Overview
Erythrocyte Sedimentation Rate (ESR) is the measurement, after 1 hour, of the sedimentation of red cells when blood is allowed to stand in an open ended glass tube mounted vertically on a stand. It is a simple, inexpensive screening test for an underlying acute phase response in the body.
This lesson also covers the reticulocyte count — a reflection of erythropoietic activity used to assess bone marrow response and monitor treatment of anemia.
Learning Objectives
After this lesson you will be able to…- Describe the methods used for measuring ESR
- Enlist the factors influencing the sedimentation of red cells
- Discuss the significance of measuring ESR
- Describe the reticulocyte count and its significance
Clinical Story
Why This MattersA 60-year-old woman presents with joint pain, stiffness, and unexplained fatigue. Her physician suspects an inflammatory condition like rheumatoid arthritis and orders an ESR test. The lab technologist sets up a Westergren pipette, and after exactly 1 hour, reads a markedly elevated ESR — supporting the diagnosis and providing a baseline to monitor her response to treatment over time.
Core Concepts
Red cell sedimentation depends on the difference in specific gravity between red cells and plasma. As red cells are denser, they settle, producing a settling force, while plasma's upward movement produces a retarding force. Normally these forces are nearly equal, so little settling occurs. When red cells form rouleaux (stacked aggregates), their mass increases and the fall rate increases, raising ESR. Rouleaux formation is normally limited by the negative charge on red cells.
Sedimentation occurs in three stages: rouleaux formation, constant-speed sinking of aggregates, and final slowing as cells pack at the tube bottom.
Key factors include: Rate of rouleaux formation (increased by fibrinogen, acute phase proteins, immunoglobulins; retarded by albumin); Ratio of red cells to plasma/hematocrit (anemia increases ESR); Type of red cells (sickle cells, spherocytes decrease ESR as they don't form rouleaux); Plasma viscosity (increases ESR); Length of tube (longer tube = higher ESR); Verticality of the tube (tilted tube causes faster, falsely elevated sedimentation); Temperature (accelerates sedimentation, hence ESR is always measured at room temperature, 18-25°C).
ESR is a useful screening test for an underlying acute phase response. Causes of elevated ESR include tuberculosis, rheumatoid arthritis, myocardial infarction, multiple myeloma, pregnancy, neoplastic and degenerative diseases. Causes of reduced ESR include polycythemia, hypofibrinogenemia, hereditary spherocytosis, congestive heart failure, and sickle cell anemia. ESR is especially useful as an index of disease activity in tuberculosis and rheumatoid arthritis, and in diagnosing temporal arteritis and polymyalgia rheumatica.
Reticulocytes are immature red cells containing remnants of ribosomal RNA, which react with supravital dyes like new methylene blue or brilliant cresyl blue to form blue granules/filaments — visible only in unfixed vitally stained preparations. On methanol-fixed films, they appear as diffusely basophilic cells called polychromatophils. The reticulocyte count reflects erythropoietic activity: increased in hemolytic anemias (correlating with hemolysis severity), low in nutritional anemias before treatment (rising with hematinic therapy), and low in aplastic anemia or aplastic crisis.
Laboratory Principle
In the Westergren method, citrated whole blood is drawn into a long, narrow, vertical glass pipette and left undisturbed for exactly 60 minutes. Gravity causes red cells, which are denser than plasma, to settle. The height (in mm) of the clear plasma column that develops above the settled red cells is the ESR result. Conditions that promote rouleaux formation (such as elevated fibrinogen in inflammation) accelerate this settling, producing a higher ESR.
Equipment Required
Reagents & Materials
| Reagent / Material | Concentration / Grade | Purpose | Storage |
|---|---|---|---|
| Trisodium citrate | 3.8% | Anticoagulant; mixed 4:1 with blood (or 2ml blood directly into 0.5ml citrate) | Room temperature |
| New methylene blue | 1g in 100ml phosphate buffer, pH 6.5 | Supravital staining for reticulocyte count | Room temperature, protected from light |
Step-by-Step Procedure
Westergren Method
Mix the citrated blood sample well and draw it into the Westergren pipette exactly to the 0mm mark using a rubber teat.
Place the tube exactly vertical in the rack, which has a rubber cork at the bottom. Remove the teat and fix the tube with adjustable screws.
Leave the tube undisturbed for exactly 60 minutes, free from vibration and not exposed to direct sunlight.
Read the column of clear plasma above the column of sedimented red cells to the nearest mm. This is the ESR value, expressed in mm in the first hour.
Flow Diagram
Quality Control
The Westergren pipette must be clean and dry, dilute the sample just before setting up, never use mouth suction, ensure the pipette is placed exactly vertical, and set up the test at room temperature (18-25°C) — any deviation directly distorts results.
The International Council for Standardization in Hematology recommends the Westergren method as the standard reference method for ESR — automated analysers must be periodically validated against this gold standard.
Reference Values
Normal Ranges⚠️ Reference ranges may vary between laboratories. Always apply your laboratory's established reference intervals.
Clinical Interpretation
| Finding | Possible Significance | Action / Follow-up |
|---|---|---|
| Markedly elevated ESR | Tuberculosis, rheumatoid arthritis, malignancy, temporal arteritis, multiple myeloma | Correlate clinically; order CRP, further inflammatory or malignancy work-up as indicated |
| Reduced ESR | Polycythemia, hereditary spherocytosis, sickle cell anemia, congestive heart failure | Correlate with hematocrit and red cell morphology on smear |
| Low reticulocyte count with anemia | Impaired red cell production (aplastic anemia, marrow failure) | Investigate bone marrow function; consider marrow examination |
Common Errors & How to Avoid Them
Cause: If the tube is tilted, sedimentation occurs more quickly due to blood streaming down the sloped wall, falsely elevating the ESR.
Prevention: Always confirm the Westergren pipette is placed exactly vertical in the rack before timing begins.
Cause: Sedimentation is accelerated as temperature increases, so testing outside room temperature gives unreliable results.
Prevention: Always set up ESR at room temperature (18-25°C), away from direct sunlight or heat sources.
Cause: Preparing the citrate dilution well before testing can alter sedimentation properties.
Prevention: Always dilute the blood sample just before setting up the ESR test.
Laboratory Tips from the Bench
Always read the ESR at exactly 60 minutes — reading too early underestimates and too late overestimates the result, since sedimentation rate is not perfectly linear throughout the hour.
For reticulocyte counting, choose an area of the smear where cells are well spread and staining is even — count at least 1000 red cells under oil immersion for an accurate percentage.
Remember "FAT" for factors raising ESR: Fibrinogen ↑, Acute phase proteins ↑, Temperature ↑ — all three accelerate rouleaux formation and sedimentation.
Important Notes
While fibrinogen, acute phase proteins, and immunoglobulins promote rouleaux and raise ESR, albumin has the opposite effect and retards rouleaux formation, lowering ESR — an important nuance when interpreting borderline results in patients with liver disease or malnutrition.
The same immature red cells are called "reticulocytes" when seen with supravital staining (new methylene blue) and "polychromatophils" when seen as diffusely basophilic cells on a methanol-fixed Romanowsky-stained smear — same cell, different staining technique.
Interactive Quiz
Test Your KnowledgeFlashcards
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Clinical Case Study
Apply Your KnowledgeMrs. Sharma presents with a 3-month history of symmetric joint pain in her hands and wrists, morning stiffness lasting over an hour, and low-grade fatigue. Her physician suspects rheumatoid arthritis and orders ESR along with other tests.
An ESR of 58 mm/1st hour is markedly elevated above the normal female range of 10-20mm. Combined with her clinical presentation of symmetric joint pain and prolonged morning stiffness, this strongly supports an inflammatory process such as rheumatoid arthritis. ESR will serve as a useful marker to monitor disease activity and treatment response over time.
- →ESR is a non-specific screening test — it indicates inflammation but does not diagnose the specific cause
- →Serial ESR measurements are valuable for monitoring disease activity and response to therapy in rheumatoid arthritis
- →ESR must always be correlated with clinical findings and other laboratory tests for accurate diagnosis
Frequently Asked Questions
Women normally have a slightly lower hematocrit than men, meaning a relatively higher proportion of plasma to red cells. This higher plasma proportion promotes greater rouleaux formation, resulting in a naturally higher baseline ESR in women compared to men.
ESR is a non-specific marker that depends on plasma protein changes, which can take days to develop. Additionally, conditions like sickle cell anemia or hereditary spherocytosis can mask an expected ESR rise because abnormal red cell shapes resist rouleaux formation regardless of inflammation present.
Automated analysers use pre-evacuated tubes (no manual filling needed), give results in about 20 minutes instead of 60, allow multiple samples to be processed simultaneously, and can store data electronically — though they rely on the same fundamental Westergren principle for accuracy.
Quick Revision
10-Minute ReviewKey Takeaways
- ESR measures the sedimentation of red cells over exactly 1 hour
- The Westergren method is the internationally standardized reference technique
- Rouleaux formation, driven by fibrinogen and acute phase proteins, drives ESR elevation
- ESR is non-specific but valuable for screening and monitoring inflammatory/infectious conditions
- Sickle cells, spherocytes, and polycythemia lower ESR despite ongoing disease processes
- Reticulocyte count is a key marker of bone marrow erythropoietic activity
Competency Checklist
Track Your MasteryReferences
- Dacie JV, Lewis SM. Practical Haematology. 11th ed.
- International Council for Standardization in Haematology. ESR Reference Method.
- NIOS Vocational Course — Hematology and Blood Bank Technique, Lesson 8.