HMA Lab Prelims PDF

WEEK 3: VENIPUNCTURE IMPORTANT NOTE: When identifying a patient, ask his or her full name PRE-ANALYTICAL PHASE and date of birth...

WEEK 3: VENIPUNCTURE IMPORTANT NOTE: When identifying a patient, ask his or her full name PRE-ANALYTICAL PHASE and date of birth - Larger amount of blood for chemistry and other CLSI recommendation – ask patient to spell hematological analyses are obtained by venipuncture from his/her last name the median basilic or median cephalic veins of the Check the ID band or bracelet if applicable antecubital fossa and from the dorsal surface of the hand or 3 way ID – patient’s verbal statement, checking of foot in adults or the external jugular vein or femoral veins in the ID band, visual comparison of the labeled infants. specimen band before leaving the bedside Sleeping patients – wake person gently; do not ANALYTICAL PHASE startle patient as it will affect test results Sterile syringe Unconscious patients - Ask a relative or the Sterile needle (gauge 21) patient’s nurse or physician to identify the patient Tourniquet and record the name of that person Cotton Infants and children – a nurse or relative may 70% Isopropyl alcohol identify the patient Evacuated tubes Micropore tape PREPARING THE PATIENT Yellow bag - Explain procedure; Never attempt to explain the purpose of a test to a patient. Because a particular test can be ordered PROCEDURE: to rule out a number of different problems, any attempt to 1. REVIEW AND ACCESSION TEST REQUEST explain its purpose could mislead or unduly alarm the patient - This is the first step for the laboratory in the pre-analytical - Addressing needle phobia: (before analysis) or pre- examination phase of the testing Have the patient lie down during the procedure, process with legs elevated - Test requisition: Apply an ice pack to the site for 10 to 15 minutes to form on which test orders are entered; become part numb it before venipuncture. of a patient’s medical record and require specific Have only the most experienced and skilled information to ensure that the right patient is tested, phlebotomist perform the venipuncture. the physician’s orders are met, the correct tests are - Verify diet restrictions and latex allergy performed at the proper time under the required conditions, and the patient is billed properly. 3. POSITIONING THE PATIENT AND TOURNIQUET - Accessioning the test request APPLICATION accession is the process of recording in the order - Inpatients normally have blood drawn while lying down in received their beds When a test request is accessed it is assigned a - Outpatients at most facilities are drawn while sitting up in unique number used to identify the specimen and special blood-drawing chairs all associated processes and paperwork and connect them to the patient. TOURNIQUET APPLICATION - Tourniquet is applied: 3-4 inches above the puncture site no 2. APPROACH, IDENTIFY, AND PREPARE PATIENT longer than 1 minute - PATIENT IDENTIFICATION most important step in the - If it is closer to the site, the vein may collapse as blood is venipuncture procedure removed. If it is too far above the site, it may be ineffective Obtaining a specimen from the wrong patient can - Hand veins- the tourniquet is applied proximal to the wrist have serious, even fatal, consequences, especially bone specimens for type and crossmatch prior to blood - When the tourniquet is in place, ask patient to clench or transfusion make a fist Misidentifying a patient or specimen can be grounds for dismissal of the person responsible and - IMPORTANT NOTE: can even lead to a malpractice lawsuit against that Pumping of the fist should be prohibited causes person. veins to move; changes in blood components (hemoconcentration, potassium and ionized calcium) 1  Often harder to palpate than medical cubital vein Fairly well anchored Often the only vein felt in obese patients - Basilic vein located on the medial aspect (inner side) of the antecubital area; last choice Not well anchored and rolls easily Increased risk of puncturing a median cutaneous nerve branch or the brachial artery Not recommended unless no other vein in either arm is more prominent - M-Shaped Antecubital Veins Median vein/intermediate antebrachial vein Median cephalic vein/intermediate cephalic vein Median basilic vein/ intermediate basilic vein - OTHER SITES: 4. SELECTING THE VEIN Great Saphenous vein - Antecubital fossa – most preferred venipuncture site Femoral vein - A patient will generally have the most prominent veins in Jugular vein the dominant arm - To locate a vein, palpate (examine by touch or feel) the area by pushing down on the skin with the tip of the index finger - In addition to locating veins, palpating helps determine their patency (state of being freely open), size and depth, and the direction or the path they follow - Do not select a vein that feels hard and cord-like or lacks resilience, as it is probably sclerosed or thrombosed. Such veins roll easily, are hard to penetrate, and may not have adequate blood flow to yield a representative blood sample - CLSI recommendation - when a tourniquet is used during preliminary vein selection, it should be released and reapplied after 2 minutes VENIPUNCTURE SITES - Antecubital Fossa - Antecubital (means front of the elbow), fossa - means a shallow depression is the shallow depression in the arm that is anterior to (in front of) and below the bend of the elbow - H-Shaped Antecubital Veins is displayed by approximately 70% of the population and includes the median cubital vein, cephalic vein, and basilic vein Median cubital vein - located near the center of the antecubital fossa - preferred vein because it is typically large, close to the surface - closer to the surface and the most stationary - easiest and least painful to puncture; least likely to bruise - Cephalic vein Located in the lateral aspect (outer side) of the antecubital area; second choice 2 5. CLEAN AND AIR DRY THE SITE the vein) and a possible adverse patient reaction from - The Recommended antiseptic for cleaning a venipuncture additives can occur if tube blood is in contact with the site is 70% isopropyl alcohol, which is typically available in needle. sterile, prepackaged pads referred to as alcohol prep pads. - Clean the site with a circular motion, starting at the point where you expect to insert the needle, and moving outward in ever-widening concentric circles (circles with a common center) until you have cleaned an area approximately 2 to 3 inches in diameter. - IMPORTANT NOTE: Allow site to dry naturally, do not dry the alcohol with non sterile gauze, do not fan or blow the site The evaporation and drying process helps destroy microbes, prevents specimen hemolysis from alcohol contamination, and avoids a burning sensation when the needle is inserted 6. PREPARE EQUIPMENT 7. ANCHORING AND NEEDLE INSERTION - To anchor antecubital veins, grasp the patient’s arm with your free hand, using your fingers to support the back of the arm just below the elbow. - Place your thumb a minimum of 1 to 2 inches below and slightly to the side of the intended venipuncture site and pull the skin toward the wrist - The bevel of the needle should be facing up; Position the needle above the vein so it is lined up with it and paralleling or following its path - ANGLE OF INSERTION: 15-30 degrees (depending on the depth of the vein) 10. REMOVE NEEDLE AND PLACE GAUZE - After the last tube has been removed from the holder or an 8. ESTABLISH BLOOD FLOW, RELEASE TOURNIQUET, adequate amount of blood has been collected (if you are ASK PATIENT TO OPEN FIST using a syringe), fold a clean gauze square into fourths and - To establish blood flow when using the ETS system, the place it directly over the site where the needle enters the collection tube must be advanced into the tube holder until skin. Hold the gauze lightly in place but do not press down the stopper is completely penetrated by the needle on it until the needle is removed. - If the vein has been successfully entered, blood will begin - Apply pressure to the site for 3 to 5 minutes or until the to flow into the tube. If you are using a syringe, a flash of bleeding stops. Failure to apply pressure or applying blood in the syringe hub indicates that the vein has been inadequate pressure can result in leakage of blood and successfully entered hematoma formation - Blood flow into the syringe is achieved by slowly pulling - Do not ask the patient to bend the arm up. The arm should back on the plunger with your free hand be kept extended or even raised (Ecchymoses/sis). 9. FILLING OF TUBES 11. DISCARD COLLECTION UNIT, SYRINGE OR - Following the order of draw, place ETS tubes in the holder NEEDLES and advance them onto the needle. ETS tubes fill - OSHA regulations prohibit cutting, bending, breaking, or automatically until the tube vacuum is exhausted or lost. A recapping blood collection needles or removing them from syringe is filled manually by slowly and steadily pulling back tube holders after use on the plunger until the barrel is filled to the appropriate level. 12. LABELLING OF TUBES - Keep the arm in a downward position so that blood fills ETS - Tubes must be labeled in the presence of the patient tubes from the bottom up and does not contact the needle in immediately after blood collection, never before the tube holder reflux (flow of blood from the tube back into - Information included: 3  Patients first and last name Patient’s identification number (inpatient) or date of birth (outpatient) Date and time of collection Phlebotomist’s initials Pertinent additional information, such as “fasting” 13. OBSERVE SPECIAL HANDLING INSTRUCTIONS - Place specimens that must be cooled (e.g. ammonia) in crushed ice slurry - Put specimens that must be kept at body temperature (e.g., cold agglutinin) in a 37°C heat block or other suitable warming device Wrap specimens that require protection from light (e.g., bilirubin) in aluminum foil or other light-blocking material or place them in a light-blocking container 14. CHECK PATIENT’S ARM AND APPLY BANDAGE - Examine the venipuncture site to determine if bleeding has stopped. (Bleeding from the vein can continue even though it has stopped at the surface of the skin) - Instruct the patient to leave the bandage on for a minimum of 15 minutes, after which it should be removed to avoid irritation - IMPORTANT NOTE: If bleeding has not stopped, the phlebotomist must apply pressure until it does. If the patient continues to bleed beyond 5 minutes, the appropriate personnel such as the patient’s physician or nurse should be notified ADDITIONAL NOTES EVACUATED TUBE SYSTEM - It is a closed system in which the patient’s blood flows through a needle inserted into a vein and then directly into a collection tube without being exposed to the air or outside contaminants - Involves the use of multi-sample needles, adapter, follows the “order of draw” for evacuated tubes - Most common and efficient system that is preferred by CLSI for collecting blood samples THE ORDER OF DRAW - Refers to the order in which tubes are collected during a multiple-tube draw or are filled from a syringe. - CLSI recommends the following order of draw for both ETS collection and in filling tubes from a syringe: 1. Blood culture tubes (yellow stopper) 2. Coagulation tube (light blue stopper) 3. Serum tube with or without activator (red, gold, red-gray marbled, orange, yellow-gray stopper) 4. Heparin (green or light green stopper) 5. EDTA tube (lavender or pink stopper) 6. Sodium fluoride with or without EDTA or oxalate (gray stopper) 4 FAILED VENIPUNCTURE - If you are unable to obtain a specimen on the first try, evaluate the problem and try again below the first site, on the opposite arm, or on a hand or wrist vein - If the patient’s veins are small or fragile, it may be necessary to use a butterfly or syringe on the second attempt (twice) - If the second attempt is unsuccessful, ask another phlebotomist to take over - Unsuccessful venipuncture attempts are frustrating to the patient and the phlebotomist. 5. MASTECTOMY - Blood should never be drawn from an arm on the PROBLEM SITES DURING VENIPUNCTURE same side as a mastectomy (surgical breast 1. BURNS, SCARS, AND TATTOO removal) without first consulting the patient’s - Healed burn sites and other areas with extensive physician. scarring may have impaired circulation and can - Lymph node removal can cause lymphostasis therefore yield erroneous test results. (obstruction or stoppage of normal lymph flow). - Newly burned areas are painful and also - Impaired lymph flow makes the arm susceptible to susceptible to infection. Tattooed areas can have swelling, called lymphedema, and to infection. impaired circulation, may be more susceptible to infection, and contain dyes that can interfere with 6. OBESITY testing. - Veins on obese patients may be deep and difficult 2. DAMAGED VEINS to find - Some patients’ veins feel hard and cord-like and 7. VASCULAR ACCESS DEVICE (VAD’s AND SITES) lack resiliency because they are occluded or obstructed ARTERIAL LINE - These veins may be sclerosed (hardened) or - is a catheter that is placed in an artery. It is most thrombosed (clotted) from the effects of commonly placed in a radial artery and is typically inflammation, disease, or chemotherapy drugs. used to provide accurate and continuous measurement of a patient’s blood pressure. - NEVER APPLY tourniquet or perform venipuncture on an arterial line 3. EDEMA - Edema is swelling caused by the abnormal accumulation of fluid in the tissues. It sometimes results when fluid from an IV infiltrates the surrounding tissues 4. HEMATOMA ARTERIOVENOUS SHUNT, FISTULA OR GRAFT - swelling or mass of blood (often clotted) that can be - is the permanent surgical connection of an artery caused by blood leaking from a blood vessel during and vein by direct fusion (fistula), resulting in a or following venipuncture bulging vein, or with a piece of vein or tubing (graft) - Venipuncture through an existing hematoma is that creates a loop under the skin. painful and can result in collection of a specimen - typically created to be used for dialysis, commonly that is contaminated with hemolyzed blood from joins the radial artery and cephalic vein above the outside the vein and unsuitable for testing wrist on the underside of the arm - NEVER APPLY tourniquet or perform venipuncture on a fistula 5 8. HEPARIN OR SALINE LOCK 2. LATE LOCAL COMPLICATION - is a catheter or cannula connected to a stopcock or Thrombosis a cap with a diaphragm (thin rubber-like cover) that - is an abnormal vascular condition in which provides access for administering medication or thrombus develops within a blood vessel of the drawing blood body. - IMPORTANT NOTE: A 5-mL discard tube should be Thrombophlebitis drawn first when blood specimens are collected - is inflammation of a vein often accompanied by a from either type of device clot which occurs as a result of trauma to the vessel wall 3. LATE GENERAL COMPLICATION HEMOLYSIS - Using a needle that is too small - Pulling a syringe plunger back too fast - Expelling the blood vigorously into a tube 9. INTRAVENOUS SITES - Forcing the blood from a syringe into an evacuated - An intravenous line, referred to simply as an IV, is a tube catheter inserted in a vein to administer fluids - Shaking or mixing the tubes vigorously - It is preferred that blood specimens not be drawn - Performing blood collection before the alcohol has from an arm with an IV dried at the collection site - Collecting blood in an IV LINE: Collect blood below HEMATOMA the IV line; Stop IV line for 2 minutes - The vein is fragile or too small for the needle size - When both arms are involved in therapy and the IV - The needle penetrates all the way through the vein cannot be discontinued for a short time, a site - The needle is partly inserted into the vein below the IV line should be sought; the initial - The needle is removed while the tourniquet is still sample (5 mL) drawn should be discarded. on. - Collection of blood below the IV line must be written - Excessive probing Pressure is not adequately on the laboratory requisition form to inform the staff applied after venipuncture in the chemistry section - IV FLUID CONTAMINATION: An increase of WEEK 3: CAPILLARY PUNCTURE infused substances such as glucose, chloride, PRE-ANALYTICAL PHASE potassium and sodium, with a decrease in urea and - Depending on the laboratory tests to be performed, small or creatinine big amounts of blood are needed. Few drops of blood are used for tests such as bleeding and clotting time COMPLICATIONS OF VENIPUNCTURE determinations, blood typing, blood smear preparation, etc. 1. IMMEDIATE LOCAL COMPLICATION which may be obtained from a skin or capillary puncture Hemoconcentration utilizing the ear lobe or tip of finger in adults and the big toe - is an increase in the number formed elements in or heel in infants. blood resulting either from a decrease or increase (hemodilution) in plasma volume ANALYTICAL PHASE Failure of blood to enter the syringe/vacutainer tube Materials: - Excessive pull of plunger Sterile blood lancet - Piercing the other pole of the vein 70% Alcohol - Transfixation of vein (moving of the vein) Cotton or gauze pad - Incorrect bevel position (bevel down) Capillary tube/Microhematocrit tube - Absence of vacuum Microcollection containers (microtainer) Syncope (fainting) Sealant (clay) - is the transient loss of consciousness due to lack of oxygen in the brain and results in an inability to stay LANCETS AND INCISION DEVICES in an upright position - Sterile, disposable, sharp-pointed or bladed instrument that - If a seated patient feels faint, the needle should be either punctures or makes an incision in the skin to obtain removed immediately, the patient's head should be capillary blood specimens lowered between the legs and the patient should be - Selection depends on the age of the patient, collection site, instructed to breath deeply volume of specimen required, and the puncture depth 6 needed to collect an adequate specimen without injuring - Often referred to as “bullets” because of their size and bone shape - Laser lancets Vaporizes water in the skin to produce a small hole in the capillary bed without cauterizing delicate capillaries. no risk of accidental sharps injury, and no need for sharps disposal CAPILLARY TUBES/MICROHEMATOCRIT TUBES - disposable, narrow-bore plastic or plastic-clad glass capillary tubes that fill by capillary action and typically hold 50 to 75 uL of blood (0.05 mL –0.075 mL) - Length: 75 mm or 7.5 inches; Internal bore: 1.3 mm - Red mark: Heparin (ammonium heparin) - Blue mark: no additive SEALANT - Plastic or clay sealants that come in small trays are used to seal one end of microhematocrit tubes (5-6 mm or 4-6 mm) MICROCOLLECTION CONTAINERS - Also called microtubes special small plastic tubes; used to collect the tiny amounts of blood obtained from capillary punctures 7 PROCEDURE: 1. REVIEW AND ACCESSION TEST REQUEST 2. PATIENT IDENTIFICATION 3. POSITIONING OF THE PATIENT 4. SELECTION OF APPROPRIATE PUNCTURE SITE - Site must be warm, pink or normal color, and free of scars, cuts, bruises, or rashes. - It should not be cyanotic (bluish in color), edematous (swollen), or infected - Swollen or previously punctured sites should be avoided, because accumulated tissue fluid can contaminate the specimen and negatively affect test results. Specific locations for capillary puncture include fingers of adults and heels of infants PUNCTURE SITES ADULTS AND OLDER CHILDREN: (> 1yr old) - Palmar surface of the distal or end segment of the 5. WARMING OF THE SITE middle or ring finger of the non-dominant hand (less - Warming the site increases blood flow as much as seven calloused) times (causes arterialization); important when performing - Site must be perpendicular to the grooves in the heel stick on newborns whorls (spiral pattern) of the fingerprint - IMPORTANT NOTE: INFANTS/YOUNG CHILDREN: (< 1 yr old) Increased blood flow makes specimens easier and - Medial Lateral plantar surface of the heel faster to obtain and reduces the tendency to - Puncturing of the bone can cause osteomyelitis and compress or squeeze the site, which can osteochondritis contaminate the specimen with tissue fluid and hemolyze red blood cells Essential when collecting capillary pH or blood gas specimens (arterialization) - To avoid burning patients, the devices provide a uniform temperature that does not exceed 42°C - Warming is done for 3-5 minutes using a washcloth, towel, or diaper that has been moistened with comfortably warm water or using a commercial heel warming device 6. PREPARE PATIENT APPLY ANTISEPTIC 7. PUNCTURING THE SITE - Finger puncture: Grasp the patient’s finger between your non-dominant thumb and index finger. Hold securely in case of sudden movement. Place the lancet device flat against the skin in the central, fleshy pad of the finger, slightly to the side of center and perpendicular to the fingerprint whorls - Heel puncture: Grasp the foot gently but firmly with your non-dominant hand. Encircle the heel by wrapping your index finger around the arch and your thumb around the bottom. Wrap the other fingers around the top of the foot. Place the lancet flat against the skin on the medial or lateral plantar surface of the heel - PUNCTURE DEPTH: 1 yr old): can hemolyze the specimen and also keep the Available veins are fragile or must be saved for blood from forming a well-rounded drop. other procedures such as chemotherapy Several unsuccessful venipunctures have been 9. FILLING AND MIXING OF TUBES/CONTAINERS performed and the requested test can be collected - Continue to position the site downward to enhance blood by capillary puncture flow and apply gentle, intermittent pressure to tissue The patient has thrombotic or clot-forming surrounding a heel puncture site or proximal to a finger tendencies puncture site The patient is apprehensive or has an intense fear - IMPORTANT NOTE: of needles Do not use a scooping motion against the surface There are no accessible veins (e.g. the patient has of the skin and attempt to collect blood as it flows IVs in both arms or the only acceptable sites are in down the finger. Scraping the scoop against the scarred or burned areas) skin activates platelets, causing them to clump, and To obtain blood for POCT procedures such as can also hemolyze the specimen glucose monitoring - For infants and very young children (< 1 yr old): 10.PLACE GAUZE AND APPLY PRESSURE Infants have a small blood volume; removing quantities of blood typical of venipuncture or arterial 11.LABEL AND OBSERVE SPECIAL HANDLING puncture can lead to anemia. According to studies, INSTRUCTIONS for every 10 mL of blood removed, as much as 4 mg of iron is also removed 12.CHECK THE SITE AND APPLY BANDAGE Large quantities removed rapidly can cause cardiac - The site must be examined to verify that bleeding has arrest. Life is threatened if more than 10% of a stopped. If bleeding persists beyond 5 minutes, notify the patient’s blood volume is removed at once or over a patient’s nurse or physician. If bleeding has stopped and the short period (iatrogenic anemia) doctor’s fault patient is an older child or adult, apply a bandage and advise Obtaining blood from infants and children by the patient to keep it in place for at least 15 minutes. venipuncture is difficult and may damage veins and surrounding tissues. ADDITIONAL NOTES Puncturing deep veins can result in hemorrhage, CAPILLARY PUNCTURE venous thrombosis, infection, and gangrene. - Also called as: Dermal puncture, skin puncture An infant or child can be injured by the restraining - Procedure in which the skin is punctured with a lancet to method used while performing a venipuncture. obtain blood in the capillaries/capillary bed in the dermal Capillary blood is the preferred specimen for some layer of the skin for laboratory testing tests, such as newborn screening tests. COMPOSITION OF CAPILLARY BLOOD: CAPILLARY PUNCTURE IS NOT APPROPRIATE Arterial blood, Venous blood, Interstitial/Tissue fluid WHEN/CONTRAINDICATION: Increased: Glucose, WBC Patient is severely dehydrated Decreased: total proteins, calcium, potassium, Shock (increased leakage of plasma) hemoglobin, hematocrit, platelets Poor circulation IMPORTANT NOTE: hemolysis and tissue fluid Tests that requires more amount of blood: ESR, contamination can increase potassium levels Coagulation studies, blood cultures (potassium is inside RBC’s) - Blood collection preferred for: infants, very small children, CAPILLARY PUNCTURE ORDER OF DRAW: elderly, obese, or severely burned patients - Specimens must be collected quickly to minimize the - Is employed if the test requested requires a small amount effects of platelet clumping and micro-clot formation and to of blood ensure that an adequate amount of specimen is collected before the site stops bleeding. Hematology specimens are 9 collected first because they are most affected by the clotting Centrifuge process. Serum specimens are collected last because they EDTA anticoagulated blood (this procedure needs are supposed to clot. about 3.0 mL of whole blood) - The CLSI order of draw for capillary specimens is as follows: Blood gas specimen (sample is arterial blood) Slides (unless the specimen is placed in an EDTA tube) EDTA microcollection tubes (affected by clotting process and platelet aggregation) Other additives (Heparin) Serum microcollection tubes (allowed to clot) - IMPORTANT NOTE: Puncture/incision releases tissue ` thromboplastin/tissue factor which activates coagulation SCOOPING: activates platelet clumping and hemolysis EXCESSIVE MILKING can cause hemolysis WEEK 4: HEMATOCRIT DETERMINATION PRE-ANALYTICAL PHASE - Hematocrit is often referred to as the packed cell volume, volume of erythrocytes or reading of packed cells - This test measures the proportion of red cells to plasma in the peripheral blood but not in the entire circulation. It gives the number of millimeters of packed red blood cells/100 millimeter of blood or simply expressed as volume percent - Reported as: PCV (%) or EVF (L/L) - Normal values: At birth: 45-60% Females: 36-48% Males: 40-55% HEMATOCRIT - Packed cell volume (PCV) - volume of packed RBCs that occupies a given volume of whole blood - Used in evaluating and classifying various types of anemia according to red cell indices - Automated hematocrit: HCT = (MCV x RBC count) / 10 PROCEDURE: 1. Mix the blood 2. With the use of a long stem pipette, fill the Wintrobe tube to the 10 mark. 3. NOTE: No air bubbles should be present on the surface of the blood, it may be removed by touching it with a tissue paper or with a pipette. If the bubbles are present in the middle of the tube, aspirate the blood and proceed to step METHODS FOR HEMATOCRIT DETERMINATION no. 2 again. 4. Centrifuge the blood for 30 minutes. 1. MACROHEMATOCRIT METHOD (manual) 5. Read the height of the packed red blood cells on the scale MATERIALS at the right side of the tube, which is graduated from 0-10 cm Wintrobe tube with rubber caps from the bottom to top, read upward. Long stem pipette/Pasteur pipette 10 IMPORTANT NOTE: - If the tube has been filled to the tenth mark, the above calculation may be eliminated by following each line or mark on the Wintrobe tube that represents 1% of the hematocrit. Therefore simply count the number of lines from the bottom of the tube to the level of the packed red blood cells. The number of lines is the value of your hematocrit reading. MACROHEMATOCRIT METHOD - Less commonly used; time consuming - Required increased blood volume; higher amount of 11000 to 12000 rpm for 4 to 5 min trapped plasma - Centrifugation: 30 minutes at 2,000- 2,300 x g - TRAPPED PLASMA: plasma trapped in the RBC portion after centrifugation Increases hematocrit by: 1-2 or 1-3% Not affected by automated tests 2. MICROHEMATOCRIT METHOD (manual) MATERIALS Heparinized capillary tube (RED MARK/BAND) Microhematocrit centrifuge Microhematocrit reader Sealant Lancet PROCEDURE: 1. After necessary preparations, make a capillary puncture and produce a rounded drop of blood. 2. In a horizontal position, put one end of the capillary tube on the drop of blood and fill the tube about ⅔ full. NOTE: The tube will be filled by capillary action. If using tubes with a colored ring at one end, fill from opposite ends. 3. Seal one end of the capillary tube with the clay by placing the dry end of the tube into the clay in a vertical position. 4. After sealing with wax, seal it again with wax. 5. Assemble the tube in a microhematocrit centrifuge in such a way that the unsealed end is nearest the center of the centrifuge. 6. Spin at 10, 000 to 15, 000 rpm for 5 minutes. 7. Using the microhematocrit reading device, determine the HCT. 11  NOTE: Buffy coat should not be included in the reading. 8. The reading on the window of the hematocrit reader corresponds to the hematocrit value. MICROHEMATOCRIT METHOD - requires small amount of blood; more commonly used - Aka: ADAM’s method - Specimen: Whole blood, Capillary blood - Sealer/Clay: 4-6mm long - Centrifugation: 5 mins for 10,000-15,000 x g MICROHEMATOCRIT (RODAK): 1. Fill two plain capillary tubes approximately three-quarters full with blood anticoagulated with EDTA or heparin. Mylar wrapped tubes are recommended by the National Institute for Occupational Safety and Health to reduce the risk of capillary tube injuries. Alternatively, blood may be collected into heparinized capillary tubes by skin puncture (required wiping first drop of blood). Wipe any excess blood from the outside of the tube. 2. Seal the end of the tube with the colored ring using nonabsorbent clay. Hold the filled tube horizontally and seal by placing the dry end into the tray with sealing compound at ERRORS IN HEMATOCRIT DETERMINATION a 90-degree angle. Rotate the tube slightly and remove it from the tray. The plug should be at least 4 mm long. 3. Balance the tubes in a microhematocrit centrifuge with the clay ends facing the outside away from the center, touching the rubber gasket. 4. Tighten the head cover on the centrifuge and close the top. Centrifuge the tubes at 10,000 g to 15,000 g for the time that has been determined to obtain maximum packing of RBCs. Do not use the brake to stop the centrifuge. 5. Determine the hematocrit by using a microhematocrit reading device (reading must be done within 10 minutes). Read the level of RBC packing; do not include the buffy coat (WBCs and platelets) when taking the reading. 6. The values of the duplicate hematocrits should agree within 1% (0.01 L/L). IMPORTANT NOTE: The time to obtain maximum packing of red blood cells should be determined for each centrifuge. Duplicate microhematocrit determinations should be made using fresh, well-mixed blood anticoagulated with ethylenediaminetetraacetic acid (EDTA). Two specimens should be used, with one of the 3. AUTOMATED METHOD: Hematocrit is computed by specimens having a known hematocrit of 50% or (MCV x RBC count) higher. Starting at 2 minutes, centrifuge duplicates at 30-second intervals and record results. When the hematocrit has remained at the same value for two consecutive readings, optimum packing has been achieved, and the second time interval should be used for microhematocrit determinations. 12  WEEK 4: HEMOGLOBIN DETERMINATION 2. KENNEDY’S AND WONG’S/ASSENDELFT METHOD PRE-ANALYTICAL PHASE - Principle: Chemical method - Hemoglobin is the main content of the red blood cells which - Measures iron bounded to hemoglobin is responsible for transporting oxygen from the lungs to the - 1 gram of hemoglobin = 3.47mg of iron tissues and CO2 from the tissues to the lungs to be eliminated to the outside 3. PHYSICAL METHOD (RULE OF THREE) - Hemoglobin is a conjugate protein that consists of 2 - Applicable only to patients that have normocytic, portions, the globin (a simple protein) and heme (organic normochromic RBCs compound in iron). Each hemoglobin molecule consists of - Hemoglobin: RBC count is multiplied to 3 the one molecule of globin (four globin chains) and four - Hematocrit: Hemoglobin is multiplied to 3 molecules of heme +/- 3 - Hemoglobin may be found in the blood plasma; such a condition is referred to as hemoglobinemia. When the free hemoglobin in the plasma reaches a concentration between 30 and 300 mg/100mL of blood, hemoglobin is detected in the urine; such a condition is called hemoglobinuria - In the normal adult, a hemoglobin concentration is as follows: MALE: 13.5-17.5 g/dL FEMALE: 12.0-16.0 g/dL - Value may vary according to age, sex, and locality 4. COPPER SULFATE METHOD - Abnormal value as in hyperchromia (increased value) is - Principle: Gravimetric found in polycythemia, dehydration and in changing from low - Based on the specific gravity of copper sulfate (CuSO4) – to high altitude 1.053 - Hypochromia (decreased value) is seen in anemia - Semi-quantitative; for mass assays (blood donation drives/bloodletting) - Drop of blood is placed into the copper sulfate solution and observed whether it will sink within 15 seconds - Sink: hemoglobin is the same as the specific gravity of copper sulfate and has a value of >12.5 g/dl - Float: hemoglobin value is