Fingerprinting Cardiolipin in Leukocytes by Mass Spectrometry for a Rapid Diagnosis of Barth Syndrome

This method generates a cardiolipin finger printing of leukocytes by MALDI-TOF mass spectrometry and allows the measurement of the ratio between monolysocardiolipin and cardiolipin for a rapid diagnosis of Barth syndrome. The main advantage of this technique is the detection of diagnostic lipid species by a single round of mass spectrometry immediately after the isolation of leukocytes from one milliliter of blood. These and the eye sensitivity and specificity make this assay a suitable diagnostic test to be integrated into the routine work of clinical laboratories to screen for Barth syndrome.

Begin by placing blood samples on an orbital shaker for 10 minutes. Next, to 0.9 milliliters of whole blood in a 1.5 milliliter tube, add 0.1 milliliters of 20%dextran. Now pipette and disperse the suspension gently 20 times while avoiding air bubbles and let RBCs sediment for about one hour at room temperature.

Using a syringe, collect and transfer the yellow supernatant to a 15 milliliter tube and then centrifuge at 400 times G for 15 minutes at room temperature. After discarding the supernatant, resuspend the pellet containing mainly leukocytes and residual RBCs in 0.6 milliliters of ice cold double distilled water. After approximately 15 seconds, add 0.2 milliliters of 0.6 molar potassium chloride to the cell suspension to restore the correct osmolarity.

The short osmotic shock will lyse RBC and leave leukocytes intact. Then adjust the final volume to 2.5 milliliters with single strength PBS. Next, centrifuge the suspension at 400 times G for 15 minutes at room temperature.

This is followed by discarding the supernatant and rewashing the leukocyte pellet with 2.5 milliliters of single strength PBS. Repeat centrifuge and discard the supernatant as described previously and resuspend the pellet containing leukocytes in 200 microliters of sterilized double distilled water. Freeze the suspension at minus 80 degrees Celsius or directly perform lipid extraction.

Start the extraction by transferring 20 microliters of leukocytes suspension to a 1.5 milliliter tube and spin at 16, 000 times G for 30 seconds. After discarding the supernatant, add 10 microliters of chloroform to the remaining pellet and repeatedly pipette to promote lipid extraction. Now, add 10 microliters of 9-aminoacridine matrix solution to the pellet in chloroform.

Pipette and disperse repeatedly to mix. Spin the solution containing lipids and chloroform and 9-aminoacridine matrix solution at 16, 000 times G for 30 seconds. Finally, deposit the supernatant as droplets of 0.35 microliters on the MALDI target to be analyzed and let the droplets air dry at least for 10 to 15 minutes.

For lipid analysis, acquire mass spectra of samples in triplicates on a MALDI-TOF mass spectrometer. After calibration with lipid standards, set the analyses in the negative ion mode and optimize the detection mass-to-charge ratio range from 200 thomson to 2, 000 thomson for small molecule analysis. Keep the laser fluence 5%above the threshold to have a good signal-to-noise ratio.

For each mass spectrum, apply an average of 2, 000 single laser shots. Acquire spectrum in reflector mode using delayed pulsed extraction. Apply gated matrix suspension to 400 thomson to prevent detector saturation.

The figure shows that the defects in the TAFAZZIN gene typically determine a lipid profile specific to Barth syndrome indicated by the appearance of monolysocardiolipin and immature cardiolipin forms together with the reduction of mature cardiolipin forms in the cardiolipin fingerprint. Further, the figure also shows that MALDI-TOF/MS analyses of leukocytes of control subjects typically exhibit only two species of mature cardiolipin. The figure shows that monolysocardiolipin plus immature cardiolipin on mature cardiolipin ratio for Barth syndrome patients was higher than for healthy subjects and heart failure patients.

Here, the control groups and Barth syndrome patients are separated by more than one order of magnitude, showing that this method has a strong diagnostic ability for Barth syndrome. To ensure a good assay result, it is crucial to carefully follow all the protocol steps from the leukocytes isolation to the monolysocardiolipin-to-cardiolipin ratio calculation. MALDI-TOF mass spectrometry is useful for obtaining lipid profiles of small amounts of various biological samples to identify lipid biomarkers or pathological alterations.