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>