The overall goal of this procedure is to provide a standardized protocol for preparing and processing dried blood spots to be subsequently used in immunoassays and in molecular techniques. This is accomplished by first taking capillary blood and transferring it to a filter card. The second step is to dry blood spots overnight and to obtain six millimeter punches out of the circles of the filter card.
Next, the ian of dried blood from the punches is achieved by immersion in buffer on a shaker for a minimum of four hours. The hemolytic EITs are then freed from debris by centrifugation. The final step is to analyze the cleared EITs for serological and molecular markers of infections with the Hepatitis B, hepatitis C, and human immunodeficiency viruses by commercially available immunoassays and molecular techniques.
The idea of using plot collected on a filter card made of cellular originated a century ago. Nevertheless, people using this method for the first time will often struggle even today because the entire pre-analytical phase, which comprises the preparation and processing of dried blood spots for their final analysis, has not been standardized for almost all applications of dried blood spot testing. Given this background, we suggest a comprehensive standardized step-by-step protocol for the preparation and processing of dried blood spots to be subsequently used in immune assay and molecular techniques.
Visual demonstration of this methodology seems to be crucial as several steps like taking off capillary blood. It's correct. Transfer to the filter cards or the preparation of appropriate punches from the crowds are not easy to learn, and consequently, individuals who are using the technique for the first time will undoubtedly benefit from visual representation.
Capillary blood will be taken from Nico's finger by a technician from our laboratory demonstrating the procedure of processing. The obtained blood spots will be by technician ko, who is working already for many years in our laboratory for molecular biological diagnostics For skin puncture. First put on a pair of disposable latex rubber gloves.
Before skin puncture, the patient should warm his or her hands. The finger is massaged an greatly to enrich the blood flow towards top puncture site. Then clean the skin of the Palmer side of the tip distal phalanx of the third or fourth finger of the non-writing hand with a suitable disinfectant puncture the skin by a single use safety lancet.
The fingers should be held in such a position that gravity facilitates the collection of blood on the fingertip. Wipe off the first drop of blood with a gauze pad because it may contain excess tissue fluids. After massaging the finger again to increase blood flow at the puncture site, transfer the following drop to one of the circles of a filter card without touching the surface directly with the fingertip.
Let the next large drop of capillary blood form on the fingertip and collect it in the next circle. Continue this procedure until all necessary circles are filled or blood flow stops during the collection. Allow the blood to be soaked into the texture of the filter by capillary forces only.
Do not squeeze or milk the finger excessively if the blood flow is not sufficient to fill all the required circles of the filter card. If blood flow stops, place a bandage on the fingertip, perform a second skin puncture on another finger if more blood is needed for the examination to dry the blood spots, put the filter cards on a clean paper towel in a biohazard safety cabinet and let them dry, preferably overnight at room temperature. In the absence of any external source of heat, when the drying process is complete, the blood spots have a uniformly dark brownish color and no red areas are visible anymore for storage.
Put the filter paper card in a single gas impermeable zipper bag containing one to two desiccant sachets to protect the specimens from moisture. Transfer this bag to a freezer with a temperature of minus 20 degrees Celsius or lower as soon as possible. If freezers are not available under field conditions, storage at minus four degrees Celsius or even at ambient temperature is feasible for up to 14 days.
Punch out one spot with a single use six millimeter device from each blood soaked circle of the grade 9 0 3 filter card transfer, all punched dried blood spots from a single patient to one well of the 12. Well plate fill the well with phosphate buffered saline containing 0.05%tween 20 and 0.08%Sodium azide. Repeat these steps to obtain a second series of dried blood spot EITs in order to perform molecular analyses.
Then put the cell culture plate on a laboratory shaker and let the punch dried blood spots gently elute for a minimum of four hours or preferably overnight. The next day, the spots are almost free from blood and hemolytic supernatants have formed. Transfer these EITs to micro centrifuge tubes, then subject them to centrifugation for two minutes at 10, 500 times G to free.
The supernatants from any debris that had formed during Ellucian centrifuged EITs are now ready to be used for the intended analyses. The first series of EITs is transferred into cups for serological testing. The second series is transferred to micro tubes for PCR analysis.
Investigate the EITs for markers of hepatitis B virus, hepatitis C virus, and HIV infection using commercially available assays and follow the respective manufacturer's instructions carefully. Finally, the results of the serological testing on the fully automated analyzer are displayed on the screen of the system. The effectiveness of the protocol suggested for preparation and processing of dried blood spots was first evaluated by analyzing 1, 762 coupled serum dried blood spot pairs with respect to hepatitis B virus surface antigen determinations.
The assay protocol was modified because with a cutoff value of 0.05 international units per milliliter, 56.2%of the EITs were reactive compared to the results obtained from Sarah. This corresponded to a false positive rate of 14.7%in hepatitis B virus surface antigen testing. Receiver operating characteristic analysis of the data indicated that an increase of the threshold to 0.15 international units per milliliter would result in an ideal separation of Hepatitis B virus surface antigen positives from hepatitis B virus surface antigen negatives when investigating serum dried blood spot pairs for the presence of hepatitis B virus surface antigen, only two discrepant were obtained.
The two falsely negative Sarah originated from patients under antiviral treatment. The qualitative determination of H-B-V-D-N-A by real-time PCR from whole blood EITs yielded a sensitivity of 93%Thus seven samples with low serum H-B-V-D-N-A concentrations were not detected. Only four falsely negative anti HCV results were determined from dried blood spot EITs.
The respective C were very probably taken from patients whose infections had long since been resolved. The analytical sensitivity of 100%for H-C-V-R-N-A determinations from dried blood spot EITs indicated the excellent sensitivity of the isothermal transcription mediated amplification approach, which was used for H-C-V-R-N-A amplification. The detection of anti HIV antibodies in dried blood spot EITs with a fully automated system also resulted in an analytical sensitivity of 100%This video shows that it is nowadays possible to reliably detect serological and molecular markers of hepatitis B, hepatitis C and HIV infections in alls of tight blood spots, even when a comparatively high elution volume is used and modifications of the essay protocols established for serum or plasma investigations are not considerably changed.
However, the communicated results also confirmed the inherent limits of the dried blood spots technique. In some cases. The outlined protocol was also tested under field conditions on 534 active drug users in the German cities of Berlin and Essen during the pilot phase of the study drugs and chronic infectious diseases.
The results obtained led to a minor modification of the testing algorithm for the main phase of the drug study in six additional large German cities.
