Hema-Lab-Trans PDF - MLS 12a Hematology 1 (Lab)

MLS 12a: HEMATOLOGY 1 (LAB) 1stSEMESTER | S.Y 2023-2024 Specimen Collection and Handling | Red Blood Cell Count | Reticulocyte Count MODULE 1: SAFETY PRACTICES IN warming the site can increase the blood flow HEMATOLO...

MLS 12a: HEMATOLOGY 1 (LAB) 1stSEMESTER | S.Y 2023-2024 Specimen Collection and Handling | Red Blood Cell Count | Reticulocyte Count MODULE 1: SAFETY PRACTICES IN warming the site can increase the blood flow HEMATOLOGY LABORATORY sevenfold - warm the site with a commercial heel Skin Puncture warmer or a warm washcloth Mix of arterial and venous blood - temperature no greater than 42° C Increased pressure in the arterioles yields a - no longer than 3 to 5 minutes specimen enriched in arterial blood clean the skin puncture site Contains interstitial and intercellular fluid ○ 70% isopropyl alcohol ○ allow it to air-dry Microsampling Povidone-iodine should not be used refers to blood collection by skin puncture ○ because of possible specimen Blood obtained is also known as contamination - capillary blood ○ which could falsely elevate levels - peripheral blood - potassium Skin punctures are often performed in - Phosphorus - infants less than 6 months of age - Uric acid - young children - adults who have poor veins Precautions with Skin Puncture - severely burned 1. Finger or heel must be securely immobilized. - elderly patients with fragile veins 2. Heel punctures in infants should not be Sites of Skin Puncture: made more than 2 mm deep margin of the earlobe because of the risk of bone injury and palmar surface of the finger possible infection (osteomyelitis) plantar surface of the heel and big toe 3. Premature infants puncture device that makes an Collection Site for Skin Puncture incision with even less depth is Infants under 1 year old preferred ○ surface of the heel 4. Phlebotomist should not puncture an area ○ lateral (outside) that is: ○ medial (inside) ○ swollen ○ plantar (bottom) ○ bruised Make Little People Happy ○ infected (Medial, Lateral, Plantar, Heel) ○ already been punctured Children older than 1 year old and adults 5. Phlebotomists should not perform skin ○ Palmar surface of the distal portion of puncture in patients with edema, dehydration, the third (middle) or poor peripheral circulation o because ○ Third or fourth (ring) finger on the specimen integrity and test accuracy may be non-dominant hand compromised NOTE: 6. The first drop of blood should be wiped away 1) puncture on the finger should be made with a clean gauze pad perpendicular to the fingerprint lines to prevent contamination of the 2) fingers of infants should not be punctured specimen with tissue fluid because of the risk of serious bone injury to facilitate the free flow of blood Amoronio | Baltazar | Jayme | Montero | Palencia | Yongque  MLS 12a: HEMATOLOGY 1 (LAB) 1stSEMESTER | S.Y 2023-2024 Specimen Collection and Handling | Red Blood Cell Count | Reticulocyte Count Key tips: Warm the puncture site. Warming increases For Children and Adults: recommended depth is the blood flow sevenfold. 2.4mm - 3mm to hit the capillary bed Use commercial warmer or warm wash-cloth. For Infants: 1.66mm and not deeper Cleanse the puncture site with 70% isopropyl alcohol using concentric circles, working from Equipments with Skin Puncture the inside to the outside. Allow skin to air-dry. Devices for skin puncture contain sterile Perform the puncture. Puncture depth should lancets that puncture or sterile blades that not exceed 2mm. make a small incision in the skin. Wipe away the first drop of blood. The lancet or blade is spring-loaded in the Collect the specimens and mix as needed. device, and when activated by the Elevate the puncture site and apply pressure phlebotomist, pierces the skin. until bleeding has stopped. Devices are single use, disposable, and have Label the specimens with the required retractable blades in information. Compliance with OSHA safety standards. Handle the specimen appropriately. Containers for collecting blood from skin Thank the patient and parents. puncture include capillary tubes and Dispose of all of the puncture equipment and microcollection tubes. bio-hazardous materials. Capillary tubes of various sizes are available Complete paperwork and indicate “skin with or without heparin. puncture collection. Microcollection tubes are preferred and are available with or without additives. Reasons for Specimen Rejection The cap colors on microcollection tubes A laboratory result is only as good as the correspond with the color coding system for integrity of the specimen provided. evacuated tubes. Specimens are rejected for conditions that The order of draw, however, is different for may result in identification errors or microcollection tubes. inaccurate results. Patient identification does not match with Procedure requisition form and label on specimen cup Obtain and examine the requisition form. Unlabeled or mislabeled specimens Assemble equipment and supplies. Specimen is hemolyzed Greet the patient; identify the patient by Specimen was collected at the wrong time having the patient verbally state his or her Specimen was collected at the wrong tube name and confirm with patient’s identification Specimen is clotted number (i.e., SSS, IDs, identification Specimen is contaminated with intravenous bracelet). fluid Verify that any dietary restrictions have been Specimen is lipemic met (e.g., fasting), ask for medical conditions, if any, and check for sensitivity to latex. NOTE: Wait for 10 to 15 minutes after the Position the patient and the parents or intravenous line has been removed before extracting designated holder as necessary. blood from a patient who previously had an Put on gloves. intravenous line Organize equipment and supplies. Explain the procedure. Select the puncture site. Amoronio | Baltazar | Jayme | Montero | Palencia | Yongque  MLS 12a: HEMATOLOGY 1 (LAB) 1stSEMESTER | S.Y 2023-2024 Specimen Collection and Handling | Red Blood Cell Count | Reticulocyte Count Evacuated Tubes Stopper Color: Orange Additives: Thrombin Evacuated Tubes - are used with both the ETS and Number of Inversions: 8 inversions the syringe method of obtaining blood specimens. Department: Chemistry Evacuated tubes are available from a number of Stopper Color: Yellow different manufacturers and come in various sizes Additives: Sodium Polyanethol Sulfonate (SPS) and volumes ranging from 1.8 to 15 mL. Number of Inversions: 8 inversions Department: Microbiology COMMON STOPPER COLORS, ADDITIVES, AND DEPARTMENTS INVOLVED Stopper Color: Yellow Additives: Acid Citrate Dextrose (ACD) Stopper Color: Light Blue Number of Inversions: 8 inversions Additives: Sodium Citrate Department: Blood Bank/ Immunohematology Number of Inversions: 3 to 4 inversions Department: Coagulation ORDER OF DRAW Stopper Color: Red (glass) Additives: None Department: Chemistry, blood bank, Serology/Immunology Stopper Color: Red (plastic) Additives: Clot Activator Number of Inversions: 5 inversions MODULE 2: EQUIPMENT AND APPARATUS IN Department: Chemistry HEMATOLOGY LABORATORY Stopper Color: Red/Black (tiger) | Gold | Red/Gold Additives: Clot Activator and Gel Separator Manual Blood Cell Count Number of Inversions: 5 inversions Department: Chemistry RBC and WBC Thoma Pipette Stopper Color: Green Non-automatic, to contain (TC) pipettes for Additives: Lithium heparin | Sodium heparin | blood cell counting Ammonium heparin RBC Thoma pipette Number of Inversions: 5 to 10 inversions - Used for manually counting Department: Chemistry erythrocytes (platelets) WBC thoma pipette Stopper Color: Lavender (including microcollection - Used for manually counting containers) | Pink | Tan | Pearl leukocytes (white blood cells) Additives: Ethylenediaminetetraacetic acid (EDTA) - Has white background with blue Number of Inversions: 8 to 10 inversions calibration marks Department: Hematology and Blood Bank Stopper Color: Gray Additives: Sodium fluoride and potassium oxalate (8 - 10 inversions) | Sodium fluoride and EDTA (5 - 10 inversions) | Sodium fluoride Department: Chemistry Amoronio | Baltazar | Jayme | Montero | Palencia | Yongque  MLS 12a: HEMATOLOGY 1 (LAB) 1stSEMESTER | S.Y 2023-2024 Specimen Collection and Handling | Red Blood Cell Count | Reticulocyte Count Features RBC Thoma WBC Pipette Thoma Steps in Manual Dilution for RBC Counting Pipette 1. Make sure to invert the EDTA vacutainer tube 8 times before aspirating the blood sample. Bead color red white 2. Pinch the rubber tubing attached to the short Bulb size larger smaller stem of the thoma pipette. Size of bore smaller larger 3. Submerge the tip of the pipet and aspirate calibrations 0.5, 1, 101 0.5, 1, 11 blood sample until it reaches the 0.5 mark. Volume of the 100 10 bulb NOTE: - The size of bore diameter of the RBC Thoma pipette is larger since it requires larger volume for RBC counting. 4. STEPS IN CLEANING RBC THOMA PIPETTE 5. Wipe the stem of the pipet with dampened gauze or lint-free cloth. 1. Discard in the waste container the NOTE: avoid touching the tip for the wiping remaining solution in RBC Thoma pipette. material may absorb the blood aspirated. 2. Aspirate hypochlorite solution and 6. Using the plunger of the syringe, slowly discard it in the waste container. aspirate the diluent up to the 101 mark for 3. Aspirate distilled H2O and discard in the RBC count. waste container. NOTE: Make sure there are no bubbles 4. Lastly, Aspirate alcohol and discard it in inside the bulb for this will cause wrong the waste container. Then air dry. Dilution. Diluting Fluids used for RBC count HEMOCYTOMETER 1. Hayem’s or Gower’s RBC Diluting fluid Aka counting chamber - used as diluting fluid to count Specimen slide which is frequently used to red blood cells. diluting fluid is determine concentration: isotonic with blood, hence - Blood cells hemolysis does not take place. - Sperm cells 2. Normal Saline Solution (0.85-0.90% Has a grid laser-etched into the glass with an NaCl) area of 9mm2 - used when no other RBC diluting fluid is 9mm2 = length 3mm x width 3mm. available - For practice dilutions Has a cover glass held at place at a specified height usually at 0.1 mm. Amoronio | Baltazar | Jayme | Montero | Palencia | Yongque  MLS 12a: HEMATOLOGY 1 (LAB) 1stSEMESTER | S.Y 2023-2024 Specimen Collection and Handling | Red Blood Cell Count | Reticulocyte Count 7. Cover the tip at the end of the calibrated stem using the thumb and gently remove the 1. After expelling 3-5 drops of the sample, rubber tubing. position the pipet at 30-35 degrees against the hemocytometer with the tip of the pipet 8. With the thumb still covering the tip of the touching the edge of the coverslip where it calibrated stem, cover the tip of the short meets the chamber floor. stem with the middle finger of the same hand Shake the pipette gently for 30-45 seconds 2. Slowly load the sample until the chamber is completely filled. Repeat charging once 9. If the pipette shaker is available, shake the counting chambers are overfilled, underfilled pipet for 5 minutes. or with bubbles. 10. Expel 3-5 drops of the solution from the 3. Charge the upper and lower chamber. pipet before charging to the hemocytometer. NOTE: if the first 3-5 drops are not expelled 4. Place the hemocytometer inside a moist RBC count is falsely decreased due to the chamber for 10 minutes before counting. To concentration of RBCs in the bulb. let the cells settle first before viewing under Sampling to a Hemocytometer/Charging the microscope. (Open type Spencer Improved Neubauer) - the most common hemocytometer used today has two platforms: upper and lower chamber The platforms are separated and surrounded by a canal, which is also known as the H-moat. NOTE: sample should not reach or overflow to the H-moat or else the results will be erroneous. NOTE: Placing the hemocytometer inside the moist - V-slashes. This is the part where the sample chamber will also prevent the evaporation of the is charged. sample while waiting for the cells to settle. Place the coverslip to the platform of the counting chamber and the distance between the cover slip MICROSCOPIC QUANTIFICATION OF CELLS and the chamber is 0.1mm. 1. Under LPO, locate and scan the counting area if the cells are evenly distributed. RBCs are counted on 5 intermediate squares of the middle large square. 2. After locating and scanning the counting area, Switch to HPO and start counting the The central large square for RBC and platelet count cells on the upper most part of the is divided into 25 smaller squares (area of each counting area. smaller square = 1mm2 divided into 25 is 0.04mm2) NOTE: RBCs are counted on the 5 smaller squares inside the central large square. RBC counting: 4 small corner squares, 1 small central squares; on the top and left area of a single To count the cells, the basic rules are: square - count all cells seen inside the square CHARGING A HEMOCYTOMETER - count all cells touching the top and left margin line of the counting area Amoronio | Baltazar | Jayme | Montero | Palencia | Yongque  MLS 12a: HEMATOLOGY 1 (LAB) 1stSEMESTER | S.Y 2023-2024 Specimen Collection and Handling | Red Blood Cell Count | Reticulocyte Count NOTE: In cases where there are two margin lines, the reference line is the inner line. References Range/Values: In cases where there are three margin lines, the Adult Male: 4.20 - 6.00 x10¹²/L (X10⁶/uL) reference line is the middle line. Adult Female: 3.80 - 5.20 x10¹²/L (X10⁶/uL) MODULE 3: RETICULOCYTE COUNT The Reticulocyte Count is an important diagnostic tool because it reflects the amount of effective red blood cell production in the bone marrow. Reticulocytes are young erythrocytes that have matured enough to have lost their nuclei but not their cytoplasmic RNA. Follow the crenellation line technique or the battlement method in counting to prevent Reticulocyte is the last immature erythrocyte stage recounting cells. It spends 2 to 3 days in the bone marrow and 1 day in the peripheral blood before 3. Count on both upper and lower chambers. developing into a mature erythrocyte. Once done, find the average of both The reticulocyte count is used to assess the chambers and record it as total cells counted. erythropoietic activity of the bone marrow Hence, an accurate reticulocyte count can BASIC CALCULATION OF RESULTS help the physician in determining if the patient’s bone marrow is producing enough red blood cells. Other terms for reticulocyte: Proerythrocyte Polychromatophilic macrocyte NOTE: If the dilution factor is not mentioned, use 200 Granulophilocyte as the diluting factor. Equipments: TO SOLVE FOR RBC COUNT: 1. Capillary tubes or Vacutainer tubes 2. Glass slides 3. Wright or Wright-Giemsa stain 4. Microscope, lens paper, and immersion oil 5. Miller ocular disc (optional) Principle: Supravital stains (New Methylene Blue, Brilliant Cresyl Blue) bind, neutralize and cross-link RNA. They cause the ribosomal and residual RNA to co-precipitate with few remaining mitochondria and NOTE: Express the results in Conventional Unit ferritin masses in living young erythrocytes to form (CU) and SI unit. microscopically visible, dark-blue clusters and Amoronio | Baltazar | Jayme | Montero | Palencia | Yongque  MLS 12a: HEMATOLOGY 1 (LAB) 1stSEMESTER | S.Y 2023-2024 Specimen Collection and Handling | Red Blood Cell Count | Reticulocyte Count filaments (reticulum). An erythrocyte which is still possessing its RNA is referred to as a reticulocyte. NOTE: Supravital stains are used to stain living cells extracted from the body. Specimen: Whole Blood anticoagulated with EDTA Procedure for Performing Reticulocyte Count: 1. Mix equal amounts of blood and New Reticulocytes in Dry Method using New Methylene Blue Methylene Blue stain (2-3 drops or 50µ𝐿 each) in a clean, dry test tube (Red top). 2. Incubate at room temperature for 10-15 minutes or in a water bath at 37℃ for 5-10 minutes. 3. Remix the preparation after incubation. 4. Prepare blood smears (wedge of coverslip method) 5. Count reticulocytes seen within 1000 RBCs under OIO in areas where cells are close together but not touching each other. Reticulocytes in Wet Method using New Methylene Blue 6. Calculate the reticulocyte count. Or the number of reticulocytes counted can NOTES: be multiplied by 0.1 (100/1000) to obtain the Two types of performing reticulocyte count - result. Wet and Dry method In dry method the smear is viewed under OIO Count the number of reticulocytes in 1000 RBC’s MILLER DISC - Designed to reduce labor-intensive process brought by large numbers of red cells. - The disc is composed of two squares, with NOTE: Incubating before smearing the sample the area of the smaller square measuring 1/9 allows the cells to settle. the area of the larger square. - The disc is inserted into the eyepiece of the microscope and the grid - Alternatively, square B may be in the center of square A Amoronio | Baltazar | Jayme | Montero | Palencia | Yongque  MLS 12a: HEMATOLOGY 1 (LAB) 1stSEMESTER | S.Y 2023-2024 Specimen Collection and Handling | Red Blood Cell Count | Reticulocyte Count RBCs = counted in the smaller square - Howell-Jolly bodies are round nuclear fragments Reticulocytes = counted in the larger square and are usually singular. - Heinz bodies are multiple in 1 cell and can be found The calculation formula for percent reticulocytes is: only in supravital stain while Howell-Jolly bodies are singular in 1 cell and can be found using a Wright Giemsa stain Howell-Jolly Bodies SOURCES OF ERROR AND COMMENTS 1. If a patient is very anemic or polycythemic, the proportion of dye to blood should be adjusted accordingly. - Anemic = small RBCs - Polycythemic = high RBCs 2. Blood stain are not mixed before smearing. Remember that the specific gravity of the reticulocyte is lower than mature erythrocytes, and if not mixed reticulocytes - Pappenheimer bodies are hemosiderin in the settle at the top of the mixture during mitochondria whose presence can be confirmed with incubation. an iron stain, such as 3. Moisture in the air, poor drying of the slide, or both may cause areas of the slide to Prussian blue appear refractile, and these areas could be - All other inclusions are not visible when using confused with reticulocytes. The RNA Prussian blue remnants in a reticulocyte are not refractile. 4. Other RBC inclusions that stain supravitally include Heinz, Howell-Jolly, and Pappenheimer bodies. - Heinz bodies represent precipitated hemoglobin, usually appear round or oval, and tend to cling to the cell membrane Heinz bodies Amoronio | Baltazar | Jayme | Montero | Palencia | Yongque  MLS 12a: HEMATOLOGY 1 (LAB) 1stSEMESTER | S.Y 2023-2024 Specimen Collection and Handling | Red Blood Cell Count | Reticulocyte Count II. ABSOLUTE RETICULOCYTE COUNT CALCULATION Traditionally, the reticulocyte count has been expressed as a percentage of the total number of REFERENCE RANGE circulating erythrocytes (e.g., 1%) Patients with a hematocrit of 35% = 2% to 3% However, this value may be erroneous because Patients with a hematocrit of less than 25% = 3% fluctuation in the percentage may be caused by a to 5% change in the total number of circulating erythrocytes - The count is increased to compensate for anemia - rather than a true change in the number of circulating - The corrected reticulocyte count depends on the reticulocytes. degree of anemia. To account for variations caused by erythrocyte quantity, expression of reticulocytes in absolute rather than proportional terms is becoming the preferred method of reporting. The correction for anemia is helpful for clinical interpretation, and several different methods are used. The CLSI proposes that the correction for anemia, the corrected reticulocyte count, be made mathematically by correcting the observed reticulocyte count to a normal packed RBC volume (hematocrit) PRINCIPLE The absolute reticulocyte count (ARC) is the actual number of reticulocytes in 1 L of whole blood. SAMPLE CALCULATION NOTE: RBC count should be in SI unit III. CORRECTED RETICULOCYTE COUNT PRINCIPLE In specimens with a low hematocrit, the percentage of reticulocytes may be falsely elevated because whole blood contains fewer RBCs. A correction factor is used, with the average normal hematocrit considered to be 45%. Amoronio | Baltazar | Jayme | Montero | Palencia | Yongque