Collection and Processing of Routine Specimens

Blood is often used as the specimen for clinical chemical test, followed by urine, and a few other body fluids (such as saliva, cerebrospinal fluid, hydrothorax and ascites, and amniotic fluid). The content of chemical components in specimens may be changed due to improper specimen collection and processing, thus affecting the accuracy of test results. Therefore, the effects of these factors should be taken into account and tried to eliminate when taking specimens.

I、Blood Specimen

The blood collection tube (BCT) is divided into three types: common tube, anticoagulant tube and procoagulant tube. Common anticoagulants are sodium citrate, EDTA-K2 or EDTA-Na2 and heparin, etc. The choice of proper BCT depends on different testing items.

1.Blood Collection Site:

In order to ensure the accuracy of blood test results, the anterior elbow vein or median vein is generally selected for venous blood collection, and the arm vein and ankle vein may be used when necessary, but the parts with skin lesions should be avoided. Blood collection by jugular vein can be adopted for children. In addition, it can be collected from femoral vein, great saphenous vein, subclavian vein and paraumbilical vein when necessary.

2.Collection Method:

For the determination of blood glucose, blood lipid, protein, uric acid, inorganic ions, and enzymes, blood is collected and injected into a clean test tube, and then centrifuged for serum extraction after being placed at room temperature for 1-2 hours. For the determination of fibrinogen, blood is collected and injected into a test tube containing a specific anticoagulant, and immediately mixed for plasma extraction.

3.During Collection:

No blood should be collected on the side of the patient’s arm where the infusion, transfusion or intravenous injection of certain drugs is taking place. This will result in some test values being too high or too low. The proper collection site should be the anterior elbow vein of contralateral arm.

4.Notes:

a. The proper blood collection tube is compulsory.
b. The needle for blood collection should not be too thin, so as not to increase blood K+.
c. The tourniquet should not be applied for too long during venous blood collection, so as not to cause intravascular hemolysis.
d. A large number of bubbles generated during extraction should not be put into the test tube, so as not to cause hemolysis.
e. No blood should be collected on the side of the patient’s arm where the transfusion is taking place, or even via the front-end indwelling needle while the patient is being infused. For patients implanted with indwelling needle, blood collection from other sites should be taken as far as possible, because the needle is filled with heparin for anticoagulation.
f. The anticoagulant tube must be reversed back and forth for 3-5 times.
g. Serum specimen should be free of hemolysis, and timely centrifuged for serum extraction. It should be sealed and stored at 4 °C, if tested on the alternate day.
h. For blood gas and pH determination, the arterial blood should be collected and free from leakage.
i. Specimens for bacterial culture should be taken using aseptic techniques to prevent pollution.

★ After specimen collection, the test tube or container must be labeled with the test request number, patient’s name, and should be verified on site.

II、Urine Specimen

It is divided into morning fasting urine, random urine, timed urine (2h, 3h, 12h, 24h, etc.), afternoon urine, postprandial urine, and urine when symptoms are typical. In principle, it is better to use no preservative in urine. If 12h or 24h urine is required, refrigeration is preferred and appropriate preservatives are added according to the test purpose. 17 hydroxyl, 17 ketone, VMA test specimens must be preserved with concentrated hydrochloric acid.

III、Bone Marrow

After disinfection as required by bone marrow puncture, wet the needle tube with the appropriate anticoagulant to 5ml scale, discharge excess anticoagulant and air, and then extract the bone marrow and put it in anticoagulation tube as required for further testing. It should be stored and transported at 4 °C. After specimen collection, the test tube or container must be labeled with the test request number, patient’s name, sex, age, transplant status (sex of donor if any), clinical diagnosis, state of bone marrow extraction (dry or not), and should be verified on site.

IV、Cerebrospinal Fluid

The cerebrospinal fluid specimen is collected by clinicians using puncture, and stored in special tubes or containers for timely testing. It should be stored and transported at 4 °C.

V、Hydrothorax and Ascites

They should be extracted and stored in a clean, dry, empty bottle for further testing within 6 hours. The anticoagulant should be added when necessary.

Collection and Processing of Special Specimens

I.Blood Viscosity

1.Preparation:

The patient needs to be on an empty stomach. Blood collection after infusion is prohibited.

2. Specimen Collection:

The patient should be seated and blood collected from the anterior elbow vein.

3. Type and Quantity:

A tube of 4-5ml heparin anticoagulant, and a tube of 2ml sodium citrate (1: 4) anticoagulant.

4. Specimen Preservation:

Blood specimen is collected and left standing for 20 minutes at room temperature for further testing. It should be stored at room temperature (15–25°C) after sealing the container for no more than 4 hours.

II. Insulin/C-peptide

1. Preparation:

Three days before the testing, the daily food glucose content should be not less than 150 g, and the normal activity be maintained. Drugs (such as caffeine, oral contraceptives, and salicylates) that may affect the testing should be discontinued within 3 days. The patient should not eat for 10-16 hours before the testing.

2. Specimen Collection:

The common clinical method is to collect the blood on an empty stomach in the morning, and then collect once every 0.5, 1, 1.5, and 2 hours after meals.

3. Type and Quantity:

No additive tube, 2ml whole blood.

4. Specimen Preservation:

It should be store at 2-8 °C.

III.RAAS Test for Hypertension

1. Preparation:

PRA is generally tested 2 weeks after discontinuing drugs that affect renin levels in the body, such as blockers, vasodilators, diuretics, steroid hormones and liquorices, and 3 weeks after discontinuing drugs that cause slow metabolism such as reserpine. Patients who should not be discontinued should take guanethidine and other antihypertensive drugs that have less effect on PRA. In addition, patients should appropriately reduce their salt intake 3 days before testing, since it will affect their body level. Therefore, it is advisable to determine the urinary sodium content 24 hours before blood collection at the same time, for reference when analyzing the results.

2. Specimen Collection：

5ml of blood should be collected from the cubital vein, and quickly injected into the special anticoagulant tubes, respectively, and shaken evenly.

3. Type and Quantity：

The special anticoagulant tube should be used for blood collection, plasma separation, and (2ml) specimen testing.

4. Specimen Preservation:

The specimen may be stored for 2 months in a cryogenic refrigerator at -20 °C.

5. Notes:

a. Requirements for blood collection: The special test tube (3ml) should be received from our center in advance, and stored at 4 °C for one week. Lying position: in the morning, patients do not get up on an empty stomach or lay down for 2 hours, and 5 ml of blood is collected. After the needle is removed, the blood specimen should be injected into the special test tube (3ml) and heparin anticoagulant tube (2ml), respectively, and shaken gently without severe shock and stored at 4 °C immediately. Standing position: patients remain standing or walking for 2 hours. The collection method is the same as that in lying position. The specimen should be submitted for testing immediately.
b. The test results will be affected by the failure to timely separate plasma from blood specimen, repeated freeze-thaw and hemolysis, and the use of expired anticoagulant tubes.

IV.Sex Hormone Testing

Notes：
a. Fasting is not required for sex hormone testing.
b. Sex hormone drugs (including progesterone and estrogen) cannot be used for at least one month before testing, otherwise the results are unreliable (except for those requiring re-examination of sex hormone after treatment).
c. The basic sex hormone level should be known. Testing should be conducted on the 2nd to 5th day of menstruation; the 3rd day is optimal.
d. If patients are not sure whether vaginal bleeding is menstruation, six items of sex hormones should be tested at the same time to prevent misdiagnosis.

V. Prenatal Screening for Chromosomal Diseases

Notes：

1.The request form must include the pregnant woman’s age, weight, height, date of last menstruation, accurate gestational week (calculated by B ultrasound or last menstrual period), blood collection time, smoking status, insulin treatment for diabetes, single or multiple births. The pregnant woman herself should sign after reading the screening instructions; otherwise, no risk assessment can be made.
2.For prenatal screening for chromosomal diseases (trisomy 21, trisomy 18, neural tube defects) (AFP, β-HCG), the blood should be collected at 14-22 weeks of gestation.

VI.HR-HPV Testing

1. Preparation：

No vaginal douche or intravaginal drug or sexual behavior is allowed within 48 hours. It is prohibited to apply the acetic acid or iodophor before testing. For women with normal menstruation, the specimen should be collected during non-menstrual period, and the optimal testing time is 10-18 days after menstruation.

2. Specimen Collection:
a. A vaginal dilator is placed to expose the cervix, and a cotton swab is used to wipe off excessive secretions from the cervix orifice. A special collecting brush is placed at the junction of the cervical orifice and mucosa and rotated in one direction for 4–5 times.
b. The collecting brush is placed into a special elution tube, and the excess is broken at the incision.
c. The lid is tightened and submitted for testing.

3. Type and Quantity:

The preservation solution of objects brushed from the cervix should be avoided from leaking out while collecting; otherwise the test result will be affected by insufficient specimen quantity.

4.Specimen Preservation:

Specimens should not be stored for more than 12 hours at room temperature, 7 days at 2-8 °C and 3 months at -20 °C. Repeated freezing and thawing should be avoided.

VII.TCT Testing

1. Preparation：

No sexual behavior is allowed three days before testing. The drugs for external use should be discontinued and external genitalia should be washed before testing.

2. Specimen Collection：

The special collector should be used for specimen collection. For patients who have more mucus and blood on the surface of cervix, the cotton swab should be used to gently wipe off the mucus and blood, and the cervical brush should be used against the cervical orifice and rotated for 5-7 times. After collecting, the collecting brush is placed into the preservation solution bottle, and the excess is broken at the incision. After that, the lid is tightened, the patient’s name is labeled on the bottle body, and the request form is filled out for submission.

3. Specimen Type:

Cervical exfoliated cells

4. Specimen Submission and Preservation:

After collecting, the specimen should be submitted timely for testing; if unavailable, it may be stored in a refrigerator at 4 °C.

5. Notes：
a. When collecting specimen with the collector, the long brush at the front end of the collector should be inserted into the cervical canal to collect the cervical epithelium and transitional epithelial cells. Collecting should be done gently and with moderate force, which is too much easy to damage the cervix, and too little to collect enough epithelial cells for diagnosis. 
b. The patient’s name should be clearly written on the submission form and specimen bottle. The request form should be filled in details, especially age, menstrual condition, medical history summary and clinical diagnosis. In case of special circumstances and requirements, please indicate them so as to make a correct diagnosis.

VIII、Collection and Processing of Bacterial Culture Specimens

1. Preparation：

The antibiotics should be discontinued for at least one week before collecting specimen for this item.

2. Sterile Collection：
1.Lower respiratory tract secretion (sputum): When collecting the first sputum in the morning, the mouth should be rinsed repeatedly, and the sputum should be forcibly expectorated after several deep breaths, without any saliva. The sputum should be stored in a refrigerator at 4 °C for further test. 
2.Urine (midstream urine): About 3ml of midstream urine should be collected with the help of nursing staff, placed into the sterile test tube, and stored in a refrigerator at 2-8 °C for further testing. 
3.Pus: The cotton swab stained with normal saline should be used to collect pus as much as possible, and then hung upside down in a sterile test tube. 
4.Blood: For patients suspected of having bacteremia and septicemia, blood specimen should be collected for culture before antibiotic use as far as possible, or collected at the lowest concentration of antibiotics (if any) in the body. Blood specimen is 5-10ml for adults and 2-3ml for children each time. 
**The following three steps are strictly followed for skin disinfection (for patients who are not allergic to iodine):①70% alcohol is used to wipe the venipuncture site for more than 30 s;；②1%-2% iodine tincture is applied for 30s or 10% iodine for 60s, and then disinfected in a circle from the puncture point outward until the diameter of the disinfected area reaches more than 3 cm;③70% alcohol is used for deiodination. Patients, who are allergic to iodine, are disinfected with 70% alcohol for 60 s and blood is collected after the alcohol evaporates and dries. 
**The culture flask should be sterilized according to the following steps:①70% alcohol is used to wipe the rubber plug of blood culture flask for 60 s;②Before injecting the blood into a blood culture flask, the sterile gauze or sterile cotton swab should be used to remove residual alcohol from the rubber plug surface. All actions from drawing blood to transferring it into the flask should be done quickly to prevent blood clotting. Specimen transport: The blood specimen should be submitted for testing immediately after collection; if unavailable, it should be stored at room temperature or in an incubator at 35–37 °C, and never refrigerated. 
5.Puncture fluid (hydrothorax and ascites, pericardial fluid, articulat cavity fluid, and hydrocele): After sterile extraction, the fluid is injected into the sterile test tube containing heparin. The test tube is gently reversed for more than 10 times to uniformly mix heparin with the puncture fluid for anticoagulation. After that, it should be stored in a refrigerator at 4 °C.
6.Bile: 10ml of bile is collected by the doctor in a sterile method, and injected into a sterile test tube.
7.Cerebrospinal fluid: 3-5 ml of cerebrospinal fluid is collected in a sterile method and kept warm in a sterile test tube for further testing. (Please bring your own sterile test tube)
8.Vaginal, cervical and prostate secretions: They should be collected by clinicians, and stored in sterile test tubes for future testing.

IX.Collection and Processing of Venereal Disease Culture Specimens

1.Secretion mycoplasma (ureaplasma urealyticum) culture + drug sensitivity.
Mycoplasma has poor resistance to dry and heat conditions, so the specimen should be collected and transferred into a culture medium for further testing as soon as possible. It may be stored for 8 hours at room temperature, and for 24 hours at 4 °C.
Male: A particularly fine sterile swab is dipped with a little sterile saline, and inserted 2 to 2.5cm into the urethral orifice to get the secretions. After collecting specimen, the swab should be inserted immediately into the culture medium for further testing.
Female: A cotton swab stained with a little sterile normal saline is used to obtain a single-layer columnar epithelial cell 1-2cm from the inner orifice of the cervical canal. The swab should not touch the vaginal wall. After collecting specimen, the swab should be inserted immediately into the culture medium for further testing.

2.Secretion Chlamydia Testing：
For male patients, the swab is inserted 2-4cm into the urethra, rotated for 3-5 seconds and removed. The swab should be placed into a sterile test tube for further testing. 
For female patients, another swab or cotton ball is used to remove cervical secretions before collecting, and then collecting swab is inserted into the cervical canal until the cotton is inserted completely, rotated for 15-20 seconds and removed. The outer cervix and vaginal wall should not be touched. The swab should be placed into a sterile test tube for further testing.
Preservation condition: The specimen should be stored at 2-8 °C after collection, and submitted for testing within 24 hours.

3.Gonococcus Culture + Drug Sensitivity：
*For male patients in acute stage of urethritis, the sterile swab stained with a little normal saline is used to get purulent secretions. For patients in the non-acute stage, the small sterile cotton swab stained with a little normal saline may be gently inserted 2-4 cm into the urethra, rotated and removed. 
* For female patients, the sterile cotton swab is used to remove cervical secretions, another cotton swab stained with normal saline is inserted 1cm into the cervix, and rotated for secretions removal.
After collecting specimen, the cotton swab should be inserted into the gonococcus transport medium for further testing.
Preservation condition: The specimen should be stored in culture medium at normal temperature, and submitted for testing within 24 hours.

4.Candida Culture + Drug Sensitivity：
Preparation: The drugs should be discontinued for at least one week prior to specimen collection.
Specimen type: Genital secretions.
Specimen collection: For patients suspected of vaginal candidiasis requiring culture and drug sensitivity test, the cotton swab stained with normal saline is used to get a large number of bean curd residue-like leucorrhea or suspicious leucorrhea, and placed upside down in a sterile test tube for further testing.

★Please inform our center in advance for microbiological tests and tests requiring special test tubes, so as to provide the corresponding special tube in time for further testing as soon as possible.

X.Routine Cytological Testing

Please fill in the request forms for routine cytological testing or special cytological testing in detail.
1.Hydrothorax and ascites and pericardial effusion testing: 3.8% sodium citrate (1:10 ratio) should be added into the specimens for anticoagulation. In summer, 10% formalin or 95% alcohol (1:10 ratio) should be added for anti-corrosion. The optimal specimen size should be 100-200ml.
2.Sputum and urine testing: The sputum should be fresh from the first deep cough in the morning, without too much saliva, nasal secretions, food and saliva. The sputum specimen should be placed and sealed in a clean cup or wax cardboard box and other containers for further testing, free from spillover or leakage. Urine volume should be collected for 12-24 hours, and supernatant should be discarded, and 100ml-200ml precipitate should be left for further testing; preservatives should be added if necessary. In addition, the special cell preserving fluids for sputum, bronchoalveolar lavage fluid, and urine can be available for further testing.
3.Tumor puncture specimens and bronchial brushing specimens testing: After collecting, 2 to 4 smears can be taken, and directly submitted for testing after being naturally dried. In addition, the special cell preserving fluids for FNAB, TBS diagnosis can be available for further testing.

XI. Collection and Processing of Histopathological Examination Specimens

General pathological specimen processing: After the surgical specimen is isolated, it is quickly immersed in 10% neutral formalin, and subjected to sampling, dehydration, paraffin embedding, slicing, HE staining (hematoxylin-eosin staining), and image viewing. The pathological application form must be submitted together with the specimen. The application form contains all necessary contents such as patient information, medical history, number and location of specimens, etc. In addition, corresponding special staining and immunohistochemistry may also be selected according to the need of pathological diagnosis.

1.Endoscopic and Puncture Specimens
Small biopsy tissues such as laparoscope, fiberoptic bronchoscopy, gastroscopy and enteroscopy or small puncture tissues of each organ should be quickly fixed in 10% neutral formalin solution after collecting, otherwise the tissues may be easily dehydrated and dried to affect the diagnosis. The corresponding locations should be indicated, such as gastric antrum.

2.Biopsy Tissue Collecting
a.The appropriate collecting site should be selected. The tissue with obvious lesions should be excised in every possible way. In the case of tumor tissue, the excised part must contain the surrounding normal tissue (the region around a tumor may reflect the interface between adjacent tissues). In case of ulcer tissue, its surrounding and underlying tissues should be excised for further testing. 
b.When collecting specimens, extrusion and cauterization should be avoided by all means. The collecting knife should be sharp and operated correctly, so as to avoid excessive extrusion of tissues, which may lead to severe deformation of cell structure and morphology to affect the diagnosis, or even cause unnecessary re-collecting, causing unnecessary pain to patients and prolonging the diagnosis time.
c.After collecting, the cervical biopsy specimens should be labeled with the corresponding hourly position, and then stored separately.
d.After collecting, the tissue specimens should be immediately fixed in 10% neutral formalin solution.

3.Surgical and Whole-organ Large Tissue Specimens
a.All tissues excised during surgery should be submitted for testing, and the integrity of the specimens should be maintained (surgical specimens with a diameter of more than 5cm may be cut open without breaking, so as to keep the overall structure of the specimen from being damaged, and help formalin to permeate all the specimens. Otherwise, it is easy to cause the degeneration inside the specimen caused by inability to fix in time, affecting the diagnosis. In case of lymph node, it should be dissected and placed in a container). After the specimen is isolated, it should be immersed in 10% neutral formalin within 30min, and submitted for testing together with the pathological application form.
b.The inlet of container containing specimens should not be too small, to ensure the specimen is not deformed and may be removed smoothly. The container should be sealed to avoid insufficient concentration due to evaporation of the fixed liquid, thus affecting the fixation effect. 
c.All parts of tissues should be immersed completely in the fixed liquid, so as to ensure adequate tissue fixation (the amount of fixed liquid should be more than 5 times of that of the specimen). Neutral formalin has strong permeability to tissues, long preservation time and good fixation effect. The fixation with 95% alcohol should be avoided as far as possible, or it will lead to excessive sclerosis of tissues, which will affect the biopsy and pathological diagnosis. In addition, 75% alcohol or normal saline should not be used, since they cannot be used for tissue fixation. The special fixed liquid is available, if there is any special request.

Collection and Processing of Specimens in Blood Item

I. Matters Needing Attention for Routine Specimens of Hematologic Tumor Detection (PCR, Flow and Genetic)

1.Bone marrow specimens are not strictly required to be inspected for some items. If peripheral blood can only be inspected for some reasons, then WBC>5×109/L in peripheral blood; there is no limit in cell population of flow cytometric immunophenotyping.
2.For a patient who pays the first visit to a doctor and is suspected to suffer from acute leukemia or multiple myeloma, as long as the proportion of abnormal cells or primitive cells in peripheral blood reaches above 20%, peripheral blood can be used for flow immunophenotyping detection; as long as the proportion of abnormal cells or primitive cells in peripheral blood reaches above 30%, karyotype analysis as well as detection of related FISH items and related detection of fusion genes can be done. For a patient who is suspected to suffer from chronic myelogenous leukemia (CML), as long as juvenile cells appear in peripheral blood, peripheral blood can be used for flow immunophenotyping detection. For a patient who is suspected to suffer from chronic lymphocytic leukemia, peripheral blood can be directly used for flow immunophenotyping detection. Flow platform-type leukemic micro-residual disease can only be detected with bone marrow (peripheral blood can be also used for detection in special cases).
3.Tissue specimens are best used for detection of relevant items in lymphoma and bone marrow can be used next but peripheral blood cannot be used for detection (unless it is confirmed that metastasis occurs).

II. Methods for Collection of Bone Marrow

1.Collection of bone marrow specimens suitable for bone marrow cytology detection, bone marrow karyotype detection, molecular biological detection and cytochemical staining detection.
2. Operating steps:
a.Position: the puncture point of the anterior superior iliac spine is located 1-2 cm behind the anterior superior iliac spine; the puncture point of the anterior superior iliac spine is located at both sides of sacral vertebrae and the protruding part of the upper buttocks. The sternal puncture point is located in the manubrium sterni or gladiolus equivalent to the site between the 1st intercostal space and the 2nd intercostal space. The puncture point of the interspinous process is located in the protruding part of the lumbar spinous process.
b.Methods:
1) After routine disinfection, wear sterile gloves, cover a sterile hole towel and use 2% lidocaine for local cutaneous, subcutaneous and periosteal anesthesia. 
2) Fix the puncture needle holder to the appropriate length (about 1.0 cm for sternal puncture and about 1.5 cm for ilium puncture). Fix the index finger and the middle finger of the left hand at the site of puncture and hold the needle in the right hand and thrust it vertically into the bone surface (for sternal puncture, an angle of 30°-40° should be maintained between the needle body and the bone surface). When the point of the needle touches the sclerotin, the puncture needle will be rotated around the long axis of the needle body, slowly piercing the sclerotin. When the resistance is felt to disappear and the puncture needle is fixed in the bone, it shows that it has entered the marrow cavity. 
3) Pull out the core of the needle, place it in a sterile plate, and connect a dry 10 ml or 20 ml syringe (the cylinder wall of the syringe shall be a heparin or EDTA anticoagulant moistened tube) and extract with appropriate force. That is, a small amount of red bone marrow liquid enters the syringe and the amount of bone marrow extracted should be the required amount. Transfer the extracted specimens into the corresponding anticoagulant tubes for preservation. 
4) Drip drops of extracted bone marrow on several glass slides and these several glass slides are respectively examined for karyocyte count, morphological and cytochemical staining. 
5) After extraction is completed, reinsert the core of the needle; take the sterile gauze with the left hand, place it at the hole of the needle and pull out the puncture needle with the core of the needle together with the right hand. Immediately cover the gauze on the hole of the needle, press for 1~2 minutes and then fix the gauze with an adhesive plaster.
3. Matters needing attention 
a. Do a blood coagulation examination. For patients with bleeding tendency, special attention should be paid to the process of operation. Hemophiliacs are prohibited to do a blood coagulation examination. 
b. The puncture needle must be dry to avoid hemolysis. 
c. After the needle enters the sclerotin, avoid swinging too much to avoid breaking; the sternal puncture should not be overexerted to prevent penetration of the medial bone lamellas. 
d. The amount of extracted liquid should not be excessive for cell morphology examination. 
e. If a patient is suspected to suffer from sepsis, after the smear test, a syringe is inserted to extract 1.0ml of bone marrow fluid for bone marrow culture. 
f. After bone marrow fluid is extracted, it is necessary to do a smear test immediately, or else it will coagulate very soon to cause the failure of the smear test.  
g. After collecting specimens, specimens must be immediately sent for detection. Transportation of specimens and storage before experiment: low temperature condition of 2-8 °C.

III. Qualitative, Quantitative and Mutant Specimens of Fusion Genes in Hematologic Diseases Sent for Detection

1. It is required to use the testing item of bone marrow specimen, collect 2-3 ml of bone marrow according to the conventional collection method of bone marrow (aseptic operation), place it in the EDTA anticoagulant tube (purple cap), and quickly reverse it for several times to prevent the specimen from coagulating. The use of heparin as an anticoagulant is strictly prohibited because it inhibits PCR amplification and it is very difficult to remove completely in the process of nucleic acid extraction.
2. Refrigerate at 4 °C and deliver preferably within 24 hours.
3. Please fill in a special application form and annex with primary diagnosis, chemotherapy history, medical history, treatment history as well as results of cytological examination of bone marrow and peripheral blood routine and cytological examination.
4. If a bone marrow specimen is not available, the doctor wants a peripheral blood examination to know about the state of the illness. Requirements of a peripheral blood specimen: 2-3 ml peripheral blood, EDTA anticoagulant (purple-cap tube), white blood cell count>5×109/L and the proportion of primitive cells or abnormal cells reaches above 20%. However, since the total number of cells and the number of abnormal cells in bone marrow are both greater than those in peripheral blood, the reported results may not accurately reflect the state of the illness and the reference value of the report needs to be considered by doctors. A doctor shall be invited to fill out the application form in details. Please annex with primary diagnosis, chemotherapy history, medical history, treatment history as well as results of cytological examination of bone marrow, peripheral blood routine and cytological examination.
5. If the whole blood or bone marrow specimen is used for DNA extraction and detection, it can be stored at 4 °C for a short time; if the whole blood or bone marrow specimen is used for RNA detection, it should be delivered as soon as possible after blood is collected, or otherwise it will be easy for RNA to degrade.
6. If coagulation or hemolysis occurs, heated or frozen specimens and other specimens anticoagulated by other methods are all disqualified specimens and specimens need to be collected again; specimens should be aseptic and should not be close to the ice bag in transport.

IV. Specimens Sent for Detection in Relevant Testing items on Flow Platform in Hematologic Diseases

1. Requirements of Bone Marrow Specimens
1.1 For the testing items with bone marrow specimens, it is required to collect 2-5 ml of bone marrow according to the conventional bone marrow collection method (aseptic operation), put the bone marrow into the heparin anticoagulant tube (green cap) or EDTA anticoagulant tube (purple cap) and reverse and mix it gently to prevent the specimen from coagulating.
1.2 It shall be refrigerated at 4 °C and sent for detection within 48 hours.
1.3 Please fill out the special application form and annex with primary diagnosis, chemotherapy history, medical history, treatment history as well as results of cytological examination of bone marrow, peripheral blood routine and cytological examination.
1.4 If coagulation or hemolysis occurs or the cell counting is too low (lower than 1×105 /ml), heated or frozen specimens and other specimens anticoagulated by other methods are all disqualified specimens and specimens need to be collected again; specimens should be aseptic and should not be close to the ice bag in transport.
1.5 Please annex Bone Marrow Morphology Report if there is one such report and please annex bone marrow smear (white) if there is no such report.
1.6 Please fill out the application form for detection. For detection of a minimal residual disease, please annex with results of immunotyping detection in preliminary diagnosis.
2. Requirements of Peripheral Blood Specimens
2.1 Collect 3-5 ml of venous blood, place in heparin anticoagulant tube (green cap) or EDTA anticoagulant tube (purple cap), and reverse and mix gently.
2.2 It shall be refrigerated at 4 °C and sent for detection within 48 hours.
2.3 If coagulation or hemolysis occurs or the cell counting is too low (lower than 1×105 /ml), heated or frozen specimens and other specimens anticoagulated by other methods are disqualified specimens and specimens need to be collected again; specimens should be aseptic and should not be close to the ice bag in transport.

V. Specimens in Relevant Testing items on Genetic Platform in Hematologic Diseases Sent for Detection

1. Requirements of Bone Marrow Specimens
1.1 For the testing items with bone marrow specimens, it is required to collect 3-5 ml of bone marrow according to the conventional bone marrow collection method (aseptic operation), put it into the heparin anticoagulant tube (green cap) and reverse rapidly for several times to prevent the specimen from coagulating. EDTA is strictly prohibited as an anticoagulant.
1.2 It shall be refrigerated at 4 °C, it is best to deliver it within 24 hours, and the longest time is no more than 48 hours.
1.3 Please fill out the special application form and annex with primary diagnosis, chemotherapy history, medical history, treatment history as well as results of cytological examination of bone marrow, peripheral blood routine and cytological examination.
1.4 If a bone marrow specimen is not available, the doctor wants a peripheral blood examination to know about the state of the illness. Requirements of a peripheral blood specimen: 3-5 ml peripheral blood, heparin anticoagulant (green-cap tube), white blood cell count>5×109/L, the proportion of primitive cells or abnormal cells reaches above 30%. However, since the total number of cells and the number of abnormal cells in bone marrow are both greater than that in peripheral blood, the reported results may not accurately reflect the state of the illness and the reference value of the report needs to be considered by doctors. If the purpose of the detection is to monitor curative effect, the use of peripheral blood specimens will be meaningless. A doctor shall be invited to fill out the application form in details. Please annex with primary diagnosis, chemotherapy history, medical history, treatment history as well as results of cytological examination of bone marrow, peripheral blood routine and cytological examination.
1.5 If coagulation or hemolysis occurs, heated or frozen specimens and other specimens anticoagulated by other methods are disqualified specimens and specimens need to be collected again; specimens should be aseptic and should not be close to the ice bag in transport.
2. Requirements of Peripheral Blood Specimens
2.1 Collect more than 5 ml of venous blood, place in heparin anticoagulant tube (green cap), and reverse and mix gently; there is no special requirement for white blood cell count and the white blood cell count cannot be lower than normal range (4.0×109/L-10.0×109/L).
2.2 It shall be refrigerated at 4 °C and sent for detection within 24 hours.
2.3 If specimens are in hypercoagulable state, hemolysis occurs, cells are fragile or the cell counting is too low (lower than 1×105 /ml), heated or frozen specimens and other specimens anticoagulated by other methods are disqualified specimens and specimens need to be collected again; specimens should be aseptic and should not be close to the ice bag in transport.
2.4 A doctor shall be invited to fill out the application form in details. Please annex with primary diagnosis, medical history as well as results of peripheral blood routine examination.

Collection and Processing of Solid Tumor Specimens

I. Gene Mutation Specimens Sent for Detection

Paraffin Specimens

It is used to detect somatic mutation of tumor tissue. In order to ensure the success rate of genomic DNA extraction and normal detection cycle from paraffin specimens, please prepare specimens sent for detection according to the following requirements:
1. The thickness of sections is 5-10mm and the number of sections depends on the area of cross section of tumor tissue. For example, for the size of an adult’s thumb cover, 6~8 sections can meet the needs of DNA extraction; if the area of tumor tissue sections is small, proportionally increase the number of sections.
2. In order to ensure that the section tissue contains a sufficient proportion of tumor cells, the department of pathology should conduct HE staining on the tumor tissue of the section in advance to ensure that there is more than 70% of tumor tissue in the section and there is less than 10% of necrotic tissue. If there is no condition for staining, you can meanwhile submit a white smear of neighboring sections for detection and we’ll test it for you.
3. After slicing is completed, stretching and pasting is not required and clean tweezers are directly used to transfer tissue sections into a 1.5ml clean centrifugal tube.
4. Please provide two tubes of specimens for detection as far as possible, one tube for detection and the other tube as backup material.
5. Specimens shall be stored and transported at room temperature.
6. In order to ensure the success rate of DNA extraction from paraffin specimens, please send paraffin specimens within 2~3 years for detection as far as possible.

Fresh Tissue Specimens

It is used to detect somatic mutation of tumor tissue.
1. Fresh tumor tissue specimens should be ≥100mg and the proportion of tumor tissue with no necrosis should be above 70%.
2. After the operation, first wash the tissue mass twice with normal saline and then place the tumor tissue of the appropriate size in a labeled cryogenic vial. The tumor tissue shall be quickly frozen and delivered at low temperature (in an ice box) within 8 hours. If it cannot be delivered in time, it must be stored in liquid nitrogen or a -70°C refrigerator within 20 minutes separately and delivered at low temperature within two weeks.
3. The tumor tissue after biopsy or the surgically excised tumor tissue can also be placed in 10% neutral formalin solution (the time of fixation is no longer than 4 hours) and the fixed tissue specimen can be stored and transported at normal temperature.

Peripheral Whole Blood Specimen

Peripheral whole blood specimen is used to detect a patient’s germinal mutation or an individual’s gene polymorphism (SNP).
1. Collect 3-5 ml of venous blood, place in an EDTA anticoagulant tube (purple cap) and reverse and mix it gently.
2. Attention must be paid to the selection of anticoagulants. EDTA-Na2, potassium oxalate or sodium citrate is generally used and the use of heparin is strictly prohibited because it inhibits PCR amplification and it is difficult to be completely removed in the process of nucleic acid extraction.
3. It shall be refrigerated at 4 °C and delivered within 24 hours.
4. If the peripheral whole blood cannot be delivered in time after collection, it is better not to be directly frozen at -20 °C. It is suggested to prepare into peripheral blood mononuclear cells (PBMC). There are two primary ways. One is to use lymphocyte separation solution for separation and preparation. The other is to use the erythrocyte lysate to lyse the erythrocytes in the whole blood. After washing with normal saline for several times, mononuclear cells can be obtained at the bottom of the tube. If nucleic acid is not extracted temporarily, the peripheral blood mononuclear cells can be stored in a refrigerator at -20 °C for a long time.

Plasma Specimen

Plasma specimens are mainly used for the detection of free DNA in plasma tumors.
1. Collect 3-5 ml of venous blood (sterile operation), place in an EDTA anticoagulant tube (purple cap), leave it standing at room temperature for 30 minutes, centrifuge at 3000 rpm for 10 minutes and get supernatant in a clean centrifugal tube.
2. It shall be refrigerated at 4 °C and delivered within 24 hours.
3. If the plasma specimens cannot be delivered in time, they can be stored at -20 °C for a short period (1~2 weeks) and shall be stored at -70 °C for a long period; attention should be paid to avoid DNA damage due to shear forces of repeated freezing and thawing, and the frozen plasma specimens should be delivered at as low a temperature as possible (in ice boxes) for future detection.
4. If there is no condition to centrifuge, it can also be equivalent to the requirement of detection in peripheral whole blood, and it can be delivered as soon as possible within 4 hours by placing it in the EDTA anticoagulant tube, and then we can separate the plasma for you.

Body Fluid (Hydrothorax) Specimen

For body fluid specimens from patients with malignant tumors (such as malignant hydrothorax from patients with lung cancer), most of the tumor cells are cast-off cells sourced from tumor tissues after centrifugation to gain tumor cells, which can be used to detect somatic mutations in tumor tissues. This is of great significance for some patients with postoperative recurrence or advanced metastases and no chance to receive operation.
1. After routine thoracentesis, extract body fluid specimens such as hydrothorax and gently inject into the collection container. After thoracentesis, leave body fluid specimens standing for 2 hours. Take 10~15 mL of hydrothorax near the bottom of the container and place in a closed centrifugal tube or glass bottle.
2. It shall be refrigerated at 4 °C and delivered within 24 hours.

II. Specimens Sent for Detection in FISH Detection

Fresh Tissues

1. The tissue should be fixed with neutral formalin and stored at room temperature and the fixation time of the specimen should not exceed 4 days. If the fixation time is more than 4 days, we will ask the detection unit to sign a trial application or send the specimen again.
2. The tissue sent for detection should be cancer tissue. We will do quality inspection on the specimens sent for detection by HE staining. If there is no cancer issue in the specimen sent for detection, the count of cancer cells is less than count capacity or necrosis occurs in a large number of cancer cells, we will notify the hospital to receive detection and the hospital to receive detection will decide whether to send specimens again or quite detection.
3. Specimens shall be stored in neutral formalin stationary liquid and delivered at normal temperature within 48 hours.

Paraffin Block

1. The Department of Pathology of the hospital to receive detection shall use neutral formalin to fix the tissue and the fixation time of the specimen shall not exceed 4 days. If the fixation time is more than 4 days, we will ask the detection unit to sign a trial application or send the specimen again.
2. The paraffin block sent for detection should be a cancer tissue paraffin block. We will do quality inspection on the specimens sent for detection by HE staining. If there is no cancer issue in the specimen sent for detection, the count of cancer cells is less than count capacity or necrosis occurs in a large number of cancer cells, we will notify the hospital to receive detection and the hospital to receive detection will decide whether to send specimens again or return the specimen to be sent for detection.
3. Please indicate the type, time of fixation, storage and transportation at normal temperature of stationary liquid at an obvious location in the detection application form.

Paraffin Section

1. The tissue should be fixed with neutral formalin and stored at room temperature and the fixation time of the specimen should not exceed 4 days. If the fixation time is more than 4 days, we will ask the detection unit to sign a trial application or send the specimen again.
2. The thickness of sections shall not be more than 4 μm. It is required to use “+” sections or dedicated immunohistochemical exfoliation-proof glass slides (APES glass slides). FISH detection will fail since ordinary white smears may exfoliate in the process of detection. If sections to be inspected are ordinary white smears, the detection unit is required by us to sign a trial application.
3. 6 “+” sections and 1 white smear are required for 1p/19q FISH detection; 4 “+” sections and 1 white smear are required for FISH testing items for other solid tumors.
4. The tissue sent for detection should be cancer tissue. We will do quality inspection on the specimens sent for detection by HE staining. If there is no cancer issue in the specimen sent for detection, the count of cancer cells is less than count capacity or necrosis occurs in a large number of cancer cells, we will notify the hospital to receive detection and the hospital to receive detection will decide whether to send specimens again or return the specimen to be sent for detection.
5. Please mark the type of stationary liquid, time of fixation and thickness of sections in a prominent position on the detection application form.
6. Sections shall be securely placed in a professional section box and stored and transported at room temperature.

Urine

1. The specimen to be sent for detection shall be fresh urine no less than 200 ml. After extraction, urine and preservation solution should be mixed in the following proportion: urine: preservation solution * = 2:1 (V/V) and the mixed liquor should be stored at room temperature.
2. If there are too few cast-off cells in the urine to meet the count requirement, we will inform the hospital to receive detection and the hospital to receive detection will decide whether to re-send the specimen or return the specimen to be sent for detection.
3. The specimen shall be stored at temperature and delivered within 72 hours.