ERYTHROCYTES AND HEMOGLOBIN
PART A: ERYTHROCYTES
4.1 Definition of Erythrocytes
Erythrocytes, commonly known as red blood cells (RBCs), are specialized blood cells responsible primarily for transporting oxygen from the lungs to body tissues and participating in carbon dioxide transport from tissues to the lungs.
Mature human erythrocytes have a distinctive biconcave disc shape. They lack a nucleus and most intracellular organelles, providing more space for hemoglobin and enabling considerable cellular deformability.
General characteristics of mature erythrocytes
| Characteristic | Description |
|---|---|
| Shape | Biconcave disc |
| Diameter | Approximately 7–8 µm |
| Thickness | Approximately 2–2.5 µm at the periphery and about 1 µm centrally |
| Nucleus | Absent |
| Mitochondria | Absent |
| Main intracellular protein | Hemoglobin |
| Energy production | Primarily anaerobic glycolysis |
| Average lifespan | Approximately 120 days |
| Main site of production | Red bone marrow |
| Main site of removal | Macrophages in the spleen, liver, and bone marrow |
Why are erythrocytes biconcave?
The biconcave shape provides several advantages:
- Increased surface-area-to-volume ratio: Facilitates rapid gas exchange.
- Short diffusion distance: Allows efficient oxygen and carbon dioxide diffusion.
- Deformability: Enables passage through narrow capillaries and splenic microcirculation.
- Efficient hemoglobin distribution: Supports oxygen transport throughout the circulation.
The RBC membrane consists of a lipid bilayer, integral membrane proteins, and a cytoskeletal network that maintains shape and flexibility.
4.2 Functions of Erythrocytes
4.2.1 Oxygen transport
The principal function of erythrocytes is oxygen transport.
Oxygen enters the blood in the pulmonary capillaries and binds reversibly to hemoglobin.
In the lungs, oxygen binds to hemoglobin. In peripheral tissues, oxygen is released according to local oxygen tension and metabolic requirements.
4.2.2 Carbon dioxide transport
Erythrocytes participate in transporting carbon dioxide from tissues to the lungs through three main mechanisms:
- Dissolved carbon dioxide in plasma
- Carbon dioxide bound to globin as carbaminohemoglobin
- Bicarbonate ions formed through carbon dioxide hydration
Most carbon dioxide is transported in blood as bicarbonate.
4.3 Erythropoiesis
Definition
Erythropoiesis is the process by which erythrocytes are produced and mature from hematopoietic stem cells.
In healthy adults, erythropoiesis occurs predominantly in the red bone marrow.
It is regulated mainly by tissue oxygen availability, erythropoietin, the bone marrow microenvironment, and the availability of essential nutrients.
Sites of erythropoiesis
The principal sites change during development:
| Developmental period | Major site |
|---|---|
| Early embryonic period | Yolk sac |
| Fetal period | Predominantly fetal liver |
| Later fetal development | Increasing contribution from bone marrow |
| After birth | Bone marrow |
4.3.1 Stages of erythropoiesis
The developmental sequence is:
Hematopoietic stem cell
↓
Multipotent progenitor
↓
Myeloid progenitor
↓
Megakaryocyte-erythroid progenitor
↓
BFU-E
↓
CFU-E
↓
Proerythroblast
↓
Basophilic erythroblast
↓
Polychromatophilic erythroblast
↓
Orthochromatic erythroblast
↓
Reticulocyte
↓
Mature erythrocyteBFU-E means burst-forming unit-erythroid, while CFU-E means colony-forming unit-erythroid. These are erythroid progenitor populations, not mature blood cells.
The proerythroblast is the first conventionally recognizable erythroid precursor on routine morphological examination.
4.3.2 Regulation by erythropoietin
Erythropoietin (EPO) is the principal hormonal regulator of erythropoiesis.
It is produced predominantly by specialized interstitial cells in the kidneys in response to reduced tissue oxygen availability.
Mechanism
Reduced tissue oxygen availability
↓
Renal oxygen sensing
↓
Increased EPO production
↓
EPO enters circulation
↓
Stimulation of erythroid progenitors
↓
Increased survival and proliferation
↓
Increased RBC production
↓
Increased oxygen-carrying capacityEPO acts particularly on erythroid progenitors, promoting their survival, proliferation, and differentiation.
Clinical significance
Reduced renal EPO production is an important mechanism contributing to anemia in chronic kidney disease.
Increased EPO production may occur in response to hypoxia, such as at high altitude or in some chronic hypoxemic conditions.
4.3.3 Nutritional requirements for erythropoiesis
Normal erythropoiesis requires adequate nutrients.
| Nutrient | Function | Effect of deficiency |
|---|---|---|
| Iron | Heme synthesis | Iron-deficiency anemia, commonly microcytic and hypochromic |
| Vitamin B12 | DNA synthesis | Megaloblastic anemia |
| Folate | DNA synthesis | Megaloblastic anemia |
| Vitamin B6 | Heme synthesis | May contribute to sideroblastic anemia |
| Protein/amino acids | Globin synthesis | Impaired hemoglobin production |
| Copper | Iron metabolism and transport | May contribute to anemia |
Iron is necessary for heme synthesis, whereas vitamin B12 and folate are essential for normal DNA synthesis and nuclear maturation.
4.3.4 Other regulatory factors
Erythropoiesis is also influenced by:
- Bone marrow stromal cells
- Cytokines and growth factors
- Inflammatory mediators
- Renal function
- Hormonal status
- Iron availability
- Hepcidin-mediated iron regulation
Inflammation can impair erythropoiesis by suppressing erythroid activity, altering iron availability, and reducing the effectiveness of EPO.
4.4 Stages of Erythrocyte Maturation
4.4.1 Proerythroblast
The proerythroblast is the earliest routinely recognizable erythroid precursor in the bone marrow.
Morphological characteristics:
- Large cell, approximately 15–20 µm.
- Large, round nucleus.
- Fine chromatin.
- One or more prominent nucleoli.
- Deeply basophilic cytoplasm.
- High nuclear-to-cytoplasmic ratio.
The blue cytoplasm results from abundant ribosomal RNA required for protein synthesis.
4.4.2 Basophilic erythroblast
This stage is also called the early normoblast.
Morphological characteristics:
- Smaller than the proerythroblast.
- Round nucleus.
- Chromatin begins to condense.
- Nucleoli are generally no longer visible.
- Deeply basophilic cytoplasm.
- Continued cellular proliferation and hemoglobin synthesis.
4.4.3 Polychromatophilic erythroblast
This stage is also called the intermediate normoblast.
Morphological characteristics:
- Further reduction in cell size.
- Increasingly condensed nuclear chromatin.
- Gray-blue or slate-colored cytoplasm.
- Increasing hemoglobin content.
4.4.4 Orthochromatic erythroblast
This stage is also known as the late normoblast.
- Smaller cell.
- Very condensed, dark nucleus.
- Pink cytoplasm due to increased hemoglobin.
4.4.5 Reticulocyte
A reticulocyte is an immature, anucleate erythrocyte that contains residual ribosomal RNA.
- No nucleus.
- Slightly larger than a mature erythrocyte.
- May appear bluish-gray on a routine Romanowsky-stained blood film.
- Residual RNA can be demonstrated using supravital stains.
Common supravital stains include new methylene blue and brilliant cresyl blue.
Reticulocytes complete their maturation in the circulation, generally over approximately one to two days.
4.4.6 Mature erythrocyte
The mature erythrocyte is a biconcave, anucleate cell containing a high concentration of hemoglobin.
Its lack of a nucleus and mitochondria supports efficient oxygen transport and allows it to rely primarily on anaerobic glycolysis for ATP production.
General changes during erythrocyte maturation
Cell size ↓
Nuclear size ↓
Chromatin condensation ↑
Hemoglobin content ↑
Ribosomal RNA ↓
Nucleus → expelled
Cellular organelles → progressively lostLaboratory significance: Premature release of immature erythroid cells or nucleated RBCs into peripheral blood may occur in severe anemia.
PART B: HEMOGLOBIN
4.6 Definition of Hemoglobin
Hemoglobin (Hb) is an iron-containing conjugated protein present in erythrocytes. Its principal function is to transport oxygen from the lungs to peripheral tissues.
Hemoglobin also contributes to carbon dioxide transport and acid–base regulation.
Hemoglobin consists of two major components:
- Globin: The protein component.
- Heme: The iron-containing, non-protein prosthetic group.
The iron within functional heme is in the ferrous state (Fe²⁺), which permits reversible oxygen binding.
Importance of hemoglobin
Hemoglobin concentration is a major laboratory parameter for evaluating anemia and erythrocytosis. A reduced hemoglobin concentration decreases the oxygen-carrying capacity of blood.
4.7 Hemoglobin Terminology
Several terms describe different forms of hemoglobin.
4.7.1 Oxyhemoglobin
Oxyhemoglobin forms when oxygen binds reversibly to ferrous heme iron. Oxyhemoglobin is the principal oxygen-carrying form in arterial blood.
4.7.2 Deoxyhemoglobin
Deoxyhemoglobin is hemoglobin without bound oxygen. It is present in venous blood and in tissues where oxygen has been released.
4.7.3 Carbaminohemoglobin
Carbaminohemoglobin forms when carbon dioxide binds to amino groups on the globin portion of hemoglobin. It contributes to carbon dioxide transport from tissues to the lungs.
4.8 Chemistry and Structure of Hemoglobin
4.8.1 Molecular structure
Normal adult hemoglobin A (HbA) is a tetramer composed of four globin chains:
It contains:
- Two alpha-globin chains
- Two beta-globin chains
- Four heme groups
- Four ferrous iron atoms
Each globin chain is associated with one heme group.
4.8.2 Heme structure
Heme consists of protoporphyrin IX combined with ferrous iron (Fe²⁺). The iron atom is coordinated within the porphyrin ring and participates in reversible oxygen binding. The heme group is essential for the oxygen-transport function of hemoglobin.
4.8.3 Globin chains
Globin chains are polypeptides encoded by globin genes. Different combinations of globin chains produce different hemoglobin types.
| Hemoglobin type | Globin-chain composition | Significance |
|---|---|---|
| HbA | α₂β₂ | Major adult hemoglobin |
| HbA₂ | α₂δ₂ | Minor adult hemoglobin |
| HbF | α₂γ₂ | Major fetal hemoglobin |
HbF has a higher oxygen affinity than HbA under physiological conditions. This property facilitates oxygen transfer from maternal to fetal blood across the placenta. In healthy adults, HbA predominates, while HbA₂ and HbF occur in smaller proportions. Their exact proportions vary with age and clinical conditions.
4.8.4 Oxygen binding and release
Hemoglobin exhibits cooperative oxygen binding.
When one oxygen molecule binds, the affinity of the remaining binding sites for oxygen increases. This produces the characteristic sigmoidal oxygen–hemoglobin dissociation curve.
Oxygen release is influenced by:
- Oxygen partial pressure
- Carbon dioxide partial pressure
- Blood pH
- Temperature
- 2,3-bisphosphoglycerate (2,3-BPG)
Increased temperature, increased carbon dioxide, decreased pH, and increased 2,3-BPG generally shift the dissociation curve to the right, facilitating oxygen release to tissues.
4.9 Hemoglobin Metabolism
Hemoglobin metabolism includes two related processes:
- Hemoglobin synthesis
- Hemoglobin degradation and recycling
4.9.1 Hemoglobin synthesis
Hemoglobin synthesis occurs primarily in developing erythroid cells in the bone marrow. It requires the coordinated production of heme and globin.
Heme synthesis
Heme synthesis involves a series of enzymatic reactions beginning with glycine and succinyl-CoA. The pathway occurs partly in mitochondria and partly in the cytoplasm.
A simplified sequence is:
Glycine + Succinyl-CoA
↓
δ-ALA formation
↓
Porphyrin pathway
↓
Protoporphyrin IX
↓
Addition of Fe²⁺
↓
HemeThe enzyme ferrochelatase catalyzes the insertion of ferrous iron into protoporphyrin IX during the final step of heme synthesis.
Globin synthesis
Globin chains are synthesized on ribosomes in developing erythroid cells. The alpha and beta chains subsequently assemble with heme to form functional hemoglobin.
Requirements for normal synthesis
- Iron for heme
- Amino acids for globin
- Functional globin genes
- Enzymes required for heme synthesis
- Adequate erythroid cell development
Defects in globin-chain synthesis can produce thalassemia, whereas structural changes in globin chains can produce abnormal hemoglobin variants.
4.9.2 Hemoglobin degradation
Mature erythrocytes circulate for approximately 120 days. As they age, changes in membrane integrity and cellular metabolism make them more susceptible to removal by macrophages. Senescent RBCs are removed primarily by macrophages in the:
- Spleen
- Liver
- Bone marrow
Hemoglobin is broken down into globin and heme.
Globin degradation
Globin is broken down into amino acids, which can be reused for protein synthesis.
Heme degradation
Heme is separated into iron and porphyrin-derived components.
Iron is recycled, while the porphyrin ring is converted into bile pigments.
Diagram: Hemoglobin degradation
Old erythrocyte
↓
Hemoglobin
↓
┌──────┴──────┐
↓ ↓
Globin Heme
↓ ↓
Amino acids ┌───┴────┐
↓ ↓ ↓
Recycled Iron Porphyrin
↓ ↓
Recycled Biliverdin
↓
Bilirubin
↓
Albumin transport
↓
Liver
↓
Conjugated bilirubin
↓
Bile
↓
Intestine
↓
Urobilinogen
┌─────┴─────┐
↓ ↓
Stercobilin Reabsorption/
in feces urinary excretionBilirubin metabolism
- Macrophages degrade heme and generate biliverdin.
- Biliverdin is converted into unconjugated bilirubin.
- Unconjugated bilirubin circulates bound to albumin.
- Hepatocytes take up bilirubin and conjugate it, primarily with glucuronic acid.
- Conjugated bilirubin is secreted into bile.
- Intestinal bacteria convert bilirubin-derived compounds into urobilinogen and related metabolites.
- Stercobilin contributes to the brown color of feces, while urinary urobilin contributes to the yellow color of urine.
Iron recycling
Iron released from heme is conserved and reused.
It may be:
- Stored in macrophages as ferritin or hemosiderin.
- Transported by transferrin.
- Delivered to the bone marrow for new hemoglobin synthesis.
Clinical significance: Increased hemoglobin breakdown may raise unconjugated bilirubin and contribute to jaundice. Reduced iron availability can impair hemoglobin synthesis and lead to iron-deficiency anemia.
4.10 Hemoglobin Compounds
Hemoglobin can exist in several forms depending on the substances bound to it and the oxidation state of its iron.
These forms are important in physiology, clinical biochemistry, and laboratory diagnosis.
4.10.1 Physiological and abnormal hemoglobin derivatives
| Hemoglobin compound | Formation or defining feature | Clinical significance |
|---|---|---|
| Oxyhemoglobin | Oxygen binds reversibly to heme iron | Normal oxygen transport |
| Deoxyhemoglobin | Oxygen is released from hemoglobin | Normal physiological form in tissues |
| Carbaminohemoglobin | CO₂ binds to globin | Contributes to CO₂ transport |
| Carboxyhemoglobin | Carbon monoxide binds to heme iron | Increased in carbon monoxide exposure |
| Methemoglobin | Heme iron is oxidized from Fe²⁺ to Fe³⁺ | Cannot bind oxygen normally |
| Sulfhemoglobin | Sulfur becomes incorporated into the heme-related structure | Abnormal oxygen transport; may cause cyanosis |
| Glycated hemoglobin (HbA1c) | Glucose becomes nonenzymatically attached to hemoglobin | Used to assess average glycemic exposure over approximately 2–3 months |
Important distinction: HbA1c
HbA1c is not an oxygen-binding derivative formed by replacing the iron's oxidation state. It is produced by nonenzymatic glycation of hemoglobin.
Its measurement is used in the diagnosis and monitoring of diabetes mellitus, subject to the limitations of the assay and conditions affecting erythrocyte lifespan.
Hemoglobin derivatives and laboratory diagnosis
Carboxyhemoglobin and methemoglobin are measured using appropriate co-oximetry methods when clinically indicated. Standard pulse oximetry may give misleading results in these conditions.
Routine hemoglobin estimation and specialized measurement of hemoglobin derivatives are different laboratory procedures.
4.12 Normal Hemoglobin Concentration
Approximate adult reference intervals are shown below.
| Population | Hemoglobin concentration |
|---|---|
| Adult males | Approximately 13.5–17.5 g/dL |
| Adult females | Approximately 12.0–15.5 g/dL |
These values are illustrative. The laboratory's validated reference intervals should be used for reporting and interpretation. Age, pregnancy, altitude, and other physiological factors can affect the expected concentration. AccessMedicine
Clinical interpretation
Reduced hemoglobin may occur in:
- Iron-deficiency anemia
- Vitamin B12 or folate deficiency
- Chronic kidney disease
- Hemolytic anemia
- Acute or chronic blood loss
- Bone marrow disorders
Increased hemoglobin may occur in:
- Dehydration-related hemoconcentration
- Chronic hypoxia
- High-altitude adaptation
- Polycythemia vera
- Certain secondary erythrocytosis conditions
Hemoglobin concentration should always be interpreted alongside the clinical history, CBC, and relevant additional investigations.
4.13 Hematocrit (HCT) or Packed Cell Volume (PCV)
Definition
Hematocrit (HCT) is the fraction of whole blood volume occupied by erythrocytes, usually expressed as a percentage.
It is also known as the packed cell volume (PCV).
For example, a hematocrit of 45% means that approximately 45% of the measured blood volume is occupied by erythrocytes.
Methods of measurement
Hematocrit can be determined by:
- Microhematocrit centrifugation
- Automated calculation from RBC count and MCV, depending on the analyzer
Microhematocrit principle
When anticoagulated blood is centrifuged in a capillary tube, the RBCs pack at the bottom, while plasma remains above them.
The packed RBC column is measured relative to the total blood column.
\[ \boxed{ HCT(\%)= \frac{\text{Packed RBC column length}} {\text{Total blood column length}} \times100 } \]A thin layer called the buffy coat, containing leukocytes and platelets, lies between the packed RBCs and plasma.
Example
If the packed RBC column is 22.5 mm and the total blood column is 50 mm:
\[ HCT=\frac{22.5}{50}\times100 \]\[ \boxed{HCT=45\%} \]4.14 Correlation of Hemoglobin, Hematocrit, and RBC Count
Hemoglobin concentration, hematocrit, and RBC count are related measurements, but they describe different properties of blood.
| Parameter | What it measures | Common units |
|---|---|---|
| Hemoglobin | Concentration of hemoglobin in whole blood | g/dL |
| Hematocrit | Fraction of blood volume occupied by RBCs | % or L/L |
| RBC count | Number of erythrocytes per unit volume | ×10⁶/µL or ×10¹²/L |
4.14.1 Relationship between the three parameters
In a typical normocytic, normochromic blood sample, these parameters tend to change in the same direction.
For example, a reduction in RBC mass commonly results in reduced hemoglobin concentration and hematocrit.
However, the relationship is not fixed because:
- RBC size varies.
- Hemoglobin content per cell varies.
- Plasma volume changes.
- RBC shape and hydration can change.
Therefore, the three measurements must be interpreted together.
4.14.2 The approximate rule of three
In selected samples with relatively normal RBC size and hemoglobin content, the following approximation may be useful:
\[ \boxed{Hb(g/dL)\times3\approx HCT(\%)} \]For example:
\[ Hb=15\text{ g/dL} \]\[ HCT\approx15\times3=45\% \]This is a rough teaching rule, not a diagnostic formula. It may not hold in microcytic, macrocytic, hypochromic, or otherwise abnormal samples.
4.14.3 Relationship to RBC indices
The RBC indices are calculated using hemoglobin, hematocrit, and RBC count.
Mean Corpuscular Volume (MCV)
MCV represents the average volume of an erythrocyte.
\[ \boxed{ MCV(fL)= \frac{HCT(\%)\times10} {RBC(10^6/\mu L)} } \]Mean Corpuscular Hemoglobin (MCH)
MCH represents the average amount of hemoglobin in an individual erythrocyte.
\[ \boxed{ MCH(pg)= \frac{Hb(g/dL)\times10} {RBC(10^6/\mu L)} } \]Mean Corpuscular Hemoglobin Concentration (MCHC)
MCHC represents the average concentration of hemoglobin within the packed RBC volume.
\[ \boxed{ MCHC(g/dL)= \frac{Hb(g/dL)\times100} {HCT(\%)} } \]These indices help characterize the RBC population and support the classification of anemia.
4.14.4 Worked Example: Correlation of CBC Parameters
Consider the following hypothetical laboratory results:
| Parameter | Result |
|---|---|
| Hemoglobin | 12 g/dL |
| Hematocrit | 36% |
| RBC count | 4.0 × 10⁶/µL |
Step 1: Calculate MCV
\[ MCV=\frac{36\times10}{4.0} \]\[ \boxed{MCV=90\text{ fL}} \]The MCV is within the typical adult normocytic range.
Step 2: Calculate MCH
\[ MCH=\frac{12\times10}{4.0} \]\[ \boxed{MCH=30\text{ pg}} \]Step 3: Calculate MCHC
\[ MCHC=\frac{12\times100}{36} \]\[ \boxed{MCHC=33.3\text{ g/dL}} \]Interpretation
The calculated indices indicate a normocytic, normochromic RBC population. Whether the hemoglobin concentration represents anemia depends on the patient's relevant reference interval and clinical context.
4.14.5 How RBC Size Affects the Relationship
Consider two hypothetical patients with similar hemoglobin concentrations but different RBC counts.
| Parameter | Patient A | Patient B |
|---|---|---|
| Hemoglobin | 10 g/dL | 10 g/dL |
| RBC count | 3.5 × 10⁶/µL | 5.0 × 10⁶/µL |
| Hematocrit | 30% | 30% |
| Calculated MCV | 85.7 fL | 60 fL |
| Calculated MCH | 28.6 pg | 20 pg |
Patient A has a normocytic pattern, while Patient B has a microcytic pattern.
This illustrates why RBC count alone cannot determine the severity or morphological type of anemia.
4.15 Clinical Correlation: Common Patterns
| Condition | Hemoglobin | RBC count | Hematocrit | Typical interpretation |
|---|---|---|---|---|
| Iron-deficiency anemia | Decreased | Often decreased | Decreased | Commonly microcytic and hypochromic |
| Thalassemia trait | Normal or decreased | Often relatively preserved or increased | Normal or decreased | Microcytosis disproportionate to the reduction in Hb |
| Acute blood loss | May initially remain unchanged | May initially remain unchanged | May initially remain unchanged | Values can fall after fluid redistribution or replacement |
| Hemolytic anemia | Decreased | Often decreased | Decreased | Reticulocyte response depends on marrow function and timing |
| Dehydration | May increase | May increase per unit volume | May increase | Hemoconcentration |
| Polycythemia vera | Often increased | Often increased | Often increased | Requires appropriate diagnostic evaluation |
These are common patterns, not absolute rules. Clinical interpretation should incorporate RBC indices, reticulocyte count, blood-film findings, iron studies, and other tests as indicated.
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