Assessment of Erythrocytes, Leukocytes, and Platelets

Basic Laboratory Assessment of Erythrocytes, Leukocytes, and Platelets

Overall Quantitative Measurements

• Erythrocytes (RBCs), leukocytes (WBCs), and platelets (PLTs) are quantified using automated instrumentation.
• Manual counts may be required in cases of low counts or quality issues.
• Manual hemoglobin determinations and centrifuge-based microhematocrit measurements serve as quality control or backup methods.
• RBC indices included in CBC:
  • MCV (Mean Corpuscular Volume)
  • MCHC (Mean Corpuscular Hemoglobin Concentration)
  • Additional measurements:
  • Reticulocyte information
  • Red cell distribution width (RDW)
  • Blood cell histograms

Manual Erythrocyte, Leukocyte, and Platelet Counts

• Blood specimens are diluted with specific diluents to a precise ratio.
• Cells are counted using a hemacytometer, which has ruled chambers marked in square millimeters.
• To count WBCs and PLTs accurately, RBCs must be eliminated from the count.
• Most lysing agents operate on the principle of osmotic pressure:
  • An aliquot of blood mixed in a hypotonic solution leads to cell lysis.

Hemoglobin Measurement in the Laboratory #1

• Hemoglobin (Hb) can be determined separately or as part of a CBC.
• Typically analyzed using instrumentation but can also be measured manually.
• The cyanmethemoglobin method is commonly used for both manual and automated determinations:
  • Modified Drabkin's reagent (containing potassium cyanide) is mixed with the blood specimen.
  • The mixture is incubated to lyse cells and then measured in a spectrophotometer at 540 nm.

Hemoglobin Measurement in the Laboratory #2

• Errors in Hb measurement can stem from specimen integrity issues:
  • Elevations can occur in:
  • Lipemic blood samples
  • Icteric blood samples
  • Hemolyzed samples
  • High WBC counts or RBCs containing Hb S or C can also cause elevated results.

Hemoglobin Measurement in the Laboratory #3

• Strategies to resolve measurement issues include:
  • Saline (Plasma) Replacement: Used for icteric, lipemic, or hemolyzed plasma.
  • Centrifugation: Clear supernatant is transferred to a cuvette for measurement, addressing high WBC count interference.
  • Specimens with Hb C or S can be diluted 1:2 with distilled water, with results adjusted by the dilution factor.

Hematocrit (Packed Cell Volume) #2

• Automated hematocrit results are obtained via multiparameter instruments, calculated from individual MCVs and red cell counts.
• Not impacted by plasma trapped in the RBC column.
• Errors in manual hematocrits:
  • Specimen-related:
  • Inadequate filling of EDTA tubes leading to false decreases in Hct due to RBC shrinkage.
  • Technical:
  • Overcentrifugation or improper sealing of test capillary tubes.
• Patients with clinical RBC disorders (e.g., macrocytic or sickle cell anemia) may show falsely elevated Hct levels.

Red Blood Cell Indices #2

• Sources of error in indices:
  • MCV: Elevated due to autoagglutination in cold agglutinin disease or paraproteinemia, hyperglycemia causing osmotic swelling of RBCs, or leukocytosis.
  • MCH: Hyperlipidemia leads to elevated MCH and increased Hb values.
  • MCHC: Influenced by conditions such as hyperlipidemia, autoagglutination, leukocytosis, hereditary spherocytosis, hemolysis, and ictericia.

Red Cell Distribution Width (RDW)

• Measures cellular volume heterogeneity, indicating variability in RBC sizes within a sample.
• Early indicator for anemia, changes appear sooner during nutritional deficiencies such as iron deficiency anemia.
• Useful in distinguishing between iron deficiency anemia and beta thalassemia in microcytic anemias.
• Can also assist in identifying RBC fragmentation.

Absolute Reticulocyte Count

• Represents the actual count of reticulocytes per liter or microliter of blood.
• Calculation formula:
  \text{Absolute reticulocyte count} = \frac{[% \text{reticulocyte count} \times \text{total erythrocyte count}]}{100}
• A count less than 100 \times 10^9/L indicates an inappropriately low erythropoietic response to anemia.

Assessment of Bone Marrow Response

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

Classification and Laboratory Assessment of Anemias

Overview

• Anemia is defined by:
  • Hb concentration or Hct below the reference range for age, gender, and geographical location.
• Functional anemia is characterized as decreased oxygen-carrying capacity of RBCs leading to hypoxia.
• Potential underlying disorders may include:
  • Liver disease
  • Alcohol toxicity
  • Hypothyroidism
  • Myelodysplasia

Clinical Signs and Symptoms of Anemia

• Symptoms result from reduced oxygen delivery to tissues:
  • Relate to decreased hemoglobin concentration and rate of hemoglobin reduction.
• Common symptoms include:
  • Fatigue
  • Shortness of breath
  • Skin pallor

Classification of Anemias

• Based on red cell morphology (by Wintrobe's classification):
  • Size-based categories:
  • Macrocytic
  • Normocytic
  • Microcytic
• Pathophysiological classifications include:
  • Impaired RBC production (e.g., insufficient or ineffective erythropoiesis)
  • Increased RBC destruction (e.g., hemolysis)
  • Blood loss (acute or chronic)

Pathophysiology of Anemia

• Categories based on impact on hematopoietic stem cells:
  1. Impaired Red Cell Production:
  • Aplastic anemia
  • Myelodysplastic anemia
  • Malignant metastases
  1. Accelerated Red Cell Destruction:
  • Acquired: Hemolytic processes
  • Inherited: Genetic defects affecting RBCs.
  1. Blood Loss:
  • Acute hemorrhage
  • Chronic conditions (e.g., iron deficiency anemia)
  • Bone marrow damage from chemicals or radiation.

Laboratory Assessment of Anemias #1

• Laboratory investigation includes:
  • Quantitative and semiquantitative measures of erythrocytes:
  • Decreased hemoglobin concentration
  • Reduced packed cell volume (microhematocrit)
  • Altered erythrocyte concentration
  • Variations in RBC indices (MCV, MCH, MCHC)

Laboratory Assessment of Anemias #2

• Clinical features to assess:
  • Weakness
  • Fatigue
  • Pallor
• Complete blood count with differential, RBC indices, and morphology, reticulocyte count is critical.
• Hemoglobin levels:
  • Males: < 13.0 g/dL
  • Females: < 12 g/dL
• Classification by RBC indices yields:
  • Low MCV, MCHC:
  • Suggestions of microcytic, hypochromic anemia (e.g., iron deficiency, thalassemia)
  • Typical of maturation defects
  • Normal MCV, MCHC:
  • Normocytic, normochromic (e.g., bone marrow disorders)
  • High MCV:
  • Macrocytic conditions (e.g., Vitamin B12 deficiency, folate deficiency)

Laboratory Assessment of Anemias #4 – Basic Assessment of Anemias

• Reference ranges (adults):
  • MCV: 80 to 96 fL
  • MCHC: 33% to 36%
  • MCH: 27.5 to 33.2 pg

Laboratory Assessment of Anemias #6

• Additional assessments might include:
  • Bone Marrow Examination: Reveals M:E (myeloid:erythroid) ratios.
  • Fetal Hemoglobin (Hb F) Concentration: Evaluating potential hemoglobinopathies and thalassemias.
  • Smear Tests: Thin and thick for assessing malarial or babesial parasites.
  • Platelet Count: Evaluating healing efficiency post-trauma.
  • Reticulocyte Count: Monitoring red cell production.
  • Sickle Cell Testing: Screening for sickling hemoglobinopathies.
  • G6PD Assays: For enzyme deficiencies.
  • Hemoglobin Electrophoresis: Identifying hemoglobinopathies and thalassemias.

Laboratory Assessment of Anemias #7

• Other lab procedures that assist in diagnosis:
  • Antibody Screening: Identifying immune-related RBC destruction causes.
  • Direct Antiglobulin Test (AHG): Screening for immune RBC destruction.
  • Bilirubin Levels: Help in determining RBC destruction.
  • Folic Acid and Vitamin B12 Assays: Identifying megaloblastic anemia due to nutrient deficiencies.

Laboratory Assessment of Anemias #8

• Haptoglobin Level Measurement: Identifies intravascular hemolysis.
• Lactic Dehydrogenase (LDH): Determining intravascular hemolysis.
• Serum Iron and TIBC: Measuring serum iron levels and its binding capacity.
• Occult Blood Testing: Identifies GI bleeds as a potential source of blood loss.
• Urobilinogen Screening: Detects general hemolysis without distinguishing further.

Acute and Chronic Blood Loss Anemia and Anemias Associated with Systemic Disorders

Acute Blood Loss Anemia #1

• Etiology:
  • Typically results from traumatic conditions, such as accidents or severe injury.
  • May occur during or after surgery.

Acute Blood Loss Anemia #2

• Physiology:
  • Acute blood loss does not create immediate anemia; rapid loss (>20% of circulating blood volume) leads to shock and cardiovascular complications.
  • The body compensates by expanding circulatory volume, resulting in subsequent anemia.

Acute Blood Loss versus Chronic Blood Loss #1

• Laboratory Findings:
  • Acute blood loss results in changes identifiable within 24 to 48 hours; chronic blood loss spans months to years.

Acute Blood Loss versus Chronic Blood Loss #2

• Physiological Adaptations:
  • Distinct adaptations exist for acute versus chronic blood loss and related physiology.

Acute Blood Loss Anemia #4

• Laboratory Findings:
  • In the healthy patient:
  • Peripheral blood film should appear normochromic and normocytic within 24 hours.
  • Increased reticulocyte counts due to enhanced erythropoiesis develop around 3 to 5 days post-bleeding, peaking by day 10.

Acute Blood Loss Anemia #5

• Laboratory Findings (cont.):
  • Total WBC count normalization occurs around 2 to 4 days.
  • Morphological changes revert back to normal in over 2 weeks.
  • Red cell profile returns to previous values longer than 2 weeks.

Clinical Features of Acute Hemorrhage in Healthy Young Adults (TABLE 12.1)

Chronic Blood Loss Anemia #7

• Etiology:
  • Related to conditions like:
  • Gastrointestinal (GI) tract issues
  • Heavy menstruation
  • Urinary tract abnormalities

Chronic Blood Loss Anemia #8

• Blood loss occurs gradually over months, resulting in slow regeneration of red blood cells.
• Clinical and hematological features differ from those seen in acute bleeding.
• Reticulocyte counts may be normal or slightly increased.

Laboratory Findings for Chronic Blood Loss Anemia #9

• Noticeable anemia develops after iron stores exhaust.
• Chronic bleeding results in iron deficiency, leading to newly formed cells becoming hypochromic and microcytic.
• The white blood cell count often remains normal or decreases slightly, with increased platelets initially, then decreased later in severe deficiency.

Chapter 13: Bone Marrow Failure Syndromes

Introduction

• Aplastic anemia represents a category of hypoproliferative disorders with reduced blood cell production.

General Characteristics of Bone Marrow Syndromes

• Cytopenia with hypocellular marrow:
  • May impact all three cell lines (pancytopenia in constitutional aplastic anemia).
  • May affect two lines (e.g., PNH).
  • May involve a single lineage (e.g., DBA).
• Cytopenias can stem from premalignant conditions or myelofibrosis.

Laboratory Findings in Bone Marrow Failure Syndromes

• Cytopenias can vary from mild to severe.
• Macrocytosis can be observed, often covered by other deficiencies.
• May see elevated Hb F levels.
• Dysplastic changes include micromegakaryocytes and other abnormalities.

Acquired Aplastic Anemia

• Idiopathic aplastic anemia occurs in individuals with no known exposure history.
• Iatrogenic forms may result from specific chemicals or drug exposure.
• Constitutional aplastic anemia results from genetic predispositions.

Aplastic Anemia Iatrogenic Causes

• Include substances such as:
  • Benzene derivatives
  • Insecticides
  • Antibiotics
  • Other drugs and radiation.

Etiology of Aplastic Anemia (BOX 13.1)

• Direct Toxicity:
  • Iatrogenic causes include radiation and chemotherapy, along with identifiable drugs.
• Immune-Mediated Causes:
  • Iatrogenic instances (e.g., transfusion-associated hemolysis) and idiopathic cases are included.

Pathophysiology: Aplastic Anemia

• Immune-mediated pathophysiology involves T cell activation and telomere repair gene mutations.
• Hematopoietic failure relates to insufficient pluripotent stem cells and inadequate growth and development factors.

Clinical Features of Aplastic Anemia

• Symptoms reflect the level of deficiency:
  • Bleeding from thrombocytopenia.
  • Increased infection risk from neutropenia.
  • Anemia-related signs include fatigue and pallor.
• Splenomegaly and lymphadenopathy typically absent.
• Long-term survival can be associated with higher malignancy risks (e.g., leukemic, solid tumors). Potential risks include PNH and myeloid malignancies.

Laboratory Findings #2 in Aplastic Anemia

• Pancytopenia occurs with involvement of all cell lines.
• Underlying conditions could decrease RBCs impacting varying levels of cellularity and morphology.
• Diagnosis is confirmed with at least two PB values below critical thresholds.

Treatment of Aplastic Anemia

• Immunosuppressive therapy effectiveness relates to organ damage and ability to regenerate tissues.
• Stem cell transplantation provides potential for remission.

Constitutional Bone Marrow Failure Syndromes #1

Fanconi’s Anemia:

• Congenital disorder leading to progressive pancytopenia.
• Diagnosis typically in children aged 5 to 10.
• Confirmed through chromosome studies revealing increased breakage.
• Clinical features may include short stature and various abnormalities.

BM Failures Involving a Single Cell Lineage

• Pure Red Cell Aplasia (PRCA):
  • Causes range from transient infections to congenital anomalies like Diamond-Blackfan anemia.

Acute Erythroblastopenia of Childhood

• More common in children under 8, often post-viral infections.
• Generally self-limiting with an acute course.

Acquired Pure Red Cell Aplasia (PRCA)

• Defined by the selective failure of RBC production;
• Associated with thymomas, reticulocytopenia, and a cellular marrow devoid of erythroid precursors.

Diamond-Blackfan Anemia (DBA) #1

• Characterized by a heterogeneous group of disorders associated with:
  • Hematopoetic failure
  • Proapoptotic hematopoiesis
  • Increased cancer predisposition.

Diamond-Blackfan Anemia (DBA): Pathophysiology

• Congenital mutations lead to defects in ribosomal protein production, inducing apoptosis in erythroid progenitors.

Laboratory Manifestations in DBA

• Diagnostic Criteria:
  • Anemia appears before the first birthday.
  • Normal or reduced neutrophil counts, variable platelet counts, often macrocytosis.
  • Normal marrow cellularity with few erythroid precursors.

Diamond-Blackfan Anemia (DBA): Treatment

• Roughly 75% respond to steroid treatment, with long-term survival rates around 65%, although many require ongoing treatment.