Diagnosis; Mutation Confirmation

Abnormal results are associated with a disorder of peroxisomal fatty acid oxidation. The PDL laboratory currently has data on more than 200 control cell lines, more than 50 cell lines from X-linked adrenoleukodystrophy males, mutation proven heterozygotes for ALD, peroxisomal biogenesis disorders, and the single enzyme defects of peroxisomal fatty … Abnormal results are associated with a disorder of peroxisomal fatty acid oxidation. The PDL laboratory currently has data on more than 200 control cell lines, more than 50 cell lines from X-linked adrenoleukodystrophy males, mutation proven heterozygotes for ALD, peroxisomal biogenesis disorders, and the single enzyme defects of peroxisomal fatty acid oxidation, D-bifunctional and Acyl-CoA oxidase. View more

Establish or confirm diagnosis

Guidance for management

X-linked adrenoleukodystrophy, peroxisomal biogenesis disorders (Zellweger spectrum disorders), peroxisomal D-Bifunctional enzyme deficiency, and peroxisomal acyl-CoA- oxidase deficiency.

Is research allowed on the sample after clinical testing is complete? Help
If the specimen is collected with a signed copy of our current IRB that states that the cultures may be retained for possible research, then the cells are frozen and stored in liquid nitrogen and later given to qualified researchers who request the cells.

Sample Negative Report Help
Sample Negative Report

Sample Positive Report Help
Sample Positive Report

Method 1: Harvest 1 T-25 confluent flask with trysin, wash the cells twice with PBS, and homogenize the pellet in 100ul distilled water. Take a 5ul aliquot for protein analysis. Extract the remaining cultured cell homogenate with 2:1 chloroform adding 1ug of C19:0 and C27:0 fatty acids as internal standards. … Method 1: Harvest 1 T-25 confluent flask with trysin, wash the cells twice with PBS, and homogenize the pellet in 100ul distilled water. Take a 5ul aliquot for protein analysis. Extract the remaining cultured cell homogenate with 2:1 chloroform adding 1ug of C19:0 and C27:0 fatty acids as internal standards. Dry the lipid extract and add to the dried lipid extract 1.5ml 1N methanolic HCL and heat at 75 deg C. for 3 hours. Cool and extract the fatty acid methyl esters using 3 ml hexane. Dry the hexane extract and add 50ul hexane, transfer to injection vials and inject 1ul on 50 meter DB-1 capillary column, identify the fatty acids, C19:0, C27:0, C22:0 and C26:0 by retention time found by calibrating the column with known fatty acid methyl ester standards and measure the fatty acids by flame ionization against the internal standards C19:0 and C27:0. Method 2: Harvest 1 T-25 confluent flask with trysin, wash the cells twice with PBS, and homogenize the pellet in PBS. Take a 5ul aliquot for protein analysis. Take 25ul of cell homogenate and add to the separate tubes containing either 1-C14 labled C16:0 fatty acid, or 1-C14 labled C24:0 fatty acid. Incubate for 1 hour, add 10 volumes 2:1chloroform:methanol to form two phases and take an aliquot of the water soluble products in the upper phase and transfer to scintillation vial, dry, add scintillation cocktail and measure the radioactivity in the Beckman scintillation counter. C16:0 fatty acid is oxidized mainly in the mitochonria, whereas C24:0 fatty acid is oxidized primarily in the peroxisome. Results are reported as a ratio of radioactivity of C24:0/C16:0. View more

Duplicate analysis using the same method.

C26:0 and C22:0 fatty acid measurements and beta oxidation C16:0 and C24:0 enzyme assays are performed using cultured cells: skin fibroblasts, amniocytes, or chorionic villi.

Tests performed
Entire test performed in-house

More than 50 control cells lines were analyzed. More than 50 cell lines from disorders of peroxisomal fatty acid oxidation were analyzed. The results were compiled and used to establish the reference ranges.

Females at risk of X-linked adrenoleukopdystrophy can have equivocal results, and 20% false negative. Recommend sequence analysis of the ABCD1 gene for a more accurate diagnosis.

Is proficiency testing performed for this test? Help
Yes

Method used for proficiency testing: Help
Inter-Laboratory

PT Provider: Help
Metabolic Diseases Laboratory in Amsterdam

Description of PT method: Help
Cells of known patients and controls were coded and exchanged. Analyses were done by similar methods in each laboratory. The results were reviewed and compared. The records of these exchanges are kept in the laboratory.

Description of internal test validation method: Help
A same normal control cell line and the same abnormal cell line are assayed with each set of diagnostic samples. The results of the normal control and abnormal cells are compared with previous values and results must be within 20% of the mean established for each cell line in order … A same normal control cell line and the same abnormal cell line are assayed with each set of diagnostic samples. The results of the normal control and abnormal cells are compared with previous values and results must be within 20% of the mean established for each cell line in order to accept the values obtained on the cells received for diagnosis. View more

Software used to interpret novel variations Help
internal proprietary software

Laboratory's policy on reporting novel variations Help
All results are reported by telephone and fax to the referring laboratory or physician. If the sample is from a female at risk for being a carrier of X-linked adrenoleukodystrophy, then recomend sequence of the ABCD1 gene.