📚 Haematology Study Notes
Comprehensive Study Guide for Medical Laboratory Science
🔬 1.1 Cleaning of Glasswares and Safety Precaution in the Laboratory
1.1.1 Introduction to Laboratory Glassware
Proper glassware maintenance is fundamental to obtaining accurate and reliable laboratory results. Contaminated or improperly cleaned glassware can lead to:
- Hemolysis of red blood cells
- Interference with serological reactions
- Inaccurate test results
- Cross-contamination of samples
1.1.2 Types of Laboratory Glassware Used in Haematology
Test Tubes & Pipettes
Used for sample collection and storage. Must be free from detergent residues that cause hemolysis.
Slides & Cover Glass
Critical for blood film preparation. Require meticulous cleaning and must be scratch-free for microscopic examination.
Beakers & Flasks
Used for chemical preparation and reagent storage. Must be properly sterilized to prevent contamination.
1.1.3 Step-by-Step Glassware Cleaning Protocol
Immediate Rinsing
Rinse glassware immediately after use with tap water to remove any residual blood or chemicals. This prevents dried material from adhering to the glass surface.
Soaking
Soak contaminated glassware in appropriate solution:
- General contamination: 5% bleach or 3% Lysol solution for 12-24 hours
- TB suspicion: 1% sodium hypochlorite
- Serological glassware: Dichromate solution for 12-24 hours
Washing with Detergent
Use nonionic, metal-free, non-alkaline detergent. Scrub with soft brush (plastic or wooden handle). For hematology glassware, avoid detergents entirely to prevent hemolysis of RBCs.
Rinsing
Rinse thoroughly:
- General glassware: 3-4 rinses with tap water
- Sensitive tests: 8-12 rinses with distilled water
- Serological glassware: Multiple rinses to remove all acids and alkalis
Drying
Dry in hot air oven at 110°C maximum or allow air-drying. Ensure complete dryness before storage.
Sterilization
Autoclave at 15 lbs pressure for 20 minutes at 121°C for new glassware or contaminated items requiring sterilization.
1.1.4 Special Cleaning Procedures
Slides used for blood film examination require special care:
- Wash with distilled water immediately after use
- Soak in glacial acetic acid for 10 minutes
- Rinse thoroughly with distilled water
- Wipe dry with clean paper towels
- Store in 70% ethanol until use
1.1.5 Safety Precautions in the Hematology Laboratory
Personal Protective Equipment (PPE)
| Equipment | Purpose | When to Use |
|---|---|---|
| Laboratory Coat/Gown | Protect clothing and skin from splashes and contamination | Always during work |
| Gloves (Latex/Nitrile) | Barrier against bloodborne pathogens | When handling blood/body fluids |
| Eye Protection (Goggles/Face Shield) | Protect eyes and face from splashes | During centrifugation, chemical handling |
| Respiratory Protection (Mask/Respirator) | Protect from airborne pathogens | During aerosol-generating procedures |
| Closed-toe Shoes | Protect feet from spills and broken glass | Always in laboratory |
Standard Precautions
- Hand Hygiene: Wash hands frequently with soap and water, especially before eating and after handling specimens
- No Eating/Drinking: Strictly prohibited in the laboratory to prevent ingestion of pathogens
- No Smoking/Cosmetics: Avoid applying makeup or lipstick in the lab
- Proper Handling of Sharps: Use sharps containers, never recap needles, use safe needle disposal devices
- Spill Management: Clean up spills immediately using appropriate disinfectants (0.5% bleach)
Biohazard Management
| Hazard Type | Risk Level | Control Measures |
|---|---|---|
| Bloodborne Pathogens (HIV, HBV, HCV) | High | Standard precautions, proper disposal, vaccination |
| Sharps Injuries (Needles, Glass) | High | Safe handling, immediate disposal, incident reporting |
| Chemical Exposure | Medium-High | Proper storage, fume hood use, appropriate ventilation |
| Biological Aerosols | Medium | Biosafety cabinet use, avoiding aerosol generation |
Emergency Procedures
- Needle/Sharp Injury: Encourage bleeding, wash thoroughly, report immediately, seek medical evaluation
- Splash to Eyes/Mouth: Rinse immediately with water for 15 minutes, seek medical attention
- Skin Contact: Wash with soap and water, apply antiseptic, report to supervisor
- Chemical Spill: Follow chemical-specific procedures, use absorbent materials, report incident
🩸 1.2 Collection and Preservation of Different Samples for the Laboratory
1.2.1 Importance of Proper Sample Collection and Preservation
The quality of laboratory results depends significantly on proper sample collection and preservation. Improper handling can lead to:
- Hemolysis of blood cells
- Altered test results
- Rejection of samples
- Inappropriate clinical decisions
- Need for repeat testing (increased cost and time)
1.2.2 Blood Sample Collection Methods
Venipuncture (Gold Standard)
- Minimal contamination
- Adequate volume for multiple tests
- Preferred for most hematological tests
- Apply tourniquet above elbow (2-3 inches)
- Palpate and identify vein
- Clean site with 70% alcohol (circular motion, center outward)
- Allow to air dry (prevents hemolysis)
- Insert needle at 15-30° angle
- Collect in appropriate tube (fill to mark)
- Release tourniquet and withdraw needle
- Apply pressure to puncture site
Capillary Puncture
Used for: Infants, children, patients with difficult veins
- Finger tips (adults and older children)
- Heel (infants and newborns)
- Ear lobe
- Clean site with 70% alcohol
- Allow to air dry
- Puncture with sterile lancet
- Wipe away first drop with gauze
- Collect free-flowing blood in capillary tubes
1.2.3 Blood Collection Tubes and Anticoagulants
| Tube Color | Additive | Volume Ratio | Tests Performed | Special Instructions |
|---|---|---|---|---|
| Red Top (Serum) | None (Clot Activator) | 5:1 (blood:additive) | Chemistry, Serology, Immunology | Allow to clot 30 min, centrifuge |
| Purple/Lavender Top | EDTA (K₂EDTA or K₃EDTA) | 1:10 | Complete Blood Count (CBC), Blood films | Mix gently 8-10 times immediately |
| Blue Top | Citrate (3.2% or 3.8%) | 1:9 | Coagulation studies (PT, APTT, FDP) | Fill to mark, mix gently, transport vertically |
| Green Top | Heparin (Lithium or Sodium) | 1:11 | Plasma chemistry, Blood cultures | Mix thoroughly, centrifuge before separation |
| Gray Top | Potassium Oxalate/Sodium Fluoride | 1:9 | Blood glucose, Lactate | Mix immediately and thoroughly |
| Yellow Top | Sodium Polyanethol Sulfonate (SPS) | 1:9 | Blood cultures | Mix gently, use within 3 hours |
1.2.4 Sample Preservation Techniques
Temperature Control
🌡️ Room Temperature (18-25°C)
For: Coagulation studies, Blood cultures, Certain immunology tests
Duration: 1-4 hours
❄️ Refrigeration (2-8°C)
For: Most chemistry tests, Immunology samples, Long-term storage
Duration: Up to 24-48 hours
🧊 Freezing (-20°C)
For: Extended storage, Special tests, Research samples
Duration: Weeks to months
❄️❄️ Cryopreservation (-70°C or below)
For: Long-term storage, Liquid nitrogen storage
Duration: Years
Chemical Stabilization
- Anticoagulants (EDTA, Citrate, Heparin): Prevent clotting for extended shelf life
- Preservatives: Sodium fluoride prevents glycolysis and bacterial growth
- Cryoprotectants: DMSO (dimethyl sulfoxide) prevents ice crystal formation during freezing
Light Protection
- Light-sensitive tests (bilirubin, beta-carotene) require opaque containers
- Store in brown/amber bottles or wrapped containers
- Keep away from direct sunlight
1.2.5 Sample Rejection Criteria
- Hemolyzed (presence of free hemoglobin)
- Clotted when anticoagulant was required
- Insufficient volume for testing
- Contaminated (visible particles or growth)
- Unlabeled or mislabeled
- Expired collection tube
- Wrong collection tube used
- Improper mixing or inversion ratio
- Damaged container
- Prolonged time since collection without proper storage
1.2.6 Sample Transportation
- Use leak-proof containers or transport boxes
- Maintain appropriate temperature during transport
- Label all samples clearly with patient identification
- Keep samples upright when possible
- Use ice packs for refrigerated samples if transport time > 1 hour
- Complete transportation within 2-4 hours of collection
- Document time of collection and time of receipt
🧪 1.3 Preparation of Chemicals and Different Strains for the Hematological Tests
1.3.1 Role of Chemicals and Reagents in Haematology
Chemicals and reagents are essential components of hematological testing. They include anticoagulants, stains, buffers, and culture media. Proper preparation and standardization ensure:
- Accurate and consistent test results
- Reproducible staining and reactions
- Extended shelf life of reagents
- Prevention of contamination
1.3.2 Common Chemicals Used in Haematology
Anticoagulants
| Anticoagulant | Chemical Formula | Mechanism | Use | Concentration |
|---|---|---|---|---|
| EDTA (Ethylenediaminetetraacetic acid) |
C₁₀H₁₆N₂O₈ | Chelates calcium, prevents coagulation cascade | CBC, Blood films, Hematology procedures | 1.5-2.2 mg/mL blood |
| Sodium Citrate | C₆H₅O₇Na₃ | Binds calcium, inhibits thrombin formation | Coagulation studies (PT, APTT) | 3.2% or 3.8% (1:9 ratio) |
| Lithium Heparin | C₁₂H₁₆LiNO₁₁S | Potentiates antithrombin III | Plasma chemistry, Drug levels | 50-100 USP units/mL |
| Potassium Oxalate | K₂C₂O₄ | Precipitates calcium | Glucose, Special chemistry tests | Varied depending on use |
Stains and Staining Solutions
Wright-Giemsa Stain
Composition: Eosin Y and methylene blue
Use: Blood film examination, WBC differential, RBC morphology
Preparation: Dissolve powdered stain in methanol, filter, store in amber bottle
Prussian Blue Stain
Composition: Ferric ferrocyanide
Use: Iron detection in RBCs, Sideroblast identification
Preparation: Mix ferric chloride and potassium ferrocyanide solutions
Leishman Stain
Composition: Methylene blue and eosin
Use: Blood films, Platelets and WBC examination
Preparation: Similar to Wright-Giemsa, may be quick or conventional
Supravital Stains
Examples: Brilliant Cresyl Blue, New Methylene Blue
Use: Reticulocyte counting, Heinz body detection
Preparation: Dissolve in saline, filter sterilize, store at 4°C
Buffers and Diluting Solutions
| Solution | Composition | pH | Use |
|---|---|---|---|
| Phosphate Buffer | Na₂HPO₄ and NaH₂PO₄ | 7.0-7.4 | Stain preparation, Cell dilution |
| Normal Saline | 0.9% NaCl in distilled water | 6.5-7.0 | Cell dilution, RBC counting, Washing |
| Hayem’s Solution | HgCl₂, NaCl, Na₂SO₄ mixture | 6.5-7.0 | RBC dilution, Hemocytometry |
| Turk’s Solution | Acetic acid, Methyl violet, Saline | Acidic | WBC dilution, RBC lysis |
1.3.3 Reagent Preparation Guidelines
General Principles
- Use analytical grade chemicals only
- Prepare solutions in distilled or deionized water
- Maintain proper pH using appropriate buffers
- Filter sterilize when necessary to prevent contamination
- Label all reagents with date prepared and expiration date
- Store in appropriate containers (amber/brown for light-sensitive reagents)
- Maintain proper temperature for storage
- Document preparation method and lot numbers
Stain Preparation Steps
Weigh and Dissolve
Weigh required amount of powdered stain and dissolve in appropriate solvent (usually methanol for most blood stains) with gentle stirring over 30-60 minutes
Filter
Pass through filter paper (Whatman No. 1) or 0.22 µm membrane filter to remove undissolved particles and ensure clarity
Bottle and Label
Transfer to amber/brown bottles to prevent light exposure. Label with stain name, concentration, date prepared, and expiration date
Storage
Store at room temperature away from direct sunlight. Most stains remain viable for 6-12 months if properly stored
1.3.4 Bacterial and Culture Media Preparation
Culture Media Types
| Media Type | Composition | Purpose | Preparation Method |
|---|---|---|---|
| Nutrient Agar | Peptone, beef extract, NaCl, agar | General bacterial culture, QC organisms | Dissolve components, autoclave at 121°C, 15 min |
| Blood Agar | Nutrient agar + 5% sheep blood | Selective culture, Hemolysis detection | Prepare nutrient agar, cool to 50°C, add blood, pour plates |
| MacConkey Agar | Peptone, bile salts, lactose, indicator | Gram-negative bacteria differentiation | Dissolve, autoclave, pour into plates |
| Saline Suspension | 0.9% NaCl, bacteria culture | QC organism standardization | Suspend colonies in saline, adjust turbidity |
Quality Control Organisms
Strains commonly used for haematological QC:
- Staphylococcus aureus (ATCC 25923): Positive control for staining, culture methods
- Escherichia coli (ATCC 25922): Gram-negative control, hematology analyzer QC
- Streptococcus pneumoniae: Blood film control, culture verification
- Candida albicans: Fungal detection control
1.3.5 Chemical Storage and Safety
- Temperature: Store chemicals at appropriate temperature (room temperature unless specified)
- Light Protection: Use amber bottles for light-sensitive chemicals
- Humidity Control: Keep in dry environment to prevent degradation
- Segregation: Keep incompatible chemicals separate (acids away from bases)
- Labeling: Clear labels with chemical name, concentration, date, hazard warnings
- Inventory: Maintain chemical inventory log with expiration dates
- Disposal: Follow institutional disposal procedures for expired chemicals
✓ 1.4 Quality Control in the Laboratory
1.4.1 Understanding Quality Control (QC)
Quality Control is the process of assessing and monitoring the analytical performance of laboratory procedures to ensure reliable and accurate test results. It is a continuous cycle of testing, analysis, and corrective action.
- Detect and prevent analytical errors
- Ensure consistent accuracy of test results
- Identify instrument malfunctions early
- Validate analytical methodology
- Ensure patient safety through reliable results
- Maintain laboratory accreditation standards
1.4.2 Internal Quality Control (IQC)
IQC involves the systematic, day-to-day monitoring of the accuracy and precision of laboratory procedures using specially prepared control samples.
Components of IQC
Control Materials
Types:
- Commercial control bloods
- Known value samples
- Prepared reagent solutions
- Quality control standards
Control Levels
Typically 3 levels:
- Normal range
- Slightly abnormal
- Significantly abnormal
Frequency
QC runs performed:
- At start of each shift
- After instrument maintenance
- When reagents changed
- Periodically during testing
Documentation
Essential records:
- QC run results
- Corrective actions
- Instrument maintenance logs
- Reagent lot numbers
IQC Statistical Methods
| Method | Concept | Calculation | Use |
|---|---|---|---|
| Mean (Average) | Central tendency | ΣX / n | Determine expected value |
| Standard Deviation (SD) | Measure of dispersion | √[Σ(X-mean)² / n-1] | Define acceptable limits |
| Coefficient of Variation (CV) | Relative precision | (SD / mean) × 100% | Compare method precision |
| Accuracy | Closeness to true value | Measured vs. Expected value | Verify calibration |
| Precision | Reproducibility | Measured by CV or SD | Assess method variability |
Levey-Jennings Chart
How to construct:
- Plot QC results on Y-axis (values)
- Plot time on X-axis (days/runs)
- Draw horizontal line for mean
- Draw lines for ±1 SD, ±2 SD, ±3 SD
- Plot each QC run result
- Look for trends, shifts, or out-of-control points
Decision rules:
- 1-3s rule: One result > 3 SD = OUT OF CONTROL
- 2-2s rule: Two consecutive results > 2 SD same side = OUT OF CONTROL
- R-4s rule: Range of two controls > 4 SD = OUT OF CONTROL
1.4.3 External Quality Control (EQC / External Quality Assessment)
EQC involves participating in external quality assessment schemes to compare laboratory performance with other peer laboratories and identify knowledge gaps.
Benefits of EQC Programs
- Benchmarking: Compare results with other laboratories
- Performance Assessment: Identify analytical weaknesses
- Accreditation: Most standards require participation in EQC
- Staff Training: Identifies need for personnel training
- Method Validation: Confirms new methods are performing acceptably
- Equipment Evaluation: Compare different instrument performances
EQC Process
Participate in Scheme
Laboratory enrolls in external quality assessment program (e.g., EQAS, proficiency testing programs)
Receive Samples
Receive coded reference samples (usually monthly or quarterly) with unknown values
Test and Report
Perform tests using routine methods and report results to EQA provider within specified timeframe
Receive Assessment
EQA provider returns performance report showing your result vs. peer results and assigned value
Evaluate and Correct
Review performance, identify errors, implement corrective actions if needed
1.4.4 Hematology Analyzer Quality Control
Analyzer Calibration
Frequency:
- Daily (if analyzer so requires)
- Weekly for stable systems
- After maintenance or reagent changes
- When results appear incorrect
Materials: Reference calibrators provided by manufacturer
Parameters to Monitor
| Parameter | Description | Normal Range (Example) | Clinical Significance |
|---|---|---|---|
| WBC Count | White Blood Cell count | 4.5-11.0 × 10⁹/L | Infection, immune status, leukemia |
| RBC Count | Red Blood Cell count | 4.5-5.5 × 10¹²/L (male) | Anemia, polycythemia |
| Hemoglobin (Hb) | Oxygen carrying capacity | 13.5-17.5 g/dL (male) | Oxygen delivery, anemia detection |
| Hematocrit (Ht) | % RBC volume in blood | 41-53% (male) | Plasma vs cell volume ratio |
| MCV | Mean Cell Volume | 80-100 fL | RBC size classification |
| PLT Count | Platelet count | 150-400 × 10⁹/L | Bleeding risk, clotting disorder |
1.4.5 Manual Blood Smear Review (MBSR)
Purpose: Microscopic verification of analyzer results and detection of abnormalities
When to perform:
- All routine samples (delta check)
- Abnormal analyzer results
- Critical values
- Patient follow-up samples
What to examine:
- RBC morphology (shape, color, inclusions)
- WBC differential count
- Platelet assessment
- Abnormal cells or parasites
1.4.6 Troubleshooting Common QC Problems
| Problem | Possible Causes | Corrective Action |
|---|---|---|
| Results out of control limits | Analyzer malfunction, reagent degradation, calibration error | Repeat QC, recalibrate, check reagent expiration, contact service |
| Upward or downward trend | Reagent deterioration, gradual calibration drift | Replace reagent, recalibrate, check storage conditions |
| High coefficient of variation | Poor precision, sample dilution errors, instrument wear | Check dilution accuracy, perform instrument maintenance |
| Delta check failures | Patient condition change, sample error, analyzer error | Review patient history, recollect sample, retest |
1.4.7 Documentation and Records
- Daily QC run sheets with results and time
- QC material lot numbers and expiration dates
- Calibration records and materials used
- Instrument maintenance logs
- Corrective action reports
- EQA/Proficiency testing results and responses
- Staff training records
- Trend analysis charts (Levey-Jennings)
🩸 1.5 Formation and Development of Erythrocytes, Leucocytes, Thrombocytes
1.5.1 Overview of Hematopoiesis
Hematopoiesis is the production and development of blood cells. All blood cells develop from a single cell type called the hematopoietic stem cell (HSC) in the bone marrow. This process is continuous throughout life, producing approximately:
- 200 billion erythrocytes per day
- 100 billion leukocytes per day
- 100 billion platelets per day
- Erythropoietin (EPO): Hormone regulating RBC production; secreted by kidneys in response to hypoxia
- G-CSF and GM-CSF: Colony-stimulating factors promoting granulocyte and monocyte development
- Thrombopoietin (TPO): Growth factor regulating platelet production
- Cytokines: IL-3, IL-6, SCF; various interleukins promoting cell differentiation
1.5.2 Erythropoiesis (Red Blood Cell Formation)
Site of Erythropoiesis
Primary: Bone marrow (specifically the red marrow)
Secondary: Liver and spleen (during stress or disease)
Stages of Erythrocyte Development
| Stage | Cell Name | Morphological Features | Duration | Nuclear Status |
|---|---|---|---|---|
| 1. Progenitor | BFU-E, CFU-E | Blast-like cells; colony-forming units | Variable | Nucleated |
| 2. Proerythroblast | Pronormoblast | Large nucleus, abundant cytoplasm, prominent nucleoli, basophilic cytoplasm | 1-2 days | Nucleated |
| 3. Early Basophilic Erythroblast | Early Normoblast | Smaller nucleus, more condensed chromatin, deep blue cytoplasm (RNA rich) | 1-2 days | Nucleated |
| 4. Intermediate Basophilic Erythroblast | Intermediate Normoblast | Further size reduction, visible nuclear membrane, mixed staining (blue-gray) | 1-2 days | Nucleated |
| 5. Late Basophilic Erythroblast | Late Normoblast | Small dense nucleus, pink-blue cytoplasm (increasing Hb content) | 1-2 days | Nucleated |
| 6. Orthochromatic Erythroblast | Orthochromatic Normoblast | Tiny dense nucleus (pyknotic), pink/acidophilic cytoplasm (Hb filled) | 1-2 days | Nucleated |
| 7. Reticulocyte | Reticulocyte | No nucleus, residual RNA (seen with supravital stain as precipitate) | 1-2 days in marrow, 1-2 days in blood | Anucleate |
| 8. Mature RBC | Erythrocyte | Biconcave disc, 7-8 µm diameter, no nucleus, pink staining | 120 days lifespan | Anucleate |
Morphological Changes During Erythropoiesis
Nucleus Changes
- Decreases in size
- Becomes more condensed
- Chromatin becomes pyknotic
- Eventually extruded
Cytoplasm Changes
- Initially basophilic (RNA rich)
- Progressively becomes acidophilic
- Hemoglobin accumulates
- Organelles disappear
Reticulocyte Development
Characteristics:
- Larger than mature RBCs (8-10 µm)
- Contain RNA residues visible with supravital stains (Brilliant Cresyl Blue, New Methylene Blue)
- Appear as precipitate threads/filaments (“reticulae”)
- Retain slight hemoglobin synthesis capability
- Normal count: 0.5-2% of total RBCs
Clinical significance:
- Elevated in hemolytic anemia (compensatory response)
- Elevated after hemorrhage (bone marrow response)
- Decreased in bone marrow failure (aplastic anemia)
- Used as marker of RBC production activity
1.5.3 Leucopoiesis (White Blood Cell Formation)
Pathways of Leucopoiesis
Leucopoiesis follows two main pathways from the hematopoietic stem cell:
Myeloid Pathway
Produces:
- Granulocytes (neutrophils, eosinophils, basophils)
- Monocytes
- Macrophages
Primary factor: GM-CSF, G-CSF
Lymphoid Pathway
Produces:
- T lymphocytes (thymus-dependent)
- B lymphocytes (marrow-dependent)
- Natural Killer cells
Primary factor: IL-7, IL-15
Granulopoiesis (Neutrophil Development)
| Stage | Cell Name | Morphological Features | Duration | Location |
|---|---|---|---|---|
| 1. Blast | Myeloblast | Large cell, large nucleus, scanty cytoplasm, 1-2 prominent nucleoli | ~24 hours | Bone marrow |
| 2. Promyelocyte | Promyelocyte | Large cell, prominent Auer rods possible, numerous cytoplasmic granules | ~24 hours | Bone marrow |
| 3. Myelocyte | Myelocyte (Neutro, Eo, Baso) | Smaller nucleus, specific granulation begins, no nucleoli visible | ~4 days | Bone marrow |
| 4. Metamyelocyte | Metamyelocyte | Kidney bean-shaped nucleus, mature granulation, reduced cytoplasm | ~2 days | Bone marrow |
| 5. Band Cell | Band Neutrophil | Non-segmented nucleus (band-like or horseshoe shape) | ~1 day | Bone marrow to blood |
| 6. Mature | Segmented Neutrophil | 3-5 lobed nucleus, abundant fine granules, mature appearance | ~120 hours total, several days in circulation | Blood and tissues |
Monocytopoiesis (Monocyte Development)
- Monoblast: Similar to myeloblast; primarily in bone marrow
- Promonocyte: Intermediate stage; begins cytoplasmic differentiation
- Monocyte: Mature form; large nucleus, abundant cytoplasm, released to blood
- Macrophage: Tissue form; exits blood to settle in tissues; functions in phagocytosis
Lymphopoiesis (Lymphocyte Development)
T Cell Development (Thymic pathway):
- Early lymphoid progenitor cell → enters thymus
- Thymocyte maturation (positive and negative selection)
- Naive T cells exit thymus to lymph nodes, spleen
B Cell Development (Marrow pathway):
- Early lymphoid progenitor cell → bone marrow
- Pro-B cell → Pre-B cell → Immature B cell
- Mature B cells released to secondary lymphoid organs
1.5.4 Thrombocytopoiesis (Platelet Formation)
Process of Platelet Production
Megakaryoblast Stage
Pluripotent stem cell differentiates under influence of TPO into megakaryoblasts (large cells with high proliferative capacity)
Megakaryocyte Maturation
Megakaryocytes undergo endomitosis (nuclear multiplication without cytokinesis), resulting in polyploidy (up to 128n DNA content). Multiple rounds without cell division produce increasingly large cells.
Cytoplasmic Maturation
Megakaryocyte develops extensive demarcation membranes and accumulates platelet-specific proteins (GPIIb/IIIa, GP1b, von Willebrand factor, fibrinogen). Cytoplasm fills with alpha and dense granules.
Platelet Release (Thrombopoiesis)
Demarcation membranes delineate individual platelets. Megakaryocytes extend pseudopodia into sinusoids, releasing mature platelets into blood circulation (8-12 days after differentiation from stem cell).
Megakaryocyte Morphology
| Stage | Cell Size | Nuclear Appearance | Cytoplasm |
|---|---|---|---|
| Megakaryoblast | 15-20 µm | Large, round, vesicular, prominent nucleoli | Basophilic, scant |
| Promegakaryocyte | 20-40 µm | Large, multiple lobes developing | Basophilic, increasing |
| Megakaryocyte | 40-150 µm | Massive, multilobed nucleus (6-16 lobes) | Abundant, granular, pink (demarcation membrane visible) |
Platelet Characteristics
- Size: 2-4 µm diameter (discoid)
- No nucleus: Fragments of megakaryocyte cytoplasm
- Lifespan: 7-10 days in circulation
- Count: 150,000-400,000 per µL
- Functions: Hemostasis, thrombosis, wound healing, inflammation
- Daily production: ~100 billion platelets
1.5.5 Regulation of Hematopoiesis
Humoral Factors (Cytokines and Hormones)
| Factor | Source | Targets | Primary Actions |
|---|---|---|---|
| Erythropoietin (EPO) | Kidneys (90%), Liver (10%) | BFU-E, CFU-E, Proerythroblasts | Stimulates RBC production; responds to hypoxia |
| Thrombopoietin (TPO) | Liver (primary), Kidney | Megakaryoblasts, Megakaryocytes | Stimulates platelet production |
| G-CSF | Endothelial cells, Fibroblasts, Monocytes | Myeloid progenitors, Neutrophils | Promotes granulocyte differentiation and release |
| GM-CSF | T cells, Fibroblasts, Endothelial cells | Multi-lineage progenitors | Stimulates granulocyte and monocyte production |
| IL-3 | T cells | Pluripotent stem cells | Multi-lineage colony formation |
| SCF (Stem Cell Factor) | Bone marrow stroma | Hematopoietic stem cells | HSC proliferation and survival |
Feedback Regulation
- Oxygen saturation: High O₂ → decreased EPO production → decreased RBC formation
- Platelet count: High platelet levels → decreased TPO action → decreased megakaryocyte formation
- Neutrophil count: High neutrophil levels → decreased G-CSF and GM-CSF → slowed granulopoiesis
1.5.6 Clinical Correlation and Abnormalities
Erythropoiesis Disorders
| Condition | Mechanism | RBC Features | Cause |
|---|---|---|---|
| Microcytic Anemia | Decreased RBC size (MCV < 80 fL) | Small RBCs, pale appearance | Iron deficiency, thalassemia, chronic disease |
| Macrocytic Anemia | Increased RBC size (MCV > 100 fL) | Large RBCs, elevated reticulocytes | B12 deficiency, folate deficiency, bone marrow stress |
| Polycythemia | Excessive RBC production | Increased Hb, Ht, RBC count | High altitude, chronic hypoxia, EPO-secreting tumors |
Leucopoiesis Disorders
- Leukopenia: Decreased WBC production; risk of infection
- Leukocytosis: Increased WBC production; infection response or leukemia
- Acute Leukemia: Uncontrolled blast proliferation; blocks normal differentiation
- Chronic Leukemia: Excessive mature cell production; slowly progressive
Thrombocytopoiesis Disorders
- Thrombocytopenia: Platelet count < 150,000/µL; increased bleeding risk
- Thrombocytosis: Platelet count > 400,000/µL; increased clotting risk
- Immune Thrombocytopenic Purpura (ITP): Autoimmune destruction of platelets
- Disseminated Intravascular Coagulation (DIC): Excessive platelet consumption
1.5.7 Bone Marrow Examination
Indications: Diagnosis of hematologic disorders, evaluation of anemias, leukemias, platelet disorders
Specimens:
- Bone marrow aspirate (liquid suspension of marrow cells)
- Bone marrow biopsy (tissue core showing marrow architecture)
Common findings evaluated:
- Cellularity (percentage of marrow that is active)
- M:E ratio (myeloid to erythroid cells)
- Presence of dysplasia or abnormal cells
- Fibrosis or infiltration