Red Blood Cell Evaluation in Anemia, Biocd 1556 Presentation

Lecture 6

Dr. Jeff Kurkewich

BIOCD 1556 Lecture 6 Dr. Jeff Kurkewich Red Blood Cell Evaluation in Anemia Overview Blood is a complex tissue composed of various cell types suspended in plasma, a nutrient- rich fluid filled with proteins and electrolytes. While plasma analysis provides valuable health information, examining the blood cells themselves yields crucial insights into a patient's physiological state. This lecture centers on laboratory assessment of red blood cells (RBCs), focusing on their overall levels and calculated indices. These measurements are integral to the complete blood count (CBC), a foundational diagnostic tool. Anemia is characterized by an abnormally low concentration of hemoglobin in the blood. It typically arises from either the premature destruction of RBCs, insufficient production of new RBCs, or both. To understand anemia's origins, we will explore hematopoiesis, the process by which all blood cells are formed. Mastery of these mechanisms is essential for identifying how disruptions lead to anemia.

Learning Goals

Terminal Objective

Grasp the biochemical mechanisms, assessment strategies, and management principles of anemias.

Enabling Objectives

• To analyze hematopoiesis as the molecular and cellular foundation of blood cell production, with a focused examination of erythropoietic maturation stages and their regulatory mechanisms
• Understand and interpret the following laboratory values: HGB, RBC, RDW, MCV, MCH, MCHC, observed reticulocyte count (OR), and corrected reticulocyte count (CRC).
• Distinguish between anemias caused by excessive RBC loss versus insufficient RBC production, using CBC data for differentiation.
• Compare anemias resulting from impaired hemoglobin synthesis to those from defective DNA synthesis and use laboratory findings for diagnosis.
• Identify the most informative laboratory tests for diagnosing anemias due to iron, folate, and vitamin B12 deficiencies.

Hematopoiesis and Erythropoiesis

Blood cells are relatively short lived within the body and need to be continually replenished throughout life. Each day, the average adult produces hundreds of billions of new blood cells through a process known as hematopoiesis. All blood cells originate from multipotent hematopoietic stem cells (HSCs) in the bone marrow, which undergo a series of sequential cell fate decisions before ultimate differentiating into one of three main blood cell categories:

• Erythrocytes: Erythrocytes, otherwise known as red blood cells (RBCs), serve as oxygen and carbon dioxide transporters, lacking nuclei, with a lifespan of ~120 days. Their biconcave shape and flexibility optimize gas exchange and capillary passage.
• Leukocytes: Leukocytes, otherwise known as white blood cells (WBCs), are key players in immune defense.
• Megakaryocytes: Megakaryocytes are cells responsible for making thrombocytes, or platelets, which are essential for blood clotting.

Hematopoietic cell fate decisions are regulated by growth factors and cytokines, allowing the body to adapt to demands such as infection or blood loss. Disruptions in hematopoiesis can result in a spectrum of blood disorders, including anemia, bleeding disorders, and blood cancers.Critical transcription factors for blood development Runx-1 Pluripotent stem cells Scl/tal-1 Lmo-2 Stem cell class LT-HSC MII Required for production, Tel survival, or self-renewal Self- renewal of HSCs Bmi-1 Gfi-1 GATA-2 ST-HSC CMP CLP Multipotent progenitors Ikaros PU.1 MEP GMP Notch- TCF-1-5 PU.1 GATA-1 GATA-2 FOG-1 C/EBPa GATA-2- GATA-1-5 Committed precursors GATA-1 FOG-1 Gfi-1b EKLF GATA-1 Gfi-1b GATA-1- C/EBP& XBP-1- Mature cells RBC Megakaryocyte Mast cell Eosinophil Neutrophil Monocyte/ Macrophage B lymphocyte T lymphocyte Fli-1]_L Nf-E2 e Platelets Hematopoiesis overview. Source: Orkin, S. H., & Zon, L. I. (2008). Hematopoiesis: An evolving paradigm for stem cell biology. Cell, 132(4), 631-644. https://doi.org/10.1016/j.cell.2008.01.025

Red Blood Cell Biology

• Erythrocytes are flexible, biconcave cells lacking a nucleus, with a lifespan of about 120 days. They are packed with hemoglobin for oxygen transport and store most of the body's iron. Note: The removal of the nucleus in the RBC maximizes the amount of hemoglobin they can store, increasing the amount of oxygen they can deliver.

Regulation of RBC Production

• Medullary Hematopoiesis: RBC production primarily occurs in bone marrow.
• Extramedullary Hematopoiesis: Under certain conditions (e.g., marrow failure), other organs (liver, spleen) may resume blood cell production.

E2A EBF Pax-5 Bcl11a (Evi9) GATA-3-5 Gfi-1-

• Erythropoietin (EPO): The kidney senses hypoxia (low oxygen) and releases EPO, stimulating the bone marrow to increase RBC production. This response is critical during hypoxic stress, such as high-altitude exposure, where increased RBC output compensates for reduced oxygen availability.

Erythropoiesis: The RBC Maturation Process

• Erythropoiesis: Erythropoiesis is the production of red blood cells (erythrocytes) within the body. This process begins with hematopoietic stem cells (HSCs), which are guided toward the erythroid lineage primarily through signaling by the cytokine erythropoietin (EPO). As these precursor cells mature, they undergo several stages of differentiation before ultimately becoming fully functional erythrocytes capable of transporting oxygen throughout the bloodstream.
• Maturation Stages:

1. Early erythropoiesis: Multipotent HSCs differentiate into committed erythroid progenitors. This includes the erythroid burst forming unit (BFU-E) and the erythroid colony forming unit (CFU-E).
2. Terminal erythroid differentiation: This stage begins with a cell type known as the proerythroblast (ProE), which will undergo a series of sequential mitotic divisions giving rise to more differentiated precursors. These sequential divisions are accompanied by decreases in cell size, increases in hemoglobin production, and increased chromatin condensation, and will ultimately enucleate and become a reticulocyte.
3. Reticulocyte maturation: Reticulocyte maturation into the erythrocyte (RBC) is the final stage. During this stage, intracellular organelles are lost, the surface area decreases, and the rearrangement of the cytoskeleton takes place.

• Unidirectionality: Once a cell differentiates beyond a certain stage, it cannot revert to an earlier form.
• Continuous Renewal: Old or damaged RBCs are removed by macrophages, primarily in spleen, and replaced by new cells.

Bone Marrow Peripheral circulation Pyrenocyte I Progenitors Differentiating Erythroblast Precursors I HSC BFU-E CFU-E BasoE OrthoE Erythrocyte ProE PolyE Reticulocyte 1) Early Erythropoiesis 2) Terminal Erythroid Differentiation 3) Reticulocyte Maturation Source: Ginzburg, Y., An, X., Rivella, S., & Goldfarb, A. (2023). Normal and dysregulated crosstalk between iron metabolism and erythropoiesis. eLife, 12, e90189

Requirements for Normal Erythropoiesis

• Healthy bone marrow microenvironment
• At least one functioning kidney (for EPO production)
• Adequate nutrition, especially iron (for hemoglobin synthesis), vitamin B12, and folate (for DNA synthesis)

Anemia: Definition and Clinical Features

Definition and Epidemiology

• Anemia is defined as a hemoglobin concentration below the normal reference range.
• It is common in the U.S., affecting roughly 1 in 77 people, with higher prevalence in females and certain populations (e.g., elderly, those with chronic illness).

Clinical Manifestations

• Common signs include pallor (notably in the palms, nail beds, face, or eye conjunctiva), dizziness, and fatigue.

Pathophysiological Categories

• Anemia is a disorder, not a disease. It can result from:
• Excessive RBC loss (hemorrhage or hemolysis)
• Inadequate stimulation of erythropoiesis (e.g., kidney disease, marrow failure)
• Impaired RBC production (nutritional deficiencies affecting hemoglobin or DNA synthesis)

Laboratory Evaluation of Erythropoiesis

CBC and RBC Indices

A CBC, or complete blood count, is a common blood test that measures and evaluates the different types and numbers of cells in your blood. This includes red blood cells (RBCs), white blood cells (WBCs), and platelets. The test also measures hemoglobin concentration, hematocrit (the percentage of blood made up of red blood cells), and various red blood cell indices that provide information about the size and hemoglobin content of red blood cells.

Measured Parameters

Parameter Description Typical Value RBC count Number of erythrocytes per microliter ~5 x 106/uL Hemoglobin (HGB) Hemoglobin concentration ~15 g/dL Hematocrit (HCT) Percentage of blood volume as RBCs ~45% Mnemonic-Rule of 3: For CBC results, the rule of three mnemonic helps to understand the relationship between RBC count, hemoglobin concentration (g/dL), and hematocrit (HCT). The number of RBCs multiplied by 3 is approximately equal to the total hemoglobin (HGB) concentration (g/dL), and the total hemoglobin concentration multiplied by 3 is approximately equal to the total hematocrit (%).

• RBC (millions) × 3 ~ HGB (g/dL)
• HGB (g/dL) × 3 ~ HCT (%)

Hematocrit Explained

4 100% Plasma Leukocytes and platelets (buffy coat) Erythrocytes = 45% Hematocrit = 45% 0 0% Whole blood can be separated in the laboratory through simple centrifugation. This yields three distinct phases, including the plasma, the buffy coat (which includes the leukocytes and platelets), and the erythrocyte layer. This erythrocyte layer can be expressed as a % of whole blood, which is known as the hematocrit. Source: https://www.medicine.mcgill.ca/physio/vlab/bloodlab/hemat_n.htm

Classification, Causes, and Diagnosis of Anemia

Calculated Corpuscular Indices (morphology assessment)

Index Meaning Reference Range Interpretation <80: microcytic 80-100: normocytic Mean corpuscular volume (MCV) The average volume of the RBCs 80-100 fL >100: macrocytic Mean corpuscular hemoglobin (MCH) Average amount of hemoglobin in individual RBCs 26-32 pg <33.4: hypochromic, Mean corpuscular hemoglobin concentration (MCHC) Average amount of hemoglobin in individual RBCs relative to the volume of the cell 33.4-35.5 g/dL 33.4-35.5: normochromic

• MCH and MCHC usually parallel MCV values.
• MCHC rarely exceeds the reference range due to solubility limits of hemoglobin.

Reticulocyte Count

• The reticulocyte count measures the number of immature RBCs. For healthy adults, the reference range is 0.5%-2.5%.
• In anemic patients, the bone marrow should produce more reticulocytes to increase the number of RBCs and HGB.
• Reticulocyte count is typically expressed as a percent (and not total number), which can be misleading in anemic patients. The corrected reticulocyte (CR) count adjusts for decreased HCT in anemia, reflecting true bone marrow response.