6.

Collect Sample
6.1 Primary sample collection
6.1.1 Introduction:
Laboratory specimen is a biological specimen taken by sampling, that is, gathered matter of a
medical patient's tissue, fluid, or other material derived from the patient used for laboratory
analysis to assist differential diagnosis or staging of a disease process. Quality laboratory reports
dependents upon Proper specimen collection and handling prior to the arrival of the specimen to
the laboratory.
Laboratory tests contribute vital information about a patient‟s health. Correct diagnostic and
therapeutic decisions rely, in part, on the accuracy of test results. Adequate patient preparation,
specimen collection, and specimen handling are essential prerequisites for accurate test results.
The accuracy of test results depends on the integrity of specimens.
6.1.2 Preparation for sample collection.
Prior to each collection, review the appropriate test description, including the specimen type to
be collected, volume, procedure, collection materials, storage and handling instructions.
Verbally reassure patient prior to collection of blood and or any body fluid that requires invasive
procedures. Verify the patient and the requisition match using at least 2 patient identifiers.
Patients may be asked to state their name and Age. (Phlebotomists should say “Please tell me
your name” rather than “Are you Ms. Shekhar?” Some patients may be hard of hearing, in a
phase of dementia, or on medications and not always give accurate or appropriate answers.)
The phlebotomist may need to ask the patient‟s caregiver for identity in some instances.
Have all supplies and equipment needed at phlebotomy department prior to collection. Supplies:
Gloves Vacutainer for various blood test Spirit Swab Tourniquet Dry Cotton Swab
Syringes (3/5/10 mL) Handy Plast
Multisampling needles Labeling Marker Zippo Bags Urine/Stool Container
Wooden Spatula Sterile container Lab Investigation Form
When more than one blood specimen is required, multisampling needles and vacuum tubes make
blood collection simpler and more efficient.
6.1.3 Patient preparation for laboratory tests
In order to guarantee the quality of laboratory test results, the patient should cooperate by
preparing themselves for the sample collection, as well as its transportation. There are several
medical tests available in laboratories. Fortunately for patients, most of these tests do not require
special preparation. For example, for tests such as CBC (Complete Blood Count), renal function
test or liver function tests can be taken at any time. Very few laboratory tests require the patient
to follow a specific set of instructions before collecting the sample. Example of these are fasting,
taking the test at a certain time of the day like cortisol test, following an specific diet for certain
period e.g. OGTT, do not smoke, and/or perform a special cleaning,
6.1.3.1 Fasting blood sugar (FBS)
- For a fasting blood sugar test, do not eat or drink anything other than water for at least 8
hours before the blood sample is taken.
6.1.3.2 Postprandial (post lunch) blood sugar:
- For a 2-hour postprandial test, patient need to collect blood exactly 2 hours after a time of
last regular lunch or breakfast whichever is heavier and/or for diabetic patient after regular meals
of taking diabetic medication. e.g. If Mr. Shah has finished his lunch at 12.00 noon he needs to
complete his blood collection at 2.00 pm. Mr. Shah will require to reach the lab at 1.50 pm, i.e.
10 minutes early to avoid registration delays. Also Mr. Shah cannot have any food between
12.00 noon and 2.00 p.m. time period. He can have water and his usual medicines.
6.1.3.3 Oral Glucose Tolerance Test
- Patient should be fasting for 8 hours (no food or drink, except for water). A fasting blood
specimen will be drawn and tested. Patient will then be given a glass of glucose drink (75 grams
of D glucose in 200 to 300 ml of drinking water and drink within 5-10 minutes) then blood will
be drawn each hour for 2 hours. Advice for patient not to drink and eat in between 2 hours except
water.
6.1.3.4 Lipid Profile
- Blood should be collected after a 12 hour fasting (no food or drink, except water). For the
most accurate results, the patient has to in normal diet from at triglyceride test.
• 24 hours prior no heavy physical activity
• 1 hour prior no smoking
• 15 minute prior seated and relaxed
• For urine test: Mid stream urine sample is preferred.
Note: Patient preparation for other special test (Outsource test) kindly follows the instruction
fromhttp://www.lalpathlabs.com/testmenu.aspx .
6.1.4 Sample collection containers
During venipuncture, do not have the patient clench and unclench the fist repeatedly (pumping).
This will cause a shift in fluid between the vein and the surrounding tissue. This can lead to
changes in concentration of certain analytes e.g. calcium. To facilitate making the vein more
prominent, the patient may be asked to clinch fist tightly. Also, never leave a tourniquet on the
arm for more than 2 minute without releasing it. This can cause discomfort to the patient and
may also cause hemoconcentration leading to erroneous results.
Container Required Composition Tests
Lavender-Top Contains liquid K3 Complete blood cell count (CBC), ESR,
EDTA or K2 EDTA. Platelets counts, Blood grouping, Malaria
parasite, Microfilaria, ACTH test,

Yellow Gel Tube ( Yellow Top) Contains clot activator Biochemistry tests, Serological test,
and gel for separating Immunological tests, Hormonal tests,
serum from cells, but not Enzyme assay. Cancer marker
anticoagulant.

3.2% Trisodium Citrate ( Blue Contains 3.2% sodium Coagulation studies, PT, APTT, FDP, D-
Top) citrate. (Anticoagulant: dimer, Coagulation factor assay,
Blood ratio must be 1:9

Heparin Tube (Green Top) Contains sodium heparin Chromosomal studies, Karyotyping,
or lithium heparin.

Sodium Fluoride Tube ( Grey Contains sodium fluoride.Blood Glucose test.

Top)

Red Top Plane Tube ( Red Top) Contains no anticoagulant Biochemistry tests, Serological test,
or preservative Immunological tests, Hormonal tests,
Enzyme assay.
Clean, Dry, leak proof wide ---------- Routine urine stool test
mouth
plastic container
Sterilized, Clean, Dry, leak ---------- All culture and sensitivity tests
proof
wide mouth plastic container
Clean, Dry, leak proof wide ---------- Sputum AFB test.
mouth
plastic container

1.1.1 Principle of sample labeling:

Proper patient identification and sample labeling is very important for Pathology Lab. Thus,
everything in lab that contains sample (specimen) must be labeled with unique identification
number for that particular patient. So that, it can be traced which sample belongs to which
patient. It is very important that this identification must be unique for each patient (sometimes
even, each test, as in case of Fasting and Postprandial (PP) Blood glucose).
1. When patient come to laboratory for testing, ALWAYS reconfirm the identity of patient
by
a) Asking the name of the patient
b) Verify the name in the PMS – Patient Movement Slip (and/or prescription when available)
2. Identify the tests ordered by scanning barcode reader or double clicking patient‟s name to
open PMS in software. Cross check the tests ordered in prescription if available or by
asking the patient.
3. Register the patient in Laboratory investigation form (SC/ADM/PL/LIF/##). following
these principles:
a) Fill the required information of patient according to the laboratory investigation form. For
example:
NAME: ……………………………. Age/Sex: ………M / F Date :……/……/..........( DD/MM/YY)

Lab No …………………………. Pin No …………Board No: …………

Consultant Doctor's …………………Clinical History:

…………………………………
b) Lab number is already printed in Laboratory investigation form (from 1 to 999) only we
have to add PMS number along with Lab number by giving dash ("-") for example: (1-
2019 where 1 is lab number and 2019 is PMS number)
c) This PMS number is automatically generated by software (is unique for every patient) and
printed in PMS and displayed in PMS in Software. For example first patient will get serial
number 01, and if his PMS number is 2019, then lab number (label) will be “01- 2019”. In
 case of software failures then write the date (instead of PMS number) after the serial
number. For example if 1st patient came on 22nd June 2013, then lab number (label) will be
“01-22”. (Please note: 01 is serial number, 22 is today's date 22nd).
d) After filling the required information of patient circle the requested test on laboratory
investigation form.
e) In case of Stool and urine test strictly circle that the container is given or not to patient for
sample collection (if given circle to yes if not circle to No). And note the time and date of
sample received in laboratory.
f) In case of customer will come again to collect the container (2 nd time or 3rd time) please
mention it in the form as 2nd time or 3rd time. While receiving the container make sure that
right container was received. In case of customer had taken 2nd container, but 1st container.
4. Once this lab number (label) is generated and requested test is noted then take necessary
containers required for sample collection and use that number for labeling each container.
Always label containers before collecting the sample and while patient is present in the lab.
Always use permanent markers to label. Always write in clear handwriting.
5. In case of Fasting blood sugar sample, letter “F” is added at the end and for postprandial
blood sugar sample collection –add letter “PP” at the end. Blood sugar sample without these
letters are considered as Random.
6. Never accept any sample without labeling in the laboratory.
1.1.6 Department wise collection of the specimen
6.1.6.1 Hematology Department:
Most commonly performed tests in LAB:

S.N Test S.N Tests

o o
1 Haemoglobin 6 Total platelets counts
2 Total counts 7 Red Blood Cell Counts
3 Differential count 8 Erythrocyte sedimentation Rate (ESR)
4 PCV/HCT 9 Malaria antigen
5 Red cell indices (MCV, MCH, MCHC) 10 Bleeding Time
Clotting Time

Note: For Routine haematological test No special preparation is required. Random blood
sample is acceptable.
6.2.2 Sample collection (blood):
Introduction:
A blood test is a laboratory analysis performed on a blood sample that is usually extracted
from a vein in the arm using a needle, or via finger prick. Multiple tests for specific blood
components are often grouped together into one test panel called a blood panel or blood work.
Blood tests are often used in health care to determine physiological and biochemical states,
such as disease, Mineral content, pharmaceutical drug effectiveness, anda organ function.
Typical clinical blood panels include a basic metabolic panel or a complete blood count.
Venipuncture is useful as it is minimally invasive way to obt in cells and extracellular fluid
(plasma) from the body for analysis.
6.2.3 Blood Collection Site Selection
The three veins in this area are the first choice for blood collection.
 The median cubital vein is the first choice for blood collection.
This vein is usually large, visible, well anchored and does not bruise easily.
 The cephalic vein is the second choice for blood collection.
This vein is not as well anchored and is usually more difficult to find.
 The basilic vein is the third choice for blood collection. This vein tends to roll
away and bruise more easily.
6.2.2 Diagrammatic representation of blood collection procedure:
6.2.4 Collection Procedure (Venipuncture):
1. Verify the patients name with the PMS by asking the name of the patient and verify the tests
required. Reconfirm the tests by asking the patient or by seeing the doctor‟s prescription.
2. Register the patient in “laboratory investigation form (SC/KTM/PL/LIF/##)”
3. Prepare the required material for sample collection and label the container according to the
principle of sample labeling. Use gloves, mask for personal and laboratory safety.
4. Check the syringe; check to be sure that the syringe works smoothly by pressing the piston.
The piston must be pushed firmly to the bottom of the cylinder to prevent injection of air into
the vein, this can be fatal.
5. Approach the patient in a friendly and calm manner. Provide comfort as much as possible,
and gain the patient's cooperation and confidence.
6. Position the patient - The patient should sit in a chair, lie down or sit up in bed. Hyperextend
the patient's arm; select the venipuncture site.
7. Apply the tourniquet 3-4 inches above the selected puncture site. Do not place too tightly or
leave more than 2 minutes. The patient should make a fist without pumping the hand.
8. Prepare the patient's arm using spirit prep (70% alcohol as disinfection). Cleanse in a
circular fashion, beginning at the site and working outward. Allow to air dry.
9. Grasp the patient's arm firmly using your thumb to draw the skin tight and fix the vein.
The needle should form a 30 to 45 degree angle with the surface of the arm. Swiftly insert the
needle through the skin and into the lumen of the vein. Avoid trauma and excessive probing. If
first prick was not successful, then apologize to the patient and try in the other site of the same
arm or the other arm. If second prick is also not successful, then again apologies to the patient
and call your senior.
10. Always apologies to the patient for any discomfort and pain due to blood collection.
11. When the blood draw is finished, remove the tourniquet.
12. Remove the needle from the patient's arm using a swift backward motion.
13. Press down with the dry cotton swab once the needle is out of the arm, applying adequate
pressure at least for 5 minutes to avoid formation of a hematoma. When bleeding is stopped
apply handy-plast on the puncture site. If patients are on anticoagulation or bleeding disorder it
might take 15 minutes or more to stop bleeding.
14. Dispose of contaminated materials and needles in special disposal containers.

6.2.5 Blood collection for culture and sensitivity:

Vigorously cleanse the skin over the venipuncture site in a circle approximately 5 cm in diameter
with 70% alcohol. Scrubbing should continue for 30 seconds.
1. Starting in the centre of the circle, apply 10% povidine iodine (betadine) in ever widening
circles until the entire circle is saturated with iodine. (Chlorhexidine swabs which are 2%
chlorhexidine Gluconate and 70% Isopropyl Alcohol can be used in place of iodine.)
2. Leave the iodine (or chlorhexidine) on the venipuncture site to act for 60 seconds.
3. Again cleanse the iodine rubbed area over the venipuncture site in a circle approximately 5
cm in diameter with 70% alcohol.
4. Do not touch the venipuncture site after preparation and prior to phlebotomy.
6.2.7 Control bleeding:
1. Patient press cotton for minimum 2 min to stop bleeding
2. do not bend the arm to avoid hematoma
3. Raise the arm above the heart level for approximately 3–5 min to control bleeding, if
necessary.
6.2.8 Capillary puncture:
1. Position the patient. Hyperextend the patient's arm
2. The best locations for finger sticks are the 3rd (middle) and 4th (ring) fingers of the non-
dominant hand. Do not use the tip of the finger or the
center of the finger. Avoid the side of the finger where there is less soft tissue, where vessels and
nerves are located, and where the bone is closer to the surface.
3. Prepare the patient's fingertip using alcohol prep. Cleanse in a circular fashion, beginning
at the site and working outward. Allow to air dry.
4. Using a sterile lancet, make a skin puncture just off the center of the finger pad. The
puncture should be made perpendicular to the ridges of the fingerprint so that the drop of blood
does not run down the ridges.
5. Collect drops of blood into the collection device by gently massaging the finger. Avoid
excessive pressure that may squeeze tissue fluid into the drop of blood.
6. Have the patient hold a small sprit cotton swab over the puncture site for minimum 5
minutes to stop the bleeding.
7. Dispose of contaminated materials/supplies in designated containers.
8. Mix all appropriate tubes
9. Deliver specimens promptly to the laboratory working benches.

6.2.9 Complication during or after blood collection:

6.2.10 Blood hemolysis
Hemolysis is the destruction of the encasing membrane of the red blood cell and subsequent
release of hemoglobin in to the plasma.
6.2.10.1 Causes of hemolysis
Incorrect venipuncture technique
1. Collecting blood from a narrow vein
2. When a vein collapses can also cause some hemolysis.
3. Alcohol don„t dry (it cause lysis to RBCs membrane)
4. Using excess EDTA or moisten EDTA as anticoagulant
5. Slow drawing blood from the vein
6.2.10.2 Effects of hemolysis
1. Hb color interferes with colometric measurement.
2. Glucose decrease due to glycolysis by RBCs enzymes
3. Bilirubin decrease due to color interference
4. Increased potassium, LDH and GOT activity due to leakage from RBCs
5. Increased phosphorus as organic esters in the cell is hydrolyzed
6.2.10.3 Hematoma
A hematoma is just a bruise like any other bruise (known as obvious bruising), and can appear
even with the most skillful of phlebotomists.
 6.2.10.3.1 Causes of hematoma
1. Penetration of the vein
2. Slow flow may cause hematoma
3. Bending the arm after sampling
4. Patients with bleeding disorders and those taking anticoagulants therapy.
6.2.10.3.2 How to avoid hematoma?
1. Use the major superficial veins
2. Do not penetrate the vein from the side
3. Remove tourniquet before removing the needle
4. Do not bend arm after sampling
5. Apply pressure by cotton to the venipuncture site
6.2.10.3.3 Management of hematoma:
It will go away; you can apply ice on and off after any blood-draw and use the affected arm as l i
t tle as possible.
6.3 PARASITOLOGY DEPARTMENT (URINE AND STOOL)
Most commonly performed tests in LAB:
S.No Test S.No Tests
1 Urine Routine and microscopic 8 Urine Chyle test
2 Urine Sugar 9 Urine Pregnancy test
3 Urine Albumin 10 Stool routine and microscopic
examination
4 Urine PH 11 Stool occult blood test
5 Urine Specific Gravity
6 Urine Ketone
7 Urine bile pigment (Bilirubin,
Urobilinogen)

6.3.1 Urine sample collection for routine analysis and/or culture

The first morning sample is preferable for urine routine and culture test but random sample can
be acceptable. For culture obtain a sterile container from the laboratory. Please avoid touching
the inside of the container and/or the lid in order to maintain sterility.
1. Label the container if the patient will give the sample in the same day then ask
the patient to bring the sample back to the lab.
2. If he/she will give it later on then let them take the container labeled with
patient‟s full name and ask to bring the sample in the lab with the PMS along.
3. Explain the patient to collect the clean caught mid-stream urine sample.
4. Culture samples should be processed within one hour of the sample collection for
appropriate results. If there is delay, the specimen should be refrigerated for 2-4
hours.
5. To be sure if the sample is given or not request patient to inform any of the lab
staff regarding the delivery.
6. After receiving the sample in lab sample received time and date should be
recorded in lab investigation form by laboratory staffs.
6.3 Specimen rejection criteria:
1. Hemolysis - If a specimen is hemolyzed, it should be recollected, if appropriate. If it is not
possible or practical to recollect a hemolyzed specimen, the physician must be notified of
the condition of the specimen and it will be noted on the test results that the specimen was
hemolyzed.
2. Clotted whole blood specimens – If a specimen is clotted, i.e. complete blood count (CBC)
or coagulation studies, it will be rejected.
3. Blood collected in expired collection device.

4. Inappropriate collection device/specimen type.

5. Unlabeled specimens.

6. Leaking/broken specimen container.

7. Gross bacterial contamination of specimen.

8. Quantity of specimen not sufficient for testing.

9. Specimen not submitted at the proper temperature and time.

10. Specimens not submitted in a sterile container for culture & sensitivity test.

11. Dried-out swabs.

12. Pooled specimens.

 13. Urine specimen for culture is not submitted immediately if not in preservative tube or
refrigerated.
14. Stool specimen contaminated with barium (white area, chalky, heavy) or oil for the
examination of ova and parasites.
6.3.2 Handling Unacceptable Specimen’s
1. Upon receipt of an unacceptable specimen in the Laboratory, immediately inform the nurse
or physician that the specimen will not be processed and request a freshly, properly
collected specimen be sent to the laboratory. And, explains the reason for the rejection.
1. Counsel the patient:
Counsel the patient about the following:
2. Report preparation time
3. Report dispatch time
4. Where to collect the report
5. Sometimes due to sample problems patient might have to give the sample again
6. Counsel about the tests which are outsourced to outside labs
7. Counsel the patient if again come for sugar PP so, how to prepare for PP?
2. Storage of the Sample:
2.1 Sample storage after performing test
1. Never discard the sample as soon as you complete the test as sometimes test might be added
or repeated. So store the sample at least for 24 hours of the test performed. Biochemistry
sample are stored on the test tube rack and placed on refrigerator for 24 hours.
2. Hematological sample are store on the hematological sample storage container and placed
on refrigerator for 24 hours.
3. Parasitological sample (stool, urine) should not be stored for long time after completing test
as they are very infectious and do not give accurate result if these tests are repeated from the
same sample. If we need to repeat the test of parasitology after a long time of collection then
ask patient politely for the repeat sample to repeat the test.
4. Sample of the culture are also not stored for a long time after performing test as they are
very infectious and don’t give accurate result if they have to be repeated. Culture specimen
should be store for 1 hr. after collection, so culturing process should be done within 1 hr.
after that it should be discarded.

2.2 Biohazard precautions:

When collecting a blood sample, the operator should, wherever possible, wear disposable plastic
or thin rubber gloves especially if he or she has any cuts, abrasions or skin breaks on the hands.
Care must be taken to prevent injuries when handling syringes, needles and lancets. Disposable
syringes, needles and lancets should be used and they should not be reused. They should be
placed (without separating needles from syringes) in puncture-resistant container for disposal or
subsequent decontamination. Specimens should be sent to the laboratory in individual closed
plastic bags, separated from the request forms to prevent the contamination should there be any
leakage from the specimens.