CONTENTS
1. INTRODUCTION
2. LABORATORY OPERATION
2.1. TEST PROMPT AND ENTRİES
2.2. RULES FOR TAKING SAMPLES OF THE TESTS WORKED
2.2.1. Preliminary rules for pre-sampling tests
2.2.2. Patient preparation guidelines before sample collection
2.2.3. Labeling of samples
2.2.4. Rules for sampling
2.2.4.1. Taking samples
2.3. TRANSFER OF SAMPLES
2.4. LABORATORY ACCEPTANCE, REJECTION, SEPARATION OF SAMPLES
2.4.1. Preliminary assessment in sample reception areas
2.4.2. Example Rejection/Acceptance Criteria
2.4.3. Statistical analysis of rejected samples
2.4.4. Initiation of corrective and preventive actions
2.5. ANALYSIS OF TESTS
2.5.1. Internal quality control studies
2.5.2. Running the tests
2.6. SAMPLE STORAGE PHASE
2.7. CONFIRMATION OF RESULTS
2.8. REPORTING RESULTS
2.9. STORAGE OF RESULTS
1. INTRODUCTION
In this guide, the operation of the Detagen Genetic Diseases Evaluation Center, the tests, the process from the examination entry to the sending of the results, the analytical quality studies of the laboratory, the working methods of the tests, the working times, the sample type / sample container selection according to the test, the acceptance / rejection criteria of the samples, the reporting of the results. time, normal values, panic values, if any, and briefly the clinical benefits of the tests were given.
2. LABORATORY OPERATION
2.1. TEST PROMPT AND ENTRIES
In outpatients who come to our center, after the patient is examined by our geneticist, the test that the doctor deems appropriate is marked on the test request form and the test request is made. For samples coming from outside, it can be done by the relevant doctor at the sending center by marking them on the test request form or the samples marked on the test request form are taken.
For test entries, the patient is selected from the patient list in the LIMS system and the "sample information" menu is selected. The name of any test/tests requested will appear here and a test can be added from the Add Test option. After the test materials taken from the patients or the test samples from outside reach the laboratory, they are received by the sample acceptance personnel. Samples that reach the center by cargo from outside are delivered by filling in the cargo delivery form. After the necessary pre-processes, the relevant personnel accepts or rejects the sample according to the rejection/acceptance criteria.
2.2. RULES FOR TAKING SAMPLES OF THE TESTS WORKED
2.2.1. Preparation Rules for Pre-Sampling Tests
It is indicated if preliminary preparation is required for the tests performed in the laboratory.
2.2.2. Patient Preparation Rules Before Sample Collection
- For the blood sample, the appropriate sample tube is selected according to the tests, the patient's name, surname and the desired test birth date are written on the selected tubes.
- While the patient is resting in the blood collection chair, the desired tests are checked and a short history is taken from the patient. If there is no inappropriate situation, blood collection is performed.
- Before the blood draw of the babies, the baby is placed on a stretcher, while the baby is expected to calm down and adapt to the environment, the family is informed about the blood collection process. If it is tried once, if it is not successful, it is directed to a center with a baby nurse to have blood drawn.
2.2.3. Labeling of Samples
Lab number and test name are written on all samples taken.
2.2.4. Rules Regarding Sampling
2.2.4.1. Taking Samples
It is aimed to collect the samples of the patients who applied to the laboratory under the most suitable conditions and to prepare them for the study.
A) Collecting Blood Samples - Venous Blood Collection
1. The patient should be in a lying or sitting position during blood collection.
2. For blood collection, preference should be given to the upper extremity veins.
3. The needle tip should be chosen as wide as possible.
4. The tourniquet should not be stuck on the arm for more than 30 seconds.
5. The tourniquet should be released after insertion of the needle into the vein.
6. Vigorous aspiration of blood into the tube should be avoided during blood collection with the syringe.
7. Care should be taken to ensure that the blood fills up to the marked line during blood collection into vacuum tubes containing anticoagulants.
8. Immediately after blood is drawn into tubes containing anticoagulants, the tube should be gently inverted and mixed carefully. Shaking should be strictly avoided.
1) Whole blood with EDTA
1. Blood is taken into 2 ml K3 EDTA glass tubes with purple caps.
2. In order to prevent clots from forming in the tubes, as soon as the blood is taken, it is mixed by gently inverting it 5-6 times. During blood collection, care should be taken to fill the blood up to the marked line. Samples should be stored in the refrigerator until they are studied.
2) Whole Blood with Heparin
1. The blood sample is taken into green capped tubes with lithium heparin.
2. In order for the blood sample to be mixed with lithium heparin, it must be filled completely up to the marked line and very slowly inverted 5-6 times to ensure full contact of the blood with the anticoagulant.
B. Amniotic Fluid
1. At least 15-20 ml of amniotic fluid should be taken.
2. The sample should be transported to the laboratory immediately and studied immediately. If not possible, it can be stored at 2-8 °C for a maximum of 3 days.
C. Chorionic Villus Sampling
At least 20 -30 mg of chorionic villus biopsy sample should be taken.
The sample should be transported to the laboratory immediately in a tube containing sterile transport medium and should be studied immediately. In cases where it is not possible, it can be stored at 2-18 ° for a maximum of 3 days.
D. Evacuation Material
Examples that can be submitted in abortion materials:
Placental biopsy: At least 1 cm3 piece should be taken from the region close to the umbilical origin (fetal origin).
Skin biopsy: Approximately 5 mm2 piece should be taken from the back, legs and hips.
Cord biopsy: About 2 cm piece should be taken
Amnion biopsy: At least 2 cm2 piece should be taken from the area closest to the umbilical cord.
The sample should be immediately transported to the laboratory in a tube containing sterile transport medium or in a sterile falcon and studied immediately. In cases where it is not possible, it can be stored at 2-18 ° for a maximum of 3 days.
E. Bone Marrow
Approximately 2-3 ml of bone marrow sample is taken into green capped tubes with lithium heparin.
In order for the bone marrow sample to be mixed with lithium heparin, it must be filled up to the marked line and very slowly inverted 5-6 times to ensure full contact of the blood with the anticoagulant.
F. Fibroblast Skin Biopsy
At least 1 cm3 biopsy sample should be taken.
The sample should be transported to the laboratory immediately in a tube containing sterile transport medium and should be studied immediately. If not possible, it can be stored at 2-8°C for a maximum of 3 days.
G. Paraffin block sections
For molecular tests;
1. 2-3 sections approximately 10 μm thick should be prepared from approximately 15 mg (17μl) paraffin blocks.
2. Samples should be sent as blocks or in two separate 1.5 ml eppendorf tubes.
3. Samples should be sent at room temperature (18-24°).
For molecular cytogenetic tests;
1. For the FISH test, sections of paraffin block sections with a thickness of about 4-6 μm taken on a slide and fixed with formaldehyde will be accepted..
2. At least 2 slides should be sent.
3. Samples should be sent at room temperature (18-24°).
Samples in which fixative other than formaldehyde was used during the preparation of the blocks and samples in insufficient amount will not be included in the study.
2.3. TRANSFER OF SAMPLES
| SAMPLE TYPE |
QUANTITY (Minimum) |
METHOD |
SHIPPING METHOD |
HEAT |
REJECT CRITERIA |
| Peripheral Blood |
2-5 ml |
Cytogenetics (conventional, FISH) |
Heparin tube (green cap) |
2-18 °C |
Frozen, coagulated, incorrectly tubed samples, samples sent 72 hours after collection, insufficient quantities of samples |
| Molecular genetics (DNA/RNA) |
EDTA tube (purple cap) |
18-24 °C |
| Bone marrow |
2-5 ml |
Cytogenetics (conventional, FISH) |
Heparin tube (green cap) |
2-18 °C |
Frozen, coagulated, wrong tube samples, insufficient amount of samples |
| Molecular genetics (DNA/RNA) |
EDTA tube (purple cap) |
18-24 °C |
| Amniotic fluid |
15-20 ml |
Cytogenetics (conventional, FISH) |
A suitable sterile syringe should be used, the expiration date should be observed or it can be transferred to a sterile 15 ml falcon. |
18-24 °C |
Frozen samples, wrong syringe (black tip), samples without patient identification information, insufficient amount of samples |
| Molecular genetics (DNA/RNA) |
| Chorionic villus sampling |
20-30 mg |
Cytogenetics (conventional, FISH) |
Container or falcon containing sterile transport medium |
18-24 °C |
Frozen samples, non-sterile media, samples in alcohol or formal or ethanol, insufficient quantities of samples |
| molecular genetics (DNA/RNA) |
| Abort material |
1-2 cm3 |
Cytogenetics (conventional, FISH) |
Container or falcon containing sterile transport medium |
18-24 °C |
Frozen samples, non-sterile media, samples in alcohol or formal or ethanol, insufficient quantities of samples |
| Molecular genetics (DNA/RNA) |
Tube or falcon containing sterile transport medium |
| Paraffin blocks or sections |
2-3 μm Or Paraffin block |
Cytogenetic FISH |
Sections taken on slides 2-3 μm at least two slides |
2-18 °C |
Samples in which fixatives other than formaldehyde were used during the preparation of the blocks, samples in insufficient amount |
| Molecular genetics (DNA/RNA) |
In addition to the tumor sections, the section marked with the tumor region |
| Peripheral blood (RNA recovery) |
10-15 ml (3 EDTA tubes) |
Real-time PCR; translocations in hematological malignancies |
Blood samples taken into 3 EDTA tubes together with the complete blood count results of the patients |
18-24 °C |
Frozen, coagulated, wrong tube, insufficient amount of samples |
| Serum |
2-5 ml |
Real Time PCR |
Gel tube with red cap |
2-18 °C |
Frozen, coagulated, incorrectly tubed samples, samples sent 72 hours after collection, insufficient quantities of samples |
| Vaginal/cervical samples |
Swap |
Real Time PCR |
Special tube for swap, in transport solution or dry |
18-24 °C |
Samples in wrong tube, samples sent 72 hours after collection |
| Swabs taken from the throat |
Swap |
Real Time PCR |
Special tube for swap, in transport solution or dry |
18-24 °C |
Samples in wrong tube, samples sent 72 hours after collection |
| NOTE |
In addition to the identity and indication information of the patients, the INFORMED CONSENT FORM about the procedure to be performed should be filled in by the patient and the physician and sent to our department with the sample. Samples without patient consent will not be reported before the consent form reaches our center. |
2.4. DELIVERY, ACCEPTANCE, REJECTION AND SEPARATION OF SAMPLES TO THE LABORATORY
2.4.1. Preliminary evaluation in sample delivery/reception areas
Samples coming to the laboratory are registered according to the Rejected Sample Control Instruction at the Sample Acceptance Unit, and the registration and acceptance procedures are evaluated according to the Sample Acceptance / Rejection Criteria List. The registered samples are delivered to the laboratory personnel responsible for the sample in the relevant Laboratory Department. In this process, the samples are reviewed again according to the Sample Acceptance and Rejection Criteria List and received from the sample delivery area by the laboratory personnel. In the preliminary evaluation; By looking at the test requests made from LIMS, the suitability of the sample container, sample amount, labeling, etc. control is done. Inappropriate samples are rejected from the system and a new sample is requested. If the return of rejected samples is requested, it is returned with
Faulty Sample Return Report.
2.4.2. Example Rejection/Acceptance Criteria
SAMPLE ACCEPTANCE CRITERIA:
Peripheral blood sample: For cytogenetic and molecular cytogenetic analyzes, at least 2-5 cc of peripheral blood sample should be taken into a heparin green capped tube. Samples should be transported at 2-18 ° transport temperature for these tests and to reach the laboratory within 72 hours after collection. For molecular genetic (DNA/RNA) analysis, at least 2-5 cc of peripheral blood sample should be taken into a purple capped tube. For molecular genetic tests, it should be transported at 18-24 ° transport temperature (room temperature) and to reach the laboratory within 72 hours after receipt.
Bone marrow sample: For cytogenetic and molecular cytogenetic analyzes, at least 2-5 cc bone marrow samples should be taken into a heparin green capped tube. Examples for these tests
It should be transported at a transport temperature of 2-18 ° and to reach the laboratory within 72 hours after receipt. For molecular genetic (DNA/RNA) analyzes, at least 2-5 cc bone marrow sample should be taken into an edta purple capped tube. For molecular genetic tests, it should be transported at 18-24 ° transport temperature (at room temperature) and to reach the laboratory within 72 hours after receipt.
Amniocentesis example: Approximately 15-20 cc of amniocentesis sample sent for cytogenetic, molecular cytogenetic and molecular genetic examination should be taken into a sterile suitable injector. Black sealed injectors should not be used. The sample should be transported at 18-24 ° transport temperature (room temperature) and reach the laboratory within 72 hours after collection.
Example of chorionic villus biopsy: For cytogenetic, molecular cytogenetic and molecular genetic analysis, approximately 20-30 mg of chorionic villus biopsy sample sent should be placed in sterile transport medium. The sample should be transported at 18-24 ° transport temperature (room temperature) and reach the laboratory within 72 hours after collection.
Sample of biopsy from evacuation material: For cytogenetic, molecular cytogenetic and molecular genetic examination, approximately 1-2 cm3 of the evacuated material should be taken into a sterile transport medium. The sample should be transported at 18-24 ° transport temperature (room temperature) and reach the laboratory within 72 hours after collection.
Examples that can be submitted in abortion materials:
Placental biopsy: At least 1 cm
3 piece should be taken from the region close to the umbilical origin (fetal origin).
Skin biopsy: Approximately 5 mm
2 piece taken from the back, leg or hip should be taken.
Cord biopsy: About 2 cm piece should be taken
Amnion biopsy: At least 2 cm
2 piece should be taken from the area closest to the umbilical cord.
Skin biopsy sample: For cytogenetic, molecular cytogenetic and molecular examination, approximately 1 cm3 of the skin biopsy sample sent should be placed in sterile transport medium. The sample should be transported at 18-24 ° transport temperature (room temperature) and reach the laboratory within 72 hours after collection.
Solid tissue biopsy (tumor) sample Approximately 5-10 mg of skin biopsy sample sent for cytogenetic, molecular cytogenetic and molecular examination sterile transport
should be included in the medium. The sample should be transported at 18-24 ° transport temperature (room temperature) and reach the laboratory within 72 hours after collection.
Paraffin block sections: For molecular cytogenetic tests, sections taken on slides should be taken as at least two slides, in addition to one hematoxylin stained section in tumor sections and the tumor region marked. The sample should be transported at 18-24 ° transport temperature (room temperature) and reach the laboratory within 72 hours after collection.
Obtaining RNA from peripheral blood At least 10-15 cc of peripheral blood sample should be taken into a purple capped tube for RNA extraction. Samples should be transported at 18-24 °C transport temperature (room temperature) and to reach the laboratory within 72 hours after collection.
Vaginal/cervical samples: It should be shipped with dry swap or special transport solution.
Swabs taken from the throat: Dry swap
Serum: 2-5 ml Gel tube with red cap 2-18 °C
Samples should be sent with the complete blood count results of the patients. In addition to the identity and indication information of the patients, the Informed Consent Form for the procedure to be performed should be filled in by the patient and the physician and sent to our department along with the sample.
Sample acceptance hours Monday-Friday (weekdays) between 9.00-18:00. Samples that have reached the laboratory at the appropriate time and conditions are accepted. The sample that has been selected for the test according to its indication is accepted. Samples in which the patient's identity information, request form and consent form are filled in correctly and completely are accepted.
SAMPLE REJECTION CRITERIA
1. Samples sent in the wrong sample container/tube will not be accepted.
2. Samples that have reached the laboratory after more than 72 hours will not be accepted.
3. Coagulated blood samples will not be accepted in studies conducted with peripheral blood.
4. Samples with damaged sample tubes/sample containers will not be accepted.
5. Frozen samples will not be accepted.
6. Samples that are not taken in the amounts recommended above will not be accepted.
7. If the samples (amniocentesis, chorionic villus biopsy, evacuation material, solid tissue, skin biopsy) to be taken into sterile medium are taken and sent under non-sterile conditions, the samples will not be accepted.
8. If tissue samples (chorionic villus biopsy, evacuation material, solid tissue, skin biopsy) have been taken in alcohol or formol, the samples will not be accepted.
9. Samples in which fixatives other than formaldehyde are used will not be accepted in the studies to be made from paraffin block sections.
10. Samples sent with the wrong indication will not be accepted (decided in consultation with the doctor).
11. Samples in which the patient's identity information, request paper and consent form are filled in incorrectly or incompletely will not be accepted.
2.4.3. Statistical analysis of rejected samples
If the sample is requested to be returned, via the Wrong Sample Return Report; If sample return is not requested, work is done on the Inappropriate Sample Registration Form. Laboratory faulty sample follow-up is done monthly.
2.4.4. Initiation of corrective and preventive actions
Sampling and transport trainings related to the Laboratory Preanalytical Process are the basic corrective and preventive actions.
2.5. ANALYSIS OF TESTS
2.5.1. Internal quality control studies
Internal quality control studies are carried out in accordance with the quality control rules defined in the Test Working Instruction of the relevant method.
2.5.2. Running the tests
Each test is studied in accordance with the Device Usage Instructions and Test Operating Instructions.
2.5.3. Reporting panic values
Tests with panic value are specified in the Panic Value List. These are the detection of pathological results in samples taken for prenatal diagnosis and preimplantation diagnosis (amniocentesis, chorionic villus sampling, cordocentesis, balstomer/trophectoderm biopsy). In these cases, if pathological results are detected, the clinician who requested the test is contacted to get more information about the patient's clinic, and the report is approved by informing the doctor about the test result. Panic value notification is recorded after meeting with the relevant physician and the result is sent to the relevant doctor and institution via e-mail immediately.
2.5.4. External quality control studies
On the calendar days determined by the external quality control program of which we are a member, external quality control samples are studied together with patient tests for tests included in the program. External quality control studies are carried out according to the Quality Control Program Procedure.
2.6. SAMPLE STORAGE PHASE
1. Samples are stored according to the times and conditions specified in the Test Working Instructions. It is also stated in the Sample Storage Periods List.
2. For molecular genetic tests, DNA-RNA is stored at < -15ºC for 5+ years and the primary sample of the patient is stored at 2-8ºC for 3 months under appropriate conditions. After the procedures are completed, the blood samples are destroyed in accordance with the instructions.
3. For molecular infection tests, DNA is stored at < -15ºC for 3 months and the primary sample of the patient is stored at +2-8ºC for 1 month under appropriate conditions. After the procedures are completed, the blood samples are filled in the "Document/Sample Disposal Report" and destroyed in accordance with the "Waste Management Procedure". DNA samples of the stored samples (child sick, ex sick child) are kept indefinitely for the possibility of further genetic testing.
4. For cytogenetic and molecular cytogenetic tests, cell pellets in primary samples such as heparinized blood, bone marrow sample of the patient (+2-8ºC), preparations at <-15ºC for 5 years and CDM images are stored indefinitely until a genetic diagnosis report is written. If there is excess primary sample in tissue cultured samples, it is stored according to the Sample Storage Periods List.
2.7. CONFIRMATION OF RESULTS
The findings obtained are evaluated by the relevant personnel and the result is presented to the Medical Genetic Specialist. After the result is approved, a report is prepared by the Patient Admission and Reporting Staff.
2.8. REPORTING RESULTS
1. After the tests performed in the laboratory are analyzed, they are approved by the responsible physician and after approval, the Patient Admission and Reporting staff prepares a report. Approved reports are directed to the attention of the relevant patient/institution/doctor and shared via LIMS.
2. Emergency FISH test requests in prenatal diagnosis samples are studied and approved within 3-4 days.
3. Patient name and surname, Lab No/Protocol No, TR Identity Number, Age and gender, Department Name, Doctor Name, Institution Name, Request Date and time, Date and time of Acceptance to the Laboratory, Date and time of Approval, Date of report in laboratory result reports and time, Test Name, Result, Unit, Reference Values, Approving physician and hospital address and contact information.
4. The reports of the results in the panic value list are sent immediately after informing the doctor.
5. Pathological results are reported with detailed information about possible risks and consequences.
6. In accordance with the Regulation on Genetic Diseases Evaluation Centers, gender is not specified in prenatal (prenatal) and preimplantation genetic diagnosis reports, except for defects in sex chromosomes and gender-related diseases.
7. The kit, method and device used are updated immediately in case of change. Reference intervals are always determined and updated by following the national and international literature and reviewing the prospectus information.
8. Patients can receive their test results in writing from our institution. In case of request, they can also reach the results via e-mail.
9. Patient data (reports and analysis images) reported in accordance with the genetic diagnosis legislation are archived and stored for 30 years.
2.9. STORAGE OF RESULTS
Patient data (reports and analysis images) reported in accordance with the genetic diagnosis legislation are archived and stored for 30 years.
