Erythropoiesis & RBC Maturation PDF

Blood  Cell  Formation,  Maturation  and  Differentiation   ERYTHROPOIESIS     It  takes  about  18-­21  days  to  produce  an  RBC  from  stimulation  of  the  earliest  erythroid   progenitor  to  release  from  the  BM.     Criteria  for  the  identification  of  the  erythroid  precursors  ...

Blood  Cell  Formation,  Maturation  and  Differentiation   ERYTHROPOIESIS     It  takes  about  18-­21  days  to  produce  an  RBC  from  stimulation  of  the  earliest  erythroid   progenitor  to  release  from  the  BM.     Criteria  for  the  identification  of  the  erythroid  precursors     Nuclear  chromatin  pattern     Nuclear  diameter     N:C  ratio     Presence/absence  of  nucleoli     Cytoplasmic  color     Rules  (as  the  cell  matures)     Diameter  of  the  cell  decreases.     N:C  ratio  decreases.     Nuclear  chromatin  becomes  coarser,  clumped  and  condensed.     Nucleoli  disappear.     Cytoplasm  changes  from  blue  to  gray-­blue  to  salmon-­pink.     Basophilia  or  blueness  is  due  to  acidic  components  that  attract  the  basic  stain  (methylene   blue).  The  degree  of  cytoplasmic  basophilia  correlates  with  the  amount  of  ribosomal  RNA.   Pinkness   called   eosinophilia   or   acidophilia   is   due   to   the   accumulation   of   more   basic   components  that  attract  the  acid  stain,  eosin.     N:C  ratio  –  visual  estimate  of  what  area  of  the  cell  is  occupied  by  the  nucleus  compared   with  the  cytoplasm     RBC  Maturation  Series  (pronormoblastic/rubriblastic/erythroblastic  nomenclature)     1.   PRONORMOBLAST   RUBRIBLAST   PROERYTHROBLAST   a.   Earliest  morphologically  recognizable  erythrocyte  precursor   b.   1%  in  the  BM   c.   12-­20  um   d.   N:C  ratio  =  8:1   e.   1-­3  nucleoli   f.   Chromatin  (purple-­red)  is  fine  and  uniform.   g.   Deep/dark  blue  cytoplasm  (ribosomes)  with  no  granules     2.   BASOPHILIC  NORMOBLAST   PRORUBRICYTE   BASOPHILIC  ERYTHROBLAST   a.   1-­4%  in  the  BM   b.   10-­15  um   c.   N:C  ratio  =  6:1   Blood  Cell  Formation,  Maturation  and  Differentiation   d.   Centrally  located  nucleus  with  0-­1  nucleoli   e.   Chromatin  (deep  purple-­red)  is  coarsening/begins  to  condense.   f.   Cytoplasm  is  less  blue  but  intensely  basophilic.     3.   POLYCHROMATOPHILIC  NORMOBLAST   RUBRICYTE   POLYCHROMATOPHILIC  ERYTHROBLAST   a.   10-­20%  in  the  BM   b.   10-­12  um   c.   N:C  ratio  =  4:1   d.   Eccentric  nucleus  with  no  nucleoli   e.   Chromatin  shows  significant  clumping.   f.   Last  stage  capable  of  cell  division   g.   Begins  to  produce  hemoglobin,  resulting  in  murky  gray-­blue  cytoplasm   (mixture  of  pink  and  blue;;  polychromatic  =  many  colors)     4.   ORTHOCHROMATOPHILIC  NORMOBLAST   METARUBRICYTE   ORTHOCHROMATOPHILIC  ERYTHROBLAST   a.   5-­10%  in  the  BM   b.   8-­10  um   c.   N:C  ratio  =  1:2   d.   Eccentric  nucleus  with  small,  fully  condensed  (pyknotic)  nucleus   e.   No  nucleoli   f.   Pale  blue  to  salmon-­pink  cytoplasm   g.   Hemoglobin  synthesis  decreases     h.   Last  stage  with  nucleus  (moves  to  the  periphery  and  is  ejected  from  the   cell  -­>  Howell-­Jolly  bodies     5.   RETICULOCYTE   a.   1%  in  the  BM   b.   8-­10  um   c.   No  nucleus   d.   Salmon-­pink  cytoplasm   e.   Last  stage  to  synthesize  hemoglobin   f.   Last  stage  in  the  bone  marrow  prior  to  release  to  the  peripheral  blood   g.   Can  be  best  seen  using  supravital  stains     6.   MATURE  ERYTHROCYTE   a.   6-­8  um  (ave  7.2  um)   b.   Round,  biconcave  discocyte   c.   Salmon-­pink  with  central  pallor       Blood  Cell  Formation,  Maturation  and  Differentiation   ERYTHROKINETICS  (dynamics  of  RBC  production  and  destruction)     Erythron   –   collection   of   all   stages   of   erythrocytes   throughout   the   body;;   entirety   of   erythroid  cells  in  the  body     RBC  mass  –  refers  ONLY  to  cells  in  the  circulation     Hypoxia   –   stimulus   to   RBC   production;;   too   little   tissue   oxygen;;   detected   by   the   peritubular  cells  of  the  kidneys  which  will  produce  EPO     Erythropoietin  (EPO)     mol  wt.  =  34  000  Daltons     MAJOR  stimulatory  cytokine  (growth  factor)  for  RBC  production     amount  is  regulated  to  sufficiently  replace  RBCs  that  undergo  senescence  (death   due  to  old  age)     trauma,  increased  RBC  destruction  and  diminished  O2-­carrying  capacity  influence   EPO  production     signal  transduction:  sends  intracellular  messages  to  RBCs  (binds  to  receptor  on   the  surface  of  cells  ad  creates  a  cascade  or  “program”  that  will  lead  to  cell  division,   maturation  and  more  RBCs  entering  the  circulation     JAK2  signal  transducer  (Janus-­activated  tyrosine  kinase  2)     three  major  effects:  early  release  of  reticulocytes  from  the  BM,  prevents  apoptosis,   reduced  time  needed  for  cells  to  mature  in  the  BM     puts  more  RBCs  into  the  circulation  at  a  faster  rate  than  without  its  stimulation     Early  release  of  reticulocytes     EPO   induces   changes   in   the   adventitial   cell   layer   to   increase   the   width   of   the   spaces  to  allow  for  movement  of  RBCs  (held  in  the  marrow  because  they  contain   receptors   for   adhesive   molecules   -­>   EPO   downregulates   or   suppress   these   receptors   for   RBCs   to   be   loosened   from   adhesion,   allowing   early   exit   -­>   shift   reticulocytes)     Limited  in  effectiveness:  precursors  in  the  BM  (storage  pool)  become  depleted     Inhibition  of  apoptosis     Increasing  the  number  of  cells  that  will  mature  into  circulating  erythrocytes     Apoptosis  –  programmed  cell  death;;  intentional  cell  wasting     When  there  is  a  steady-­state  demand  for  RBCs,  early  progenitors  are  allowed  to   die,  but  in  cases  of  increased  demand,  RBC  progenitors  are  given  a  head-­start,   preventing  apoptosis  or  death.     EPO  indirectly  removes  the  apoptosis  induction  signal.   o   Fas   –   death   receptor   found   on   the   membrane   of   progenitor   cells;;   ligand:   FasL  (present  on  more  mature  cells)   o   When   EPO   levels   are   low,   so   is   cell   production   because   hypoxia   is   not   present;;   thus;;   early   precursors   are   allowed   to   undergo   apoptosis.   This   happened   when   FasL-­bearing   precursors   cross-­link   with   Fas-­marked   immature  precursors  which  are  stimulated  to  undergo  cell  death.  As  long  as   Blood  Cell  Formation,  Maturation  and  Differentiation   more  mature   cells   with  FasL   are  present   in  the   marrow,   erythropoiesis   is   subdued.  If  FasL-­bearing  cells  are  depleted,  EPO  is  produced  and  younger   Fas+  precursor  are  allowed  to  develop  which  increases  RBC  output  by  the   marrow.     Direct  EPO  rescue  from  apoptosis   o   When   EPO   binds   to   its   receptor   on   the   CFU-­E,   one   of   the   effects   is   to   reduce   production   of   Fas   ligand;;   thus,   younger   cells   avoid   the   apoptotic   signal  from  the  older  cells.     Reduced  marrow  transit  time     Increasing  the  rate  at  which  surviving  precursors  can  enter  the  circulation   o   Increased  rate  of  cellular  processes   o   Decreased  cell  cycle  times  =  reduced  time  for  cells  to  mature;;  fewer  mitotic   divisions     Increased  hemoglobin  production     Bone  marrow  egress  =  loss  of  adhesive  molecules     Cessation   of   division   =   cell  division   takes   time,   delay   in   the   entry  of   cells   to  the   circulation     Stress  reticulocytes  –  due  to  early  marrow  exit     Other   stimuli   to   erythropoiesis:   hormones   (testosterone,   pituitary   and   thyroid   hormones)     Erythrocyte  Destruction     RBC  life  span:  120  days     Senescence   (old   age   or   cellular   aging)   –   brought   about   by   loss   of   glycolytic   enzymes  (RBC  lacks  mitochondria)     Macrophage-­Mediated  Hemolysis  (Extravascular  Hemolysis)     Deteriorating  glycolytic  processes  =  reduced  ATP  production;;  low  glucose     Oxidation  of  membrane  lipids  and  proteins     Imbalance  between  intracellular  and  extracellular  ions  –  effect  on  the  membrane’s   selective  permeability  (water  enters  the  cell,  losing  its  discoid  shape)     Spherical  RBCs  are  rigid  and  cannot  squeeze  though  narrow  vessels,  trapped  and   ingested  or  salvaged  by  macrophages.     Macrophages   are   able   to   recognize   senescent   cells   and   distinguish   them   from   younger  cells.     Iron  is  removed  and  stored  as  ferritin.     Mechanical  Hemolysis  (Fragmentation  or  Intravascular  Hemolysis)     Mechanical  or  traumatic  stress  –  rupture  of  cell  membrane  while  the  cell  is  in  the   peripheral  circulation  (inside  the  blood  vessels)