Hematology Lab - 05 Reticulocyte Count

HEMATOLOGY LABORATORY MATERIALS RETICULOCYTE COUNT • OUTLINE • • • • • Reticulocytes Reticulocyte Count Supravital Stain Sources of Error Reticulocytosis • • • Absolute Reticulocyte Count (ARC) Corrected Reticulocyte Count (CRC) Reticulocyte Production Index (RPI) RETICULOCYTE COUNT RETICULOCYTES • • • • • The last immature erythrocyte stage o Last stage normally present in the blood o Wright stain - polychromatic erythrocyte o Supravital stain - reticulocyte Stained darkly with RNA remnants and organelles (mitochondria & ribosomes) Assess erythropoietic activity of bone marrow Spend 2-3 days in Bone marrow, and 1 day in peripheral blood before developing into a mature RBC. Reference Range: o Newborn: 1.5-5.8% o Adult: 0.5-1.5% RETICULOCYTE COUNT • • • • Index for evaluating effectiveness of erythropoiesis. o Erythropoiesis - occur in bone marrow “Poor Man’s Bone Marrow” o Assess bone marrow response to stimulus without studying bone marrow Importance: 1. Confirm diagnosis of pernicious anemia by therapeutic response 2. Determines response of pernicious anemia patient to vitamin B12 therapy 3. Aids in diagnosis of lead poisoning & hemolytic anemia 4. To determine whether there is normal RBC regeneration 5. To aid in the prognosis of acute hemorrhage. An increase reticulocyte count indicates RBC regeneration. Principle: Whole Blood is stained with supravital stain. Any non-nucleated erythrocyte that contains two or more particles of blue-stained granulofilamentous material is a reticulocyte. o Polychromatophilic erythrocyte are only reported o Blue filaments - precipitated inclusions (remnants, organelles, iron stores • • • • • • • • Specimen: EDTA-anticoagulated whole blood or capillary blood EDTA tube Cotton balls 0% isopropyl alcohol Binocular microscope Calibrated Miller Disk Supravital Stain (New Methylene Blue, Brilliant Cresyl Blue) o New Methylene Blue - more preferred Glass slides Cedarwood oil (for OIF) DRY METHOD • • • Procedure: 1. Mix equal amounts of EDTA whole blood and supravital stain (2-3 drops each). 2. Incubate for 3-10 minutes at room temperature 3. Remix the preparation ▪ Reticulocyte has tendency to settle at the top (due to low specific gravity) ▪ Inability to remix may cause false decrease 4. Prepare a blood smear ▪ Allow slide to dry 5. Count 1000 RBC/Reticulocyte under OIF. ▪ RBC and reticulocyte are counted as one Formula: Sample Problem: 23 reticulocytes are counted per 1000 RBC observed. o 23 Reticulocytes o 977 RBC WET METHOD • • Rapid method of Schilling, Osgood-Wilhelm, and Sabin o Smear is not allowed to dry Procedure: 1. Place a drop of freshly filtered stain on a clean glass slide. 2. Add an equal volume of blood. Mix by stirring. 3. Cover the mixture with a clean coverslip lined with Vaseline. 4. Proceed with counting. (same counting as dry method) MILLER DISC METHOD • Miller Disc – an optical aid inserted into the eyepiece of microscope. The disc ruling is composed of a center square (Square A) containing a secondary square (Square B). o Square B – 1/9 of the total area of Square A • • • The method was developed to simplify counting by minimizing the number of cells counted. Procedure: 1. Prepare blood smear using dry method. 2. Count reticulocytes in the entire Square A 3. Count at least 112 RBCs in Square B ▪ RBC & reticulocytes are counted as 1 Formula: ABSOLUTE RETICULOCYTE COUNT (ARC) • • Actual number of reticulocytes in 1 liter of whole blood Computation: • • Reference Range: 25-75 x 109/L Sample Problem: If patient’s reticulocyte count is 3.6% and the RBC count is 3.20 x1012/L. What is the ARC? SUPRAVITAL STAIN • • Mature RBC – gray blue Reticulocytes – gray blue cells with deep blue filamentous web or granules (2 or more) CORRECTED RETICULOCYTE COUNT (CRC) • • SOURCES OF ERROR • • • • • • Failure to mix blood and stain completely Presence of refractile artifacts Increased blood glucose level Presence of pappenheimer bodies, Heinz bodies, and Howell-Jolly bodies. o These are also stained with supravital stain. • • RETICULOCYTOSIS • • • Increased reticulocytes in the blood Seen in: 1. Hemolytic Anemia 9. Blood intoxication 2. Lead Poisoning 10. Kala-azar 3. Iron Deficiency Anemia 11. Erythroblastic anemia 4. Acute and Chronic Blood 12. Sickle cell anemia Loss 13. Relapsing fever 5. Thalassemia 14. Leukemia 6. Sideroblastic Anemia 15. Myeloproliferative 7. Malaria disorder 8. Parasitic Infections 16. Splenic tumor o Anemia - decrease in Hgb; marrow compensates by releasing reticulocytes RETICULOCYTE PRODUCTION INDEX (RPI) • • CHANGES IN RETICULOCYTE COUNT • • Physiologic Increase in Reticulocyte Count o Pregnancy o At birth o Menstruation o High altitudes Decreased Reticulocytes: o Aplastic anemia - blood cells are not produced by bone marrow o Bone Marrow Failure o Acute benzol poisoning o Chronic infections aka Reticulocyte Index or Hematocrit Correction In specimens with low Hematocrit, the percentage of retics may be falsely elevated because whole blood contains fewer RBCs. Correction Factor: 45% Computation: • aka Shift Correction Provides further refinement of the CRC Measures erythropoietic activity when stress/shift reticulocytes are present. o Stress/Shift Reticulocyte - premature reticulocytes that are released from the bone marrow to compensate for anemia Formula: Maturation Index: HEMATOCRIT (%) MATURATION INDEX (days) 40 – 45 1 35 – 39 1.5 25 – 34 2 15 – 24 2.5 <15 3 Sample Problem: A patient with reticulocyte count of 3.6% has a hematocrit level of 29.6%. Compute for CRC and RPI.