:  Blue ring – no AC  Red ring – heparinized  Green ring – heparinized  Skin puncture site must be warmed (increase BF):  Dry heat or paper towel w/ warm H2O (39-42C)  Flicking w/ index finger until flushing is observed  Chemicals (trafutil paste Histamine creams)  Arterial blood from capillary puncture may yield unreliable results if:  Systolic is less than 95mmHg  Cardiac output is severely restricted  Vasoconstriction  Puncture depth: 0.85-2mm deep; 1.75-3mm length VACUTAIJNER/ EVACUATED TUBES: Color coded based on AC (2,5,7 &10ml) 1. Yellow (blood culture tubes) 2. Red (no AC—obtain serum) 3. Blue (Na citrate) 4. Green (heparin) 5. Lavender (EDTA) 6. Others--Gray (Fluoride-glucose det)  Gel vs non gel: serum or plasma separator tubes maybe unacceptable for some analytes (therapeutic drug) Color Additive Action Use Lavende EDTA Chelates Hematologic r calcium assays, lead  Versene assays, CEA (disodium determination salt) & cell counts  Sequestren e (dipottasium salt) Red none Allow blood Most to clot chemistry, immunologic & blood bank tests Red/ None; Allows blood Most gray or separator to clot; chemistry red material barrier tests black between cells & serum Orange thrombin Accelerated STAT serum clot test Blue Buffered Binds Ca Coagulation citrate assays like PT & APTT Black Buffered Binds Ca Westergren sodium ESR citrate Gray Glucose  NaF/  Inhibits determination K2C2O4 glycolytic enzymes enolase &  Iodoacetat     

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acts as AC  Inhibits glyceraldeh ydes 3phosphate dehydrogen ase Yellow Citrate Preserves Blood culture dextrose RBC Green Heparin (Na+. Inhibits Active & Li+ or NH4+) thrombin methemoglob BGA, in ammonia CO-Hb  Anticoagulant interference:  Dilution errors—oxalates: highly osmotic  Inhibition of plasma enzyme activities—Fluoride (enzyme poison) - EDTA: chelates metallic enzyme activators - Oxalates: inhibits AMS, LD & ACP - Citrate: inhibits AMS  Oxalates, citrate & EDTA: lower plasma Ca levels  False increase in electrolyte analyses due to AC in salt form SPECIMEN HANDLING & PROCESSING Serum:  20-30 mins: ideal clotting time  Preferred than plasma: 1. interfering substance are co-precipitate during clotting,LPL 2. optically clearer 3. free from AC interference  Reach lab w/in 45 mins  Agitation avoided  Amber containers for photolabile substances  Transport in ice (4C): BGA, rennin, enzymes & catecholamines Specimen Interference:  Lysis of cells (hemolyzed serum)  Leakage if IC substances  Lysis of RBC = laking/ hemolysis (in vivo or in vitro)  In vitro hemolysis is due to (common): use of vacuum tubes vigorous mixing use of too narrow/ roo wide needle bores effect of ROH centrifugation & separation steps  Hemolysis is visible only not until a 200mg/L of Hb level  Ictersia (Icteric serum)  Intensely yellow serum sample (elevated Bb value)  Jaundice: Bb greater than 430uM (25mg/L)  Bb interferes w/ test using dyes & turbidity test  Interference due to Bb may be minimized: sample blanking or dual wavelength method (Allen correction method)  Lactescene (Lipemic serum)  Obtained normally after a meal (elevated exogenous chylomicrons)  Characterized by milky or highly turbid serum  Lactescene appears when TAG level reaches 4.6mm (4g/L)  Corrected by ultracentrifugation of serum sample  Grounds for rejecting specimen  Inadequate sample ID

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 Insufficient volume of specimen collection  Inappropriate collection tube  Hemolysis  Improper transportation  Interferences Things to remember:  All materials: dry & sterile (avoid hemolysis)  Never puncture/ draw blood from vein where IV medication is running (false low)  Site of puncture: thoroughly clean (avoid Thrombophlebitis)  Tourniquet: not tied too tight (constrict arteries & veins)  Tourniquet: always released before withdrawing needle from vein  Remove needle from adaptor of syringe & allow blood to flow gently down sides of tube  Container: stoppered; AC tubes inverted 6-10x  Reusable syringes: rinsed w/ tap H2O  Needles: sharp container

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