The erythrocyte sedimentation rate (sedimentation rate, sed rate, or ESR for short) is a commonly performed hematology test that may indicate and monitor an increase in inflammatory activity within the body caused by one or more conditions such as autoimmune disease, infections, or tumors.[1] The ESR is not specific for any single disease but is used in combination with other tests to determine the presence of increased inflammatory activity. The ESR has long been used as a "sickness indicator" due to its reproducibility and low cost. Over many decades, several methods have evolved to perform the test. However, the reference method for measuring the ESR proposed by the International Committee for Standardization in Haematology (ICSH) and also by various national authorities is based on that of Westergren, a century ago.[2] Newer automated systems using closed blood collection tubes and automatic readers have been introduced into laboratories to decrease the biohazardous risk to operators and decrease the time it takes to perform the ESR.[3]

The erythrocyte sedimentation rate (ESR) is a commonly used hematological test that assesses the rate at which red blood cells (RBCs) settle in a column of anticoagulated blood over a one-hour period - a process called sedimentation. It is an indirect measure of inflammation.

PATHOPHYSIOLOGY OF ESR

In inflammatory conditions such as infections, cancers, or autoimmune diseases, plasma protein levels (especially acute-phase proteins) increase. These proteins reduce the natural negative charge repulsion between RBCs, allowing them to form stacks known as rouleaux (like coins), which settle faster, increasing the ESR.

Rouleaux formation is facilitated by the discoid shape of RBCs and the presence of positively charged plasma proteins (e.g., prothrombin, fibrinogen, C-reactive protein, haptoglobin, complement proteins).[4] These are part of the acute-phase response, mostly regulated by the liver in response to tissue injury or inflammation. The first discovered acute-phase protein was C-reactive protein (CRP), which, along with others, increases in both acute and chronic inflammatory states, contributing to higher ESR.[5,6]

However, some conditions can lower the ESR. For example, polycythemia increases blood viscosity, reducing sedimentation. Hemoglobinopathies like sickle cell disease and conditions like spherocytosis impair rouleaux formation due to abnormal RBC shapes, also leading to a decreased ESR. Thus, ESR is influenced by both elevations in plasma proteins and the physical characteristics of red blood cells.

The medical records of 200 patients presenting with anemia and elevated erythrocyte sedimentation rate (ESR), who attended the Outpatient Department at laboratory, department of pathology, PDU medical college & hospital - Rajkot, between 5 May 2025 and 6 June 2025, were retrospectively reviewed and analyzed. The Westergren ESR test begins with a routine blood draw, typically performed by a healthcare professional. After cleaning the skin over a vein, a needle is inserted to collect blood, which is then transferred to a special tube and the puncture site is covered. Blood is collected in a black-top vacuum tube containing 3.2% sodium citrate (preferred) or a lavender-top EDTA tube (acceptable). The sample must be collected in its own dedicated tube due to volume requirements and cannot be shared with other tests.[1]

The standard method for determining ESR is the Westergren method. In the lab, the anticoagulated whole blood is transferred into a Westergren tube, which is placed vertically. These tubes, made of either glass or plastic, have an internal diameter of 2.5 mm and a length between 190 mm and 300 mm. After one hour, red blood cells settle to the bottom under the influence of gravity, leaving a clear layer of plasma at the top. The distance the red cells fall is measured in millimeters per hour (mm/hr) and reflects the ESR value.

For the complete blood count (CBC) and peripheral smear examination, a fresh, well-mixed EDTA-anticoagulated blood sample was used. A clean glass slide was prepared by placing a small drop of blood near one end, which was then spread using a second slide held at an angle of approximately 30–45 degrees. The spreader slide was swiftly and smoothly advanced to produce a thin, even film with a feathered edge. The prepared smear was allowed to air-dry completely and subsequently stained with a Romanowsky-type stain, such as Leishman or Giemsa stain, to facilitate detailed morphological evaluation of red cells, white cells, and platelets under light microscopy.[1]

INCLUSION CRITERIA

1. Erythrocyte sedimentation rate (ESR) exceeding 40 mm/hr at the time of sample collection.
2. Age 5 years and above

EXCLUSION CRITERIA

1. Clotted blood samples
2. ESR value below 40mm/h
3. Diluted blood samples

INTERFERING FACTORS

Technical factors, such as seasonal variations in room temperature, time from specimen collection, tube orientation and inclination, and vibration, can affect the results.[7,8]

Factors increase ESR:

• Higher room temperature
• Decreased blood viscosity
• Direct sunlight
• Tilted, Improper filling, vibrations of ESR tube

Factors decrease ESR:

• A blood sample allowed to sit too long before starting the test will cause RBC sphering and decrease the ESR value.

Clotted blood sample

The test should be run within two hours of collection. Using ESR tubes with inconsistent internal boreholes can be sensitive to RBC clumps and may lead to variations in the ESR results. Icteric blood samples (drawn from patients with liver disease) will produce a dark yellow plasma that may be difficult to differentiate from the sedimented RBCs by direct inspection. Likewise, hemolysis (damaged erythrocytes) will cause hemoglobin to leak into the plasma and turn it red, making it difficult to differentiate from the sedimented RBCs.

1. CORRELATION OF ESR WITH HEMOGLOBIN CONCENTRATION- NEGATIVE CORRELATION

This graph [Figure 1] illustrates the comparative distribution of erythrocyte sedimentation rate (ESR) and hemoglobin concentration across a sample population. A clear inverse trend can be observed: as ESR values increase, hemoglobin concentrations tend to remain comparatively lower and more consistent. This pattern highlights the typical negative correlation between ESR and hemoglobin—individuals with elevated ESR often demonstrate lower hemoglobin levels, which is consistent with the physiological principle that anemia contributes to an elevated ESR due to reduced hematocrit and increased plasma proportion, facilitating faster sedimentation of red blood cells. Conversely, when hemoglobin levels are higher, ESR values are generally lower, reinforcing the interpretation that robust red cell mass resists aggregation and slows sedimentation. This graphical depiction supports the conclusion that hemoglobin concentration should be considered when evaluating ESR results to avoid misinterpretation of inflammatory status.

1. THE DISTRIBUTION OF DIFFERENT MORPHOLOGICAL TYPES OF ANEMIA

 The pie chart

Figure 2] illustrates the distribution of different morphological types of anemia observed in the study population. The majority of cases, accounting for 53.0%, were classified as normochromic normocytic anemia, indicating that over half of the anemic patients had red blood cells of normal size and hemoglobin content, a pattern commonly associated with anemia of chronic disease or acute blood loss. The second most prevalent type was hypochromic microcytic anemia, comprising 45.5% of the cases, which typically reflects iron deficiency anemia or thalassemia traits characterized by smaller and less hemoglobin-rich red blood cells. A very small fraction of patients, only 1.5%, exhibited macrocytic anemia, which is generally linked to deficiencies in vitamin B12 or folate, or to certain hematological disorders. This distribution highlights that normochromic normocytic and hypochromic microcytic patterns predominate in the studied cohort, suggesting that iron deficiency and chronic disease processes are the principal underlying causes of anemia in this group.

1. CORRELATION OF ESR WITH NORMOCHROMIC NORMOCYTIC ANEMIA

 This Figure 3 displays the distribution of ESR values in patients diagnosed with normochromic normocytic anemia. The data points reveal that the majority of ESR measurements in this subgroup cluster around moderate to high levels, predominantly ranging between 40 and 80 mm/hr. This pattern indicates that even in normochromic normocytic anemia—which often reflects anemia of chronic disease or inflammatory conditions—ESR tends to be elevated, likely due to the underlying inflammatory processes rather than red cell morphology alone. Overall, this distribution supports the observation that normocytic normochromic anemia is frequently associated with an increased inflammatory burden, as reflected by persistently raised ESR values.

The histogram depicts the distribution of ESR values in patients diagnosed with hypochromic microcytic anemia. The majority of cases show moderately to markedly elevated ESR levels, with most values clustering between 40 mm/hr and 80 mm/hr. This pattern indicates that in iron deficiency or microcytic anemias, ESR remains elevated, likely due to the reduced red cell mass and enhanced plasma protein effects, which facilitate rouleaux formation and increase sedimentation rate. This supports the inverse correlation between hemoglobin levels and ESR in this subgroup as well.[Figure 4]

1. CORRELATION OF ESR WITH MACROCYTIC ANEMIA

This Figure 5 illustrates the correlation of ESR values in patients with macrocytic anemia. Although macrocytic anemia was the least common type in the study, the data show a gradual increase in ESR values across the small sample. This suggests that even in macrocytic anemia—typically due to vitamin B12 or folate deficiency—ESR can be moderately to markedly elevated, reflecting underlying inflammatory or nutritional etiologies contributing to the anemia.

A total of 200 patients presenting with an erythrocyte sedimentation rate (ESR) greater than 40 mm/h in conjunction with anemia were enrolled in this study. Among these, 142 patients (71%) were female, while 58 patients (29%) were male. The mean age of the female patients was 46.42 ± 10.0 years, whereas the mean age of the male patients was 44.74 ± 11.1 years. The average ESR value recorded was 58.13 ± 10.2 mm/h for females and 56.9 ± 10.5 mm/h for males. The ESR is typically higher in females than males and increases gradually with age.

The mean hemoglobin concentration was 10.75 ± 1.60 g/dL in the female group and 10.6 ± 1.52 g/dL in the male group. With respect to the morphological classification of anemia, normochromic normocytic anemia was the most common subtype, identified in 53% of the cases. Hypochromic microcytic anemia was observed in 45.5% of the patients, while macrocytic anemia constituted the smallest proportion, affecting only 1.5% of the study population.

Kanfer and Nicol (1997) demonstrated a significant inverse correlation between hemoglobin concentration and ESR even among non-anemic individuals (P < 0.001). In comparison, our study similarly established a statistically significant inverse relationship, with a P value of <0.003, further supporting the consistency of this association across different patient populations^[9]. Similarly, Arora et al. (2018) observed that ESR rises substantially in cases of severe anemia (Hb < 6 g/dL) and shows slight increases at very high hemoglobin levels, indicating that extreme deviations in hemoglobin influence ESR markedly.^[10] Additionally, a large cross-sectional study published in BMJ Open (2019) confirmed that hemoglobin is an independent determinant of ESR, with lower hemoglobin levels consistently associated with higher ESR values, and age and sex further modulating this relationship.^[11] Together, the present graph and these supporting studies reinforce the conclusion that normochromic normocytic anemia—typically linked to chronic disease—is frequently accompanied by elevated ESR, highlighting the importance of interpreting ESR results in conjunction with hemoglobin levels and patient context.

A recent single-center retrospective study conducted in Turkey in 2023 examined the disease distribution among 300 adult patients with anemia and an erythrocyte sedimentation rate (ESR) exceeding 50 mm/hr.^[12] In this cohort, the mean ESR was approximately 80 mm/hr, while the mean hemoglobin concentration was about 10.5 g/dL. These results align closely with the present study, which demonstrated a mean ESR of 62.5 mm/hr and a mean hemoglobin concentration of 10.6 g/dL. Consistent with previous findings, this analysis confirmed a clear inverse relationship between ESR and hemoglobin levels, whereby higher ESR values were observed in patients with lower hemoglobin concentrations.

This correlation underscores the frequent coexistence of significant anemia with markedly elevated sedimentation rates, reflecting the inflammatory or chronic disease processes commonly encountered in such patient populations.

Table 1: Comparative Analysis Key Studies Demonstrating the Inverse Relationship between Hemoglobin and ESR

The findings of this study reaffirm the well-established inverse relationship between erythrocyte sedimentation rate (ESR) and hemoglobin concentration, highlighting that lower hemoglobin levels are consistently associated with elevated ESR values. This relationship arises from hematological changes inherent to anemia, which facilitate red blood cell aggregation and accelerate sedimentation. The morphological distribution of anemia observed further supports this correlation, with normochromic normocytic anemia being the most prevalent, followed closely by hypochromic microcytic anemia, indicating that chronic disease processes and iron deficiency are the predominant contributors to anemia within this population. These results underscore the importance of interpreting ESR in conjunction with hemoglobin concentration and red cell indices to accurately assess the presence and extent of underlying inflammation and to avoid potential diagnostic misinterpretation.

Our study has demonstrated a clear and significant correlation between hemoglobin concentration and erythrocyte sedimentation rate (ESR), consistent with the findings reported in the existing literature. Various factors, including infection, age, pregnancy, and smoking, may influence this relationship and should be considered during interpretation.

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