1. MLAB 1415- Hematology Keri Brophy-Martinez  Chapter 5: The Erythrocyte Part One

2. 2 Erythropoiesis: Production and maturation of erythrocytes in the bone marrow Rubriblast Pronormoblast Proerythroblast Prorubricyte Basophilic normoblast Basophilic erythroblast Metarubricyte Orthochromatic normoblast Orthochromatic erythroblast Reticulocyte Diffusely basophilic erythrocyte Polychromatophilic erythrocyte Mature RBC Erythrocyte Discocyte Rubricyte Polychromatic normoblast Polychromatic erythroblast

3. Maturation of Erythrocytes  Erythropoietin (EPO) The growth factor that stimulates RBC production Released in response to decreased levels of oxygen in the body tissues Produced and released by the kidneys With normal levels of EPO stimulation and normal red cell lifespan, about 1% of the red cells in the blood are newly released red cells called reticulocytes. Aged rbc’s are primarily removed by the spleen.

4. Production and Affects of EPO

5. Reference Ranges for RBC  Reference ranges varies with Sex Age Geographic location  Normal RBC in adults Male4.5-5.5 x 10 6 /µl Female4.0 - 5.0 x 10 6 /µl Infants and children - normals vary by age

6. Maturation Sequence of Erythrocytes  Stem cell - an unspecified cell that gives rise to a specific specialized cell, such as a blood cell Multipotential and cannot be identified morphologically Can self-renew and differentiate  CFU-GEMM: granulocyte, erythrocyte, monocyte, megakaryocyte  BFU-E: burst forming unit  CFU-E : colony forming unit Stem Cell CFU-GEMM BFU-E CFU-E Mature RBC EPO

7. Terminology Systems  Normoblast Pronormoblast Basophilic normoblast Polychromatophilic normoblast Orthochromatophilic normoblast Reticulocyte Erythrocyte  Rubriblast Rubriblast Prorubricyte Rubricyte Metarubricyte Reticulocyte Erythrocyte

8. 8 Erythropoiesis  As cell develops see structural changes: decrease in cell volume, condensation of chromatin, decrease in nuclear to cytoplasmic ratio, loss of nucleoli, decrease of RNA in cytoplasm, decrease in mitochondria, and gradual increase in hemoglobin synthesis.  Nucleus eventually extruded.

9. Maturation Sequence of Erythrocytes  Rubriblast (Pronormoblast) Size = 14-20 µm Cytoplasm  Deeply blue (basophilic)  Scant amount, may have a perinuclear halo  No granules Nucleus  Large and round  Reddish-purple with fine chromatin  1-2 nucleoli  may be bluish N:C ratio ( nuclear: cytoplasmic)  8:1

10. Maturation Sequence of Erythrocytes  Prorubricyte (basophilic normoblast) Size = 10-16µm Cytoplasm  Deeply basophilic indicating RNA activity needed to produce hemoglobin  No granules Nucleus  Round, large  Chromatin more clumped  No nucleoli N:C ratio = 6:1

11. Maturation Sequence of Erythrocytes  Rubricyte (polychromatic normoblast ) Size = 10-12µm Cytoplasm  Blue-gray to pink-gray (pink indicates that hemoglobin production can be seen)  Slight increase in amount Nucleus  Round and smaller  Chromatin more clumped, irregular  No nucleoli  N:C ratio = 4:1

12. Maturation Sequence of Erythrocytes  Metarubricyte - Nucleated RBC (orthochromic normoblast) Size: 8-10 µm Cytoplasm  Pinker indicating larger amounts of hemoglobin production  Increased amount Nucleus  Tightly condensed chromatin (pyknotic)  No nucleoli  Mitosis ends at this stage (no more DNA synthesis)  Nucleus is extruded at end of this stage N:C ratio = 1:1

13. Maturation Sequence of Erythrocytes  Reticulocyte (diffusely basophilic or polychromatophilic erythrocyte Size: 8-10µm Cytoplasm  Diffusely basophilic due to residual RNA  Stain with new methylene blue to see fine reticulum strands  Hemoglobinization is not complete No nucleus present Present in circulation for 1-2 days

14. Lab Methods  New Methylene Blue is a supravital stain it is used to stain reticulocytes. They cannot be identified as reticulocytes from Wright’s stain.

15. Maturation Sequence of Erythrocytes  Mature erythrocyte Size = 7-8µm Volume = 80-100 fL Cytoplasm  Pink/red  Biconcave shape Nucleus - none Present in circulation for about 120 days

16. References  Diggs, L., Strum, D., & Bell, A. (1975). The Morphology of Human Blood Cells. North Chicago: Abbott laboratories.  McKenzie, S. B., & Williams, J. L. (2010). Clinical Laboratory Hematology. Upper Saddle River: Pearson Education, Inc.