Evaluation of a finger prick blood collection method for the seroepidemiology of hepatitis B*

Bulletin of the World Health Organization, 56 (5): 791-796 (1978) Evaluation of a finger prick blood collection method for the seroepidemiology of hepatitis B * W. W. BOND, N. J. PETERSEN, M. S. FAVERO, C. A. SCHABLE, B. L. MURPHY, K. R. BERQUIST, & J. E. MAYNARD A finger prick-swab method of blood specimen collection was qualitatively and quanti- tatively compared with the conventional venipuncture method for HBsAg and anti-HBs determinations by radioimmunoassay (RIA). The new method consisted of pricking the finger, collecting 0.1-0.2 ml of blood with a cotton-wool swab, and eluting the swab in I ml of 1 % bovine albumin in saline containing 0.1 % sodium azide. Using chimpanzees seropositive for HBsAg or anti-HBs, comparisons were made ofRIA results of: (a) whole blood, haemolysed blood, serum, and plasma; (b) paired finger prick samples andserum; (c) dilutions offinger prick samples and serum; and (d) different volumes of blood on swabs. Field studies were carried out at two institutions where hepatitis B was hyperendemic to compare results from pairedfinger prick and serum specimens assayed by the RIA and haemagglutination techniques. The laboratory studies showed that swab RIA values for anti-HBs were significantly lower than serum values and that for HBsAg, swab values were significantly higher than serum values. In HBsAg tests, the field studies showed 100 % agreement between the two methods; in anti-HBs tests, the finger prick method showed 85 % agreement with positive sera. Because of the logistics of collecting and processing blood serum, the finger prick-swab technique may be a valuable aid in large-scale sero- epidemiological surveys for hepatitis B. Viral hepatitis type B is a serious international health problem and determining the prevalence and distribution of hepatitis B surface antigen (HBsAg) and its antibody (anti-HBs) is essential to the epidemiological study of the disease. The logistic, material, and personnel problems involved in conventional large-scale seroepidemiological surveys are manifest even when dealing with urban or predominantly adult populations; difficulties increase considerably in rural areas or when dealing with children or parents reluctant to submit their children to venipuncture. In those situations, an underesti- mate of the epidemiological pattern of the disease may result. We have developed and evaluated a new specimen collection method that is rapid, economical, efficient, and should be widely acceptable to popula- tions involved in seroepidemiological studies of hepatitis B. * From the Hepatitis Laboratories Division (WHO Collaborating Centre for Reference and Research on Viral Hepatitis), Center for Disease Control, US Public Health Service, Department of Health, Education, and Welfare, 4402 North Seventh Street, Phoenix, AZ 85014, USA. METHODS AND RESULTS The technique involves taking blood by finger prick using a blood lancet, 0.1-0.2 ml of blood being collected on a cotton-wool swab (the swabs used should be capable of absorbing at least 0.2 ml of blood). The blood is then immediately eluted by vigorous swirling for 15-20 s in a screw-cap 1-dram (, 3.7-ml) vial with 1 ml of 1% w/v bovine serum albumin in saline (BSAS) containing 0.1% w/v sodium azide as a preservative; fluid in the swab is expressed by pressing and rotating the swab on the inside lip of the vial. The swab should appear dry after expression of the fluid and should have very little visible blood remaining. The added preservative in the eluent is convenient for unrefrigerated transport of field specimens because it prevents microbial degradation of HBsAg or anti-HBs. It was observed that about 0.1 ml of blood collected on a swab and eluted in 1 ml of BSAS results in a small amount of clot formation which subsequently hinders pipetting from the vial; a larger volume of blood results in a correspondingly larger clot. The 3745 -791 - 792 W. W. BOND ET AL. clot can be conveniently removed by freezing the specimen to aid clot shrinkage and using the end of a wooden swab stick to express excess fluid from the clot. A series of laboratory and field studies were designed to test the feasibility of specimen collection on swabs. In the laboratory studies, swab eluents were assayed for HBsAg and anti-HBs using Ausria 11-125 and Ausab a radioimmunoassays (RIA), respectively. Field studies were conducted to compare qualitatively and quantitatively results obtained from paired finger prick-swab and conventional venipuncture (serum) specimens assayed by both RIA and haemagglutination techniques for HBsAg and anti- HBs. The objectives of the laboratory studies were: 1. To compare the relative differences in RIA results from whole blood, haemolysed blood, serum, and plasma, using chimpanzees seropositive for HBsAg or anti-HBs. 2. To compare finger prick-swab RIA results with conventional RIA testing of serum in terms of sample ratio units (SRU), using chimpanzee blood positive for HBsAg. SRU values are determined by dividing the RIA counts per minute (CPM) of a sample by the mean CPM of seven replicates of negative control serum. Specimens with SRU values greater than 2.1 are considered positive. 3. To compare quantitatively finger prick-swab results and conventional serum assay results (chim- panzee specimens) of triplicate, serial 2-fold dilutions of swab eluents and serum positive for HBsAg or anti-HBs, using the proportional response region (see Fig. 1) of the RIA kits. 4. To determine the quantitative effect of sample volume (0.05, 0.1 and 0.15 ml) on RIA sample ratio unit results, using a simulated finger prick-swab technique with chimpanzee blood positive for HBsAg or anti-HBs. Two field trials were conducted to evaluate the finger prick-swab technique of serological specimen collection. Tests were performed on specimens collected for diagnostic purposes during routine epidemiological investigations of two separate outbreaks of viral hepatitis. In the first trial, the following serological tests were conducted on paired serum and finger prick-swab specimens from 36 patients: (a) for HBsAg, Ausria II (RIA sample ratio a Abbott Laboratories, North Chicago, IL 60064, USA. 4. -'-., ~~~~~SLOPE' -0.631 " CORRELATION COEFFICIENT'-0.G6 1') 1 **XMEAN Of I5 REPLICATES 4. 1,40 1 ,6 310 SINGLE DETERMINATION$ NON--PROP TIONALt RESPONSE REGION 2 3.5 2.0 1.5 2.0 2.5 5.0 .5 40 4S 5. LOGN DITLUTION Fig. 1. Qualitative and quantitative response of the Ausria II radioimmunoassay for HBsAg. Serum diluted in 1% bovine serum albumin in saline. units) and reverse passive haemagglutination (Aus- cell, + or - ) on serum and swab eluents, and (b) anti-HBs for serum samples and swab eluents using Ausab (RIA sample ratio units) and passive haemag- glutination (PHA) determinations (HBsAg/adw- coated erythrocytes). In the second field trial, 73 paired serum samples and finger prick-swab eluents were obtained and the same type analysis as described above was carried out. Additionally, for RIA determinations, a direct serum dilution in BSAS (0.1 into 1 ml) was included as a control to approxi- mate the dilution of blood in the finger prick-swab technique. For all laboratory studies, whole and heparinized blood samples were obtained from chimpanzees No. 109 (HBsAg positive) and No. 709 (anti-HBs positive); in part 1 chimpanzees No. 926 (HBsAg positive) and No. 810 (anti-HBs positive) were also used. Whole blood was immediately diluted serially 2-fold in BSAS; plasma and serum were obtained from heparinized and whole blood, respectively, and haemolysed blood was prepared from the heparinized specimens by freeze-thawing three times. Dilutions in BSAS were assayed with appropriate RIA kits for HBsAg (overnight incubation method) or anti-HBs, and linear regression analyses were performed on data points in the proportional response regions of each dilution curve. Fig. 1 shows the results using an Ausria II RIA kit when log,, CPM of serum dilutions were plotted against the log10 dilution; results using Ausab RIA kits were similar. Extrapolations of the best-fit lines allowed determination of the "end-point titre " of each EVALUATION OF A BLOOD COLLECTION METHOD FOR HEPATITIS B specimen, i.e., the points at which the best-fit lines crossed the lower positive limits of the assays (2.1 times the negative kit serum mean). The results in Table 1 show that with HBsAg, the descending order of end-point titre was plasma>serum>whole or haemolysed blood; with anti-HBs, the order was not well defined but the serum had the highest end-point titre in both series, with whole blood, haemolysed blood, and plasma being approximately equal within each series. Table 1. Comparison of RIA end-point titres for whole blood, haemolysed blood, plasma, and serum positive for either HBsAg or anti-HBs End-point titre Chimpanzee HNo. Wobloe molysed Plasma Serum blood 109 (HBsAg) 43 651 22 908 114815 79432 926 (HBsAg) 537031 588843 1 288249 1 202264 709 (anti-HBs) 245 200 219 339 810 (anti-H1Bs) 8.9 8.9 9.7 18 In part 2, ten 0.1-ml portions of whole blood were immediately pipetted onto glass slides and, to simulate the finger prick sampling, each portion was absorbed with a cotton-wool swab. All swabs were eluted in 1-ml portions of BSAS and subsequently assayed by RIA. The remaining blood samples were allowed to clot to obtain serum; 10 replicates of each serum were assayed conventionally and in parallel with the swab eluents, as shown in Table 2. With HBsAg-positive blood, swab eluents gave significantly higher SRUs than serum when com- pared by the Student t test (P = 0.005). Swab eluent SRUs also showed a lower coefficient of variation than those of serum replicates. Conversely, with anti-HBs-positive blood, swab SRUs were significant- ly lower than the serum SRU values (P < 0.0005) and the swab SRU coefficient of variation was highest. From this it was concluded that the finger prick-swab technique might present problems of interpretation of results in seroepidemiological surveys of anti-HBs. In part 3, three 0.1-ml portions of HBsAg- and anti-HBs-positive blood were pipetted onto glass slides. As far as possible, all the blood in each por- tion was absorbed with separate swabs and each sample was eluted in 1 ml BSAS. The blood was Table 2. Quantitative comparison of RIA sample ratio units (SRU) for the finger prick-swab technique (blood) and the conventional technique (serum) Swab SRU Serum SRU HBsAg 63, 59, 65, 65, 58 42, 37, 50, 53, 54 64, 56, 64, 66, 51 57, 51, 38, 52, 45 Mean = 61 Mean = 48 CVa = 8.1 % CV = 14.5 % Anti-HBs 18,14,16,18,12 94, 94, 99, 94, 96 20,10,16,16,13 88, 92,88,99,87 Mean = 15 Mean = 93 CV=19.7% CV=4.6% a CV = coefficient of variation. allowed to clot to obtain serum, and three 0.1-mI portions of each serum were added to 1 ml of BSAS. All swab eluents and serum dilutions were sub- sequently diluted serially 2-fold in BSAS to obtain end-point RIA titres, and the quantitative results are shown in Table 3. Statistical analysis indicated that for both HBsAg and anti-HBs, the serum end- point titres were significantly higher than the swab titres (P = 0.005 and P < 0.0005, respectively) with mean serum HBsAg titres being 1.98 times and mean serum anti-HBs titres 2.6 times higher than swab samples. As in part 2, this experiment showed that the initial dilution involved in the finger prick- swab method (about 10 fold) has a greater quan- titative effect on the anti-HBs assay than on the assay for HBsAg. In part 4, five replicate portions of 0.05, 0.1, and 0.15 ml of whole blood were pipetted onto glass Table 3. Quantitative (end-point titre) comparison of the finger prick-swab technique (blood) with the conventional RIA technique (serum) Swab 1 Swab 2 Swab 3 Serum 1 Serum 2 Serum 3 HBsAg 33 884 25 118 26 915 67 608 48 977 53 703 Mean = 28 639 Mean = 56 763 CVa =16.2% CV = 17.1 % Anti-HBs 631 832 776 1995 1995 1820 Mean = 746 Mean = 1937 CV=13.9% CV=5.2% a CV = coefficient of variation. 793 W. W. BOND ET AL. slides. All portions were absorbed with separate swabs and eluted in 1-ml volumes of BSAS for RIA; the results are shown in Table 4. The mean SRU values increased with volume for both HBsAg and anti- HBs; however, the statistical significance for volume Table 4. Evaluation of sample volume on HBsAg and anti-HBs sample ratio units (SRU) values obtained by the finger prick-swab technique Blood H BsAg Anti-HBs volume Mean swab Mean swab (ml) SRU cv a SRU cv 0.05 46 20 % 21 12% 0.10 50 11 % 45 5.0% 0.15 55 5.5 % 58 9.5 % a CV = coefficient of variation, N = 5. was greater with anti-HBs. The mean SRU value for 0.1-ml portions of anti-HBs-positive blood was significantly greater than the mean value for 0.05-ml portions (P = 0.001). For HBsAg, the 0.15-ml value was significantly greater than the 0.05-ml value, but the 0.1-ml versus 0.05-ml and 0.15-ml versus 0.1-ml values were not significant. It was also noted that swabs with 0.1 ml of blood produced compara- tively small amounts of clot when eluted in 1 ml of BSAS. This experiment demonstrated the importance of obtaining as much blood as possible (not less than 0.15 ml) in the finger prick-swab method, especially for anti-HBs assays, and that the relative amounts of blood collected could be estimated by observing the amount of clot in eluent tubes. The results of the two field trials are presented in Table 5. In trial I, clots observed in only one quarter of the eluent tubes indicated that somewhat less than 0.1 ml of blood was collected on the majority of swabs. Nevertheless, there were two major observations: (a) there was 100% agreement between the results of RIA and the haemagglutina- tion technique for HBsAg when the specimens were collected by either venipuncture or the finger prick- swab method, and (b) for anti-HBs, there was a 70% positive-negative agreement between sera and swab eluents when tested by RIA, while there was only a 40% agreement when serum RIA and swab eluent PHA techniques were compared. The Ausab Ta ble 5. Finger prick-swab field trials H BsAg Anti-HBs Ausria SRU Auscell (RPHA) Ausab SRU PHA Serum Swab Serum Swab Serum Swab Serum Swab Trial / a No. positive 9 9 9 9 20 14 15 8 Percentage of total 26 26 26 26 59 41 44 24 100 % agreement 70 % agreement 40 % agreement Trial // b No. positive 18 18 18 18 53 45 41 37 Percentage of total 25 25 25 25 73 62 56 51 100 % agreement 85 % agreement 70 % agreement a HbsAg, N = 35; anti-Hbs, N = 34. b N = 73. 794 EVALUATION OF A BLOOD COLLECTION METHOD FOR HEPATITIS B RIA appeared to be more sensitive than PHA for the detection of anti-HBs in serum. Additionally, haemo- lysis in swab eluents made the interpretation of PHA results extremely difficult. In field trial II, the data were similar to those from the first trial, with HBsAg determinations showing 100% agreement among the test parameters. The majority of swab eluent tubes showed visible clots indicating that larger amounts (> 0.1 ml) of blood were collected than in field trial I. This observation is reflected in the improved agreements among anti- HBs test parameters, i.e., 85 % agreement between serum and swab Ausab values and 70% agreement between serum Ausab and swab PHA values. DISCUSSION The laboratory evaluation of the finger prick-swab method of specimen collection for HBsAg and anti- HBs serological surveys showed that: (a) quantitative results using RIA kits varied depending on the use of whole blood, haemolysed blood, plasma, or serum; (b) when compared with the respective undiluted serum SRU values, the swab values for HBsAg were significantly higher and those for anti-HBs were significantly lower; end-point titres of swab specimens were significantly lower than titres of respective sera in both cases; and (d) the amount of blood on swabs was much more critical with anti-HBs than with HBsAg. Laboratory and field tests comparing HBsAg and anti-HBs determinations on serum samples and paired finger prick-swab eluents indicated that the finger prick-swab technique is an acceptable method of specimen collection when venipuncture is impos- sible or impractical. In fact, recognizing the time, equipment, personnel, and logistics involved in the collection and processing of blood serum, the finger prick-swab technique may be a valuable aid in future large-scale seroepidemiological surveys. We found no instance in 108 paired serum samples and finger prick-swabs where a serum was HBsAg- positive and the swab eluent negative when assayed by the Ausria II RIA technique. When tested by Ausab RIA for anti-HBs, we found 70-85 %o positive- negative agreement between swab eluents and serum depending on the amount of blood collected on the swabs. In future surveys, this discrepancy could be improved in several ways, as indicated by preliminary laboratory studies: 1. The negative cut-off point of the Ausab RIA could be re-evaluated and readjusted. The dis- crepancies between serum and swab positivities in the field trials occurred when the serum SRU values were low, ranging from 2.2 to 29. In several instances there was a low level of anti-HBs with simultaneous presence of HBsAg and, owing to the sensitivities of the RIA techniques, there is reasonable doubt that an SRU value of 2.1 is a sufficiently high level for a positive-negative cut-off. 2. The amount of blood collected on swabs could be increased. In three recent serological surveys conducted in the United States of America and abroad by our laboratory, 35% of the populations studied had relatively low anti-HBs levels by RIA (serum SRUs < 30). Since the laboratory data pre- sented in this report revealed a high significance between the amount of blood on swabs and re- sulting SRU values for anti-HBs, as much blood as possible should be absorbed by each swab. Also, laboratory studies have shown as much as a 50% sample loss owing to inadequate elution of swabs; only a 9% sample loss was observed when swabs were eluted as directed. 3. Two swabs could be eluted in each specimen vial. Preliminary studies have shown 50-95% in- creases in RIA responses by eluting two swabs instead of one. 4. The RIA volume could be increased from 0.2 ml to 0.4 ml. This procedure alone would effect approx- imately a 50% increase in RIA response. When two swabs per patient are collected and 0.4 ml volumes are assayed, the RIA responses are increased by factors of 1.2-1.4 when compared to the usual method. 5. Quantitative positive controls could be used to determine the relative sensitivity of each RIA kit. When using the positive control serum provided with each Ausab kit, we have observed compar- atively low sensitivities after the Ausab kits have passed the third week of the 1-month shelf life. Kits of this age should not be used for assays of finger prick specimens for anti-HBs. We feel that the 85% positive-negative agreement between the swab eluents and serum as demonstrated in the second field trial is sufficient for comparative seroepidemiological studies of anti-HBs. Combined with the described methods of assay improvement, the finger prick-swab technique may provide valid data from otherwise unobtainable specimens, even in populations with generally low anti-HBs titres. 795 W. W. BOND ET AL. ACKNOWLEDGEMENTS The authors wish to thank Dr M. T. Schreeder, Dr A. E. Denes, and Dr S. H. Hindman for assistance in the collection of specimens. RtSUMt EVALUATION D UNE METHODE DE PRtLEVEMENT PAR PIQURE DU DOIGT AUX FINS D'IUDES StRO-EPIDEMOLOGIQUES DE L 'HEPAITE B Une methode de prelevement par piqfure et tamponne- ment du doigt a subi une comparaison qualitative et quantitative avec le procede classique de la ponction veineuse destin6e a prelever du sang en vue de la d6ter- mination par titrage radio-immunologique (TRI) de l'HBsAg et de l'anti-HBs. Cette nouvelle m6thode consiste, apres piqOre du doigt, a recueillir 0,1 a 0,2 ml de sang a l'aide d'un tampon de coton qui est elue dans 1 ml de solution saline de s6rum-albumine bovine a 10% contenant 0,1 % d'azoture de sodium. Chez des chim- panzes dont le serum contenait de l'HBsAg ou de I'anti- HBs, nous avons compare les resultats de TRI pratiques 1) sur le sang total, le sang hemolyse, le serum et le plasma, 2) sur des paires d'echantillons de serum obtenu apres ponction veineuse et de sang preleve par piqure du doigt, 3) sur des dilutions de serum et de sang respecti- vement obtenus par les memes procedes, et 4) sur des tampons imbibes de differents volumes de sang. Des etudes pratiques ont et6 effectuees dans deux etablisse- ments ou l'hepatite B etait hyperendemique, afin de comparer les resultats fournis par les techniques de TRI et d'hemagglutination appliquees a des paires d'echantil- lons de sang pr6lev6 par piqure du doigt et de serum provenant de ponction veineuse. Les etudes experimen- tales ont montre que les valeurs trouvees pour les TRI d'anti-HBs effectues sur des tampons etaient notablement plus faibles que les valeurs mesurees dans le strum et, pour l'HBsAg, que les resultats des examens effectues sur tampons etaient sensiblement plus eleves que les valeurs trouvees dans le serum. Dans les epreuves relatives a l'HBsAg, les etudes pratiques ont montre une concor- dance de 100% entre les deux m6thodes, et dans les epreuves portant sur l'anti-HBs la methode de piqure du doigt a foumi des resultats concordants a 850% avec la positivite des serums. Les resultats des epreuves d'hemag- glutination sont difficiles a interpreter par suite de l'hemolyse qui survient dans les prelevements operes par piqure du doigt. Les exigences de la collecte et du traite- ment du serum sanguin expliquent que la technique de la piqure suivie de tamponnement du doigt puisse constituer une aide utile dans les vastes enquetes sero-6pidemio- logiques sur l'hepatite B. 796