Diagnosis; Mutation Confirmation

For the plasmalogen synthesis assay, we have assayed more than 200 controls including other non-peroxisomal genetic disorders, more than 400 samples from the peroxisomal biogenesis disorders, and more than 100 patients with rhizomelic chondrodysplasia punctata. The results of peroxismal plasmalogen synthesis help to define the diagnosis and give an estimate … For the plasmalogen synthesis assay, we have assayed more than 200 controls including other non-peroxisomal genetic disorders, more than 400 samples from the peroxisomal biogenesis disorders, and more than 100 patients with rhizomelic chondrodysplasia punctata. The results of peroxismal plasmalogen synthesis help to define the diagnosis and give an estimate as to the clinical severity (phenotype and prognosis). View more

Establish or confirm diagnosis

patients with rhizomelic chondrodysplasia punctata and peroxisomal biogensis disorders

Is research allowed on the sample after clinical testing is complete? Help
If the patient or family signed a copy of our IRB that permits that research be done on the cells, we would store the cells in liquid nitrogen and provide coded anonymized cells to qualified researchers on request.

Sample Negative Report Help
Sample Negative Report

Sample Positive Report Help
Sample Positive Report

The first two steps in the pathway of plasmalogen synthesis occur in the peroxisome, while later steps occur in the microsome. The in vivo assay in cultured cells uses the double-substrate, double isotope method which permits a comparison of the peroxisomal incorporation of 14C- hexadecanol versus the microsomal incorporation of … The first two steps in the pathway of plasmalogen synthesis occur in the peroxisome, while later steps occur in the microsome. The in vivo assay in cultured cells uses the double-substrate, double isotope method which permits a comparison of the peroxisomal incorporation of 14C- hexadecanol versus the microsomal incorporation of the 3H-hexadecyl-glycerol. Grow two T25 flasks of cultured cells in MEM plus 10% FBS to at least 50% confluence, but no more than 80% confluence. Remove media and wash cells with Hank’s buffer. Label with 2 ml of fresh medium containing 10% FBS and 250,000 DPM of 3H substrate and 250,000 DPM of 14C substrate. Incubate for 18 hours or overnight at 37 0C . Harvest cells into small screw-top glass test tubes and store frozen at –20 0C or assay immediately.Prewash a LK-50 Linear – K Silica gel TLC plate, 20x20 cm (Whatman Cat# 4855-821) plate in methanol (approx 200 ml) in a glass tank and air dry for 45 minutes. Activate plate in a 104 0C oven for 10 minutes.Make 200 ml of a 100:1 ether: MilliQ water (200 ml ether: 2ml MilliQ water) and 200 ml of a 95:5 petroleum ether:ether (190ml pet ether: 10 ml ether). Pour each into separate TCL tanks (#1 and #2 respectively) lined with chromatography paper. Let the solutions saturate the paper before use. Resuspend cell pellets in 0.5 ml of MilliQ water and vortex. If pellet does not break up easily, sonicate briefly. Add 2.5 ml of 2:1 chloroform:methanol, vortex, and let the samples stand at room temperature for 30 minutes. Centrifuge the samples for 10 minutes at 2500rpm (Beckman swinging bucket centrifuge) at room temperature.Discard the upper phase and interphase by suctioning off, using a disposable glass Pasteur pipette. Dry down the lower phase under nitrogen. Resuspend in 0.5 ml of 2:1 chloroform methanol, vortex and dry down again. Spot 30 ul of unlabeled phosphotidylethanolamine “carrier” (2ug/ml) on each lane of the plate and allow to dry. It is best to do this two times with 15 ul of the carrier. Resuspend samples in 45 ul of 2:1 chloroform methanol and vortex. Spot one sample per lane overtop of the “carrier” spot on each lane and allow to dry briefly. Develop the plate in Tank #1 (ether:water) TWICE, allowing the plate to dry for 10 minutes between each developing time. Each developing time is approximately 30 minutes. Dry the plate using the hair dryer set on “cold”. Put the plate in a HCl tank (vapor phase only!) for 10 minutes. Make sure the plate does not come in contact with the liquid HCl. The tank is good for about one month each time it is prepared with fresh HCl. Dry the plate with the hair dryer for 30 minutes. Draw a pencil line across the plate, 10 cm from the bottom of the plate. Develop the plate in Tank #2 ((pet ether:ether) until the solvent front reaches the 10-cm line (about 6 minutes). Dry the plate with the hair dryer for 15 minutes. Place the plate into a tank of iodine vapor until yellow bands develop (about 30-60 sec). Outline the plasmalogen band with a pencil (the most prominent band at 5 cm from the bottom of the plate). Be consistent each time you mark this band! After decoloring the plate with a hair dryer, moisten the lanes with MilliQ water, one at a time. Do not wet the lower “spotting area” unless the atmosphere is very dry and the plate dust is flying everywhere. For each lane, scrape the area above and below the marked band, (including the lower “spotting area”) and put into a scintillation vial (Vial “B”). Scrape the marked band into a separate vial (Vial “A”) and place it BEFORE vial B in the scintillation rack. Add 10 ml of Betaflour to each vial, and count for 10 minutes in the Beckman LS6500 scintillation counter. Use “User #1”Calculation: Total 3H = 3H DPM “A” + 3H DPM “B” Plasmalogen 3H as % of Total 3H = (3H DPM “A”/Total 3H) x 100 Total 14C = 14C DPM “A” + 14C DPM “B” Plasmalogen 14C as % of Total 14C = (14C DPM “A”/Total 14C) x 100 Ratios of Plasmalogen 3H as % of Total 3H : Plasmalogen 14C as % of Total 14C and Plasmalogen 14C as % of Total 14C : Plasmalogen 3H as % of Total 3H are calculated View more

Duplicate flasks of cells are assayed. If results do not check, then the cells are analyzed again. ( It is also possible to measure the plasmalogen content of the cultured cells by capillary gas chromatography; however, the cells must be grown in delipidated fetal calf serum.)

Enzyme assays are performed using cultured cells: skin fibroblasts, amniocytes, or chorionic villi.

Tests performed
Entire test performed in-house

For the plasmalogen synthesis assay, we have assayed more than 200 controls including other non-peroxisomal genetic disorders, more than 400 samples from the peroxisomal biogenesis disorders, and more than 100 patients with rhizomelic chondrodysplasia punctata.

The radioactive substrates are available only by custom synthesis and must be tested before use.

Is proficiency testing performed for this test? Help
Yes

Method used for proficiency testing: Help
Inter-Laboratory

PT Provider: Help
Biochemical Genetics Laboratory in Amsterdam, Netherlands

Description of PT method: Help
Several coded cell lines including mild and severe plasmalogen synthesis defects are assayed by each laboratory. As the two laboratories have dissimilar methods for the peroxisomal plasmalogen synthesis assay, the interpretation of the results for each cell line are compared.

Description of internal test validation method: Help
The same normal and abnormal control cells lines are assayed with each set of diagnostic cell lines. Values for each of the control cell lines must fall within 20% of the mean of values obtained on 10 separate analyses using the same lot #s of radioactive substrates.

Software used to interpret novel variations Help
proprietary internal sofware

Laboratory's policy on reporting novel variations Help
In this assay, if the values for the peroxisomal plasmalogen synthesis are in the borderzone between normal and abnormal values, we would state this in the interpretation.(There are some peroxisomal disorder patients that have mild decreases in peroxismal plasmalogen synthesis because of mild PEX gene mutations.)