Hemoglobin and Blood Indices

Introduction

• Hemoglobin and blood indices are central to understanding how red blood cells transport respiratory gases and reflect hematological health. This chapter explores hemoglobin types, normal concentrations, HbA1c, and key red-cell indices used in the evaluation and classification of anemia.

Hemoglobin

• Hemoglobin is a conjugated protein in red cells and forms more than 90 percent of their dry weight.
• It transports oxygen from lungs to tissues and carbon dioxide from tissues to lungs.
• It also participates in regulation of nitric oxide levels in circulation.
• Oxygen binding to hemoglobin shows variable affinity, which is essential for efficient gas exchange.
• The oxygen dissociation curve is sigmoid due to cooperative binding of oxygen.
• Oxygenated hemoglobin releases hydrogen ions, while deoxygenated hemoglobin binds them.
• The concentration of hemoglobin within red cells is high, approximately 34 grams per deciliter.
• This high concentration enhances oxygen-carrying capacity of blood.

Structure and Synthesis of Hb

• Hb is synthesized in the precursors of red cells during their development in the bone marrow. It appears in the early normoblast stage and attains maximum concentration in the late normoblast stage.

Structure

• Hemoglobin consists of two components: heme and globin.

Heme

• Heme is a porphyrin compound with four pyrrole rings forming protoporphyrin.
• A central iron ion binds oxygen reversibly.
• During red cell destruction, iron is efficiently recycled for new hemoglobin synthesis.
• The porphyrin portion is converted into bilirubin and excreted.

Globin

• Globin is a protein composed of four polypeptide chains arranged in pairs.
• Each chain is associated with one heme group, forming a functional unit.
• After hemoglobin breakdown, globin is degraded into amino acids.
• These amino acids are reused for protein synthesis in the body.

Synthesis of Hb

• Hemoglobin synthesis requires coordinated formation of heme and globin.
• Heme synthesis occurs mainly in mitochondria, while globin is synthesized on ribosomes.
• Succinyl coenzyme A combines with glycine to form δ-aminolevulinic acid through ALA synthase.
• δ-aminolevulinic acid is converted into porphobilinogen and then into protoporphyrin IX.
• Ferrous iron is inserted into protoporphyrin IX to form heme.
• Globin chains are synthesized as polypeptides and fold into functional units.
• Heme combines with globin chains to form the complete hemoglobin molecule.

Types of Hemoglobin

• Hemoglobins can be broadly divided into normal and abnormal types (Table 14.1).

Normal Hemoglobins

• Hemoglobin A (Hb A) is the predominant form in adults, comprising about 97 percent.
• It consists of two alpha and two beta chains.
• It begins to appear during fetal life and becomes dominant after birth.
• Adult levels are usually established by six months of age.

Hemoglobin A₂ (Hb A₂)

• Hemoglobin A2 (Hb A2) is a minor adult hemoglobin.
• It contains two alpha and two delta chains.
• Its concentration is low at birth and increases to about 2–3 percent in adults.
• Levels may rise in certain anemias, especially those affecting globin synthesis.

Fetal Hemoglobin (Hb F)

• Hemoglobin F (Hb F) is the major hemoglobin during intrauterine life.
• It is composed of two alpha and two gamma chains.
• It constitutes about 70–90 percent of hemoglobin at birth.
• Its level declines after birth and reaches about 1 percent in adults.
• Hb F has higher oxygen affinity, facilitating placental oxygen transfer.
• Increased levels may be seen in some hemoglobin disorders and leukemias.

Hemoglobin Bart’s (Hb Bart’s)

• Hemoglobin Bart’s consists of four gamma chains.
• It is present in small amounts during fetal life.
• Its concentration increases in severe thalassemia

Table 14.1: Types of Hb.

Category	Types
Normal hemoglobin	Adult (Hb A, Hb A2), fetal hemoglobin (Hb F), embryonic hemoglobins
Abnormal hemoglobin	Structural variants (Hb S, Hb C, Hb D, Hb E) and unstable hemoglobins

Embryonic Hemoglobins

• Embryonic hemoglobins are present only during early stages of development.
• They are replaced by fetal and later adult hemoglobins as development progresses.
• Three main types are recognized:
• Gower 1 consists of two zeta and two epsilon chains.
• Gower 2 consists of two alpha and two epsilon chains.
• Portland hemoglobin consists of two zeta and two gamma chains.
• These forms support oxygen transport in early embryonic life.

Abnormal Hemoglobins

• Abnormal hemoglobins arise due to genetic alterations in globin chains.
• Important variants include Hb S, Hb C, Hb D, and Hb E.
• These are associated with inherited hemoglobin disorders.
• Hb S contains a substitution of valine for glutamic acid in the beta chain.
• It is responsible for sickle cell disease and causes altered cell shape.
• Unstable hemoglobins denature easily and form Heinz bodies in red cells.
• They are seen in certain congenital hemolytic anemias.

Normal Values, Functions and Variations

Normal Values

• Hemoglobin concentration in adult males ranges from 14 to 18 grams per deciliter.
• In adult females, it ranges from 12 to 16 grams per deciliter.
• Newborns have higher values, about 16 to 22 grams per deciliter due to relative hemoconcentration.
• Levels decline to 9 to 14 grams per deciliter by two to four months of age.
• By childhood, values stabilize around 12 to 14 grams per deciliter.
• A slight reduction may occur in older individuals after 50 years of age.

Functions of Hb

• Hemoglobin transports oxygen from lungs to tissues by forming oxyhemoglobin.
• It carries carbon dioxide from tissues to lungs as carbamino compounds.
• Each gram of hemoglobin carries approximately 1.34 milliliters of oxygen.
• Thus, oxygen-carrying capacity depends directly on hemoglobin concentration.
• Iron in heme binds oxygen reversibly, allowing efficient oxygenation without chemical oxidation.
• Each hemoglobin molecule can bind four oxygen molecules.
• Hemoglobin also acts as an important buffer, helping maintain blood pH.
• It participates in regulation of nitric oxide, influencing vascular tone.
• Hemoglobin imparts the characteristic red color to blood.

Variations in Hb Concentration

Conditions that Decrease Hb Concentration

Physiological

• Physiologically, hemoglobin levels are lower in children and females compared to adult males.
• During pregnancy, values decrease due to hemodilution from increased plasma volume.

Pathological

• Pathologically, hemoglobin decreases in various types of anemia.
• Relative reduction may occur in conditions causing fluid retention and dilution of blood.
• Increased antidiuretic hormone activity can produce such dilutional effects.

Conditions that Increase Hb Concentration

Physiological

• Physiologically, hemoglobin rises at high altitude due to hypoxia-induced erythropoiesis.
• Newborns show higher values because of increased red cell mass.
• Excessive sweating may cause a relative increase due to hemoconcentration.

Pathological

• Pathologically, hemoglobin increases in dehydration from diarrhea or vomiting.
• Chronic hypoxic states, such as lung disease or congenital heart disease, elevate levels.
• Primary increase occurs in polycythemia, where red cell production is excessive.

Hemoglobin Ligands (Complexes)

• Hemoglobin binds not only oxygen but also various gases and molecules.
• Normal complexes include carbaminohemoglobin and nitrosohemoglobin.
• Abnormal complexes include carboxyhemoglobin, methemoglobin, and sulfhemoglobin.
• Cyanmethemoglobin is used in laboratory estimation of hemoglobin.
• Glycated hemoglobin reflects long-term blood glucose levels.

Nitrosohemoglobin

• Nitric oxide is a physiological ligand that regulates vascular tone via cyclic guanosine monophosphate.
• It promotes vasodilation and inhibits platelet aggregation.
• Hemoglobin binds nitric oxide and forms nitrosohemoglobin.
• This binding reduces nitric oxide availability and limits its signaling effects.
• Free hemoglobin in plasma can enhance nitric oxide removal.
• Excess scavenging may lead to vasoconstriction.

Carbaminohemoglobin

• Carbaminohemoglobin is formed when carbon dioxide binds to hemoglobin as a physiological ligand.
• Carbon dioxide reacts with the globin component, not with the heme group.
• This interaction occurs mainly at the terminal amino groups of globin chains.
• It plays a significant role in the transport of carbon dioxide from peripheral tissues to the lungs.
• Approximately 20–25% of carbon dioxide is transported in this combined form in blood.

Carboxyhemoglobin

• Carboxyhemoglobin is produced when carbon monoxide binds to hemoglobin at the heme iron.
• Hemoglobin has nearly 200 times greater affinity for carbon monoxide than for oxygen.
• Even low concentrations of carbon monoxide can significantly reduce oxygen binding.
• The formation is reversible, but dissociation occurs slowly compared to oxygen release.
• In healthy individuals, levels remain very low, typically less than 1%.
• In smokers, levels may increase substantially, leading to impaired oxygen delivery and tissue hypoxia.

Methemoglobin

• Methemoglobin is formed when the iron in hemoglobin is oxidized from the ferrous (Fe²⁺) to the ferric (Fe³⁺) state.
• This altered form is unable to bind oxygen reversibly, reducing oxygen-carrying capacity.
• Normally, methemoglobin constitutes less than 1% of total hemoglobin due to enzymatic reduction systems.
• Exposure to oxidizing agents, certain drugs, or toxins can increase its formation.
• The condition, termed methemoglobinemia, can be partially reversible through cellular enzymatic mechanisms.

Cyanmethemoglobin

• Cyanmethemoglobin is formed when cyanide ions bind to methemoglobin.
• In this form, hemoglobin iron is already in the ferric state, facilitating cyanide binding.
• The compound is also known as hemiglobincyanide and is relatively stable.
• This reaction is reversible under controlled conditions.
• It is clinically significant in laboratory estimation of hemoglobin concentration using standard methods.

Sulfhemoglobin

• Sulfhemoglobin is produced following exposure to sulfur-containing drugs or toxic chemicals.
• In this derivative, sulfur binds to the porphyrin ring, not directly to iron.
• Although iron remains in the ferrous state, oxygen affinity is markedly reduced, nearly 100 times lower than normal hemoglobin.
• The formation is irreversible, and affected erythrocytes retain it throughout their lifespan of approximately 120 days.
• Elevated levels can lead to persistent cyanosis due to reduced effective oxygen transport.

Glycosylated or Glycated Hemoglobin

• Glycated hemoglobin is formed when glucose binds to hemoglobin through a nonenzymatic reaction in circulating blood.
• This process occurs continuously and reflects the average blood glucose level over time.
• Hemoglobin is particularly susceptible because of its prolonged lifespan of about 120 days in erythrocytes.
• The clinically important fraction is hemoglobin A1c, a subtype of adult hemoglobin.
• In this form, glucose attaches to the terminal valine residue of the beta chain.
• The extent of glycation depends directly on the prevailing concentration of blood glucose.
• In healthy individuals, hemoglobin A1c usually remains below 5% of total hemoglobin.
• Values between 5.7% and 6.4% indicate increased risk of impaired glucose regulation.
• A value of 6.5% or higher is considered diagnostic of diabetes mellitus according to accepted criteria.
• Levels rise in conditions with chronic hyperglycemia, including diabetes mellitus and certain endocrine disorders.
• Measurement of hemoglobin A1c provides a reliable index of long-term glycemic control, rather than short-term fluctuations.

In Oxidative Stress

• Glycation is the nonenzymatic attachment of glucose to proteins, including hemoglobin.
• This term distinguishes it from enzymatic glycosylation, which occurs in cellular organelles such as the endoplasmic reticulum and Golgi apparatus.
• Glycated hemoglobin forms gradually in erythrocytes exposed to circulating glucose.
• The process is promoted by elevated glucose levels and increased free radical activity.
• Reactive oxygen species enhance molecular interactions, facilitating glycation.
• Therefore, glycated hemoglobin reflects both chronic hyperglycemia and oxidative stress status.

Applied Physiology

Hb Destruction

• Hemoglobin destruction occurs following breakdown of red blood cells in the macrophage system or circulation.
• Hemoglobin is separated into heme and globin components.
• Globin is degraded into amino acids and reused in the body’s metabolic pool.
• The heme portion undergoes enzymatic cleavage by heme oxygenase, producing biliverdin.
• Biliverdin is subsequently reduced to bilirubin by biliverdin reductase.
• Bilirubin is transported to the liver for further metabolism and excretion.
• Excessive hemolysis increases bilirubin production, leading to hemolytic jaundice.
• In severe cases, free hemoglobin appears in urine, resulting in hemoglobinuria.

Hemoglobin Estimation

• Hemoglobin estimation is one of the most commonly performed laboratory investigations in clinical practice.
• It is routinely carried out in outpatient evaluation and for hospitalized patients, including bedside assessment.
• Measurement of hemoglobin is essential before surgical procedures to evaluate patient safety and oxygen-carrying capacity.
• It is preferred over total red blood cell count for detecting anemia, as it directly reflects oxygen transport capacity.
• Anemia is diagnosed when hemoglobin concentration falls below the normal reference range for age and sex.
• Regular assessment helps in early detection, monitoring, and management of hematological and systemic disorders.

Blood Indices

• Blood indices are calculated from hemoglobin concentration, packed cell volume, and red blood cell count.
• These indices provide quantitative information about red cell size, hemoglobin content, and concentration.
• The principal indices include mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration.
• Additional parameters include mean corpuscular diameter and red cell distribution width, which assess variability in cell size.
• These measurements are essential for the classification and evaluation of anemia.

Mean Corpuscular Volume

• Mean corpuscular volume represents the average volume of a red blood cell and is expressed in femtoliters.
• It is calculated using packed cell volume and red blood cell count with appropriate unit conversion.

$$MCV = \frac{Hematocrit \times 10}{Red\ cell\ count\ (millions/mm^3)}$$

• For example, if hematocrit is 40% and red cell count is 5 million per cubic millimeter, the mean corpuscular volume is 80 femtoliters.
• The normal adult range of mean corpuscular volume is 78 to 96 femtoliters.
• This index indicates the size of red blood cells and helps categorize anemia.
• Values below 78 femtoliters indicate microcytic cells, often seen in iron deficiency states.
• Values within the normal range indicate normocytic cells, commonly observed in acute blood loss or chronic disease.
• Values above 96 femtoliters indicate macrocytic cells, typically associated with vitamin deficiency or bone marrow disorders.
• Thus, blood indices provide critical insight into red cell morphology and underlying pathophysiological processes.

Mean Corpuscular Hemoglobin

• Mean corpuscular hemoglobin represents the average mass of hemoglobin in a single red blood cell and is expressed in picograms.
• It is calculated from hemoglobin concentration and red blood cell count.

$$MCH = \frac{\mathrm{Hemoglobin\ (g/dL)} \times 10}{\mathrm{Red\ cell\ count\ (millions/mm^3)}}$$

• For example, with hemoglobin of 14 grams per deciliter and red cell count of 5 million per cubic millimeter, mean corpuscular hemoglobin is 28 picograms.
• The normal range is 27 to 33 picograms.
• Reduced values occur in hypochromic microcytic anemia, while increased values are seen in macrocytic anemia.

Mean Corpuscular Hemoglobin Concentration (MCHC)

• Mean corpuscular hemoglobin concentration indicates the average concentration of hemoglobin within packed red blood cells.
• It reflects the degree of hemoglobinization and is expressed in grams per deciliter or percentage.

• For example, with hemoglobin of 15 grams per deciliter and hematocrit of 45 percent, mean corpuscular hemoglobin concentration is 33.3 percent.
• The normal range is 33 to 37 grams per deciliter.
• Values above 40 percent are usually due to technical error, as physiological limits prevent higher concentrations.

• Low values, often 20 to 25 percent, indicate hypochromic states with reduced hemoglobin content.

Color Index (CI)

• Color index is the ratio of hemoglobin percentage to red blood cell percentage.

$$CI = \frac{\mathrm{Hemoglobin}\%}{\mathrm{Red\ blood\ cell}\%}$$

• One hundred percent hemoglobin equals 14.8 grams per deciliter, and one hundred percent red blood cells equals 5 million per cubic millimeter.
• Normal range is 0.85 to 1.10.
• Values below normal indicate hypochromic anemia.