Laboratory Assessment of Blood Cells and Anemias

Basic Laboratory Assessment of Erythrocytes, Leukocytes, and Platelets

Overall Quantitative Measurements

  • Erythrocytes, leukocytes, and platelets are quantified using automated instrumentation.
    • Automated methods improve efficiency and accuracy in blood component measurement.
  • Manual counts may be necessary due to low counts or quality issues with automated systems.
  • Manual methods include:
    • Determinations of hemoglobin
    • Centrifuge-based measurement of microhematocrit (quality control strategy or backup method).
  • RBC indices—a standard part of the CBC (Complete Blood Count)—include:
    • Mean Corpuscular Volume (MCV)
    • Mean Corpuscular Hemoglobin Concentration (MCHC)
    • Additional indices included:
      • Reticulocyte information
      • Red Cell Distribution Width (RDW)
      • Blood cell histograms

Manual Erythrocyte, Leukocyte, and Platelet Counts

  • Blood specimens are diluted with specific diluents to an exact ratio and counted in a hemacytometer.
    • Hemacytometer: A chamber with accurately ruled areas measured in square millimeters for counting cells.
  • Cell Counting Process:
  • Since RBC counts are typically higher than WBC and platelet counts, RBC counts must be eliminated to accurately count WBC and PLT counts.
  • Most lysing agents operate using the principle of osmotic pressure:
    • Exposing blood aliquots to hypotonic solutions causes cells to lyse.

Hemoglobin Measurement in the Laboratory

Hemoglobin Determination

  • Hemoglobin (Hb) can be measured separately or as part of a CBC.
  • Analysis is typically conducted using automated instrumentation but can also be done manually.
    • Commonly used manual method: Cyanmethemoglobin method
      • Uses modified Drabkin’s reagent with potassium cyanide mixed with blood.
      • The mixture is incubated to induce lysing, and measured with a spectrophotometer at 540 nm.

Sources of Error in Hemoglobin Measurement

  • Elevations may occur in cases of:
    • Lipemic samples
    • Icteric samples
    • Hemolyzed blood samples
    • High WBC counts
    • RBCs containing Hb S or C
  • Errors stem from issues with specimen integrity.

Problem-Solving Strategies

  • Saline (plasma) replacement methods for icteric, lipemic, or hemolyzed samples.
  • Centrifugation can clarify specimens, allowing the clear supernatant to be measured.
  • For specimens with Hb C or S, dilute 1:2 with distilled water, and multiply results by the dilution factor.

Hematocrit (Packed Cell Volume)

Automated Hematocrit Measurement

  • Automated hematocrit results derived from multiparameter instruments calculate from individual MCVs and red cell counts.
    • Not affected by trapped plasma in the RBC column.

Sources of Error in Manual Hematocrit Measurements

  • Errors may arise from:
    • Specimen errors (e.g., inadequate filling of EDTA tube leads to RBC shrinkage)
    • Technical errors (e.g., over-centrifugation, improper sealing of capillary tubes)
    • Clinical RBC disorders (e.g., macrocytic anemia or sickle cell anemia) can falsely elevate Hct levels.

Red Blood Cell Indices

Common Sources of Error

  • Mean Corpuscular Volume (MCV):
    • Autoagglutination (e.g., cold agglutinin disease) falsely elevates MCV and lowers Hct values.
    • Hyperglycemia leads to osmotic swelling of RBCs, falsely elevating MCV.
    • Leukocytosis can also spuriously elevate MCV.
  • Mean Corpuscular Hemoglobin (MCH):
    • Hyperlipidemia falsely elevates MCH and Hb values.
  • Mean Corpuscular Hemoglobin Concentration (MCHC):
    • Elevated by hyperlipidemia, autoagglutination, leukocytosis, hereditary spherocytosis, hemolysis, and ictericia.

Red Cell Distribution Width (RDW)

  • RDW quantifies cellular volume heterogeneity, reflecting the range of RBC sizes in a sample.
  • Useful for early classification of anemia: first to become abnormal in nutritional deficiencies (e.g., iron deficiency anemia).
  • Helps characterize microcytic anemias, distinguishing between iron deficiency anemia and beta-thalassemia.
  • Identifies RBC fragmentation.

Absolute Reticulocyte Count

  • Represents the actual number of reticulocytes in 1 L or 1 µL of blood.
  • Calculation:
    Absolute \, reticulocyte \, count = \frac{\% reticulocyte \, count \times total \, erythrocyte \, count}{100}
  • If the absolute reticulocyte value is less than 100 \, x \, 10^9/L, it indicates an inappropriately low erythropoietic response to anemia.

Assessment of Bone Marrow Response

  • Normal bone marrow activity:
    • Reticulocyte Production Index (RPI) = 1
  • Increased hemolysis or destruction:
    • RPI = 3 to 7
  • Bone marrow damage or suppression:
    • RPI < 2

Classification and Laboratory Assessment of Anemias

Overview of Anemia

  • Defined by lowered Hb concentration or Hct below reference range for age, gender, and geographical location.
  • Functional anemia: Decrease in RBCs' oxygen-carrying capacity leading to hypoxia.
  • Anemia may signify underlying disorders:
    • Liver disease
    • Alcohol toxicity
    • Hypothyroidism
    • Myelodysplasia

Clinical Signs and Symptoms

  • Symptoms arise from the reduced oxygen delivery to tissues, related to lowered Hb concentration.
  • Common signs include:
    • Fatigue
    • Shortness of breath
    • Skin pallor
  • Signs may reflect the rate of Hb reduction and blood volume loss.

Classification of Anemias

  • Classification as per red cell morphology (originally proposed by Wintrobe):
  • Categories based on erythrocyte size include:
    • Macrocytic
    • Normocytic
    • Microcytic
  • Pathophysiological classification includes:
    • Impaired RBC production (insufficient/ineffective erythropoiesis)
    • Increased RBC destruction (hemolysis)
    • Blood loss (acute or chronic)

Pathophysiology of Anemia

Categories and Factors Impacting Anemia
  • Impaired Red Cell Production:
    • Factors impacting hematopoietic stem cells or developmental issues:
      • Aplastic anemia
      • Myelodysplastic anemia
      • Malignant metastases
  • Accelerated Red Cell Destruction:
    • Hemolytic Processes:
      • Acquired or inherited (hereditary mechanisms)
  • Blood Loss:
    • Acute (e.g., trauma) or chronic (e.g., iron deficiency anemia)
    • Caused by chemicals or radiation affecting bone marrow

Laboratory Assessment of Anemias

Basic Measurements
  • Measurement involves quantitative and semiquantitative analyses:
    • Decreased hemoglobin concentration
    • Reduced packed cell volume (microhematocrit)
    • Decreased erythrocyte concentration
    • Altered RBC indices (MCV, MCH, MCHC)
Clinical Signs and Symptoms of Anemia
  • Weakness, fatigue, pallor
  • Complete Blood Count (CBC) includes:
    • Differential with RBC indices and morphology
    • Reticulocyte count
  • Male Hb threshold: < 13.0 g/dL
  • Female Hb threshold: < 12 g/dL
Classification by RBC Indices
  • Low MCV, MCHC:
    • Refers to microcytic, hypochromic conditions, often indicative of maturation defects (e.g., iron deficiency anemia)
  • Normal MCV, MCHC:
    • Normocytic, normochromic, associated with hypoproliferation (bone marrow disorders, chronic diseases)
  • High MCV:
    • Macrocytic conditions usually due to maturation defects (Vitamin B12 deficiency, folate deficiency, excessive alcohol intake, hypothyroidism)
Supplementary Assessment of Anemias
  • Bone marrow examination may reveal abnormal myeloid:erythroid (M:E) ratio.
  • Evaluation of fetal hemoglobin concentration for hemoglobinopathies/thalassemias.
  • Examination of blood smears for parasitic infections.
  • Platelet counts to assess healing post-trauma.
  • Reticulocyte counts to evaluate red cell production and identify anemia mechanisms.
Additional Laboratory Procedures
  • Antibody screening and identification tests to determine immune causes of RBC destruction.
  • Direct antiglobulin (AHG) test for RBC destruction identification.
  • Bilirubin measurement to assess RBC destruction (both intravascular and extravascular hemolysis).
  • Nutritional assays for folic acid, vitamin B12.
  • Haptoglobin level to identify intravascular hemolysis.
  • Lactic dehydrogenase (LDH) to detect hemolysis.
  • Occult blood testing for gastrointestinal bleeding sources.

Acute and Chronic Blood Loss Anemia

Acute Blood Loss Anemia

Etiology
  • Occurs due to traumatic conditions (accidents, severe injuries) or post-surgical events.
Physiology
  • Acute blood loss does not produce immediate anemia; significant blood loss reduces total blood volume, resulting in shock and cardiovascular issues.
  • Body compensates for blood loss through circulatory volume expansion, leading to subsequent anemia.
Laboratory Findings
  • Blood film should return to normochromic and normocytic appearance within 24 hours.
  • Reticulocyte count increases due to erythropoiesis; transient macrocytosis peaks around 10 days post-hemorrhage.
Clinical Features
  • Symptoms correlate with degree of blood loss; ranges from asymptomatic (10-20% loss) to shock (more than 40% loss).
    • Clinical presentation includes light-headedness, hypotension, tachycardia, and potential for irreversible shock.

Chronic Blood Loss Anemia

Etiology
  • Associated with conditions such as gastrointestinal tract issues, heavy menstruation, or urinary tract abnormalities.
Physiology
  • Involves gradual blood loss over time without immediate disruption of blood volume.
  • Unlike acute loss, clinical symptoms are less prominent; RBC regeneration occurs slowly and reticulocyte counts may be normal or slightly elevated.
Laboratory Findings
  • Noticeable anemia develops only after iron stores are depleted; blood cells become hypochromic and microcytic.
  • White blood cell counts typically normal; platelet counts may increase or decrease in severe iron deficiencies.

Bone Marrow Failure Syndromes

Introduction

  • Aplastic anemia is characterized by reduced growth of blood cells, known as hypoproliferative disorders.

General Characteristics

  • Cytopenia with hypocellular marrow affects all three cell lines, leading to pancytopenia (e.g., constitutional aplastic anemia).
    • Two cell lines affected (e.g., paroxysmal nocturnal hemoglobinuria) or single lineage (e.g., Diamond-Blackfan anemia).

Laboratory Findings

  • Wide range of cytopenias; some may exhibit macrocytosis, potentially masked by iron deficiency. Elevated Hgb F is common alongside dysplastic changes.

Acquired Aplastic Anemia

  • Idiopathic aplastic anemia occurs without established risk factors (chemical exposure, viral infection).
  • Causes may be iatrogenic or constitutional, indicating a genetic predisposition to bone marrow failure.

Iatrogenic Factors

  • Include agents like benzene, pesticides, antimetabolites, and specific antibiotics associated with bone marrow suppression.

Pathophysiology

  • Immune-mediated mechanisms involve Type 1 cytotoxic T cells, where mutations in telomere repair genes are implicated.
  • Hematopoietic failure occurs due to insufficient pluripotent stem cells or environmental factors hampering hematopoiesis.

Clinical Features

  • Symptoms depend on degree of deficiency; bleeding, infections, and signs of anemia may present. Absence of splenomegaly and lymphadenopathy is noted.
    • High-risk factors for malignancy develop in long-term survivors.

Laboratory Assessment

  • Diagnosis involves identifying pancytopenia; typical red blood cells are normocytic normochromic with varying anisocytosis and poikilocytosis.
  • Genetic mutations affecting telomerase (TERT or TERC) may be noted.

Treatment

  • Treatments include immunosuppressive therapy, stem cell transplantation, and management of the immune response.
    • Historically fatal, outcomes have improved.

Constitutional Bone Marrow Failure Syndromes

  • Fanconi’s Anemia:
    • Genetic disorder presenting progressive pancytopenia typically diagnosed in children aged 5-10.
    • Chromosomal testing confirms condition via increased chromosome breakage.

BM Failures Involving a Single Cell Lineage

  • Pure Red Cell Aplasia (PRCA):
    • Established causes (e.g., infections, drugs, deficiency states).
    • Acute forms: Often transient following viral infection.
    • Chronic forms: Associated with thymoma or lymphoproliferative disorders.

Diamond-Blackfan Anemia (DBA)

  • Characterized by congenital disorders of hematopoiesis and BM failure.
  • Often presents with anemia prior to the first birthday.
  • Laboratory findings may indicate macrocytosis despite normal marrow cellularity.
  • Treatment may yield 75% response to steroids with long-term survival about 65%.

Conclusion

  • Understanding the comprehensive assessment of erythrocytes, leukocytes, and platelets is crucial for diagnosing various anemias and bone marrow failure syndromes. Proper laboratory techniques, awareness of physiological responses, and classification systems provide a thorough framework for blood disorders evaluation.