Thesis (MScMed)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Introduction: Flow cytometry has progressively replaced many traditional laboratory
tests due to its greater accuracy, sensitivity and rapidity in the routine clinical settings
especially clinical trails. It is a powerful tool for the measuring of chemical (the
fluorochrome we add) and physical (size and complexity) characteristics of individual
cells. As these instruments became major diagnostic and prognostic tools, the need for
more advanced quality control, standardized procedures and proficiency testing
programs increased as these instrumentations and their methodology evolve. Minor
instrument settings can affect the reliability, reproducibility and sensitivity of the
cytometer and should be monitored and documented in order to ensure identical
conditions of measurement on a daily basis. This can be accomplished by following an
Internal Quality Assurance (IQA) and/ or External Quality Assurance (EQA) program.
Currently there are no such programs available in South Africa and poorer Africa
countries. HIV is a global concern and the laboratories and clinics in these places are in
need of such IQA programs to ensure quality of their instrumentation and accurate
patient results. Quality assurance programs such as CD Chex® and UK Nequas are
available but due to bad sample transport, leave the receiving laboratories with
nightmares. It would be best if there was a laboratory in South Africa that could
provide the surrounding laboratories with stabilized whole blood samples that can be
utilized as IQA. The transport of these samples can be more efficient due to shorter
distance and thus the temperature variations limited. Aims and Objectives: The aim of Chapter one is to familiarize the reader with general
terminology and concepts of immunology. Chapter two describes in detail the impact
stabilized whole blood had on clinical immunology concerning Quality Control and
Quality Assurance. The objective of this study is to stabilize whole blood with a shelf
life of greater than 30 days to serve as reference control material for South African
Immunophenotyping. It is further an objective to use these in-house stabilized control
samples for poorer African countries as Internal Quality Assurance reference material.
It is a still further objective to stimulate various lymphocyte subsets to express
activation antigens and then stabilize these cells for more specialized immunological
test and can serve as a QC for those required samples.
Study design: In Chapter three, the method currently used to stabilize whole blood was
modified. The stability of different concentrations of a first stabilizing agent
(Chromium Chloride hexahydrate) was investigated. Incubation periods and
concentrations of paraformaldehyde as second stabilizing agent were investigated.
Blood samples from healthy individuals (n=10) were stabilized and monitored for the
routine HIV phenotypic surface antigens over a period of 40 days. These samples
(n=10) were compared on the Becton Dickinson Biosciences (BD) FACSCalibur™
versus BD FACSCount™ instrumentation. Blood samples (n=3) were stabilized and
monitored to identify phenotypic cell surface molecules for as long as possible. They
were quantified on both flow cytrometric instruments. In addition, these stabilized
samples (n=3) were investigated as control blood for calibration purposes on the BD
FACSCount™ instrument. In Chapter four, lymphocytes were isolated and activated with various stimuli to
express sufficient activation antigens such as CD25, CD69, HLA-DR and CD40
Ligand on the T helper cell surfaces. These activated antigens were analyzed on the
BD FACSCalibur™ and further stabilized to serve as possible IQA samples in future.
Results: In Chapter three, the ten individual stabilized samples had non-significant P
values (P > 0.05) for CD3, CD4 and CD8 percentages and absolute values comparing
day 3 until day 40. Comparing the BD FACSCalibur™ versus BD FACSCount™,
resulted in a R2 = 0.9848 for CD4 absolute values and a R2 = 0.9636 for CD8 absolute
values. Stabilized blood samples (n=3) were monitored for routine HIV phenotypic
markers until day 84. The cells populations were easily identifiable and could be
quantified on both BD FACSCalibur™ and BD FACSCount™ instruments.
In Chapter four; for the activation study purposes, activated T helper lymphocytes
expressed approximately 25 to 35% CD40 Ligand cell surface molecules. The
stimulant of choice was Ionomycin at a 4μM concentration. Cells were incubated for
four hours at 37 degree Celsius in a 5% CO2 environment. For CD69 surface
expression, 6 hour incubation was optimum. The stimulus of choice in this case was
4μM Ionomycin which induced 84.21% CD69 expression in the test samples. For
CD25 expression; 6 hour incubation with PHA resulted in approximately 43% of CD25
expression. For HLA-DR surface expression; 6 hour incubation with PHA resulted in
approximately 43.32% of HLA-DR expression. Activated lymphocytes expressing
CD40 Ligand showed stability until day 23. Activated Lymphocytes expressing CD69,
CD25 and HLA-DR were stabilized in the same manner and stability could be
achieved until day 16. Conclusion: This thesis was related to the preparation of control samples (IQA)
designed to simulate whole blood having defined properties in clinical laboratory
situations. In future kits can be developed with a low, medium and high control sample
for the various immunological phenotypic determinants. Another kit can be compiled
where various activation markers can be identified, quantified with a “zero”, low and
high control. These whole blood IQA kits and “activation IQA kits” can be
implemented for training of newly qualified staff, competency testing of staff, method
development, software testing, panel settings and instrument setting testing. Control
samples ideally must have a number of properties in order to be effective. For instance
stability during storage times, preferably lasting more than a few weeks,
reproducibility and ease of handling. These will provide the information on day-to-day
variation of the technique or equipment which will enhance accuracy and improve
patient care. / AFRIKAANSE OPSOMMING: Inleiding: Vloeisitometrie tegnologie het verskeie tradisionele laboratorium toetse
vervang as gevolg van beter akuraadheid, sensitiwiteit en vinniger beskikbaarheid van
resultate in ‘n kliniese omgewing, veral kliniese proewe. Vloeisitometrie is ‘n kragtige
tegniek om chemiese (fluorokroom byvoeging) en fisiese (sel grote en kompleksiteit)
karakter eienskappe van individuele selle te meet. Met die toename in gebruik en
gewildheid van hiedie instrumente, neem die behoefde toe vir gevorderde kwaliteit
kontroles, gestandardiseerde prosedures, met profesionele toets programme tesame met
metode ontwikkeling.
Klein verstellings aan instrument parameters beinvloed die betroubaarheid,
herhaalbaarheid en sensitiwiteit van ‘n sitometer en moet gemonitor (en dokumenteer)
word om identiese kondisies van leesings op ‘n daaglikse basis te verseker. Dit kan
bereik word deur in te skakel met ‘n interne kwaliteits versekerings program [IQA:
“Internal Quality Control”] en/of ‘n eksterne kwaliteits versekerings program [EQA:
“External Quality Control”] te volg. Op die oomblik is daar geen sulke kwaliteits
versekerings programme in Suid Afrika en/of in die verarmende Afrika lande
beskikbaar nie. MIV is ‘n wêreldwye bekommernis en laboratoriums en klinieke in
hierdie gedeeltes van die land verlang ‘n dringende behoefdte vir sulke “IQA”
programme om kwaliteit van instrumentasie en akkurate pasiënt resultate te verseker
wat tot beter behandeling van pasiënte lei. Kwaliteit versekerings programme soos
“CD Chex®” en “UK Nequas” is beskikbaar, maar baie probleme met verwysing na
monster integriteit as gevolg van tydsame vervoer en aflewering kondisies word
hiermee geassosieër. Die behoefte het ontstaan vir ‘n laboratorium in Suid Afrika wat direk die omliggende
laboratoriums, hospitale en klinieke kan voorsien met gestabiliseerde blood monsters
wat gebruik kan word as “IQA”. Die vervoer en aflewerings kondisies van hierdie
monsters sal aansienlik verbeter as gevolg van die korter aflewerings afstand wat direk
die beperkte temperatuur wisseling beinvloed.
Doel van studie: Die doelwit van hoofstuk een is om vir die leser ‘n inleiding te gee
tot terminologie en konsepte van immunologie en die immune sisteem. Hoofstuk twee
beskyf die impak wat gestabiliseerde heelbloed het op die kliniese immunologie met
betrekking tot kwaliteit beheer en kwaliteit versekering. Die doelwit van hierdie studie
is om heelbloed te stabiliseer sodat die rakleeftyd meer as 30 dae is en sodoende as
verwysings-materiaal kontroles vir Suid Afrikaanse immunofenotipering kan dien. Dit
is ‘n verdere doelwit om hierdie tuis-gestabiliseerde kontrole monsters te gebruik as
“IQA” verwysings materiaal in verarmende Afrika lande. Die doelwit van hoofstuk
vier is om limfosiete te stimuleer om verskeie aktiverings merkers uit te druk op hul
selmembrane en dan te stabiliseer en dié te gebruik as Kwaliteits Kontroles vir die
meer gespesialiseerde immunologiese toetse.
Studie ontwerp: Hoofstuk drie beskryf ‘n aangepaste en verbeterde metode van heel
bloed stabiliseering. Stabiliteit word ondersoek in ‘n verskyndenheid konsentrasies van
‘n primêre stabiliseerings agent (chromium chloried heksahidraat) en inkubasie
periodes met paraformaldehied as tweede stabiliseerings agent word deeglik
gedokumenteer. Bloedmonsters van gesonde indiwidië (n=10) was gestabiliseer en
gemonitor vir roetine MIV membraanoppervlak antigene oor ‘n periode van 40 dae. Hierdie monsters (n=10) was gelees en geanaliseer op ‘n BD FACSCalibur™ en
vergelyk met ‘n BD FACSCount™ vloeisitometer instrument. Drie gestabiliseerde
heelbloed monsters (n=3) was gemonitor vir ‘n periode vir so lank moontlik die
fenotipiese selmembraan molekules identifiseerbaar was en die kwantiteit bepaalbaar
was. Hierdie drie monsters was gemeet op beide instrumente. As ‘n addisionele
doelwit, was hierdie drie gestabiliseerde monsters ondersoek om as moontlike
kalibrasie materiaal (verteenwoordig ‘n normale bloedmonster) te dien vir die BD
FACSCount™ instrument in die oggende voor pasiënt monsters gelees kan word.
In hoofstuk vier was limfosiete geϊsoleer en geaktiveer met ‘n verskyndenheid
stimulante om optimale aktiveerings-antigene uit te druk op T helper selmembrane
(byvoorbeeld CD25, CD69, HLA-DR en CD40 Ligand). Hierdie geaktiveerde
monsters was geanaliseer op die BD FACSCalibur™ en daarna gestabiliseer. Na
stabilisasie van die geaktiveerde limfosiet monsters was dit gemonitor oor ‘n tydperk
so lank moontlik data plotte leesbaar en selpopulasies identifiseerbaar was. Hierdie
monsters kan dien as ‘n moontlike “IQA” toets stel vir ‘n meer gespesialiseerde
immunologiese aktiveerings kontrole doeleindes.
Resultate: In hoofstuk drie; tien individiële gestabiliseerde heelbloed monsters het
gedui op geen-beduidende P waardes (P > 0.05) vir CD3, CD4 en CD8 persentasies en
absolute waardes; gemeet vanaf DAG 3 vergelykbaar tot-en-met DAG 40. Met korrelasie statistiek en vergelyking van die BD FACSCalibur™ met die
FACSCount™ instrumente, is die volgende opgemerk; R2 = 0.9848 vir die CD4
absolute waardes en ‘n R2 = 0.9636 vir die CD8 absolute waardes. Drie gestabiliseerde
monsters (n=3) was gemonitor vir MIV roetine fenotipeering tot en met DAG 84. Die
selpopulasies was duidelik identifiseerbaar en die kwantitatief meetbaar op albei
instrumente (BD FACSCalibur™ en BD FACSCount™).
Hoofstuk vier: geaktiveerde T helper lymphosiete het 25 – 35% membraan CD40
Ligand uitgedruk op hul selmembrane. Die stimulant van keuse was ionomysien teen
‘n optimale konsentrasie van 4μM. Die optimale inkubasie tydperk was vier ure by
37°C in 5% CO2 kondisie. Ses uur inkubasie in 4μM ionomysien by 37°C in ‘n 5%
CO2 omgewing was optimal vir die CD69 selmembraan uitdrukking en het 84.21%
opgelewer. Vir CD25 selmembraan uitdrukking was die selle vir ses ure met
phietoheamagglutinin (PHA) gestimuleer by 37°C in 5% CO2 kondisie en het 43%
CD25 selmembraan uitdrukking opgelewer. HLA-DR selmembraan uitdrukking: selle
was vir ses ure saam met PHA by 37°C in 5% CO2 kondisie inkubeer en het 43.32%
opgelewer. CD40 Ligand aktivering/gestabiliseerde limfosiete het tot en met dag 23
stabiliteit getoon. Die ligand was duidelik identifiseerbaar en kwantifiseerbaar.
Geaktiveerde lymphosiete wat CD69, CD25 en HLA-DR selmembraan merkers
uitdruk het na die stabiliseerings proses stabiliteit getoon tot-en-met dag 16. Gevolgtrekking: Die doel van hierdie studie was om verwysingskontroles voor te
berei sodat dit vars heelbloed naboots met uitkenbare eienskappe vir kliniese situasies.
‘n Toets kontrolestel met verwysings materiaal vir drie vlakke (byvoorbeeld ‘n lae,
medium en hoë kontrole) absolute selwaardes en persentasies kan voorberei word vir
roetine immunologiese fenotiperings merkers (CD3/CD4/CD8/CD45). Meer
gespesialiseerde kontrolestelle vir meer spesifieke doeleindes kan opgemaak word wat
‘n verskydenheid van limfosiet aktiveringsmerkers bevat met byvoorbeeld ‘n “nul”, lae
en hoë verwysings kontrole daarin. Hierdie heelbloed kan dien as “aktiveerde interne
kwaliteits verwysings materiaal” en kan gebruik word om nuut aangestelde
laboratorium werkers en nuut gekwalifiseerde studente op te lei. Hierdie verwysings
materiaal / kontroles kan aangewend word vir bevoegdheids doeleindes (byvoorbeeld
vir SANAS akkreditasie doeleindes), vir metode ontwikkeling, vir sagteware toetsing,
vir paneel opstelling en instrument verstellings doeleindes. Die kontroles moet ‘n
verskydenheid eienskappe bevat om effektief te wees. Byvoorbeeld, stabiliteit tydens
storing, gewenslik meer as ‘n paar weke, herhaalbaar en maklik handteerbaar. Hierdie
kontroles sal inligting voorsien op ‘n daaglikse basis tydens wisseling van tegnieke of
instrumentasie wat akuraatheid beinvloed en op die ou-end direk pasiënt versorging
bevoordeel.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/21671 |
| Date | 03 1900 |
| Creators | Louw, Anne-Rika |
| Contributors | Bouic, P. J. D., Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medical Microbiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | xx, 216 leaves : ill. |
| Rights | Stellenbosch University |
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