Reason for blood specimen rejection

 Hemolysis / lipemia.
 Quantity not sufficient.
 Clots present in an anticoagulated specimen.
 Non fasting specimen when test require fasting.
 Incorrect anticoagulant.
 Improper blood collection tube.
 Short draws and wrong volume.
 Discrepancies between requisition and specimen labeling.
 Unlabeled or mislabeled specimen.
 Contaminated specimen /leaking container.

Unacceptable urine collection methods

 Sample collected into a container have detergent residue or bleach or one
that has not been adequately cleaned.
 Urine collected in bed pan that also contains faces .
 Urine squeezed out of a diaper.

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 VEIN PUNCTURE PROCEDURE
1. properly identify the patient.

2 .Assemble all necessary equipment.

Selection of an appropriate site

Visually inspect inspect both arms choose a site that has not been repeatedly
used for phlebotomy .

3 . Apply tourniquet. Do not leave tourniquet for more than one minute.

4 . To make the vein more prominent , ask the pt to make a fist with index
finger

Palpate for an appropriate vein.

The ideal site is near or slightly below the bend in the arm.

PREPARATION OF THE VEIN PUNCTURE

5 . After an appropriate site has been chosen , release the tourniquet.

6 .Using an alcohol pad saturated with 70% alcohol , clean the skin in the area

vein puncture site using a circular motion ,

clean the area from center and move out ward

Do not go back over any

area of the skin once it has been cleaned.

7. Allow site to air dry.

PERFOMING THE PUNCTURE

8 . Use one hand to hold the syringe. Position the pt arm in the slightly dawn ward position .

use one or more finger of the other hand to secure the skin and vein.

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9 . Position the blood drawing syringe at an angle about 20 degree . the bevel of the needle s
be upward.

10 . Insert the needle through skin and into the vein.

Termination of the procedure

11. The tourniquet can be released immediately before the final amount of blood is drawn.

12 . Ask the pt to open the hand .

13 . Withdraw the syringe with one hand , and immediately press dawn on the gauze pad with
the

other hand after desired amount of blood has been drawn.

14 . Inform the pt elevate the entire arm and press on the guaze pad with the opposite hand .

if the pt is unable to do this , apply pressure until bleeding ceases.

NOTE failure to apply sufficient pressure to vein puncture site could result
in hematoma ( collection of blood under skin ).

15 . Mix tube with anticoagulant by inverting several times .

Do not shake the tubes. Discard used equipment into an appropriate puncture proof
container.

16 . Label all test tubes.

17 . Clean up supplies from the work area and wash hands.

18 . If patient is an out patient , wait a few minutes after the vein puncture is complete , and

check patient doesn’t feel dizzy or nauseated before discharge

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 CAPPILLARY BLOOD COLLECTION
Selection of an appropriate site
1. The finger tip of 3rd or 4th finger , heel and big toe are appropriate site for the
2. collection of capillary blood.
The ear lobe may be used as a site of last resort in adults .
Don’t puncture the skin through the previous site.
The plantar surface ( sole ) of the heel or big toe is an appropriate site in infants or in
special cases such as burn. the ideal site in infants is the medial or lateral plantar surface
of the heel with puncture no deeper than 2 mm beneath the plantar heel skin surface
and no more than half distance at the posterior curve of the heel.
NB back of the heel should never be used b/c the danger of injuring the heel bone ,
cartilage and nerves in this area.
A .For infants younger than 12 months old the lateral or medial plantar heel surface
used.
B. For infants older than 12 months , children and adults palmar surface of 2 nd , 3rd , 4th
Finger may be used.

PREPARATION OF THE SITE

1. Hold the area to be punctured with thumb and index finger of hand.
2. Wipe the area with 70% alcohol pad and allow to air dry.
3. Wipe the area with the dry gauze . if the isn’t dry , the blood will not form a rounded
drop and will be difficult to collect.

Puncture the skin

4. Use the disposable sterile lancet once and discard it properly in the puncture proof
container.
5. Securely hold the area and puncture once ( perpendicular ) with the firm motion.
Wipe away the first drop of the blood , because the first drop is mixed with lymathic
fluid and possible alcohol.
Apply gentle pressure to area to obtain suitable specimen.

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 PRINCIPLE OF GRAM STAIN
Bacteria stain either Gram positive or Gram negative on the basis of difference in their cell wall
composition. Gram positive bacteria have a thick peptidoglycan layer and large amount of
teichic acids whereas Gram negative bacteria have a thin peptidoglycan layer and an outer
membrane composed of a lipid bilayer . the outer membrane of Gram negative organism is
damaged by decolorizer.

Specimen : clinical specimen (direct smear ) wounds , eye lesion , sterile fluids ,

body tissue and discharge.

Gram stain reagents

 Crystal violet _ intial stain.
 Iodine _ mordant ( binding agent ).
 Alcohol _ decolorizer.
 Safarinin or diluted carbol fucsin _ counter stain.
Smear preparation
Prepare a thin smear and make sure it dry before processing.
Fix the slide with methanol ( allow to dry ) or by gently passing through a flame 3 or 4x.

BASIC GRAM STAI PROCEDURE

1. Flood sear with crystal violet and wait for one minute .
2. Rinse clear with tap water and drain excess water.
3. Flood with loglo’s iodine solution for one minute .
4. Rinse with water.
5. Cover with acetone alcohol for 30 sec , add acetone alcohol if blue color remain and
wait for few seconds.
6. Rinse with water.
7. Flood the slide with counter stain for one minute.
8. Rinse and allow air dry.

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 INTERPRETATION AND REPORTING
Enumerate cells per LPF EPis PMNs.

1+ ( rare or occasional ) < 1/LPF.

2+ ( few ) 1_ 9 / LPF.

3+ ( moderate ) 10 _ 15/LPF.

4+ ( many ) > 25/LPF.

Enumerate bacteria , yeast cells & RBCs.

1+ ( rare or occasional ) < 1/OLF.

2+ ( few ) 1_ 5 /OLF.

3+ ( moderate ) 6_ 25/OLF.

4+ (many ) >25/OLF.

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 SPUTUM SPECIMEN COLLECTION
Two specimen optimal for identifying infectious cases of tuberculosis.

WHO recommendation : SPOT_ SSPOT

Allow the same day diagnosis .

1st SPOT at time of initial visit to the health facilities.

2nd SPOT 30 min to one hour after the first spot sample .

Sample collection safety.

 Instruct the patient to cover his / her mouth when coughing.

 Sample should be collected out side the LAB or clinic to minimize risk of aerosol
transmion.
 Collect sputum away from other people in a well ventilated space or designated sputum
collection area.
 Do not stand in front of patient during specimen collection while providing instruction.

Specimen collection container

 30 _ 50 ml capacity.
 Translucent or clear material.
 Sides & walls that allow easy labeling.
 Leak _ proof with screw cup.
 Wide mouth
 Label with patient name , ID and date of collection on the side of the
container.
 Never label the lid.

Patient instruction
 The best specimen comes from the lung.
 Saliva or nasal secretions are unsatisfactory.
 Specimen should not contain food or other particles
Patient should instructed with :
1. Wash your mouth with clean water to remove food or other particles.
2. Inhale deeply 2 _ 3x & breath out strongly each time.
3. Cough deeply from your chest produce sputum.
4. Place the open container close to your mouth to collect the specimen.

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 5. Wash your hand after collecting the sample.

Specimen quality
Obtain an adequate quantity of good quality sputum is critical to ensuring accurate test result.

For best result obtain 1 _ 4 ml of purulent or mucoid sputum.

STAING WITH ZIEHL NEELSEN

Principle of acid fastness : The cell wall of acid fast bacilli contain fatty acids
known as MYCOLIC acid , which makes them resistance to the action of many chemicals.
because of this , the bacilli can’t be stained with easily like in Gram’s stain .strong dye
concentration application of heat , addition of acid alcohol & longer staining time required to
stain the bacilli. once stained stained it is difficult to destain them .this property is used to
differentiate the AFB from all other materials like bacterial cells & mucus will get decolorized by
the action of strong acids ( 3% acid alcohol ) , leaving acid fast bacilli stained with primary stain ,
which is basic fuchin in case of ZN method.

PROCEDURE OF SMEAR PREPARATION

A. Use a pencil to label the forsted end of slide.
B. Open the sputum container carefully & place the lid face up on the work surface.
C. Examine the specimen to select the best portion of the sample & choose yellow
( purulent )or bloody stained particle if present.
D. Use the applicator stick to transfer selected portion of specimen to glass slide.
E. Smear the specimen over 1 x 2 cm or 2 x 3 cm area centered in the middle of slide.
F. Use the applicator stick to crush , break up , & spread out particle.
G. Use small circular motion to distribute the specimen & recup sputum container.
H. Discard applicator stick and recuped sputum container into discard container
I. Allow the smear to air dry completely.
J. Pass the slide 2 _ 3 x over the flame for 2 – 3 sec each.
K. Evaluate the fixed smear for proper thickness.

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 Procedure for staining
1. Cover the entire surface of the slide with carbol fuchsin solution . if the staining solution
drains add more stain to cover the entire slide.
2. Heat the slide with flame of an alcohol soaked cotton swab until stea rises from the
stain , leave for 3 min.
3. Rinse the slide with gentle stream of water.
4. Decolorize the smear by covering whole slide with 3% acidic alcohol & leave it for 3 min.

NB : If carbol fucsin retained in the smear , it is undecolorized , repeat the

decolorization if necessary

5. Wash the slide with gentle stream water. Tilt the slide to drain off excess water.
6. Counter stain the smear by covering the entire surface of the slide with methylene blue
solution & leave for one minute.
7. Rinse with gentle stream water.
8. Wipe under side of the smear & allow to air dry.

AFB REPORTING

Reporting Result AFB seen

Negative No AFB seen in at least 100 fields

Actual number 1_ 9 AFB /100 F

1+ 10 _ 99 AFB/ 100F

2+ 1_ 10 AFB / Field in at least 50 field.

3+ More than 10 AFB per field in at least 20 field.

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