Sample Collection

1. Patient Preparation

Patient Preparation

Many tests require specific patient preparation (i.e., fasting, diets, urine voiding). If you have questions about patient preparation for any test, please consult this Directory or call Client Services for further assistance.

Fasting Requirements

A fasting specimen is preferred for the majority of tests performed on serum, plasma or whole blood. Non-fasting specimens often contain fat particles that can interfere with many analytical procedures. Refer Common Causes of Unacceptable Blood Specimens and Inaccurate Test Results (Turbidity) in the Blood, Urine and Stool section. Fasting is defined as no consumption of food or beverage, other than water, for at least 10 to 12 hours before testing.

Provocation Tests

Some tests require the patient to ingest a substance. The most common are the Glucose Tolerance Tests where the patient drinks a solution containing glucose, and blood samples are obtained before and at various times after the drink, to measure the concentration of glucose in plasma or serum. In the standard Glucose Tolerance Tests, adults ingest 75 g (10 ounces) of a glucose solution. Children ingest an amount of glucose proportional to their body weight. (1.75 grams of glucose per kilogram of body weight, up to 75 g of glucose)

2. Proper Identification of Specimens

Specimen Labels

Each specimen submitted must have a label. This label must include at a minimum of two identical items: the patient’s name, written exactly as it appears on the test requisition (e.g., Doe, John), TRF Number (provided by the Lab in the Test Requisition Form), Patient Date of Birth, etc. Be sure that the label is securely attached. If the label is hand-written, use a ballpoint pen. Do not use a felt tip pen. When submitting a specimen in a container other than the tube used to draw the sample (e.g., transfer vials), also indicate the specimen type on the label (e.g., serum, plasma, urine, etc.). When submitting specimens for microbiological testing (e.g., cultures, bacterial antigen, microscopic examination) the nature of the sample and the specific organism(s) to be detected, if any, should be specified.

3. Test Requisition

Test Requisition

Specimens must be accompanied by a paper requisition, prepared either by hand or printed from an electronic ordering system. The requisition, at a minimum should contain the following information:

• Adequate patient identification information (e.g., name, address, telephone number, medical record number)
• Patient gender
• Patient date of birth, or age
• Name and address of the physician ordering the test
• Test(s) requested
• Date of specimen collection, when appropriate
• Source and type of specimen and time, when appropriate
• Clinical information, when appropriate

Complete the “Patient Information” and “Insurance Information” sections on the requisition. Select the tests to be performed. In the case of specimens submitted for microbiological testing (e.g., cultures, bacterial antigen, microscopic examination) the nature of the sample and the specific organism(s) to be detected, if any, should be specified. When ordering tests in a series (e.g., growth-hormone stimulation, glucose tolerance, etc.):

1. Use one test requisition.
2. Label each specimen with the patient’s name, date and time, or site (if applicable).
3. Write the number of specimens on the test requisition.
4. Submit all specimens within a series together in one specimen bag.

Improperly labelled specimens will be rejected.

4. Specimen Packaging

Specimen Packaging

The following are the minimum specimen packaging guidelines that should be followed when submitting specimens.

1. Ensure that all specimen container caps and lids are properly tightened to prevent leakage.
2. Properly complete the Test requisition form.
3. Collect the specimen(s) in a proper transport container.
4. Fold the top copy (original) of the test requisition in half widthwise (top to bottom) with the patient’s name and bar code facing out. Retain the second copy for your files.
5. The specimen bag has two pouches. Place the specimen container(s) in the rear pouch (printed side) and the test requisition in the front pouch.
6. Use single pouch for each patient specimen. Do not mix multiple patient specimen in a single pouch.

Holding and Securing Specimens

While awaiting pick-up by a Thumbay Labs messenger, maintain specimens at room temperature or on cold packs unless otherwise noted under the “Transport Temperature” or other specimen requirement in this section or in the Test Listing section. Thumbay Labs has provided lock boxes to the Thumbay Labs messengers for specimen pick-up. However, customers are responsible for the security of specimens prior to pick up.

Frozen Specimens

Frozen specimens must be transported in insulated containers surrounded by an ample amount of frozen ice packs to keep the specimen frozen until it reaches the laboratory. Thawed specimens are unsuitable for analysis. In the event a thawed specimen is received, you will be asked to resubmit an adequate specimen. If you would like more information about sending specimens to Thumbay Labs, please contact our Client Service Representative.

Transporting Specimens to Thumbay Labs

Needles, Sharp Objects or Medical Waste
Do not send any needles or other sharp or breakable objects. Do not send medical waste as a diagnostic specimen since it may violate the law and create a health hazard. Properly discard used needles or other sharp objects prior to transport. Please note for tests requiring the submission of syringes, the needle must be removed and the syringe capped before sending to the laboratory. Ensure that there is no leakage from or visible contamination outside the specimen container.

Transportation

The specimens picked up from the clients end shall be transported in an appropriate temperature condition in sample transportation bags with biohazard symbol. All specimens shall be treated as infections substances.

Thumbay Labs Bike messengers have been provided with temperature controlled transport boxes for specimen collection from the Clients.

Specimen Tracking

Thumbay Labs follow strict compliance on specimen chain of custody from the client end to specimen reaching to laboratory. When the specimens are picked up from the client end the specimen details and time of pick up shall be noted in the Chain of Custody form. Upon receipt of samples in the laboratory the sample transportation time received time and condition shall be reviewed along with number of sample picked up from client end and delivered to laboratory. Thumbay Labs comply to zero lost sample policy during transportation.

5. Specimen Collection Tubes

Blood Collection Tubes The use of vacutainers with appropriate collection devices is bly recommended so that blood volume matches with the amount of additive. Please ensure that selected collection tube is within expiry period stated on the tube.

There are universal color codes for various commercially available blood containers to indicate presence and type of anticoagulant/additive.

Specimen Labelling

Label each specimen/sample with minimum of two identifications (name/initials, TRF ID, Hospital ID, Date of Collection and Date of Birth & Gender of the patient). It is important to insure that the patient information on the label and the test request form (TRF) match with a minimum of two identifications. Samples which have a mismatch between the label and the accompanying TRF are liable to be rejected.

6. Order of draw

7. Specimen Separation And Storage Guidelines

It is important to note that when Plasma/Serum are the sample required for the test, whole blood samples should not be sent.

Whole blood: The most common test using anticoagulated whole blood is the Complete Blood Count (CBC) and blood film morphology, which should be collected using a lavender-top (EDTA) plastic vacuum tube. Other tests might require anticoagulants such as green-top (heparin) or light blue-top (sodium citrate) tube. Please follow instructions for the individual test. Collect an adequate volume of blood. Fill the tube to capacity (fill mark on the tube), since the partial filling will result in distortions caused by the osmolality of the anticoagulant. Under filled or overfilled blood collection tubes will not be accepted for testing. Immediately mix the blood thoroughly with the additives by gently inverting eight (8) times, or four (4) times when using light blue-top (sodium citrate) tubes. Incomplete mixing or delay in mixing after phlebotomy will result in microscopic partial clotting of the sample, which can cause spuriously low platelet counts. Maintain the specimen at room temperature or on cold packs before submitting to our laboratory, unless instructed otherwise by the specimen requirement information in this Directory or by the laboratory. Never freeze whole blood unless it is specifically instructed in the specimen requirement instructions. If you store cold packs in the freezer, be sure to allow sufficient time for them to warm to refrigerator temperature before placing whole blood specimens near them. To minimize the risk of hemolysis, do not place whole blood specimens in direct contact with cold packs. CBC samples should reach to the laboratory within 24 hours of collection.

Heparin-whole blood: Common use of sodium heparin tube is for chromosomal analysis of blood and/or bone marrow. This is usually for the investigation of hematologic disorders/malignancies. In case of Blood Specimen, 5-10 mL total volume is preferred but volume < 3 mL calls for rejection. In case of bone marrow minimum 2 mL is recommended. Do not use lithium heparin for any serum metal testing.

Plasma: Blood collection is to be carried out using standard Vaccutainer tubes & collection devices selected for suitable anticoagulant for the tests of interest, provided by Thumbay Labs. This avoids under filling or overfilling of tubes and in turn, ensures adequate anticoagulation. Overfilling can result into inadequate anticoagulation and can result into clot or micro a clot formation. Under filling will lead to an excess of anticoagulant and can affect quality of plasma. Mix the collected whole blood specimen in tubes containing anticoagulant by gently inverting for few times as recommended in Table-2.

Centrifuge the sample at RT, 3500 RPM or 1250 RCF for 15 min as early as possible after blood collection. It is recommended that plasma should be separated from cells within 60 min of collection. Platelet poor plasma (plasma platelet < 10,000/µL) is a mandatory requirement for all coagulation tests and would require double centrifugation i.e. recentrifugation of sample/obtained plasma for the same duration and at the same speed. Though samples are collected in Sodium Fluoride & Oxalate containing tube for Plasma Glucose, glucose levels decrease by 7-9 mg during first hour. Though many routine chemistry tests can be performed from heparinized plasma, serum is recommended. Values obtained using such plasma could be higher by 3% as compared to Serum.

Platelet Poor Plasma: Collect blood in a light blue-top (sodium citrate) tube and mix the tube by inverting the tube gently four (4) times. Centrifuge at 2,500–3,500 RPM for 15 minutes. Using a plastic pipette, remove plasma taking care to avoid the buffy coat layer containing leukocytes and platelets. Transfer to a plastic tube. Centrifuge a second time at 2,500–3,500 RPM for 15 minutes. Using a new plastic pipette, remove the plasma taking care to avoid the layer at the bottom of the tube. Transfer plasma to a properly labeled, clean plastic screw-cap vial and attach the label from the lower portion of the test requisition, if applicable. Cap firmly to prevent leakage. Write “PP PLASMA” on the plastic screw-cap vial label and on the test requisition to ensure that Serum or Plasma is separated as described below, within the required time limits.

Serum: When blood is collected in Plain Tube (Red Top) without clot activator, allow it to stand at Room Temperature (RT), keeping it vertical. It takes about 30-60 min for blood to clot completely. When blood collected in plain tube with Clot Activator (Golden cap), it takes about 15-30 min to clot completely. After ensuring that there is complete clotting, please centrifuge the sample at RT, 3500 RPM or 1250 RCF for 10-15 min; immediately after centrifugation remove separated serum using clean transfer pipette into transport vial. Separate serum from cells no later than 60 min after collection. Samples collected in SST tube require to be centrifuged. Following centrifugation it can be transported as it is. It is recommended to preserve primary tube (in which the sample was collected) till reports are released. The vial into which the serum is transferred, also called secondary tube, must be labeled identically as was the primary tube and the information must match that transcribed on Test Request Form.

8. Common Causes of Unacceptable Blood Specimens and Inaccurate Test Results

Hemolysis: Hemolysis occurs when the membrane surrounding red blood cells is disrupted and hemoglobin and other intracellular components escape into the serum or plasma. Hemolyzed serum or plasma varies in color from faint pink to bright red, rather than the normal straw color. Grossly or moderately hemolyzed specimens may necessitate a new sample for some tests. Even slight hemolysis that might not be obvious on visual examination of the serum, or plasma, may significantly alter certain test results (e.g., serum potassium, serum LD), refer to the General Test Listing section for the particular test to determine the effect of hemolysis.

Hyperbilirubinemia: Icteric serum or plasma varies in color from dark to bright yellow, rather than the normal straw color. Icterus may affect certain test results and might necessitate a new sample to assure results of diagnostic value.

Turbidity (Lipemia): We recommend that patients fast for at least 8 hours before a blood specimen is obtained. Eating prior to blood collection produces a transient presence of fatty substances (lipids) in the blood resulting in turbid, cloudy or milky serum. Moderately or grossly lipemic specimens may alter certain test results (See Fasting Requirements in the Patient Preparation and Specimen Transport section).

9. Specimens for Urine Examination

Spot/Random Urine The normal composition of urine varies considerably during a 24-hour period. Submit a first morning voided specimen whenever possible because it has a more uniform volume and concentration; its lower pH helps preserve formed elements. To reduce contamination, the specimen submitted should be a “mid-stream” sample. Urine for pregnancy testing should be first morning void, or a random specimen with a specific gravity of at least 1.010. Note the time of collection of the specimen on the test requisition and on the container label. For some urine tests, there are dietary restrictions. For others, some drugs must be avoided prior to obtaining the specimen. This information is included as part of the specimen requirements for the individual tests in the Test Listing section in this Directory. Since the concentration of urine varies widely, a convenient way of normalizing test results is to divide the result by the concentration of creatinine in the same aliquot. The amount of creatinine excreted daily in the urine is fairly constant and thus, the amount of creatinine in a spot sample is a good estimate of the fraction of the total daily urine volume that the spot sample represents. For specific urine tests that are reported as a creatinine ratio, see Test Listing in this Directory.

24-Hour Urine: Proper collection, preservation and accurate measurement of the volumes of 24-hour urine specimens are essential for accurate test results. Patients should be carefully instructed in the correct collection procedure. Printed instructions for the patient and appropriate containers are available from Thumbay Labs Client Services.
1. Unless the physician indicates otherwise, instruct the patient to maintain the usual amount of liquid intake, but to avoid alcoholic beverages.
2. During the collection period, place the 24-hour urine container (with appropriate preservatives, if applicable) provided by Thumbay Labs in a refrigerator or cool place to prevent growth of microorganisms and possible decomposition of urine constituents. See specimen requirements for the individual tests in Test Listing in this Directory for information on required preservatives, if any.
3. Have the patient empty his/her bladder in the morning into the toilet (not to be included in the 24-hour collection). Write the date and time of voiding on the container label.

4. Collect the patient’s next voiding and add as soon as possible to the 24-hour container. For trace element analysis (e.g., heavy metals), the patient should urinate directly into the 24-hour container to avoid contamination of the sample.
5. For analyses requiring the addition of 6N HCl, add the acid at the start of collection. Has the patient collects each void in a smaller container and carefully pour the urine into the 24-hour container to avoid splashing and possible acid burns.
6. Add all subsequent voiding’s to the container as in (4). The last sample collected should be the first specimen voided the following morning at the same time as the previous morning’s first voiding, which was discarded.
7. Mix the contents of the container gently but thoroughly. Examine to help ensure that the contents appear homogeneous.
8. Measure and note the total volume of urine.
9. Transfer the required aliquot to the plastic screw-cap specimen container provided by Thumbay Labs.
10. Record the total 24-hour urine volume on the specimen container and on the Test Requisition before sending to the laboratory.
11. If necessary, refrigerate the aliquot until it can be sent to the laboratory. For frozen specimens, freeze before packing in dry ice for transport. (See Frozen Serum or Plasma Specimens in this section.)
12. Ensure the lid is properly tightened to prevent leakage.

Routine Urine Culture
Patient Preparation
1. Prevention of contamination by normal vaginal, perineal and anterior urethral flora is the most important consideration for collection of a clinically relevant urine specimen.
2. Obtain early morning specimens whenever possible. There are increased bacteria in the bladder after overnight incubation. Symptomatic patients may have lower colony counts if specimens are collected late in the day.
3. The microbial load in urine may be influenced by fluid intake. Symptomatic patients may have lower colony counts if diuresis is occurring.
4. Tests for urinalysis and culture require two different preservatives; therefore, they require two separate specimens.

Patient Guidelines For Clean Catch Urine Collection
Female
Wash hands. Wash the vulvar area well from the front to the back, using a 5% green soap gauze pad or towel. Do not use benzalkonium chloride disinfectant as a residual disinfectant may cause a false negative test result. Rinse the area from front to the back, using first one moistened gauze pad or paper towel soaked with either clean, tap, sterile or distilled water, then a second and a third if necessary, to remove residual soap. Last, dry the area from the front to the back with a dry gauze pad or towel. Discard all gauze pads or towel in a wastebasket. With one hand, separate the labia and lean slightly forward so that the urine flows directly down into the toilet without running along the skin. After voiding the first portion of the urine, with the other hand, place a clean container under the stream of urine and collect the urine sample. A clean, dry plastic paper or waxed-paper cup is recommended and will not contaminate the specimen. Do not touch the inside of the container.
Male
Use a cleansing wipe, a soapy gauze pad or towel, to wash the end of the penis. Rinse, using first one gauze pad or paper towel moistened with sterile, tap or distilled water. A second or third rinse may be needed to remove soap. Use a clean gauze pad or paper towel to dry. Discard all pads and towel into the wastebasket. Begin to urinate into the toilet. After voiding the first part, place a clean container under the stream of urine and collect the rest of the urine except the very last part into the container (a clean, dry plastic paper or waxed paper cup is recommended and will not contaminate the specimen). After collection, transfer immediately into the urine transport tube, which contains a preservative.
Note:
• Collect mid-stream urine in a sterile container.
• In case of delay in transport to the laboratory, please refrigerate the sample.
• In any case, as per CLSI guidelines the specimen is required to be processed within 2 hours of collection.
• Culture must be performed before the specimen is used for routine analysis.
• Use sterile container with wide mouth and screw cap.
• Please label the container for identification by writing Name/Initials, DOB & gender, Hospital/Laboratory ID, Date of Collection.
• Sample < 4 mL is termed as QI or Quantity Insufficient and will be rejected unless it is a pediatric sample.
• Please ensure, especially for pediatric samples, that there is no fecal contamination.

Collection of Urine from Catheterized patients:
• Do not collect specimen from urine bag of catheterized patients.
• In case needle & syringe are used, please, collect/insert at the angle of 45º to the catheter.
• Do Not Use Timed Urine Specimens e.g. 24 hour specimen for Urine Routine Examination.
• Do not add any additive for preservation. Please refrigerate in case there is delay in transport.
• Refrigerated/Cool (100–150 C) transport is recommended.
• When refrigerated, it is stable for 72 hrs. However it is recommended that specimen should reach the laboratory as early as possible (within 2 hours)
• For pediatric samples (esp. infants), urine pouch is commercially available which fits onto the genital region and facilitates collection.

10. Sample Collection for Fecal Specimens

About 5 gm. of random fecal specimen from area of concern (blood, mucus etc.) is recommended when requested for leukocytes and ova-parasites (Rectal swabs also required for Enterobius vermicularis).

For Fecal Occult Blood, collect the specimen into the container for fecal specimen within 2 hrs of the void. The patient is advised to restrict consumption of meat, spinach, dates, iron supplements or other pharmaceutical agents like NSAIDs (non-steroidal anti-inflammatory drugs), vitamin C etc. for 3 days prior to the test.

11. Body Fluids for Routine Examination

Cerebrospinal fluid (CSF):
• CSF must be collected in a sterile container without preservative. About 2-4 mL volume is adequate for all routine examinations.
• Ideally three tubes must be collected and labeled in the order of collection
• CSF must be analyzed within 1 hr. of collection for routine analysis. Cell morphology gets altered if examine later than 2hrs of collection.
• CSF for Bacteriology Culture must be Stored at 20ºC- 35ºC and must not be refrigerated. Please refer to “Test Section” for test specific specimen handling information.
• Short term-Refrigerated (40-80 C) or long-term frozen (-200 C) specimens can only be used for analysis of proteins as far as routine testing is concerned.

Pleural, peritoneal, pericardial & synovial fluid – routine examination:
• Do not send specimens in Syringes, IV-infusion bottles, Transfusion bags etc.
• Do not send large volumes. Please mix well and take out an aliquot of about 10-30 mL to send to the laboratory. Please segregate well-mixed sample immediately post-collection into required tubes as directed in subsequent instructions.
• For WBC Counts please send 3-5 mL of fluid in EDTA tube. WBC Counts are stable for 24 hrs when refrigerated8 (CLSI).
• For routine chemistry (Glucose, LDH & Total Protein), please send about 5 mL of fluid in Gold top (SST) or Light Green top (Li-heparin) tube.
• For synovial fluid- ANA, Crystals, RF etc. 5 mL sample should be sent into Red top (plain) tube is recommended.
Complete a test requisition and send with patient’s specimen(s). Each container and specimen must be separately identified on the test requisition. The test requisition should reflect pertinent clinical information including patient’s date of birth, gender, clinical/pre-op diagnosis (duration, size, impression), pertinent clinical history/operative findings and/or previous surgery, specific anatomic location of tissue removed and the procedure (excision, cone, punch, etc.).

12. Sample Collection for Bacterial Cultures

General considerations for bacteriology:
• Isolation of pathogens from various sources is totally dependent upon the Quality of Specimens, which in turn depends upon selection of the specimen, aseptic precautions while collection, fulfilling of transport requirements and labeling.
• Do not submit needle-syringes or collection bags, etc. However, if it is not possible to transfer the specimen, please remove needle and seal the syringe with appropriate aseptic precautions.
• Criteria for rejection of microbiology specimens
1. Improper transport container, temperature and duration of transport.
2. Absence of mention/information on source of specimen
3. Leakage of the specimen.
4. Contamination of the specimen.
5. Expired transport-media or container.

Blood Cultures
• Label all culture bottles with the patient’s name, TRF No, date and collection time and, if a specific agent is suspected, with “Rule out ”.
• All samples to be collected prior start of antibiotics.
• Please refer to Table below

Sr. No Type of Infection No. of Sets of Blood Cultures Comments
1. Suspected acute sepsis, meningitis, osteomyelitis, arthritis, or acute, untreated bacterial pneumonia 2 ----
2. Fever of unknown origin 2 initial
2 after 24-36 hrs.
The yield beyond 3 sets of blood cultures is often negligible except in patients with suspected typhoid fever, brucellosis, and infective endocarditis.
3. Suspected early typhoid fever or brucellosis 4 over 24-36 hrs. The same venipuncture site may be used.
4. Infective Endocarditis    
a Acute 3 sets, 15 min apart during first 1-2 hrs. of evaluation.  
b Subacute 3 sets as above, if all 3 negative, 2 extra sets  
c Culture Negative After 5 negative culture reports, review the process Consult Laboratory Head.

• An aerobic bottle and an anaerobic bottle filled from a single venipuncture or collection site should be interpreted as a single culture set. For example, if a physician orders two separate sets of blood cultures, the phlebotomist would use two separate venipuncture sites, drawing one culture set from the right arm and one culture set from the left arm; in most cases, both sets may be obtained at the same time.
• Preferably, blood for culture should not be drawn through an indwelling or intra-arterial catheter.
• Draw the sample for each set of blood cultures (10 mL per bottle is optimal from adult patients and 3 to 5 mL per bottle from pediatric patients) with a needle and syringe.
• Phlebotomy for Blood Cultures 1.After palpation, scrub the venipuncture site with 70% alcohol for a minimum of 30 seconds. 2.Apply iodine/ iodophor (1–2% tincture of iodine or 10% providoneiodine) for 60 seconds in concentric circles away from the venipuncture site covering an area 1.2–2 inches in diameter. After the puncture site has been decontaminated, do not touch. 3.Decontaminate the diaphragm bottle tops by swabbing with 70% alcohol. Allow it to dry. Do not use iodine on the diaphragm tops.
• Using a syringe and needle or a “butterfly” double needle collection system, perform venipuncture and obtain 10 mL of blood (if an adult patient), 3 to 5 mL of blood (if a pediatric patient weighing 13.5–13.5 kg) and inoculate bottles (BD BACTEC® Aerobic and Anaerobic blood bottle(s)) 4.Following venipuncture, remove iodophor that can irritate the skin of patients with 70% alcohol and allow to evaporate. 5.Do not overfill bottles. Greater than 12 mL in the adult bottles and greater than 5 mL in the pediatric bottles constitute overfills.
• Keep the blood culture bottles at room temperature all the time, away from direct sunlight and/or ventilation sources, even if pick-up is delayed. Do not refrigerate or incubate the bottles.

Genital Specimens General The anatomic site determines which specimens may be appropriate for aerobic or anaerobic culture. Only genital specimens listed in the “Specimens Acceptable for Anaerobic Culture” section are appropriate for anaerobic culture. Collect genital specimens using sterile culture swab containing gel. After collection, plunge the swab into transport medium / saline in the carrying tube to prevent drying where applicable. The swab may be used to culture urethral exudate or inflammation of the vaginal area. Specimens should be stored at room temperature until and during transport to the laboratory. Do not refrigerate.

Nasopharyngeal Specimens Nasopharyngeal secretions obtained by aspiration, washings or flexible, twisted wire mini-tip culture swabs are the preferred specimens in order to avoid contamination with nasal or oral flora. To obtain specimen in culture swabs with a thin flexible wire shaft, immobilize patient’s head and insert swab through a nostril. Push forward using gentle downward pressure to keep the swab on the floor of the nasal cavity until the tip reaches the posterior wall of the nasopharynx. Rotate gently for a few seconds and remove. Submit swab in transport medium. However, sterile nasal swabs without gel may also be used for sample collection.

Throat Cultures Use a sterile aerobic culture swab to sample the back of the throat (posterior pharynx), tonsillar crypts, and between the tonsillar pillars and uvula. Avoid touching the lips, cheeks, tongue and uvula. Throat specimens should not be collected if the patient may have epiglottitis. Specimens should be stored at room temperature until and during transport to the laboratory. Do not refrigerate. For fastidious organisms it is ideal to collect the sample in an appropriate sample transport media (Amie’s/ Stuart’s transport media)

Sputum And Lower Respiratory Tract Specimens The preferred sputum specimen is an early morning expectorated sample obtained after a deep cough. Do not pool multiple samples. The patient should rinse mouth with water before sputum is collected. Instruct the patient to avoid adding saliva or nasopharyngeal discharges to the sputum sample to avoid contamination by indigenous microorganisms. Collect the expectorated sputum in the leak-proof container provided by the laboratory (sterile 50 mL conical centrifuge tube or a sterile screw-cap cup). Twist the cap securely closed after placing the specimen in the container.

13. Stool Specimens for Culture

• The organism(s) to be cultured should be specified on the test requisition.
• Collect the stool specimen in a clean, dry wide mouthed container (do not collect from toilet).
• In case of any delay in transporting it is ideal to collect and send the sample to the laboratory in a transport vial containing Cary-Blair transport medium.
• Using the collection spoon built into the lid, obtain scoops of stool from areas that appear bloody, slimy or watery and place them into the vial until the volume rises to the red line. If the stool is formed (hard), sample small amounts from each end and the middle.
• Mix the contents of the vial with the spoon, twist the cap tightly closed, and shake until the contents are well mixed.

14. Sample Collection for Fungal Cultures

Hair, Skin and Nail
• Specimens for the diagnosis of Tinea should include both representative abnormal hairs removed with forceps and scales collected by scraping. Wipe the affected area with an alcohol swab.
• Obtain skin specimens for both dermatophytosis and primary cutaneous candidiasis by scraping the active (advancing) borders of the lesion(s) with a scalpel or glass microscope slide.
• For nails, wipe the area with an alcohol swab before collecting the specimen. Take nail specimens from the proximal portion of the nail plate and subungual debris from between the nail plate and bed. Do not submit initial nail clippings (tips of nails). Nail clippings of at least 3 mm in length should be obtained. This will increase the yield of the culture.
• Do not submit specimens if the patient is currently undergoing antifungal therapy because this may result in a negative culture. If active antifungal treatment has been initiated, discontinue the treatment for 5–30 days (based on topical vs. systemic treatment) before taking the specimen.
• If the first culture is negative, a repeat culture is recommended if clinically indicated. If the culture continues to be negative, a biopsy may be indicated.
• All hair, skin, and nail specimens should be transported in sterile sample containers or petri dish or on a sterile glass microscopic slide. Store and transport at room temperature. Do not refrigerate.

15. Sample Collection for Parasitology

Blood Parasites: Malaria, Babesia Species and Trypanosoma
• At least one thick and one thin blood film smear must be submitted on two separate glass slides with one end frosted in addition to a lavender-top (EDTA) tube, as described below.
• Blood for the films should be taken from a peripheral source such as the finger during a febrile episode, at 6-hour intervals. If this cannot be done, at a minimum, submit one lavender-top tube.

16. Sample Collection for Gynecologic Cytology (PAP Smears And HPV)

• Schedule an appointment approximately two weeks (10–18 days) after the first day of last menstrual period. Menses may interfere with Pap test-interpretation.
• No use of douche for 48 hours prior to the test.
• No use of tampons, birth control foams, jellies/ lubricants or other vaginal creams or vaginal medications for 48 hours prior to the test.
• Refrain from intercourse 48 hours prior to the test.
• The clinician should not use any lubricant jelly for the examination until after the Pap has been obtained
• Testing for Human Papilloma Virus (HPV) DNA is an important adjunct to the diagnosis of pre-malignant or malignant lesions, and it can be performed on the same liquid medium used to submit cytology samples.
• Testing for N. gonorrhea and C. trachomatis can also be performed on the same liquid medium used to submit cytology samples.

17. Sample Collection For Tissue Pathology (Histopathology)

• Obtain biopsy (-ies) with care not to crush the specimen with forceps, hemostats, or other instruments. Cautery will cause heat artifact.
• After biopsy, immediately place each specimen in a tightly secured container with 10% neutral buffered formalin. Do not force a large specimen into a small container; formalin must surround the specimen for proper fixation. Formalin volume to specimen ratio should be 10:1.
• Use a separate container for each separately identified specimen.
• Label each container wall (not the lid) with patient’s last name and first initial or unique identifier and source of specimen. Place one of the peel-off labels from the test requisition onto each specimen container, if available.
• Do not freeze formalin-fixed specimens.
• Special stains and immunohistochemistry studies are done at the discretion of the pathologist. Additional charges will be incurred.
• Complete a test requisition and send with patient’s specimen(s). Each container and specimen must be separately identified on the test requisition. The test requisition should reflect pertinent clinical information including patient’s date of birth, gender, clinical/pre-op diagnosis (duration, size, impression), pertinent clinical history/operative findings and/or previous surgery, specific anatomic location of tissue removed and the procedure (excision, cone, punch, etc.).

Specimen Rejection Criteria (Histopathology)
When preparing to submit a specimen, please avoid the following conditions that will cause the specimen to be rejected or delayed in processing:
• Improper or no labeling
• Prolonged transport time beyond stability limits
• Improper container
• Leaking container: sputum, blood, body fluid, etc.
• Oropharyngeal contamination
• Multiple test requisitions submitted with the same source, day and test requested, except for blood cultures, stools for parasite exams and specimens for occult blood analysis
• Transportation temperature inappropriate for request
• Colostomy discharge, Foley catheter tip or emesis
• Use of aerobic swabs for anaerobic culture
• Swab transport systems and preservatives used beyond expiration date Quantity not sufficient for testing (QNS)

18. Sample Collection for Newborn Screening Using Blood Spot Card

Timing of screening: The first dried blood spot specimen should be collected between 24 to 72 hours of birth. Specimens should not be collected prior to 24 hours of birth except in some special circumstances (vide infra).

Special circumstances include:
• Early discharge: If the infant is to be discharged before 24 hours of age, collect the specimen prior to discharge. Inform parents that the infant must be rescreened during the second day of life.
• Transfers: If the infant requires transfer to another facility, a specimen must be collected prior to transfer, regardless of infant’s age. If a specimen cannot be collected prior to transfer due to the medical instability of the neonate, the transferring facility is responsible for informing the admitting facility of the need for specimen collection prior to any transfusion.
• Transfusion: If the infant is to receive a transfusion, every effort must be made to collect a specimen prior to transfusion, since even small transfusions may invalidate screening results. Infants receiving transfusions with no prior screening test need two collections: three days or more after the most recent transfusion and three months after the final transfusion.
• Total Parenteral Nutrition (TPN): If the infant is to receive TPN, every effort must be made to collect a specimen prior to treatment, since even small amounts of TPN may invalidate screening results.

Infants receiving TPN with no prior screening test need two collections: three days or more after the most recent TPN and three months after the final TPN.

Blood Collection: Gloves should be worn for personal safety. Care should be taken to avoid contamination of blood collection circles with antiseptic solutions, powders, lotions or other materials which may adversely affect the testing process. Before collection of blood sample on to the circle of paper, all information on specimen card is filled out completely and accurately.

To prevent specimen contamination, do not touch any of the filter paper circles before or after collection.

1.Blood sample should be collected on to the circles of Whatman 903 specimen collection card.
2.Place infant's leg in a down position to increase venous pressure (heart above the feet position).
3.Warm the heel to increase blood supply to the area by covering the puncture site for three to five minutes with a warm, moist towel.
4.Clean the heel with 70% isopropyl alcohol pad. Allow the site to air dry.
5.Puncture the heel at the edge of the plantar surface using an automated lancet with ≤2 mm tip.
6.Wipe away the first drop of blood with dry sterile gauze to remove any contamination and discard.
7.Apply surface of first filter paper circle to the next large drop of blood from puncture. Allow blood to fill and completely saturate front and back of the circle never use both the front and back of the paper to fill the circle.
8.Fill all the required circles with blood.
9.Allow the blood specimens to air-dry for at least 4 hours on a flat, nonabsorbent surface away from heat and direct sunlight.
10. Place dried specimen collection form/card in special envelope (in sealable plastic bags) and send within 24 hours to the testing laboratory.
11.The specimen collection form/card will be rejected for testing in case of incomplete information or unsatisfactory specimen collection.

NOTE: The step wise instructions for specimen collection are given on the back of the specimen cards. All these instructions are to be followed.

19. Newborn Screening Dried Blood Spot (DBS) Check

After the card has dried, check your collection against these examples. Any cards that look like the ‘not acceptable’ examples should be recollected.

A Valid DBS Specimen:

It is good sample collection and all circles filled, blood soaked in front and back of card, one application with no over-layering of blood, sample dried in the air for 4 hours.
Acceptable sample: It is not essential to fill all circles if collection is difficult or causes unnecessary trauma to the baby or parents. Three good spots are usually sufficient for screening.
A small misalignment with the circles or slight over or under filling are also acceptable for screening.

Invalid DBS Specimens:

Insufficient blood collection and blood not soaked through card properly.
Insufficient blood collection and not enough circles for screening.
Contaminated sample spots: another liquid has been in contact with the card e.g. TPN, water, urine, tea, coffee, etc
Sample not air dried properly for 4 hours and card placed in plastic bag after immediate collection.
Serum rings: due to not wiping of alcohol from heel before puncture, card contaminated with alcohol, hand lotion or water and excessive squeezing to collect sample.
Clotted or layered spots due to filling card from both sides and touching paper to blood several times.
Filter card submitted without blood.

20. General Guidelines for Laboratory Blood Specimen Collection for Neoscreen

• Do not squeeze heel of the baby to obtain blood.
• Do not use devices that contain EDTA (ethylene-di-amine-tetra-acetic acid).
• Do not apply specimen to both sides of specimen card.
• Do not expose card to heat, moisture, or direct sunlight.
• Make sure all information on specimen card is filled out completely and accurately.
• Allow the blood specimens to air-dry for at least 4 hours on a flat, nonabsorbent surface.
• Do not refrigerate specimens after collection till completely dried.
• Do not expose card to heat, moisture, or direct sunlight
• Do not put more than one specimen in the sealable plastic bag.
• Send the specimen within 24 hours of collection to the testing laboratory in cool conditions.
• Avoid leaving the specimen in a vehicle, exposed to heat and sun.