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STANDARD OPERATING PROCEDURES


Our laboratory uses only the standard methods for sample testing. We annually reviewed the methods and reference values. All test methods are documented and maintained. The standard operating procedures are written as per guidelines. They are in simple language which is understood by the technical staff performing the test. Kit procedures are attached for the estimation of glucose, serum widal and urine pregnancy test.

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HAEMATOLOGY
I. TOTAL LEUCOCYTE COUNT

a. Purpose of Examination To determine the total number of WBCs present in cubic mm of blood. b. Principle - Blood is diluted with a fluid that causes haemolysis of earythrocyts, but has no effect on leucocytes, which can then be counted in Improved Neubauer counting chamber. c. Performance specifications When the leucocytes count is low (below 4000 cells per cm of blood) the dilution must be 1 in 10 or when the leucocyte count is high the dilution must be increased by adding diluents and necessary arrangemen ts made in the calculation factor. d. Type of primary sample Whole blood or EDTA blood sample may be used. e. Type of containers and additives - .05ml of 10% aqueous solution of EDTA is dispensed in small bottles and evaporated to dryness is used in our lab. f. Equipments and reagents required 1. Binocular microscope 2. Improved Neubauer counting chamber with cover glass. 3. .02 ml pipette. 4. Small tube. 5. WBC diluting tube. g. Examination procedure steps .02 ml of blood is taken and mixed with .38 ml of WBC diluting fluid in a small tube. Mix it and allow standing for 5 minutes. The cover glass is placed on the counting chamber then applies slight pleasure to the ends of the cover glass, will reveal rainbow Newton diffraction rings. The chamber is now ready to be charged.

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The tip of the pipette is brought up to the edge of the cover glass. By gentle release of index finger pressure, fluid is allowed to run out slowly until the counting platform is just covered. The fluid is drawn into the chamber by capillary attraction. The chamber is placed in passion under the microscope stage and is allowed to sta nd for two minutes,

so that the cells may settle. Using the low power objective of microscope count the for large squares.
g . Calculation If X is the number of WBC in squares The dilution of blood is 1in 20 Number of WBCs per cubic mm of blood is Xx10x20= 50X 4 h Interferences 1. Improper mixing of blood. 2. Clots in blood 3. Improper dilution 4. Improper filling of counting chamber 5. Errors in counting I Biological reference interval 5 to 10 thousand cells per cubic mm of blood. Commonly increase the infections and leukemia. h. Document control identifiers Lynches Medical lab. Technology - Raphae
II. DIFFERENTIAL COUNT OF LEUCOCYTE

a. Purpose of Examination To differentiate different type of WBCs present in the blood after staining. b. Principle of procedure Acidic dyes unite with the basic component of the cells conversely basic stains are attracted to acidic components of the cells. This consists of the enumeration of the relative percentage of the various types WBC seen in stained films. c. Performance specifications Unripened Leishmans stain give pale nuclear staining. If the smear has a bluish to violet appearance it may be due to over staining, under washing or to alkaline staining or washing with buffer improve quality. Some of smears are under stained and they may be restrained.

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Issue No. 1 Amendment No. Issue Date: 1.7.2009 Amendment Date. Approved by : Dr. Abdul Azeez Issued by: Superintendent THQH Chavakkad

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d. Type of primary sample Whole blood or EDTA blood may be used. e. Type container and additives - .05ml of 10% aqueous solution of EDTA is dispensed in small bottles and evaporated to dryness is used in our lab. f. Equipments and reagents required 1. Binocular microscope 2. Glass slide and spreader 3. Leishmans stain 4. Buffer solution 5. Cedar wood oil 6. Cell counter 7. Examination procedure steps Take a clean grease free slide and place a drop of blood one to two cm away from one end of he slide. Using a spreader spread the blood about 30 0 angles with 3 cm length and 2 cm width with tongue shape. Allow to air dry. Place the smear on a staining rack. Cover smear with Leishmans stain and leave for 1 minute. Dilute it with double volume of distilled water keep it for 5 minutes. Wash with buffer solution. Air dry and examine under the oil immersion objective of the binocular microscope. 8. Interferences The greater the number of leukocytes counted the smaller the error. 9. Calculation Count hundred number of WBCs using the counter and write the percentage of neutrophils, basophils, eosinophils, lymphocytes and monocytes. 10. Biological reference interval Neutrophils 50 70% Basophils 0 1% Eosinophils 1 4% Lymphocytes 25 40% Monocytes 3 4% 11. Document control identifiers Lynches Medical lab. Technology- Raphel.

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III.

ESR (ERYTHROCYSTES SEDIMENTATION RATE)

a. Purpose of examination This test is used to detect the Erythrocystes Sedimentation Rate. b. Principle of procedure This test is used as an index of the presence of active deceases of many types. The rate of this process depends on a number of factors. Rouleaux formation, concentration fibrinogen and constration of alpha beta globulin in plasma. It is also used for the prognosis disease. c. Performance specifications This is the reference method for ESR. d. Type of primary sample Whole blood. e. Type of container and additive Small bottle with 3.8% Sodium citrate. f. Equipments and reagents required 1. Westegren pipette 2. Westegren stand 3. 3.8% Sodium citrate g. Examination procedure steps 1.6ml of blood is added to .4ml of 3.8%Sodium citrate. Mix well. Drawn up to the zero mark of a westgren tube. It is placed in a rack in vertical position at room temperature for one hour. Then read the end of the plasma column above he cells. h. Interferences Anaemia increases the ESR. Polycythemia decreases ESR. Direct drafts, sunlight and vibration may interfere the ESR. i. Calculation The length of the plasma column above the cells in ml per one hour is reported as the result. j. Bioligical reference interval 1. Man 3 to 5mm/hr 2. Woman 4 to 7mm/hr 3. Document control identifiers Lynches Medical lab. Technology- Raphel.

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Haemoglobin Estimation: To detect blood haemoglobin.Haemoglobin is a complex protein present in the red blood cells which gives red colour to the blood that helps to carry oxygen to various cells. Principle:Method:- Sahli s haemoglbin method. It is also known as acid haematin method. In this method Haemoglobin is converted to acid haematin by the addition of N/10 HCl and the resulting brown colour is compared with standard brown glass reference block. The intensity of brown colour depends on the amount of acid haematin ,whic h in turn depends on the amount of haemoglobin present. Meterial required:- Sahli s haemoglobinometer, N/10 HCl ,whole blood ,glass rode,Sahli s pipette. Sample used:- Capillary blood or anticoagulated venous blood. Procedure:-place N/10 HCl to the lower mark of the Sahli s tube.Draw 20 micro.ltr of blood Sahli s pipette and after wiping out all traces of blood from the out side of the pipette, transfer the blood from the pipette to the tube containing HCl. Mix well and kept for10 minutes.Gradually the fluid become brown colour. Then dilute the fluid with distilled water drop by drop.Mix it with a glass road until the colour matches with the colour of the standared.The reading should be only against natural light. Result:- The level of the fluid in tube is noted at its lower meniscus and the reading corresponding to this level on the scale is read as g/dL Normal range:Male Female
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13- 16 gm% 12- 14 gm%


Issue No. 1 Amendment No. Issue Date: 1.7.2009 Amendment Date. Approved by : Dr. Abdul Azeez Issued by: Superintendent THQH Chavakkad

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a. b.

c. d. e. f.

g.

h.

i.

j.

k.

IV. PLATELET COUNT. Purpose of examination To determin the total number of platelets present in one cmm of blood. Principle of procedure Blood is diluted with a diluting fluid that causes lysis of erythrocytes but has no effect on platelets, which can then be counted in improved Neubauer counting chamber. Performance specification Tube method is used in our laboratory Type of primary sample Whole blood is used. Container and additives - .05ml of 10% solution of EDTA is dispensed in small bottle and evaporated to dryness is used. Equipments and reagents 1. Binocular microscope 2. Imporved Neubauer counting chamber with cover glass 3. Platelet diluting fluid 4. Small testube Examination procedure steps - .02ml of blood is diluted with .38 ml of diluting fluid. Mix for 2 minutes. Then fill both sides of the counting chamber and allow the platelets to settle for 20 minutes in a petridish on the bottom of which is moist disc of filter paper. Count the number of platelets which will appear as refractile bodies in 5 squares including the central ruled area of the counting chamber. Interferences 1. Clots in sample 2. Improper dilution 3. Improper filling of counting chamber 4. Improper counting 5. Contamination of counting fluid 6. Clumping of platelets 7. Skin puncture shows low values than venous puncture. Calculation Area of 5 squares in central rules area = .2sq.mm. Volume of 5 squares in central ruled area = .2x.1 Ql = .02 Q l. Dilution of blood sample 1:20 if R is the number of platelets counted. The total number of platelets = Rx20x1/.02 = Rx20x50 = Rx1000cumm of blood Biological reference interval Normal values 1.5 to 4.5 lakhs/cmm of blood . Low platelets are found in ITP and Dengue fever. Increased platelets are found in polycythemia and spleenectomy. Document control identifiers Lynches Medical lab. Technology- Raphel.
Issue No. 1 Amendment No. Issue Date: 1.7.2009 Amendment Date. Approved by : Dr. Abdul Azeez Issued by: Superintendent THQH Chavakkad

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V.

CLOTTING TIME

a. Purpose of examination This test is used to detect the time taken for the clotting of whole blood. b. Principle for procedure Method of Lee and White. Whole blood is delivered by venipuncture to glass testubes. The time taken for clotting is expressed in minutes. c. Performance specification This test is not reliable for the detection of mild and moderate procoagulant defect and is therefore not recommended as screening test. It is till useful for control of apparen, provided that the test is carefully performed. d. Type of primary sample Venous blood. e. Type of container Disposable syringe and needle 10/1cm glass tubes. f. Equipments Stop watch. g. Procedure Venous blood is withdrawn using normal precaution, and a stop watch is started, the moment blood appears in the syringe. Deliver one ml of blood into each of 4 small testubes which is previously placed in 37 0 c water bath. After 3 minutes keep the tubes out of the water bath and tilt them individually after every 30 seconds. Avoid unnecessary agitation. This may prolong plotting time. The clotting time is taken when the tube can be inverted without its contents filling. The clotting time of each tube is recorded separately and is reported as an average of the 4 tubes. h. Quality control procedure The test must be run with a normal control. i. Interferences It is rough measure of the efficiency of the intrinsic blood clotting mechanism, and normal values do not exclude major defects. Calculation Take the average time of the 4 tubes as the clotting time j. Biological reference interval Normal value 4 minutes to 10 minutes. k. Document control identifiers Lynches Medical lab. Technology- Raphel.

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Issue No. 1 Amendment No. Issue Date: 1.7.2009 Amendment Date. Approved by : Dr. Abdul Azeez Issued by: Superintendent THQH Chavakkad

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VI.

BLEEDING TIME

a. Purpose of examination To determine the bleeding time of a patient in case of snake bites, poisoning or haemophilia. b. Principle This tests provides an estimate of the integrity of the primary haemostatic plug and thus measures the interaction between th e capillaries and platelets. A normal result means there no clinically significant platelet vascular defect. c. Performance specifications It is not positive in platelet function defect either because of mildness of the defect of the variability of the cond itions. d. Equipments and reagents Lancet, filter paper and stop watch. e. Examination procedure steps Prepare the patient by explaining the procedure. Allow him or her to relax in a comfortable position. Make a puncture on he finger tip using a sterile lancet. Start the stop watch. Then by applying gentle pressure, remove the blood oozing out with a filter paper every after 30 seconds. Note the time when no blood is picked up on the filter paper. e. Interferences The range of result will depend upon individual technique. f. Standerdise the test to avoid undue variation. g. Calculation Note the time from the stop watch. h. Biological reference interval Above 3 minutes is considered significant. i. Document control identifiers Lynches Medical lab. Technology - Raphel.
VII. PERIPHERAL SMEAR Purpose of examination To rule out the smear is normal or abnormal. Principal of procedure Same as Leishmans stain. Type of primary sample Blood Container and additives Bottle containing .05 ml of 10% evaporated EDTA. Equipments and reagents required Binocular microscope, glass slide, spreader, Leishmans stain and cedar wood oil. Examination procedure steps Stain the blood smear with Leishmans stain and examin under the oil immersion objective of the microscope. Observation RBCs normally circular, non nucleated, highly flexible biconcave disc with an average size of 7.2 micron diameter and 2.1 micro meter in thickness. They are called normocytes. Microcytes Less than 6 micrometer diameter. Macrocytes More than 8 micro meter diameter. Normochromatic - It has an evident central concavity but stains uniformely.
Issue No. 1 Amendment No. Issue Date: 1.7.2009 Amendment Date. Approved by : Dr. Abdul Azeez Issued by: Superintendent THQH Chavakkad

a. b. c. d. e. f. g.

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Hypochromatic Dicreased Hb concentration with a central pallor. Anisochromatia Cells with a morphology uniquely stained portions that is only a portion appears as hypochromic. (After blood transfusion to a Iron deficiency anaemia). Poikilocytosis RBCs with variation in shape that is spherocytes, crenation, target cells. WBCs - To rule out. lukaemia Myeloid or lymphoid. Neutrophils Normally 60 to 70% seen. Lymphocytes 20 to 40% seen. Increases in lymphoid leukemia Eosinophils Normally 5to 6% cells seen. Increased in eosinophilia. Basophils and Monocytes Increased in various conditions. Leukocytosis More than 10,000 cells is called Leukocytosis. Platelets These tiny 2 to 4 micro meter. Cytoplasmic, non nucleated bodies are present normally 1.5 to 5 lakh/ml. In normal blood Plateletes adequately seen. In abnormal picture scatterly seen. Blood parasites like Malaria and Filaria may also seen. h. Document control identifiers Lynches Medical lab. Technology - Raphel.

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URINE ALBUMIN DETECTION


Urine albumin estimation:Sulphosalicylic acid method :- The reagent used in this test is 3% sulphoalicylic acid (30 gm/litre in distilled water) Procedure :- Check the p H of the urine,and if it is alkaline or neutral add 10% acetic acid drop by drop until it is just acid . To 2.0 ml of clear urine , add an equal volume of 3% sulphosalicylic acid. Let stand for 10 minutes. The results are recorded and reported as follows: Negative Trace 1+ 2+ 3+ 4+ -No cloudiness - cloudiness just visible against a dark background . - Dense cloudness. - cloudiness with granules and definite flocculation Cloudiness with heavy flocculation Cloudiness with flocculation and precipitation .

Strip method:Principle: The test is based on the protein error of indicator principle . Anion in the specific p H Indicator attracted the by cation on protein molecule mak es the indicator further ionized,which chages its colour Materials required Midstream urine sample. Uristix.

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Procedure: . put a strip in to the sample and remove . allow to stand for 30 sec. . Compare the colour change with the colour chart Reporting:- Report from Negative ,trace, +,++,+++,++++ by comparing the strip with the colour chart given on the bottle. URINE SUGAR BENEDICTS QUALITATIVE TEST

a. Purpose of examination: To detect the approximate percentage of sugar present in the urine. b. Principle of procedure Sugar has a reducing property. CuSO 4 present in the benedicts reagent reduces the sugar present in the urine and against undergoes reduction with Ferrie chloride which produces different colours according to the degree of sugar present . c. Performance specifications Any reducing substance other than sugar produces a false +v result. In pregnancy lactosuria may also produce false +ve result. d. Type of primary sample urine e. Type of containers and additive clean, sterile wide mouthed glass bottle . f. Equipments & Reagents required 1. Boiling test tube. 2. Test tube holder. 3. Spirit lamp. 4. Benedicts reagent. 5. 5 ml. and 1ml. pipettes. g. Examination procedure steps. : Take0.5 ml. urine & 5 ml. of Benedicts reagent in a Boiling test tube. Mix well and boil it using the spirit lamp. Cool and read the colour.

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h. Observation : Blue Negative Green 0.5 Yellow 1% Orange 1.5% Brick red 2% Biological reference interval Not normally present in urine. Document control identifiers Lynches Medical lab. Technology - Raphel

Note: ALBU STIX is also used for testing albumin & sugar in urine.

MICROSCOPICAL EXAMINATION:
a. Purpose of examination To detect the presence of RBCs, puscells, Epicells casts and crystals in the urine. b. Principle of procedure The different substances present in the urine sample is separated by centrifugation and detected using the high power objective of the binocular microscope. c. Type of primary sample:- Urine collected within 1/2 an hour. d. Type of container and additives.; Ster ile, clean & dry glass bottle is used for collecting urine. e. Equipments & reagents required.: Centrifuge tube, Centrifuge, Glass slide, cover glass, Binocular microscope. f. Examination procedure steps. : Take 10 ml. of urine in a centrifuge tube and cent rifuge for 3 minutes at 3000 rpm. Discard the supernatant. Then take the deposit on a glass slide and examine under the high power objective of the binocular microscope.

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g. Observation: Three types of cells, casts and crystals may present and report the number/high power field. Epithelial cells Small round cells larger than leukocytes & caudate epithelial cells may also present. Red cells - Small, round /crenated non nucleated cells. Leukocytes 1-3/HPF is considered normal more than that WBCs should be counted per high power field and reported. Casts Hyaline casts, granular casts, pus cell casts and red cells are not normally seen in the urine. If they are found report it. Crystals - Crystals are not normally present in urine. In Acidic urine. Calcium Oxalate crystals are colorless, octahedral crystals and appear as small squares crossed by two diagonal lines. It may also seen as dumbbell shaped. Uric acid- seen as plates, prisms, sheaves and hexagons usually colored and are dissolved by heating. Urates Urates may appear as amorphous sediment which dissolved by heating. They are often appears in the form of thorn apples. Cystine highly refractile hexagonal plates similar to the plate form of uric acid crystals. They are differentiated from uric acid crystals by their solubility in hydrochloric acid. Not found in alkaline urine which is soluble. Tyrosine Usually in the shape of fine needles/sheaves. Crystals in alkaline urine. Phosphates Seen as amorphous phosphates soluble in Acetic acid. Triple phosphates Seen as prisms and coffin lids. Soluble in Acetic acid. Calcium carbonate - Appear as dumbbells, spheres or amorphous granules; they are soluble in Acetic acid. Ammonium urates Appear in round, oval or thorn apple form. They are soluble in Acids. Sulfonamide crystals may be due to drug administration. They vary in shapes. Spermatozoa may also present in normal urine Bacteria may also present in normal urine. Documents collected identifier - Lynches Medical Lab. Technology -Raphel.

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BILE PIGMENT
a. Purpose of examination To detect the presence of bilirubin in the urine sample. It is not normally present. b. Principle of procedure Oxidation of bilirubin is used. Bacl2 is added to the urine. A precipitate of BaSO 4 is produced on to which the bilirubin is adsorbed. It is filtered off and a drop of Fouchets Ferric chloride solution is added to the precipitate. The ferric chloride oxidizes the bilirubin to biliverdin producing a greenish blue spot. c. Performance specification Very specific test. d. Type of primary sample Urine. e. Type of containers and additives Clean dry sterilized wide mouthed glass bottles are used. f. Equipments and Reagents required 10% Barium chloride, Fouchets Reagent, large test tube, filter paper, glass funnel. g. Examination procedure steps-. To about 10ml. of urine add a few ml. of 10% Barium chloride 2 solution, mix well and filter through whatman No.1 filter paper. To the filtered precipitates add a few drops of Fouchets Reagent. A greenish blue colour Indicates the presence of bilirubin. Documents collected identifier - Lynches Medical Lab.Technology -Raphel.

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BILE SALT

a. Purpose of examination To detect the presence of Bile salt in the urine. b. Principle of procedure the presence of bile salts reduces surface tension. c. Performance specification Very specific test. d. Type of primary sample Urine e. Type of containers and additives Clean dry wide mouthed glass bottles are used to collect urine . f. Equipments & Reagents used Sulphur powder, wide mouthed bottle. g. Examination procedure steps Place the urine in the wide mouthed bottle. Just put powdered sulphur over the urine sample. If the powder sink down then the urine sample is +ve for bile salt. Documents collected identifier - Lynches Medical Lab.Technology -Raphel.
Urine acetone estimation :Method :- Rothera s test Principle :- Acetone and diacetic acid react with sodium nitroprusside [ nitroferricyanide ] in the presence of alkali to produce a purple colour . Procedure :- Take about 5 ml of urine in a test tube and saturate it with ammonium suphate , and then add one crystal of sodium nitroprussiede. Then gently add 0.5 to 1 ml of liquor ammonia down the side of the tube so that it layers on top of the urine. Report :- If acetone / diacetic acid are present , a purpile [ permanganate calomel red ] colour will form at the junction of the two layers within 30 -60 seconds. A brown colour has no significance. The results can be graded from trace to ++++ by the intensity of the colour formation .
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SEROLOGY
Sl.No. Materials or items tested Specification Specification, std. tests method or examination technique used performed
Widal Rapid slide test span diagnostic semi quantitative slide test

Range of testing/ Limit of detection


-

% CV/MG (+ -)
Internal Quality control

Serum

Kit procedure attached.

SPUTUM EXAMINATION
In our laboratory sputum examinations are done under RNTCP programme . Both macroscopic and microscopic examinations are done here The aims of sputum microscopy are to y Diagnose patients with infectious tuberculosis y Monitor the progress of tuberculosis patients who are on treatment Specimen collection:- The patients are advised to take two samples spot and early morning. Receive the patients and the laboratory form. Check the lab form for completeness and accuracy. The laboratory technician can help patients by showing genuine concern patience. Emphasis that diagnosis facilities and treatment are free and that tuberculosis can be cured simply by taking complete and regular treatment as prescribed. First the patient is registered into RNTCP register with his / her lab serial no, date of entry, name and address, age , sex, t he name referring health facility and the type of examination done[ diagnosis / follow-up ].

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Then a specific bottle supplied by RNTCP is given to the patient and ask him to take a good sputum sample. The bottle is numbered as same as to that of lab serial no which is pasted on the side of the container, never on the lid. For each patient there is a different lab no that is same for his / her both samples. The container must be wide mouthed, leak proof, clean, dry and tight- fitted lid. All sputum containers should be used only once. The patient is advised to take this sample from a deep cough from an open place far away from other people. Smear preparation :y Arrange the specimen containers in serial order. Ensure that the lab sl. No. on the sputum containers match with the no. in the lab form. y Take a clean, new, grease-free, unscratched slides and label with a diamond marker on one end of the slide. y Take a broom stick with uneven ends y Using the jagged end of broom stick prepare a thin smear of 3*2 cm in size with mucopurulent portion of sputum. It must be near to a flame. y Allow the smear to dry in air. y Fix the smear by passing over the flame for 3 to 5 times for 3 to 4 seconds each . Staining :Principle :- The presence of mucolic acid on the cell wall of bacteria resist decolourisation with acid or alcohol once stained with carbol-fuchsin in the presence of heat. So the acid fast bacilli takes the primary stains and back ground appears in the counter stains colour. Procedure:y Place the smear on a staining rack with the smear side up. y Pour 1% carbol fuchsin to cover the entire smear for 5 minutes heat until the vapours just arise.

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y y y y y y

Rinse the slides with water. Decolourise with 25% H2SO4 let it stand for 2 to 4 mts. Rinse with tap water and drain off the slides. Counterstain with 0.1% ethylene blue and stand for 30 sec. Rinse with tap water and allow to air dry. Examine under high power objective and then turn to oil immersion objective.
Reporting :No. of fields to be examined

Examination

Result

Grading

No AFB per 100 OIF

Negative

---Scanty

100

1-9 AFB per 100 OIF

Positive

[record exact no. ]

100

10-99 AFB per 100 OIF

Positive

1+

100

1-10 AFB per OIF

Positive

2+

50

More than 10 AFB per OIF

Positive

3+

20

Document No: 3 Page No : 19 Prepared by . Noorjahan.A. Lab technician , THQH Chavakkad

Issue No. 1 Amendment No. Issue Date: 1.7.2009 Amendment Date. Approved by : Dr. Abdul Azeez Issued by: Superintendent THQH Chavakkad

SECTION

HOSPITAL DIAGNOSTIC SERVICES KERALA QUALITY MANUAL Q.C.I. ESSENTIAL STANDARDS 3.1.1 Standard operating procedures PAGE

20/20

We are designating the Medical officer to approve the test results. The unused portion of the sample is kept until the result is issued to the patient. The laboratory technicians discard the unused samples as per the documented procedure

DISPOSAL OF SPECIMENS

Wastes disposal are entrusted with IMAGE services

Document No: 3 Page No : 20 Prepared by . Noorjahan.A. Lab technician , THQH Chavakkad

Issue No. 1 Amendment No. Issue Date: 1.7.2009 Amendment Date. Approved by : Dr. Abdul Azeez Issued by: Superintendent THQH Chavakkad

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