8.3 Erythrocyte Sedimentation Rate, Hemolytic Indicators & Special Erythrocyte Studies
Key Takeaways
- The erythrocyte sedimentation rate depends on rouleaux formation driven mainly by fibrinogen; the Westergren 200 mm column read at 60 minutes is the reference method, and anemia raises while polycythemia and poikilocytosis lower the value.
- A tilted tube is the most important ESR error and falsely INCREASES the result; delay beyond 4 hours, bubbles, a short fill, and excess anticoagulant all falsely decrease it.
- Hemolysis is confirmed by low haptoglobin with raised lactate dehydrogenase and raised indirect bilirubin; adding raised plasma free hemoglobin, hemoglobinuria, and a positive urine hemosiderin (Rous test) localizes it to the INTRAVASCULAR compartment.
- In the G6PD fluorescent spot test, fluorescence means the enzyme is present; testing during or soon after a hemolytic crisis gives a FALSE NEGATIVE because deficient older cells have already lysed, so retest 2 to 3 months later.
- Heinz bodies require a supravital stain (crystal violet or new methylene blue) and are invisible on Wright stain; osmotic fragility is increased in hereditary spherocytosis and decreased in thalassemia, and eosin-5-maleimide flow cytometry is the modern confirmatory test.
Erythrocyte Sedimentation Rate, Hemolytic Indicators & Special Erythrocyte Studies
The ASCP BOC laboratory testing content area names three groups of tests that fall outside the CBC and outside the leukemia work-up: hemolytic indicators (haptoglobin, lactate dehydrogenase) and the "other studies" group of erythrocyte sedimentation rate, glucose-6-phosphate dehydrogenase, and Heinz body preparations. They are individually small and collectively worth several examination items.
1. The Erythrocyte Sedimentation Rate (ESR)
Principle
The ESR measures how far erythrocytes fall through plasma in one hour in a vertical tube. Red cells normally repel one another because their sialic-acid-rich membranes carry a net negative surface charge (zeta potential). Acute-phase proteins, above all fibrinogen, and to a lesser degree immunoglobulins and alpha-2 macroglobulin, are asymmetric molecules that neutralize that repulsion and allow cells to stack into rouleaux. Rouleaux have a higher mass-to-surface-area ratio, so they sediment faster. The ESR is therefore an indirect, nonspecific measure of plasma protein change.
The Three Phases of Sedimentation
- Rouleaux formation (lag phase), roughly the first 10 minutes.
- Rapid settling, a constant-rate fall over roughly 40 minutes; this phase determines the result.
- Packing, the final 10 minutes as cells compress at the bottom.
Methods
| Method | Tube and Fill | Anticoagulant | Read At |
|---|---|---|---|
| Westergren (reference method) | 300 mm tube, 2.5 mm bore, filled to the 200 mm mark | 3.8% sodium citrate at a 4:1 blood-to-citrate ratio | 60 minutes |
| Modified Westergren | Same tube geometry | EDTA whole blood diluted with saline or citrate to the same final ratio | 60 minutes |
| Wintrobe | 100 mm tube | EDTA, undiluted | 60 minutes |
| Automated / capillary photometric | Sealed capillary, closed system | EDTA | 20 minutes or less, reported as a Westergren-equivalent value |
The long Westergren column is why it, and not the 100 mm Wintrobe tube, is the reference method: it can report markedly elevated values without the cells reaching the bottom of the tube.
Reference Intervals
Widely used adult Westergren values are 0 to 15 mm/h for men and 0 to 20 mm/h for women under 50, rising to about 0 to 20 mm/h for men and 0 to 30 mm/h for women over 50. An age-adjusted formula is also common: age divided by 2 for men, and (age + 10) divided by 2 for women. The ESR rises physiologically in pregnancy and with increasing age.
Sources of Error
| Variable | Effect on ESR | Reason |
|---|---|---|
| Tube tilted from vertical | Falsely INCREASED (a tilt of only a few degrees can raise the result substantially) | Cells slide down the lower wall while plasma rises along the upper wall |
| Temperature above 25 degrees Celsius | Falsely increased | Lower plasma viscosity |
| Vibration, drafts, direct sunlight | Falsely increased | Disturbs laminar settling |
| Delay beyond 4 hours at room temperature | Falsely decreased | Red cells become crenated (echinocytes) and stack poorly |
| Bubbles or a short fill | Falsely decreased | Effective column length is reduced |
| Excess anticoagulant / clotted specimen | Falsely decreased | Cell shrinkage or removal of fibrinogen |
| Anemia | Falsely increased | Fewer cells fall through less resistance |
| Polycythemia | Falsely decreased | Crowding impedes settling |
| Sickle cells, spherocytes, acanthocytes | Decreased | Abnormal shapes cannot form rouleaux |
Clinical Use
The ESR is nonspecific but retains real diagnostic weight in temporal (giant cell) arteritis and polymyalgia rheumatica, where values above 100 mm/h are typical, and in monitoring multiple myeloma, Waldenstrom macroglobulinemia, and chronic inflammatory disease activity. C-reactive protein rises and falls faster and has largely replaced the ESR for acute monitoring.
2. Hemolytic Indicators
When red cells are destroyed, their contents enter the plasma and are cleared by defined pathways. The pattern of results separates intravascular from extravascular hemolysis.
| Analyte | Intravascular Hemolysis | Extravascular Hemolysis | Basis |
|---|---|---|---|
| Serum haptoglobin | Markedly decreased or undetectable | Normal to mildly decreased | An alpha-2 globulin that binds free hemoglobin dimers; the complex is cleared by hepatic CD163 receptors far faster than haptoglobin is resynthesized |
| Lactate dehydrogenase (LD) | Markedly increased (isoenzymes LD-1 and LD-2, with LD-1 exceeding LD-2, the "flipped ratio") | Increased | Released from the erythrocyte cytoplasm, which is rich in LD-1 |
| Indirect (unconjugated) bilirubin | Increased | Increased | Heme catabolism by splenic and hepatic macrophages |
| Plasma free hemoglobin | Increased (normal is under about 5 mg/dL) | Normal | Only intravascular lysis releases hemoglobin directly into plasma |
| Hemoglobinuria | Present once haptoglobin is saturated and the renal threshold is exceeded | Absent | Filtered dimers appear in urine |
| Urine hemosiderin (Rous test) | Positive after several days, using Prussian blue on the urinary sediment | Negative | Reabsorbed iron is stored in sloughed renal tubular cells |
| Methemalbumin (Schumm test) | Present in severe cases | Absent | Oxidized heme binds albumin after haptoglobin and hemopexin are exhausted |
Interpretation rule for the examination: a low haptoglobin with a raised LD and a raised indirect bilirubin confirms hemolysis; adding hemoglobinuria and a positive urine hemosiderin localizes it to the intravascular compartment. Haptoglobin is an acute-phase reactant, so an inflamed patient may show a spuriously "normal" haptoglobin despite genuine hemolysis.
3. Special Erythrocyte Studies
Glucose-6-Phosphate Dehydrogenase (G6PD) Testing
- Fluorescent spot screen (the standard screen). The patient's hemolysate is incubated with glucose-6-phosphate and NADP. Functional G6PD generates NADPH, which fluoresces under long-wave ultraviolet light. No fluorescence indicates deficiency.
- Quantitative spectrophotometric assay measures the rate of NADPH generation and is used for confirmation and for female heterozygotes with mosaic expression.
- The timing trap. During and immediately after an acute hemolytic episode the oldest, most deficient cells have already lysed, and the surviving reticulocytes are comparatively enzyme-rich. Testing during a crisis or shortly after transfusion therefore yields a false-negative result. Retest 2 to 3 months after the episode.
Heinz Body Preparation
Heinz bodies are denatured, precipitated hemoglobin bound to the inner leaflet of the red cell membrane. They are invisible on a Wright-stained smear and require a supravital stain on living cells: crystal violet or new methylene blue. They appear as one to several round, deep-purple inclusions at the cell periphery. Causes include G6PD deficiency after an oxidant exposure, unstable hemoglobins, and drug or chemical oxidant injury. Splenic pitting of Heinz bodies produces the "bite cells" and "blister cells" seen on the routine smear.
Membrane and Fragility Studies
| Study | Principle | Classic Result |
|---|---|---|
| Osmotic fragility | Red cells are placed in graded hypotonic saline; lysis is measured photometrically | Increased fragility (curve shifted LEFT) in hereditary spherocytosis and immune hemolysis; decreased fragility (curve shifted right) in thalassemia and iron deficiency, where thin target cells tolerate water |
| Incubated osmotic fragility (24 h at 37 degrees Celsius) | Depletes ATP and exaggerates membrane loss | Improves sensitivity for mild hereditary spherocytosis |
| Eosin-5-maleimide (EMA) binding by flow cytometry | EMA binds band 3 and related membrane proteins | Reduced mean fluorescence in hereditary spherocytosis; now the preferred confirmatory test |
| Cryohemolysis | Hypertonic cooling stress | Increased lysis in hereditary spherocytosis |
| Sucrose hemolysis and Ham acidified serum | Complement-mediated lysis of GPI-anchor-deficient cells | Historic screens for paroxysmal nocturnal hemoglobinuria, both replaced by FLAER flow cytometry |
A Westergren erythrocyte sedimentation rate is set up correctly but the rack is later found resting against a wall so that the tubes are tilted about 5 degrees from vertical. How is the result affected and why?
A patient with a mechanical aortic valve has: haptoglobin under 5 mg/dL, lactate dehydrogenase 940 U/L, indirect bilirubin 2.6 mg/dL, plasma free hemoglobin 210 mg/dL, and a urine hemosiderin (Rous) test that is positive. Which conclusion is supported?
A 6-year-old boy of Mediterranean ancestry is hospitalized with acute hemolysis 2 days after eating fava beans. A fluorescent spot test for glucose-6-phosphate dehydrogenase performed on admission shows normal fluorescence. What is the correct interpretation and next step?
Which combination correctly pairs a special erythrocyte study with its expected result?