Haemotology Week 2 - Erythropoiesis and RBC Indices

RBC Indices

• RBC indices are used to define the size and Hb content of individual RBCs in a blood sample (along with blood film morphology) and to classify different types of anaemia.
• The 3 indices are:
  • Mean Cell Volume (MCV)
  • Mean Cell Hb (MCH)
  • Mean Cell Hb Concentration (MCHC)
• Core equations (as given in lecture):
  • $MCV = \frac{PCV}{RCC}$
  • $MCH = \frac{Hb}{RCC}$
  • $MCHC = \frac{Hb}{PCV}$
• Additional indices introduced by instruments:
  • RDW = Red cell distribution width (variation in cell volume / anisocytosis)
  • HDW = Haemoglobin distribution width (variation in Hb content)
• Purpose of indices: facilitate classification of anaemias when considered with red cell morphology.

Mean Cell Volume (MCV)

• Definition: average volume of Hb-containing RBCs.
• Clinical interpretation (size of RBCs):
  • Normocytic: MCV within reference range (~80–97 fL).
  • Microcytic: MCV < 80 fL.
  • Macrocytic: MCV > 97 fL.
• Example: If PCV = 0.45 L/L and RCC = 5.0 × 10^12/L, then
  • $MCV = \frac{0.45}{5.0\times10^{12}/\text{L}} = 90\text{ fL}$

Mean Cell Hb (MCH)

• Definition: average Hb content per RBC.
• Indicates the amount of Hb per cell; should correlate with MCV and MCHC.
• Example: If Hb = 150 g/L and RCC = 5.0 × 10^12/L,
  • $MCH = \frac{150}{5.0\times10^{12}} = 30\text{ pg}$

Mean Cell Hb Concentration (MCHC)

• Definition: average Hb concentration within RBCs; ratio of Hb mass to RBC volume.
• Indication of Hb density per RBC (not Hb per cell).
• Reference interpretation:
  • Hypochromic: MCHC < 320 g/L
  • Normochromic: MCHC 320–360 g/L
  • Hyperchromic: MCHC > 360 g/L
• Example: If Hb = 150 g/L and PCV = 0.45 L/L,
  • $MCHC = \frac{150}{0.45} = 333.3\,\text{g/L}$

Additional RBC indices

• RDW: quantitative measure of variation in RBC volume (anisocytosis) from the automated analyzer.
• HDW: Haemoglobin distribution width; alternative measure of Hb content variation.
• These indices help support classification of anaemias in conjunction with morphology.

Normal RBC Indices (reference ranges)

• Male: Hb 130–180 g/L; Hct 40–54%; RCC 4.5–6.5 × 10^12/L; MCH 27–32 pg; MCV 80–97 fL; MCHC 320–360 g/L; Retic count 25–85 × 10^9/L (0.2–2%)
• Female: Hb 115–165 g/L; Hct 37–47%; RCC 3.8–5.8 × 10^12/L; MCH 27–32 pg; MCV 80–97 fL; MCHC 320–360 g/L; Retic count 25–85 × 10^9/L (0.2–2%)
• Notes:
  • Reticulocyte absolute count is typically in the range ~25–85 × 10^9/L, with a relative percentage of ~0.2–2%.

Morphology and Blood Films

• Blood films (peripheral blood smear) are used to evaluate cell morphology and identify abnormalities.
• Preparation and staining methods include Wright’s, Romanowsky, May-Grünwald, and Giemsa stains.
• Practical cues for slide quality:
  • Film too thin: tail appears uneven; RBCs separated; poor field of view.
  • Film too thick: tail appears thick; RBCs stacked; WBCs poorly separated.
  • Ideal field: RBCs spaced with central pallor visible.

Blood Film Features (morphology cues)

• Normal RBCs: biconcave, uniform size with central pallor.
• Anisocytosis: variation in RBC size.
• Poikilocytosis: variation in RBC shape.
• Polychromasia: variation in Hb content (color) due to reticulocytes or reticulocyte-like cells.
• Reticulocytes on smears are identified with supravital stain revealing a mesh-like RNA network.

RBC Maturation and Erythropoiesis

• Erythropoiesis (EP): production of RBCs; initiated by erythropoietin (EPO).
• Normal production rate: ~10^12 new RBCs per day; can be accelerated under hypoxia or anaemia.
• EPO physiology:
  • Secreted by kidneys in response to hypoxia.
  • Stimulates progenitor cells (late BFU-E and CFU-E) to speed up maturation from pronormoblast to normoblast to reticulocyte to RBC.
• Stages of maturation:
  • Pluripotent stem cell → BFU-E → CFU-E → pronormoblast → normoblast → reticulocyte → mature RBC
• Key regulatory factors for EPO production:
  • Enhanced oxygen retention by Hb
  • Low atmospheric oxygen
  • Anaemia
  • Structural defects in Hb (reduced O2 carrying capacity)
  • Cardiac or lung defects with compromised renal circulation

EPO Clinical Relevance

• Plasma EPO levels help differentiate polycythemia etiologies:
  • High EPO: secondary polycythemia
  • Low EPO: polycythemia vera (primary)
  • Normal EPO in some anaemias (kidney disease-other conditions)
• EPO is used therapeutically for anemia due to:
  • Renal disease or inflammatory bowel disease
  • Myelodysplasia after cancer therapy

Erythropoiesis Timeline (from kidney to RBC)

• EPO stimulates progenitors to speed maturation: pronormoblast → normoblast → reticulocyte → RBC
• Visual progression (from Hoffbrand): pronormoblast → normoblast → reticulocyte → mature RBC
• The amplification/maturation sequence can be summarized as:
  • pluripotent stem cell → late BFU-E → CFU-E → pronormoblast → normoblast → reticulocyte → mature RBC

Erythroblast and Normoblast Stages

• Erythroblasts (normoblasts) display various stages of development, as shown in standard diagrams.
• First recognizable RBC lineage cell: pronormoblast.
• In bone marrow, DNA is present in nucleus; in mature RBCs there is no nucleus.
• Reticulocytes are immature RBCs released after 2–3 days in bone marrow, maturing to RBCs within ~24 hours in circulation.

Reticulocytes

• Definition: immature RBCs; lack a nucleus but contain cytoplasmic RNA.
• Reticular (mesh-like) network of rRNA is visible with supravital staining.
• Origin: produced in bone marrow; released into circulation after 2–3 days in marrow; mature in ~24 hours.
• Normal ranges:
  • Absolute reticulocytes: ~50–150 × 10^9/L
  • Relative reticulocytes: ~1–3%

Reticulocyte Counts (diagnostic utility)

• Reticulocyte count reflects the amount of effective erythropoiesis.
• Clinical use:
  • Monitor treatment effectiveness in anaemia (iron or vitamin deficiency tends to increase retics if responding).
• Methods:
  • Manual reticulocyte counts using supravital stains (e.g., methylene blue, brilliant cresyl blue) that bind RNA to form blue precipitates.
  • Count retics per 1000 RBCs; report as % retics and absolute count.
  • Manual counting error ~±25%; automated counts error ~±0.1%.
• Significance of reticulocyte trends:
  • Increased retics: acute or chronic blood loss; haemolytic anaemia; sideroblastic anaemia; thalassemia; response to deficiency treatment.
  • Decreased retics: aplastic anaemia; myelofibrosis; BM suppression.

Supravital Staining (for reticulocytes)

• Stains used: new methylene blue, brilliant cresyl blue.
• Stains react with RNA to form visible blue filaments/granules.

RBC Abnormalities and Morphology

• Abnormal EP or Hb synthesis can cause visible changes on blood films.
• Common suffixes:
  • Anisocytosis: variation in cell size.
  • Poikilocytosis: variation in shape.
  • Chromasia/colour variation: polychromasia indicates variation in Hb content.
• Common abnormal morphologies include:
  • Spherocytes, elliptocytes, osteoclast-like cells (occasional historical terms), stomatocytes, acanthocytes, echinocytes, burr cells, target cells, teardrop cells, pencil cells, microcytes, macrocytes, macrocytic cells, hypochromic cells, hyperchromic cells.
  • Sickle cells, schistocytes (fragmented cells), burr cells, bite cells, basophilic stippling, Heinz bodies, Howell-Jolly bodies, Cabot rings, malaria parasites.
• Inclusions and fragments:
  • Howell-Jolly bodies (nuclear DNA remnants in RBCs)
  • Basophilic stippling
  • Siderotic granules (Pappenheimer bodies)
  • Heinz bodies (Hb inclusions)
  • Nucleated RBCs (In-vivo circulating NRBCs)

Normal vs Abnormal RBCs (quick reference)

• Normal RBC appearance vs various abnormalities (macrocytes, microcytes, target cells, stomatocytes, elliptocytes, sickle cells, acanthocytes, echinocytes, schistocytes, bite cells, Howell-Jolly bodies, basophilic stippling, etc.).
• Examples and associations are provided in Hoffbrand and related texts; refer to Fig. 2.16 in Hoffbrand’s Essential Haematology for visual correlations.

RBC Membrane and Metabolism

• RBC membrane: lipid bilayer with a membrane skeleton of integral proteins and surface antigens.
• Function of membrane: maintain biconcave shape, deformability, and integrity under shear stress.
• RBC metabolism is essential to maintain shape, osmotic stability, and redox balance.
  • ATP production via glycolysis (Embden-Meyerhoff pathway) maintains membrane integrity and ion pumps.
  • NADPH production via the hexose monophosphate shunt (pentose phosphate pathway) maintains glutathione in its reduced form (GSH), protecting Hb and proteins from oxidative damage.
  • Methaemoglobin reductase pathway reduces methaemoglobin back to functional Hb.
  • Rapoport-Luebering pathway modulates 2,3-DPG, which regulates Hb-O2 affinity.

Key Pathways in RBC Metabolism ( Glycolysis and Related Shunts )

• Embden-Meyerhoff glycolytic pathway: glucose → lactate with net production of ATP and NADH.
• 2,3-Bisphosphoglycerate (2,3-DPG) through the Luebering-Rapoport shunt regulates Hb oxygen affinity.
• Methaemoglobin reductase pathway reduces methaemoglobin to Hb.
• Hexose-monophosphate shunt (HMP shunt, pentose phosphate pathway): generates NADPH for maintaining intracellular reducing potential; crucial for protection against oxidative damage.
• Key enzyme for NADPH generation: Glucose-6-phosphate dehydrogenase (G6PD).
• Net effects: ATP supports cell shape/osmotic stability; NADPH supports antioxidant defense via glutathione system.

Figure reference (conceptual details)

• The Embden-Meyerhoff pathway converts glucose to lactate, producing 2 ATP per glucose molecule and generating NADH.
• The Luebering-Rapoport shunt regulates 2,3-DPG concentration and thereby Hb-O2 affinity.
• G6PD is the rate-limiting enzyme of the HMP shunt, generating NADPH to maintain glutathione (GSH/GSSG) balance.

Erythropoiesis and Developmental Stages (Detailed)

• Erythropoiesis initiation and regulation:
  • Hormonal control by erythropoietin (EPO) from kidneys in response to hypoxia.
  • Continuously produces ~10^12 RBCs daily; can be accelerated when needed.
  • Maturation requires metals, vitamins, amino acids, and hormones.
• Maturation sequence (amplification and maturation from pluripotent stem cells):
  • Pluripotent stem cell → BFU-E → CFU-E → pronormoblast → normoblast → reticulocyte → mature RBC.
• Erythropoietin (EPO) specifics:
  • Stimulated by hypoxia, enhanced Hb O2 retention, low atmospheric O2, anaemia, Hb defects, cardiac or lung abnormalities.
  • Kidney-derived EPO speeds up maturation by acting on late BFU-E and CFU-E progenitors.
• Clinical relevance of EPO:
  • Diagnosis: plasma EPO levels help differentiate normal vs abnormal erythropoiesis (e.g., high in secondary polycythemia, low in polycythemia vera).
  • Therapeutic use: treat anemia of renal disease or inflammatory bowel disease; adjunct in myelodysplasia after cancer therapy.

Maturation Stages (in detail)

• Stages include pronormoblasts, erythroblasts (normoblasts), reticulocytes, and mature RBCs.
• The progression is diagrammed in Hoffbrand’s texts (e.g., Fig. 2.3 and Fig. 2.5): amplification from pronormoblast to mature RBC via multiple divisions and cytoplasmic/nuclear maturation.
• Nuclear changes: DNA present in nucleus during early stages; gradually lost as cells mature into reticulocytes and RBCs.
• In BM vs peripheral blood:
  • In BM: DNA present in nucleus; RNA in cytoplasm.
  • In blood: No nuclear DNA; RNA remnants may be present in reticulocytes observed with supravital stains.

Reticulocytes: Practical and Diagnostic Aspects

• Reticulocytes are immature RBCs with cytoplasmic RNA but no nucleus.
• Supravital staining reveals a reticular network of RNA (mesh-like).
• Production and maturation timeline:
  • Produced in BM; released after 2–3 days; mature within ~24 hours in circulation.
• Normal ranges (summary):
  • Absolute: 50–150 × 10^9/L
  • Relative: 1–3%
• Diagnostic value:
  • Retic counts reflect effective erythropoiesis and are used to monitor therapy in deficiency anaemias.
• Practical reticulocyte counting:
  • Manual: supravital stains (e.g., methylene blue) with counts per 1000 RBCs; results reported as % retics and absolute count; manual error ~±25%.
  • Automated: smaller error margin (~±0.1%), but depends on analyzer performance.

RBC Abnormalities: Classification and Examples

• Abnormal erythropoiesis or Hb synthesis can cause a range of RBC morphologies:
  • Anisocytosis, poikilocytosis, microcytosis, macrocytosis, elliptocytosis, ovalocytosis, spherocytosis, sickle cells, schistocytes, helmet cells, target cells, burr cells, teardrop cells, stomatocytosis.
  • Hypochromasia and anisochromasia indicate variation in Hb content.
  • Polychromasia indicates mixed Hb content (often due to reticulocytes).
• RBC inclusions and fragments:
  • Howell-Jolly bodies, cabot rings, basophilic stippling, iron inclusions, Heinz bodies, siderotic (Pappenheimer) bodies, malaria parasites in RBCs.
• Selected examples and associations (illustrative):
  • Target cells: iron deficiency, liver disease, post-splenectomy, haemoglobinopathies.
  • Stomatocytes: liver disease, alcoholism.
  • Acanthocytes: liver disease, abetalipoproteinemia, renal failure.
  • Elliptocytes/Ovalocytes: hereditary elliptocytosis or megaloblastic processes.
  • Spherocytes: hereditary spherocytosis, autoimmune haemolytic anaemia, sepsis.
  • Sickle cells: SCD.
  • Teardrop cells: myelofibrosis with extramedullary haemopoiesis.
  • Basophilic stippling and Howell-Jolly bodies as inclusions in various anemias.

Clinical and Laboratory Implications

• How to interpret results in context:
  • Combine RBC indices (MCV, MCH, MCHC) with blood film morphology to categorize anaemias (e.g., microcytic hypochromic vs macrocytic normochromic).
  • Reticulocyte counts help distinguish between underproduction vs loss/destruction anemias.
  • EPO levels aid in differentiating polycythemia etiologies and guiding therapy.
• Practical diagnostic flow:
  • Step 1: Full Blood Count (FBC) and differential WBC count.
  • Step 2: Blood film review for morphology.
  • Step 3: Reticulocyte count to gauge erythropoietic activity.
  • Step 4: Consider bone marrow assessment (aspirate/trephine) if indicated.

Bone Marrow Assessment (Brief)

• Bone marrow aspiration and trephine biopsy provide histology for hematopoietic activity and cellularity.
• Useful when peripheral blood findings are inconclusive or when marrow pathology (e.g., aplastic anaemia, myelofibrosis) is suspected.
• Images and figures in Hoffbrand’s text illustrate marrow aspirates and cellular morphology (not reproduced here).

Quick Review Prompts (from Lecture 2)

• Erythropoiesis is regulated by the hormone , secreted mainly by the in response to _.
• The stages of RBC maturation: pluripotent stem cell → late BFU-E → CFU-E → _ → normoblast → _ → mature RBC.
• The major glycolytic pathway by which RBC generates ATP and NADH is the .
• Reticulocytes can be manually counted after treating the blood sample with ___ such as methylene blue.
• Anisocytosis refers to RBC variations in , _ refers to variations in shape. Pale staining RBCs are described as ___.
• RBC indices are used to define the _ and ____ content of RBCs. The 3 indices are: , , and _.
• Equations for MCV = , MCH=, MCHC=__

A Question to Ponder

• Can we tell if someone has ineffective erythropoiesis?

Peripheral content (not core exam material)

• The transcript contains a news/feature section about MicroToons and Dr. Rina Fu related to outreach in STEM education. This material is not central to hematology exam content.
• If you are studying for an exam, focus primarily on the hematology-specific slides above; peripheral content can be skimmed or ignored for test preparation.