Reacii Ag-Ac care utilizeaz

componente marcate

RFC principiu, aplicaii

RSN principiu, aplicaii
Teste de imunitate celular, IDR la
tuberculin.
REFACERI
 The complement fixation test

is a test that can be used to detect the presence

of either specific antibody or specific antigen in
a patient's serum, based on whether
complement fixation occurs. It was widely used
to diagnose infections, particularly with
microbes that are not easily detected by culture
methods, and in rheumatic diseases. However,
in clinical diagnostics labs it has been largely
superseded by other serological methods such
as ELISA and by DNA-based methods of
pathogen detection, particularly PCR.
Reacia de fixare a complementului (RFC)

reacie al cror rezultat este vizualizat cu un artificiu tehnic, un

sistem hemolitic indicator.

acest sistem indicator se foloseste deoarece Ag este sub form

macromolecular (reprezentat de corpi microbieni de dimensiuni
foarte mici)

complexul Ag-Ac format nu este vizibil cu ochiul liber

RFC a fost imaginat nc din anul 1901 de Jules Bordet

RFC se poate folosi att pentru identificarea Ag ct i a Ac

(diagnosticul serologic)

In diagnosticul serologic, cea mai cunoscut metod rmne nc

RFC Bordet-Wasserman, utilizat n diagnosticul serologic al
sifilisului.
STEPS
The complement system is a system of serum proteins that react with
antigen-antibody complexes. If this reaction occurs on a cell surface,
it will result in the formation of trans-membrane pores and therefore
destruction of the cell. The basic steps of a complement fixation test
are as follows:
Serum is separated from the patient.
Patients naturally have different levels of complement proteins in their
serum. To negate any effects this might have on the test, the
complement proteins in the patient's serum must be destroyed and
replaced by a known amount of standardized complement proteins.
The serum is heated in such a way that all of the complement proteinsbut
none of the antibodieswithin it are destroyed. (This is possible because
complement proteins are much more susceptible to destruction by heat than
antibodies.)
A known amount of standard complement proteins are added to the serum.
(These proteins are frequently obtained from guinea pig serum.)
The antigen of interest is added to the serum.
Sheep red blood cells (sRBCs) which have been pre-bound to anti-sRBC
antibodies are added to the serum. The test is considered negative if
the solution turns pink at this point and positive otherwise.
 Reacia de fixare a
complementului (RFC)
Complementul:
se leag de complexul imun
lizeaz hematiile din sistemul hemolitic
 Reacia de fixare a
complementului (RFC)
Reacie pozitiv: lipsa hemolizei
hematiile se depun la fundul
eprubetei
 Reacia de fixare a
complementului (RFC)
Reacie negativ: hemoliz,
complementul
lizeaz hematiile din complexul imun
 RFC - interpretare
Se verifica rezultatele pentru martori:
Hemoliza: martor pentru serul de cercetat,
martor pentru Ag
martor pentru C
martor pentru ser sigur negativ
Testul este negativ = in tuburile cu ser de
cercetat nu exista Ac, astfel apare hemoliza
Testul este pozitiv = in tuburile cu ser de cercetat
exista Ac, nu apare hemoliza, hematiile se depun
in buton in partea inferioara a tubului
 interpretation
If the patient's serum contains antibodies against the
antigen of interest, they will bind to the antigen in
step 3 to form antigen-antibody complexes. The
complement proteins will react with these complexes
and be depleted. Thus when the sRBC-antibody
complexes are added in step 4, there will be no
complement left in the serum. However, if no
antibodies against the antigen of interest are present,
the complement will not be depleted and it will react
with the sRBC-antibody complexes added in step 4,
lysing the sRBCs and spilling their contents into the
solution, thereby turning the solution pink.
 Reacia de fixare a
complementului (RFC)
Dg serologic al infectiilor cu Mycoplasma
pneumoniae
Dg serologic al infectiei cu Chlamydia spp
Dg serologic al infectiei cu Treponema
pallidum
Dg serologic al infectiei cu Leptospira spp
Dg serologic al infectiei cu Borrelia spp
Dg serologic al infectiei cu Brucella spp
Anti-streptolysin O (ASO or ASLO) is the antibody made against
streptolysin O, an immunogenic, oxygen-labile streptococcal hemolytic
exotoxin produced by most strains of group A and many strains of
groups C and G Streptococcus bacteria. The "O" in the name stands for
oxygen-labile; the other related toxin being oxygen-stable streptolysin-
S. The main function of streptolysin O is to cause hemolysis (the
breaking open of red blood cells) in particular, beta-hemolysis.
Antistreptolysin O titer (AS(L)O titer or AS(L)OT) is a measure of the
blood plasma levels of antistreptolysin O antibodies used in tests for the
diagnosis of a streptococcal infection or indicate a past exposure to
streptococci.
The ASOT helps direct antimicrobial treatment and is used to assist in the
diagnosis of scarlet fever, rheumatic fever, and
post infectious glomerulonephritis .
A positive test usually is >200 units/mL, but normal ranges vary from
laboratory to laboratory and by age.
Positive ASO test confirms past infection; thus its useful to support the
diagnosis of the poststreptococcal illness when its suspected, like
poststreptococcal glomerulonephritis, pediatric autoimmune
neuropsychiatric disorders associated with streptococcus (PANDAS), and
rheumatic fever. ASO titers may be negative in up to 20% of patients
who develop acute rheumatic fever.
 RSN dg serologic reactia
ASLO
Se utilizeaza in dg retrospectiv al infectiilor streptococice sau pt dg
bolilor poststreptococice; determinam titrul de anticorpi anti
streptolizina O.
PRINCIPIU
SLO are efect hemolitic asupra hematiilor de berbec.
Daca in serul pacientului exista anti-SLO actiunea hemolitica a SLO e
neutralizata. Prin dilutii in tuburi se determina titrul ASLO.
TEHNICA
1. O cantitate constanta de SLO + serul pacientului in dilutii binare in
tuburi
2. Incubam 15minute la 37 C
3. Se adauga suspensia de hematii
4. Incubam 45 minute la 37 C
5. Centrifugam si pastam tuburile la frigider pana a doua zi
INTERPRETARE
Titrul reactiei ASLO e dat de cea mai mare dilutie de ser in care
lipseste complet hemoliza. Titrul normal este 200 UI
 Reactia ASLO - Interpretare
Pornind de la dilutia initiala a serului
1/10, titrul ASLO va fi:
12 50 100 125 166 250 333 etc
Titrul reactiei ASLO este dat de cea
mai mare dilutie de ser la care
lipseste complet hemoliza
Pentru Romania: titru normal: 200
250 UI
RSN dg serologic reactia
ASLO

125 25
12 50 100 166
0

Titrul reactiei ASLO este dat de cea mai

mare dilutie de ser la care lipseste
complet hemoliza
RSN dg serologic reactia
ASLO

125 25
12 50 100 166
0

Care este titrul ASLO in aceasta situatie? Este un

titru normal?
 IDR cu PPD - Principiu
Tuberculina = amestec de Ag proteice mycobacteriene
Evalueaza reactivitate dpdv al RIC
Dupa infectia cu mycobacterii => sensibilizarea si
proliferarea unor populatii de LT
Injectarea tuberculinei => stimuleaza LT si declanseaza
un RIC si manifestari de tip HS IV=> vasodilatatie
+edem+infiltrat limfocite, monocyte, bazolfile, neutrofile
LT Ag-specifice prolifereaza ->limfokine->acumulare alte
cellule
Raspuns maxim: la 48 ore
Infiltratul cellular = induratie = reflecta
hipersensibilitatea de tip intarziat
Eritemul = reactive inflamatorie acuta produsa de
vasodilatatie = NU va fi interpretata ca reactive pozitiva.
 IDR cu PPD - Principiu
Infiltratul cellular = induratie =
reflecta hipersensibilitatea de tip
intarziat
Eritemul = reactive inflamatorie
acuta produsa de vasodilatatie = NU
va fi interpretata ca reactive pozitiva.
IDR cu PPD - Principiu
Limfocitele sensibilizate ating un
nivel adecvat pentru a conduce la un
rapsuns interpretabil in in 2-4
saptamani de la infectia cu
M.tuberculosis
Sensibilitatea poate persista mai
multi ani, dar reactivitatea scade cu
varsta.
 Se
injecteaza Testarea imunitatii
ID 0.1 ml de tip celular
PPD. IDR la PPD

Apare o bula cu
diametru de 5
mm.
La persoanele
infectate apare
eritem-edem-
induratie.

Citire finala la 72
ore.
 Se masoara
induratia.

In Romania
reactia e O valoare
pozitiva mai mare de
daca 10 mm
diamertul poate arata
induratiei infectia cu
este mai tuberculoza,
mic sau egal boala care
cu 9 mm. necesita
tratament.
Test pozitiv
 IDR cu PPD Materiale si
reactivi
1891 Old tuberculin (Koch) -> 1901 New tuberculin -> 1941
PPD-S (Seibert)
PPD-RT 23 lot de tuberculina purificata in 1958 in
Danemarca
Obtinere: precipitarea proteinelor din filtratul mediului de
cultura concentrate la cald cu solutie 50% sulfat de amoniu.
Se utilizeaza 2 UI/doza PPD-RT 23 = 5 UI/doza PPD-S
In Romania se utilizeaza PPD IC-65 produsa de INCDMI
(~PPD-RT 23)

*UI = activitatea biologica continuta in 0,000028 mg PPD-S

(0,0002 mg PPD + 0,00008 mg saruri)
 IDR cu PPD Tehnica de
lucru
Injectare intradermica Mantoux
Se verifica produsul de injectat
Seringa cu volum maxim 1 ml si diviziuni 1/10 ml + ac de
injectare id
Se agita fiola pt omogenizare (tuberculina se absoarbe pe
pereti)
Se aspira cantitate suficienta de solutie pt a putea elimina
orice bula de aer
Se pozitioneaza acul ai bizoul sa fie aliniat diviziunilor
seringii
Pe fata anterioara a antebratului, la unire 1/3 medie cu 1/3
superioara antiseptizam cu alcool minim 3 minute o zona
 IDR cu PPD Tehnica de
lucru
Se cuprinde zona aleasa in palma, intinzand pielea cat mai
uniform intre extremitateaa inferioara a eminentei tenare si
hipotenare pe de o parte si cele 4 degete strans lipite pe de
alta parte
Patrundem cu acul aproape tangent la suprafata antebratului,
cu bizoul in sus, pana acesta este situate in derm
Eliberam tegumentul si fixam seringa presand-o pe antebratul
subiectului, avand grija sa nu se acopere diviziunile seringii
pentru a putea urmari cantitatea injectata
Injectam strict 0,1 ml daca s-a injectat correct intradermic,
apare o bula usor denivelata cu margini abrupte cu aspect de
coaja de portocala si diametru de 5-8 mm
In lipsa aparitiei acestei bule se repeta injectarea la 3-5 cm sau
la antevratul opus.
 IDR cu PPD Citirea
rezultatului
Se face la 72 ore, in lumina naturala
Se recomanda citirea la 24 ore (surprinde HS tip umoral), 48
ore si final al 72 h
Se identifica o zona eritemato-violacee care circumscrie zona
de injectare
Se apreciza tactil cu pulpa policelui/inelarului zona de
infiltratie = reliefata ce corespunde inflamatiei (edemm
limfocite, macrofage, PMN)
Masuram diametrul maxim al zonei de infiltratie
Notam intennsitatea infiltratiei tip Palmer:
I. induratie ferma sau flictena
II. Induratie elastic
III. Induratie depresibila
IV. fara infiltratie aparenta
!! Citirea - subiectiva => dubla citire in orb
 Classification of the Tuberculin Skin Test Reaction

Aninduration of 5 Aninduration of 10 Aninduration of 15

or more or more or more
millimetersis millimetersis millimetersis
considered positive in considered positive in considered positive in
-HIV-infected persons -Recent immigrants any person, including
-A recent contact of a (< 5 years) from high- persons with no
person with TB prevalence countries known risk factors for
disease -Injection drug users TB. However, targeted
-Persons with fibrotic -Residents and skin testing programs
changes on chest employees of high- should only be
radiograph consistent risk congregate conducted among
with prior TB settings high-risk groups.
-Patients with organ -Mycobacteriology
transplants laboratory personnel
-Persons who are -Persons with clinical
immunosuppressed conditions that place
for other reasons them at high risk
(e.g., taking the -Children < 4 years of
What Are False-Positive
Reactions?
Infection with nontuberculosis
mycobacteria
Previous BCG vaccination
Incorrect method of TST
administration
Incorrect interpretation of reaction
Incorrect bottle of antigen used
What Are False-Negative Reactions?
Cutaneous anergy (anergy is the inability to react to
skin tests because of a weakened immune system)
Recent TB infection (within 8-10 weeks of exposure)
Very old TB infection (many years)
Very young age (less than 6 months old)
Recent live-virus vaccination (e.g., measles and
smallpox)
Some viral illnesses (e.g., measles and chicken pox)
Incorrect method of TST administration
Incorrect interpretation of reaction
 IDR cu PPD

Cui se poate efectua IDR cu PPD? - >

oricarei persoane
(cu exceptia celor ce au avut o reactive
severa la un IDR cu PPD anterior)

* La 4-6 saptamani de la vaccinarea cu un

virus viu atenuat sau in aceeasi zi cu vaccinul

Se poate repeta? -> Da

 Biopreparate produse
imunobiologice
Seruri hiperimune
Vaccinuri
imunomodulatori
Biopreparate Seruri imune
Omologe sau heterologe
Heterologe imunizarea unor animale sanatoase (cai)

1. Imunoglobuline standard donator/placenta

->sepsis
2. Imunoglobuline specifice (hiperimune) de la
pers.imunizate cu un anumit antigen
-> tetanus, rabie, varicela, HerpesZoster, CMV, VSR
3. Seruri immune heterologe de la animale imunizate
-> patogenie determinate de exotoxine: difterie,
tetanus, botulism, cangrena gazoasa
 Difterie:
tratamentul
incepe in
cazul
suspiciunii
clinice
administrare
de
antitoxina
difterica.
 Vaccinuri
1. Corpusculare = include corpi vii atenuati sau
inactivati prin actiunea unor factori fizici sau chimici
a.) Vaccinuri vii attenuate: BCG, Poliomielitic Sabin, ROR
b) Vaccinuri cu virusuri omorate: Poliomielitic (Salk),
Hepatita A
2. Subunitare: inginerie genetica siguranta
VHB
Vaccinul acellular pertussis
3. Vaccinuri care contin anatoxine
DTP
DT
dT
ATPA
 Vaccinuri vii atenuate
Avantaje vaccinuri vii:
Capacitatea de a disemina de la vaccinati la contacti
Imunogenitate crescuta
Imunitate durabila
Rasouns imun impotriva tuturor antigenelor

Dezavantaje vaccinuri vii:

reversie la virulenta
Interfera cu virusuri care impart acelasi habitat
Instabilitate la transport/stocaj
Reactii severe la imunosupresati
 https://
www.youtube.com/watch?v=bR86G-i
trTQ