Apresentação NRL MMP SCC Versao Final

8/17/2019 Apresentação NRL MMP SCC Versao Final

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Somatic Cell Count in

cow’s raw milk byreference method

Portugal and Turkey

Cooperation Training

sessions in the

framework of the

National Program of

Waste Monitoring

November rd !"#$

%N%&'( )*%STS&

Polo do +umiarCristina &lei,oManuela 'ida

INIAV – UEISTSAPortuguese NRL-MMP



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8 P"ant and Anima" reeding and #enetics

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Somatic Cells

CountPortugal and Turkey Cooperation

Training sessions in the framework of the National Program of Waste

Monitoring

%N%&'( )*%STS& Polo do +umiar(

rd November !"#$



• +eukocytes released from the blood to combatudder infection( and thereby( prevent or reduceinRammation >mastitis?

4rom 1reek Somatik=s U body( from the body

Macrophage>D"V?

Neutrophiles>#"V?

+ymphocytes>!OV?

• *pithelial cells presents in lownumber >"K!V?

!at are Somatic (e""sF



• Re"eased from t!e ,"ood to com,at udderinfectionG

– in res%onse to a %!ysica"G• c!emica" or

– infeccious agression

– and t!ere,yG to %re)ent or reduce inammation/mastitis0

• $actors t!at contri,ute to increases S(( on mi"

inc"ude9 – su,-c"inica" mastitisG

– ad)ance in stage of "actationG

– "actation num,erG

– stress and %oor nutrition5

!at are Somatic (e""sF



• S(( is a s%eci4c "a,oratory test – eC%resses t!e num,er of somatic ce""s %er mi""i"iter

of mi"

• !en ana"yJed indi)idua""y – met!od of diagnosis of Su,c"inica" Mastitis

• !en ana"yJed in t!e tan – indicati)e of t!e Kua"ity of ra mi"5

T!e meaning of S(( in mi"



SCC tank (cells/mL) % breast quarters % production losses

!!.!!! 6 !

"!!.!!! 16 6

1.!!!.!!! 3 18

1."!!.!!! #8 $

Source9 NM$ 1>

• S(( is an indicator of mi" Kua"ity

T!ere is a direct re"ations!i% ,eteen – !ig! S(( and mi" com%osition

– S(( and !ig! rates of infection of cos ,reastKuarters

S(( and mi"



• S(( is an indicator of mi" Kua"ity – !ig! )a"ue of S(( t!ere is an increase of some mi"

com%onents• sodiumG c!"oridesG %H

• serum %roteinG "actoferrinG immunog"o,u"insand

– Undesire,"e decrease of ot!er com%onents• $atG

• "actoseG• caseinG

• ca"ciumG %otassiumG etc5

S(( and mi"



(om%onents/gO1mL0

S(( C 15 ce""OmLMain

c!ange 1 ;@ @ -15 Q15

Lactose :G :G<: :G> :G;1Reduction

of synt!esis

(asein ;G1 ;G< ;G>@ ;G;@

$at =G<: =G> =G@1 =G1=

Serum %rotein G1 G; 1G1 1G=1

Passageof ,"ood

Serum a",umins G; G;@ G;= G=@(!"oridesG ;1 G1 G> G1;1 G1:<Sodium G@< G>; G1 G1@Potassium G1<= G1 G1=@ G1@<%HG ;1 >G> >G> >G >G

Source: Schallibaum, 2001

S(( and mi"



• (!eeses9 – decrease in industria" yie"d

– increased ater content in t!e c"ot

– negati)e c!anges in t!e sensory %ro%erties

– "o rate of !ardening of c"ot and defects of teCture

– !ig! so"id "oss in serum

– increased time for c"ot formation

S(( and Kua"ity in mi" andmi" %roducts



• Podered mi" – c!ange of t!erma" sta,i"ity

– reducing t!e %eriod of usefu" "ife

– undesira,"e a)ors in t!e 4nis!ed %roduct

• utter – sensoria" Kua"ity im%aired9 rancid and rusty a)or

• UHT mi" – Reducing t!e time for start of ge"atiniJation

• ogurt – grot! in!i,ition of mo used in t!e manufacture of

t!e %roduct

S(( and Kua"ity in mi" andmi" %roducts



%ndustryXs concern with the conse9uences of mastitisin animals

reduces the concentration of milk components>casein( mostly?

reduces the industrial yield

reduces shelfKlife of dairy products aects product’s 9uality oered to consumers

5amage for everyone from the farmer tothe consumer

*conomical

+ossesY

T!e meaning of S(( in mi"



• E( Regu"ation ;<:O;@ /modi4ed ,y E(Regu"ation 1>>:O;>0 de4nes t!e testingmet!ods for ra mi" to ,e used to c!eccom%"iance it! t!e "imits for tota" ora and

somatic ce""s count "aid don in Regu"ation@=O;: – reference met!ods - EN IS2 :== for T$ or EN IS2

1==>>-1 for S((G

– a"ternati)e met!ods - conditions detai"ed in E(Regu"ation ;<:O;@5

• #i)en t!e or"oad to im%"ement t!e referencemet!ods for T$ and S((G routine contro"s /onc!ecs0 are current"y %erformed in Euro%e ino)er!e"ming ma&ority - if not uniKue"y - ,y

S(( met!ods and EURegu"ation

! d d



• To c!ec com%"iance it! "ega" "imits of E(Regu"ation @=O;: t!e resu"ts of t!einstrumenta" met!ods !a)e to ,e con)erted andeC%ressed into t!e unit of t!e reference met!od5

• T!is reKuires t!e esta,"is!ment of a con)ersioneKuation ,eteen t!e instrumenta" and t!ereference met!odG !ic! !as ,een identi4ed as acritica" %oint for t!e im%"ementation of t!e

instrumenta" met!od5• T!e EURL MMP is conducting at Euro%ean "e)e"

t!e !armoniJation of nationa" con)ersioneKuations ,eteen t!e a"ternati)e and EN IS2

:== reference met!odG for T$ in ra mi"5


S(( ! d d



• Micro,io"ogica" criteria !a)e ,een 4Ced onRegu"ation @=O;:9 – for ra mi" in Section I /(!a%ter IG III0 and

– for dairy %roducts /(!a%ter IIG III - criteria for t!e

use of ra cos mi" for furt!er %rocessing05 – T!ey inc"ude criteria on somatic ce"" count for ra

cos mi"5

• T!e Regu"ation ;<:O;@ modi4ed ,y Regu"ation

1>>:O;> inc"udes t!e descri%tion of testingmet!ods for ra mi" and !eat-treated mi"5

• inc"uding t!e reference met!od for somatic ce""count as e"" as conditions for t!e use of a"ternati)emet!ods /if t!ey are )a"idated against EN IS2 1==>>-

1 in accordance it! t!e %rotoco" of t!e Standard EN


S(( ! d d EU



• T!e *) !as set t!e "egis"ati)e "imit of Z H"" ,#" SCCIm+

• T!e %ermitted S(( "imit )aries internationa""yG ,ut%ressure to reduce S(( furt!erG e5g5 onus for ; C 1= ce""sOmL

• It is considered ,y some researc! t!at mi"constituents a,andon t!eir %!ysio"ogica" rangesat S(( Q1 C 1= ce""sOmL and t!at infection is%resent at S(( Q 1 C 1= ce""sOmL




• WNorma"X T!res!o"d – S(( – 15 ce""sOmL

• T!res!o"d )a"ue to indicate t!e %resence ora,sence of inammation or mastitis – 15 ce""s OmL S(( ;@5ce""s OmL

• Main %at!ogenic micro-organisms» Staphylococus aureus, Streptococcus agalactiaeG co"iforms

• increase of somatic ce""s to 155

ce""sOmL

• Pat!ogenic micro-organisms» Corynebacterium bovis, coagulase-negative staphylococci

• are res%onsi,"e for a)erage scores of S((

S(( in mi"



• *N %S. #DD *numeration of somaticcells part #6 microscopic method

S(( – t!e reference met!od

Microscopic

method>referencemethod?

$"uoro-o%to-e"ectronic

met!od /a"ternati)emet!od0

$ossomatic $( /uses ocytometry tec!no"ogy0



• Scope – EN IS2 1==>> %art 1 s%eci4es a microsco%ic

met!od /reference met!od0 for t!e counting ofsomatic ce""s in ,ot! ra and c!emica""y %reser)ed

mi"5

– Is a%%"ica,"e for t!e counting of somatic ce""s incos mi"

– T!is met!od is suita,"e for %re%aring standard testsam%"es and determining reference met!od )a"ues

*N %S. #DDK#*numeration of somatic cells part #6 microscopicmethod



• Principle – A test %ortion of mi" to ,e eCamined is s%read o)er

a s"ide to form a smear5

– T!e smear is dried and t!en t!e ce""s are stainedit! Neman-Lam%ert modi4ed dye

– Su,seKuent"yG t!e stained ce""s are counted using amicrosco%e5

– T!e num,er of ce""s counted in a de4ned area are

mu"ti%"ied ,y a oring factorG to gi)e t!e num,erof ce""s %er mi""i"itre 5




• Preparation of test sample – Prior to testingG store t!e test sam%"es at a tem%erature of

: Y( Z ; Y(5

– Ana"yse t!e test sam%"es it!in > ! after sam%"ing5• %n our laboratory( the samples are stored in an

inverted position to avoid fatty substances adheringto the lids

– Heat t!e test sam%"e in a ater ,at! set at : Y(5

– MiC t!e test sam%"e carefu""y5 – (oo" t!e sam%"e to t!e tem%erature at !ic! t!e

micro%i%ette !as ,een ca"i,rated >room temperature?

– If necessary di"ute test sam%"es /it! an estimatedsomatic ce"" count of Q 1 ce""sOmL0 in a

%!os%!ate ,u6er so"ution to o,tain a somatic ce"" count of




• Preparation of the smear• Prepare( for each test sample( at least two

smears and count the best one >eg a smearnot damaged by the dyeing process?

– *i% t!e s"ides in et!ano" / of )o"ume fraction @?0 – $"ame and coo"

– Using t!e micro%i%ette 1 µL of t!e test sam%"e/e)entua""y di"uted0

– P"ace t!e miCture on a c"ean s"ide it! an area of 1cm;

• %n our lab we use slides only once We do notwash them

– Using t!e need"eG s%read t!e test sam%"e e)en"y

o)er t!e entire area de4nedG !i"e ensuring t!at




• Staining – *i% t!e dried smear on t!e s"ide in t!e modi4ed

Neman-Lam%ert stain so"ution for at "east 1@ min5

– Modi4ed Neman-Lam%ert stain so"ution is com%osed9

» Et!ano" [ Tetrac!"oroet!ane !eated in a ater ,at! at >@\(5» Met!y"ene ,"ue added under a fume cu%,oard and carefu""y miC

» MiCture coo"ed in a refrigerator to :\(5

» #"acia" acetic acid added and carefu""y miC again t!e so"ution5

» 2,tained so"ution is %assed t!roug! an a%%ro%riate 4"ter into anairtig!t ,ott"e5

» $i"ter t!e Neman-Lam%ert stain so"ution again ,efore use• %n our lab we dip the slide " min We use Newman’s stain

solution modi7ed commercialy available

– *ry t!e smear at am,ient tem%erature5

– *i% t!e smear gent"y in ta% ater unti" a"" sur%"us dye is

as!ed aay5




• 5etermination6 reading optimi<ation >#? – Using t!e microsco%e G count t!e ce"" nuc"ei in t!e

o,tained smear of 4e"ds entire"y 4""ed it! mi"smear on"y5

• We advise to check microscopes once a year byan e,ternal service provider

– (!oose t!e ,est magni4cation /from @] to 1]0G in order to !a)e an a)erage maCimumnum,er of ; ce""s in eac! 4e"d5

• %n our lab the best magni7cation is H"" [

– T!e training and si"" of t!e ana"yst is t!e maincrucia" success factor for a %ro%er %erformance oft!e met!od

• freKuent eCecution of t!e met!od andartici ation in inter"a,orator tria"s




• 5etermination6 reading optimi<ation >!?» T!e ce""s %ossess a stained nuc"eus5

» T!e ce""s genera""y are ^m or "arger5

» *o not count ce""s "ess t!an : ^m

» (ount fragments on"y if more t!an @ ? of

nuc"ear materia" is )isi,"e5» (ount ce"" c"usters as oneG un"ess t!e nuc"ear

unit/s0 is /are0 c"ear"y se%arated5

» (e""s in mi" are distri,uted according to aPoisson distri,ution

» T!e minimum num,er /N0 of ce""s to ,e

counted in re"ation to t!e ce"" count "e)e"G inorder to arri)e at t!e "isted coe_cient of)ariationG are "isted in Ta,"e 15

» $or a %ro%er eCecution of t!e met!odG it isessentia" t!at t!e "isted minimum num,er ofce""s ,e counted5

» $ie"ds and stri%s to ,e counted s!a"" ,e c!osenso as to o,tain a re%resentati)e count for t!e

Table # KMinimumnumber >N? of cells





• Counting in sucessive 7elds >#? – (ount nuc"ei in successi)e 4e"dsG in )ertica" stri%s in

regu"ar"y s%aced 4e"ds – a0 $or a circu"ar s!a%eG start from a su_cient distanceG

dLG from t!e "eft side of t!e !oriJonta" diameterG so asto a""o counting of a minimum of @ 4e"ds from t!e to%of t!e stri%5 A distanceG dLG of G@ mm is suita,"e forcircu"ar s!a%e5

• ,0 P"ace t!e u%%er or "oer edge of t!e 4e"dcircumference tangentia""y onto t!e interna"u%%er or "oer ,order of t!e tem%"ate /t!e"atter s!ou"d not a%%ear in t!e 4e"d05 In t!ecase of an unco)ered surface near t!e,order of t!e tem%"ateG ad&ust t!e 4e"d to

t!e ,order of t!e smear5




• Counting in sucessive 7elds >!?• c0 After !a)ing counted t!e 4rst 4e"dG mo)e t!e

o,&ecti)e it! a 4Ced s%ace distanceG dHG don or u%to t!e neCt graduation in t!e direction of t!e "oer oru%%er edge and count t!e ne 4e"d5 A distanceG dHGof 1 mm is considered a%%ro%riate5

d0 $rom t!e "ast 4e"d countedG re%eat t!eo%eration in c0G unti" t!e o%%osite side/,ottom or to%0 of t!e stri% is reac!ed5 Ift!e "oer or u%%er ,order a%%ears and it4""s "ess t!an !a"f t!e 4e"d surfaceGcounting is %erformed after mo)ing u% t!eo,&ecti)e unti" t!e ,order disa%%ears againcom%"ete"y from t!e 4e"dG !ic! t!en on"y

co)ers smear5




• Counting in sucessive 7elds >?• e0 T!en mo)e t!e o,&ecti)e to t!e rig!t ,y a

distanceG d /d ` ; mm0G and start a nestri% in t!e o%%osite direction /to% or ,ottom05

• f0 Re%eat from ,0 to e0 unti" t!e rig!t side of t!e

tem%"ate is reac!ed5• g0 In case an insu_cient num,er of 4e"ds is

counted /see Ta,"e 10G su%%"ementary 4e"ds can,e counted5 To do t!isG focus t!e microsco%eo,&ecti)e on ot!er "ocations /e5g5 ,y c!angingt!e starting %"ace andOor ada%ting t!e ste%-,y-ste% mo)ing distances0 so as to o,taina%%ro%riate 4e"d num,ers t!at arere%resentati)e of t!e entire smear5

• !0 (a"cu"ate t!e resu"t5



• Calculation of results >#? – (!ec t!e 11G; mm diameter of t!e smearG using

t!e graduations and )ernier of t!e microsco%e5

– (a"cu"ate t!e tota" concentrationG cG of ce""s ,y

using t!e fo""oing eKuation9• c is t!e tota" concentrationG eC%ressed in num,er of

ce""s %er mi""i"itre

• 5c is t!e diameterG in mi""imetresG of t!e smear5

• Nt is t!e tota" num,er of ce""s counted

• 5f is t!e diameterG in mi""imetresG of t!e microsco%e

4e"d

• Ws is t!e idt!G in mi""imetresG of t!e smear

• +s is t!e "engt!G in mi""imetresG of t!e smear

• Nf is t!e num,er of 4e"ds counted com%"ete"y

• 'm is t!e )o"umeG in mi""i"itresG of t!e test sam%"e

smeared

• d is t!e di"ution factor used

• /If no di"ution is reKuiredG d ` 1 it! a 191 di"utionG d

` G@50





• Calculation of results >!? – 4or our lab

• *c 115; mm

• *f of microsco%e it! o,&ecti)e : ` 5:> mm

or• *f of microsco%e it! o,&ecti)e 1 ` 51 mm

– e determine t!e )a"ue of t!e diameter of t!emicrosco%e 4e"d /*f0 ,y a ca"i,rated micrometer

• Vm ` 51 mL – oring $actor it! o,&ecti)e : ` > 1=;

– oring $actor it! o,&ecti)e 1 ` =; <1>

S. DD




• *,pression of results – EC%ress t!e test resu"ts in !o"e 4gures of

t!ousands• for eCam%"e9 eC%ress :1 @> ce""sOm" as :;

ce""sOm"

– Precision• T!e )a"ues for t!e re%eata,i"ity and re%roduci,i"ity

ere deri)ed from t!e resu"t of an inter"a,oratorytest carried out in accordance it! IS2 @<;@-1 and

IS2 @<;@-;5 *etai"s of t!is inter"a,oratory test aresummariJed in AnneC A of t!e Standard5

» T!e mean )a"ues for eac! "e)e" ere res%ecti)e"y9

» Le)e" 1G sam%"es A and 9 ;:@ ce""sOm"» Le)e" ; sam "es ( and *9 :@@ ce""sOm"

study for cos mi" in)o")ing

1 "a,oratories and 1=countries

*N %S. #DD #




• /epeatability – T!e a,so"ute di6erence ,eteen to inde%endent

sing"e test resu"tsG o,tained using t!e same met!od

• on identica" test materia"

• in t!e same "a,oratory

• ,y t!e same o%erator

• using t!e same eKui%ment

• it!in a s!ort inter)a" of timeG• i"" in not more t!an @ ? of cases ,e greater t!an t!e

)a"ues gi)en in Ta,"e ;9

*N %S. #DD #




• /eproducibility • T!e a,so"ute di6erence ,eteen to sing"e test

resu"tsG o,tained

– using t!e same met!od

– on identica" test materia"

– in di6erent "a,oratories

– it! di6erent o%erators

– using di6erent eKui%mentG

– i"" in not more t!an @ ? of cases ,e greater t!ant!e )a"ues gi)en in Ta,"e =9



• #i)en t!e de4ciencies of t!e referencemicrosco%ic met!od for S(( in ra mi" /"ac ofre%roduci,i"ity0 and t!e "imited num,er of"a,oratories using itG it is of utmost im%ortance to

de)e"o% (erti4ed Reference Materia"s /(RMs0

• (RMs are needed to ca"i,rate instrumenta"met!odsG most"y used for routine ana"yses of S((

in ra mi"G as to !a)e com%ara,"e S(( ana"ysesconducted it!in eac! Euro%ean country and,eteen di6erent Euro%ean countries5

• bR(OIRMM /#ee"G e"gium0 in coo%eration it! t!e

Met!od u%date - (RMs

Met!od u%date - Training



• T!e EURL MMP /E* Unit0 organiJed once a yeara training session on !o to count somatic ce""sin ra cos mi" ,y t!e reference met!od EN IS21==>>-15

• A;-day session inc"ude ,ot! a %resentation of t!ereference met!od EN IS2 1==>>-1 and a %ractica"session9 – smear %re%arationG

– smear stainingG – s"ide countingG

– ca"cu"ation and eC%ression of resu"ts5

Met!od u%date - Trainingsessions

SCC in milk K *N %S. #DD#



M*T-.5 P/%NC%P+* &5'&NT&1*S I5%S&5'&NT&1*S

2%tica" microsco%y

/EN IS2 1==>>1 –10

T!e ce""s are 4Ced ong"ass s"ideG stained

and counted using t!emicrosco%e

reference met!od"a,oriustiring /induces counting

errors0reKuires )ery e"" trainedtec!nicians"itt"e accuracy

ased on

ocytometrytec!no"ogy G/eKui%ament$ossomatic0/EN IS2 1==>>1 –;0

counts andc!aracterises %artic"es

and ce""s !i"e t!eyare oing5 (ountce""s t!at !a)eminimum intensity ofuorescence due tot!e staining of nuc"eic

*NA

Rotine met!od

aut!omatic met!odmedium and !ig! yie"d easy %rocessing!ig! %recision!ig! ca%acity ana"yser9@ sam%"esO!our

SCC in milk K *N %S. #DD#part # vs part !



nstituto &acional de n'estia*o +r,ria e -eterin,ria ..

Av. a !e"#blica, $ui%&a o 'ar(u)*, 2780+157 eira*, -or&ual

/el : 351 21 440 3500 a : 351 21 440 3666.i%iav."&

0ank 2ou or 2our

attention4

ri*&i%a Aleiocri*&i%a.aleioi%iav."&Portugal and Turkey Cooperation Training sessions in the framework of the National

Program of Waste Monitoring

INIAVG UEISTSA5 Po"o do LumiarG No)em,er =tr ;1@



AnneCes

*N %S. #DD #




*N %S. #DD #




*N %S. #DD #




*N %S. #DD #




Documents

Apresentação NRL MMP SCC Versao Final