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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.

Document No: 3 Issue No. 1 Amendment No.Page No : 1 Issue Date: 1.7.2009 Amendment Date.Prepared by . Noorjahan.A. Lab technician , THQH Chavakkad

Approved by : Dr. Abdul Azeez Superintendent THQH Chavakkad

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HOSPITAL DIAGNOSTIC SERVICES KERALA QUALITY MANUAL Q.C.I. ESSENTIAL STANDARDS

SECTION 3.1.1 Standard operating procedures PAGE 20/01



HAEMATOLOGY

I. TOTAL LEUCOCYTE COUNT

a. Purpose of Examination – To determine the total number of WBC’s 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 arrangements 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 microscope2. 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 stand 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 WBC’s per cubic mm of blood is Xx10x20= 50X

4 h Interferences –

1. Improper mixing of blood.2. Clots in blood3. Improper dilution4. Improper filling of counting chamber5. 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 WBC’s 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.



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c. Performance specifications – Unripened Leishman’s 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.

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 spreader3. Leishman’s stain 4. Buffer solution5. Cedar wood oil6. Cell counter7. 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 300 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 Leishman’s 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 WBC’s 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%



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Monocytes 3 – 4%11. Document control identifiers – Lynches Medical lab. Technology- Raphel.

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 pipette2. Westegren stand3. 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 interval1. Man – 3 to 5mm/hr2. Woman – 4 to 7mm/hr3. Document control identifiers – Lynches Medical lab. Technology- Raphel.



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I. HAEMOGLOBIN ESTIMATION

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 ,which 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 13- 16 gm%

Female 12- 14 gm%



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IV. PLATELET COUNT. a. Purpose of examination – To determin the total number of platelets present in

one cmm of blood. b. 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.

c. Performance specification – Tube method is used in our laboratoryd. Type of primary sample – Whole blood is used.e. Container and additives - .05ml of 10% solution of EDTA is dispensed in small

bottle and evaporated to dryness is used.f. Equipments and reagents

1. Binocular microscope2. Imporved Neubauer counting chamber with cover glass3. Platelet diluting fluid



4. Small testubeg. 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.

h. Interferences1. Clots in sample2. Improper dilution3. Improper filling of counting chamber4. Improper counting5. Contamination of counting fluid6. Clumping of platelets7. Skin puncture shows low values than venous puncture.

i. Calculation – Area of 5 squares in central rules area = .2sq.mm. Volume of 5 squares in central ruled area = .2x.1l = .02 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

j. 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.



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k. Document control identifiers – Lynches Medical lab. Technology- Raphel.

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 370 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.Document No: 3 Issue No. 1 Amendment No.Page No : 8 Issue Date: 1.7.2009 Amendment Date.Prepared by . Noorjahan.A. Lab technician , THQH Chavakkad


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k. Document control identifiers – Lynches Medical lab. Technology- Raphel.

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 the 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 conditions.

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 a. Purpose of examination – To rule out the smear is normal or abnormal.b. Principal of procedure – Same as Leishman’s stain.c. Type of primary sample – Bloodd. Container and additives – Bottle containing .05 ml of 10% evaporated EDTA.e. Equipments and reagents required – Binocular microscope, glass slide, spreader,

Leishman’s stain and cedar wood oil. f. Examination procedure steps – Stain the blood smear with Leishman’s stain and

examin under the oil immersion objective of the microscope.g. Observation – RBC’s 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.



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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 – RBC’s with variation in shape that is spherocytes, crenation, target cells. WBC’s - 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 -No cloudiness

Trace - cloudiness just visible against a dark background .

1+ - Dense cloudness.



2+ - cloudiness with granules and definite flocculation

3+ - Cloudiness with heavy flocculation

4+ - 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 makes the indicator further ionized,which chages its colour

Materials required

Midstream urine sample.



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Uristix.

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. CuSO4 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. Benedict’s 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 – 0.5Yellow - 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.; Sterile, 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 centrifuge

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 BaSO4 is produced on to which the bilirubin is adsorbed.

It is filtered off and a drop of Fouchet’s 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, Fouchet’s 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 Fouchet’s 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.



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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 .

SEROLOGY

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

Diagnose patients with infectious tuberculosis 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.



Sl.No.

Materials or

items tested

Specification tests

examination performed

Specification, std. method or

technique used

Range of testing/ Limit of detection

% CV/MG

(+ -)

1 Serum Widal

Rapid slide test span diagnostic

semi quantitative slide test

-Internal Quality control

First the patient is registered into RNTCP register with his / her lab serial no, date of entry, name and address, age , sex, the 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 :-

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.

Take a clean, new, grease-free, unscratched slides and label with a diamond marker on one end of the slide.

Take a broom stick with uneven ends 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. Allow the smear to dry in air. 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:-

Place the smear on a staining rack with the smear side up. Pour 1% carbol fuchsin to cover the entire smear for 5 minutes heat until the vapours

just arise.



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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 :-



Examination

Result Grading

No. of fields to be examined

No AFB per 100 OIF Negative ---- 100

1-9 AFB per 100 OIF Positive

Scanty

[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



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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



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