Hemolysis area: A new parameter of erythrocyte osmotic fragility for screening of thalassemia trait

Abstract

Background: One-tube osmotic fragility test (OFT) is widely used for screening thalassemia traits. Interobserver variation may occur with 0.36% NaCl-based OFT due to the naked eye result reading style.

Purpose: The purpose of this study was to establish and evaluate the novel numerical OFT-based parameter, so-called hemolysis area (HA), in screening thalassemia traits.

Materials and methods: The portable spectrophotometer was invented capable of calculating the HA values. The HA values were then compared among 69, 156, and 19 blood samples having positive, negative, and suspicious 0.36% NaCl-based OFT results, respectively; 109 and 135 blood samples having mean corpuscular volume (MCV) ≤80 fL and >80 fL, respectively; and 138 and 106 blood samples having mean corpuscular hemoglobin (MCH) ≤27 pg and >27 pg, respectively. In addition, the HA values were compared in 166 blood samples having different globin gene genotypes. Finally, the HA cutoff value was determined by receiver operation curve (ROC) analysis.

Results: The HA values in samples having positive, suspicious, and negative 0.36% NaCl-based OFT were 33.3 ± 14.4, 42.9 ± 10.5, and 65.3 ± 13.4, respectively; in sample having MCV ≤80 fL and >80 fL were 43.1 ± 19.6 and 63.8 ± 14.5, respectively; and in samples having MCH ≤27 pg and >27 pg were 46.7 ± 20.1 and 64.8 ± 14.2, respectively. The HA values in normal, hemoglobin E, SEA-α thalassemia 1, and β-thalassemia traits were 67.1 ± 12.6, 36.4 ± 13.9, 20.2 ± 4.8, and 18.6 ± 1.1, respectively. All were significantly different. ROC analysis established 52.4 as the HA cutoff that had comparable effectiveness to the conventional screening tests.

Conclusion: The new HA value was effective and could be an alternative choice for screening thalassemia traits.

Keywords: Hemolysis; kinetics; osmotic fragility test; thalassemia heterozygote; thalassemia screen.

Figure 1

The newly invented portable spectrophotometer and the on-screen menu (a) as well as the on-screen test result (b) showing changes of light transmission (%T), optical density, and hemolysis (HmFr) of the tested sample. The numerical results are also shown at the top of the screen in panel B. HmFr = (OD₀–OD_t)/OD₀ where OD₀ was OD at 0 s and OD_t was OD at 1^st–120^th s). HemeArea or hemolysis area = ΣHmFr taken at 1 s interval for 120 s. (Note: HmFr (120) could also be used as an osmotic fragility test selection parameter, similar to benchtop.)

Figure 2

Appearance of the 0.36% NaCl-based osmotic fragility test results as visualized by naked eyes. The result is written under each tube in which the reddish-turbid RBC suspension is reported as positive, reddish-clear red blood cell solution as negative. The tube with RBC suspension having the appearance between positive and negative result is reported as suspicious

Figure 3

Relationship of hemolysis area and mean corpuscular volume values (a) and comparison of hemolysis area values in blood samples having mean corpuscular volume >80 fL and ≤80 fL (b). Mean values are placed at the middle of each error bar. “Sig.” indicates statistically significant difference after Student's t-test analysis

Figure 4

Relationship of hemolysis area and mean corpuscular hemoglobin values (a) and comparison of hemolysis area values in samples having mean corpuscular hemoglobin >27 pg and ≤27 pg (b). Mean values are placed at the middle of each error bar. “Sig.” indicates statistically significant difference after Student's t-test analysis

Figure 5

Comparison of hemolysis area values in samples having positive, negative, and suspicious 0.36% NaCl-based osmotic fragility test results. Mean values are placed at the middle of each error bar. “Sig.” indicates statistically significant difference after Student's t-test analysis. Note that hemolysis area values in osmotic fragility test-negative group are much higher than those in osmotic fragility test-positive and osmotic fragility test-negative groups

Figure 6

Comparison of hemolysis area values among blood samples having different thalassemia mutations. Mean values are placed at the middle of each error bar. “Sig.” and “Nonsig” indicate statistically significant and nonsignificant difference, respectively, after Student's t-test analysis

Figure 7

Receiver operation curve for determination of cutoff values of hemolysis area that will be used to screen thalassemia trait from normal or nonclinically significant thalassemia. The point where the hemolysis area cutoff value of 52.4 unit is placed is arrowed. Area under curve stands for area under curve

Figure 8

The hemolysis area values and results of 0.36% NaCl-based osmotic fragility test in blood samples having different thalassemia genotypes. The data were presented in box plots with a line of 52.4 cutoff point drawn. The full descriptions of “SEA,” “b^41/41,” “b¹⁷,” “E,” and “neg” can be seen in the text. Note that thalassemia trait with positive and suspicious 0.36% NaCl-based osmotic fragility test results all had the hemolysis area values of <52.4