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Inclusions, sickling screens, erythrocytosis and the ESR

15 min

Skills you'll build4 skills
  • 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

At the bench: Relate red cell indices and morphology to the anemia workup

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?

Keep going to see the reasoning.

Right. Keep going to follow the reasoning.

Keep going to see the reasoning.

Keep going to see the reasoning.

Get the idea

Use the explanation to check your first decision.

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 confirms the iron.1 Reporting iron granules without that stain can mistake another inclusion for the iron-loaded cells of sideroblastic anemia, so clustered granules are stained before they are reported.

Five photographs of red cells. Three Romanowsky-stained fields: a red cell holding one round, dense, dark purple dot beside a neutrophil; a polychromatic red cell with fine purple dots spread evenly through it; red cells holding small clusters of dark purple granules. A crystal violet field shows pale red cells, two of them with purple clumps at the cell edge marked by arrows. A Perls Prussian blue field shows pale red cells holding clusters of blue-green granules.
On the Romanowsky-stained film, a Howell-Jolly body is one round dot, stippling spreads through the whole cell and Pappenheimer granules appear in a small cluster.7

Hemoglobin variant screening

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 cannot distinguish them, and every positive result goes on to electrophoresis or high-performance liquid chromatography (HPLC).2,3 A negative screen also does not exclude a sickling hemoglobin in severe anemia, in a young infant with a large Hb F fraction, or after recent transfusion.2

Erythrocytosis workup

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) is the next test. 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. Skipping the EPO step sends a patient with a plausible secondary cause to molecular testing that may not be needed.4

ESR specimen and technique

The Westergren reference method requires blood diluted 4:1 with citrate or saline. A coagulation tube has a 9:1 ratio, which is wrong for this method.5 Anemia raises the result without any change in inflammation. A tilted tube, vibration, or high temperature also raises it.5,6 Interpret every ESR with the hematocrit.

Follow the bench demonstration

Sources for this step
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
  7. Palmer L, Briggs C, McFadden S, et al. ICSH recommendations for the standardization of nomenclature and grading of peripheral blood cell morphological features. Int J Lab Hematol. 2015;37(3):287-303. doi:10.1111/ijlh.12327

Watch one

Read each decision and its explanation.

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. There is 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 distinguishes 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 makes polycythemia vera likely enough to test for.

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

Practice this skill

Your turn

Problem 1 of 3 · Hints available

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

Incorrect. The positive screen already shows a reactive result. Any interpretation beyond that requires confirmation.

Correct. The solubility screen cannot distinguish trait from disease. 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 · Hints available

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 result with a low EPO is followed by exon 12 testing.

Incorrect. Pulse oximetry can read falsely normal 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 test distinguishes a primary process from a secondary one before any gene is tested.

Review Investigation sequence

Problem 3 of 3 · On your own

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

The Westergren method requires blood diluted 4:1 with citrate or saline. A 9:1 coagulation tube has 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 has 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

Read the case evidence, then make each decision. The step is complete without 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 is needed to confirm 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 need the stain to identify the inclusion.

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 requires 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 has 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. The specimen is rejected, and a correctly collected specimen is requested.

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 distinguish them.

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 distinguish 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

None of the three findings can be reported as it stands. The clustered granules need a Prussian blue stain before they are reported as iron. The ESR specimen has a 9:1 ratio, so it is rejected. The positive sickle screen needs fractionation before trait or disease is reported.

For reference

Sources checked