Material and method 1-material Tris (Sigma-Aldrich, Germany). Albumax II (Gibco,USA).

RPMI 1640 (Gibco,USA). Gentamicin (china). 1 M HEPES (Gibco,USA). Hanks’ balanced salt solution (Gibco,USA). Acridine orange (10 mg/mL) or Giemsa 5%. CD36 (FITC -100T) immunostep (Campus Miguel de Unamuno 37007 SALAMANCA, Spain). LDL bio system (C/ Costa Brava 3008030 Barcelona, Spain). HbA1C bio system (C/ Costa Brava 3008030 Barcelona Spain). 27% sorbitol in PBS,filter-sterilized, stored at 4°C (Sigma- Spain). 5% sorbitol in PBS, filter-sterilized, stored at 4 °C (Sigma- Spain). 15% AB+ serum 1-1 Equipment: 1- Glass desiccator (e.g., candle jar) (china) 2- Cell culture flask (china) 3- Candles (china) 4- Sterile pipettes (china) 5- Sterile tubes (china) 6- Glass slides & coverslips(surgiemd medical supplies, China) 7- Face mask (chagjunmedical ,Shanghai ,China) 8- Surgical cap( Changjun medical -china) 9- Sterile disposable swab 70% isopropyl (KAWAMOTO CORPOATION, China). 10-Disposable syringe 5ml/cc (yun don medical supply, China)
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Class II safety cabinet (HOOD.Inverted microscope (Olympus .Spain) 5. 2.Method 2.2 Instruments 1.Spectrophotometer (Bio system . parasitized P. 6. oxidative LDL.F RBCS. Serial No: . china) 2.AH 30133.Centrifuge ( lab Tech. 2.5 Sampling: Venous blood sample in heparin containers for culture and OxLDL.5 ml (BRAND GMBH. (Flow Cytometer Beckman Coulter Model. Germany) 12-Vacoteaner tube with heparin anticoagulant (China) 13-Gloves (Grip Pro PD. The samples were mixed well and tested within 6 hour.ESCO Singapore).CO2 incubator (37 °C) (Thermo electron co.3 Study area: This study was conducted in Khartoum state. 2. EPIX XL mm.Flow cytometer machine – type.1 study type and design: This study is cross section 2. and in EDTA for HbA1c & random sugar with 0. 0.4 Study Population: The study was conducted among diabetic patient attending In Jabir Abo Eleiz diabetic center. 2.2 Study variables: CD36. Germany) 3.04 mg anticoagulant holding 2-5 ml of blood. USA).11-Autmatic pipette 1.ltd. 2.Malaysia) 1. HbA1c.Germany) 4. Samples was classified into four groups: 2 .

3. falciparum (Culture) with high concentration oxLDL more than 170 mg / dl (43) And was tested for CD36. C = part of test = 0. Group II: Amount of 15 samples was cultured with plasmodium falciparum as CD36 positive control.(T-test.05 SS = 22 × 0.6 Sample size: For group III and IV according to the prevalence of diabetes Mellitus abu alizz center for diabetics the sample size was determined from known equation: ( ( ) ( ) ) Z= standard error of test= 2.05)2 Sample size will be 53 samples and will be collected from known diabetic patients With Hb A1C more than 8%.966 = 53 (0. Group IV: Amount of 15 sample diabetic patient with P.034× 0. P = prevalence of diabetes.Group I: Amount of 15 samples was collected from apparently healthy People free from any disease as negative control. 2.) 3 .Data analysis: Statistical package for social science (SPSS) was used for data Analysis . Group III: Amount of 15 sample from known diabetic patients (Hb A1C more than 8%) and was tested for CD36.

43 g RPMI 1640 powder (Gibco) 25 mL 1 M HEPES solution or 6 g HEPES (Gibco) 2 g NaHCO3 0.1 Malaria culture P.4 Laboratory procedure: 3.0 mL to 20 mL). Use within 10 days. falciparum: Preparations: Prepare malaria culture medium (MCM) was prepared and cell wer washed use Tris-buffered Hanks’(TH). -Albumax complete medium: 10. 3.Erythrocytes were washed 3 times in TH or RPMI 1640 to remove CPD. and leukocytes if present. The verbal of the consent was taken from Patients and take the permission from medical management of Jaber Abu Ezz Diabetes Center and selected individual after being informed with all objectives of the study and its health impact in the future. Filter-sterilize. Dilute to 5% hematocrit with cMCM in small flasks of 25cm2 (0. serum. In vitro cultures in tissue-culture flasks: . Manufacturing control and control sample werused in each test.2 mL of packed cells to 4 mL of cMCM) or in 75-cm2 flasks (1.1 Quality control: All reagents and test equipment wer controlled according to The instructions in the procedures manual.3.4.2 Ethical consideration: Ethical clearance will be obtaining from the Ethical Committee Board of the Tropical Medicine Resarch Institute. 3. store at -20 °C. 4 .5 mL gentamicin (from 50 mg/mL stock) 5 g Albumax II Add distilled water to 1 liter.

Let it stand for 5 min.-Add parasites to an appropriate parasitemia.Add 2 volumes of 5% sorbitol over 10 min. Produce low oxygen by burnt out candle and place the jar at 37 C °. slowly stir the tube constantly on a vortex. shake it lightly. . -Replace the MCM every day (not necessary the day after subcultivation).Slowly add 2 volumes of 27% sorbitol. discard the supernatant. .Transfer thawed parasites into a round-bottomed 13-mL tube (Max. .250 rpm on a table-top Beckman centrifuge) for 3 min. -Subculture the cultures 2 times/week. Continue culturing as usual. packed RBC’s to 5% hematocrit. . -Put the flask in a candle jar and loosen the screw cap. -Let it stand for 5 min.mL/vial).Let it stand for 5 min. . the first volume over 8 min. .4.2 OX-LDL 5 . 3. Procedure: -Remove a vial from liquid nitrogen and thaw it quickly in a 37 °C water bath. . discard the supernatant. .Change the medium the next day.Centrifuge the tube at 400 ×g for 3 min. Centrifuge the tube at 400 ×g and discard the supernatant. . .Centrifuge the tube at 400 ×g (1.Resuspend the pellet in 15% AB+ serum in MCM.Add 2 volumes of 5% sorbitol over 8 min. The wash may be repeated once. the second over 5 min. Add washed.Add 1 to 2 mL of MCM over 1 min.

Specimen type.02 QC 2. the cost per test can be kept to the minimum. Procedure: Blank Reagent solution (ml) Standard (ml) Test sample /QC (ml) 2. However. However.0 0.Principle: LDL Cholesterol esters in serum are hydrolysed by cholesterol esterase. The specimen is stable for a week at 2 .0 0.0 0. if a laboratory employs a photometer requiring 6 . which can be measured at 515nm/ yellow green filter. Set spectrophotometer / filter photometer to zero using blank at 510 nm / yellow green filter and measure the absorbance of standard. Since economical use of the reagent is possible with this protocol. which is then converted to water and oxygen by the enzyme peroxidase. Remove from water bath and cool to room temperature. A fasting blood sample is preferred for lipid profile test. if LDL cholesterol alone has to be analysed. a random sample can also be used. collection and storage: Serum or plasma can be used.02 Mix well. Para aminophenazone (4 aminophenazone) takes up the oxygen and together with phenol forms a pink coloured quinoneimine dye.80C and at least for 3 months at -200C. LDL cholesterol is then oxidized by cholesterol oxidase to the corresponding ketone liberating hydrogen peroxide. Incubate at 370C in a water bath for 5 minutes or at room temperature (25-35C0) for 15 minutes.02 Test 2. This protocol was designed for spectrophotometers / filter photometers that require a minimum volume of reaction mixture in the cuvette of 1ml or less.0 Standard 2. test and QC.

‫في البروبوزال‬ 3. and test sample/QC mentioned under #6 be increased proportionately. standard. 200 mg/dl) can be used and cholesterol in patients’ samples can be calculated using the formula.hemoglobins are retained by a cationic exchange resin. 5 ml. Inform the requesting physician of the problem (52). Do not report results from specimens with suspected interference. ( ) Normal range and interpretation Limitations: Hemolysis and lipaemia cause elevated cholesterol levels.4. Serum bilirubin >5mg/dl and ascorbic acid > 10 mg/dl also cause elevated cholesterol levels. Therefore a single standard (viz. In the author’s laboratory it is from 20 to 500 mg/dl. 7 .3 HbA1C: HbA1C principle: After preparing the hemolysate. Calculation and calibration graph: Linearity for calibration graph has been well documented by several kit companies. where the labile fraction is eliminated. it is advised that the volumes of reagent.a large volume of reaction mixture for measurement. viz.

Column Preparation (Notes 2 and 3) 4. Separation and Reading of HbA1c fraction 8 . push the upper disc down to the resin surface taking care not to compress it. and is quantified by direct photometric reading at 415 nm. Let the column drain completely to waste. This hemolysate will be used in steps 6 and 11. Hemoglobin A1C is stable for 7 days at 2-8ºC.Hemoglobin A1c(HbA1c) is specifically eluted after washing away the hemoglobin A1a+b fraction1 (HbA1a+b).Pipette into a test tube: Blood Regent 1 50µL 200µL 3. 5. Heparin or EDTA may be used as anticoagulants.Shake thoroughly and let it stand at room temperature for 10-15 Minutes. Hemolysate Preparation and Labile Fraction Elimination 1-Bring the columns and reagents to room temperature (21-26ºC) Not 1 2.Using the flat end of a pipette. Sample: Whole blood collected by standard procedures.Remove the upper cap of the column and then snap the tip off the bottom.

Shake thoroughly and read the absorbance (A) of the HbA1c fraction at 415 nm against distilled water (AHbA1c). Reading of Hb TOTAL 11.Shake thoroughly and read the absorbance (A) at 415 nm against distilled water (AHb TOTAL). Calculation 9 .Pipette: Reagent (2) 2.Place the column over a test tube and add: Reagent (3) 4.0 mL 50 µL 12.0ml Let the column drain to waste 9.Carefully pipette on the upper filter: Hemolysate 50 µL Let the column drain to waste 7.6.Pipette into a test tube: Reagent (3) Hemolysate 12. The absorbance is stable for at least one hour.0ml Collect the eluate (HbA1c Fraction) 10. The absorbance is stable for at least one hour.In order to drain any sample residue left above the upper disc pipette: Reagent (2) 200 µL Let the column drain to waste 8.

5 6. using the following formulas: %HbA1C-NGSP = 0.8-6.4 >6.7-7.4-6.5.4.2 4.8.1 Degree of control Non Diabetic Goal Good control Action suggested 10 .7 6.3 7.( reference in bio system ) CCT /NGSP 4. The following formula is deduced for the calculation of the concentration: The results obtained with the present method can be converted into equivalent to a US National Glycohemoglobin Standardization Program certified method (NGSP) or equivalent to the International Federation of Clinical Chemistry standardized method (IFCC).1 >9.0 >8.4 Bio system 4.94 x %HbA1C-BioSystems – 2.0 -6.0-4.0 6.24 %HbA1C-IFCC = 0.0 IFCC 2.09 Reference value The following cut-off points have been established by the Diabetes Control and Complications Trial Research Group (DCCT) and have been adopted by many countries for a reference population (Non diabetic) and for the evaluation of the degree blood glucose control in diabetic patients2.The HbA1c percentage in the sample is calculated using the following general formula: (A HbA1C ×V HbA1C ) ÷ (A Hb total ×V Hb total ) ×100=%HbA1c The volume of HbA1c (VHbA1c) is 4 mL.3.2. the volume of Hb total (VHbTOTAL) is 12 mL.0-6.8 4.3-9.86 x %HbA1C-BioSystems + 0.

Since large numbers of cells are analyzed in a 11 . Light scatter is dependent on the internal structure of the cell and its size and shape. Absorbed light of the appropriate wavelength may be reemitted as fluorescence if the cell contains a naturally fluorescent substance or one or more fluorochrome-labeled antibodies are attached to surface or internal cell structures. The suspension of cells or particles is aspirated into a flow cell where. The resulting electrical pulses are digitized. 8 ‫)في ورقة الفلو‬ the end result is quantitative information about every cell analyzed. Fluorescein isothiocyanate (FITC).4. Various immunoflurescent dyes or antibodies can be attached to the antigen or protein of interest. The light is either scattered or absorbed when it strikes a cell. surrounded by a narrow fluid stream. analyzed.(7. they pass one at a time through a focused laser beam. Optical filters are essential to block unwanted light and permit light of the desired wavelength to reach the photodetector.3. Light and/or fluorescence scatter signals are detected by a series of photodiodes and amplified.4 Flow cytometer: Principle: Prepared single cell or particle suspensions are necessary for flow cytometric analysis. Texas red. and phycoerythrin (PE) are the most common fluorescent dyes used in the biomedical sciences. Fluorescent substances absorb light of an appropriate wavelength and reemit light of a different wavelength. and the data is stored. and displayed through a computer system.

Fluor 488-conjugated antibody) diluted in buffer containing ethidium bromide 10. . goat anti-human IgG (Fc.) .000/sec). RT. This reduces cell loss in the assay. .Add 50 µL PRBC suspensions per well containing test serum to achieve desired serum concentration (usually 1/10 or 1/20).1% casein or 1% FCS added.Wash PRBC three times in PBS with 0.1-0. .Add 50 uL of tertiary antibody (FITC-conjugated anti-secondary IgG.2% hematocrit) . Drain wells after incubation.Incubate 30 mins at RT.Re suspend PRBC to 1-2 x 107 cells/mL (0.Add 50 uLof secondary antibody(eg.Sp. . then wash 3 times. -Use parasitised red blood cells (PRBC) from in vitro culture not more than 10% parasitaemia. Procedure: Procedure (Beeson et al. . or Alexa.For all incubations and washes.20 ug/mL. 1999) Buffers to use in the assay . either RPMI-HEPES or PBS can be used. .1% casein or 1% FCS prior to using in the assay. . synchronous at mid-late stage pigmented trophozoites (not schizonts).Incubate 30 mins at RT. with either 0.Pre-coat the required number of wells of a U-bottom 96 well microtitre plate with 0. to block non-specific binding. then wash 3 times. statistically valid information about cell populations is quickly obtained.1% casein or 1% FCS in PBS for 30 mins.Incubate 30 mins at RT in the dark wash 3 times 12 .short period of time (>1.

Acquisition time should be sufficient to count 500-1000 ethidium bromide positive cells· For the ethidium bromide negative and positive populations. . There should be a clear separation of ethidium bromide positive cells (PRBC) from negative cells (RBC).Gate RBC population. Proportion of PRBC labelled as positive relative to the mean+3SD of the fluorescence of RBC. giving the absolute difference in fluorescence..Resuspend cells in 200 ul of buffer and transfer to flow cytometry tubes Analysis by flow cytometry. giving the fold difference in fluorescence levels · 2. Antibody binding can be expressed as: 1. . mean (geometric) in fluorescence channel 1 of:  PRBC (ethidium bromide positive cells) minus that of RBC (EtBr negative cells). . determine the mean fluorescence intensity in channel 1 (antibody binding) of the cell population and the proportion of cells positive for antibody binding. or  PRBC divided by that of RBC. 13 .Plot cells by fluorescence in channel 1 (for FITC or Alexa-Fluor) against fluorescence in channel 2 (for EtBr).

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