ESR

What is the ESR?

• The erythrocyte sedimentation rate (ESR) is a simple, inexpensive laboratory test that serves as a general “sickness index” in conjunction with the patient’s clinical history and physical examination findings.¹
• The erythrocyte sedimentation rate is a measure of red cell aggregation.
• The ESR measures the the distance that erythrocytes have fallen after one hour in a vertical column of anticoagulated blood under the influence of gravity.²

What is the history behind the ESR?

• Historically, the sedimentation of the blood was used by ancient Greek physicians to detect the presence of certain bad bodily “humors”.
• In two papers (1894 and 1897), Polish physician Edmund Faustyn Biernacki reported a method for measuring the ESR. He used glass cylinders of his own design. Blood, with sodium oxalate added to prevent coagulation, was stored at room temperature and readings were taken after 1 and 24 h. Results were expressed as per cent of the length of the erythrocyte column after 24 h.³
• According to a review by Grzybowski and Sak,⁴ Biernacki presented 5 important conclusions from his observations:
  • The blood sedimentation rate is different in different individuals.
  • Blood with fewer blood cells sediments faster.
  • Blood sedimentation rate depends on the level of plasma fibrinogen.
  • In febrile diseases (rheumatic fever included) with high levels of plasma fibrinogen the ESR is increased.
  • In defibrinated blood the sedimentation process is slower.

• In 1918 and 1921,⁵ Robert Robin Sanno Fåhræus, a Swedish physician, defined many of the important characteristics of this test, and reported increased ESR in pregnant women and in patients suffering from various inflammatory states.⁶

• In 1921, Alf Vilhem Albertsson Westergren:
  • Refined the technique of measuring the sedimentation rate.
  • Used a vertical tube 2.5 mm in diameter on all sedimentation rates to standardize the volume of blood used.
  • Substituted sodium oxalate with sodium citrate as an anticoagulant agent used for ESR measurement.
  • Described elevated ESR in patients with pulmonary tuberculosis.
• Rapidly became a common screening test worldwide for acute phase proteins and chronic diseases.
• Over time, the ESR has become largely substituted by other so-called acute phase reactants, and is sometimes considered an old-fashioned or outdated medical laboratory procedure.

How is the ESR test carried out?

• Wintrobe’s method:
  • Uses a narrow glass tube closed at the lower end only.
  • The Wintrobe tube has a length of 11 cm and internal diameter of 2.5 mm.
  • Blood is anticoagulated in oxalate or EDTA.
  • The main difference from the Westergren method is that the tube is only 11 cm long (vs. 20-30 cm long for the Westergren tube); the shorter column makes this method less sensitive than the Westergren method because the maximal possible abnormal value is lower.
• Westergren method:
  • The most commonly used method.
  • Recommended (with modifications) by the International Council for Standardization in Haematology (ICSH) as the gold standard reference method for ESR determination.
  • Venous blood is mixed 4:1 with 3.2% sodium citrate solution and drawn into in a colorless glass or plastic tube with a minimum sedimentation scale of 20 cm and a minimum bore of 2.55 mm.
  • The tube is fixed vertically in a Westergren stand at room temperature.
  • At the end of 1 h, the distance from the lowest point of the surface meniscus to the top level of the red cell sediment is recorded as the ESR in mm/h (distance from the top edge to the blood cell layer).
• Semi‐automated and automated methods for ESR measurement:⁷
  • Two types:
    • Modified Westergren methods:
      • Methods with minor modifications to the gold standard Westergren method.
      • Modifications include:
        • Use of tubes of different length or diameter than endorsed by published recommendation.
        • Shorter assay time (15 or 30 minutes)
        • Use of no diluent or different diluents than recommended by ICSH
    • Alternate ESR methods for those instruments not based on Westergren method principles:
      • Centrifugation
      • Use of photometric rheology to measure rouleaux formation
  • Surveys indicate that worldwide two-thirds of all laboratories now use modified or alternate ESR test methods for the measurement of the ESR.⁸

What are the three phases of erythrocyte sedimentation?

• The sedimentation curve is sigmoidal and comprises three phases: the lag phase, sedimentation phase, and packing phase. Under low shear flow, erythrocytes dispersed in plasma form rouleaux through interactions with inflammatory proteins such as fibrinogen and immunoglobulins and grow as aggregates over time, increasing the sedimentation rate.
• The three phases in sequence are:
  • Lag phase (lasts about 10 min):
    • Erythrocytes dispersed in plasma form one-dimensional coin stacks (rouleaux).⁹
    • Rouleaux then form aggregates by gathering in two- to three-dimensions over time (as rouleaux grow large they form rings and branching aggregates).
    • The aggregate size increases according to the plasma concentration of fibrinogen or globulin and decreases as the hematocrit increases.
    • Late in this phase, rearranges to form spheres of equal size. This is a sudden change. It can be observed with the naked eye in the Westergren sedimentation tube: the homogeneous cell suspension suddenly becomes granular in appearance, with the spheres visible at the sides of the tube.
  • Sedimentation phase (lasts about 40 min):
    • The red cells form fairly stable aggregates which eventually begin to fall out of solution, leading to a relatively constant sedimentation rate.
    • The rate of sedimentation directly proportional to the square of the radius of the aggregate (Stokes’ Law).
  • Packing (deceleration) phase (lasts about 10 min):
    • RBC aggregates that have fallen out of solution become packed as they settle in the bottom of the testing tube.
    • The rate of fall of subsequent red cells is reduced by the deposition of erythrocytes at the bottom of the tube, therefore slowing the measured sedimentation rate.

Phase	AKA	Duration	Mechanisms/process
Phase 1	Lag phase	10 min	Erythrocyte aggregates are being formed, velocity of sedimentation is increasing.
Phase 2	Sedimentation phase	40 min	erythrocyte aggregates are sinking, the velocity is constant
Phase 3	Packing phase	10 min	the velocity is decreasing and drops towards zero

What factors affect rate of erythrocyte settling?

• The ESR depends on the balance between factors promoting sedimentation and factors hindering sedimentation.
  • The major factor promoting sedimentation is aggregation of red blood cells and formation of rouleaux. Rouleaux formation is determined by:
    • The bridging forces of macromolecules
      • For rouleaux formation to occur, macromolecules must be present in the suspending medium.
      • Macromolecules actually bridges two erythrocytes and only elongated macromolecules of sufficient length can serve this function.
      • Fibrinogen (and immunoglobulin) but not albumin meets the shape requirements to cross-link erythrocytes. Therefore, sedimentation will not take place in serum.
    • The characteristics of the red blood cells
  • The major factors hindering sedimentation are
    • The repulsive negatively charged red cell membranes (red cells repel each other because of their negative surface charge from sialic acid residues).
    • The shear forces and viscosity of the plasma.

• Plasma properties
  • Increased amounts of large asymmetric positively charged plasma proteins (macromolecules) such as fibrinogen and to a lesser extent gamma globulins:¹⁰
    • Counteract the natural repellent zeta potential of erythrocytes, which are normally monodisperse, causing them to be forced closer together than normal.
    • As a result, the RBCs are more prone to rouleaux formation (a type of regular, ordered aggregation in which cells adhere face to face and resemble coins in a stack, creating long cylindrical objects that are commonly branched),¹¹ which increases the rate at which they settle, thus leading to an elevated ESR result.
  • Hypoalbuminemia – albumin retards, and hypoalbuminemia permits, more rapid erythrocyte sedimentation.
  • Plasma viscosity – marked hyperviscosity may impede the fall of the aggregates and hence slow the sedimentation rate.

• Red cell properties
  • RBC number
    • Polycythemic individuals have decreased ESR values
    • Patients with anemia exhibit increased ESR values
  • RBC size
    • Macrocytic RBCs settle faster (increased ESR values) due to a smaller surface-to-volume ratio.
    • Microcytic RBCs tend to settle slower (decreased ESR values).
  • RBC shape – because their altered shape impedes rouleaux formation, the presence of RBC poikilocytes leads to decreased ESR; examples include:
    • Sickle cells
    • Acanthocytes
    • Spherocytes

•  Technical variables 
  • Increased ESR:
    • Tilted ESR tube
    • High room temperature
  • Decreased ESR:
    • Short ESR tubes
    • Low room temperature
    • Delay in test performance (>2 hours)
    • Clotted blood sample
    • Excess anticoagulant
    • Bubbles in tube

Let’s look at some primary data

What is a normal ESR value?

Normal ESR commonly is defined in men as age in years divided by 2 and for women, age in years plus 10 ¹²

What conditions are associated with altered ESR levels?

Decreased ESR	Increased ESR
Polycythemia: Polycythemia vera Secondary polycythemia	Anemia
Conditions associated with microcytosis	Conditions associated with macrocytosis
Poikilocytes: Sickle cell disease Acanthocytosis	NA
Hypo- or afibrinogenemia: DIC HLH	Elevated fibrinogen: Infection Inflammation Cancer Pregnancy
Hypo- or agammaglobulinemia	Hypergammaglobulinemia: MGUS Multiple myeloma Polyclonal gammopathy IVIG therapy

Various published lists of differential diagnosis:

What causes extreme elevation in erythrocyte sedimentation rate?

• Extreme elevations of 100 mm/h or greater are typically associated with:
  • Malignancy
  • Infection
  • Inflammation
• Cohort study of 4807 patients with extreme ESR values examined at Mayo Clinic, Rochester, Minnesota:
  • Causes included:
    • Infection 40%
    • Autoimmune disorders 38%
    • Malignancy 36%.
  • Extreme ESR elevations did not correlate with the CRP level.
  • There were no correlations between extreme elevations in ESR and age or race.