HEMA-LAB-MODULE2-PDF - Hematology Laboratory Module 11: Reticulocyte Count

HEMATOLOGY LABORATORY MLS 3A MODULE 11: RETICULOCYTE COUNT INTRODUCTION RETICULOCYTES Reticulocyte count or the “retic count” As t...

HEMATOLOGY LABORATORY MLS 3A MODULE 11: RETICULOCYTE COUNT INTRODUCTION RETICULOCYTES Reticulocyte count or the “retic count” As the erythrocyte develops, the nucleus becomes more Reticulocyte is the last immature erythrocyte stage and more condensed and is eventually lost It spends 2 to 3 days in the bone marrow and 1 day in After the loss of the nucleus, an immature erythrocyte the peripheral blood before developing into a mature (reticulocyte) remains in the bone marrow for 2 to 3 days erythrocyte before entering the circulating blood The reticulocyte count is used to assess the - Reticulocytes are the immediate precursor of mature erythropoietic activity of the bone marrow erythrocytes Hence, an accurate reticulocyte count can help the - First, they stay in the bone marrow for 2 to 3 days physician in determining if the patient’s bone marrow is before they go to circulating blood and they spent producing enough red blood cells another day in the circulation before they mature completely During this period in the bone marrow and during the rst UNIT MAP day in the circulation, this immature erythrocyte is referred to as a reticulocyte - Other terms for reticulocyte: Proerythrocyte Polychromatophilic macrocyte Granulophilocyte Although the reticulocyte lacks a nucleus, it contains various organelles, such as mitochondria, and an extensive number of ribosomes The formation of new ribosomes ceases with the loss of the nucleus in the late metarubricyte - However, while RNA is present, protein and heme synthesis continues During reticulocyte maturation, the RNA is catabolized, and the 1 fi HEMATOLOGY LABORATORY MLS 3A MODULE 11: RETICULOCYTE COUNT ribosomes disintegrate Normal erythropoiesis corrects for a shorter life span by - RNA is catabolized since there is already no nucleus in increasing the production rate, which the reticulocyte the reticulocyte, there are no new ribosomes that are count measures produced, so when RNA is catabolized, ribosomes - Shortened life span = increased erythropoiesis = disintegrate that means gaubos lang ang RNA and increased reticulocytes ribosomes after few days. - No problem in bone marrow so the body can Reticulocyte to mature erythrocyte compensate therefore, increased erythropoiesis - Loss of ribosomes and mitochondria and full - Decreased RBC production = decreased erythropoiesis hemoglobinization of the cell marks the transition An elevated reticulocyte count accompanies a from the reticulocyte stage to full maturation of the shortened RBC survival erythrocyte Reticulocytosis indicates that the body is trying to maintain homeostasis I. RETICULOCYTE COUNT (RELATIVE VALUE) REFERENCE RANGE The reticulocyte count procedure is frequently performed Adults: 0.5% to 2.0% in the clinical laboratory Newborn infants: 2.5% to 6.0% Indicator of the rate of erythrocyte production - This value falls to the adult range by the end of the Usually, the count is expressed as a percentage of total second week of life (2 weeks after birth) erythrocytes Indication of a shorter-than-normal erythrocyte survival PRINCIPLE - Based on the deduction that the total red blood cell Whole blood, anticoagulated with EDTA, is stained with a (RBC) mass in a steady state is equal to the number of supravital stain, such as new methylene blue. Any new RBCs produced, multiplied by the 120-day life nonnucleated erythrocyte that contains two or more particles span of individual cells of blue-stained granulo lamentous material after new When the RBC mass falls (decreased RBC mass, it is the methylene blue staining is de ned as a reticulocyte. (Figure result of decreased RBC production or a shortened life 11.1) span - The reticulocyte count will tell if the reason of decreased RBC mass is due to decreased RBC Figure 11.1: Reticulocytes under production or shortened life span an oil immersion lens (peripheral blood x1000) 2 fi fi  HEMATOLOGY LABORATORY MLS 3A MODULE 11: RETICULOCYTE COUNT NOTE: 2. Mix well. Place in a clean brown bottle that is properly Reticulocyte cannot be di erentiated from mature labeled with the name of the reagent, date of erythrocyte under Wright’s stain unless they exhibit preparation, and name of the individual who prepared polychromasia the solution. There are other types of supravital stains such as brilliant 3. Filter solution daily or immediately before use to cresyl blue, neutral red, janus green that can be used for remove any precipitate. reticulocyte count - The precipitates can be mistaken as RNA remnants Supravital stain can keep the reticulocytes in its live form of reticulocytes, that is why it is very important to Blue-stained granulo lamentous material are the RNA lter the solution daily remnants EQUIPMENT 1. Capillary tubes SPECIMEN 2. Glass slides Whole blood 3. Wright or Wright-Giemsa stain - Anticoagulated with either EDTA or heparin is suitable 4. Microscope, lens paper, and immersion oil Capillary blood 5. Miller ocular disc (optional) - Drawn into heparinized tubes or immediately mixed with stain may also be used PROCEDURE The test should be performed promptly after blood 1. Mix equal amounts of blood and new methylene blue collection stain (2 to 3 drops, or 50 mcL each) and allow to - To keep the reticulocytes alive incubate at room temperature for 3 to 10 minutes. Stained smears retain their color for a prolonged period - For example, when you add 2 drops of blood, you are also going to add 2 drops of new methylene blue stain REAGENT - It is needed to incubate in order for reticulocytes to New methylene blue solution - Other name = Methylene Blue N take up the stain 2. Remix the preparation. - We need to remix the preparation because the This solution is prepared as follows: sediments are settled at the bottom and the 1. Weigh out 0.5 g of new methylene blue N, 1.4 g of reticulocytes are settled above the matured RBCs potassium oxalate, and 0.8 g of sodium chloride. Place and we get the solution, it is equally distributed these chemicals in a 100-mL volumetric ask and 3. Prepare two wedge lms. dilute to the calibration mark with distilled water. 3 fi fi fi ff fl HEMATOLOGY LABORATORY MLS 3A MODULE 11: RETICULOCYTE COUNT 4. In an area in which cells are close together but not MILLER DISC touching, count 1000 RBCs under the oil immersion Designed to reduce labor-intensive process brought by objective lens. Reticulocytes are included in the total large numbers of red cells RBC count (i.e., a reticulocyte counts as both an RBC The disc is composed of two squares, with the area of the and a reticulocyte). smaller square measuring 1/9 the area of the larger square 5. To improve accuracy, have another laboratorian count The disc is inserted into the eyepiece of the microscope the other smear; values should agree within 20%. and the grid in Figure 11.2 is seen 6. Calculate the reticulocyte count: RBCs = counted in the smaller square - Or the number of reticulocytes counted can be Reticulocytes = counted in the larger square multiplied by 0.1 (100/1000) to obtain the result Selection of the counting area is the same as described earlier A minimum of 112 cells should be counted in the small square - Because this is equivalent to 1008 red cells in the large square and satis es the College of American Pathologists (CAP) hematology standard for a manual reticulocyte count based on at least 1000 red cells - RBCs in the smaller square (B) is counted and reticulocytes are counted in the larger square (A) NOTE: - To consider that the count is correct, count at least 112 Two types of performing reticulocyte count cells in the small square; it means that there are 1000 - Wet and Dry method RBCs in the entire square In this procedure, dry method is used - 9 is multiplied because In wet procedure, we add a drop on the glass slide and we square B is 1/9ths of the placed cover slip entire square (there are 9 In dry method, we make a smear small squares in the entire - The smear is viewed under OIO large square) - Count the number of reticulocytes in 1,000 RBCs It is best to used Miller Disc’s in counting the reticulocytes Figure 11.2: The “1000” is the number of RBCs counted Miller ocular disc. Alternatively, square B may be in the center of square A 4 fi HEMATOLOGY LABORATORY MLS 3A MODULE 11: RETICULOCYTE COUNT The calculation formula for percent reticulocytes is: Howell-Jolly bodies are round nuclear fragments and are usually singular. - Heinz bodies are multiple in 1 cell and can be found only in supravital stain while Howell-Jolly bodies are singular in 1 cell and can be found using a Wright Giemsa stain SOURCES OF ERROR AND COMMENTS Pappenheimer bodies are hemosiderin in the mitochondria whose presence can be con rmed with 1. If a patient is very anemic or polycythemic, the proportion of dye to blood should be adjusted an iron stain, such as accordingly. Prussian blue - Anemic = small RBCs - All other inclusions - Polycythemic = high RBCs are not visible when 2. An error may occur if the blood and stain are not using Prussian blue mixed before the smears are made. The speci c gravity of the reticulocytes is lower than that of the Figure 11.3: Reticulocytes (A) and Heinz mature erythrocytes, and reticulocytes settle at the bodies (B) stained with supravital top of the mixture during incubation. stain (peripheral blood ×1000) - To acquire the correct representation of the solution for smearing 3. Moisture in the air, poor drying of the slide, or both II. ABSOLUTE RETICULOCYTE COUNT may cause areas of the slide to appear refractile, and Traditionally, the reticulocyte count has been expressed as these areas could be confused with reticulocytes. The a percentage of the total number of circulating RNA remnants in a reticulocyte are not refractile. erythrocytes (e.g., 1%) 4. Other RBC inclusions that stain supravitally include However, this value may be erroneous because uctuation Heinz, Howell-Jolly, and Pappenheimer bodies. in the percentage may be caused by a change in the total Heinz bodies represent precipitated hemoglobin, number of circulating erythrocytes rather than a true usually appear round or oval, and tend to cling to the change in the number of circulating reticulocytes cell membrane (Figure 11.3). To account for variations caused by erythrocyte quantity, - Both Heinz bodies and reticulocytes appear in expression of reticulocytes in absolute rather than supravital stain and both do not appear in Wright proportional terms is becoming the preferred method of Giemsa stain reporting 5 fi fi fl HEMATOLOGY LABORATORY MLS 3A MODULE 11: RETICULOCYTE COUNT The correction for anemia is helpful for clinical contains fewer RBCs. A correction factor is used, with the interpretation, and several di erent methods are used average normal hematocrit considered to be 45%. The CLSI proposes that the correction for anemia, the corrected reticulocyte count, be made mathematically by CALCULATION 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. CALCULATIONS REFERENCE RANGE Patients with a hematocrit of 35% = 2% to 3% Patients with a hematocrit of less than 25% = 3% to 5% - The count is increased to compensate for anemia - The corrected reticulocyte count depends on the degree of anemia IV. RETICULOCYTE PRODUCTION INDEX PRINCIPLE NOTE: Reticulocytes that are released from the marrow RBC count should be in SI unit prematurely are called shift reticulocytes These reticulocytes are “shifted” from the bone marrow to the peripheral blood earlier than usual to compensate for III. CORRECTED RETICULOCYTE COUNT anemia Instead of losing their reticulum in 1 day, as do normal PRINCIPLE reticulocytes, these cells take 2 to 4 days to lose their In specimens with a low hematocrit, the percentage of reticula reticulocytes may be falsely elevated because whole blood 6 ff HEMATOLOGY LABORATORY MLS 3A MODULE 11: RETICULOCYTE COUNT When erythropoiesis is evaluated, a correction should be The patient’s hematocrit is used to determine the made for the presence of shift reticulocytes if there is a report of polychromasia in the morphology. Normal (nonshift) reticulocytes become mature RBCs 1 day after entering the bloodstream and thus represent 1 day’s production of RBCs in the bone marrow Cells shifted to the peripheral blood prematurely stay longer as reticulocytes and contribute to the reticulocyte count for more than 1 day appropriate correction factor (reticulocyte maturation time For this reason, the reticulocyte count is falsely increased in days): when polychromasia is present, because the count no longer represents the cells maturing in just 1 day CALCULATION On many automated instruments, this mathematical The reticulocyte production index (RPI) is calculated as adjustment of the reticulocyte count has been replaced by follows: the measurement of immature reticulocyte fraction. NOTE: The reticulocytes remain in the bone marrow for 2-3 days before entering the circulating blood because it takes 2-4 days for the reticulocytes to lose their reticulum If the reticulocytes shift early to the peripheral blood, the reticulocyte count is falsely increased because we only count the reticulocyte that stays in the bone marrow for 1 day However, early pa sila nagkadto sa bone marrow ang mga reticulocytes from 3-4 days ago is also counted, that’s why it is falsely increased Memorize the correction factor 7  HEMATOLOGY LABORATORY MLS 3A MODULE 11: RETICULOCYTE COUNT REFERENCE RANGE - Decreased erythropoietin = decreased rate of > 3 = adequate bone marrow response erythropoiesis = decreased reticulocytes < 2 = inadequate erythropoietic response c. Chemotherapeutic or radiation-induced hypoproliferation - The stem cells or other healthy cells are being CONDITIONS WITH DECREASED AND INCREASED destroyed therefore the amount of reticulocytes is RETICULOCYTES decreased Increased Reticulocytes The bone marrow is responding well, can compensate with the need of the body, and produce more RBCs. a. Hemolytic anemia and blood loss - There is no problem with the bone marrow or the erythropoiesis - The problem is with the survival rate of the RBCs. Therefore, the production of the reticulocytes is not hindered NOTE: b. After treatment of pernicious anemia, folic acid Decreased Reticulocytes de ciency, or iron de ciency There is a problem with the production of red blood - Vitamins and minerals are given to the body cells therefore you already have the ingredients for the The problem could be in the bone marrow or in the production of the RBC thus the body can kidneys compensate by producing more RBC Decreased reticulocytes = decreased erythropoiesis a. Aplastic crises of hemolytic anemia - The bone marrow is already overwhelmed and cannot produce more RBCs therefore there is decreased reticulocytes b. Decreased erythropoiesis - Ex. Chronic Kidney Disease (CKD) 8 fi fi  HEMATOLOGY LABORATORY MLS 3A MODULE 11: RETICULOCYTE COUNT ADDITIONAL NOTES (MA’AM ELSA) - 50,000 in the BM normally 3. Release of reticulocytes RETICULOCYTE - EPO comes from kidney will signal the BM to start Polychromatophilic erythrocyte producing RBC lineage and release stored - Juvenile reticulocytes Di use basophilic 4. Reticulocyte increased immediately Proerthyrocyte - So that it will compensate the lacking of RBC in the Granuloreticulocyte circulation - Granulo lamentous - Reticulum is blue IMPORTANCE TO PERFORM Found in the BM and peripheral blood Measures the erythropoietic index of the BM activity Takes 2 to 4 days to mature - To know if the BM produces RBCs In 24 hours, some of them will be mature Report the result in percentage Contains 2-3 reticulum Reticulocyte can’t be detected by wright stain because it is alcoholic NORMAL VALUES: - Methanol is increased Newborn: increased value of reticulocyte; active BM Supravital stain are used - Conventional unit in percent or SI - New methylene blue or methylene blue N should not - Relative value in percent be confused with methylene blue used in bacteriology, - There must be a “N” to di erentiate if it is vital stain GRANULOPHILOCYTE - Methylene blue in bacteriology is a simple stain - Reticulum in color blue except with Janus Green - BCB = Brilliant cresyl blue: most common in - Usually, New methylene blue or brilliant cresyl blue is Hematology used - Bromcresyl blue: in Clinical Chemistry - Janus Green, neutral red FACTORS IN DETERMINING IN CIRCULATION Reticulocyte is requested for clearer picture of bone 1. Speed in release marrow activity - normal condition the normal birth rate of cells esp rbc Routine testing is 1% meaning if 1% gn kwa sang spleen kay senescent ang RBC and ang bm ma kwa man 1% in AUTOANALYZER order to compensate It detects RNA and maturity of reticulocyte 2. 1% 9 ff fi ff  HEMATOLOGY LABORATORY MLS 3A MODULE 11: RETICULOCYTE COUNT It also includes stain such as auramine O, thiosyl orange - If iron aggregate ma retain sa RBC as blue and thio avin - Siderotic granules if stained with Prussian blue but pappenheimer if stained with Wright’s stain. IMMATURE RETICULOCYTE FACTOR (IRF) c. Intracellular parasite Detects di erent stages of reticulocytes. - Rx. malaria and babesia - They have granules INCLUSIONS IN RBC THAT CAN BE EASILY CONFUSED - Can be seen in the Wright’s stain but much better in the Giemsa stain WITH RETICULOCYTE a. Howell Jolly METHODS IN PREPARATION RETICULOCYTE COUNT - A DNA, Wright’s stain, it has a similar look especially in A. WET METHOD granular form - Reticulocyte: RNA, vital stain, have laments 1. 1:1 drop, kung taas 2:2 and stand for 10 minutes para ma take up ang stain b. Heinz Bodies 2. Put it in the slide and cover slip - Especially in dealing with G6PD, kung may de ciency - 2-4 mm lang ang drop para ma avoid leakage kay the Heinz bodies will be developed ma budlay mag identify - It doesn’t have lament 3. Press down and use cotton to remove excess - Vital stain is used too 4. Count - Di er with reticulocyte since reticulocytes have - Oil immersion is used lament - Can be seen with thalassemia, G6PD, unstable B. DRY METHOD hemoglobin, if the babies eat chocolate or ice cream, it 1. We make a smear and let it dry will lead to formation of Heinz bodies - Kung ara sa RBC kag mag agi sa spleen, e kagat sang 2. After drying, oil immersion is used to count 3. 1000 RBC is counted splenocyetes or pit and it is called bite cells - Provide a veil in the pitted RBC that’s why Coomassie Brilliant blue is used FLUORCYTOMETRIC METHOD c. Pappenheimer bodies Flowcytometric principle is used - Iron inclusion/aggregate Routine testing in highly industrialized countries - Prussian blue or pearls blue is used FLUORESCENCE METHOD Fluorochrome dye is used 10 fi ff fl ff fi fi fi  HEMATOLOGY LABORATORY MLS 3A MODULE 11: RETICULOCYTE COUNT Thio avine is used kay ga tapik na sya sa RNA sa Hb S, Hb C reticulocyte kag ma ourescence sya - Unstable hemoglobin, the RBC is degraded Best method because it is précised and accurate result is given EXTRACELLULAR RBC is not involved MILLER DISC METHOD On the eyepiece, not on the stage WARM IG Manually will give a better result IGG,SLE, RA, AUTOIMMUNE DISORDERS, PCH, - PHYSIOLOGIC INCREASE OF RETICULOCYTE COLD Newborn IgM, infectious mononucleosis, viral, - Because their BM are very active Menstruation UREMIA - Women release more or less 35 ml of blood, it must be Defect in kidney compensated by the BM High BUN, uric acid, creatinine. - When you lose blood, you lack oxygen and this will Forms uremic debris- caused mild hemolysis because gina trigger the kidney to produce EPO guba ya ang RBC, survival rate of RBC decreases - It will signal the BM to produce RBC lineage and Phenolic acid substances attach to RBC and destroys release stored reticulocytes RBC - In cases like this, it is easy to produce reticulocytes Decreased EPO Pregnancy - Pallor is shown because iron is used by the baby INCREASED RETICULOCYTE COUNT THERAPY - Higher reticulocytes to compensate the blood loss Iron - Iron supplement or folic acid is given Vitamin B12 Folic acid PATHOLOGIC INCREASE OF RETICULOCYTES Kung ang retic nag taas kay ga take sang supplement, or DECREASED RETICULOCYTE COUNT may blood loss, hemolytic anemia hemolysis due to Defective hemoglobin synthesis like: destruction of RBC or shprtening of RBC life span - Thalassemia: abnormality in globin chain, - Alpha thalassemia: gene deletion of alpha chain HEMOGLOBINOPATHIES - Beta thalassemia: Warm Ig there is gene mutation Abnormal Hb Replacement of the marrow by cancer cell 11 fl fl HEMATOLOGY LABORATORY MLS 3A MODULE 11: RETICULOCYTE COUNT - Focal or invasive (neighboring tissue) or metastatic (circulate in the blood) normal marrow site is occupied Failure of the BM to produce associated with aplastic (partial or complete cessation of blood production) like: - Fanconi: BM will not produce those elements, pantocytopenia Diamond black fan - RBC lineage is a ected only, normal platelet and WBC Data given is hematocrit you don’t need to change the percentage - Male: 45 constant - Female: 42 constant BIRTH RATE 1% in circulation; 1% in BM - Normal RPI 2 - Considered normal Less than 2 - There must be a failure, or incymopathies, More than 2 Hemolytic mechanism 3 and above - Assess the anemia or not entertain the hemolytic anemia 12 ff HEMATOLOGY LABORATORY MLS 3A MODULE 11: RETICULOCYTE COUNT 13