2. Containing the sample for serum separation and transport.

Very few serological tests are conducted today on the poultry farm, and fewer still on whole blood. The exception would be the whole blood plate agglutination testing for Salmonella pullorum, which is still carried out in some countries. In other cases the objective is to obtain a sample of serum of good quality (without bacterial growth or haemolysis) and more than sufficient in volume to carry out, and repeat if necessary, all of the required tests. There are a number of options which may be used to contain such samples.

Figure 2.1. Serum separation tends to be proportional to the surface area of the clot. If collecting in tubes or vials they should be kept slanted until clotted.

2a. Glass or plastic vials. These are the traditional containers for blood samples. 5 ml vials should be sufficient for most purposes. For these and all other containers cited here a chemical treatment (e.g. 1% polysiloxane fluid) is highly beneficial from the point of view of enhancing the speed of serum separation and serum yield. Immediately after the blood is placed in the vial it should be firmly stoppered and laid horizontally to encourage the formation of a long thin clot (Figure 2.1).

2b. Micro-titre plates. Whittemore and Williams (1972) described the use of micro-titre plates for transport and storage of serum samples derived from conventionally collected blood samples. Williams (1973) took this one step further by describing a method of collecting the whole blood directly into the micro-titre plate. The disadvantages of these techniques are:

• I. Maximum blood or serum volume of 0.3 mls
• II Difficulty in effectively sealing plate, hence samples should be transported deep frozen, on dry ice.

2c. Plastic Straws The use of thin flexible plastic straws for collecting whole blood samples (Figure 2.2) has been described (McMullin, 1982a). The main advantages of this system are:

• I. A long thin clot is produced without tilting or inclination, maximizing serum yield.
• II. Most of the container volume is used for blood, minimizing the space required for packing samples and minimizing the loss of serum adherent to the container.}

Figure 2.2. Because of the long thin clot produced in blood collected in siliconised plastic straws serum separation tends to be rapid

2d Plastic card

The use of a multi-compartment plastic card for the storage of sera has been described (McMullin, 1982c). This allows groups of sera of various sizes to be easily held together. The volume of sera used can be carried by altering the height of the card, while the size of the group of sera can be altered by using cards of different lengths. The samples are easily identified by writing directly on the card with a marker. The same cards if siliconized can also be used to collect whole blood. Recent experience has shown, for example, that it is perfectly feasible to collect blood in the field via a straw directly into the plastic card. This eliminates the need to hold, manipulate, close or identify the straws . However this is associated with somewhat slower separation of serum,and greater difficulty of serum manipulation in the laboratory.

Figure 2.3. Multi-compartment plastic card allows frozen storage of a large number of individually identifiable serum samples in a small space. This photograph shows whole blood samples in the card with satisfactory serum separation. The normal use of card in our laboratory is, however, for separated serum.

A system which possibly gives the best of both worlds involves taking the blood sample into a simple open-ended plastic straw whose end is then doubled over and placed in one of the divisions in the card. After sufficient serum separation, the straw is manipulated in such a manner as to allow the serum to leak out into the cell of the card. The card can then be sealed and sent to the laboratory. This technique can be used to completely eliminate re-labeling of samples associated with serum separation. It is to be recommended that anyone interested in applying such methods in serological testing examine these and other possible combinations to determine suitability (both for field and lab.) for the various applications envisaged.

Regardless of the type of container used the samples should be maintained at room temperature until the blood has clotted (usually within 1 hour). The samples may then be refrigerated (not frozen) until dispatch to the laboratory. One person at each site should be trained to take the samples required whenever necessary. This avoids the use of specialized bleeding crews and reduces risk of disease transmission between farms on personnel or fomites.

It is most important that the reception of the samples in the laboratory be organized in such a way as to avoid excessive re-labeling and minimize labour in the storage of samples. We have found micro-titration plates appropriate for short-term storage of serum samples (Williams,1973), and hollow plastic cards can be used for long-term storage or where larger volumes are involved (McMullin, 1982c). All of these methods may be usefully integrated in an efficient and flexible sample-handling system for sera for routine testing.

3. Filter paper disks Another method of submitting samples for serological testing is to absorb blood samples on filter paper (Beard and Brugh, 1977, Brugh and Beard, 1980). These authors recommended the collection of whole blood directly on to strips of filter papers. By stapling 3 strips together in the form of a star up to 6 birds may be sampled. The ends of the strips can be bent upwards to avoid the blood-soaked paper coming in contact with the supporting surface. The samples are eluted by cutting 2 4.8 mm disks using a conventional paper punch and placing them in 200 mcl of phosphate-buffered saline (PBS, pH 7.2) in a micro-titre plate. The samples are agitated for 1 hour at room temperature and held overnight at 4 C. The resulting eluate was found to be equivalent to a serum dilution of 1:10 when used in HI tests for Newcastle Disease and Avian Influenza.

A modification of this method was tested (McMullin, 1979) using filter paper (Frama Qualitative Filter paper, Sao Paulo, Brazil). Blood samples were collected from 12 adult broiler parent chickens vaccinated against Newcastle Disease using both plastic straws and filter papers. The first set of eluates were prepared by cutting 5mm disks shortly after the papers had dried. Either 2 or 4 disks from each paper were stored in a micro-titre plate well at -20 C. The disks were stored in a plastic bag and left on the laboratory bench at room temperature (18-26 C.) overnight. The papers were not exposed to direct sunlight. After 24 hours a further set of disks were cut from the papers and added to the wells of the plate. Fifty mcl of PBS was added to each well, this being 12.5 mcl/disk for those wells with 4 disks and 25 mcl/disk for those with 2 disks. Elution was carried out in the refrigerator at 4 C. for 1 hour. The objective was to obtain an eluate of high titre suitable for use in the HI test. The HI test was carried out using a micro-titre method in which the first dilution tested was 1:4. Table 1 shows the results obtained, which suggest that in this system an eluate obtained with 12.5 mcl/disk was equivalent to a serum dilution of 1:8. The eluates obtained from 25 mcl/disk were equivalent to a serum dilution of 1:16. Even these highly concentrated eluates would not be very sensitive in the detection of low HI titres, however elution with agitation or over a more prolonged period might improve the antibody concentration in eluate. It should be noted that, in this experiment, simply holding disks for 24 hours at room temperature caused a drop of Log2 0.6 in the mean titre.

Table 1. Newcastle Disease HI titres for 12 different samples comparing serum with filter paper elutions (2 or 4 disks per 50 mcl eluting fluid, shortly after drying and 24 hours later.

The Central Veterinary Laboratory (Weybridge, U.K.) stocks pre-punched filter papers, each consisting of a quarter segment of a circle with 6 small disks attached to it. The following are the instructions provided by the CVL (Nicholas, 1992) for the use of paper disks in blood sampling:

"Each small disk should be completely saturated with blood. The papers should be held separately until dried (photographic slide boxes are ideal for this purpose). The samples should be dried as soon as possible. When returned to a laboratory they are normally dried at 37 C. for 30 minutes (the time can be extended to a maximum of 2 hours). When the disks are dry (dark red in colour and dry to the touch) they should be stored in clip-seal plastic bags at 4 C. Avoid dispatching to the testing laboratory at a week-end. When it is intended to run the tests 2 disks from each bird are selected and each pair is placed in a separate flat-bottomed micro-titre plate containing 100 mcl of PBS (pH 7.2). Elution of blood from the disk is allowed to proceed for 30 minutes at room temperature, with agitation during the first 10 minutes. Individual wells need to be checked for satisfactory elution by observing the liquid colour. Any samples which have not eluted well should be recorded as this will affect the test results. For the purpose of further testing the eluate produced in this system is considered to be equivalent to a 1:25 dilution of serum. It will need to be further diluted in an appropriate sample diluent to carry out a specific Elisa test (i.e. if the required dilution is 1:500 then the eluate will be diluted 1:20 prior to testing)."

This system has been used mainly for screening samples using an Elisa for Salmonella enteritidis . However we have recently carried out a small validation experiment using these papers to run Chick Anaemia Virus (CAV) Elisa tests. Six 46 week old broiler parent chickens were sampled by wing vein puncture. Blood was collected into siliconized plastic straws and on CVL paper disks. The samples were returned to the laboratory within 3 hours of collection and were dried according to the above instructions. Both the straws and papers were then held in the refrigerator at 4 C. for 48 hours. The sera were then separated and placed in a micro-titre plate. An eluate was prepared using 2 disks for each sample according to the standard CVL method. Both sera and eluates were frozen in the micro-titre plate at -20 C. The remaining paper disks were separated in 2 groups, each in a clip-seal plastic bag. One was stored in the refrigerator at 4 C. and the other was left at room temperature (on laboratory bench, no direct sunlight). After 4 days eluates were prepared in the same fashion as the initial sample and stored frozen in the micro-titre plate until the tests were run. It was noted that the eluates from the "room-temperature" disks were relatively pale. The tests were run according to our standard procedure for the assay. It was assumed that eluate represented a serum dilution of 1:50 so it was further diluted 1:10 for running the test.

Table 2. Chick Anaemia Virus Elisa Serum-to-positive ratios for 6 different samples, serum collected conventionally and eluate produced in 3 different ways.

The serum-to-positive ratios for the 6 samples are shown in table 2. All 6 sera produced strongly positive reactions. The fact that the first elutions produced reactions which were, on average, twice as strong as the sera suggests that the elutions are closer to being equivalent to 1:25 serum dilution rather than 1:50. There was an appreciable weakening in the reactions when the disks were held for 4 days at 4 C. (S/P dropped from 1.63 to 1.13. However, storage of the disks at room temperature (about 18 C.) led to a very marked reduction in antibody activity, although 3 sera were still just within the positive range (S/P => 0.2). Analysis of variance of these data confirmed that there was a highly significant overall treatment effect (F=46.01,p<0.001). As might be expected the comparison between the freshly frozen eluate and elution after holding disks at room temperature also indicated a highly significant effect (F=132.36, p<0.0001).

These results are consistent with the findings on the use of disks for ND HI tests (Table 1 above) and with other experience we have had in the use of the disks for Salmonella enteritidis Elisa tests. This accumulated experience suggests that antibody titres in paper disks do not remain stable after drying, as judged by the results obtained from eluates. The effect occurs during storage at 4 C. but is much more marked at 18 C. This may be due to degenerative changes in the antibody molecules. Alternatively it may be due to the formation of insoluble complexes which prevent their elution. If the latter is the case then it may be possible to avoid this problem by the use of an improved elution procedure.

Hassan, J.O. et alii (1990) have used a similar disk-based system to test samples using an indirect Elisa to detect Salmonella typhimurium antibodies. In this case serum was dried on to filter paper strips and held refrigerated for four weeks. This resulted in little change in Elisa antibody titre. Treatment of such strips with phenol or chloroform vapour had little or no effect on the anti body titre. Avakian and Dick (1985) carried out a detailed comparison between whole-blood samples collected on filter-paper strips and sera collected in the normal manner using an Elisa test to measure Pasteurella multocida antibody.

Whole blood was collected on filter-paper strips, dried for 48 h at room temperature, and then stored in sealed plastic bags at 4C. Eluates of whole blood, obtained by overnight elution of two 4.8 mm disks in 200 mcl of buffered saline at 4 C, were compared with conventionally harvested serum for antibody activity by Elisa. Paired samples, taken from the same bird at the same time, showed no significant difference in antibody activity as measured by absorbance when the disk-elution process itself was consid ered to be a 1:20 dilution. It was concluded that eluates of blood, derived from whole blood dried on filter-paper strips, may be used as an alternative to serum in Elisa for measuring P. multocida antibody activity

In summary we can say that whole blood samples collected and dried on filter papers have been found capable of providing eluate samples which can be used in a variety of serological tests.

The balance of advantages and disadvantages may be stated as follows:

Advantages:

• 1. Ease of collecting a small volume sample
• 2. Elimination of risk of sample spillage
• 3. Reduced risk of bacterial growth in sample

Disadvantages

• 1. An eluate equivalent to neat serum is not obtainable (this is still required for rapid plate agglutination tests)
• 2. Samples are haemolyzed
• 3. Elution requires more laboratory time than serum separation
• 4. Antibody activity in a disk will decrease on storage. The rate of loss of titre will depend on time and temperature.

The work on filter paper samples which has been done more recently places greater emphasis on standardized methods of drying and low-temperature (4 C.) storage of samples. Our experience suggests that filter papers need to be handled and stored at least as carefully as liquid samples. It is unlikely that filter papers can produce reliable quantitative results when samples need to be transported any distance. In order to minimize loss of titre elutions should be prepared as soon as the samples are received and stored frozen until tested. Even under ideal conditions the use of filter papers is bound to add an extra source of variability to the results (due to small variations in up-take of blood into the paper and in elution of antibodies from the paper). For these reasons it is important to validate a specific sample handling system against normal serum samples and to reserve the filter paper technique to situations in which large differences in antibody activity are being sought (screening and qualitative tests).