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Inclusions, sickling screens, erythrocytosis and the ESR

15 min

  • Choose an iron stain to confirm suspected Pappenheimer bodies
  • Report a positive Hb S solubility screen without calling trait or disease
  • Choose between a repeat count, EPO, and JAK2 V617F for a raised hemoglobin
  • Recognize how citrate ratio, anemia, tube tilt, and temperature change a Westergren ESR

Read the full reference

Try first

Try first

Small, dark, clustered granules are seen inside several red cells on a Wright-stained film. Do you report iron granules from this alone?

The next section explains it.

Right. The next section explains why.

The next section explains it.

The next section explains it.

Get the idea

Confirm iron before you report it

Pappenheimer bodies, Howell-Jolly bodies, and basophilic stippling can all look dark on a Wright-stained film. Only Pappenheimer bodies contain iron, and a Prussian blue stain is what confirms it.1 Reporting iron granules without that stain can mistake another inclusion for the iron-loaded cells of sideroblastic anemia, so a suspicious clustered granule pattern is stained before it is named.

A positive screen goes on to fractionation

A positive Hb S solubility screen shows that a sickling hemoglobin is present in some amount. Trait and sickle cell disease both give a positive result, so the screen alone cannot separate them, and every positive result goes on to electrophoresis or high-performance liquid chromatography (HPLC).2,3 A negative screen does not exclude a sickling hemoglobin either, in severe anemia, in a young infant with a large Hb F fraction, or after recent transfusion.2

Confirm persistence before you test genes

A raised hemoglobin is confirmed against prior results before anything else is ordered, because plasma-volume loss, hypoxia, and other causes can raise it without a true increase in red-cell mass.4 Serum erythropoietin (EPO) comes next: a subnormal result supports a primary process, and a normal or increased result supports a secondary cause. JAK2 V617F is tested when polycythemia vera is plausible, and skipping the EPO step sends a plausible secondary cause straight to molecular testing it may not need.4

The tube and the technique change the ESR

The Westergren reference method needs blood diluted 4:1 with citrate or saline. An ordinary coagulation tube carries the wrong 9:1 ratio for this method.5 Anemia raises the result on its own, without any change in inflammation, and a tilted tube, vibration, or high temperature raises it further.5,6 Read every ESR with the hematocrit.

References
  1. Keohane EM, Preston MM, Mirza KM, Walenga JM, eds. Rodak's Hematology: Clinical Principles and Applications. 7th ed. Elsevier; 2025. Accessed September 26, 2026. https://www.us.elsevierhealth.com/rodaks-hematology-9780323936507.html
  2. Mayo Clinic Laboratories. Sickle solubility, blood (SDEX). Clinical Information, Cautions, and Method Description. Accessed September 26, 2026. https://www.mayocliniclabs.com/test-catalog/Overview/9180
  3. Bender MA, Carlberg K. Sickle cell disease. In: Adam MP, Bick S, Mirzaa GM, et al, eds. GeneReviews. University of Washington, Seattle; 2003. Revised February 13, 2025. Accessed September 26, 2026. https://www.ncbi.nlm.nih.gov/books/NBK1377/
  4. Noumani I, Harrison CN, McMullin MF. Erythrocytosis: diagnosis and investigation. Int J Lab Hematol. 2024;46(suppl 1):55-62. doi:10.1111/ijlh.14298
  5. Jou JM, Lewis SM, Briggs C, Lee S-H, De La Salle B, McFadden S. ICSH review of the measurement of the erythrocyte sedimentation rate. Int J Lab Hematol. 2011;33(2):125-132. doi:10.1111/j.1751-553X.2011.01302.x
  6. Kratz A, Plebani M, Peng M, Lee YK, McCafferty R, Machin SJ. ICSH recommendations for modified and alternate methods measuring the erythrocyte sedimentation rate. Int J Lab Hematol. 2017;39(5):448-457. doi:10.1111/ijlh.12693

Watch one

A repeat CBC confirms a hemoglobin of 18.6 g/dL, unchanged from a result 3 months ago. The patient has no history of dehydration, smoking, high altitude, or lung or heart disease. What is the next test?

  1. Confirm persistence: the repeat CBC matches the result from 3 months ago.

    A persistent increase is confirmed against prior results before further testing is ordered.

  2. Review the history: no dehydration, smoking, altitude, or cardiopulmonary disease to explain a secondary cause.

    Ruling out plasma-volume loss and hypoxia narrows the differential before a specific test is chosen.

  3. Order serum erythropoietin (EPO) as the next test.

    Serum EPO separates a primary from a secondary process before molecular testing is ordered.

  4. A subnormal EPO result would support a primary process and would make JAK2 V617F testing the next step.

    A subnormal EPO is what makes polycythemia vera plausible enough to test for.

Order serum EPO next. A subnormal result would support ordering JAK2 V617F testing.

Your turn

Problem 1 of 3

A sickle solubility screen is positive. Which conclusion requires additional hemoglobin evaluation?

Incorrect. That observation is already supplied. Interpretation beyond the screening endpoint needs confirmation.

Correct. The solubility screen cannot separate these states. Hemoglobin identification and quantitation are required.

Incorrect. Turbidity is the stated screening endpoint. It does not determine genotype or quantify hemoglobin fractions.

Hint
  1. The screen has one endpoint: turbid or clear.
  2. Trait and disease both turn the solution turbid.

Review Hemoglobin variant testing

Problem 2 of 3

A persistent hemoglobin increase is confirmed on repeat CBC with no plasma-volume explanation. Which result best separates a primary from a secondary erythrocytosis at the next step?

Correct. A subnormal erythropoietin (EPO) supports a primary process, and a normal or increased EPO supports secondary causes, with overlap in both directions. JAK2 V617F is tested when polycythemia vera is plausible, and a V617F-negative, low-EPO pattern directs exon 12 testing.

Incorrect. Pulse oximetry can look reassuring during carbon monoxide exposure. Co-oximetry measures carboxyhemoglobin when exposure is possible.

Incorrect. Thrombocytosis, leukocytosis, and basophilia support polycythemia vera, and marrow panmyelosis with the formal criteria completes that classification. Serum EPO is the step that separates primary from secondary causes.

Hint
  1. Persistence has already been confirmed in this scenario.
  2. One result separates a primary process from a secondary one before any gene is tested.

Review Investigation sequence

Problem 3 of 3

A Westergren ESR is requested, and the specimen arrives in a 9:1 sodium citrate coagulation tube. What do you do?

The Westergren method needs blood diluted 4:1 with citrate or saline. A 9:1 coagulation tube carries the wrong ratio for the reference method.

Ran a Westergren ESR from a 9:1 coagulation citrate tube

The Westergren method needs blood diluted 4:1 with citrate or saline, or collected directly at 4:1. A 9:1 coagulation tube carries the wrong ratio for the reference method. Anemia also raises the ESR without a change in inflammation, so read the result with the hematocrit.

A 9:1 tube cannot be corrected after the draw, because the anticoagulant and blood volumes are already fixed at the wrong ratio. A new specimen collected or diluted at 4:1 is needed.

The blood-to-citrate ratio inside the tube was fixed at collection. Adding saline afterward cannot recreate the specimen the reference method requires.

Review Erythrocyte sedimentation rate

Use it

  • A man, MRN 0392718, splenectomized 5 years ago after trauma, reports fatigue and joint pain.
  • The emergency department orders a CBC with film, an ESR, and a sickle solubility screen.
  • The film shows several red cells with small, clustered, dark granules.
  • The ESR specimen arrives in a 9:1 sodium citrate coagulation tube.
  • The sickle solubility screen returns positive.
Decision 1 of 3

What do you do with the clustered granules seen on the film?

A Prussian blue stain is needed to confirm iron before the granules are reported as Pappenheimer bodies, especially in a splenectomized patient where they are a recognized finding.

Splenectomy makes Pappenheimer bodies more likely, but the same dark appearance can come from Howell-Jolly bodies or basophilic stippling. A Prussian blue stain still confirms which one is present.

Reported iron granules without a Prussian blue stain

Pappenheimer bodies, Howell-Jolly bodies, and basophilic stippling can all look dark on a Wright stain. Only Pappenheimer bodies contain iron, and Prussian blue stains them blue. Reporting iron granules without the stain can mistake Howell-Jolly bodies or stippling for the iron-loaded cells of sideroblastic anemia.

Howell-Jolly bodies are usually single per cell. Clustered granules in a splenectomized patient still need the stain to settle which inclusion is present.

Review Red-cell morphology on the stained film

Decision 2 of 3

What do you do with the ESR specimen?

The 9:1 ratio is wrong for the Westergren reference method. A specimen collected or diluted at 4:1 is needed before the test can be run.

Sodium citrate is the correct anticoagulant, but the 9:1 blood-to-citrate ratio is wrong for this test. The reference method needs a 4:1 ratio.

Ran a Westergren ESR from a 9:1 coagulation citrate tube

The Westergren method needs blood diluted 4:1 with citrate or saline, or collected directly at 4:1. A 9:1 coagulation tube carries the wrong ratio for the reference method. Anemia also raises the ESR without a change in inflammation, so read the result with the hematocrit.

A result run at the wrong ratio does not measure what the reference method measures. Rejecting it and requesting a correctly collected specimen is the recognized way to handle a specimen at the wrong ratio.

Review Erythrocyte sedimentation rate

Decision 3 of 3

The sickle solubility screen returns positive. What do you report?

The screen shows that a sickling hemoglobin is present in some amount. Trait and sickle cell disease both give a positive result, so electrophoresis or HPLC is needed to tell them apart.

The solubility screen cannot distinguish trait from disease. Reporting trait from the screen alone skips the fractionation that measures the hemoglobin fractions.

Used the solubility screen to distinguish trait from disease

A positive solubility screen shows that a sickling hemoglobin is present in some amount, and trait and sickle cell disease both give positive results. Reporting trait or disease from the screen skips the electrophoresis or HPLC that measures the fractions.

A positive screen cannot by itself separate disease from trait. Fractionation by electrophoresis or HPLC is needed before either is reported.

Used the solubility screen to distinguish trait from disease

A positive solubility screen shows that a sickling hemoglobin is present in some amount, and trait and sickle cell disease both give positive results. Reporting trait or disease from the screen skips the electrophoresis or HPLC that measures the fractions.

Review Hemoglobin variant testing

The clue that settles this case is that none of the three findings can be reported on its own: the clustered granules need a Prussian blue stain before they are called iron, the ESR specimen's 9:1 ratio means the result cannot be trusted regardless of what it reads, and the positive sickle screen needs fractionation before trait or disease is named.

Keep

Sources checked