Korean J Lab Med.  2010 Aug;30(4):339-344. 10.3343/kjlm.2010.30.4.339.

Flow-Assisted Differential Diagnosis of Hemolytic Anemia with Spherocytosis: A Case Report

• Won DI

• KMID: 1781638
• DOI: http://doi.org/10.3343/kjlm.2010.30.4.339

Abstract

In patients with hemolytic anemia associated with spherocytosis, differential diagnosis has to be made whether the hemolysis is immune-mediated or of non-immune origin. We report a case of hereditary spherocytosis in a 12-yr-old male child, in whom flow-assisted diagnosis was made. In this case, diagnosis was not determined because routine laboratory workups for hereditary spherocytosis yielded discrepant
RESULTS
positive osmotic fragility test, positive direct antiglobulin test, and normal result in the red cell membrane protein sodium dodecyl succinimide polyacrylamide gel electrophoresis. However, all flow cytometry-based tests, such as osmotic fragility, direct antiglobulin, and eosin 5-maleimide binding test, yielded results compatible with hereditary spherocytosis. Additionally, in family study, the results of eosin 5-maleimide binding test suggested his disease being hereditary. In cases with diagnostic difficulties, flow cytometry may be used as an alternative tool, which can provide additional information in the differential diagnosis of hemolytic anemia with spherocytosis.

Keyword

Hemolytic anemia; Hereditary spherocytosis; Flow cytometry

MeSH Terms

Anemia, Hemolytic/complications/*diagnosis
Child
Coombs' Test
Diagnosis, Differential
Eosine Yellowish-(YS)/analogs & derivatives/chemistry
Erythrocytes/immunology/metabolism
Flow Cytometry
Humans
Male
Osmotic Fragility
Spherocytosis, Hereditary/complications/*diagnosis

Figure

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  Fig. 1. Peripheral blood smear at 12 yr of age. Microspherocytes accounts for ~50% of red cells. A few nucleated red cells and many polychromatic macrocytes are seen (Wright-Giemsa stain, ×200).

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  Fig. 2. Osmotic fragility test at 10 yr of age. The patient (thick lines) showed increased osmotic fragility as compared with a healthy control (dashed lines) in both immediate and 24 hr-incubated samples. Reference ranges are indicated in thin lines. The initial osmotic fragility test performed at 6 yr of age had also showed same patterns.

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  Fig. 3. Laboratory findings associated with hemolytic anemia during last 2 yr. Steroid therapy was tried in every admission.

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  Fig. 4. Flow cytometric osmotic fragility test at 12 yr of age. The events after DW spiking are indicated in red. Post DW spiking, a main subset of the patient showed both a less increased FSC and a less decreased SSC than that of a healthy control (white arrows). On the patient's FSC/SSC plot, a subsidiary subset showing largely increased FSC appears to be reticulocytes (yellow arrow on the right). The control/patient ratio of residual red cells was 4.68 (89%/19%), which was determined as increased osmotic fragility (cutoff 3.0). Abbreviations: FSC, forward scatter; SSC, side scatter; DW, deionized water.

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  Fig. 5. Direct antiglobulin test by flow cytometry at 12 yr of age. Patient's MFIs were below the cutoff MFIs on both the anti-IgG and anti-IgM histogram of red cells (4.97 and 4.99, respectively), and these values are within normal limits. Abbreviation: MFI, mean fluorescence intensity.

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  Fig. 6. Eosin 5-maleimide (EMA) binding test at 12 yr of age. (A) Fluorescence microscopy. EMA-bound red cells show green fluorescence. (B) Flow cytometry. Patient's MFI (443) was below the cutoff (523-3×21=460). Abbreviations: FSC, forward scatter; SSC, side scatter; MFI, mean fluorescence intensity, EMA, eosin 5-maleimide.

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  Fig. 7. Genogram of the patient's family. The results of hematologic and EMA binding test are provided. His elder brother also showed significantly decreased MFI value compared to the cutoff value in this batch of test. As EMA binding test for the patient's family was performed in a different batch from the patient, direct comparison of MFI values between the patient's in Fig. 6 and his family's in this figure is not possible. Abbreviations: EMA, eosin 5-maleimide; MFI, mean fluorescence intensity.

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