Upload
mercedes-corp
View
218
Download
1
Tags:
Embed Size (px)
Citation preview
Blood Sampling with Filter Paper Strips Developed under Poultry Management Technique Improvement Project andIts Availability in Haemagglutination Inhibition Test for Newcastle Disease
Case Study
Koike Ikuo1
1 ex-JICA Expert, (D.V.M., M.A)
Technical Consultant
< Current Weak Points in Management of ND Vaccination > particular for small-scale farming
(vaccination in the farms)
On the schedule
Vaccine efficacy
make money
losing money
?
Vaccine efficacy
Protective immuneresponse
Virus neutralization (VN)
Haemagglutination Inhibition (HI)
Blood
sampling
Enzyme-linked immunosorbent assay(ELISA), Agar gel precipitation test,etc.
< Assessment >
Haemagglutination Inhibition (HI) Test for Assessment of Immune Response after Vaccination
< Factors >
Blood sampling
-Sample-blood/serum,
-Bleeding tool-
syringe & needle,filter paper & punctureneedle,cotton soaked withDisinfectant (alcohol +povidon-iodine)
-Materials for HI test-
•chicken red blood cell(CRBC) suspension,• Phosphate buffer solution(PBS),• Haemagglutination(HA) antigen suspension,• serum
-Instrument for HI test-< micro titration >
• dropper & diluter(25 ul & 50 ul )• microplate with 96 wells of V bottom• multichannel micropipette (25 ul & 50 ul )• microplate with 96 wells of U bottom• reservoirs for CRBC, PBS, HA
-Other Instrument/ Tool-
•centrifuge, tubes, tips,pipette, cylinder for measuring,
<Blood Sampling with Filter Paper>
-Case in Japan-
Dr.Kenzo Nobuto(pioneer in Japan )
Available commercially
30 mm18 mm
10 mm
5 mm
blood absorbing area (strip type)
13mm
30 mm
20 mm
(disc type)
-thickness:0.3mm-
•Newcastle Disease (HI)•Toxoplasmosis (Indirect Agglutination)•Canine Distemper (VN)•Japanese Encephalitis (HI, VN)•Mycoplasmosis(AG)•Hog Cholera (VN), etc.
Application to;
Cost Comparison Between FPS and Disposable Syringe in Bleeding for ND HI Test
Item
FPS(Filter Paper Strip)
Materials
Filter Paper of Whatman,
Cost
10-12US$ for100 papers of 32 cmin diameter
Samples Available
About 9,000.
Disposable Syringe
Plastic and attachedNeedle(2.5-3 ml)
6 US$ for100 pieces / box
100
(6 x 90=540 US$) 9000
(Plastic Waste)
Plastic pollution
Approach to Verify Applicability of the FPS
Determination of FPS and Amount of PBS(-) for Elution of Serum from FPS
Field TestComparison of Eluate from FPS and Original Serum
Statistical AnalysisVariance Analysis, Check of Correlation
<Requirements to Filter Paper for ND HI Test >
1. Uniform material and quality in every batch of product.2. Available at local dealers.
Screening work in view of absorbing efficiency and matching with a puncher
Maker: Whatman, Cat. No. :1113320Final selection
Puncher : for punching out disks of regular size, 6mm diameter
About 40 mm
20 mm
5 disks punched out with the puncher
300 ul PBS Correspond to 1 :10 dilutionof serum
Cat. No. :1113320; 1113 as ID, 320 as diameter (mm)
Local Site
Japan
< Filter Paper Strip and the Puncher for Blood Sampling >
< Major Equipment and Materials Necessary for ND HI Test >
<filter paper & puncher> < microtubes or small test tubes > <balance,10mg measurable>
< reagents > < 0.6%CRBC & HA antigen > <multichannel pipette,tips & microplate>
One set in the local veterinary hospital,
Preparation for Bleeding with Filter Paper Strips
1. Cut out the strip from the filter paper
Strip
About 90 strips are cut out from a filter paper of 32 cm in diameter
2. Needle and Alcohol-Iodine cotton
Alcohol: Ethanol (70-80%) for DisinfectionIodine:Povidone-Iodine is recommended
Alcohol : Iodine=5 : 1
3. Fine wire or String with Nods
(used to hang FPS for drying)
< Bleeding Procedure with FPS >
1. Disinfection of the
bleeding spot with
(wing vein region)
alcohol-iodine cotton
2. Puncture the wing vein with the needle
3.Soak the blood into the FPS
4. Leave the FPS dry with hanging, avoiding exposure to sun
6. Keep the FPS in a sunproof box etc.
5. Disinfect the needle with alcohol-iodine cotton for next bleeding
NaCl ----------------------------- 7.4 g NaH2PO4 · H2O---------------- 0.45 g
Na2HPO4· 12 H2O-------------2.4 g
Distilled water----------------- 1,000 ml pH 7.0
<Phosphate buffered saline(PBS)>
<Alsever’s solution for stock of chicken red blood cells > C6H5O7Na3 (Sodium citrate) ----- 8.0 g
NaCl --------------------------------- 4.2 g Glucose------------------------------20.5 g C6H8O7H2 (Citric acid)-------------0.55 g
Distilled water---------------------1,000 ml pH 6.1
< Procedure for extracting blood from the FPS >
1. Data record 2. Punch out 5disks from each FPS
(soaking in PBS of small tubes)
(soaking in PBS of the microtube)
PBS:300 ul/ tube,Soaking time:60 minutes or more,
< Working Flow Chart of Blood Sampling with Filter Paper Strips and ND HI Test >
Statistical Analysis
Purpose: Verification of No Significant Difference
Statistically Between
Eluate of Filter Paper Strip and Original Sera
Variance Analysis of Two Groups
Verification of No Significant Difference between Two Means in the Distribution of Variance Ratio
at Probability of 5 % (0.05)
HI titer of original serum (1:10 fold dilution)
HI
An
tib
od
y T
iter
PBS(ul) for elution
100 200 300 400
: sample FPS soaked with bloodIn above case, 300 ul shall apply as provisory volume of
PBS for elution of serum to be equivalent to no more than 1:10 fold dilution of its original serum.
Test for Optimum Volume of PBS for Elution of Serum from FPS
Distribution of ND HI Antibody Titers in Sera Recovered from Filter Papers and Original One Separated from Whole Blood in Field Survey
ND HI Titer (Reciprocal of Serum Dilution )
0*(5)**
1(10)
2(20)
3(40)
4 5 6 7 8 9 10 11
7 1 1 5 10 6 2 2 2 1
Sera from F.P.S
6 1 1 6 8 6 3 3 1 2
*: indicates exponent x in a series of serum dilution of 1/5 x 2-x ,
**: indicates reciprocal of dilution fold of serum, (5) means antibody titer 5, F.P.S: Filter Paper Strip, Mean indicates the mean of HI titer expressed with exponent x of serum dilution
< Formula for Statistical Analysis Relating to the Subject >
1.
12
n
Ss yy
n
yyS yy
2
2
1. Variance ( s2 )
n= number of samples
2. Standard deviation ( s )
SD = = s 2s
3. Standard error ( SE )
n
sSE
4. Variance ratio
21
22
0 s
sF 2
122 ss
(degree of freedom: f1=n1 – 1, f2=n2 – 1, )
5. Mean of two variances
2
)1()1(
21
2221
212
nn
nsnss
)( 21
21
2
2
210 nn
nn
s
yyF
6. Significant difference between two means in the distribution of the variance ratio
(degree of freedom: f1 = 1, f2 = n1 + n2– 1 )
Group No. of samples (n)
Mean(y)
Variance(S2
1,S22)
Standard Deviation (S)
Standard Error (SE)
Original sera 37 5.378 9.575 3.094 0.508
Sera from FPS
37 5.621 9.908 3.147 0.517
575.9
908.90 F =1.034 < 1.726 < F36, 36 (0.05) < 1.776
F0 < F36, 36 (0.05)
3737
3737
741.9
378.5621.5 2
0
F = 0.112 <3.960 < F1, 72 (0.05) <4.001
F0 < F1, 72 (0.05) ( f1= 1, f2 =37 +37–2=72 )
The variance ratio between two groups
Significant difference between two groups’ means
No significant difference between two groups’ means at a probability of 0.05
Analysis with statistical method
(0.05: probability 5%)
±Std. Dev.
±Std. Err.
Mean
Rec
ipro
cal o
f ser
um d
ilutio
n in
dex
1
2
3
4
5
6
7
8
9
10
ORI_SERA FP_SERA
Fig.1 HI antibody titer distribution in original sera and one from filter paper
3D Range Graph (sera x filter p sera.STA 2v*37c)
Analogous Distribution of HI Antibody Titers in Eluate of Filter Paper and Original Sera
Check of Correlation Between Sera of FPS and Original One
Coefficient of Correlation: r
n
xxSxx
2
2
n
yySyy
2
2
n
yxxySxy
to =
r n - 2
1 – r2
( Degree of Freedom= n – 2 )
<Calculation>
r = 0.981
to = 29.9129.91 > t 35(0.001) = 3.591
Correlation:0.98
Error rate: 0.1 %
1. Drying the FPS soaked with blood should be made in the shade out of the direct sunbeam.
2. The FPS soaked with blood should not be exposed to volatile chemicals or volatilizing gas.
3. After completion of drying, the FPS should be kept into envelopes (plastic bags) or other appropriate storage box(container) for transportation. Cooling condition is preferable.
4. The FPS may be available at 4 for a few weeks and ℃should be examined within the period, but if mold frequently grows under some environment, it should be used under a week. For longer storage, minus 20 or lower temperature is available.℃
Notes for Handling of Filter Paper Strips Soaked with Blood
<Blood Sampling in the Local Site>