According to the pneumonia treatment protocol for novel coronavirus infection (trial version 4) released on the 28th, respiratory or blood specimens should be collected for novel coronavirus nucleic acid testing, or viral gene sequencing for suspected cases. How is specimen collection performed? What are the key points of operation? How can health care workers do self-protection?
On the same 28th, the National Health and Wellness Commission released the "Pneumonia Prevention and Control Program for Novel Coronavirus Infection (3rd Edition)", and also released as an annex the "Technical Guidelines for Laboratory Testing of Pneumonia with Novel Coronavirus Infection (3rd Edition)", which provides detailed instructions on the specific requirements and procedures for specimen collection.
Specimen collection target
Suspected cases of pneumonia with novel coronavirus infection, patients with suspected aggregated cases.
Others who require diagnosis or differential diagnosis of novel coronavirus infection.
or other environmental or biological materials that require further screening tests (e.g., traceability analysis).
Specimen collection requirements
1. Technical personnel engaged in specimen collection for novel coronavirus testing should be trained in biosafety (training qualified) and have the appropriate laboratory skills.
Sampling personnel personal protective equipment (personalprotectiveequipment, PPE) requirements: N95 and above protective masks, goggles, one-piece protective clothing, double-layer latex gloves, waterproof boot covers; if contacted with patient blood, body fluids, secretions or excretions, the outer latex gloves should be replaced in a timely manner.
2. Specimens of hospitalized cases are collected by the medical and nursing staff of the hospital.
3. Specimens from close contacts are collected by the local designated CDC and medical institutions.
4. According to the needs of laboratory testing, sampling can be combined with the disease process several times.
Specimen collection types
Each case must collect acute respiratory specimens (including upper respiratory specimens and lower respiratory specimens); in severe cases, priority is given to collecting lower respiratory specimens (such as bronchial or alveolar lavage fluid, etc.); in cases with symptoms of eye infection, conjunctival swab specimens need to be collected; in cases with symptoms of diarrhea, stool specimens need to be retained. Collection can be done according to the clinical presentation and sampling interval.
Other study materials are collected according to design requirements.
Specimen types.
1. upper respiratory tract specimens: including pharyngeal swabs, nasal swabs, nasopharyngeal extracts, etc.
Lower respiratory tract specimens: including deep cough sputum, respiratory tract aspirates, bronchial lavage, alveolar lavage, and lung tissue biopsy specimens.
3. Blood specimens: Try to collect anticoagulated blood in the acute phase within 7 days after the onset of the disease. The collection volume is 5 ml, and fasting blood is preferred. It is recommended to use vacuum blood collection tubes containing EDTA anticoagulant to collect blood.
4. Serum specimen: Try to collect double copies of serum in the acute phase and recovery phase. The first serum should be collected as early as possible (preferably within 7 days after the onset of the disease), and the second serum should be collected in the 3rd to 4th week after the onset of the disease. The collection volume is 5 ml and the use of vacuum blood collection tubes without anticoagulant is recommended. Serum specimens are mainly used for the determination of antibodies and to confirm the infection status of the case from the serum antibody levels. Nucleic acid testing is not performed on serum specimens.
5. Conjunctival specimens: Conjunctival swabs are collected in cases with symptoms of ocular infection.
6. Stool specimens: Patients with diarrhea symptoms should have stool specimens collected.
Specimen collection method
1. Pharyngeal swab: wipe the bilateral pharyngeal tonsils and posterior pharyngeal wall simultaneously with 2 plastic rod swabs with polypropylene fiber heads, dip the swab head into a tube containing 3 ml of virus preservation solution (isotonic salt solution, tissue culture solution or phosphate buffer can also be used), discard the tail and screw the cap tightly.
2. Nasal swab: Gently insert 1 polypropylene fiber-tipped plastic rod swab into the nasal tract at the nasal palate, leave for a moment and then slowly rotate to withdraw. Take another polypropylene fiber-tipped plastic rod swab to collect the other side of the nostril in the same way. The above two swabs were dipped into the same tube containing 3 ml of sampling solution, the end was discarded and the cap was screwed on tightly.
3. Nasopharyngeal extractions or respiratory extractions: Mucus is extracted from the nasopharynx or respiratory secretions from the trachea using a collector connected to a negative pressure pump. Insert the collector head into the nasopharynx or trachea, connect the negative pressure, rotate the collector head and withdraw slowly to collect the extracted mucus, and rinse the collector once with 3ml of sampling solution (the collector can also be replaced by a pediatric catheter connected to a 50ml syringe).
4. Deep coughing of sputum: Ask the patient to cough deeply and then collect the coughing sputum in a 50ml screw-top plastic tube containing 3ml of sampling fluid.
5. Bronchial lavage fluid: Insert the collector head into the trachea (about 30cm deep) through the nostril or tracheal socket, inject 5ml of saline, connect the negative pressure, rotate the collector head and withdraw it slowly. Collect the extracted mucus and rinse the collector once with the sampling fluid (a pediatric catheter connected to a 50 ml syringe can also be used to collect instead).
6. Alveolar lavage fluid: After local anesthesia, insert the fiberoptic bronchoscope through the mouth or nose through the pharynx into the bronchus of the middle lobe of the right lung or the lingual segment of the left lung, fit its tip into the opening of the bronchial branches, and slowly add sterilized saline through the tracheal biopsy hole, 30-50 ml each time, total 100-250 ml, should not exceed 300 ml.
7. Blood specimen: It is recommended to use a vacuum blood collection tube containing EDTA anticoagulant to collect 5ml of blood specimen, leave it at room temperature for 30 minutes, centrifuge it at 1500-2000rpm for 10 minutes, and collect plasma and blood cells in sterile screw-top plastic tubes respectively.
8. Serum specimen: collect 5ml of blood specimen by vacuum negative pressure blood collection tube, let stand at room temperature for 30 minutes, centrifuge at 1500-2000rpm for 10 minutes, and collect serum in sterile spiral plastic tube.
9. stool specimens: If the patient shows diarrhea symptoms early in the course of the disease, 3-5 ml of stool specimens are retained.
10. Conjunctival swab specimen: After the conjunctival surface is gently wiped with a swab, the swab head is put into the sampling tube and the tail is discarded, and the tube cover is suspended tightly.
Other materials: collected according to the design requirements specification.
Specimen packaging
Specimens are collected and dispensed in a biosafety cabinet in a biosafety level II laboratory.
1. All specimens should be placed in appropriately sized sample collection tubes with screw caps with gaskets inside and freezing resistance, tightened. Indicate the specimen number, type, name and date of sampling on the outside of the container.
2. Seal the sealed specimens in plastic bags of appropriate size, one specimen per bag. The specimen packaging requirements should comply with the corresponding standards of the Technical Rules for the Safe Transport of Dangerous Goods by Air.
3. If external specimen transportation is involved, the specimen should be packed in three layers according to the type of specimen, according to Class A or Class B infectious substances.
Specimen preservation
Specimens used for virus isolation and nucleic acid detection should be tested as soon as possible. Specimens that can be tested within 24 hours can be stored at 4°C; specimens that cannot be tested within 24 hours should be stored at -70°C or below (if no -70°C storage conditions, then stored temporarily in a -20°C refrigerator).
Serum can be stored at 4℃ for 3 days, and below -20℃ for long term storage.
Specimens should be stored separately in special storage or cabinets. Repeated freezing and thawing of specimens should be avoided during transport.
Specimen delivery
Specimens should be sent to the laboratory as soon as possible after collection. If specimens need to be transported over long distances, it is recommended to use dry ice and other refrigeration methods for preservation.
1. Sending specimens up
Specimens from aggregated cases in each province (autonomous region and municipality directly under the central government) should be sent to the Institute of Viral Disease Prevention and Control of the Chinese Center for Disease Control and Prevention for testing and review, with the sample delivery form.
