HEMATOLOGY LABORATORY 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 MATERIALS • 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 : SUPRAVITAL STAIN • Mature RBC – gray blue • Reticulocytes – gray blue cells with deep blue filamentous web or granules (2 or more) 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 2. Lead Poisoning 3. Iron Deficiency Anemia 4. Acute and Chronic Blood Loss 5. Thalassemia 6. Sideroblastic Anemia 7. Malaria 8. Parasitic Infections 9. Blood intoxication 10. Kala-azar 11. Erythroblastic anemia 12. Sickle cell anemia 13. Relapsing fever 14. Leukemia 15. Myeloproliferative disorder 16. Splenic tumor o Anemia - decrease in Hgb; marrow compensates by releasing reticulocytes 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 ABSOLUTE RETICULOCYTE COUNT (ARC) • Actual number of reticulocytes in 1 liter of whole blood • Computation : • Reference Range : 25-75 x 10 9 /L • Sample Problem : If patient’s reticulocyte count is 3.6% and the RBC count is 3.20 x10 12 /L. What is the ARC? CORRECTED RETICULOCYTE COUNT (CRC) • 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 : RETICULOCYTE PRODUCTION INDEX (RPI) • 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.