The Erythrocyte Sedimentation Rate (ESR) is a widely utilized, non-specific laboratory test that measures the rate at which red blood cells (RBCs) in a column of anti-coagulated blood settle over a specific period, typically one hour. While not diagnostic of any particular disease, an elevated ESR often indicates the presence of an inflammatory or infectious process, autoimmune disease, or certain cancers. Conversely, a low ESR can also provide clinical insight. Understanding the precise methodologies for ESR determination is crucial for obtaining accurate and reliable results, which directly impact patient management.

Understanding Erythrocyte Sedimentation Rate (ESR)

The principle behind ESR is simple: under normal conditions, red blood cells, due to their negative surface charge, repel each other and are suspended uniformly in plasma. In inflammatory states, the concentration of acute phase proteins, particularly fibrinogen and immunoglobulins, increases in the plasma. These proteins neutralize the negative charge on the RBC surfaces, causing them to aggregate into rouleaux (stacks of coins). These larger, heavier rouleaux settle faster by gravity, thus increasing the ESR.

The Westergren Method: The Gold Standard

The Westergren method is the most commonly used and widely accepted method for ESR determination globally, often considered the “gold standard” due to its sensitivity, especially for detecting elevated ESR values.

Principle: The Westergren method measures the rate of sedimentation of red blood cells in a precisely calibrated, long, narrow tube over one hour, using a diluted blood sample. The dilution with an anticoagulant (typically sodium citrate) prevents clotting and minimizes rouleaux formation before measurement, ensuring a more standardized sedimentation.

Equipment Required:

1. Westergren pipette/tube: A specialized glass tube, 300 mm long, with a uniform internal bore of 2.55 mm, graduated in millimeters from 0 at the top to 200 at the bottom.
2. Westergren rack/stand: A perfectly vertical stand to hold the Westergren tube upright during the sedimentation period.
3. Stopwatch or timer: For accurate timing of the one-hour sedimentation period.
4. EDTA whole blood sample: Collected via venipuncture.
5. Anticoagulant/Diluent: 3.8% Trisodium Citrate solution or 0.85% physiological saline.
6. Pipettes: For precise measurement and transfer of blood and diluent.
7. Mixing device: Mechanical rotator or gentle manual mixing.
8. Biohazard waste container.
9. Personal Protective Equipment (PPE): Gloves, lab coat, safety glasses.

Specimen Requirements:

• Anticoagulant: The blood sample must be collected into an evacuated tube containing K2EDTA (ethylenediaminetetraacetic acid) as the anticoagulant.
• Sample Stability: The ESR test should be performed within 4 hours of blood collection if stored at room temperature (18-25°C) or within 12 hours if refrigerated at 4°C. If refrigerated, the sample must be brought to room temperature before testing.
• Dilution: For the Westergren method, the EDTA whole blood is diluted with 3.8% Trisodium Citrate solution (or 0.85% NaCl) in a ratio of 4 parts blood to 1 part diluent. This specific dilution is critical for accurate results.

Step-by-Step Procedure:

1. Patient Preparation & Blood Collection:
   • No special patient preparation is typically required, though certain medications can affect results.
   • Collect venous blood using standard venipuncture techniques into an EDTA vacutainer tube. Ensure adequate mixing by gently inverting the tube 8-10 times immediately after collection to prevent clotting.
2. Sample Preparation (Dilution):
   • Gently invert the EDTA blood sample several times to ensure thorough mixing.
   • Into a clean, dry test tube or a specialized ESR mixing vial, accurately combine 0.5 mL of 3.8% Trisodium Citrate solution with 2.0 mL of the well-mixed EDTA whole blood. This maintains the 1:4 diluent-to-blood ratio.
   • Alternatively, some systems use pre-filled citrate tubes where blood is added directly to achieve the correct ratio.
   • Mix the diluted blood thoroughly for at least 30 seconds using a mechanical rotator or by gentle inversion. Avoid vigorous shaking, which can cause hemolysis or air bubbles.
3. Filling the Westergren Tube:
   • Carefully draw the diluted blood mixture into the Westergren pipette up to the “0” mark (the top graduation).
   • Ensure there are no air bubbles trapped in the column of blood. If bubbles are present, expel the sample and refill the tube.
   • Wipe any excess blood from the outside of the tube.
4. Placement in Rack:
   • Immediately place the filled Westergren tube vertically into the Westergren rack, ensuring it is perfectly upright and free from vibrations. The rack should be placed on a level surface away from direct sunlight or heat sources.
   • Improper tilting, even by a small degree, can significantly affect the ESR value.
5. Timing the Sedimentation:
   • Start the stopwatch immediately after placing the tube in the rack.
   • Allow the tube to stand undisturbed for exactly 60 minutes (one hour) at room temperature (18-25°C).
6. Reading the Results:
   • After precisely 60 minutes, read the ESR by observing the clear plasma column formed above the red blood cells.
   • Record the distance (in millimeters) from the “0” mark (top of the plasma meniscus) to the top of the packed red blood cell column. Do not include the buffy coat (white blood cells and platelets layer) in the reading.
   • Values are expressed in mm/hour.

Interpretation of Results: Normal ESR values vary with age and gender. While specific ranges may differ slightly between laboratories, typical ranges are:

• Males under 50 years: 0-15 mm/hour
• Males over 50 years: 0-20 mm/hour
• Females under 50 years: 0-20 mm/hour
• Females over 50 years: 0-30 mm/hour
• Children: 0-10 mm/hour
• Newborns: 0-2 mm/hour

Elevated ESR indicates an increased inflammatory response, infection, autoimmune disease (e.g., rheumatoid arthritis, lupus), certain cancers (e.g., multiple myeloma, lymphoma), or chronic kidney disease. Decreased ESR can be seen in conditions like polycythemia, sickle cell anemia, or spherocytosis.

Advantages of Westergren Method:

• Widely accepted and standardized.
• Highly sensitive for detecting elevated ESR values, especially those greater than 100 mm/hr, as the longer tube allows for greater sedimentation.
• Results are less affected by anemia due to dilution.

Disadvantages of Westergren Method:

• Requires a larger blood volume (though often collected for other tests).
• Potential for dilution errors if not performed carefully.
• Requires specific, longer tubes and a dedicated rack.

The Wintrobe Method

The Wintrobe method is an older technique for ESR estimation and is less sensitive than the Westergren method for very high ESR values. However, it offers the advantage of simultaneously determining the Packed Cell Volume (PCV) or Hematocrit (Hct) from the same sample.

Principle: The Wintrobe method measures the sedimentation rate of red blood cells in an undiluted anticoagulated blood sample placed in a shorter, narrower tube over one hour.

Equipment Required:

1. Wintrobe tube: A specialized glass tube, 110 mm long, with a uniform internal bore of 3 mm, graduated with two scales: one from 0-100 mm downwards for ESR, and another from 0-100 mm upwards for PCV.
2. Wintrobe rack/stand: A perfectly vertical stand to hold the Wintrobe tube upright.
3. Stopwatch or timer.
4. EDTA whole blood sample: Collected via venipuncture.
5. Pasteur pipette or syringe with needle: For accurate filling of the narrow tube.
6. Biohazard waste container.
7. Personal Protective Equipment (PPE).

Specimen Requirements:

• Anticoagulant: K2EDTA is the preferred anticoagulant.
• Sample Stability: The ESR test should be performed within 4 hours if stored at room temperature or 12 hours if refrigerated.
• No Dilution: Unlike the Westergren method, the Wintrobe method uses undiluted whole blood.

Step-by-Step Procedure:

1. Patient Preparation & Blood Collection:
   • Collect venous blood into an EDTA vacutainer tube using standard venipuncture.
   • Gently invert the tube 8-10 times immediately after collection to ensure thorough mixing and prevent clotting.
2. Filling the Wintrobe Tube:
   • Using a clean Pasteur pipette or a syringe with a long needle, carefully draw up the well-mixed, undiluted EDTA blood.
   • Insert the pipette/needle to the very bottom of the Wintrobe tube.
   • Slowly expel the blood from the pipette/syringe, gradually withdrawing the pipette/needle as the tube fills, ensuring no air bubbles are trapped.
   • Fill the tube precisely to the “0” mark (the top graduation). Overfilling or underfilling can lead to inaccurate results.
   • Wipe any excess blood from the outside of the tube.
3. Placement in Rack:
   • Immediately place the filled Wintrobe tube vertically into the Wintrobe rack, ensuring it is perfectly upright and free from vibrations. The rack should be placed on a level surface away from direct sunlight or heat sources.
4. Timing the Sedimentation:
   • Start the stopwatch immediately after placing the tube in the rack.
   • Allow the tube to stand undisturbed for exactly 60 minutes (one hour) at room temperature (18-25°C).
5. Reading the Results:
   • ESR Reading: After precisely 60 minutes, read the ESR by observing the clear plasma column. Record the distance (in millimeters) from the “0” mark (top of the plasma meniscus) to the top of the packed red blood cell column using the scale that reads downwards. Do not include the buffy coat.
   • PCV (Optional but commonly done): After the ESR reading, the tube can be centrifuged to determine the Packed Cell Volume. Read the volume of packed red cells using the scale that reads upwards from the bottom of the tube.

Interpretation of Results: Normal Wintrobe ESR values are generally slightly lower than Westergren values due to the shorter tube and undiluted blood. Typical normal ranges are:

• Males: 0-9 mm/hour
• Females: 0-20 mm/hour

The clinical significance of elevated or decreased Wintrobe ESR is similar to that of Westergren.

Advantages of Wintrobe Method:

• Requires a smaller volume of blood.
• The same tube can be used to determine PCV/Hematocrit after ESR reading.
• No dilution step, reducing potential for dilution errors.

Disadvantages of Wintrobe Method:

• Less sensitive for significantly elevated ESR values (e.g., >60 mm/hr) because the shorter tube causes the RBCs to pack quickly, limiting further sedimentation measurement.
• More prone to clotting if the blood sample is not properly anti-coagulated or if there’s a delay in testing.
• Plasma layer is smaller, making it harder to read the meniscus accurately in some cases.

Comparison of Westergren and Wintrobe Methods

The Westergren method is generally preferred for routine ESR testing due to its better sensitivity and standardization, while the Wintrobe method finds niche use where both ESR and PCV are required from a single sample, or when dealing with limited blood volumes.

Factors Influencing ESR Results

Both physiological and technical factors can significantly influence ESR results, leading to false positives or negatives.

Physiological Factors:

• Increased ESR: Anemia (especially macrocytic), inflammation, infection, pregnancy (after 1st trimester), older age, female gender, renal disease, liver disease, certain medications.
• Decreased ESR: Polycythemia, sickle cell anemia, spherocytosis, microcytosis (severe), extreme leukocytosis, severe hypofibrinogenemia, high bile salt concentration.

Technical Factors:

• Temperature: Testing outside the 18-25°C range can alter results (higher temp increases ESR, lower temp decreases).
• Tube Tilt: Even a slight tilt of the ESR tube can significantly increase the sedimentation rate.
• Vibration: Any movement or vibration during the 60-minute period will invalidate the test.
• Air Bubbles: Trapped air bubbles in the blood column can disrupt sedimentation.
• Clotting: Incomplete mixing with anticoagulant or delays can lead to micro-clots, affecting sedimentation.
• Incorrect Anticoagulant/Ratio: Using the wrong anticoagulant or an incorrect blood-to-anticoagulant ratio.
• Delayed Testing: Old blood samples can yield inaccurate results due to red cell shape changes.

Quality Control and Safety Precautions

1. Quality Control (QC):

• Regularly check the calibration of Westergren and Wintrobe tubes.
• Utilize commercial ESR control materials (low, normal, high) to monitor the accuracy and precision of the procedure. Perform QC at least daily or per laboratory protocol.
• Maintain accurate temperature control in the testing area.
• Ensure proper training and competency of personnel performing the test.

2. Safety Precautions:

• Always treat blood samples as potentially infectious biohazards.
• Wear appropriate Personal Protective Equipment (PPE), including gloves, lab coat, and safety glasses, when handling blood and blood products.
• Dispose of all used tubes, pipettes, and contaminated materials in designated biohazard waste containers.
• Follow strict laboratory safety guidelines for sharps disposal and spill management.

Conclusion

The estimation of Erythrocyte Sedimentation Rate using either the Westergren or Wintrobe method remains a valuable, albeit non-specific, tool in clinical diagnostics. While automated ESR analyzers are becoming more common, understanding these manual methods is foundational for laboratory professionals. Adherence to precise procedural steps, careful attention to technical details, and awareness of influencing factors are paramount to ensuring the accuracy and reliability of ESR results, thereby contributing to effective patient care.