Clinical Specimens (1) - CLS 251 Lecture 3 PDF

Clinical Specimens CLS 251 Lecture 3 Mohammad A. Alfhili, Ph.D.  Outline For each body fluid, understand three aspects: 1. Physiology – how the fluid is formed and what role(s) it plays 2. Collection – how the fluid is collect...

Clinical Specimens CLS 251 Lecture 3 Mohammad A. Alfhili, Ph.D.  Outline For each body fluid, understand three aspects: 1. Physiology – how the fluid is formed and what role(s) it plays 2. Collection – how the fluid is collected for testing 3. Storage/disposal – how the fluid is stored in the lab and how it is disposed of List of body fluids discussed: 1. Blood 2. Urine 3. Cerebrospinal fluid 4. Semen 5. Serous fluids 6. Gastric fluid 7. Synovial fluid 8. Amniotic fluid 9. Stool Blood  Plasma Serum Liquid portion of blood – Liquid formed off clotted contains all coagulation blood – no coagulation factors factors and fibrinogen or fibrinogen Coagulation factors and No interference additives interfere with results Immediate centrifugation 20 min required for clot formation Hazy, yellow Clear, yellow More protein (fibrinogen) More K+ (off platelets)  Phlebotomy Phlebotomy is the procedure of blood collection (Phleb = vein; tomy = cut): Patient ID, diet, exercise, and tests required Isopropyl alcohol (70%) is the sterilizing agent used in form of wipes. Tourniquet restricts blood flow and makes veins more visible. It’s placed above the puncture site. In case of IV fluid infusion, use the other hand or ask the nurse to stop the infusion for 2 min, place the tourniquet below the IV site and draw from below the site. If neither is possible, draw blood and discard the first 5 mL. Note this on the requisition form. Pierce at 45o with bevel side up. Infants – fingerstick sampling by heel stick or scalp veins. Avoid areas with scars, wounds, burns, hematoma, edema… Place a gauze or cotton ball over the puncture site, then remove the needle as you firmly position the gauze over the wound (firmly to prevent hematoma). Place a bandage over the wound, and label tubes with patient’s number.  The medial cubital vein is preferred because it is large, close to the surface of the skin, and sufficiently anchored to the tissue (doesn’t move) for a successful venipuncture. Avoid the cephalic, median, and basilic veins because of their tendency to bruise easily.  Evacuated/Vacuum Tubes A disposable, double-pointed needle (one side is plastic-covered, and the other is rubber-covered) is attached to a hub, and inserted into the vein at 45o with the bevel side up. Remove tourniquet to avoid hemoconcentration (↑ concentration of large molecules, AST, iron, and cells) which occurs if the tourniquet remains on during blood flow due to hydrostatic pressure that causes water and filterable elements to leave the extracellular space. While keeping the needle in place, use the other hand to fill a vacutainer tube. Multiple tubes can be filled. The vacuum in the tube causes blood to move through the needle and into the tube (if no blood is drawn, the tube may have no vacuum). Remove the tube before withdrawing the needle (as there may still be some suction left, causing pain upon withdrawal).  Needle & Syringe The rear end of a normal or butterfly needle is attached to a syringe (5 or 10 ml) and the needle is inserted into the vein while the other hand holds the syringe. Butterfly helps with difficult sticks, such as small veins and veins located in difficult-to-reach areas. Once blood starts flowing, raise the plunger slowly to prevent hemolysis. When needed amount is obtained, remove the tourniquet and the needle while still raising the plunger (to prevent blood spelling). Needle gauge (diameter) is important: o Large 16-gauge needle – blood donation o Standard 21-gauge needle – routine phlebotomy o Small 23-gauge needle – venipuncture of small veins (hemolysis risk) o Smaller needles are used for IV injections Syringes are preferred over an evacuated tube system as they allow for control over the suction pressure on the vein by slowly withdrawing the plunger.  Phlebotomy Complications 1. Syncope: If the patient indicates that they feel faint, the phlebotomy procedure must be immediately stopped. Quickly remove the needle and release the tourniquet. If the patient in sitting, lower the patient’s head, have the patient take deep breaths, and apply cool wet cloths/compresses to the back of the neck. A drink of cold water is often helpful. If the patient faints and collapses, lower the patient to the floor to a supine position. Apply cold compresses till recovery. 2. Hematoma: Blood leaking into the surrounding tissues from around the needle. Causes: a. Failure to insert the needle far enough into the vein b. Bending the arm while applying pressure c. Excessive probing to obtain blood d. Failure to remove the tourniquet before removing the needle e. Applying inadequate pressure on the site after removing the needle If swelling is observed around the area of the needle, the phlebotomy procedure must be immediately stopped. Remove the needle and tourniquet. Apply a firm pressure to the site for a minimum of 2 min.  Red Top Tubes Plain tube. Blood physiologically clots in about 60 min, and serum is obtained by centrifugation. Suitable for: Chemistry, serology, and blood bank (RBCs are preserved, too). Plastic red top tubes contain silica, and are thus not recommended for blood banking.  Lavender (Purple) Top Tube Anticoagulant: Ethylenediaminetetraacetic acid (EDTA). Form of anticoagulant: Liquid tripotassium (K3EDTA) or spray-dried dipotassium (K2EDTA). Mechanism of action: EDTA chelates calcium which is required for blood coagulation. Gently invert 8 times upon collection. Suitable for: Hematology (preserves cells; no platelet clumping, compatible with staining). Not suitable for: 1. Chemistry (chelates ions; false low results). 2. Coagulation (interferes with factor V and thrombin-fibrinogen reaction). Must be completely filled to avoid excess EDTA which may shrink RBCs. Pink Top tubes are used for blood banking as labels contain information required by the American Association of Blood Banks (AABB). They contain spray-dried K2EDTA, and must be inverted 8 times.  Light Blue Top Tube Anticoagulant: 3.2% or 3.8% Sodium citrate (Na₃C₆H₅O₇). Mechanism of Action: Binds to calcium. Blood/Anticoagulant Ratio: 9 to 1 (e.g., 4.5 ml blood + 0.5 ml sodium citrate). This percentage preserves the labile coagulation factors. Therefore, tubes must be completely filled, and the tubes are REJECTED if incompletely filled. Tubes must be inverted 3-4 times. Suitable for: Coagulation tests. Polycythemia: o For patients with polycythemia or hematocrit >55%, sodium citrate must be lowered because high citrate interferes with coagulation tests. Always using 3.2% is recommended for this reason. Fibrin-degradation products: A special blue top tube with thrombin and a soybean trypsin inhibitor to immediately clot blood. Westergren sedimentation rate: A special black top tube with sodium citrate at a blood/anticoagulation ratio of 4:1.  Green Top Tube Anticoagulant: Heparin (sodium, lithium, or ammonium ion). Mechanism of Action: Inhibits thrombin (binds to antithrombin III). Suitable for: Chemistry (e.g., STAT electrolytes) Not suitable for: Hematology (interferes with blood film by imparting a dark background). Interference by the salt with the corresponding test must be avoided (e.g., Li heparin for Li measurement). In general, Li heparin shows the lowest interference. Tubes must be inverted 8 times. Light green (black/green) tubes: o Contain Li heparin + separator gel and are called plasma separator tubes (PSTs) o Suitable for K+ testing (prevent K+ release by platelets during clotting). o Must be inverted 8 times.  Gray Top Tube Anticoagulant: Potassium oxalate (C2K2O4) or K2EDTA. Antiglycolytics: Sdoium fluoride (NaF) or lithium iodoacetate (C2H2ILiO2). Mechanism of Action: C2K2O4 and K2EDTA prevent clotting by binding to Ca2+. Suitable for: Chemistry (i.e. glucose and ethanol). Not suitable for enzyme assays. Not suitable for: Hematology (oxalate destroys cellular morphology). For ethanol measurement: o NaF inhibits microbial growth which could produce alcohol as a metabolic end product. o Potassium oxalate may be used if plasma is needed.  White Top Tube Anticoagulant: Spray-dried dipotassium (K2EDTA) and separation gel. Also called: Plasma preparation tubes (PPTs). Suitable for: Molecular diagnostics (e.g., PCR) and chemistry (myocardial infarction and ammonia levels) Tubes must be inverted 8 times.  Royal Blue Top Tube Anticoagulant: None or sodium heparin to conform to diverse tests. Suitable for: Toxicology, trace metal, and nutritional assessment tests. All of these studies measure metals at very low concentrations which could be present in other tubes. Royal blue top tubes are virtually metal free. If anticoagulant is present in the tube, it must be inverted 8 times. Tan top tubes: o Na heparin in glass tubes; K2EDTA in plastic tubes o Contain very little lead (