Optimization of Enzyme Essays to Enhance Reliability of Activity Measurements in Leukocyte Lysates for the Diagnosis of Metachromatic Leukodystrophy and Gangliosidoses

Abstract

(1) Lysosomal storage diseases are rare inherited disorders with no standardized or commercially available tests for biochemical diagnosis. We present factors influencing the quality of enzyme assays for metachromatic leukodystrophy (MLD) and gangliosidoses (GM1; GM2 variants B and 0) and validate the reliability and stability of testing in a retrospective analysis of 725 samples. (2) Patient leukocytes were isolated from ethylene-diamine-tetra-acetic acid (EDTA) blood and separated for subpopulation experiments using density gradient centrifugation or magnetic cell separation. Enzyme activities in whole leukocyte lysate and leukocyte subpopulations were determined. (3) The enzyme activities in leukocyte subpopulations differed significantly. Compared to lymphocytes, the respective enzyme activities were 2.31-4.57-fold higher in monocytes and 1.64-2.81-fold higher in granulocytes. During sample preparation, a considerable amount of the lysosomal enzymes was released from granulocytes. Nevertheless, with the sample preparation method used here, total leukocyte count proved to be more accurate than total protein amount as a reference unit for enzyme activities. Subsequent analysis of 725 individuals showed clear discrimination of enzyme activities in patient samples (48 MLD; 21 gangliosidoses), with a sensitivity of 100% and specificity of 98-99%.

Keywords: gangliosidoses; lysosomal storage disease; metachromatic leukodystrophy; sphingolipidoses.

Figure 1

Presentation of blood cells on the ADVIA-120 blood cell counter before and after preparation of leukocytes via dextran or Histopaque separation. (a–c) Perox-channel: x-axis, myeloperoxidase (MPO) activity; y-axis, side scatter. (d–f) Baso-channel: x-axis, chromatin density (high angle signal 5–15°); y-axis, cell volume (low angle signal). (a,d) Normal cell distribution in whole blood measured in the perox-channel (A: neutrophil granulocytes, B: monocytes, C: lymphocytes; the small number of eosinophilic granulocytes are located in the area below A) and in the baso-channel (a: lymphocytes and monocytes, b: neutrophilic and eosinophilic granulocytes). (b,e) Distribution of cell populations after dextran sedimentation. The neutrophil granulocyte population in the perox-channel shifted partly into the monocyte gate due to MPO release, whereas the population of granulocytes (neutrophilic and eosinophilic granulocytes) was unchanged in the baso-channel (measuring nucleus characteristics). (c,f) Distribution of separated granulocyte fraction after Histopaque isolation (>96% purity). The granulocyte fraction in the perox-channel showed a shift into the monocyte gate similar to that after dextran sedimentation. The population in the baso-channel was not influenced.

Figure 2

Correlation between release of MPO and other lysosomal enzymes from neutrophilic granulocytes into the supernatant: (a) Localization of the neutrophilic population (ADVIA 120, Histopaque separation) after different degranulation conditions: A: incubation without any additives; B: incubation with cytochalasin B + N-fMLP; C: incubation with granulocyte macrophage colony-stimulating factor (GM-CSF) + cytochalasin B + N-fMLP; (b) correlation between MPO (myeloperoxidase) amount (ELISA) and the activities of arylsulfatase A (ARSA), β-hexosaminidase A (HEX A), β-hexosaminidase A+B (HEX A+B) and β-galactosidase (β-Gal) in the supernatant of granulocyte suspensions after degranulation conditions as in the upper graph.

Figure 3

Enzyme activities in normal cohorts: Frequency distributions of enzyme activities measured in the normal cohort show a normal Gaussian distribution. Set minimal permissible values are indicated as dashed vertical lines. (a) The average arylsulfatase A activity (ARSA) was 1.27 ± 0.46 E_514nm/10⁶ leukocytes (n = 474), and 2.3% of the samples were a false pathological with measured values below the minimal permissible value (0.4 E_514nm/10⁶); (b) The average β-galactosidase activity (β-Gal) was 1.36 ± 0.44 E_405nm/10⁶ leukocytes (n = 369), and 0.5% of the samples were false pathological with measured values below the minimal permissible value (0.5 E_405nm/10⁶ leukocytes); (c) the average β-hexosaminidase A activity (HEX A) was 0.81 ± 0.27 E_405nm/10⁶ leukocytes (n = 432), and 0.7% of the samples were a false pathological with measured values below the minimal permissible value 0.3 E_405nm/10⁶ leukocytes); (d) the average β-hexosaminidase A and B activity (HEX A+B) was 3.78 ± 0.80 E_405nm/10⁶ leukocytes (n = 434), and 0.2% of the samples were a false pathological with measured values below the minimal permissible value 0.8 E_405nm/10⁶ leukocytes.

Figure 4

Boxplot of normal and pathological cohort for each analyzed lysosomal enzyme. Set minimal permissible values are indicated as dashed horizontal lines; ○: outliers. (a) Arylsulfatase A activity (ARSA) in normal (n = 474) and pathological cohort (n = 48); (b) β-galactosidase activity (β-Gal) in normal (n = 369) and pathological cohort (n = 3); (c) β-hexosaminidase A activity (HEX A) in normal (n = 432) and pathological cohort (n = 14); (d) β-hexosaminidase A and B activity (HEX A+B) in normal (n = 434) and pathological cohort (n = 4).

Figure 5

Contingency tables for ARSA, β-Gal, HEX A and HEX A+B diagnostics. With the help of contingency tables enzyme activity assays for arylsulfatase A (ARSA) (a), β-galactosidase (b), β-hexosaminidase A (c), and β-hexosaminidase A+B (d), were evaluated for their sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy.