Name: dried blood spots - preparing and processing for use in immunoassays and in molecular techniques
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This communication is based on the results previously published in Virology Journal17 in open access mode under the terms of the Creative Commons Attribution License (<a href="http://creativecommons.org/licenses/by/2.0">http://creativecommons.org/licenses/by/2.0</a>) with BioMed Central as a licensee. The authors gratefully acknowledge the fruitful collaboration in the framework of piloting the study &#8220;Drugs and Chronic Infectious Diseases&#8221; (&#8220;DRUCK Study&#8221;) with U. Marcus, W. Cai, W. Zhang, and R. Zimmermann from the Robert Koch-Institute (Berlin, Germany) and are thankful to S. Moyrer for taking the photographs assembled in Figure 1 as well as to D. F. Whybrew, Ph.D, (G&#246;ttingen, Germany) for correcting the the manuscript. They also express their appreciation for skilful technical assistance to J. Ackermann, E. Bayrambasi, U. B&#252;ttner, S. Dziubek, I. Jakobsche, B. Krellenberg, H. Krohn, P. Kusimski, A. Metcalf, J. Piejek, S. Saar, M. Schr&#246;ter and K. Seidel. This study was supported in part by a grant of the German Ministry of Health to the National Reference Centre for Hepatitis C.

Since the protocol is designed for use in medical diagnostics, its application must follow the principles of the Declaration of Helsinki19. For the first part of the results presented in this communication, both a separate agreement by the patients and approval by an ethics committee seemed to be dispensable for two reasons: (1) “Upon admission” to Essen University hospital, “every patient provides written consent to all necessary biochemical, bacteriological, and virological investigations.”17 (2) “All samples used throughout the evaluation of DBS testing were sent to the Institute of Virology in the process of routine clinical diagnostics. Thus, none of the specimens was collected specifically for the purpose of the study, not a single additional venipuncture was performed and none of the materials was tested for any parameter other than those required by the physicians in the course of the normal diagnostic work-up.”17 The study “Drugs and Chronic Infectious Diseases” (DRUCK Study”)18, in which DBS testing was finally used in the German cities of Berlin and Essen to evaluate people who actively inject drugs, was approved by the Federal Commissioner for Data Protection and Freedom of Information (Berlin, Germany) and as well as by the ethics committee of the Medical University Charité, (Berlin, Germany).
1. Collection of Blood
<ol>
	<li>Venipuncture14, 15, 20, 21
	<ol>
		<li>Put on disposable latex rubber gloves, and clean the area of the intended puncture site (preferably the median cubital vein in the antecubital fossa) with an appropriate disinfectant, e.g., 70% isopropyl alcohol.</li>
		<li>Apply a tourniquet 4 – 6 inches above the putative puncture site to distend the veins. Guide the needle into the patient’s vein and, once it is in place, gently fill the connected blood tube, which contains EDTA as an anticoagulant.</li>
		<li>As soon as venipuncture is complete, release the tourniquet and withdraw the needle. Then, press a dry gauze pad on the puncture site, which subsequently can be held in place by a bandage.</li>
	</ol>
	</li>
	<li>Skin puncture (Figure 1A)13 - 15, 20, 22, 23
	<ol>
		<li>Put on a pair of disposable latex rubber gloves.</li>
		<li>Before skin puncture, the patient should warm his/her hands. The finger is massaged anterogradely the enrich the blood flow towards to puncture site.</li>
		<li>Clean the skin of the palmar side of the tip distal phalanx of the third or fourth finger of the non-writing hand with a suitable disinfectant, e.g., 70% isopropyl alcohol. Puncture the skin by a single-use safety lancet. The finger should be held in such a position that gravity facilitates the collection of blood on the fingertip.</li>
		<li>When collection of capillary blood by skin puncture is complete, place a bandage on the finger tip.</li>
	</ol>
	</li>
</ol>
2. Preparation of Blood Spots
<ol>
	<li>Preparation from blood collected by venipuncture15
	<ol>
		<li>Spot the collected anti-coagulated (EDTA) whole venous blood on the filter cards as soon as possible. Do not prepare dried blood spots more than 24 hr after venipuncture.</li>
		<li>Put all the information necessary for the identification of the patient on the filter card. One card should be spotted only with the blood of a single individual.</li>
		<li>Put on disposable latex rubber gloves.</li>
		<li>Gently invert the blood collection tube 2 – 4 times and subsequently open the stopper carefully.</li>
		<li>Aspirate 50 µl of whole venous blood using a pipette with a disposable tip. Transfer the blood to the center of one circle without touching the filter paper directly with the tip of the pipette. Try to fully saturate the circle.</li>
		<li>Repeat this procedure to fill all required circles of the card.</li>
	</ol>
	</li>
	<li>Preparation from blood collected by skin puncture (Figures 1B and 1C)13 - 16, 23
	<ol>
		<li>Wipe off the first drop of blood with a gauze pad because it may contain excess tissue fluids. Massage 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. Allow the blood to be soaked into the texture of the filter by capillary forces only.</li>
		<li>Let the next large drop of capillary blood form on the finger-tip and collect it in the next circle. Continue this procedure until all necessary circles are filled or blood flow stops.</li>
		<li>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 finger-tip. Perform a second skin puncture on another finger if more blood is needed for the examination.</li>
	</ol>
	</li>
</ol>
3. Drying of Blood Spots
<ol>
	<li>To dry the blood spots, put the filter cards on a clean paper towel in a biohazard safety cabinet and let them dry, preferably O/N (but for at least 4 hrs), at RT 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 (Figure 1D)13, 15, 16.</li>
</ol>
4. Storage and Transportation of Dried Blood Spots (DBS)
NOTE: Processing of the blood spots can be interrupted after drying. The filter cards can now be stored13 - 16, 23.
<ol>
	<li>For storage, put the filter paper card in a single, gas-impermeable zipper bag, containing 1 to 2 desiccant sachets to protect the specimens from moisture (Figure 1E). Optionally, add a humidity indicator card.</li>
	<li>Transfer this bag to a freezer with a temperature of -20 °C or lower as soon as possible. If freezers are not available under field conditions, storage at -4 °C or even at ambient temperature is feasible for up to 14 days.</li>
	<li>Transport frozen DBS specimens on dry ice. For filter cards initially kept at ambient temperature, use a triple packaging system, which consists of the zipper bag(s) as the inner container(s) as well as an inner and an outer envelope. No content markings are required on the outer envelope for shipment by regular mail but the international biohazard symbol must be affixed to the primary inner container16.</li>
	<li>Exclude the filter cards from further processing if the desiccant packs and/or the additional humidity indicator card changes to a pink color.</li>
</ol>
5. Elution of Dried Blood Spots13, 15, 16, 23
<ol>
	<li>Punch out one spot with a single-use 6 mm device from each blood-soaked circle of the Grade 903 filter card (Figure 1F). Transfer all punched dried blood spots from a single patient to one well of the 12-well plate.</li>
	<li>Fill the well with phosphate-buffered saline containing 0.05% Tween 20 and 0.08% sodium azide (Figure 1G). Adapt the volume of added buffer to the minimal respective requirements of the assay used for subsequent analysis of dried blood spots eluates.</li>
	<li>Repeat these steps to obtain a second series of dried blood spot eluates in order to perform molecular analyses.</li>
	<li>Put the cell culture plate on a laboratory shaker and let the punched dried blood spots gently elute for a minimum of 4 hr or, preferably, O/N (Figure 1H).</li>
	<li>The next day, the spots are almost free from blood and hemolytic supernatants have formed (Figure 1I). Transfer these eluates to microcentrifuge tubes. Then, subject them to centrifugation for 2 min at 10,500 x g (Figure 1J) to free the supernatants from any debris that had formed during elution (Figure 1K).</li>
</ol>
6. Analysis of Eluates
<ol>
	<li>Centrifuged eluates are now ready to be used for the intended analyses. Investigate the eluates for markers of hepatitis B virus, hepatitis C virus and HIV infection using commercially available kits and follow the respective manufacturers’ instructions carefully (Figure 1L).</li>
</ol>
<img alt="Cell culture process diagram; multi-day experiment showing petri dishes, centrifuge, imaging system." src="/files/ftp_upload/52619/52619fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Graphical summary of the protocol proposed in this communication for preparation and processing of dried blood spots to be used in immunoassays and by molecular techniques. Whole venous blood and capillary blood obtained by either venipuncture or puncture of the skin by a lancet (A) may serve as specimens for dried blood spot analysis. After transfer of the blood to the circles of a Grade 903 filter card (B, C), the samples should be dried preferably O/N at ambient temperature in a biohazard safety cabinet to form spots of an evenly brownish color without any interspersion of red areas (D). When drying of the blood spots is complete and storage is necessary, the filter cards can be packaged in gas-impermeable zipper bags containing 1 – 2 desiccant sachets (E). Further laboratory processing of DBS comprise the generation of punches by a 6 mm single use device from the center of the circles (F, G), the elution of the punches in a PBS-based buffer for a minimum of 4 hr or, preferably, O/N on a shaker (H), the recovery of the eluates (I), and finally centrifugation of the laboratory cups (J) to free the DBS eluates from any debris that might have originated during the elution process (K). Subsequently, the DBS eluates are ready for analyses, which were performed by a fully automated platform (L) in order to detect serological markers of HBV, HCV and HIV infections, respectively. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52619/52619fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

In the past, RSR received an honorarium for delivering lectures and financial support for his scientific work from Abbott Diagnostics. NG and OM declare that they have no conflicting interests.

DBS have been used for 100 years2 but, surprisingly, there is still no general consensus about their preparation and processing. To date, a sufficient standardization of this important pre-analytical phase has only been achieved in the field of newborn screening14, whereas a variety of different protocols exists for all other applications of DBS testing5,&#160;16,&#160;23. To overcome this remarkable heterogeneity, a comprehensive step-by-step instruction for preparing and processing DBS to be utilized in immunoassays and by molecular techniques is presented in this communication and&#160;evaluated with regard to its effectiveness for detecting markers of HBV, HCV and HIV infections. The focus of the following discussion is primarily placed on the different steps of the suggested protocol.
In the history of DBS testing many different filter paper cards have been used2 but today only two commercial sources are approved by the FDA as class II medical devices for blood collection5,&#160;16. These filter card systems are highly uniform and have very similar absorption characteristics so that analytical results obtained from capillary blood prepared on either of them do not differ by more than 4%&#160;&#8211;&#160;5%28. Not surprisingly, Masciotra and co-workers29 therefore detected HIV-1&#160;RNA equally well with a qualitative assay after elution from blood collection cards from different sources. Given these data, it can be virtually excluded that any of the discrepancies observed when testing coupled serum/DBS pairs for markers of HBV, HCV and HIV infections17 was caused by the choice of the filter card alone. However, when accurate quantification of an analyte is indispensable, source-to-source variation may no longer be negligible and more sophisticated techniques, e.g., perforated DBS (PDBS) as a method for microsampling30 or the preparation of dried serum spots (DSS)31 may have to be applied instead of conventional DBS testing10.
Since only small quantities of blood are used for DBS testing (one drop of capillary blood consists of approximately 50 &#181;l)5,&#160;16, variations in the sample volume are crucial and, admittedly, at least some of the discrepancies between serological results and participant&#8217;s self-reported HBV and HCV status recorded during the piloting of the &#8220;DRUCK Study&#8221; are attributable to this variable. The single most important factor for minimizing &#8220;fluctuations&#8221; of the sample volume is undoubtedly a correct technique of capillary blood collection, which can only be achieved by careful and ongoing training of the technical personnel22. Furthermore, as a measure of quality control, all laboratories working with DBS should have already initiated procedures for identifying and subsequently excluding DBS specimens that have to be considered as unsatisfactory or invalid16.
Studies conducted primarily in the context of HIV32 and newborn screening33 have shown that high humidity may lead to a degradation of analytes, but hitherto no consensus has been reached regarding the question of how long DBS should be air-dried. An interval of at least 4 hr or preferably O/N is proposed in this communication, which therefore adopted conditions that were used in the vast majority of all relevant publications32,&#160;34 Data on storage of DBS to be subsequently used for HBV and HCV testing are conflicting. In 1981, Villa and co-workers35, who applied contemporary analytical techniques, reported that storage at RT did not affect the results of HBV analyses during the entire observation period of 180 days if antibody titers were &#62;&#160;1/1,000, but that DBS became borderline-positive or even negative after 15 days when titers in serum were only 1/100. Storage at -20&#160;&#176;C or 4&#160;&#176;C did not result in a substantial improvement. In contrast, a study conducted thirty years later36 tested replicates of a HBV-positive sample, and the authors found that anti-HBc as well as anti-HBs antibodies were stable for up to 183 days at RT, whereas HBsAg under the same conditions became false-negative already after 63 days. With regard to HBV&#160;DNA detection from DBS eluates, the concentration of the viral nucleic acid was stable for at least seven days at 37&#160;&#176;C37 or proved to be &#8220;resistant&#8221; to storage at RT for up to three weeks38. Testing for anti-HCV antibodies using two commercially available third generation immunoassays39 provided accurate results for a period of 117 days using DBS samples stored at -20&#160;&#176;C, 2&#160;&#8211;&#160;8&#160;&#176;C, and 20&#160;&#8211;&#160;25&#160;&#176;C, respectively. Storage at -20&#160;&#176;C, however, resulted in the lowest variation of optical densities. Applying a fourth generation anti-HCV ELISA, i.e., Monolisa HCV-Ag-Ab-ULTRA, in the context of DBS testing, Larrat et al.40 observed a sharp decrease in analytical specificity after storing the DBS specimens for more than three days at RT. On the other hand, precise testing results were obtained with the same kit utilizing samples deposited for 60 days under various conditions (-20&#160;&#176;C, 2&#160;&#8211;&#160;8&#160;&#176;C, and 22&#160;&#8211;&#160;26&#160;&#176;C) by Brandao and co-workers41. Observations on the decline of HCV&#160;RNA concentrations in DBS under various storage conditions range from no significant alteration at RT for up to one year42 to a tenfold change after four weeks at ambient temperature43. Taking this rather conflicting data on the stability of HBV and HCV antigens, nucleic acids and antibodies into account, it seemed reasonable to retract in the proposed protocol to a consensus, which was defined earlier for the storage of DBS specimens to be used for HIV testing15,&#160;30: For short-term deposition (up to two weeks) antigens, viral nucleic acid, and antibodies are regarded as stable at RT, whereas optimal storage for longer periods is at frozen conditions.
As a rule, three parameters should be considered when designing an elution protocol: (1) the elution buffer; (2) the duration and temperature of elution; and (3) the elution volume23. In the vast majority of all relevant publications phosphate-buffered saline (PBS) was used for eluting DBS, and most authors added a protein, e.g., bovine serum albumin (BSA) or Tween 20, a surfactant, in order to improve the assay signal by stabilizing proteins, as they go into solution, and simultaneously blocking non-specific binding sites23. Only a few reports, which directly compare different elution buffers in the context of DBS testing, are available. Villar et al.36, e.g., recorded almost equivalent elution capacities for all buffers used, but PBS/BSA 0.5% resulted in the lowest level of non-specific reactivity. A very similar observation was made by Croom and co-workers44 when applying the specimen diluent of the Genetics Systems rLAV EIA for the elution of anti-HCV antibodies from DBS. Since the risk of sample degradation is exceedingly low in the first hours after preparation of DBS (see above), it was decided to incubate the spots O/N at ambient temperature and to support the elution process by gentle end-over-end mixing. This approach has the advantage that specimens punched out the previous day can be directly transferred to routine diagnostics early the next morning23. The volume of the elution buffer should be adapted to the minimal respective requirements of the assays used for subsequent analyses in order to keep the dilution factor as low as possible. However, a careful optimization of the elution conditions for every single analyte was not possible in the preceding evaluation because a high sample throughput had to be guaranteed in a comparatively short time during the &#8220;DRUCK Study&#8221;17. Consequently, the rather unfavorable volume of 1,000&#160;&#181;l of PBS-based buffer was used in order to complete the whole elution process for all parameters in two separate operations.
This approach one the one hand fails &#8220;completely in the anti-HBc/anti-HBs system for those individuals infected with HIV due to the low antibody concentrations; &#8230; and it required molecular biological procedures with optimal analytical sensitivity with regard to HBV&#160;DNA and HCV&#160;RNA tests.&#8221;17 On the other hand, the high elution volume proved to be in no way disadvantageous for the determination of HBsAg, anti-HCV, and anti-HIV by the kits used throughout the evaluation. &#8220;The detection of HBsAg positive materials from whole blood eluates succeeded with a sensitivity of 98.6% to a similarly high degree as in previous studies, which had in part used a much smaller elution volume of 100&#160;&#181;l, 250 &#181;l, or 600&#160;&#181;l, or 500&#160;&#181;l&#8221;17, 36,&#160;45,&#160;46. The sensitivity of 97.8% determined in the investigation of 179 serum/DBS pairs for anti-HCV antibodies corresponded to the results of already existing reports24,&#160;44,&#160;47,&#160;48,&#160;49, which had worked with an elution volume that was lower by a factor of 5 to 10. &#8220;In addition, the protocols for anti-HCV detection had been appropriately optimised &#8230; by stipulating their own cut-off points&#8221;17,&#160;24,&#160;48,&#160;49 &#8220;or by increasing the sample volumes from 20&#160;&#181;l to 100&#160;&#181;l.&#8221;17,&#160;49 Finally, the analytical specificity and sensitivity (100% each) established for anti-HIV detection were equal or superior to the performance characteristics of other immunoassays specifically adapted to DBS testing50,&#160;51 &#8220;or a stepwise procedure with the combined use of several anti-HIV tests&#8221;17,&#160;52. &#8220;They, indeed, also exceeded the performance record of an assay that had been specially developed and optimised for the detection of anti-HIV antibodies in DBS eluates (Q-Prevent HIV 1&#160;+&#160;2 DBS kit).&#8221;17,&#160;53
Taken together, the comprehensive step-by-step protocol presented in this communication proved to be a feasible and user-friendly tool for preparing and processing DBS and can, thus, be used reliably in diagnostic virology. It allows approaches using automation&#160;54 and due to its excellent performance characteristics has the potential to serve as a kind of foundation-stone for a future generally accepted consensus protocol in the global field of DBS testing in laboratory medicine.

The idea of using blood collected on a paper card made of cellulose is ascribed to Ivar Christian Bang (1869 &#8211; 1918), the father of modern clinical microanalysis1,&#160;2. In 1913, Bang determined glucose from eluates of dried blood spots (DBS)3 and, later, also performed nitrogen measurements using the Kjeldahl method with this filter paper technique2. Subsequently, several investigators reported on the use of DBS for serological testing to diagnose syphilis2. As early as in 1924, Chapman summarized the advantages of DBS testing when he particularly stressed four items, which are still valid today: (1) compared to conventional venipuncture, less blood volume is required and this fact was most important in pediatric diagnostics; (2) blood collection is simple, non invasive, and inexpensive; (3) the risk of bacterial contamination or hemolysis is minimal; and (4) DBS can be preserved for long periods with almost no deterioration of the analytes2,&#160;4. Besides its use in testing for syphilis, further early applications of the DBS technique included, e.g., the detection of antibodies against measles, mumps, poliovirus, parainfluenza virus, and respiratory syncytial virus (RSV) in 19532, the identification of Shigella in feces dried onto filter paper and shipped by regular mail from Indonesia to Leiden in the Netherlands as well as the detection of antibodies to Schistosoma in DBS taken in endemic areas and analyzed more than three months later5. In 1963, fifty years after Bang&#8217;s original communication2,&#160;6, Guthrie finally published his famous method for the diagnosis of phenylketonuria from DBS obtained by a heel prick from newborns7,&#160;8.
Although from that time onwards, DBS were regarded as a commonly applicable method for collecting, storing, transporting, and analyzing a variety of human body fluids5, their use in diagnostics still remained predominantly focused on the diagnosis of infections especially in resource-limited settings and the systematic screening of newborns for inherited metabolic disorders for decades9,&#160;10. Since 2005, however, a variety of new and innovative DBS applications have begun to emerge. This resulted in an almost exponential increase in the number of respective scientific publications on DBS from about 50 to almost 450 annually at present. Among the emerging applications are such diverse fields as toxico- and pharmacokinetic studies, metabolic profiling, therapeutic drug monitoring, forensic toxicology, or environmental contamination control10,&#160;11.
DBS testing has, thus, made a triumphal march through clinical laboratory diagnostics during the past 100 years2. As in clinical chemistry12, however, during this march pre-analytical variables were not adequately considered for many years. Indeed, even today, after such eminent activities as the CDC&#8217;s filter paper evaluation project13 or the formulation of a national standard for blood collection on filter paper in the framework of newborn screening14, the pre-analytical phase is still largely undervalued in most of the other fields, in which DBS testing is applied5,&#160;10.
Given this background, a comprehensive step-by-step protocol14&#160;-&#160;16 for use in immunoassays and in molecular techniques, which covers all of the essential steps, is suggested for preparing and processing DBS in the following communication: (1) collection of blood; (2) preparation of blood spots; (3) drying of blood spots; (4) storage and transportation; (5) elution of DBS; and finally (6) analyses of DBS eluates. The effectiveness of the protocol was first evaluated with 1,762 coupled serum/DBS pairs for detecting hepatitis B virus (HBV) surface antigen (HBsAg), antibodies to HBV core antigen (anti-HBc), antibodies to HBV surface antigen (anti-HBs), HBV&#160;DNA, antibodies to the hepatitis C virus (HCV) (anti-HCV), HCV&#160;RNA, and human immunodeficiency virus (HIV)&#160;1-p24-antigen/anti-HIV 1/2 using either a fully automated platform or sensitive qualitative nucleic acid tests.17. In a second step, the protocol was utilized in the pilot study &#8220;Drugs and Chronic Infectious Diseases&#8221; (&#8220;DRUCK Study&#8221;) which was conducted by the Robert Koch-Institute in close collaboration with the National Reference Centre for Hepatitis C on active drug users in the German cities of Berlin and Essen18.

<table><tbody><tr><td>Latex rubber gloves</td><td>Hartmann</td><td>#4049500041515</td><td></td></tr><tr><td>70% isopropyl alcohol</td><td>Bode Chemie</td><td>#9768050</td><td></td></tr><tr><td>Mulifly safety cannula</td><td>Sarstedt</td><td>#85.1638</td><td></td></tr><tr><td>EDTA blood collection tube</td><td>Sarstedt</td><td>#02.1066.001</td><td></td></tr><tr><td>Adhesive bandage</td><td>Hartmann</td><td>#2994163</td><td></td></tr><tr><td>Dry gauze pad</td><td>Hartmann</td><td>#143213</td><td></td></tr><tr><td>Singel-use safety lancet</td><td>Owen Mumford</td><td>#3802421</td><td></td></tr><tr><td>Filter paper card</td><td>Whatman/sigmaaldrich</td><td>#10534612</td><td>Filter paper card Grade 903</td></tr><tr><td>Disposable pipette tips</td><td>Starlab</td><td>#S1126-7810, #S1120-8810</td><td></td></tr><tr><td>Zipper bag</td><td>Flexico</td><td>#20219</td><td></td></tr><tr><td>Mini desiccant bag</td><td>Tropack</td><td>#-</td><td></td></tr><tr><td>Disposable Punch</td><td>pfmmedical</td><td>#48601</td><td>6.0 mm Disposable Biopsy Punch</td></tr><tr><td>Disposable Forceps</td><td>Servoprax</td><td>#H7301</td><td></td></tr><tr><td>12 Well Cell Culture Plate</td><td>Greiner</td><td>#665180</td><td></td></tr><tr><td>PBS Buffer</td><td>Gibco</td><td>#14190-136</td><td></td></tr><tr><td>Tween 20</td><td>Applichem</td><td>#A1389.0500</td><td></td></tr><tr><td>Sodium Azide</td><td>Merck</td><td>#66880250</td><td></td></tr><tr><td>Safe-Lock Tubes, 1.5 ml</td><td>Eppendorf</td><td>#30120086</td><td></td></tr><tr><td>ARCHITECT HBsAg (Quantitative)</td><td>Abbott</td><td>#6C3642</td><td></td></tr><tr><td>ARCHITECT anti-HBc II</td><td>Abbott</td><td>#8L4425</td><td></td></tr><tr><td>ARCHITECT anti-HBs</td><td>Abbott</td><td>#7C1825</td><td></td></tr><tr><td>ARCHITECT anti-HCV</td><td>Abbott</td><td>#6C3725</td><td></td></tr><tr><td>ARCHITECT HIV Ag/Ab Combo</td><td>Abbott</td><td>#4J2727</td><td></td></tr><tr><td>MagNA Pure 96 DNA and Viral NA Large Volume Kit</td><td>Roche</td><td>#6374891001</td><td></td></tr><tr><td>artus HBV LC PCR Kit</td><td>Qiagen</td><td>#4506063 (24 tests), #4506065 (96 tests)</td><td></td></tr><tr><td>VERSANT HCV TMA Assay IVD</td><td>Siemens Healthcare</td><td>#2554311</td><td></td></tr></tbody></table>

dried blood spots - preparing and processing for use in immunoassays and in molecular techniques

The idea of collecting blood on a paper card and subsequently using the dried blood spots (DBS) for diagnostic purposes originated a century ago. Since then, DBS testing for decades has remained predominantly focused on the diagnosis of infectious diseases especially in resource-limited settings or the systematic screening of newborns for inherited metabolic disorders and only recently have a variety of new and innovative DBS applications begun to emerge. For many years, pre-analytical variables were only inappropriately considered in the field of DBS testing and even today, with the exception of newborn screening, the entire pre-analytical phase, which comprises the preparation and processing of DBS for their final analysis has not been standardized. Given this background, a comprehensive step-by-step protocol, which covers al the essential phases, is proposed, i.e., collection of blood; preparation of blood spots; drying of blood spots; storage and transportation of DBS; elution of DBS, and finally analyses of DBS eluates. The effectiveness of this protocol was first evaluated with 1,762 coupled serum/DBS pairs for detecting markers of hepatitis B virus, hepatitis C virus, and human immunodeficiency virus infections on an automated analytical platform. In a second step, the protocol was utilized during a pilot study, which was conducted on active drug users in the German cities of Berlin and Essen.

The effectiveness of the protocol suggested for preparation and processing of DBS was first evaluated by analyzing 1,762 coupled serum/DBS pairs for markers of HBV, HCV, and HIV infection.17. For this purpose, DBS were prepared from 100 µl whole venous blood (cf. points 2.1.1 – 2.1.6 of the preceding protocol) and were eluted with 1,000 µl of PBS-based buffer, each, (cf. points 5.1 – 5.5 of the aforementioned protocol) to complete the process for all seven parameters in two separate operations. Such an approach without careful optimization of the elution conditions for every single analyte seemed to be unavoidable because a rather high sample throughput was expected in a comparatively short time during the forthcoming field study “Drugs and Chronic Infectious Diseases” (“DRUCK Study”).
All measurements adhered to the manufacturer’s recommendations in the course of DBS analyses but had to be modified with respect to HBsAg and anti-HBs determinations to compensate either for the effect of hemolysis or the result of dilution. With a cut-off value of 0.05 IU/ml, HBsAg testing of DBS eluates lead to a rate of false-positives of 14.7% (52 out of 354) when compared to the serum samples (Figure 2A). Receiver operating characteristic (ROC) analysis25, 26 of the data indicated that an increase of the threshold to 0.15 IU/ml would result in an ideal separation of HBsAg-positives from HBsAg-negatives (0.986 [sensitivity], 0.000 [1- specificity], Figure 2B). On the other hand, with a cut-off of 10 IU/l for quantification of anti-HBs antibodies a rate of false-negative measurements of 14.2 % (47 out of 331) was recorded (Figure 2C). Hence, following ROC analysis again, this cut-off was lowered to 1.5 IU/l to achieve an optimal discrimination of values (0.917 [sensitivity], 0.007 [1- specificity], Figure 2D).
<img alt="Hepatitis B antigen/antibody frequency histogram. Bar graphs compare different IU/ml cut-offs." src="/files/ftp_upload/52619/52619fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. HBsAg and anti-HBs testing in DBS eluates (modified from17). The ROC-guided increase of the cut-off value for HBsAg quantification from 0.05 IU/ml (A) to 0.15 IU/ml (B) reduced the rate of false-positive results from 14.7 % to 0 %. Similarly, ROC analysis of anti-HBs concentrations suggested a decrease of the respective threshold from 10 IU/l (C) to 1.5 IU/l (D), thereby completely eliminating false-negatives, which initially accounted for 14.2 % of all determinations. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52619/52619fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The results of the coupled serum/DBS analyses are summarized in Table 117.No non-specificity was recorded in the course of DBS testing for any of the seven analytes.
When DBS eluates were investigated for the presence of HBsAg, i. e. the serological key parameter of both acute and chronic infection with HBV, an analytical sensitivity of 98.6 % was determined. HBsAg concentrations in the two falsely-negative sera were 749 IU/ml and 6 IU/ml, respectively. Anti-HBc antibodies were successfully detected in 176 out of 204 (i. e 86.3 %) DBS eluates. Twenty-two out of the 28 specimens which were not detected by the measurements originated from individuals co-infected with HIV. Thus, if HIV-positive individuals were excluded from the calculation, a sensitivity of 97.1 % was achieved for retrieving anti-HBc antibodies from eluted dried whole venous blood. Similar observations held true for the determination of anti-HBs antibodies. Even after adjustment of the cut-off value of the assay to 1.5 IU/l, nine discrepant serum DBS results occurred. In the patients not co-infected with HIV, serum anti-HBs concentrations of 11 – 26 IU/l were found, which were too low to allow for detection after elution of the dried blood.
Anti-HCV antibodies are indicative of either an acute or chronic or resolved infection. Only four (i. e. 2.2 %) falsely-negative anti-HCV results were determined from DBS eluates and further serological and molecular analyses clearly demonstrated that the respective sera were very probably taken from patients, whose infections had long since been resolved.
The detection of anti-HIV antibodies in DBS eluates with the fully automated system was a completely smooth process which yielded an analytical sensitivity of 100 %.
“Mean HBV DNA concentration from whole blood eluates was performed on 100 specimens (mean DNA concentration: 1,573,898 IU/ml, range: &lt; 357 - &gt; 17,860,000 IU/ml) and yielded a value of 93.0 %. Thus, seven samples with low serum HBV DNA concentrations between 409 and 3,643 IU/ml were not detected by the DBS testing. Of 100 sera which had proven to be HCV RNA positive (mean HCV RNA concentration: 1,415,944 IU/ml, range: 2,479 - &gt;7,692,000 IU/ml), corresponding whole blood aliquots were available. The comparative investigation resulted in an analytical sensitivity of 100 % for HCV RNA determinations from DBS eluates.”17
To test the outlined protocol for preparing and processing DBS under field conditions, it was used in close collaboration with the Robert Koch-Institute (Berlin, Germany) during the pilot phase of the study “Drugs and Chronic Infectious Diseases”, which was conducted on active drug users in the German cities of Berlin and Essen18. All 534 participants enrolled (433 men and 101 women) underwent capillary blood sampling as described in detail in sections 2.2.1 – 2.2.3 of the preceding protocol, and DBS were, again, eluted by addition of 1,000 µl of PBS-based buffer.
Two individuals were diagnosed to be chronically infected with HBV, and 39 showed the serological pattern of a resolved HBV infection. Furthermore, fifteen participants were positive for anti-HBs (status after vaccination) and eight were found to be positive for anti-HBc alone. The anti-HCV prevalence among the participants was 57.3 % (N = 193) in Berlin and 73.0 % (N = 144) in Essen. From the antibody-positive samples 65 % (N = 125) and 62 % (N = 89) were also viremic. Twenty-five out of the 534 individuals tested positive for anti-HIV. Nineteen of the infections were already known and 24 of these individuals were co-infected with HCV.
The results obtained by DBS testing were carefully compared with the study participants’ anamnestic data regarding the self-reported HBV and HCV status. This comparison in conjunction with the results of testing coupled serum/DBS pairs led to minor modifications of the testing algorithm for the main phase of the “DRUCK Study”, which will enrol active drug users in six additional large German cities: “Individuals infected with HIV will be always tested for the presence of HBV DNA. Participants whose DBS eluates are positive for anti-HBc or anti-HBs should be subjected to a venipuncture during a second consultation in order to definitely clarify their anti-HBc/anti-HBs status and, finally, all DBS eluates will be screened for HCV RNA regardless of the results of anti-HCV testing.”17
<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Parameters</td>
			<td>Coupled sera/DBS eluates (N)</td>
			<td>Discrepant results (N)</td>
			<td>Reason for discrepancies between testing serum and DBS eluates</td>
		</tr>
		<tr>
			<td>HBV</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HBsAg</td>
			<td>299</td>
			<td>2</td>
			<td>Two patients with chronic HBV infection. Both were under antiviral treatment and one of them was co-infected with HIV. HBsAg concentrations in serum: 749 IU/ml and 6 IU/ml, respectively.</td>
		</tr>
		<tr>
			<td>Anti-HBc</td>
			<td>305</td>
			<td>28</td>
			<td>In serum, twenty-two patients had a resolved HBV infection. Six individuals showed the serological finding “anti-HBc alone”. Twenty-two of the patients were co-infected with HIV. From the “HBV resolvers”, twenty were assessed as “anti-HBs positive alone” by DBS testing and, thus, were falsely classified as “condition after vaccination”.</td>
		</tr>
		<tr>
			<td>Anti-HBs</td>
			<td>310</td>
			<td>9</td>
			<td>The four patients not co-infected with HIV had serum anti-HBs concentrations of 11 ‑ 26 IU/l, which were too low to allow for antibody detection after elution of the dried blood spots.</td>
		</tr>
		<tr>
			<td>HBV DNA</td>
			<td>150</td>
			<td>7</td>
			<td>The respective sera had been obtained from seven patients with low serum HBV DNA concentrations ranging from 409 IU/ml to 3,643 IU/ml.</td>
		</tr>
		<tr>
			<td>HCV</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-HCV</td>
			<td>339</td>
			<td>4</td>
			<td>These four sera had been taken from patients, with long since resolved HCV infections.</td>
		</tr>
		<tr>
			<td>HCV RNA</td>
			<td>150</td>
			<td>0</td>
			<td> –</td>
		</tr>
		<tr>
			<td>HIV</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HIV 1-p24/anti-HIV 1/2</td>
			<td>209</td>
			<td>0</td>
			<td> –</td>
		</tr>
	</tbody>
</table>
Table 1: Results obtained from 1,762 DBS, which were prepared and processed from whole venous blood following the protocol proposed in this communication, compared to the findings in coupled serum samples as a reference.

Watch this Scientific Journal Video about Dried Blood Spots - Preparing and Processing for Use in Immunoassays and in Molecular Techniques at JoVE.com

Dried Blood Spots - Preparing and Processing for Use in Immunoassays and in Molecular Techniques

The preparing and processing of dried blood spots (DBS) for their final analysis are still poorly standardized for most diagnostic applications. To overcome this shortcoming, a comprehensive step-by-step protocol is suggested and subsequently evaluated with regard to its effectiveness for detecting markers of viral infections.

The idea of collecting blood on a paper card and subsequently using the dried blood spots (DBS) for diagnostic purposes originated a century ago. Since ...

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40.0K Views.  University of Duisburg-Essen. The preparing and processing of dried blood spots (DBS) for their final analysis are still poorly standardized for most diagnostic applications. To overcome this shortcoming, a comprehensive step-by-step protocol is suggested and subsequently evaluated with regard to its effectiveness for detecting markers of viral infections.

Video: Dried Blood Spots - Preparing and Processing for Use in Immunoassays and in Molecular Techniques

Enzyme-linked Immunospot Assay (ELISPOT):  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Adaptive immunity is an important component to clearance of intracellular pathogens. The ability to detect and quantify these responses in humans is an important diagnostic tool. The enzyme-linked immunospot assay (ELISPOT) is gaining popularity for its ability to identify cellular immune responses against microbial antigens, including immunosuppressed populations such as those with HIV infection, transplantation, and steroid use. This assay has the capacity to quantify the immune responses against specific microbial antigens, as well as distinguish if these responses are Th1 or Th2 in character. ELISPOT is not limited to the site of inflammation. It is versatile in its ability to assess for immune responses within peripheral blood, as well as sites of active involvement such as bronchoalveolar lavage, cerebral spinal fluid, and ascites. Detection of immune responses against a single or multiple antigens is possible, as well as specific epitopes within microbial proteins. This assay facilitates detection of immune responses over time, as well as distinctions in antigens recognized by host T cells. Dual color ELISPOT assays are available for detection of simultaneous expression of two cytokines. Recent applications for this technique include diagnosis of extrapulmonary tuberculosis, as well as investigation of the contribution of infectious antigens to autoimmune diseases.

Enzyme-linked Immunospot Assay ELISPOT:  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Identification of microbial targets of adaptive immunity in idiopathic diseases can be accomplished by the use of the enzyme-linked immunospot assay.

Adaptive immunity is an important component to clearance of intracellular pathogens.  The ability to detect and quantify these responses in humans is an ...

Immunology and Infection

Dried Blood and Serum Spots As A Useful Tool for Sample Storage to Evaluate Cancer Biomarkers

Blood sample quality is crucial to ensure accurate downstream analyses such as real-time PCR or ELISA. Correct storage of biological materials is the starting point to achieve reproducible and reliable results. All samples should be treated in the same way from blood collection to storage. Depending on the analyses to be performed, whole blood and serum samples should be stored at -20 &#176;C or -80 &#176;C until use. Blood/serum samples should also be aliquoted to avoid multiple freeze-thawing. Another important issue is the sample conditions during shipment from one laboratory to another. If dry ice is not available or the shipment takes longer than a few days, alternative approaches are needed. One option is to use filter paper for blood collection. Here, we propose a method for blood and serum sample collection that takes advantage of dried blood spots (DBS) and dried serum spots (DSS). We developed the procedure to extract DNA from DBS for the downstream evaluation of some single nucleotide polymorphisms (SNPs) by real time PCR. We also optimized an ELISA assay starting from proteins eluted from DSS. This method can be used with other ELISA assays or procedures evaluating proteins.

This protocol describes a simple and useful method to store peripheral blood and serum/plasma for downstream analyses such as single nucleotide polymorphism (SNP) evaluation and ELISA assay.

Blood sample quality is crucial to ensure accurate downstream analyses such as real-time PCR or ELISA. Correct storage of biological materials is the ...

Cancer Research

Dried Blood Spot Collection of Health Biomarkers to Maximize Participation in Population Studies

Biomarkers are directly-measured biological indicators of disease, health, exposures, or other biological information. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spots meet this need, and can be collected in the field with high response rates. These elements are particularly important in longitudinal study designs including interventions where attrition is critical to avoid, and high response rates improve the interpretation of results. Dried Blood Spot sample collection is simple, quick, relatively painless, less invasive then venipuncture, and requires minimal field storage requirements (i.e.&#160;samples do not need to be immediately frozen and can be stored for a long period of time in a stable freezer environment before assay). The samples can be analyzed for a variety of different analytes, including cholesterol, C-reactive protein, glycosylated hemoglobin, numerous cytokines, and other analytes, as well as provide genetic material. DBS collection is depicted as employed in several recent studies.

Biomarkers are directly-measured biological indicators of disease or health. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spot (DBS) collection meets this need, can be collected in the field with high response rates&#160;and analyzed for a variety of biomarkers.

Biomarkers are directly-measured biological indicators of disease, health, exposures, or other biological information. In population and social sciences, ...

Medicine

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Extraction of Tissue Antigens for Functional Assays

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune diseases and cancer2. Our aim is to investigate the antigens recognized by human T cells in the autoimmune disease type 1 diabetes 1,3,4,5. To analyze human T-cell responses against tissue where the antigens recognized by T cells are not identified we developed a method to extract protein antigens from human tissue in a format that is compatible with functional assays 6. Previously, T-cell responses to unpurified tissue extracts could not be measured because the extraction methods yield a lysate that contained detergents that were toxic to human peripheral blood mononuclear cells. Here we describe a protocol for extracting proteins from human tissues in a format that is not toxic to human T cells. The tissue is homogenized in a mixture of butan-1-ol, acetonitrile and water (BAW). The protein concentration in the tissue extract is measured and a known mass of protein is aliquoted into tubes. After extraction, the organic solvents are removed by lyophilization. Lyophilized tissue extracts can be stored until required. For use in assays of immune function, a suspension of immune cells, in appropriate culture media, can be added directly to the lyophilized extract. Cytokine production and proliferation by PBMC, in response to extracts prepared using this method, were readily measured. Hence, our method allows the rapid preparation of human tissue lysates that can be used as a source of antigens in the analysis of T-cell responses. We suggest that this method will facilitate the analysis of adaptive immune responses to tissues in transplantation, cancer and autoimmunity.

A simple protocol for preparing extracts of human tissue to be used as a source of antigens in functional T-cell assays is described. This method allows T-cell responses to tissue-derived antigens to be measured in vitro.

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune ...

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.
The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes1, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.
We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.

We are describing a new method of isolating genomic DNA from whole blood collected for plasma/serology. After plasma collection, the compacted blood is usually discarded. Our novel method represents a significant improvement over existing methods and makes DNA and plasma available from a single collection, without requesting additional blood.

Laboratory tests can be done on the cellular or  fluid portions of the blood. The use of different blood collection tubes  determines the portion of the ...

Preparation of Peripheral Blood Mononuclear Cell Pellets and Plasma from a Single Blood Draw at Clinical Trial Sites for Biomarker Analysis

Analysis of biomarkers in peripheral blood is becoming increasingly important in clinical trials to establish proof of mechanism to evaluate effects of treatment, and help guide dose and schedule setting of therapeutics. From a single blood draw, peripheral blood mononuclear cells can be isolated and processed to analyze and quantify protein markers, and plasma samples can be used for the analysis of circulating tumor DNA, cytokines, and plasma metabolomics. Longitudinal samples from a treatment provide information on the evolution of a given protein marker, the mutational status and immunological landscape of the patient. This can only be achieved if the processing of the peripheral blood is carried out effectively in clinical sites and samples are properly preserved from the bedside to bench. Here, we present an optimized general-purpose protocol that can be implemented at clinical sites for obtaining PBMC pellets and plasma samples in multi-center clinical trials, that will enable clinical professionals in hospital laboratories to successfully provide high quality samples, regardless of their level of technical expertise. Alternative protocol variations are also presented that are optimized for more specific downstream analytical methods. We apply this protocol for studying protein biomarkers against DNA damage response (DDR) on X-ray irradiated blood to demonstrate the suitability of the approach in oncology settings where DDR drugs and/or radiotherapy have been practiced as well as in preclinical stages where mechanistic hypothesis testing is required.

This protocol details clinically implementable preparation of high quality PBMC and plasma biosamples at the clinical trial site that can be used for translational biomarker analysis.

Analysis of biomarkers in peripheral blood is becoming increasingly important in clinical trials to establish proof of mechanism to evaluate effects of ...

Optimized Whole Blood Immunoassays for Analyzing Fungal and Viral Immunity

Whole Blood Assay with Dual Co-Stimulation for Antigen-Specific Analysis of Host Immunity to Fungal and Viral Pathogens

Rapid and resource-efficient sample processing, high throughput, and high robustness are critical for effective scientific and clinical application of advanced antigen-specific immunoassays. Traditionally, such immunoassays, especially antigen-specific T-cell analysis by flow cytometry or enzyme-linked immunosorbent spot assays, often rely on the isolation of peripheral blood mononuclear cells. This process is time-consuming, subject to many pre-analytic confounders, and requires large blood volumes. Whole blood-based assays provide a facile alternative with increased pre-analytic robustness and lower blood volume requirements. Furthermore, whole blood-based assays allow for the preservation of inter-cellular interactions that are not captured by assays using isolated cell subsets. Recently, a refined whole blood immunoassay with dual anti-CD28 and anti-CD49d co-stimulation for comprehensive analysis of both antigen-specific T-cell functions and complex intercellular interactions in response to various fungal and viral antigens has been proposed. This protocol provides guidance for the preparation of stimulation tubes, blood stimulation, and downstream sample processing for flow cytometry, cytokine secretion assays, and transcriptional analyses. This includes a validated and functionally equivalent, previously unpublished, low-volume protocol (250 &#181;L) to make flow cytometric and cytokine-based T-cell monitoring more accessible for studies in pediatric patients or preclinical studies in small animals (e.g., mice). Altogether, these protocols provide a versatile toolbox for complex antigen-specific immune analysis in both clinical and translational research settings.

Author Spotlight: Advancing Immune Monitoring in Critical Care Patients Using Whole Blood Assays

Whole blood-based immunoassays provide a facile and resource-efficient tool to analyze antigen-specific immunity for diagnostic and research purposes. This article provides an optimized whole blood-based protocol with dual co-stimulation for comprehensive analysis of host immunity to fungal and viral pathogens, including a low-volume version for pediatric patients and small animals.

Rapid and resource-efficient sample processing, high throughput, and high robustness are critical for effective scientific and clinical application of ...

Procedure and Key Optimization Strategies for an Automated Capillary Electrophoretic-based Immunoassay Method

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. However, similar to other antibody-based protein assays, optimization of capillary-based immunoassay parameters, such as protein concentration, antibody dilution, and exposure time is an important prerequisite to the generation of meaningful and reliable data. Measurements must fall within the linear range of the assay where changes in signal are directly proportional to changes in lysate concentration. The process of choosing appropriate lysate concentrations, antibody dilutions, and exposure times in the human bronchial epithelial cell line, BEAS-2B, is demonstrated here. Assay linearity is shown over a range of whole cell extract protein concentrations with p53 and &#945;-tubulin antibodies. An example of signal burnout is seen at the highest concentrations with long exposure times, and an &#945;-tubulin antibody dilution curve is shown demonstrating saturation. In addition, example experimental results are reported for doxorubicin-treated cells using optimized parameters.

A capillary-based immunoassay using a commercial platform to measure target proteins from total protein preparations is demonstrated. In addition, assay parameters of exposure time, protein concentration, and antibody dilution are optimized for a cell culture model system.

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. ...