Effects of Blood Collection Tubes on Determination Vitamin-A by HPLC

Methods: Blood specimen was divided into three parts, Three tubes, for Part I, No Additive tubes were drawn, for Part 2 Serum SepClot Activator tube (gel tube), and for Part 3 EDTA-coated plastic tubes. The tubes were gently mixed using ten complete inversions immediately following blood collection and centrifuged after venipuncture for 15 min and the supernatants were stored at -70°C until analysis. Vitamin A Testing was performed on healthy blood donors; the testing was performed by HPLC.


Introduction
All VACUETTE ® Serum Tubes are coated with micronised silica particles which activate clotting when tubes are gently inverted. VACUETTE ® Serum Sep Tubes contain a barrier gel that is present in the bottom of the tube. The specific gravity of this material lies between the blood clot and the serum [1 -3]. During centrifugation the barrier gel moves upward to the serum-clot interface, where it forms a stable barrier separating the serum from fibrin and cells. Serum may be aspirated directly from the collection tube, eliminating the need for transfer to another container. Serum Clot Activator tubes are made of plastic and are used for the collection of venous blood, tubes should be used with care when measuring therapeutic drug levels because the drug/hormone may diffuse from the serum into the gel, causing a reduction in measured drug level [4][5][6][7].
Tables 1 and 2 Serum Tubes, Yellow cap tubes, indicate type of blood collection tubes containing clot activator and separation gel [8][9][10]. VACUETTE ® Serum tubes are used for determinations in serum   to yield the necessary plasma volume. Gently mix the blood collection tube by inverting 8-10 times immediately after collection. If required, separate plasma from cells by centrifugation within 20-30 minutes.

Serum
Draw a sufficient amount of blood to yield the necessary serum volume. Invert tube 5-10 times to activate clotting. Allow blood to clot at room temperature for 15 minutes.

Materials and Methods
We simultaneously collected blood samples from 10 healthy volunteers (median age, 36 years; range, 23-55 years) into plastic tubes, Blood specimen was divided into three parts, Three tubes, For Part I, No Additive tubes were drawn, For Part 2Z Serum Sep Clot Activatortube, and For Part 3 potassium EDTA-coated plastic tubes, The tubes were gently mixed using ten complete inversions immediately following blood collection and centrifuged after venipuncture for 15 min and the supernatants were stored at -70°C until analysis. Vitamin A Testing were performed on healthy blood donors; the testing was performed by HPLC.

HPLC apparatus
To assay the concentration of vitamin A, run in triplicate using HPLC systemknauer with a smartline UV detector 2500, pump 1000, and manager 5000. C 18 column [250×4.6 mm (I.d.); 5 µm bead size]. The chromatographic separation was performed by a mixture of methanol, water (95:5 byvolume) at a flow rate of 2.5 mL/min; Detection was monitored at 287 nm. Data acquisition and peak purity tests were performedwith chromgate V 3.1 software. The quantitative results were expressed as ug/dl vs. control and calibrator.

Results
Our study showed that, There was no difference in the results were obtained with the serum (No Additive tube) and plasma tube (Figure 1 and Figure 2) in concentration of retinol 67.7 ug/dl, 73.8 ug/dl, but the results was 9.2 ug/dl obtained with the VACUETTE ® Serum Sep Clot Activator tubes (gel tube), and different in the Chromatogram shape, the gel separator may interfere with analysis ( Figure 3). Discrepancies in serum samples prepared in tubes with clot activator were detected [12,13].

Discussion
Laboratory test results are dependent on the quality of the specimen submitted. If there is any doubt or question regarding the type of specimen that should be collected, it is imperative that the laboratory is called to clarify the order and specimen requirements.
The pre-analytical steps are needed for blood sampling by using serum or plasma samples and the processing influence vitamin A determination by HPLC required.
The inner walls of the vacutte serum tubes have a special coating of microscopic silica particles to activate the coagulation process. Serum gel tubes contain an inert barrier gel that is present in the bottom of the tube. During centrifugation the barrier gel moves upward to the serumclot interface, where it forms a stable barrier separating the serum from fibrin and cells.

Limitation
1. Refer to the instrument assay's instructions for use for information on the correct sample material, correct storage and stability.
2. Verify the compatibility prior to use. If there is no assay compatibility, it could lead to false or invalid analysis results.
3. Vitamin D3 determination by HPLC cannot be carried out with all gel tubes without some restrictions (13,14,15).

Conclusion
Tubes are drawn in a specific order to avoid the possibility of erroneous test results caused by carryover of an additive. If Serum tube-with or without clot activator or gel. This tube is either a red top tube or a gold top tube depending on manufacturer and tube additive; it should be drawn as the first tube. We recommended that only serum and EDTA -plasma samples be used for Vitamin A determination by HPLC and cannot be carried out with all gel tubes.