LABORATORY SPECIMEN COLLECTION & HANDLING

1. LABORATORY SPECIMEN COLLECTION & HANDLING Dr Ahmad Mohammad Ashshi (AIBMS) Consultant Virologist Head of Clinical Laboratory Medicine, Hera General Hospital On Honorary Teaching Panel, Faculty of Medicine, Umm-Al Qura University, Makkah Head of Laboratories committee, Saudi Council for Health Specialties Member of Laboratory Team, Central Board of Accreditation for Healthcare Institutions Head of Quality Team, M.O.H, Makkah.

2. Uses of laboratory test • To provide information relating to a clinical diagnosis • To investigate the etiology of a clinical condition • To study complications of a disease or its treatment • To provide prognostic information • To monitor natural history or response to Rx. for a • Condition • To detect subclinical disease (screening) • For Research Purposes

3. Proper procedures must be followed to assure the integrity of the specimen and its safe and expedient delivery to the laboratory as well as its processing once in the laboratory It is the job of the phlebotomist to make sure that all the guidelines of the Laboratories are met and implemented in order to deliver a suitable sample for analysis so that an accurate and precise result will be issued

4. Several essential steps are required for every successful collection procedure

5. Identify the patient. • Assess the patient's physical disposition (i.e. diet, exercise, stress, basal state). • Check the requisition form for requested tests, patient information, and any special requirements. • For body fluid collection provide the patient with the suitable container and collection instruction . • Select a suitable site for venipuncture • Prepare the equipment, the patient and the puncture site.

6. Perform the venipuncture. • Collect the sample in the appropriate container. • Recognize complications associated with the phlebotomy procedure. • Assess the need for sample recollection and/or rejection. • Label the collection tubes at the bedside or drawing area. • Promptly send the specimens with the requisition to the laboratory

7. WHAT IS THE MOST IMPORTANT THING YOU NEED FOR SPECIMEN COLLECTION???

8. Patient identification

9. Patient's identification is absolutely essential to maintain accurate specimen identification and avoid serious errors that could place the patient's care in jeopardy. •  The phlebotomist must correctly identify the patient before specimen collection. • Proper patient identification MANDATORY.

10. The two unique identifier will be • The patient’s full name • Medical record number

11. Patient’s Preparation • Specimens collected during this "fasting" period will yield the most reliable results. • Patients should be informed that urine and some of the blood specimens are generally collected during the early morning hours after waking and before eating, usually about 8 to 12 hours after the last ingestion of food or drink

12. Patient’s Preparation,cont. • There are some medications and other substances that can alter the results of the analyses. That’s why, the lab staff should be informed about the kind of medication ( e.g. Factor s Assay) • There are certain tests performed after administration of drugs / substances such as: glucose tolerance tests (GTT) performed after glucose administration or dynamic tests (of stimulation or inhibition) for different hormones ( e.g. provactive test for growth hormone

13. urine collection • Random Urine specimen (drug abuse) • First-morning (microscopic examination, b-HCG, • Clean -catch specimen (MSU ) For urine culture • 24 hours urine For quantitative chemical determinations in urine (such as creatinine, protein, calcium, magnesium, sodium, potassium,) • Suprapubic specimen especially for infant • Urine collected from children collection bags with hypoallergenic skin adhesive • Catheter specimen

14. Patient’s Preparation factors Therapeutic Drug Monitoring: different pharmacologic agents have patterns of administration, body distribution, metabolism, and elimination that affect the drug concentration as measured in the blood. Many drugs will have "peak" and "trough" levels that vary according to dosage levels and intervals. Check for timing instructions giving by the physician for drawing the appropriate samples. Stress: May cause transient elevation in white blood cells (WBC's) and elevated adrenal hormone values (cortisol and catecholamines). Anxiety that results in hyperventilation may cause acid-base imbalances, and increased lactate.

15. Patient’s Preparation factors ,cont. Effects of Exercise: Muscular activity has both transient and longer lasting effects. The creatine kinase (CK), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and platelet count may increase

16. Patient’s Preparation factors ,cont. Postural • Change in posture from supine to erect or sitting causes a shift in fluid from the intravascular to the interstitial space of about 12%. • Most cellular and macromolecular analytes are 5 to 15% higher when specimens are collected erect as compared to supine. • Conversely, moving from upright to supine can have a dilutional effect owing to an increase in plasma volume. • Postural effects are accentuated in patients with edema and low • plasma protein values.

17. Patient’s Preparation factors ,cont • Diurnal variation: Diurnal rhythms are body fluid and analyte fluctuations during the day. • For example : • Serum cortisol levels are highest in early morning but are decreased in the afternoon. • Serum iron levels tend to drop during the day. • Some sex hormones need special timing as regard cycle : • (1)FSH,LH (3rd day of cycal {follicular phase}) • (2)Progesterone (21 day of cycle{ luteal phase}) • Other Factors: Age, gender, and pregnancy have an influence on laboratory testing. Normal reference ranges are often noted according to age.

18. Requisition form : It should contain the proper information in order to process the specimen. : A requisition form must accompany each sample submitted to the laboratory. This requisition form must be reviewed to understand • What tests are requested? • What specimens are to be collected? • What containers are required to collect the specimens? • When the specimens are to be collected? • What is the amount of specimen necessary for testing? • What is the collection order of tubes?

19. The essential elements of the requisition : form are • Patient's surname, first name, and middle initial. • Patient's Medical record Number . • Patient's date of birth, sex and nationality. • Requesting physician's complete name, signature and stamp. • Diagnosis. • Source of specimen. This information must be given when requesting microbiology, cytology, fluid analysis, or other testing where analysis and reporting is site specific. • Date and time of collection. • Indicating the test(s) requested.

20. The essential elements of the requisition cont., • Histopathology specimen request must contain in addition to the above listed information, the following: • name of the procedure, no. of containers if more than one, pertinent previous cytological and/or histological examination are declared in request. • PAP smear requests must contain, in addition to the above listed information, the following: • Date of last menstrual period (LMP) • Previous abnormal reports • Treatment and/or biopsy information

21. EQUIPMENT: • Evacuated Collection Tubes -. • Needles - The gauge number indicates the bore size: the larger the gauge number, the smaller the needle bore. Needles are available for evacuated systems and for use with a syringe, single draw or butterfly system. • Holder/Adapter - use with the evacuated collection system. • Tourniquet - Wipe off with alcohol and replace frequently.

22. EQUIPMENTcon., • Alcohol Wipes - 70% isopropyl alcohol. • Povidone-iodine wipes/swabs - Used if blood culture is to be drawn. • Gauze sponges - for application on the site from which the needle is withdrawn. • Gloves - can be made of latex, rubber, vinyl, etc.; worn to protect the patient and the phlebotomist. • Syringes - may be used in place of the evacuated collection tube for special circumstances. • Adhesive bandages / tape - protects the venipuncture site after collection

23. Collection Tube Additives • Heparin • EDTA • Citrate • Clot Activator • Serum Separator

29. Blood should NEVER be poured from one tube to another since the tubes can have different additives or coatings. • Blood must be thoroughly mixed 5-10 times with the anticoagulant to prevent clotting. DO NOT SHAKE THE TUBE. • Serum Separator tubes should also be inverted. There is a clot activator present and inversion expedites the clotting process

30. Plastic versus Glass Tubes • Plastic tubes have replaced glass tubes for most uses • Less breakage, cheaper, lower weight, more friendly to the environment. • Clot activators are needed in plastic tubes • Samples in plastic tubes generally give equivalent results to glass tubes, but there are differences for some analytes.

31. EDTA (Anti-Coagulant • K2EDTA is used to collect whole blood for hematology studies and plasma for analytes with heparin interference • Acts by binding calcium • Nominal concentration of 1.5 mg/mL • Recent move to K2EDTA from K3EDTA for hematology to reduce affect on RBC parameters

32. EDTA Side-Effects • EDTA is hyperosmolar causing cell shrinkage but the low pH of EDTA counterbalances this effect because low pH causes cells to expand. • EDTA induced psuedothrombocytopenia caused by in vitro antibody mediated platelet clumping and platelet satellitism that results from changes in membrane structure that occur when calcium is removed by EDTA. • Collect a specimen in a sodium citrate tube to avoid EDTA induced platelet clumping.

33. Heparin (Anti-Coagulant) • Used to collect whole blood or plasma • Binds to anti-thrombin III to inhibit Xa, IXa, and thrombin • Nominal concentration of 12 – 30 U/mL • Heparin binds calcium so ionized calcium must be collected using “Calcium Titrated” or “Electrolyte Balanced” heparin

34. Citrate (Anti-Coagulant) • Citrate is used for collection of coagulation tests • Acts by binding calcium • Nominal concentration of 3.2% (0.109 mol/L) • Recent move to 3.2% from 3.8% to get more consistent results for Prothrombin Time, particularly for more sensitive reagents • Tubes must be properly filled to within +/- 10% of assigned collection volume

35. Clot Activator • Shortens Time to Clot • In vitro activation of clotting system to enhance clot formation • A silica clot activator is attached to the tube wall with a silicone surfactant • Requires mixing for optimal affect • Time to clot is 15-30 minutes instead of 1 hour

36. Serum Separator Gel • Separates Serum from Cells • Polymer gel with specific gravity between that of serum and cells • Migrates and forms a barrier during centrifugation • Separation of cells from serum stops metabolic and • hemolytic effects • Certain analytes and therapeutic drugs may bind to gel over time

37. Labelling the Sample A properly labelled sample is essential so that the results of the test match the patient. The key elements in labelling are: • Patient's surname, first and middle. • Patient's ID number. • NOTE: Both of the above MUST match the same on the requisition form. • Date, time and initials of the phlebotomist must be on the label of EACH tube

38. Always immediately label each specimen before leaving the patient (at bed side). • Never: Pre-Label blood specimen tubes before drawing blood. • Never give the specimen to someone else to label. • Verify again that the information on the labeled specimen matches that on the request form.

39. Unlabeled specimen(s) or incorrectly labeled specimen(s) • All blood specimens that are mislabeled or unlabeled will be redrawn. • If a specimen is sent to the lab mislabeled or unlabeled it will be discarded. • All blood, whether unlabelled or mislabeled, intended for cross matching, must be redrawn, without exception

40. If the specimen is irreplaceable the ordering physician must attest to the correct patient’s identity and must authorize (on the specimen relabeling form) affixation of the correct label by an attendance nurse.

41. Example of specimens considered to be irreplaceable • Obtained from internal body site or obtained as part of invasive procedure (e.g. Bronchoscopy specimen, bone marrow or CSF). • Critically timed specimens (e.g. prior to antibiotics or dosing schedules). • Neonatal specimens. CORD BLOOD specimens that are received with the mother's information on the label can be relabeled with the baby's labels. A nurse must do the relabeling.

42. Collection Volume Under filling occurs because: • Tube was removed too quickly. • Tube slips back from vacutainer needle. • Air drawn in from butterfly or connector tubing. • Expired tubes with low vacuum (rare)

43. Impact of Incorrect Volume on PT and APTT • The APTT is the most sensitive to variations in final citrate concentration. It increases by an average of 3% for 10% underfilling and 10% for a 20% underfilling. • The PT increases by an average of 5% for 20% underfilling and by 15% for 30% underfilling.

44. Order of Draw

45. Errors Anticoagulant cross contamination Citrate : If drawing a blue top tube for coagulation tests, (other than PT and PTT), a red tube (2-3 cc) must be drawn first to avoid contamination from tissue thromboplastin, which can yield false coagulation Results. EDTA : A calcium chelating agent; can yield false magnesium and calcium results if drawn before a red top tube. Can falsely elevate potassium results if drawn before a greentop tube. Oxalate : Interferes with cell membranes

46. Reasons for a Rejected Specimen • No Test Requisition Form (TRF) • Test not written on TRF • Submitted wrong specimen for test requested • Quantity Not Sufficient (QNS) • Missing or inadequate identiification • Clotted

47. Avoiding Common Errors in Specimen Collection • Insufficient quantity • Hemolysis • Failure to use the correct container for specimen collection • Inaccurate and incomplete patient guidance • Failure to label a specimen correctly and to provide all pertinent information • Failure to tighten specimen container lids, resulting in leakage and/or contamination of specimen

48. ADDITIONAL CONSIDERATIONS

49. To prevent a hematoma: • Puncture only the uppermost wall of the vein • Make sure the needle fully penetrates the upper most wall of the vein. (Partial penetration may allow blood to leak into the soft tissue surrounding the vein by way of the needle bevel) • Remove the tourniquet before removing the needle • Use the major superficial veins • Apply pressure to the venipuncture site • Avoid a probing, traumatic venipuncture

50. To prevent hemolysis (which can interfere with many tests): • Mix tubes with anticoagulant additives gently 5-10 times. • Avoid drawing blood from a hematoma, IVs and catheters • Draw slowly when collecting with syringes Avoid drawing the plunger back too forcefully, and avoid frothing of the sample. • Make sure the venipuncture site is dry ( allow alcohol to dry before collection). • Avoid a probing, traumatic venipuncture • Use a larger bore needle (20 gauge) • Draw from antecubitalfossa rather than the hand • •