Principles of Method: Total hexosaminidase is measured from the hydrolysis of the synthetic substrate 4-methylumbelliferyl-N-acetyl-a-D-glucosamine, which releases fluorescent 4-methylumbelliferone when acted upon by a b-hexosaminidase. Hexosaminidase A (hex A) is determined as a percentage figure from the amount of activity left after samples are heated for 3h at 50° C. This procedure leads to loss of hex A which is heat-labile but not of hex B or intermediate isoenzymes. Hex A can also be assayed with the more specific substrate 4-methylumbelliferyl-N-acetyl-b-D-glucosamine-6-sulphate (MUGS). The percentage of hex A is measured using a semi-automated procedure with the Cobas Bio centrifugal analyser, whereas hex A with the sulphated substrate is usually measured manually, although an automated procedure is also available. An auxiliary method used for prenatal diagnosis depends on observing a “halo” of fluorescence around fragments of chorionic villi incubated in the presence of MUGS.
Uses and Limitations of Method: Hex A is the deficient enzyme in Tay-Sachs disease and variants, whereas total hexosaminidase deficiency (hex A plus hex B) occurs in Sandhoff disease and variants. The hexosaminidases are composed of two different subunits, a and b, coded by genes HEXA and HEXB. Hex A is ab and hex B is bb. A minor component, hex S, is aa and does in fact occur in Sandhoff disease. Tay-Sachs disease is common in the Jewish population and heterozygotes are screened for by measuring hex A using the heat inactivation method. The most convenient specimen is serum but this is not suitable during pregnancy or when the patient is taking the contraceptive pill or has certain inflammatory diseases; in these circumstances leucocytes are used. In practice both serum and leucocytes are assayed wherever possible for all subjects being tested for carrier status. A semi-automated assay is used for carrier testing in the SAS laboratory because of the increased precision of this method. Heterozygotes for Sandhoff disease can also be detected by this procedure (they have low total hexosaminidase but high percentage hex A). The disease states, Tay-Sachs or Sandhoff, are tested for more reliably using a manual method, employing a sulphated substrate for hex A which is not hydrolysed by hex B. This has advantages because some variant Tay-Sachs patients (referred to as B1 variants) do not have low hex A by the heat inactivation method. The sulphated substrate may sometimes be of value for Tay-Sachs carrier identification and can be used for following up inconclusive subjects. Prenatal diagnosis of Tay-Sachs and Sandhoff disease is possible using chorionic villi or amniotic cells. Manual methods only are used for this. For second-trimester amniocentesis enzymology directly on amniotic supernatant fluid is possible but results are always confirmed by testing cultured cells.
b-hexosaminidase (total and A) is elevated in plasma and serum from patients with I-cell disease and pseudo-Hurler polydystrophy .
Assay of total hexosaminidase and of hex A (towards MUGS) is included in our lysosomal enzyme screening procedure for patients with a neurodegenerative disorder.
Specimen Requirements: For carrier testing: serum from 5 ml clotted blood plus 10 ml lithium heparin unseparated blood sample, for isolation of white cells.
PLEASE SUPPLY DRUG DETAILS (INCLUDING CONTRACEPTIVE PILL) AND STATE WHETHER PREGNANT AND WHETHER JEWISH/NON-JEWISH.
For disease state testing: Blood. 5 ml lithium heparin (orange capped tube) unseparated and unfrozen. Send at room temperature to arrive at the laboratory within 24h of venepuncture.
THE LABORATORY RECOMMENDS USE OF A COURIER SERVICE OR ROYAL MAIL SPECIAL DELIVERY FOR SENDING ALL SPECIMENS TO THE LABORATORY.
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