Erythropoiesis and Reticulocytes

● TOPIC OUTLINE A. B. C. D. E. F. G. H. I. J. ● ● ● ● Erythropoiesis Reticulocytes 1. Reticulocyte count i. Methods of counting reticulocytes ii. Methods of reporting reticulocyte counts Erythrocytes 1. RBC count 2. RBC Metabolic Pathways Red Blood Cell Anomalies 1. Anisocytosis 2. Anisochromia 3. Poikilocytosis 4. RBC inclusion bodies Hemoglobin 1. Hemoglobinometry 2. Hemoglobin electrophoresis 3. Hemoglobin Synthesis 4. Hemoglobin derivatives Hematocrit and ESR 1. Hematocrit determination Related Topics 1. Rule of Three 2. Erythrocyte Indices 3. Erythrocyte sedimentation rate General Overview of Anemia 1. Morphologic Classifications 2. Pathophysiologic Classification Hemoglobinopathies ThalassemiaS ERYTHROPOIESIS ERYTHROPOIESIS → process of RBC formation (takes place inside the bone marrow) 50% red marrow, 50% yellow marrow Site of formation: red marrow Related terms: ○ ERYTHRON → Total mass of rbc circulating in the peripheral blood and the bone marrow rbc precursors ○ RBC MASS → erythrocytes in the circulation, total population present inside the circulation (blood vessel only) ○ ERYTHROKINETICS → a term that describes the dynamics of rbc creation and destruction ■ Lifespan of RBC: 120 days ○ INEFFECTIVE ERYTHROPOIESIS ■ Production of defective erythroid precursor cells (These defective RBC precursors often undergo apoptosis (programmed cell death) in the bone marrow before they have a chance to mature to the reticulocyte stage.) ■ Happens inside the RED BONE MARROW, no longer circulate in the blood vessel = patients suffer anemia ■ Examples of conditions include ● Vitamin B12 deficiency → MACROCYTIC, NORMOCHROMIC ANEMIA ● Folate deficiency → MACROCYTIC, NORMOCHROMIC ANEMIA ○ ○ ○ ○ ○ Thalassemia → MICROCYTIC, HYPOCHROMIC ● Sideroblastic anemia→ MICROCYTIC, HYPOCHROMIC ANEMIA INSUFFICIENT ERYTHROPOIESIS → Decrease in the number of rbc precursors in the bone marrow (resulting in low rbc production = ANEMIA) ■ Examples of conditions: ● iron deficiency → MICROCYTIC, HYPOCHROMIC ANEMIA ● renal disease → NORMOCYTIC, NORMOCHROMIC ANEMIA ● acute leukemia → NORMOCYTIC, NORMOCHROMIC ANEMIA PROGENITOR CELLS → immature hematopoietic cell that is committed to a cell line but CANNOT be identified morphologically (makikita pero hindi makikilala) ■ EX. BFU-E, CFU-E PRECURSOR CELLS → immature hematopoietic cells that is morphologically IDENTIFIABLE as belonging to a given cell line (makikita mo na, makikilala mo pa) ■ EX. Rubriblast, Prorubricyte CD71→ EARLIEST marker of erythroid differentiation; transferrin receptor (in the erythroid cells) CD34→ classical marker of the HEMATOPOIETIC STEM CELLS HORMONES RELATED TO ERYTHROPOIESIS HORMONE REMARKS Erythropoietin ● Chief stimulatory cytokine for (EPO) RBCs ● Major hormone that stimulates the production of erythrocytes ● Thermostable, non dialyzable, glycoprotein hormones ● Primary cell / organ source: ○ Organ: KIDNEYS ○ Cells: Peritubular interstitial cells EXAMPLE BOARD EXAM QUESTION: What do you call the hormone produced by the kidneys for RBC production? A) Erythropoiesis B) --C) --D) Erythropoietin ● Primary target cells: BFU-E and CFU-E ○ Affected by erythropoietin ● Normally, EPO is released from the kidney into the blood in response to hypoxia (too little tissue oxygen) ● Three major effects: ○ ● ● GROWTH HORMONE ● ● TESTOSTERONE ● ● PROLACTIN ESTROGEN ● ● ● ● ● ● ● ● Allowing early release of reticulocytes from the bone marrow ○ Reducing the time needed for cells to mature and the bone marrow ○ Preventing apoptotic death (note: apoptosis rescue is the MAJOR way in which EPO increases rbc mass) Some of the current or potential therapeutic applications: ○ Anemia of chronic renal disease ○ Autologous donation blood collection ○ Anemia and hiv infection to permit use of zidovudine (AZT) “BLOOD DOPING” → Some athletes illegally use epo injections to increase oxygen carrying capacity of their blood ○ To enhance endurance and stamina ○ can lead to deadly arterial and venous thrombosis Produced by: Pituitary gland Stimulates erythropoiesis directly in the BM Produced by: Testes Stimulates erythropoiesis indirectly (stimulates kidneys to produce more erythropoietin in the BM) Male hormone Produced by: Pituitary gland Stimulates erythropoiesis directly Produced by: Ovaries Inhibits erythropoiesis indirectly (pinipigilan ang process of RBC production; has an effect on kidneys - inhibits the production of more erythropoietin = BM of women less stimulated to produce RBC) Bakit magkaiba RBC COUNT in males and females? ○ Because of estrogen hormones in females (inhibits erythropoiesis) = lower RBC ct. ○ Testosterone stimulates kidneys to stimulate BM to produce MORE red cells Female hormone RBC STAGES OF MATURATION BFU-E → burst forming unit-erythroid ● CFU-E → colony forming unit-erythroid NTR: ● BFU-E & CFU-E are committed erythroid progenitor cells (makikita pero di makikilala) ● It takes about 18 to 21 days for the BFU-E to mature to an erythrocyte, of which approximately 6 days are spent recognizable precursors in the bone marrow. NOMENCLATURE SYSTEMS RUBRI NORMOBLAST ERYTHROBLAST Rubriblast Pronormoblast Proerythroblast Pro rubricyte Basophilic Basophilic Normoblast OR erythroblast OR Early normoblast Early erythroblast Rubricyte Polychromatophi Polychromatophili lic normoblast c erythroblast OR OR Intermediate Intermediate Normoblast Erythroblast Metarubricyte Orthochromatic Orthochromatic normoblast OR erythroblast OR Late normoblast Late erythroblast Reticulocyte Reticulocyte Reticulocyte Mature Mature Mature erythrocyte erythrocyte erythrocyte * not a precursor *for as long you’re using supravital stain, Reticulocyte will be called the same; it will differ in name when using Wright’s stain NTR: 5 RBC Precursors under RUBRI: Rubriblast, prorubricyte, rubricyte, metarubricyte, reticulocyte 3 nomenclature: Rubriblast, Normoblast, Erythroblast Polychromatic and polychromatophilic are the same When reticulocytes are found in a Wright-stained smear: ● Polychromatophilic erythrocytes ● Diffusely basophilic erythrocytes ------------------------------------------------------------------------------PRO = “before” META= “after” STAGES 1. RUBRIBLAST (PRONORMOBLAST) ● This cell (rubriblast) gives rise to 2 prorubricyte. ● Earliest recognizable erythroid precursor using the light microscope ● Capable of mitosis= produces 2 prorubricyte each ● Moderately basophilic with nucleoli ● Additional information: N:C RATIO → 8:1 ○ ● ● A morphological feature used to identify and stage RBC and WBC precursors ○ A visual estimate of what area of the cell is occupied by the nucleus compared with the cytoplasm ○ If the areas occupied by the nucleus and the cytoplasm or approximately equal the N:C ratio is 1:1 ○ If the nucleus takes up < 50% of the area of the cell, the ratio is lower ( eg. 1:5 or less than 1) ○ Is the nucleus takes up > 50% of the area of the cell, the ratio is higher (eg. 3:1 or 3) Basophilia → pertains to the blueness (BUGHAWWW) of a particular part of the cell and is due to the acidic components that attract the basic stain (example: methylene blue) ○ Degree of cytoplasmic basophilia correlates with the quantity of ribosomal rna Eosinophilia or acidophilia → pertains to the pinkness of a particular part of the cell and is due to the accumulation of more basic components that attract the acid stain eosin ○ As the rbc matures, the eosinophilia of the cytoplasm correlates with the accumulation of hemoglobin. SIZE NUCLEOLI NUCLEUS CYTOPLASM 12 to 20 um 2. ● ● ● ● ● ● ● 1 to 2 Round or slightly oval, thin nuclear membrane, central or slightly eccentric Small and amount, moderately basophilic, homogeneous NC ratio= 8:1 PRORUBRICYTE (BASOPHILIC NORMOBLAST) The prorubricyte gives rise to 4 rubricyte Capable of mitosis= produces 4 prorubricyte No nucleoli and has cell chromatin The color of the cell is BLUE Most helpful criteria in distinguishing the prorubricyte from the rubriblast ○ 1) Coarser chromatin ○ 2) Absence of nucleoli (pertains to the material found inside the nucleus) Last stage with nucleolus First stage of hemoglobinization (Hb synthesis) ○ Hemoglobin -> eosinophilic material (pink) ○ Although it starts to creates Hb, it cannot be seen under the microscope because of small amount SIZE NUCLEOLI NUCLEUS CYTOPLASM 10-15 um 0-1 round, thin nuclear membrane, smaller, slightly eccentric NC ratio= 6:1 Appears more abundant than in normal blast because of smaller nucleus 3. RUBRICYTE (POLYCHROMATIC/ INTERMEDIATE NORMOBLAST) ● Each of this cell (rubricyte) gives rise to 2 metarubricyte ● “POLY” = many; many colors (CHROMA) ● May be confused with the lymphocyte ○ LYMPHOCYTE ■ Nucleus → CRUSHED VELVET ■ Cytoplasm → SKY BLUE/ “ROBIN EGG” BLUE ○ RUBRICYTE ■ Nucleus → CHECKERBOARD ■ Cytoplasm → MUDDY or GRAY ● LAST stage capable of mitosis ● First stage in which the cytoplasm becomes PINK ○ Cytoplasm of rubricyte = GRAY (because of the mixture of pink and blue) SIZE NUCLEOLI NUCLEUS CYTOPLASM Round and Basophilic to 10-12 um none smaller, pick nuclear membrane, eccentric nucleus NC ratio= 4:1 4. ● ● ● ● ● diffusely lilac in color (muddy or gray), depending on hemoglobin content METARUBRICYTE (ORTHOCHROMATIC/ LATE NORMOBLAST) Nucleus is extruded at this stage, and the cell becomes a reticulocyte. Last stage that the RBC has its nucleus ORTHO- “the same” ○ Mature red blood cell = cytoplasm: saHmon pink Other names: (total number of names: 7) ○ Nucleated RBC ○ Pyknotic erythroblast ○ Acidophilic normoblast PYRENOCYTE ○ ○ ○ Enveloped extruded nucleus Engulfed by bone marrow macrophages Frequently, small fragments of the nucleus are left behind if the projection is pinched off before the entire nucleus is enveloped ■ The fragments inside the cell are called HOWELL-JOLLY BODIES when seen in the RBCs in the circulation ■ Howell-Jolly bodies are typically removed from the rbcs by the splenic macrophage pitting crosses once the enter the circulation ■ Pag nawala spleen = more HJ bodies in circulation ■ SPLENECTOMY- surgical removal of the spleen SIZE NUCLEOLI NUCLEUS CYTOPLASM 8-10 um none Pyknotic salmon pink (dense mass of the degenerated NC Ratio = 1:2 chromatin) 5. RETICULOCYTE (DIFFUSELY BASOPHILIC ERYTHROCYTE) SIZE 7-8 um ● NUCLEOLI & NUCLEUS None CYTOPLASM Salmon pink (with a central pallor occupying ⅓ of the cell’s diameter) RETICULOCYTES RETICULOCYTE COUNT RETICULOCYTE ○ Immature, non-nucleated RBC which contains >2 blue stained, granulofilamentous materials (RETICULUM) after staining (supravital stains) ■ In Wright stain, reticulum CANNOT be seen that’s why it’s called Diffusely basophilic erythrocytes ○ Continues to generate hemoglobin (last stage) ○ Normal maturation time for reticulocytes in blood: 1 DAY ○ Production of reticulocytes: 50x10^9/L/day *wright-stained ● ● ● Last young stage of RBC that appear in the circulation Last stage of hemoglobin synthesis Young RBCs containing residual RNA (last immature erythrocyte stage) ● Spends 2-3 days in the bone marrow and 1 day in the peripheral blood before developing into a mature RBC ● Shift Cells (POLYCHROMATOPHILIC MACROCYTE) ○ Seen in cases of increase RBC production ● Stress Reticulocytes (MACRO RETICULOCYTES) ○ Seen in more severe conditions (ex. Hemolytic anemia SIZE NUCLEOLI & CYTOPLASM NUCLEUS Cytoplasm still with small 8-10 um None amounts of RNA = polychromasia (mixed pink and blue staining with Golgi apparatus remnants and residual mitochondria that allows continued aerobic metabolism and hemoglobin generation 6. ● ● ● ● ● ● MATURE ERYTHROCYTE Shape: biconcave disk Thickness: 1.5 to 2.5 um Average life span: 120 days Number of erythrocytes produced from each rubriblast: 16 Normal ratio of RBCs to WBCs is approximately 600:1 and the normal ratio of RBCs to Platelets is approx. 15:1 Adult RBC contains no mitochondria (no protein or Hb synthesis) ● ● RETICULOCYTE COUNT ○ Permits effective assessment of RBC production by the bone marrow ○ A measure of EFFECTIVE ERYTHROPOIESIS REFERENCE RANGES: ○ Adults: 0.5 to 1.5% ○ Newborns: 1.8 to 5.8% (by 1-2 weeks of age, reference values are the same as for adults) ○ Increased (↑) reticulocyte counts (reticulocytosis) aka: POLYCHROMASIA or POLYCHROMATOPHILIA ■ Considered as the first sign of accelerated erythropoiesis and observed in hemolytic anemias (RBCs are continually lysed in circulation), individuals with iron deficiency anemia receiving iron therapy, thalassemia, sideroblastic anemia, and in acute and chronic blood loss. ■ When RBC mass is lessened, the BM is stimulated to produce more RBCs at a faster rate (accelerated erythropoiesis) = manifest as increased retics (young rbc) ○ Decreased (↓) reticulocyte counts (reticulocytopenia): ■ Observed in aplastic anemia and and conditions in which the bone marrow is NOT producing RBCs