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 Disclaimer This document contains content developed by IQMH. IQMH’s work is guided by the current best available evidence at the time of publication. The application and use of this document is the responsibility of the user, and IQMH assumes no liability resulting from any such application or use. This document may be reproduced without permission for non-commercial purposes only and provided that appropriate credit is given to IQMH. Copyright The reader is cautioned not to take any single item, or part thereof, of this document out of context. Information presented in this document is the sole property and copyright of the Institute for Quality Management in Healthcare (IQMH). The logos and/or symbols used are the property of IQMH or other third parties. © Institute for Quality Management in Healthcare. All rights reserved. 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. Continue e-Learn Module Confidence. Elevated. © Institute for Quality Management in Healthcare 18 That’s Not Correct Try Again 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. Continue with e-Learn Module Confidence. Elevated. © Institute for Quality Management in Healthcare 28 That’s Not Correct Try again 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. Continue with e-Learn Module Confidence. Elevated. © Institute for Quality Management in Healthcare 31 That’s Not Correct Try again 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. Continue with e-Learn Module Confidence. Elevated. © Institute for Quality Management in Healthcare 34 That’s Not Correct Try again Confidence. Elevated. © Institute for Quality Management in Healthcare 35 Question 6 For this patient, what is the