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Sample Collection Method & Handling Precautions

Sample Collection Specimen Handling Precautions

Blood
Urine
Stool
Body fluid
Hair
QuantiFERON-TB Plus
NK cell activation induced interferon-gamma
Oligomerized amyloid beta test, AlzOn (OAβ test)
Congenital anomaly screening
NIPT (Non-Invasive Prenatal Test, SeeNIPT)
Inborn errors of metabolism (screening)
Lysosomal storage disease screening

Lysosomal storage disease screening

Blood spot collection method

1)	Puncture the outer part of the newborn’s heel with a lancet to make the blood flow.
2)	After wiping the first drop of blood with gauze, aollw a large droplet of blood to for. Dispense blood samples inside the four circles in the blood spot paper and allow the blood to soak through to the other side.
3)	Be careful not to press the heel too forcefully, as it mat cause abnormal test findings due to the presence of tissue fluid.
4)	Note that iodine, alcohol, petroleum jelly, faces, urine, or hand lotion on the hand during collection may affect the test results.

Adult blood collection method

1)	When collecting blood using a syringe, dispense approximetely 50-75 μL of whole blood at the center of the blood spot on the paper before coagulation and aloow it to soak well on the paper. Wait until the blood spreads throughout the circle. * Reference : The Journal of the international Federation of Clinical Chemistry and Laboratory Medicine (eJIFCC 2016. 27(4):288-317)
2)	Adult blood spot collection method

Rub your hands for approximately 20-30 seconds to warm the fingers.
Massage the tip of the selected finger.
Open the protective cap of the lancet.
Fimly place the lancet against the side of the selected finger and press until a clicking sound is heard.
Massaege the bleeding finger.
Dispense the first drop of blood.

Massage the finger punctured with the lancet again and wait until the second drop of blood is dispensed.
Let the second drop of blood fail inside the first circle of the card without tounching the paper
Fill the second circle using the same method as #8.
Do not let the finger touch the paper when filling the circle with blood.
Appropriate cillection is when a single drop of blood fills the circle and one drop does not touch another drop.

* Source : UMCG(Clinical Pharmacy and Pharmacology dried vlood spot procedure)

Inborn errors of metabolism (screening)

Preaparations

1)	Check to ensure that the identification tag on the newborn’s wrist or ankle matches the examinee information indicated in the test request form.
2)	Accurately record all details on the blood spot sample.Accurately record all details on the blood spot sample.
3)	Wash hands thoroughly and wear clean gloves(use powder-free gloves with no foreign substances).

Pretreatment for blood spot sample collection

1)	Wrap the newborn’s heel with your hand or warm wash cloth (≤42°C) for 3-5 minutes to increase blood flow.
2)	To increase blood flow in the foor, place the newborn’s leg at the same height as, or below, the heart
3)	Wash hands thoroughly and wear clean gloves(use powder-free gloves with no foreign substances).Wash hands thoroughly and wear clean gloves(use powder-free gloves with no foreign substances).

Blood spot collection method

1)	Use a sterilized lancet.
2)	Punture the outer part of the newborn’s heel with the sterilized lancet.

3)	After wiping the first drop of blood with sterilized gauze, use your thumb to gently press the newborn’s heel. When a blood drop forms, realease pressure and place one side of the blood spot paper on the blood spot paper on the blood drop formed on the heel. Check to ensure that blood has soaked throught to the back of the blood spot paper.
4)	Collec sufficient blood to ensure that all four circles of the blood spot paper have been filled and ensure that blood has soaked through to both sides of the blood spot paper.
5)	After collecting the sample, raise the newborn’s foot above the level of the heart. Use sterilized gauze to gently press the collection site until the bleeding stops (do not use a bandage).
6)	Place the blood spot paper on a flat surface and aloow it to dry for at least 3 hours (avoid direct sunlight).
7)	Once the blood spots have dried completely, place the paper in the vinyl sample bag and sent it for testing.

Examples of acceptable/unacceptable collection of blood spot

Please be careful during blood collection, as inappropriate blood sampling can cause inaccurate (false-positive) results.

Sample	Details	Sample	Details
			Insufficient drying : Must dry for at least 3 hours
	Acceptable sample : Circle in the paper is completely filled and blood has soacked through both sides of the paper		Imprint of barcode ink from the papers sticking together before the sample har dried completely
	Insufficient blood : Blood does not fill the circle		Contamination
	Insufficient blood : Blood does not soak through to the back side		Formation of a ‘serum ring’ due to separation of serum from RBC caused by incomplete drying
	Too much blood : When there is too much blood		Collection of multiple, small blood samples
	Oversaturation due to scratching or smearing of the blood spot paper		Collection through a device such as a capiliary tube will result in uneven coverage and poor absorption
	Multiple drops of blood in a single circle		Apllying the blood drops multiple times using a capiliart tube
	Dilution, discoloration, contamination : May be diluted by alcohol or woter during collection (such factor may vinterfere with the analysis)		Blood not being absorbed evenly on the paper due to coagulation

Congenital anomaly screening

Required information

Please ensure the following details are provided on the test request form for congenital anomaly screening test :

· Date of birth
· Body weight
· Gestational age (LMP (last maenstrual period), BPD (biparietal diameter))
· Nuchal translucency (NT) if the test is for early pregnancy
· Previous medical history (history of giving birth to a child with Down syndrome or neural tube defect; insulin-dependent diabetes mellitus)
· Number of fetuses
· Race
· Smoking status
· In-vitro fertilization (IVF)
· When requesting a retest, please attach the test result from the first attempt along with any other findings.

Types of tests by gestational age

First trimester(10w0d - 13w6d)	First double marker	PAPP-A, hCG, NT
Second trimester(14w0d - 22w6d)	Triple test	AFP, hCG, uE3
	Quad test	AFP, hCG, uE3, inhibin-A
	NTD	AFP, AFP(AF)
First+Second trimester	Intergrated test	First trimester (PAPP-A, NT) Second trimester (AFP, hCG, uE3, Inhibin-A)
First+Second trimester	Sequential test	First trimester (PAPP-A, NT) Second trimester (AFP, hCG, uE3, Inhibin-A)
pregnancy(10-24w)	NIPT	Fetal DNA

Oligomerized amyloid beta test, AlzOn (OAβ test)

Collection method and precautions

1)	Write the patient’s name on three labels of the blood collection kit and affix one each on an Na-heparin tube and two polypropylene(PP) tubes (1.5mL).
2)	Use a 21G needle to collect blood in the Na-heparin tube. (Do not use a syringe as the plastic on the inner wall of the syringe may cause agglomeration of beta-amyloid, resulting in false negative results.)
3)	After collecting the whole blood, gently invert 8-10 times or use a roll mixer to ensure that the blood mixes thoroughly with the anticoagulation.
4)	Whole blood can be stored at room temperature for up to 1 hour after collection and plasma should be separated by centrifugation within 1 hour (during centrifugation, spin down slowly)
5)	After centrifugation, transfer the plasma to two PP tubes(1.5mL) using the polypropylene filter tip and pipette provided in the blood collection kit or the eyedropper provided separately. (To prevent mixing with blood, collect plasma from at least 0.5mc away from the buffy coat layer. The polyprolylene tube provided l the kit must be used.)
6)	Separated plasma must be frozen for storage prior to use in the test.

Natural killer (NK) cell activation induced interferon-gamma

Collection method and precautions V

1)	If the amount sampled exceeds 0.9-1.1mL, resampling in another tube is recommended.
2)	If multiple tubes are used for blood sampling, use an NK Vue® tube first, exept if a scalp needle must be used, in which cases the tubling must be filled with whole blood to ensure that an accurate amoint of blood has been collected. In such cases, use an NK Vue® tube last.
3)	Using a roller after blooding sampling is not recommended, as it may influence the activity of NK cells. Hemolysis due to inadequate mixing may lead to inaccurate reults.
4)	Collected whole blood must not be refrigerated or cryopreserved.
5)	After collecting whole blood in exclusive NK Vue® tubes, the samples must be incubated immediately at 37℃ during 20-24 hours for pretreatment.
6)	After incubation, only the separated upper plasma is transferred to a 5mL pp tube and stored in the freezer immediately.

7) NK Vue® tube

QuantiFERON-TB Plus (TB-specific antigen-induced IFN-γ)

Collection method

1)	There should be four tubes (gray, green, yellow, and purple caps) per patient and the tubes must be kept refregerated before use.
2)	Leave the tube at room tempurature for 1 hour prior to blood collection.
3)	Write the patient’s nama and collection time on each tube.
4)	Collet 1mL in each of the four tubes, in the order of gray, green, yellow, and purple caps. Check the black mark indicating 1mL on the side of each tube. Collected blood must be 0.8-1.2mL.
5)	After collection, hold the tube vertically and gently mix it 10 times. Thoroughly mix the blood to allow the antigen coating on the wall of the tube to be dissolved sufficiently.
6)	Tubes with collected blood must be stored standing upright at room temperature (22-25°C).
7)	Transfer the tube to an incubator at 37°C ±1°C as soon as possible within 16 hours of blood collection.
8)	Incubate the tube upright at 37°C ±1°C for 16 to 24hours.
9)	Centrifuge the cultured tube at a speed of 2000-3000 x g RCF for 15 minutes to collec the plasma.

Collection method

1)	If the tube has not been cultured immediately after blood collection, invert the tube several times (approximately 10 times) just before incubation.
2)	The incubator does not require CO2 or humidification.
3)	Before collecting the plasma agter centrifugation, do not perform any action such as moving the pipette up and down or mixing the plasma.
4)	The plasma must be collected in a minimum of 150 μL.

NIPT (Non-Invasive Prenatal Test, SeeNIPT)

Required information

Please ensure the following details are provided on the test request form:

· Name and date of birth of maternal
· Body weight
· Gestational age(LMP (last menstrual period), BPD (biparietal diameter))
· In-vitro ferilizaion (IVF)
· Specific ultrasound findings
· Nember of fetuses
· Fill out the genetic testing consent form

Sample collection method for NIPT

1)	Collect at least 7.0mL in Cell-Free DNA BCT® (STRECK) tube and mix thoroughly.
2)	Keep the sample at room temperature after collection.
3)	Please seal the tube tightly to protect from leakage.
4)	Please check the ‘Specimen stability’ and pack it according to the ‘Specimen shipping guide’.

Conical tube(Sterile container)
	Additives : K3 EDTA, cell preservative Storage / After collection : Room tempeture Specimen volume : Blood 8-10mL Remarks : 1. Writhe the receipient’s name and date of birth on the container. 2. Collect the 8mL of whole blood and mix it thoroughly (Do not separate the plasma) 3. Keep the sample at room temperature and request the test (Store the sample at refregerated or frozen are prohibited).

Conical tube(Sterile container)

Additives : K3 EDTA, cell preservative Storage / After collection : Room tempeture Specimen volume : Blood 8-10mL Remarks : 1. Writhe the receipient’s name and date of birth on the container. 2. Collect the 8mL of whole blood and mix it thoroughly (Do not separate the plasma) 3. Keep the sample at room temperature and request the test (Store the sample at refregerated or frozen are prohibited).

Hair

Collection method

1)	The hair analysis kit should contain a paper scale, collection enveolpe, return envelop, and test order form.
2)	Distinfect a pair of stainless-steel scissors with alcohol swabs and fold both sides of the paper scale.
3)	Grab the appropriate amount from 3-4 sites on the back of the head and cut with the scissirs close to the scalp(for long hair, leave only 3-4cm from the scalp and cut the rest).
4)	place the cut hair specimen on the “place complete hair specimen” part(--- part) of the paper scale and collect until scale tilts. Press the oppsite side to make sure the scale tilits again.
5)	Plece the collected sample in the sample envelope and seal it with tape or guide.
6)	Fill out the required information on the test order form and sent in together with the sample envelope.

Precautions

1)	In the following cases, collect hais 2 weeks later.
· Chemically treated hair, induding, perm, and coating · Perform the test 2 weeks after discountinuing functional shapoos, including anti-hair-loss shampoo (e.g., Selsun Blue) anti-dandruff shampoo (e.g., Nizoral, Head&Shoulders), and braided-hair shampoo. · Perform the test 2 weeks after discountinuing the use of swimming pools, if used regularly.
2)	Samples can be collected even if hair gel or mousse has been applied. However, add approximately 10 more strands after the scale tilis in such cases.

Body fluid

Cerabrospinal fluid, CSF

1)	Collect 5-10mL of CSF by aspiration using a syringe.
2)	Samples are typically collected in three asepic containers. The first is for biochemistry and serology, the second container is for gram stain and culture, and the third container is for cell count
3)	If appropirate, pour the specimen into an 5mL PP tube in sterile.
4)	Please seal the tube tightly to protect from leakage.
5)	Please check the ‘Specimen stability’ and pack in according to the ‘Specimen shipping guide’. * Please store the cerebrospinal fluid (CSF) specimen at room temperature when requesting a microbiological culture test.

Body fluid

For body fluid tests using fluids other than CSF, such as pleural, ascitic, and joint fluid, use containers with EDTA added, since the samples may easily coagulate after collection.

Sample container		Test item
EDTA tube		Some body fluid analysis and chemistry yest
Plain tube		Chemistry test(not clotted), cytology test

Conical tube(Sterile container)
	Sterile container for various test items Specimen Volume : ≤ 15mL ≤ 50mL Remarks : Be careful not to contaminate the sample.

Stool

One-time stool

1)	Collect 2-3g of stool in a sterilized plastic container.
2)	If appropriate, transfer the specimen into an 50mL conical tube
3)	Please seal the tube tightly to protext from leakage.
4)	Please check the ‘Specimen stability’ and pack it according to the ‘Specimen shipping guide’.

Urine

Collection method

1)	Random urine
1) Urine should be collected in a clean, well-dried container. 2) Collect the first morning midstream urine and order the test as soon as possible, keeping the sample refrigeated before it is retrieved. * Keep the sample at room temperature when ordering the Neisseria gonorrhoeae culture test. 3) Please send approvimately 10mL of volume in two 5mL PP tube when making. 4) Please seal the tube thightly to protect from leakage. 5) Please check the ‘Specimen stability’ and pack it according to the ‘Specimen shipping guide’.
2)	24hr urine
1) Collect urine at 08:00 and continue to collect it in a 24hr urine collection bag until 08:00 the follwing day (including urine during defecation). Samples can be refrigerated for most tests, but if a preservative must be added, place the designated preservative in the 24hr urine collection bag first before collecting urine. 2) When ordering the test, mix the stored urine throughly and send approximately 10mL of urine in two 5mL PP tube. 3) The volume of stored urine must be indicated. 4) Please seal the tube thightly to protect from leakage. 5) Please check the ‘Specimen stability’ and pack it according to the ‘Specimen shipping guide’.

Urine preservation method

1)	Refrigerate (4-6℃)
Used to prevent the effects of uring a urine preservative; may result in the precipitation of urinary crystals
2)	Freeze (-20℃)
Used to quantify unstable substances (urobilinogen, bilirubin, prophobilinogen, etc.) after freezing a protion of the 24hr urine sample
3)	Chemical preservatives
Enable long-term storage by inhibiting bacterial growth, stabilizing urinary solutes, or fixing cellular components in urine

Preservative	Amoung of preservative	Characteristics
Toluene	2.0 mL / 100 mL	Does not inhibit microbial proliferation, but is good for preserving chemical components
6N HCL	2.0 mL / 100 mL	Maintains an acidic pH of urine, which can turn alkaline if left alone
Acetic acid	Acetic acid	Suitable for hormone tests
Boric acid	Boric acit	Inhibits microbial proliferation with a pH of approximately 6.0 and is used for the quantification of hormones

4)	pH adjustment
Urinary pH is generally lowered using HCL to inhibit bacterial proliferation.

Guide for using 24hr urine preservatives

Test name	24hr urine preservative
Test name	No preservative	Toluene	6N HCI	50% Acetic acid	Boric acid	Na2CO3	6N HNO3
17-Ketosteroids	○		◎
17-OHCS	○		◎
5-HIAA*		○	◎	○	○	○	○
Aldosterone	◎
Aluminum (Al)	◎		○	○			○
Arsenic (As)	◎		○	○			○
Cadmium (Cd)	◎		○	○			○
Calcium (Ca)	◎		○				○
Catecholamine*			◎
Chloride (Cl)	○	◎
Citric acid*		○	◎	○	○
Cobalt (Co)	◎
Copper (Cu)	◎		○	○			○
Cortisol	◎
Creatinine	◎
Dopamine, total*			◎
HVA (Homovanilic acid)*			◎	○
Hydroxyproline, free*			◎
Hydroxyproline, total	○
Lead (Pb)	◎	○	○	○			○
Magnesium (Mg)	◎		○
Manganese (Mn)	◎
Mercury (Hg)	◎	○	○	○			○
Metanephrine*			◎
Microalbumin	◎
Nickel (Ni)	◎		○				○
Oxalic acid	◎
Phosphorus (P)	◎
Porphobilinogen*						◎
Potassium (K)	○	◎
Protein E.P	◎
Protein, total	◎
Selenium (Se)	◎	○	○				○
Uric acid	○	◎
UUN (Urine urea nitrogen)	○	◎
VMA (Vanillylmandelic acid)*			◎
Zinc (Zn)	◎		○	○			○

(* : Preservative is mandatory,◎ : recommended,○ : possible)

Blood

Preparations for patients

1)	The test is perforemed after checking for restrictions on the intake of any prohibited foods (or drugs).
2)	Before blood sampling, it is necessary to explain to the patient about the collection of blood samples from the patietnt.
3)	Blood sampling must be performed after identifying the patient with at least two kinds of information.
Identify the patient by combining at least two of the following items of indormation : patient’s name, registration number, date of birth, and age. When asking a question, makr sure the question is open-ended. (Example : When asking for the name, you should ask “What is your name?” instead of “is your name XXX?” to get an accurate answer. Platient’s with difficulty communicating or hearing may simply answer “Yes” to the question “Is your name XXX?” believing that would indicate their willingness to cooperate.)
4)	Place the patient’s arm on the table(same height as the heart) in a relaxed state and have the palm facing upward.
5)	Use a vein on the opposite side if the patient is receiving an IV infusion.

Blood sampling time

1)	The general rule is to collect blood in the morning in a fasting state since the blood components may cause the test results to vary depending on physiological factors.
2)	For outpatients, collect blood at least 2 hours after a meal.
3)	After strenuous exercise, ensure that blood is collected after sufficient rest.
4)	When repeating the test on ohe same patient, ensure that blood is collected at the same time under the same conditions.
5)	Collect blood before antibiotic administration.
6)	Blood cannot be collected from a vein where a contrast agent has been injected.

Blood sampling volume

When ordering multiple types of tests(using whole blood, plasma, and serum) at the same time, the volume of the blood vsanples needed for each test should be calculated in advance.

Whole blood : Collect the volume needed for the test
Serum(plasma) : Collects 3 times more volume than is needed for testing

Sample containers

1)	Use sample containers that are approproate for whole blood, plasma, and serum. When a plasma or whole blood sample is needed, use a container containing an anticoagulant, such as citrate or EDTA. When a serum sample is needed, use a plain tube without anticoagulant or a SST(serem separator tube) containing a clot activator and gel.
2)	Check the expiration date on the sample container before using it.
3)	When collecting the sample into a container contaning an additive, mix thoroughly by gently inverting the container 3-10 times depending on the type of container.

Blood sampling method

When using a vacuum tube

1)	Wrap the tourniquet close to the blood collection site.
2)	Except when measuring blood alcohol concentration (use alcohol-free disinfectant), use an alcohol aponge(70% alcohol) to disinfect the blood collection site and aloow it to dry.
Do not use an iodine sponge, as it may contaminate the sample. Undried aocohol on the blood collection site may cause hemolysis of the sample.
3)	Puncture the vein while keeping the needke tilited at an angle of 15-20°.

4)	Sufficient volume shoud be collected to fill the vacuum tube. Beacuse the tube is in a vaccum state, use it without opening the cap.
There is sufficient vacuum insite the sample container to draw an appropriate volume of blood. Therefore, the appropriate volume can be collected by drawing blood until no more can be drawn due to no more vacuum. However, be careful not to open the cap, drop the container on the groud, leave it at a high temperature for a long time, or keet it beyond the expriation date, as doing so may result in insufficient blood collection due to a loss or reduction of vacuum. The expiration date must be checked and the container must be stored at an appropirate temperature (4-25°C).
5)	For continuous blood sampling, slide the second tube into the holder after the first tube has been removed from the holder.
6)	The tube with collected blood must be mixed immediately to prevent coagulation.
7)	Realease the tourniquet.
8)	After removing the container from the needle, place a dry guaze and carefully remove the needle.Notify a physician if excessive bleeding lasts for more than 5 minutes, or if blood in the resum container does not coagulate.

When using a syringe

1)	A syringe should be used for veins that may collapse easily when a vacuum tube is used for blood sampling(e.g., veins of the elderly and children that are small and may rupture easily).
2)	After removing the needle from the syringe used for blood sampling, allow the blood to slowly flow down the wall of the sample container.
Blood collected with a syringe can coagulate, so it must be transferred without delay to a vaccum tube. Blood must be drawn only by the force of vaccum. Take care not to apply excessive pressure while drawing the blood, as it may cause hemolysis and destruction of blood cells.

Blood sampling order

Check the sample for each test and use the designated sample container. When multiple types of sample container are Blood sampling order by sample container ※ The same order applies when collecting blood using a syringe.

Order	Test tube	Use of purpose	Mixing times
1	Bacteria Blood culture Bacterial culture broth - Soybeon-casein Digest Broth - Soybeon-casein Digest Broth	· Bacterial culture · Priority blood sampling to prevent contamination	8-10 times
2	Citrate tube Sodium citrate (3.2%) or CTAD	· Plasma for blood coagulation and platelet fuction tests Collect in citrate tubes first among tubes containing additives to prevent mixing with additives that may affect blood coagulation test results	3-4 times
3	Plain tube Clot activator	· Serum for chemistry, immunology, and blood bank tests	0 times
3	SST (Serum separator tube) Clot activator and gel	· Serem for chemistry, immunology, and blood bank tests collect in citrate tubes next, since silica particles in SSTs that act as clot activators should not be mixed in citrate tubes used for blood coagulation tests.	5 times
4	PST (Plasma separator tube with heparin) Lithium heparin and gel	· Plasma for chemistry tests Even if introduced into other tubes, heparin does not significantly affect the results, except for theb lood coagulation tests.	8-10 times
5	Heparin tube Lithium heparin or Sodium heparin		8-10 times
6	EDTA tube Spray dried K2 EDTA or liquid K3 EDTA	· Plasma for hematololy tests, such as CBC	8-10 times
7	Antiglycolytic tube (NaF tube) Sodium fluoride, Potassium oxalate	· Plasma for measuring blood glucose concentration.	8-10 times

The sample container should have a different-colored cap depending on whether an anticoagulant is used and the type of anticoagulant. Anticoagulants are used in tests requiring whole blood or plasma.

Serum Separation Method

When using a vacutainer


1)	After blood collection gently invert about 6 times for mixing.
2)	The whole blood should be left at room temperature for approximately 30 minutes to allow coagulation and minimize subsequent fibrin formation before centrifugation
3)	Serem can be obtained by centrifuging whole blood(3,000rpm, 10 minutes).
4)	Transfer the upper layer of serum to new 5mL PP Tube.
5)	Seal the tubre securely and send it to Seegene Medical Foundation, Korea
Cautionary * Ensure that the sample volume for each test meets the requirements. * Separate serum or plasma that connot be transported on the same day must be refrigerated. * Samples collected for general chemistry should be centrifuged within 2 hours after collection. If immediate centrifugation isn’t possible, store the samples at room temperature to prevent hymolysis that may occur when refreigerated. * Serum must be seqarated within 30 minutes after collection for a blood glucose test. If immediate separation isn’t possible, use NaF containers whenecer frasible.

Factors that may affect the test results

1)	Patient identification errors Be careful of patient identification errors, especially for blood bank tests, since such errors may have detrimental consequences.
2)	Use of a tourniquet for more than a minute This produces relatively concentrated blood, which may affect the test results due to an increased concentration of proteins and protein-bindings substances and blood from venules or capiliaries being mixed together
3)	Use of wrong sample container The correct order of cantainers used for sampling must be followed to prevent errors in test results due to the mixing of additives and the formation of microclots.
4)	Ratio of anticoaguland and blood An improper ratio may cause the formation of microclots, which can lead to error in cell counts or blood coagulation test results.
5)	Hemolysis Hemolysis may cause hemoglobin and intracellular substances to react, which may affect the test results and cause elevated values. Therefore, caution is needed when collecting blood and dispensing it into test tubes. when using a syringe, do not pull the piston too forcefully, and when collecting the blood into a tube, remove the needle and allow the blood to flow slowly down the wall of the container. centrifugatin should be performed approximately 30 minutes after blood collection.

6)	Coagulation If the anticoagulant and blood are not mixed sufficiently, inaccurate hematology results may occur due to partial clotting. Therefore, when the blood is collected in a container containing an anticoagulalant, it is necessary to mix thoroughly and send it for testing as soon as possible.
7)	Lipemic blood samples Blood samples collected after a meal may be lipemic. Such lipemic samples may cause inaccuracies in the test result(elevated values). Depending on the test items, collec blood after 12 hours of fasting.
8)	Test items affected by blood collection after a meal

9)	Sample contamination Betadine should not be used to collect blood for testing and blood must be collected after the alcohol has completely dried. The inclusion of alcohol may cause hemolysis and may produce false negatives in bacterial cultures. When collecting blood for blood cultures, it is necessary to be especially careful to prevent contamination by skin flora.
10)	Factors related to sample storage Samples that are not taseted immediately must be left at room temperature or refrigerated, depending on the ytyle of test. When refrigerated, a stopper must be placed to prevent evaporation. For long-term storage, crypreservation at°C or below is prefferred.

11)	Physiological factors - Be aware of the fact that using blood collected immediately after a patient changes position after holding another position for a logn tine may after that test results. - The duration and intensity of exercise have diffetent effects on changes in body fluids. - It is important to have a consistent blood collection time to minimize changes due to diurnal fluctuations. Generally, the best time for blood collection is in the morning in a fasting state. - Food intake causes many changes, especially a significant increase in blood glucose, iron, total lipid, and alkaline phosphatase concentration.s - After alcohol intake, the blood glucose concentration may increase temporarily in some cases, but in mose cases, there is a decrease in blood glucose concentration and an increase in the concentration of ketones, whice are metabolites of alcohol. - Nicotine from smoking may affect various test results. - After intramuscular drug administration, the concentrations of muscle anzymes such as creatine kinase and aldolase, and low-density lipoprotein increase due to muscle stimulation. - Fever causes an initial increase in the blood glucose concentration, which promotes insulin secretion and increases growth hormone, glucagon, and aldosterone concentration. - Age, gender, ethnicity, envirionmentatl foctors, the menstrual cycle, obesity, pregnanct, anc stress also effect the test results.

Effects of diurnal fluctuation, posture, and stress on test result

Test	Effects
Cortisol	Highest beween 04:00 and 06:00 and lowest between 20:00 and 00:00 (50% lower at 20:00 than at 08:00); increased by stress
ACTH	Low at night; increased by stress
Renin activity	Low at night; higher when standing than when lying down
Aldosterone	Low at nigh
Insulin	Low at nigh
Growth hormone	High in the evening and nigh
Acid pgosphatase	High in the evening and nigh
Thyroxine (T4)	Increases during exercise
Prolactin	High between 04:00 and 08:00 and between 20:00 and 22:00; increased by stress
Iron	High during the morning and decreases up to 30% throughout the day
Calcium	Decreases by 4% when lying down

Samples that need to be transported under special conditions

1)	Samples that need to be shielded from light Bilirubin, carotene, vitamin C, vitamin E, and DPD decompose when exposed to light, which does not allow accurate measurements to be obtained. Therefore, such samples must be transported while shielded from light.
2)	Samples that must be kept at 37°C Samples for cryoglobulin, cold agglutinin, and paroxysmal cold hemoglobinuria tests must be kept at 37°C during transport since they may produce false negatives at lower temperatures.
3)	Samples that must be transported in a frozen state Calcitonin (freeze immediately after collection), gastrin (freeze after stabilizing for 4 hours at 2–8°C), CH50 (freeze immediately after collection)

Safe disposal of sampling tools

Waste from medical institutions can cause environmental pollution, and thus, medical waste with concerns for secondary infection must be handled safely and hygienically, processed appropriately, and managed effectively. Needles for sampling must comply with the following rules to prevent infection.

1)	Needles must be disposed of in puncture-resistant, non-reusable sharps containers immediately after use.
2)	Recapping the needle, forcefully bending or breaking the needle, removing it from a disposable syringe, and touching it with the other hand are prohibited
3)	Used needles must be disposed of through the opening of a dedicated needle collection box.
4)	When the needle collection box is 75% full, the opening of the box must be sealed for disposal.

Diagnostic hematology
Cytogenetic analysis
Molecular diagnostic test
Cytopathology
Histopathology

Diagnostic hematology

General blood test(CBC)

1)	Collect 3mL of blood in an EDTA tube and gently mix for 8-10times immediately.
2)	If the ratio an anticoagulant(EDTA) and blood is inappropriate ir they are nit mixed throughly, clots and dilution may occur to cause errors in the results(effects on blood cell count, hematocrit lecel, cell morphlogy, and staining due to excessive anticoagulant).

Blood smear sample preparation

Preipheral blood smear is used to count the number of each type of blood cell and examine its morphlogy for abnormalities to diagnosis various blood diseases, including anemia and leukmia.

1)	Prepare two clean glass sildes.
2)	Hold one glass slide between the thumb and middle finger of the left hand dispense a drop of blood on a spot 1.5cm from the upper right-hand corner.
3)	Hold the spreader glass between the thumb and middle finger of the right hand.
4)	Make sure the drop of blood placed on the glass slide touches the back of the spreader glass and spreads evenly.
5)	While maintaining and angle of approximately 30-40℃) between the spreader slide and the glass slide, push the spreader slide toward the left thumb at a constant speed while holding up the glass slide. A slow spreading speed will cause the smear to be too thin and a fast spreading speed will cause the smear to be too thick. Be aware that when the smear is too thin, WBCs have a tendency to migrate toward the outer edges of the smear.
6)	Before placing it in the sorage box, air dry the smear slide and be careful not to contaminate the sample.
7)	The storage boxed should be wrapped in bubble wrap, secured with a rubber band or tape and placed in an outer shipping box

Blood coagulatin test : citrate blood

Because citrate removes calcium and has anticoagulation activity, it is used in blood coagulation tests such as prothrombin time(PT) and activated partial thromboplastin time(aPTT).

1)	The blood coagulation test uses blood collected in a sodium citrate tube. The sample is collected by indwelling catheter or venipunture using a 19-21G needle(23G butterfly needle for child). If vacutainers are used, the second container must be used as the coagulation test sample.
2)	Use 3.2% sodium citrate as the anticoagulant. The ratio of sample to anticoaguland should be 9:1(10.9-1.9mMol/L) and the volume of blood to be collected is indicated on the sodium citrate tube. the exact volume must be collected. An inaccurate ratio may alter the test results.
3)	After collection, immediately mix for 3-4times. If not mixed throughly in a timely manner, microclots may form in some parts of the blood sample and a prolortional decrease in clotting factors occurs, which can cause errors in test results.
4)	The collected samples were centrifuged at 2,500g for 15 minutes within 4 hours.
5)	Separate the platelet-poor plasma and transfer it to 5mL PP tube.
6)	Freeze before testing.

Platelet-poor plasma separation method The specimen must be double-centriduged to prepare a platelet-free plasma specimen(platelet coung < 10,000 μL

1)	Immediately centrifuge specimen at 2,500g for 10 minutes.
2)	Carefully remove plasma from cells, avoiding the platelet/buffy coat.
3)	Dispense into a plastic tube using a plastic transfer pipette. Do not pour off.
4)	Centrifuge aloquoted plasma at 2,500g for 10 minutes.
5)	Remove the top portion of plasma, leaving approximately 250 μL in the bottom to discard.
6)	The double-centroduged plasma should be aliquoted(1 to 2mL per aliquot) into clearly labeled plastic tubes. The numver of tests ordered will determine the aliquots needed. Generally, a 1mL aliquod per test is required, althogh test volumes may be combined up to 2mL of plasma per aliquot. Pay particulat attention to the amount of specimen required for the ordered tests. Coagulation profiles(see individual test specimen requirements) and multiple single-test orders will require multiple aliquots.
7)	After centridugation, examine the plasma for firbrin clots and pour the cellular portion through guaze to observe for small red cell clots Clooted specimens must be discarded and recollected.
8)	Specimens should be frozen at below -40℃, if possible, and sent together in the same contaiver with at least 5 lbs of dry ice. Specimens must arrive frozen.

Collection of samples for blood coagulation test : collection by indwelling catheter When collecting blood using an indwelling catheter, use 5mL of physiological saline to flush the line and discard the first 5mL of blood (or six times the line volume) to prevent contamination by heparin. If unexpected blood coagulation test results are found, test again using freshly collected blood or after removing heparin with a resin. Type of tests according to anticoagulant

Anticoagulant	Mechanism of action	Mornitor test
Wafarin(Coumadin)	· Inhibits vitamin K-releated factors : Factor II, VII, IX, X · Half-life : 40 hours	PT
Unfractionated heparin(UFH)	· Binds with antithrombin Ⅲ to block actination of factor X · Administer protamine sulfate for hemorrhagic complications	aPTT, ACT, TT, Anti-factor Xa
Low-molecular-weight heparin (LMWH)	· Dalteparin, Enozaparin, etc. · Excreted by the kidneys; caution needed in cases of renal failure · Subcutaneous injection 1-2 times per day since it has a long half-life · Partial inhibition by protamine sulfate · Better factor-Xa-antagonistic effec than UFH · Fewer homorrhagic complications · Dose-response relationship is predictable	Anti-factor Xa, aPTT test not needed
Aspirin	· Inactivates platelet function · Discontinue use for 1 week prior to surgery · Desmopressin(DDAVP) can normalize bleeding time	Platelet drug response assay

Cytogenetic analysis

1)	The specimen must be collected asrptically.
2)	Clinical information such as clinical findings and purpose of the request must be provided on the cytogenetic test request form and the genetic testing consent form.
3)	If there is a delay of more than 24 hours from sample collection to culturing, there may be a delay in reporting test results or occasionally even failure of the test.
4)	It is desirable to explain to the patient or guardian the accuracy and limitations of the test before collecting the fetal chromosomal sample and obtain consent before conducting the test.

Peripheral blood

1)	Asptically collect 5.0mL of blood(3.0mL for child) in a sodium heparin container. A coagulated sample connot be tasted. Anticoagulants other than sodium heparin have a high probability of culture failure due to reduced cell division ability.
2)	The testing method may very depending on the clinical findings and suspected diagnosis of the patient, and thus, the clinical findings and diagnosis must be indicated on the order form.
3)	Transport the sanple to prefer that all steps from sample collection to cell culture are performed within 24 hours(testing within 24-72 hours ensures good cell division, resulting in a high reporting rate within the turnaround time; if it requires more than 24 hours, refregerate the sample).
4)	Please check the ‘Speciman stability’ and pack it according to the ‘Specimen shipping guide’.

Bone marrow

1)	hemato-oncological diseases, indicate or attach the CBC results, since an appropriate number of cells need to be cultured based on the blood test results.
2)	Indicate the disease name, since the culture method must be selected according to the disease.
3)	For other conditions, the same rules apply as those for peripheral blood samples(refer to the above mentioned details).
4)	Store and transport the sample at refrigerated.
5)	Please seal the tube tightly to protet from leakage.
6)	Please check the ‘Specimen stability’ and pack it according to the ‘Specimen shipping guide’.

Aminiotic fluid

1)	To prevent the contamination of maternal cells in the sample, a small initial volume(approximately 2mL) must be discarded and 20-30mL of amniotic fluid must be collected separately and transferred to a 15mL conical tube(x2).
2)	The test should be performed starting from a gestational age of 15 weeks or older and the general rule is to have the sample arrive within 24-72 hours with caution for contamination.
3)	Culture may fail if the amniotic fluid lacks sufficient number of fetus-derived cells(no visible sediment after centridugation) or if the sample is visibly red colored(inclusion of blood) or dark colored.
1) Store and transport the sample at 2-8°C. 2) Please seal the tube tightly to prevent from leakage. 3) Please check the ‘Specimen stability; and pack it according to the ’Specimen shipping guide’.

Cord blood

1)	Because cord blood con coagulate easily, mix it several times after collection.

Heparin tube		Conical tube(50mL)
	Additives : Sodium heparin Test items : Heavy metal test(Hg, Pb, Mn, etc), chromosomal test(Chromosome study, fluorescence in situ hybridication [FISH]) Speciman volume : Blood 5-10mL Remarks : Mix thoroughly after collection to prevent coagulation.

Molecular diagnostic test

Precautions

1)	The most important aspects in molecular diagnostic tests include the use of appropriate samples and the prevention of contamination.
2)	Samples for infectious diseases must be sent to the laboratory immediately after collection. If there is a delay, urine, sputum, respiratory samples, stool, bacteria, and virus test samples must be refrigirated to prevent the proliferation of normal flora. CSF, body fluid, blood, and gonococci test samples must be stored at room temperature.
3)	Please note that hemolyzed samples used for RNA virus tests may produce unreliable test results.

Collection method

1)	Generally, EDTA containers shoul be used for all blood and bone marrow samples for molecular diagnostinc tests. One of the anticoagulants, heparin, inhibits Taq polymerase activity, whick makes PCR testing impossible.
2)	Thyroid fine needle aspiration fluid : Place tissue samples in sterile physiological saline without formalin fixation, and pour the specimen into an 5mL PP tube in sterile * Instead of the above method, you can also use a container dedicated to Liquid-based aspiration cytopathology
3)	Respiratory samples Nasopharyngeal aspirate and nasopharyngeal swab : Nasopharyngeal aspirates have a higher virus isolation rate than nasopharyngeal swabs.
4)	Throat swab : Can be used to identify adenovirus by swabbing the infected area between the posterior pharynx tonsilla area. Be careful not to touch the tongue, oral mucosa, teeth, or gums.
5)	BAL : Use the lavage fluid collected during bronchoscoly. * Transfer the BAL and Nasopharyngeal aspirate samples into a conical tube(50mL)
6)	Please seal the tube tightly to protect from leakage.
7)	Please check the ‘Specimen stability’ and pack it according to the ‘Specimen shipping guide’.

EDTA tube		Liquid-based aspiration cytopathology		PP tube(50mL)	Conical tube(50mL)
	Additives : K3 EDTA Test items : Hematology(CBC, ABO, RH, etc.), HbA1c, cellular immunity test Speciman volume : Blood 3.0mL Remarks : Mix thoroughly after collection to prevent coagulation.		Additives : Cell preservative Remarks : 1. Add the sample to the container immediately after collecting and mix thoroughly. 2. Be careful not to contaminate the sample.

Cytopathology

Precautions

1)	Smples should always be freshly preserved.
2)	When using a cytofixer(spray) for smear slide fixation, pressure may cause cell densaturation. Accordingly, spray the entire slide surface evenly from a distance of 30cm.
3)	Write the patient’s name, chart number, and collection date on the sample container and slide.
4)	Provide essential clinical information such as the patient’s name, collection site, medical history, and primary physician on the cytopathology test request form.
5)	Be careful not to daname or lose the slide.
6)	The slide holder/mailer contaning the properly labeled slodes may be used for shipping.
7)	The storage boxes should be wrapped in bubble wrap, secured with a rubber band or tape and placed in an outer shipping box.
8)	For further inquiries, contact Seegene Medical Foundation via e-mail(csglobal@mf.seegene.com).

Guide for cytopathology samples

Smaple type		Manufacturing type
Gyneco logical cytology(GY)	Veginal Cervical Endometrial	① After smearing the sample on the slide, immediately fix it in 95% ethyl alcohol for at least 2 hours. ② Liquid-based cervical cytology : after removing the stem part of the collection brush, place it in the dedicated container and seal tightly. ③ Do noy use any lubricant or talcum powder on the gloves or instrument used for sample collection. If moisture is needed, use warm saline. ④ When using a swab to collect the sample, smear a sufficient amount on the slide and fix immediately. ⑤ When using a pap brush or cytobrush to colled the sample, be careful as severe bleeding may interfere with the diagnosis. ⑥ Use of intravaginal medication or contraceptives within 24 hours of sample collection is prohibited.
Nongynaco logical cytology(Non-GY)	Sputum	① Collect sputum by a deep cough originating from the diaphragm. ② Collect the pathological part(discolored, dark brown, or bloody part) and place it on the glass slide. Smear by onerlapping two slides and fix immeniately in 95% ethly alcohol. ③ With the sputum test, the cancer dell detection rate is higher when the tests are performed over 3-5 consecutive days.
	Urine CSF	① Collect midstream urine because first morning urine is vulnerable to severe degenerative change and is concentrated. ② Mix 1-2 drops of egg albumin with the precipitate obtained by centrifugation at 1,500rpm for 5 minutest. After smearing on the slide, fix immediately in 95% ethly alcohol for at least 2 hours. ③ If a smear is not possible, add an equal fracton of ethyl alcohol to prevent cell damage.
	Pleural fluid Ascitic fluid	① Never put the sample in alcohol or formalin. (Alcohol makes the cells become round and hard. Formalin disrupts the microstructure of the nucleus.) ② Smear the precipitate obtained by centrifugation at 1,500rpm for 15 minutes on the slide and fix immediately in 95% ethyl alcohol for at least 2 hours.
	Nipple discharge	① Directly smear one drop of discharge on the slide and fix immediately in 95% ethyl alcohol for at least 2 hours.
Fine needle aspiration (FNA) cytology	Breast Thyroid Lymph nodes	① After smearing the sample, fix it immediately in 95% ethyl alcohol for at least 2 hours. ② If the sample is not sufficient, wash the needle and syringe with physiological saline. After centrifugation, smear the precipitate and fix it immediately in 95% ethyl alcohol for al teast 2 hours. ③ If there is excess sample, send in together, since any excess can be used for cell block.

Histopatholgy

How to fill out the histopathology request form

The patient’s name, date of birth, sex, hospital name, and registration number and the collection date, collection site, collection method, and nama of the test requested must be indicated. In addition, provide the patient’s medical history and clinical findings for accurate and timely diagnosis.

Fixation method

1)	Tissues undergo autolysis when separated from the human bodt. For an accurate diagnosis, fix the tissue in a fixative (10% formalin) immediately after collection.
2)	The fixative is typically 10% neutral buffered formalin. Submerge the tissue completely in a fixative volume 10-20 times that of the tissue for fixation at room temperature for 13-14 hours.
3)	The opening of the sample container should be wide. If the samples were collected from different sites or need to be differentiated, place them in different containers.
4)	If a fixative is not avaliable, weap the tissue, place each of them soaked in saline, and refregerate. Soak in a fixatixe as soon as possible.
5)	The sample container must be sealed tightly to prevent leakage of the tissue sample or fixative.
6)	For further imquiries, contact Seegene Medical Foundation via e-mail(csglobal@mf.seegene.com).

Sample type	Fixation method
Small biopsy tissues	Remoxe moisture from the biopsy tissue and attatch filter paper for fixation on 10% formaling. Ensure that the tissue does not become dry.
Large organs	Fixation of large organs may be insufficient in some cases. Place the tissue section facing downward and add a sufficient amount of faxative for fixation(10-29 times the volume of the tissue).
Lungs	Inject the fixative into the bronchus terminal to inflate the alveoli. After deflating the air, fix the specimen in the fixative.
Mucosal organs, such the digestive tract	Fix the resection sample by exposing the mucosal part and wrapping it with thin gauze. Fix the endoscopic mucosal resection sample in 10% formalin by pinning it flat on syrofoam with the mucosal surface facing upward.

Sample packaging and transport

1)	1. Sample packaging mathod : package the sample using the following three steps. 1) Primary container This is the container that holds the sample. Ensure that the fixative does not leak from the container. This container should be packed with sufficient absorbent material such that ecen if the container is damaged, all solutions are absorbed. 2) Secondary container - This is the container that surrounds the primary container to protect it. It should be sturdy so that the fixative does not leak from it. - When placing multiple primary containers inside a single secondary container, ensure that each primary container is packaged separately so that they are not in direct contact with each other. - Add sufficient absorbent material between the primary and secondary containers such that, even if the fixative is spilled during transport, it will all be absorbed and will not wet the outer packaging. 3) Outer transport Package the secondary container with an appropriate shock-absorbing material so that the outer packaging can protect the contents from external impact including physical damage that may occur during transport. The outer packaging should be at least 10x10cm. 2. Sample transport 1) Inform the transport employees that pathology samples must be transported safely and delivered within the designated time. 2) Check the type and quantity of the pathology samples and the packaging method, departure tine, storage method during transport, and desired storage temperature and write them on the invoice. 3) The transport employees must notify the consignment institution of the completion of transport.
2)	Paraffin Block : 1. During storage of the paraffing blocks, they must not be exposed to temperatures that exceed 27°C (80°F). 2. Insert the properly labeded paraffin blocks in a plastic bag or storage box. 3. Bubble-wrap the plastic baks or storage boxes, secure them with a rubber band or tape, and then place them in an outer shipping box. 4. Insert refrigerated gel packs in the outer shipping box to maintain refregerated temperature during shipping. 5. Please seal the tube tightly to protext from leakage. 6. Please check the ‘Specimen stability’ and pack it according to the ‘Specimen shipping guide’.
3)	Paraffin Block / Slide : 1. The slide holder/mailer containing the properly labeled slides may be used for shipping. 2. The storage boxes should be weapped in bubble wrap, secured with a rubber band or tape and placed in an outer shipping box.