E-Learn LAB — RCD 1708

Home , Basophilic stippling, Heinz body, Reticulocytosis, Spherocytosis, Supravital staining

e-Learn LAB — Hemoglobinopathy Based on IQMH Centre for Proficiency Testing Survey RCD-1708-WB

Confidence. Elevated. © Institute for Quality Management in Healthcare 1 Disclaimer and Copyright

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Confidence. Elevated. © Institute for Quality Management in Healthcare 2 Focus of this Presentation

• This is a case study of a hemoglobinopathy investigation to familiarize participants with the laboratory features of the unstable hemoglobins, as well as the pathophysiology of common red cell inclusions. • You will be presented with clinical information, images, and laboratory results and will be prompted with self- learning questions.

Confidence. Elevated. © Institute for Quality Management in Healthcare 3 Credits

Case and discussion provided by members of the 2017 IQMH Hematology Scientific Committee, and the IQMH Consultant Technologist.

Confidence. Elevated. © Institute for Quality Management in Healthcare 4 Clinical Information

A 14-year-old female is being assessed for gall stones.

Confidence. Elevated. © Institute for Quality Management in Healthcare 5 Laboratory Results

Result Reference Interval WBC 5.70 × 109 /L 3.50–10.50 × 109 /L RBC 5.12 × 1012/L 3.90–5.00 × 1012/L Hgb 110 g/L 108–133 g/L MCV 68.4 fL 76–90 fL RDW 17 % 11.5–15.0 % Platelet count 218 × 109 /L 130–380 × 109 /L Reticulocyte count 200 × 109 /L 22–92 x 109 /L Ferritin Normal

Confidence. Elevated. © Institute for Quality Management in Healthcare 6 Peripheral Blood

1000× Wright Giemsa Stain

Confidence. Elevated. © Institute for Quality Management in Healthcare 7 Supravital Stain

1000× Supravital Stain

Confidence. Elevated. © Institute for Quality Management in Healthcare 8 Question 1

Take a moment to consider which of the following are noteworthy on the CBC and peripheral blood smear: a) Thalassemic RBC indices (high RBC, low MCV, normal Hgb) b) Coarse basophilic stippling c) Polychromasia d) Spherocytes e) Schistocytes

Confidence. Elevated. © Institute for Quality Management in Healthcare 9 Answer

The correct answers for this question are a), b) and c)

• Thalassemic RBC indices • Coarse basophilic stippling • Polychromasia

Confidence. Elevated. © Institute for Quality Management in Healthcare 10 Discussion

• The RBC indices demonstrate the classic thalassemic indices. • Most individuals with thalassemia trait will demonstrate: – microcytosis, – normal to mild anemia and – normal to elevated RBC count.

Confidence. Elevated. © Institute for Quality Management in Healthcare 11 Discussion : Basophilic Stippling

• On the image of the Wright-Giemsa stained slide, a cell with coarse basophilic stippling is easily noted. • Coarse basophilic stippling is an indicator of impaired hemoglobin synthesis and the aggregation of ribosomes, and can be seen in numerous disease states including lead poisoning, thalassemia, and sideroblastic anemia. • The coarse granules are easily seen and distributed regularly throughout the red cell. • The International Council for Standardization in Haematology (ICSH) recommends that basophilic stippling be reported only if moderate 2+ / 5-20% or many 3+ / >20%.1

Confidence. Elevated. © Institute for Quality Management in Healthcare 12 Discussion : Polychromasia

• Polychromasia is also noted on the Wright-Giemsa stained slide, and corresponds to the reticulocyte count of 200 × 109 /L and the finding of increased reticulocytes noted on the supravital stain.

Confidence. Elevated. © Institute for Quality Management in Healthcare 13 Discussion : Spherocytes

• In the featured field, there is an identifiable spherocyte, which is a red cell with absence of central pallour and appearing slightly smaller than the surrounding red cells.

Confidence. Elevated. © Institute for Quality Management in Healthcare 14 Discussion : Spherocytes

• When spherocytes are noted in abundance, consideration should be given to conditions such as immune hemolytic anemias or hereditary spherocytosis. • When noted infrequently, the differential diagnosis is much broader, and can occur as a secondary effect of microangiopathic hemolytic anemia (“microspherocytes”) and direct damage to the red cell membrane. • Similar to the grading for basophilic stippling, the presence of spherocytes should be noted if seen in moderate 2+ / 5-20% or many 3+ / >20% amounts.1

Confidence. Elevated. © Institute for Quality Management in Healthcare 15 Discussion : Schistocytes

• A schistocyte is an umbrella term for fragmented red cells that also includes the helmet cell and keratocyte. • The classic red cell fragment has jagged cell edges with sharp angles and straight borders and lack of central pallor. • Small irregular cells with central pallor should be called non-specific poikilocytes. • The cell noted in the centre of the field would be more in keeping with a non-specific poikilocyte.

Confidence. Elevated. © Institute for Quality Management in Healthcare 16 Question 2

Based on the available information, think about which of these are the possible diagnoses. Which one is not a possibility? a) Alpha-thalassemia trait b) Beta-thalassemia trait c) Thalassemic hemoglobinopathy trait (e.g. Hb E trait) d) Iron deficiency e) Unstable hemoglobin

Confidence. Elevated. © Institute for Quality Management in Healthcare 17 That’s Correct

Iron deficiency is not a possibility.

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Confidence. Elevated. © Institute for Quality Management in Healthcare 18 That’s Not Correct

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Confidence. Elevated. © Institute for Quality Management in Healthcare 19 Discussion

• Thalessemic variant hemoglobins such as Hb E, Hb Lepore and Hb Constant Spring are structurally defective hemoglobins that are detectable on hemoglobin quantitation analysis, but have the additional feature of being produced in limited quantities; resulting in a thalassemic phenotype.

Confidence. Elevated. © Institute for Quality Management in Healthcare 20 Discussion

• The features of the common hemoglobin variants with an associated thalassemia phenotype is provided on the next few pages.2 • In a typical thalassemia or variant trait, one would expect a mild phenotype and no significant increase in the reticulocyte count.

Confidence. Elevated. © Institute for Quality Management in Healthcare 21 Hemoglobin Variants - 1 of 4

Confidence. Elevated. © Institute for Quality Management in Healthcare 22 Hemoglobin Variants- 2 of 4

Confidence. Elevated. © Institute for Quality Management in Healthcare 23 Hemoglobin Variants- 3 of 4

Confidence. Elevated. © Institute for Quality Management in Healthcare 24 Hemoglobin Variants – 4 of 4

Confidence. Elevated. © Institute for Quality Management in Healthcare 25 Discussion

• Unstable hemoglobins result most commonly from a single nucleotide substitution that affects the stability of the heme pocket in an autosomal dominant pattern. • The majority of unstable hemoglobins are difficult to detect using HPLC/capillary electrophoresis, and additional investigations are required. • This may include the demonstration of in vitro instability with the heat or isopropanol test and ultimately DNA analysis. • Unstable hemoglobins would typically present as a hemolytic anemia, with evidence of Heinz body formation on supravital stain. • At least some of these unstable hemoglobins can present with a thalassemic phenotype.3

Confidence. Elevated. © Institute for Quality Management in Healthcare 26 Question 3

For this patient, what is the expected result for LDH? a) Elevated b) Normal c) Decreased

Confidence. Elevated. © Institute for Quality Management in Healthcare 27 That’s Correct

The LDH is expected to be elevated.

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Confidence. Elevated. © Institute for Quality Management in Healthcare 28 That’s Not Correct

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Confidence. Elevated. © Institute for Quality Management in Healthcare 29 Question 4

For this patient, what is the expected result for unconjugated bilirubin? a) Elevated b) Normal c) Decreased

Confidence. Elevated. © Institute for Quality Management in Healthcare 30 That’s Correct

The unconjugated bilirubin is expected to be elevated.

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Confidence. Elevated. © Institute for Quality Management in Healthcare 31 That’s Not Correct

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Confidence. Elevated. © Institute for Quality Management in Healthcare 32 Question 5

For this patient, what is the expected result for conjugated bilirubin? a) Elevated b) Normal c) Decreased

Confidence. Elevated. © Institute for Quality Management in Healthcare 33 That’s Correct

The conjugated bilirubin is expected to be normal.

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Confidence. Elevated. © Institute for Quality Management in Healthcare 34 That’s Not Correct

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Confidence. Elevated. © Institute for Quality Management in Healthcare 35 Question 6

For this patient, what is the expected result for haptoglobin? a) Elevated b) Normal c) Decreased

Confidence. Elevated. © Institute for Quality Management in Healthcare 36 That’s Correct

Haptoglobin is expected to be decreased

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Confidence. Elevated. © Institute for Quality Management in Healthcare 37 That’s Not Correct

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Confidence. Elevated. © Institute for Quality Management in Healthcare 38 Question 7

For this patient, what is the expected result for a DAT? a) Negative b) Positive

Confidence. Elevated. © Institute for Quality Management in Healthcare 39 That’s Correct

A DAT is expected to be negative.

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Confidence. Elevated. © Institute for Quality Management in Healthcare 40 That’s Not Correct

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Confidence. Elevated. © Institute for Quality Management in Healthcare 41 Discussion

• If considered alone, the RBC indices are suggestive of thalassemia trait. • However, when interpreted together with the peripheral blood morphology, elevated reticulocyte count and supravital stain the laboratory features are suggestive of a hemolytic anemia. • In the setting of hemolysis, one would expect to find an elevated LDH, elevated unconjugated bilirubin, and decreased haptoglobin. • A positive DAT would be expected in immune-mediated causes of hemolysis, and should be looked for when this is part of the diagnostic differential. • Conjugated hyperbilirubinemia is the biochemical marker for cholestasis and indicates that there is a problem with bile flow.4 • As such, abnormalities in conjugated bilirubin are not typical of a hemolytic anemia.

Confidence. Elevated. © Institute for Quality Management in Healthcare 42 Question 8

On which preparation(s) are reticulocytes visible? a) Wright-Giemsa b) Supravital c) Neither d) Both

Confidence. Elevated. © Institute for Quality Management in Healthcare 43 That’s Correct

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Confidence. Elevated. © Institute for Quality Management in Healthcare 44 That’s Not Correct

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Confidence. Elevated. © Institute for Quality Management in Healthcare 45 Question 9

On which preparation(s) are Howell-Jolly bodies visible? a) Wright-Giemsa b) Supravital c) Neither d) Both

Confidence. Elevated. © Institute for Quality Management in Healthcare 46 That’s Correct

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Confidence. Elevated. © Institute for Quality Management in Healthcare 47 That’s Not Correct

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Confidence. Elevated. © Institute for Quality Management in Healthcare 48 Question 10

On which preparation(s) are Heinz bodies visible? a) Wright-Giemsa b) Supravital c) Neither d) Both

Confidence. Elevated. © Institute for Quality Management in Healthcare 49 That’s Correct

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Confidence. Elevated. © Institute for Quality Management in Healthcare 50 That’s Not Correct

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Confidence. Elevated. © Institute for Quality Management in Healthcare 51 Question 11

On which preparation(s) is Hemoglobin H visible? a) Wright-Giemsa b) Supravital c) Neither d) Both

Confidence. Elevated. © Institute for Quality Management in Healthcare 52 That’s Correct

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Confidence. Elevated. © Institute for Quality Management in Healthcare 53 That’s Not Correct

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• All four types of red cell inclusions are noted on a supravital stain, whilst only the Howell-Jolly body can additionally be seen on the Wright-Giemsa stain. • Supravital stains can pick up a variety of red cell inclusions, and differentiating between the different types of inclusions relies on additional morphologic features and clinical/laboratory information.

Heinz bodies can be found in which of the following conditions? a) Unstable hemoglobin b) Oxidant drug exposure c) Hb H disease d) Beta-thalassemia major e) Alpha thalassemia trait f) a) and b) g) c) and e)

Heinz bodies can be seen in patients with unstable hemoglobins, and with oxidant drug exposure.

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• Heinz bodies are precipitates of hemoglobin and can be found in small amounts in normal individuals, especially if the tested blood is allowed to sit for prolonged periods of time following a blood draw. • An increase in Heinz bodies is a classic finding in unstable hemoglobins, where the structurally defective hemoglobin is prone to precipitation. • Another common cause of Heinz body formation is oxidative stress, when the red cell’s hexose monophosphate shunt is overwhelmed and results in hemoglobin precipitation.

• Heinz bodies are characteristically found at the periphery of the red cell, seemingly attached to the membrane. • Heinz bodies vary in size from 1–3 micrometres, with the number being proportional to the rate of formation (i.e. increased number with increased rate of formation) and the size of the Heinz bodies being inversely proportional to rate of formation.5

What is the mechanism behind the formation of Hb H bodies? a) Denatured hemoglobin b) Excess alpha chains c) Excess beta chains d) Excess gamma chains e) Precipitation of unstable hemoglobin

Excess beta chains

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• Hb H bodies are formed due to a deficiency of alpha chains, resulting in an excess of beta chains (i.e. H body = β4). • Therefore they are a hallmark finding in Hb H disease (i.e. triple α-gene deletions) where they are easily detected (see image next page). • Hb H disease is characterized by symptomatic anemia, reticulocytosis and microcytosis.

Hb H bodies in Hb H disease. Note the presence of reticulocytes (black arrow), which are also detected on supravital staining.

• Detection of rare Hb H inclusions in red cells is useful in the diagnosis of alpha0-thalassemia trait, but slide assessment is time consuming and laborious and there is the possibility of both false positive and false negative results.6 • Most laboratories would confirm their findings with further DNA analysis.7 • However, the detection of Hb H bodies remains a useful rapid screening tool in a prenatal patient for determining their alpha thalassemia risk.2 • Hb H inclusions are characterized by their small size (<1 micrometre) and normal, even distribution throughout the red cell, resulting in the so-called “golf ball” cell.5

• This individual had presented in infancy with jaundice and evidence of a chronic hemolytic anemia. • DNA analysis determined that she was a carrier for Hb Sallanches and alpha thalassemia trait (-α3.7/Hb Sallanches,α).

• Hb Sallanches is an alpha chain mutation that produces borderline α-thalassemia hematologic features in the heterozygote state.3 • However, when inherited together with other alpha gene deletions or in a homozygous pattern, this results in a chronic hemolytic anemia and a non-transfusion dependent thalassemic state.

• The patient appears to be well-compensated, with normal hemoglobin, but her counts are sustained only with a robust reticulocyte count. • The history of gallstones is a common complication of individuals with chronic hemolysis, and is caused by excess bilirubin and heme pigments being released from hemolyzed red cells.

• As a group, unstable hemoglobins are relatively rare. • However, common to all is the propensity for the affected globin chain to denature more readily than Hb A. • The denatured hemoglobin forms Heinz bodies, which are detectable on supravital staining.

• Unstable hemoglobin can fall into two categories: – spontaneously unstable variants resulting in a congenital Heinz body hemolysis and – those variants that are induced after exposure to oxidative stress causing an acquired Heinz body hemolysis after ingestion of oxidative drugs, chemicals or accelerated by fever from virus or infections.

• The diagnosis of unstable hemoglobin often relies initially on a high index of suspicion upon the presentation of an individual with evidence of hemolysis. • Peripheral blood morphology and supravital staining can provide some further clues. • HPLC/CE may not detect identifiable variant and further specialized testing needs to be performed that includes demonstration of in vitro instability with either heat or isopropanol testing, and ultimately DNA analysis.

Unstable hemoglobins are an uncommon cause of hemolytic anemia, but should be kept in mind. Peripheral blood morphology can include the presence of bite cells, irregularly contracted erythrocytes, and polychromasia. Increased Heinz bodies and reticulocytes would be typical features on a supravital stain. Supportive laboratory evidence includes the presence of an elevated LDH, elevated unconjugated hyperbilirubinemia and decreased haptoglobin.

The supravital stain detects many different types of red cell inclusions. Morphologic features and clinical information will aid in differentiating between the different types of inclusions.

1. Palmer L, Briggs C, McFadden S, Zini G, Burthem J, Rozenberg G, et al. ICSH recommendations for the standardization of nomenclature and grading of peripheral blood cell morphological features. Int J Lab Hematol. 2015;37:287–303. 2. Quality Management Program—Laboratory Services. Red Cell Disorders Consensus Practice Recommendations for the Laboratory Investigation of Hemoglobinopathies [database on the Internet]. Toronto (ON): QMP–LS QView. c2012 [updated 2012 December 11; cited 2018 January 29]. Available from: https://qview.ca/qview/MainForm.aspx?dl=531291. 3. Wajcman H, Traeger-Synodinos J, Papassotiriou I, Giordano PC, Harteveld CL, Baudin-Creuza V, Old J. Unstable and thalassemic alpha chain hemoglobin variants: a cause of Hb H disease and thalassemia intermedia. Hemoglobin. 2008;32:327–49. 4. Harb R, Thomas DW. Conjugated hyperbilirubinemia: screening and treatment in older infants and children. Pediatr Rev. 2007;28:83–91. 5. Glassy EF, ed. Color Atlas of Hematology 1998, College of American Pathologists. 6. Bain BJ. Haemoglobinopathy diagnosis, 2nd edition. Malden, Mass.;Oxford:Blackwell, 2006. 7. Ryan K, Bain BJ, Worthington D, James J, Plews D, Mason A, et al; British Committee for Standards in Haematology. Significant haemoglobinopathies: guidelines for screening and diagnosis. Br J Haematol. 2010;149:35–49.

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