Chapter

 8  –  Erythrocyte  Production  and    The   terms   BFU-­‐E   and   CFU-­‐E   are  

Destruction   laboratory   descriptions   of   growth  
patterns  in  culture  media  
 As  RBCs  mature,  several  trends  in    Only   a   few   BFU-­‐E   are   present   in   the  
appearance  are  observed:   bone   marrow   and   therefore   are  
1.) Overall  diameter  of  cell  decreases   difficult  to  identify  morphologically  
2.) N:C  ratio  decreases    They   are   different   from   but   similar   to  
3.) Nuclear  chromatin  pattern   small  to  medium  sized  lymphocytes  
becomes  coarser    Increased  numbers  of  BFU-­‐E  have  been  
4.) Nucleoli  disappear   demonstrated  in  some  anemias      
5.) Cytoplasm  from  blue  to  pink    
(higher  RNA  to  higher  Hb)   Production  Sites  
 The   main   sites   of   adult   blood   cell  
 The   primary   function   of   the   production   include   the   vertebrae,  
erythrocyte  is  to  deliver  oxygen  to  the   pelvis,  ribs,  sternum,  skull  and  the  long  
tissues   and   carbon   dioxide   from   the   bones  
tissues  
 Radioactive  imaging  have  been  used  to  
 To  function  effectively,  the  body  needs   study  the  site  of  blood  cell  production  
approximately   309   X   109   circulating  
 Iron   is   the   radioisotope   of   choice  
erythrocytes   per   kilogram   of   body  
because   it   mimics   ingested   iron   which  
weight  
is  bound  to  transferrin  in  the  blood  
 The   production   and   destruction   of  
 Studies   can   be   carried   out   in   cases   of  
erythrocytes  is  kept  in  balance  
anemia  or  after  radiation  treatment  to  
 The   erythrocytes   live   for   120   days   evaluate  production  sites        
after  which  they  are  removed  from  the    
circulation   by   the   cells   of   the   RE   Erythrocyte  Maturation  
system  
 Erythropoietin   and   other   growth  
 They   are   promptly   replaced   to   factors   stimulate   BFU-­‐E   and   CFU-­‐E   to  
maintain  the  balance       differentiate  to  the  rubriblast  stage  
 
 The   rubriblast   is   the   first   precursor  
Origin  
that   can   be   recognized   by   light  
 The   erythrocytes   originate   from   a   microscopy  
pluripotent   stem   cell   called   the   colony  
 The   rubriblast   gives   rise   to   sixteen  
forming  unit-­‐S  (CFU-­‐S)  
mature   erythrocytes   through   four   cell  
 During   development,   the   cells   become   divisions  which  take  about  72  hours  
specialized   and   synthesize   proteins  
 Changes  take  place  in  the  rubriblast  as  
needed  for  function  and  survival  
it   matures   and   differentiates   from   a  
 As   the   cell   matures   the   morphology   primitive   nucleated   cell   to   a   mature  
changes.   non-­‐nucleated  cell    
 There   are   stages   in   the   maturation    
process   Ultrastructure  
 These   stages   take   place   in   the   bone    Organelles   are   present   in   the   early  
marrow   and   may   be   differentiated   by   erythrocyte   which   are   necessary   for  
light  microscopy   the   synthesis   of   hemoglobin,   and  
 The  burst-­‐forming  unit  erythroid  is  the   proteins  
earliest  erythroid  committed  cell              As   the   proteins   accumulate,   the  
 The   BFU-­‐E   is   closely   related   to   the   number   of   organelles   gradually  
CFU-­‐S  and  matures  into  the  CFU-­‐E   diminish  
  Differentiation   of   the   maturing    Polyribosomes   probably   synthesize  
erythrocyte   results   in   alterations   in   different   proteins   from   those  
morphology  and  membrane  properties   synthesized  by  the  ribosome  
 This   results   from   reorganization   of    Ribosomes  give  the  cytoplasm  of  early  
membrane  skeletal  protein  network   precursors  a  deep,  dark  blue  color  
 An   important   component   of   the    As   hemoglobin   is   formed,   the   number  
network  is  protein  4.1   of   ribosomes   diminish   and   the   blue  
 Protein   4.1   serves   as   a   critical   link   color   is   replaced   by   a   reddish   pink  
between  the  cytoskeleton  and  the  lipid   color  
bilayer          
  Other  Organelles    
Nucleus    Golgi   apparatus   is   also   present   in   the  
 The   nucleus   is   very   important   in   the   early  precursor  and  is  located  near  the  
earliest  stages  of  red  cell  development     nucleus  
 It  is  the  site  of  DNA  and  RNA  synthesis    The   Golgi   is   involved   with   protein  
and   thus   critically   involved   in   the   red   modification  within  the  cell  
cell  development  and  maturation    The  mitochondria  is  also  visible  under  
 Chromatin   contains   genetic   material   electron   microscope   as   rod   shaped  
and  is  composed  of  DNA,  histones,  and   organelles    
other  proteins    They   are   involved   with   aerobic  
 The   chromatin   is   finely   dispersed   and   generation   of   energy   for   the   maturing  
appears  condensed  or  granular   cell   and   insertion   of   ferrous   iron   into  
 The   more   condensed   heterochromatin   protoporphyrin   IX   during   heme  
are  inactive   synthesis  
 The   heterochromatin   take   on   a    Iron   is   present   in   the   cytoplasm   as  
basophilic  color  (dark  blue)  with  basic   ferritin  and  hemosiderin    
dyes    
 The   active   euchromatin   does   not   stain   Maturation  Stages:  
with  basic  dyes    Six   morphological   stages   of  
 As  the  cell  matures  chromatin  becomes   erythrocyte   maturation   may   be  
more   dense   and   metabolic   and   identified  from  a  bone  marrow  sample  
synthetic  activities  start  to  decline   with  Wright  stain  
 Finally   the   nucleus   becomes   inactive    Normal   maturation   is   dependent   on  
and  it  is  extruded  from  the  cell   intake   of   proper   nutrients   and  
 Nucleoli   are   present   in   the   rubriblast   vitamins  such  as  folate,  vitamin  B12  and  
and   they   contain   RNA,   proteins   and   iron  
DNA    Nomenclature:  
 Nucleoli  are  involved  with  synthesis  of    There   are   three   nomenclatures   used   to  
ribosomal  RNA     describe  the  six  stages  
  – Rubri  (proposed  by  the  ASCP)  
Cytoplasm   – Erythroblast   (proposed   by   Paul  
 Ribosomes   and   polyribosomes   are   Ehrlich)  
present   in   the   early   erythrocyte   – Normoblast    (normal  precursor)    
precursor    
 They   are   the   sites   of   globin   and   other   Rubriblast  (Pronormoblast)  
protein  synthesis    This   is   the   earliest   erythrocyte  
precursor   identifiable   by   light  
 microscopy   in   a   Wright   stained   bone    The   cytoplasm   is   polychromatophilic  
marrow  prep   and   more   pinkish   than   that   of   the  
 Cell  size  ranges  from  12  to  25  µm   rubricyte  
 The  nuclear:  cytoplasmic  ratio  is  high    The   nuclear   chromatin   is   dense,   coarse  
 Nucleus   usually   occupies   more   than   and  clumped  
80%  of  cell    The   nucleus   is   degenerated   and  
 The  cytoplasm  stains  basophilic  due  to   pyknotic  
high  RNA  content    The   nucleus   is   extruded   from   the   cell  
 The   Golgi   may   be   visible   near   the   at  this  stage  
nucleus  usually  pale      Sometimes   nucleus   is   not   completely  
  extruded   resulting   in   Howell-­‐Jolly  
Prorubricyte  (Basophilic  normoblast):   body  
 Slightly   smaller   (12   –   17   µm)   than   a    
rubriblast   Reticulocyte   (diffusely   basophilic  
 Nucleus   usually   occupies   75%   of   the   erythrocyte):  
cell    The   reticulocyte   is   slightly   larger   than  
 The   cytoplasm   is   basophilic   and   the   the  mature  erythrocyte  
Golgi  is  usually  visible  near  the  nucleus    The   cytoplasm   still   contains   small  
 The  nucleus  is  round  and  its  chromatin   amounts   of   RNA   which   produces  
is   dark   violet   and   coarser   and   more   varying  amount  of  polychromasia  
clumped    The   reticulocytes   are   retained   in   the  
 The  nucleoli  is  absent  and  helps  in  the   bone   marrow   for   2   to   3   days   before  
identification   release  into  the  marrow  sinisoids  
 The   prorubricyte   usually   divides   two    The  mechanism  of  release  is  unknown  
times  giving  rise  to  four  rubricytes      The   retic   contain   Golgi   apparatus  
  remnant   and   residual   mitochondria  
Rubricyte  (Polychromatophilic  normoblast):   which   permit   continued   aerobic  
 It   is   usually   smaller   than   the   metabolism    
prorubricyte  (12-­‐15  µm)    The   retic   also   contain   RNA   which   may  
 Has   a   round   nucleus   that   may   be   be   stained   supravitally   with   methylene  
eccentric   blue  or  brilliant  cresyl  blue  
 The   nucleus   is   smaller   and   the    The   RNA   precipitates   and   the   retics  
cytoplasm  becomes  more  prominent   can   then   be   counted   and   the  
 There   is   a   spectrum   of   blue   color   due   reticulocyte   production   index  
to  synthesis  of  hemoglobin   determined  
 The   RNA   and   hemoglobin   give   the    
cytoplasm   a   blue   gray   violet   color   Mature  Erythrocyte:  
called   polychromasia   or    The   mature   erythrocyte   is  
polychromatophilia   approximately  7.2  µm  in  diameter  
 The   cell   may   be   confused   with   a    It   is   a   biconcave   disc   and   hence  
lymphocyte   referred  to  as  a  discocyte  
   In  a  Wright  stain,  a  central  pale  area  is  
Metarubricyte  (Orthochromic  normoblast):   revealed  which  fades  gradually  into  the  
 This   is   the   last   nucleated   erythrocyte   reddish  pink  cytoplasm        
stage    
 It  is  slightly  smaller  than  the  rubricyte    
(8-­‐12µm)    
Structure   and   Physiology   of   the   Mature   Lipids:  
Erythrocyte    Phospholipids   and   unesterified  
 The   mature   erythrocyte   lacks   a   cholesterol   predominate   in   the   lipid  
nucleus  or  organelles   fraction  
 Components   necessary   for   function    The   phospholipids   form   the   bilayer  
and  survival  are  present   and  the  hydrophilic  polar  heads  of  the  
 The   cell   has   a   specialized   membrane   phospholipids  are  oriented  toward  the  
that   allows   for   O2   and   CO2   transport   aqueous  environments    
and  for  survival  for  120  days    The   hydrophobic   tails   of   the  
 Various   factors   contribute   to   phospholipids   are   oriented   to   the  
erythrocyte   membrane   and   interior  of  the  bilayer  
hemoglobin  maintenance      The   phospholipids   are   fluid   and   the  
 A  source  of  energy  is  required   fatty  acid  tails  move  freely  
 Membrane   shape   and   deformability    Cholesterol   plays   an   important   role   in  
are  needed       maintaining  surface  area      
   
Shape  and  Deformability:   Protein:  
 The   erythrocyte   is   a   biconcave   disc    Proteins   are   bound   to   lipids  
which   facilitates   O2   and   CO2   transport   throughout  the  membrane  
by   maximizing   ratio   of   surface   area   to    The   proteins   are   either   peripheral  
volume   proteins  or  integral  proteins  
 Allows   the   cell   to   be   flexible   and    The   peripheral   proteins   are   present   on  
deformable   the   inner   portion   of   the   membrane  
 This   allows   the   cell   to   adjust   to   small   nearest  the  cytoplasm  
vessels   in   the   microvasculature   and    The   integral   proteins   are   in   contact  
still  maintain  a  constant  surface  area   with   both   the   inner   and   the   outer  
 A   less   deformable   cell   would   be   surface  of  the  membrane  
subjected  to  fragmentation    The   integral   proteins   act   as   receptors  
  for   ions   and   molecules   needed   in   the  
Membrane  Composition  and  Structure:   cell  such  as  transferrin  and  EPO          
 The   composition   of   the   membrane    The   peripheral   proteins   include   the   α  
allows  the  cell  to   and   β   spectrin   also   called   band   1   and   2  
– Separate   the   intracellular   fluid   and  actin  
environment   of   the   cytoplasm    The   proteins   form   the   cytoskeleton   of  
from   the   extracellular   fluid   the   cell   and   regulate   membrane   shape  
environment   and  deformability  
– Selectively  pass  nutrients  and  ions    Their   linkage   is   mediated   by   protein  
into  and  out  of  cell     4.1  
– Deform  when  required    The   principal   integral   proteins   are  
 The   membrane   is   composed   of   lipids   glycoproteins   designated   glycophorin  
and   proteins   in   approximately   equal   A  and  band  3  
proportions  by  weight    They  span  the  lipid  bilayer.  
 The   difference   in   the   lipids   and    Band  3  is  an  inorganic  anion  transport  
proteins   in   the   cytoplasmic   side   and   channel    
the   plasma   side   allow   for   selective    Integral   proteins   contain   sialic   acid  
movement   of   molecules   in   and   out   of   which   gives   erythrocytes   a   negative  
the  cell   charge    
 
  The   negativity   between   cells   called    ATP   is   needed   to   maintain   membrane  
zeta   potential   cause   cells   to   repel   one   shape  and  deformability    
another   as   they   move   through   the   - Through   phosphorylation   of  
circulation   spectrin  and  calcium  chelation  
 Membrane   proteins   facilitate   - Provide  energy  for  active  transport  
movement   of   substrates   and   cofactors   of  cations  
in  and  out  cell   - And   to   modulate   the   amount   of   2,3  
 Examples  include  the  Na+,  K+  -­‐  ATPase   DPG  generated  
and  Ca2+,  Mg2+  -­‐  ATPase    There   is   a   net   yield   of   two   ATP  
 Calcium   is   involved   in   regulation   of   molecules   per   molecule   of   glucose  
and   stabilization   of   membrane   catabolized  
phospholipid  structure    2,3   DPG   is   formed   from   the   Rapoport-­‐
 High   intracellular   concentration   of   Luebering  shunt  
calcium,  cause  cell  deformability    Helps  modulate  O2  transport  in  the  cell      
   Hexose   Monophosphate   Shunt   and  
Energy  Metabolism:   Glutathione  Reduction  pathway:  
 The   cell   requires   energy   for   cell    Also   called   the   pentose   phosphate  
metabolism   and   to   preserve   the   pathway   is   an   aerobic   method   of  
membrane  integrity   erythrocyte  glycolysis  
 Various  enzymatic  reactions  in  the  cell    Processes   about   10%   of   erythrocyte  
require  energy   glucose  
 Energy   is   required   to   reduce   proteins    Purpose   is   to   provide   reducing  
and   maintain   hemoglobin   in   its   potential   by   generating   reduced  
reduced  state  for  proper  functioning     nicotinamide   adenine   dinucleotide  
 Two   site   prone   to   oxidation   are   the   phosphate  (NADPH)  
iron   atom   in   the   heme   ring   and   the    It  is  an  oxidative  pathway      
sulfhydryl   groups   on   the   globin    As   the   red   cell   ages,   changes   occur   that  
molecule   make  it  susceptible  to  destruction  
 Oxidation   of   the   normal   ferrous   state    Alteration   in   the   membrane   integrity  
to   the   ferric   state   results   in   takes  place    
methemoglobin   which   does   not   deliver    Loss   of   sialic   acid   and   lipids,   decreased  
oxygen     ATP   and   increased   Calcium   have   been  
 Normally   1%   to   3%   of   oxygen   is   implicated  in  the  aging  process  
oxidized  to  methemoglobin      At   120   days   the   erythrocytes   are  
 Oxidation   of   sulfhydryl   groups   causes   recognized   as   abnormal   and   are  
hemoglobin   precipitation   (Heinz   body   removed  by  phagocytic  cell  in  the  RES  
formation)    As  the  cell  ages  it  is  depleted  of  glucose  
 Sources  of  Energy:   and  their  surface  area  decreases  
 The   Embden-­‐Meyerhof   Pathway    The   spleen   recognizes   abnormalities   in  
(EMP):   the  cell  and  sequester  it  for  removal      
 This  is  an  anaerobic  process  for  energy    
generation  through  glucose  catabolism   Regulation  of  Erythropoiesis  
to  lactate    A   balance   between   production   and  
 About  90%  to  95%  of  glucose  used  by   destruction   keeps   the   erythrocyte  
the  cells  is  metabolized  by  the  EMP       number  constant  
 ATP  is  generated  during  the  glycolysis  
of  glucose  to  lactate  
  Production   of   erythrocyte   requires   a  
normal   functioning   competent   bone  
marrow    
 Adequate  levels  of  EPO,  growth  factors  
and   nutrients   such   as   iron,   folate,  
vitamin  B12  
 Between   3   X   109   and   8.5   X   109  
erythrocytes  are  produced  daily  
 Cytokines   or   growth   factors   play   a  
important  part  in  the  process    
 
Erythropoietin  Production  and  Regulation:  
 EPO   is   a   glycoprotein   hormone   with   a  
molecular  weight  of  34,000  
 It  is  an  erythroid  growth  factor  
 It  is  produced  in  the  kidney  
 It   is   regulated   by   renal   O2   tension  
which   when   decreased   induces  
expression   of   the   EPO   gene   and   the  
release  of  EPO  
 Prostaglandins   help   regulate   EPO  
production  
 
Growth  Factors:  
• Many   other   hormones   and   cytokines  
secreted   by   various   cells   have   been  
found  to  stimulate  erythropoiesis  
• These   cytokines   have   been   identified  
in  cell  cultures  and  they  include  
– EPO  
– Insulin  
– Growth  hormone  
– Steroid  hormone  
– Nonandrogenic  thyroid  hormone  
– IL-­‐1,  IL-­‐4,  IL-­‐6,  IL-­‐7,  IL-­‐11,  IL-­‐12,    
– G-­‐CSF  
– Macrophage   inflammatory   protein    
(MIP)  and  steel  factor  (SF)