Phlebotomy and Specimen Considerations PDF - University of Khartoum

University of Khartoum Faculty of Medical Laboratory Sciences Department of Chemical Pathology 2nd Year 2019/2020 Phlebotomy and Specimens Considerations By: Lamia Omer Osman Lecturer of Chemical Pathology Faculty of Medical Labora...

University of Khartoum Faculty of Medical Laboratory Sciences Department of Chemical Pathology 2nd Year 2019/2020 Phlebotomy and Specimens Considerations By: Lamia Omer Osman Lecturer of Chemical Pathology Faculty of Medical Laboratory Sciences University of Khartoum  Objectives of the lesson Identify and describe the types of samples used in clinical chemistry. Outline the general steps for processing blood samples. Identify different types of anticoagulants and their mechanisms of action. Describe collection procedure, handling and preservation of urine, CSF, serous, and synovial fluid. Outline the factors and variables affect test result.  Types of Samples Examples of specimens that analyzed in clinical Chemistry laboratory include: – Blood; whole blood, serum or plasma. – Urine; random, or timed sample. – Cerebrospinal Fluid (CSF). – Serous Fluid; pleural, pericardial, and peritoneal. – Synovial Fluid – Semen Fluid – Amniotic Fluid. – Faeces. – Kidney stone. Blood  Phlebotomy Phlebotomy: is the process of collecting blood, which literally translated means “to cut a vein”. Phlebotomists: are individuals have been specifically trained in blood collection techniques and are employed primarily to collect blood specimens.  Source and Composition of Blood Specimens Blood specimens can be obtained from:  Arteries.  Veins.  Puncturing the capillary bed in skin.  Source and Composition of Blood Specimens  Arterial blood: Arterial blood composition is normally uniform throughout the body. The radial, brachial, and femoral arteries are the primary arterial sites used for collection. It is primarily reserved for blood gases evaluation and pH Collection performed only by those with special training.  Source and Composition of Blood Specimens  Puncturing the capillary bed in the skin: Capillary glucose is normally higher; calcium, potassium, and total protein are normally lower. In Practice it used in situations where: – Sample volume is limited (e.g, pediatric applications) – Repeated venipunctures have result in severe vein damage – Patients have been burned and veins therefore unavailable – And also when sample is to be applied directly to a testing device in a point-of-care testing, to slide or to filter paper  1 2 3 1. The fleshy part 2. The outer 3. The fleshy of the middle of area of the portion of the the last phalanx bottom of the earlobe. of the third or foot fourth (ring) finger (a heel stick) (finger stick).  Equipments for capillary blood collection A sharp lancet is used to pierce the skin and a capillary tube is used for sample collection  Source and Composition of Blood Specimens  Venous blood: Differs most from arterial blood in its low oxygen content, and chloride, glucose, pH, CO2, lactic acid …etc. Phlebotomy, or venipuncture, is the act of obtaining a blood sample from a vein using a needle attached to a syringe or a stoppered evacuated tube. The most frequent site for venipuncture is the antecubital vein of the arm.  Venipuncture Equipments 1. Venipuncture Device: – Syringe.  Venipuncture Equipments a a 1. Venipuncture Device: –Evacuated tube a system Serum a) evacuated tube, Separator Gel (with additive, Clot separator gel or c plain) b) a special needle, b c) a safety plastic holder  Venipuncture Equipments 1. Venipuncture Device: – Intravenous lines (Butterfly)  Venipuncture Equipments 2. Tourniquets. 3. Gloves. 4. Antiseptics (70%isopropanol) to cleanse the puncture site. 5. Sterile Gauze Pads and Bandages 6. Labeling supplies. 7. Special trays to transport blood specimens. 8. Sharp Bin marked with the biohazard symbol or with red or yellow color-coding.  Techniques of venous blood collection Separator Gel Serum Separator Tube (SST)  Types of Blood Specimens Regardless of source, blood is approximately 55% fluid and 45% blood cells. Plasma Serum White Blood Blood cells (WBC) Clot & Platelets -comprised of clotting (RBC) factors (Fibrin, platets etc) -RBCs Whole Blood Whole Blood after centrifugation Whole Blood after clotting Note: clotting has been prevented and centrifugation Types of Blood Specimens  Types of Blood Specimens Whole blood  contains both cells and plasma.  It must be collected in an anticoagulant tube to keep it from clotting.  Whole blood is used for most hematology tests and many point-of-care tests (POCTs), especially in acute care and stat situations.  Types of Blood Specimens Serum:  Is normally a clear, pale yellow fluid. (nonfasting serum can be cloudy due to lipids).  Separated from clotted blood in plain container by centrifugation.  Types of Blood Specimens Plasma  Is normally a clear to slightly hazy, pale yellow fluid.  Separates from the cells when blood in an anticoagulant tube is centrifuged.  Plasma contains fibrinogen; serum does not because it was used in clot formation.  The advantage of plasma over serum is that no time is wasted waiting for the specimen to clot  Types of Blood Specimens Many chemistry tests can be performed on either serum or plasma. Some testing can also be done on whole blood. Choice depends on a number of factors: 1. Analyte to be measured: - Most hematology tests requires whole blood, blood gases, ammonia and trace elements. 2. Instrumentation used for analysis – Most automated instruments are not set up for whole blood analysis  Types of Blood Specimens 3. The way the test was developed. – Tests are often only validated on either plasma or serum 4. Turn around time TAT – Analysis of whole blood is the quickest. No waiting for clot or spinning – Plasma requires centrifugation prior to analysis – With serum, the blood must clot then you have to centrifuge  Anticoagulant and Additives in Tubes Additives: substances (gels, clotting activators or anticoagulants) that are added to tubes to alter the specimen to make it appropriate for analysis. Anticoagulant: substance that prevents blood clotting. – Heparin. – Ethylenediaminetetraacetic acid (EDTA). – Citrate. – Oxalate. – Fluoride Oxalate.  Anticoagulant and Preservative for Blood Anticoagulant Mechanism of Action Heparin. Accelerates the action of antithrombin III, which inhibits thrombin, so blood does not clot. Ethylenediaminet Act as Chelators bind to and remove free Ca2+ etraacetic acid which is required for clotting factor activation. (EDTA) Citrate Prevent coagulation by removing calcium and forming insoluble salts Oxalate Prevent coagulation by removing calcium and forming insoluble salts Fluoride Oxalate Sodium fluoride: prevent glycolysis and stabilize (Sodium fluoride glucose in plasma by inhibiting enzymes of the glycolytic pathway inhibit enolase and potassium oxalate) Potassium oxalate act as anticoagulant  Collection tubes (Vacutainer) Red-top tubes contain no Gold (and “tiger”) top anticoagulants or tubes contain a gel that preservatives forms a physical barrier Red-top tubes are used between the serum and for collecting serum cells after centrifugation – 10-15 minutes is required No other additives are to allow blood to clot present before centrifuging Gel barrier may affect – Used for blood bank some lab tests. specimens, some chemistries  Collection tubes (Vacutainer) Used for glucose measurement. Green-top tubes contain After blood collection, glucose either the Na, K, or lithium (Li) concentration decreases significantly salt of heparin. Most widely because of cellular metabolism Gray-top tubes contain either: used anticoagulant for – Sodium fluoride and potassium oxalate, chemistry tests. or – Should not be used for Na, K – Sodium iodoacetate or Li measurement Both preservatives stabilize glucose in plasma by inhibiting enzymes of the – Can effect the size and glycolytic pathway integrity of cellular blood – NaF/oxalate inhibits enolase components and not – Iodoacetate inhibits glucose-3- phosphate dehydrogenase, recommended for hematology studies  Collection tubes (Vacutainer) Lavender-top tubes Blue-top tubes contain contain the K salt of sodium citrate, which ethylenediaminetetraaceti chelates calcium and c acid (EDTA), which chelates calcium inhibits coagulation (essential for clot Used for coagulation formation) and inhibits studies. coagulation Used for hematology, and some chemistries Cannot be used for K or Ca tests.  Collection tubes (Vacutainer) Brown and Royal Blue top tubes are specially cleaned for trace metal studies Brown-top tubes are used Royal blue-top tubes are for lead (Pb) analysis used for other trace element studies (acid washed)  After Sample Processing Specimen should be submitted in a screw-cap container. Every specimen container must be adequately labeled. Label should be placed in container or tube directly not in the cap. The minimum information should include a patient’s name, identifying number and the date and time of collection. All specimens should be handled with special care and treated as if they are potentially infectious.  After Sample Processing The laboratory scientist should note the presence of any serum or plasma characteristics such as: Haemolysis. Icterus (increased bilirubin pigment) or Turbidity often associated with lipemia.  Haemolysis Is defined as the disruption of the red cell membrane and result in the release of haemoglobin and other cellular component. Slight haemolysis has little effect on most test values. Severe haemolysis cause: – Dilutional effect on constituents present at a lower concentration in RBCs than in plasma. – Elevation of level of constituents that are present at a higher concentration in RBCs than in plasma. ↑aldolase, acid phosphatase, LDH, potassium, phosphate, SGOT What the causes of haemolysis?  After Sample Processing Plasma or serum should be separated from cell as soon as possible. Samples should be analyzed within 4 hours; to minimize the effects of evaporation Samples should be properly capped and kept away from areas of rapid airflow, light, and heat. Most analytes [with the exception of ALP (increases) and LDH (decreases )]stable for 8 hours at 4°C (refrigeration) Samples may be frozen at −20°C and stored for longer periods without deleterious effects on the results. Repeated cycles of freezing and thawing, should be avoided. Urine Specimen  Type of Urine Samples Random Urine Specimen: – A sample of urine collected at any time of the day. – this type of sample may be used to detect the presence of various substances in the urine at one particular point in Urine specimen in clean the day. container – Often, no special handling is required with these samples. – Clean- dry wide mouthed container used for collection, Sterile urine container  Type of Urine Samples Early morning midstream urine – Is the most concentrated specimen – Preferred for microscopic examinations and for detection of abnormal amounts of constituents such as proteins – Although tests in the clinical chemistry laboratory don’t need sterile collection procedures, the patient’s genitalia should be cleaned before each voided.  Type of Urine Samples Timed Urine Specimen – Urine specimens are collected over a predetermined intervals of time Such as 1,4 or 24 hours. – patient showed be well instructed to collect the timed urine  Protocol for Collection of 24-hours Urine Specimen 1. Void and discard this urine (empty the bladder). 2. Start timing the 24-hour period immediately after voiding. 3. Collect all the urine voided for the next 24 hours. Keep the urine in a cool place. 4. At 24 hours, void and add this urine to the collection container. 5. Bring the urine to the lab as soon as possible.  Preservation of urine a. Refrigeration: Refrigeration of specimens is prevent bacterial growth and preserve formed elements. b. Preservatives: – Toluene or thymol: are used to inhibit growth of aerobic bacteria. – Formalin (40% formaldehyde): is a good preservative for formed elements; however, it can interfere with certain chemical tests. – HCl (6mol/l): is used to inhibit bacterial growth by lowering urine pH, and also useful for Ca , Mg and +2 +2 phosphate determination. – Sodium carbonate: is used for the preservation of porphyrins, urobilinogen and uric acid. Cerebrospinal Fluid (CSF)  CSF is the clear colorless liquid that occupies the subarachnoid space surround the brain and spinal cord. Act as mechanical barrier to cushion the brain and spinal cord against trauma. CSF is a selective filtrate of plasma.  Specimen collection for CSF Examination Lumbar puncture by well trained Physician under aseptic precautions and procedure to prevent introduction of Infection and damage of neural tissue.  The trained physician will collect only 3-5 ml into a labeled sterile screw capped container. The fluid to be collected at the rate of 4-5 drops per second. Tests are performed on STAT basis Serous Fluid  Lung, heart and internal organ surrounded by serous membrane consist of two layer (Parietal membrane and Visceral membrane). Serous fluid fills the cavity between membranes to lubricate the opposing membrane surfaces. Serous fluid is ultrafiltrate of plasma. Inflammation or infections affecting the cavities cause fluid to accumulate (effusion) Accumulation of fluid is called effusion; specifically, pleural, pericardial, and peritoneal effusion Effusion is collected and examined to classify fluid as transudate or excudate. Sample Collection and Handling Collection procedure is needle aspiration under aseptic condition; and it referred to as: Thoracentesis (pleural), Pericardiocentesis (pericardial), and Paracentesis (peritoneal). >100 mL is usually collected. Sample collected in: 1. Heparinized for microbiology and cytology, 2. EDTA for microscopy (cell counts and differential), 3. Sodium fluorescein (NaF) for glucose and lactate. Synovial Fluid  Synovial fluid, is a viscous liquid found in the cavities of the movable joints. Serves to lubricate the joint space, reduce friction between the bones during joint movement. Mechanism of formation of synovial fluid is ultrafiltration of plasma across the synovial membrane, combined with a mucopolysaccharide containing hyaluronic acid. Fluid collected and examined in order to classify bone disorders.  Specimen collection and handling Collection of a synovial fluid sample is needle aspiration arthrocentesis under aseptic conditions. The sample should be collected in a:  heparin tube for culture.  EDTA for microscopic analysis.  fluoride tube for glucose analysis.  Sample Variables Factors affecting test results: – Technical – Physiological – Pathological  Sample Variables Patient preparation. Sample collection and processing. Handling Transportation. Storage.  Sample Variables Physiologic variation refers to changes that occur within the body, such as: – Diurnal or circadian variation, – Exercise, – Diet, – Stress, – Gender, – Age, – Lifestyle. – Pregnancy. – Drugs, or – Posture underlying medical conditions (e.g., fever, asthma, and obesity),  References Teitz Fundementals of Clinical Chemistry; Sixth Edition Michael L. Bishop, Edward P. Fody, Larry E. Schoeff, Clinical Chemistry Principles, Techniques, and Correlations, 8th Edition. Chemical Pathology Lecture Notes 2011 University of Cape Town www.labtestsonline.org Thank you for your attention