V C. FagoFlowEx Kit (100 tests / testů / testov) Cat. No. / Kat. č.: ED7042 ENGLISH. 1. Intended use. 2. Introduction. 3. Principle PDF

V C FagoFlowEx Kit (100 tests / testů / testov) Cat. No. / Kat. č.: ED Intended use ENGLISH The FagoFlowEx Kit is intended for the examination of phagocytic activity of neutrophil granulocytes by

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V C FagoFlowEx Kit (100 tests / testů / testov) Cat. No. / Kat. č.: ED Intended use ENGLISH The FagoFlowEx Kit is intended for the examination of phagocytic activity of neutrophil granulocytes by measuring the respiratory (oxidative) burst after their stimulation with E. coli bacteria in human heparinized whole blood using flow cytometry. 2. Introduction Immune deficient patients are initially detected clinically by a history of recurrent infection. The responsible specific infectious agent is a guide to which part of the immune system is being affected. Deficiencies of innate immune system consist mainly of complement and phagocyte dysfunction and seriously dampen our ability to combat infections despite an apparently effective adaptive immune response. Besides diverse migration defects, the most common genetic defects of innate immune system target phagocytic and killing activities of neutrophil granulocytes, monocytes and macrophages based on myeloperoxidase (MPO) deficiency [1], chronic granulomatous disease (CGD) [2] and glucose-6- phosphate dehydrogenase (G6PD) syndromes [3]. All of these deficiencies lead to serious chronic infections of specific pathogens (bacteria, yeast and fungi) that are resistant to standard treatment. The respiratory burst is impaired in CGD, MPO, and G6PD deficiencies. Altogether these genetic disorders prevent the oxidative environment accumulation and hence the phagocytes are not capable of effectively destroying intracellular or ingested extracellular pathogens. Chronic granulomatous disease (CGD) is clinically and genetically a diverse group of hereditary diseases with deficiency in multiple enzymes of the NADPH oxidase cascade generating reactive oxygen radicals used for the pathogen killing. Approximately 1/3 of CGD patients harbor autosomal recessive mutations (CYBA, NCF1, NCF2) and two-thirds of CGD cases result from defects in the X-linked CYBB gene encoding the gp91 phox subunit of flavocytochrome b558 [4, 5]. Active NADPH oxidase of healthy individual catalyses NADPH reaction with oxygen, producing NADP + and superoxide radical O2ˉ, which undergoes a further series of reactions to produce singlet oxygen and hydrogen peroxide, later transformed by MPO. CGD patients suffer from pneumonia, abscesses of the skin, tissues, and organs, suppurative arthritis, osteomyelitis, bacteremia/fungemia and superficial skin infections such as cellulitis or impetigo [6]. Moreover, the persistent infection can lead to the formation of tissue granulomas based on overt CD4 + T cell-produced cytokines TNF and IFN. Importantly, CGD patients not only suffer from recurrent infections, but also present with excessive inflammatory, non-infectious conditions on the other hand [7]. MPO deficiency is a genetic disorder featuring deficiency, either in quantity or function, of a downstream myeloperoxidase enzyme generating hydroxyl radical and hypochlorite from the hydrogen peroxide. Hypochlorite is considered the most effective killing agent produced during a respiratory burst [8]. The G6PD enzyme is in the upstream hexose monophosphate pathway one of the major NADPH-generation processes. It is linked both to chronic nonspherocytic hemolytic anemia as well as decreased phagocyte respiratory burst [9] if the enzyme activity is low. All the above mentioned diseases related to phagocyte function illustrate their critical role in the removal and killing of pathogens while maintaining homeostasis in human organism. 3. Principle This test is based on the measurement of respiratory burst of neutrophil granulocytes after their stimulation with E. coli bacteria. During the process of bacteria ingestion, phagocytes activate the NADPH oxidase producing reactive oxidative intermediates (respiratory burst). Resulting hypochlorite ions inside phagocytes strongly oxidize dihydrorhodamine 123 (DHR123) into fluorescent rhodamine 123 which is detected by a flow cytometer. In the case of MPO deficiency, DHR123 is oxidized with less intensity by other oxidative products resulting in lower fluorescence intensity of stimulated granulocytes. A positive control sample is stimulated using PMA (Phorbol 12- myristate 13-acetate) which activates respiratory burst of granulocytes without adhesion and ingestion of the pathogen. 4. Precautions Intended for In Vitro Diagnostic use in laboratories outside USA and Canada. This CE-IVD kit is in conformity with the European In Vitro Diagnostic Medical Device Directive 98/79/EC. Blood must be collected into a tube containing heparin. Anticoagulants EDTA and citrate negatively affect results of the analysis. Blood samples should be treated within 24 hours after collection if stored at room temperature. 1/12 Stained samples should be analyzed within standardized time frame but no later than 2 hours after lysis. The flow cytometer should be calibrated on a routine basis using fluorescent microbeads to ensure stable sensitivity of detectors. Do not use reagents after expiration date stated on vial labels. Avoid DHR123 (ED7042-2) prolonged exposure to light. Avoid contamination of the reagent. Any non-performance of assay procedure may produce false results. Blood samples are considered as potentially infectious and must be handled with care. Warning: The Lysing Solution (ED7042-4) contains formaldehyde and methanol. Danger H-phrases H302+H312+H332: Harmful if swallowed, in contact with skin or if inhaled. H317: May cause an allergic skin reaction. H351: Suspected of causing cancer. P-phrases P270: Do not eat, drink or smoke when using this product. P280: Wear protective gloves / protective clothing / eye protection / face protection. P301+P312: IF SWALLOWED: Call a POISON CENTER or doctor/physician if you feel unwell. P302+P352: IF ON SKIN: Wash with plenty of soap and water. P305+P351+P338: IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. P501: Dispose of contents/container to authorized facility for dangerous wastes. 5. Reagents provided ED E.coli 5 vials containing lyophilized E. coli bacteria, 1 vial is intended for stimulation of 20 tubes. ED DHR123 5 vials containing lyophilized Dihydrorhodamine 123, 1 vial is intended for staining of 60 tubes. ED Stimulation Control 5 vials containing lyophilized PMA (Phorbol 12-myristate 13-acetate), 1 vial is intended for stimulation of 20 positive controls. ED Lysing Solution 15 ml 6. Necessary material not supplied Suitable 5ml test tubes for blood staining (e.g mm) Ultrapure demineralized water Automatic pipettes with disposable tips Vortex mixer Thermostat able to incubate test tubes at 37 C Centrifuge with rotor suitable for test tubes Flow cytometer - blue laser excitation at 488 nm, light emission at 525 nm (FITC) 7. Storage Store the FagoFlowEx Kit at 2-8 C. Expiration date is stated on a vial labels and on the box. 8. Assay procedure Use heparinized whole blood for examination (no later than 24 hours after collection). Prepare reagents according to following instructions. Preparation of reagents before assay 1. Reconstitute lyophilized E. coli bacteria using 250 l of demineralized water. Unused volume of the suspension store at 2-8 C up to 24 hours. Alternatively, the suspension can be aliquoted, frozen once, and stored at -20 C for another use. Avoid repeated freeze/thaw cycles. 2. Reconstitute lyophilized DHR123 using 650 l of demineralized water and shake gently - do not vortex. Unused volume of the reagent store at 2-8 C up to 8 hours. Alternatively, the reagent can be aliquoted, frozen once, and stored at -20 C for another use. Avoid repeated freeze/thaw cycles. Use frozen DHR123 solution within 14 days. Storage of frozen reagent may cause a decrease of Stimulation Index up to 20 %. 3. Reconstitute lyophilized Stimulation Control using 250 l of demineralized water. Unused volume of the reagent store at 2-8 C up to 24 hours. Alternatively, the reagent can be aliquoted, frozen once, and stored at -20 C for another use. Avoid repeated freeze/thaw cycles. 4. Lysing Solution is ready to use. Assay procedure For the examination of one blood sample prepare test tubes for negative control, positive control and for the sample to be stimulated with E. coli. 1. Into the tubes destined for: stimulated sample: add 10 l of E. coli suspension, negative control sample: add nothing, positive control sample: add 10 l of Stimulation Control. 2. Add 50 l of heparinized whole blood into all tubes and vortex gently. 3. Add 10 l of DHR123 into all tubes and vortex gently. 4. Incubate tubes at 37 C for 20 minutes in water bath or 30 minutes in air incubator. 5. Add 50 l of Lysing Solution into all tubes, vortex gently, and incubate for 5 minutes at room temperature. 6. Add 1 ml of demineralized water into all tubes, vortex gently, and incubate for 5-10 minutes at room temperature until the red blood cells are lysed. The fluorescence of Rhodamine 123, produced by oxidation of DHR123, is detected in FITC channel (525 nm). Since the Rhodamine 123 is quickly released from granulocytes, samples need to be measured as soon as possible (no later than 2 hours after lysis) preferable in a standardized narrow time frame. 2/12 9. Flow cytometric analysis Analyze stained samples using flow cytometer. Acquire at least 5,000 10,000 leukocytes per sample. Visualize measured data from stimulated and positive control and negative control sample in the side-scatter (SSC) versus forward-scatter (FSC) dot-plot. Set the gate around granulocytes as shown in figure 1. Ingestion of bacteria influences the position of granulocytes in a SSC-FSC dot-plot. Due to this, it is recommended to set gates individually for each blood sample. Then bring the gated granulocytes to histograms as shown in figures 2a, 2b, 2c where the X-axis represents fluorescence intensity in FITC channel (FL1). Set appropriate gates to calculate the percentage of positive and negative granulocytes and their mean fluorescence intensity (MFI). Stimulated granulocytes which undergo the oxidative burst exhibit bright fluorescence of Rhodamine Interpretation of results There are two parameters resulting from the assay that may indicate respiratory burst defects: a) Relative number of positive granulocytes which exhibited respiratory burst after the E. coli stimulation. b) Stimulation index means the MFI ratio of positive granulocytes of E. coli stimulated sample and negative granulocytes of negative control sample (see chapter 13). The stimulation index is used for approximate comparison of respiratory burst intensity between blood samples. Since the stimulation index may vary in different laboratories and instruments, each laboratory has to establish a normal range using its own test conditions. 11. Expected values Normal range of respiratory burst activity of granulocytes has been determined in 50 peripheral blood samples of healthy adults. Granulocytes with respiratory burst activity % Stimulation index of granulocytes 30 c) Rarely, low activity of glucose-6-phosphate dehydrogenase (G6PD) producing NADPH may also cause low respiratory burst of granulocytes. 2) If the positive control (stimulated by Stimulation Control) is positive and the tested sample (stimulated by E. coli) is negative, the blood sample probably contains anticoagulant EDTA or citrate. Alternatively, it can indicate the patient defect in adhesion or ingestion process of phagocytosis. 3) Low relative number of positive granulocytes indicates that blood sample is older or improperly stored as higher percentage of damaged granulocytes accumulates over time. Detection of unusual results described above indicates the suspicion of disease only which need to be confirmed by other tests. The MPO deficiency can be proved by intracellular staining of granulocytes using MAb to MPO (e.g. clone CLB- MPO-1/1, Cat. No. 1F-635-T100). Defects of NADPH oxidase (CGD) and G6PD must be confirmed by genetic analysis. Repeatability and reproducibility of the assay was determined from the data measured by five operators on six blood samples in two parallels under the same experimental conditions. Using Analysis of variance the following parameters were calculated: a) For relative number of positive granulocytes CV repeatability = 1.4 % CV reproducibility = 3.2 % b) For determination of Stimulation index 12. Limitations CV repeatability = 12.2 % CV reproducibility = 13.6 % Reproducibility of the assay strongly depends on precise working (incubation times, temperature, pipetting). Flow cytometer may produce false results if the device has not been regularly calibrated and maintained appropriately. Unusual results (potential defects of respiratory burst) 1) Lower stimulation index a) If granulocytes exhibit low respiratory burst after their stimulation with both E. coli and Stimulation Control (PMA), it can indicate suspicion of the most frequent defect - a myeloperoxidase (MPO) deficiency. The stimulation index is reduced approximately to 1/10 of normal value (Fig. 4). b) Less frequently, if granulocytes exhibit none or low respiratory burst after their stimulation with both E. coli and Stimulation Control (PMA), it can indicate chronic granulomatous disease (CGD). The intensity of the respiratory burst (Stimulation index) depends on the mutation of multiple enzymes of the NADPH oxidase cascade. If stimulated granulocytes of a female patient exhibit two subpopulations differing in the intensity of respiratory burst, it can indicate the defect in the X-linked gene. In this case, a part of granulocytes shows standard intensity, and the second part shows decreased intensity of respiratory burst (Fig. 5). 3/12 1. Použití soupravy ČESKY FagoFlowEx Kit je určený pro vyšetření fagocytární aktivity neutrofilních granulocytů měřením respiračního (oxidačního) vzplanutí po jejich stimulaci bakteriemi E. coli v lidské periferní krvi s heparinem pomocí průtokové cytometrie. 2. Úvod Pacienti s imunitním deficitem jsou v klinické praxi zachyceni na základě opakovaných infekcí. Příslušný typ infekčního agens bývá dobrým vodítkem k defektu určité složky imunitního systému. Deficity přirozené složky imunitního systému zahrnují především poruchy komplementu a fagocytózy a výrazně snižují naši schopnost odolávat infekcím, přestože adaptivní složka našeho imunitního systému zůstává nepostižena. Nejčastějšími genetickými poruchami přirozené obranyschopnosti jsou vedle různých defektů migrace buněk poruchy fagocytární a zabíječské aktivity neutrofilních granulocytů, monocytů a makrofágů, které bývají způsobeny deficitem myeloperoxidázy (MPO) [1], chronickou granulomatózou (CGD) [2], nebo deficitem glukóza-6-fosfát dehydrogenázy (G6PD) [3]. Všechny tyto nedostatky vedou k vážným chronickým infekcím specifických patogenů (bakterie, kvasinky a plísně), které jsou odolné vůči standardní léčbě. Respirační vzplanutí je narušeno u CGD, MPO a G6PD deficitů. Výsledkem těchto genetických poruch je snížení intenzity respiračního vzplanutí a proto fagocyty nejsou schopny účinně zničit intracelulární nebo pohlcené extracelulární patogeny. Chronická granulomatóza (CGD) je klinicky a geneticky různorodá skupina dědičných onemocnění s deficitem ve více enzymech kaskády NADPH oxidázy vytvářející reaktivní kyslíkové radikály. Podstatou přibližně 1/3 pacientů s CGD je autozomálně recesivní mutace (CYBA, NCF1, NCF2) a dvě třetiny případů CGD vznikají na základě vad genu CYBB kódujícího gp91 phox podjednotku flavocytochrome b558, který je lokalizován na chromozomu X [4, 5]. Aktivní NADPH oxidáza zdravého jedince katalyzuje reakci NADPH s kyslíkem a produkuje NADP + a superoxidový radikál O2 -, který prochází další řadou reakcí vedoucí k produkci singletového kyslíku a peroxidu vodíku, který je dále transformován myeloperoxidázou. Pacienti s CGD trpí zápalem plic, abscesy na kůži, tkáních a orgánech, hnisavou artritidou, osteomyelitidou, bacteremií / fungemií a povrchovými kožními infekcemi, jako je celulitida nebo impetigo [6]. Kromě toho může přetrvávající infekce vést k tvorbě tkáňových granulomů na základě přílišné produkce TNF a IFN cytokinů CD4 + T lymfocyty. Důležité je, že pacienti s CGD trpí nejen opakujícími se infekcemi, ale též nadměrnými zánětlivými neinfekčními stavy [7]. Deficit MPO je genetická porucha, která má za následek kvantitativní nebo funkční nedostatek následného generování hydroxylových radikálů a chlornanu z peroxidu vodíku enzymem myeloperoxidázou. Chlornan je považován za hlavní zabíječ patogenů při respiračním vzplanutí [8]. Enzym G6PD se účastní dráhy hexóza monofosfátu, tedy jednoho z hlavních procesů tvorby NADPH. Snížená aktivita G6PD je spojena s chronickou nesférocytickou hemolytickou anémií, ale může též způsobovat snížení respiračního vzplanutí fagocytů, pokud aktivita tohoto enzymu vlivem mutace poklesne přibližně pod 5 % normální aktivity [9]. V tomto případě jsou příznaky onemocnění podobné s CGD. Všechny výše uvedené nemoci spojené s fagocytární funkcí ilustrují důležitou roli fagocytů při odstraňování a usmrcování patogenů pro udržení homeostázy našich těl. 3. Princip Princip testu je založen na měření respiračního vzplanutí neutrofilních granulocytů po jejich stimulaci inaktivovanými bakteriemi E. coli. Při procesu ingesce bakterií je ve fagocytech aktivována NADPH-oxidáza, která spustí respirační vzplanutí. Vznikající chlornan uvnitř fagocytů silně oxiduje dihydrorhodamin 123 (DHR123) na fluorescenční produkt rhodamin 123, který je detekován průtokovým cytometrem. V případě defektu myeloperoxidázy, kdy nevzniká chlornan jako terminální produkt respiračního vzplanutí, je dihydrorhodamin 123 oxidován v menší míře ostatními reaktivními meziprodukty respiračního vzplanutí, což se projeví sníženou intenzitou fluorescence stimulovaných granulocytů. Pozitivní kontrolu představuje vzorek, který je inkubován s PMA (phorbol 12-myristate 13-acetate), který spustí respirační vzplanutí i bez adheze a ingesce patogenu granulocytem. 4. Upozornění Souprava je určena pro In vitro diagnostiku a vyhovuje požadavkům NV 453/2004 Sb., které je harmonizováno s evropskou směrnicí pro In vitro diagnostické zdravotnické prostředky 98/79/EC. Krev musí být odebrána do zkumavky s heparinem. Antikoagulanty EDTA a citrát ruší analýzu. Vzorky krve zpracujte nejdéle do 24 hodin po odběru, přičemž krev v odběrových zkumavkách musí být skladována za laboratorní teploty na kývačce. Vzorky analyzujte nejpozději 2 hodiny po lyzi ve standardizovaném časovém rámci. Průtokový cytometr pravidelně kalibrujte pomocí fluorescenčních kuliček, aby byla zajištěna stabilní citlivost detektorů. Nepoužívejte reagencie po uplynutí doby použitelnosti. Reagencii DHR123 (ED7042-2) nevystavujte dlouhodobému působení světla. Chraňte obsah vialek před kontaminací. Nedodržení doporučeného postupu analýzy může ovlivnit výsledky testů. Pracujte výhradně v ochranných rukavicích a při práci dodržujte správné postupy pro zacházení s potenciálně infekčním materiálem. Varování: reagencie Lysing Solution (ED7042-4) obsahuje formaldehyd a metanol. Nebezpečí H-věty H302+H312+H332: Zdraví škodlivý při požití, při styku s kůží a při vdechování. H317: Může vyvolat alergickou kožní reakci. H351: Podezření na vyvolání rakoviny. 4/12 P-věty P270: Při používání tohoto výrobku nejezte, nepijte ani nekuřte. P280: Používejte ochranné rukavice / ochranný oděv / ochranné brýle / obličejový štít. P : PŘI POŽITÍ: Necítíte-li se dobře, v
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