Department of Pathology & Laboratory Medicine 2401 Gillham Road Kansas City, MO 64108 http://www.childrensmercy.org/lab Client Services personnel are available from 7am to 7pm, Monday through Friday. Phone: (816) 234-3835 Fax: (816) 983-6534 Outreach Services Laboratory Manager may be reached at (816) 234-3829 After hours and weekends, please call (816) 234-3230 General Information We provide inpatient, outpatient and outreach services for the greater Kansas City community. Our team includes eight full-time pathologists and eight PhDs who provide administrative and technical oversight. Clinical laboratory staff consists of approximately 185 employees certified in various specialties by credentialing agencies such as the American Society of Clinical Pathologists (ASCP) and the National Registry of Clinical Chemists (NRCC). An additional 30 staff members provide support in: specimen processing and laboratory assistance; information systems; courier and client services. Our test menu covers a broad range of procedures, ranging from routine to highly specialized testing. Please refer to the Laboratory Test Directory for a comprehensive list of tests we perform. Children's Mercy Hospital laboratory is a member of the Regional Laboratory Alliance (RLA), a network of nine hospital-based and physician-owned laboratories, providing services in Kansas City and outlying areas throughout western Missouri and eastern Kansas. This membership enhances our services by providing courier service, expanded draw sites for specimen collection, and access to a broader menu locally of provided testing. Refer to the RLA website for additional information. All specimens must be properly labeled, packaged in specially marked biohazard bags, include a legible requisition and transported at appropriate temperatures. We encourage the use of batch lists/packing lists or other mechanism to track specimens submitted to our laboratory. Routine courier service is provided at no charge to those clients who submit a minimum number of specimens on a regular basis. Clients may contact a commercial courier service or call our Client Services to arrange for a STAT pickup; however Clients are responsible for bearing the cost of STAT courier services. CMH Lab Manual Last updated: 4/2012 Page 1 of 23 Notice To Clients Children's Mercy Hospital laboratory remains committed to compliance with all guidelines governing the submission of claims. This notice is to inform you that you must comply with all applicable laws, rules and regulations of the United States, the State(s) of Missouri and/or Kansas and any other applicable governmental agencies related to the performance, ordering and billing of laboratory services provided by the Children's Mercy Hospital laboratory including, without limitation, those laws and regulations related to medical necessity, Medicare and Medicaid billing, procedure codes (CPT), and diagnosis codes (ICD-9). Payors may deny payment for tests that are not of medical necessity. In situations where the physician feels a test is appropriate but acknowledges that it may not be medically necessary (and for all non-covered services under Kansas Medicaid), the test order should be accompanied by a properly executed Advance Beneficiary Notice (ABN). If you have questions, please contact Client Services at (816) 234-3835 during normal business hours. Billing Client - For client billing (applicable to hospitals, home health agencies and other non-physician office clients), check the box marked CLIENT. Please be certain that you are using a test request that includes your client name and account mnemonic. Patient or Insurance - For test requests submitted by physician offices, Children's Mercy Hospital laboratory has the ability to bill patients directly or bill a variety of government and commercial payors. If you would like a complete and comprehensive list of payors that the Children's Mercy Hospital laboratory can bill, please call Client Services at (816) 234-3835 to request the list. This list is periodically updated. • Patient – Please include the following required billing information: patient birth date, sex, social security number, responsible party, complete address including zip code, home and/or work phone numbers on the request form. Providing this information will avoid additional correspondence to your office. Please inform your patients that they will receive a bill for laboratory services from Children's Mercy Hospital laboratory. • Insurance – Please include the following required billing information: patient birth date, gender, social security number, responsible party, relationship to insured party, employer, name of insurance plan, type of plan (HMO, EPO, POS, etc), group name and number, and diagnosis code. Please send a copy of the front and the back side of the insurance card along with the laboratory requisition. CMH Lab Manual Last updated: 4/2012 Page 2 of 23 Administrative Team Questions concerning test appropriateness, test methodology, test interpretations should be addressed to the respective director. Process questions such as sample volumes, tube types or testing turn-around time should be directed to section supervisors/coordinators. Medical Director Associate Medical Director Administrative Director Manager- Laboratory Operations Manager- Core Laboratory Services & PI Manager- Outreach Laboratory Services Manager- Quality Systems Manager- Department Office Evening Supervisor Night Supervisor David Zwick, MD, 816-234-3234 Marilyn S. Hamilton, M.D, PhD, 816-234-3811 Toni Sheffer, MBA, MT (ASCP), 816-234-3237 Cynthia Kelley, MT (ASCP), 816-234-3023 Randah Althahabi, MS, MT(ASCP), 816-234-3907 Debra Faller, MBA, MT (ASCP), 816-234-3829 Christina Vasquez, MT (ASCP), 816-983-6783 Robin Ray-Harris MA, CAP, 816-234-3828 Brad Parker, MT (ASCP), 816-234-3831 Shirley Coupal, MA, MT (ASCP), 816-234-3831 Central Processing- Supervisor Chemistry- Assistant Director Chemistry- Supervisor Coagulation- Director Cytogenetics- Director Cytogenetics- Supervisor Decentralized/POCT Supervisor Education/Safety Coordinator Endocrinology- Assistant Director Endocrinology- Coordinator Flow Cytometry- Director Flow Cytometry- Coordinator Hematology- Director Hematology- Supervisor Histology- Supervisor Immunology- Director Immunology- Coordinator Microbiology- Director Microbiology- Supervisor Molecular Genetics- Director Nephrology- Director Nephrology- Coordinator Pathology- Surgical Pathology Director Pathology- Autopsy Services Director Pathology Pathology Pathology Reference/Sendouts- Coordinator Transfusion Services-Director Transfusion Services-Supervisor Toxicology & Biochemical Genetics- Director Toxicology- Supervisor Marinda Cooper, MT (ASCP), 816-234-6619 Angela Ferguson, PhD, 816-234-1696 Trang Nguyen, MT (ASCP), 816-234-3210 Marilyn S. Hamilton, MD PhD, 816-234-3811 Linda D. Cooley, MD, 816-802-1220 Barbara Mouron, CLSp (C.G.), 816-802-1220 Diane Burford, MT(ASCP) 816-234-3189 Sandy Claussen, MT(ASCP), CPHQ 816-802-1459 Angela Ferguson, PhD, 816-234-1696 Nancy Reddig, MT (ASCP), 816-234-3071 David Zwick, MD, 816-234-3234 Ruth Morgan, BS, 816-234-3983 David Zwick, MD, 816-234-3234 Beth Heimberger, MSA, MT (ASCP), 816-234-3112 Angela Smith, BS HT(ASCP), 816-234-3827 Charles Barnes, PhD, 816-235-1824 Marilie Stuck, MT (ASCP), 816-234-3095 Rangaraj Selvarangan, BVSc, Ph.D., 816-234-3031 Bev Doerge, BS, M (ASCP), 816-234-3386 Carol Saunders, PhD, 816-234-3588 Uri Alon, MD, 816-234-3010 Nancy Wilson, MT (ASCP), 816-234-3013 Eugenio Taboada, MD, 816-234-3234 Lei Shao, MD., 816-234-3234 Atif Ahmed, MD, 816-234-3234 Alex Kats, MD, 816-234-3234 Vivekanand Singh, MD, 816-234-3234 Vicki Smith, MT (ASCP) 816-234-3221 Lejla Music Aplenc, MD, 816-983-6980 Steve Buckley, MT(ASCP) 816-234-3232 Uttam Garg, PhD, 816-234-3803 Clint Frazee, MBA, NRCC- TC, 816-234-3295 CMH Lab Manual Last updated: 4/2012 Page 3 of 23 Accreditation / Licensure Accreditations College of American Pathologists (CAP) Main Campus 19365-01-01 South 19365-03-02 COLA Broadway 22906 Northland 15946 West 014429 American Association of Blood Banks Main Campus 2782401 Licensures Clinical Laboratory Improvement Act (CLIA) Main Campus 26D0443323 South 17D0864218 Broadway 26D2022344 Northland 26D1017316 West Other 17D0880689 Medicare Provider 263302R Missouri Medicaid 10931608 Kansas Medicaid 305190 US Nuclear Regulatory Commission 24-15513-01 Federal Drug Administration (FDA) 1974136 Website for copies of documents CMH Lab Manual Last updated: 4/2012 Page 4 of 23 Specimen Labeling Laboratory specimens must be properly labeled. A minimum of two patient identifiers MUST be included on the label (first and last name are considered jointly as one identifier). • Identifiers may include: patient name, medical record number, account number, date of birth. • Other pertinent items to include: the date and time of collection, the initials of the collector, specimen source and comments such as peak or trough, pre or post. Specimens with missing or incomplete identification are rejected. Instances when relabeling is clinically indicated require notification of the Associate Medical Director. A process for relabeling is implemented and requires the collecting individual to assume responsibility for specimen identification and relabeling. A disclaimer is placed on the report. Specimen Collection Laboratory test results are dependent on the quality of specimen collection. Clinical Laboratory Standards Institute (CLSI) guidelines serve as our standard for collections. The Laboratory Test Directory provides specimen requirements for each test. Following are general guidelines for specimen collection. Blood Collection Tubes Blood collection tubes are designated by the color of the stopper. Tube manufacturers provide description of contents on the respective labels. Please refer to these contents to ensure that the correct tube is being used. Tube Type Description Plain red top No additive Red gel/ gold Serum separator with clot activator Lavender/ purple Potassium EDTA Light blue Sodium citrate Green & black Lithium heparin with gel Green Lithium or Sodium heparin Mint Green Lithium heparin with gel Gray Sodium Fluoride/Potassium oxalate Royal blue (red label) Trace element free, without anticoagulant Royal blue (lavender label) Trace element free, with anticoagulant Tan Metal free, with & without anticoagulant Yellow small (2.5 mL) ACD solution B Yellow large (10 mL) ACD solution A CMH Lab Manual Last updated: 4/2012 Page 5 of 23 Order of Draw Based on CLSI H3-A6, the following order of draw is recommended when drawing multiple specimens for clinical lab testing during a single venipuncture in order to avoid possible test result error due to additive carryover. 1st- Blood culture tubes 2nd- Coagulation tubes containing citrate (blue closure) 3rd- Serum tubes with or without clot activator, with or without gel (red closure) 4th- Tubes containing heparin with or without gel plasma separator (green closure) 5th- Tubes containing EDTA with or without gel plasma separator (lavender closure) 6th- Tubes containing glycolytic inhibitor; potassium oxalate-sodium fluoride (gray closure) Additional guidelines • Note the EXPIRATION DATE on the tubes before drawing. Do not use expired tubes for blood collection. • Vacutainer tubes are manufactured to draw blood into the tube; do not remove the tops. Transfer devices are the approved method to fill vacutainers when collection is from a line or syringe. • Vacuum may be lost if the tube is dropped or if the top has already been pierced by a needle (repeat draw). Use a fresh tube for the collection to avoid problems due to loss of vacuum. • The volume of blood drawn should be approximately 2 ½ times the amount of serum/plasma required. • Avoid shaking blood in the tubes. Gently invert appropriate tubes 5-10 times after draw. • Always follow Standard Precautions including use of appropriate personal protective equipment when collecting and processing blood and body fluids. Urine Collection The Care Card links below provide instructions. General Instructions Clean Catch Timed Collection CMH does not recommend utilization of preservatives during timed collection. CMH Lab Manual Last updated: 4/2012 Page 6 of 23 Specimen Processing Specimen Type Some tests are affected by prolonged contact of serum/plasma with cells and may require centrifugation/separation prior to transportation. The following guidelines are provided for external facilities processing specimens before submission to CMH. Plasma Sufficient whole blood should be drawn in the anticoagulant indicated to provide the volume of plasma indicated in the individual test listing. Whole blood specimens should be inverted 5-10 times immediately after they are drawn. If required, separate plasma and red cells thru centrifugation within 20-30 minutes of the specimen draw. Please indicate on the plasma tube what anticoagulant was used, if separated from cells. Serum Sufficient whole blood should be drawn to obtain the volume of blood indicated in the individual test listing. Use a red top, tiger-top, or gold gel tube, as appropriate. Avoid hemolysis. Whole Blood Use the anticoagulant indicated for the test ordered. Mix well, but gently, by inverting the tube 5-10 times. Fasting Specimens Some tests are most accurate when the patient has been fasting. Standard fasting recommendations are 12-hours but variations may occur. See individual test for specific fasting instructions. CMH Lab Manual Last updated: 4/2012 Page 7 of 23 Specimen Rejection Specimens may be rejected for analysis if they arrive in the laboratory under conditions other than those specified in the Laboratory Test Directory. If testing can not be completed the laboratory will notify the clinician of the need for recollection. An overview of key rejection criteria is below. Please refer to our policy for additional details or contact the Laboratory at (816) 234-3230 with questions. • Unlabeled or Inappropriately Labeled Specimens must arrive at the laboratory properly labeled. Specimens will be rejected if unlabeled or inappropriately labeled. • Quantity Not Sufficient (QNS) Volume: Occasionally the serum yield from a clot will not be enough to perform the requested tests because the patient has a high hematocrit or not enough specimen was drawn. When multiple tests have been ordered and the volume is sufficient for some but not all, the ordering physician will be contacted to select and prioritize the testing to be completed. Anticoagulant mix: Vacutainer tubes with anticoagulant have minimum volume requirements to maintain correct blood to anticoagulant ratios. o Coagulation testing: the vacuum must be allowed to fill the tube o CBC or component thereof - less than 250 µL in a EDTA microtainer tube or less than 1.0 mL in a EDTA vacutainer tube will be rejected o Ionized calcium collected in sodium heparin: tube must be filled at least ½ • Hemolyzed Specimens Hemolysis is graded as slight, moderate or gross. Some testing can be completed on slight or moderate hemolyzed specimens. Chemistry tests which are most affected by hemolysis are: ammonia, bilirubin, iron, LDH, phosphorus and potassium. Coagulation testing is impacted by moderate or gross hemolysis. • Clotted Specimens Clotted specimens will not be accepted for: CBC, coagulation testing, CSF protein, cyclosporine, ESR, tacrolimus, blood gases, erythrocyte protoporphyrins and lead. Some orders may be partially completed with tests impacted by clotting withheld. • Analyte Stability Analyte stability can affect some Chemistry and Hematology testing. This is a key consideration in requests for add-on testing. o Testing can not be performed on specimens greater than 4-hours from collection for CRP, Bilirubin, LDH and KC, ESR, platelet reticulocyte, PFA and coagulation assays except PT. o Specimens left un-spun greater than 4-hours from the time of collection can not be used for glucose testing. • Inappropriate Specimens Specimen types which do not match the requirements for the ordered test. CMH Lab Manual Last updated: 4/2012 Page 8 of 23 Coagulation – Special Instructions Specimen Collection Collect specimens with minimal trauma into evacuated light blue closure tubes (containing 3.2% buffered sodium citrate), ensuring that they are filled to the correct level and mix thoroughly by gentle inversion. Devices It is recommended that blood specimens for coagulation testing be collected by venipuncture using a system that directly collects the specimen into the sodium citrate tube. If a syringe is used in collection, it is important that the blood is added to the light blue tube within 1-minute of completion of draw and the specimen is immediately and properly mixed. Venipuncture When using a winged blood collection set and a coagulation tube is the first tube needed, first draw a discard tube. The discard tube must be used to prime the tubing of the collection set, which will assure maintenance of the proper anticoagulant/ blood ratio in the first tube filled. The discard tube should be a light blue tube, and need not be completely filled. Vascular Access Devices When obtaining blood from a VAD, avoid air leaks, which may cause hemolysis and incorrect draw volumes. Collection through line previously flushed with heparin should be avoided, if possible. Mixing and Transporting • • • Mix the blood by gentle inversion 5-10 times Sample must be transported to the lab as soon as possible, testing can not be performed if lab receipt is greater than 4-hours from collection Specimens submitted from non-CMH Labs – if transport will require more than 4-hours between collection and CMH receipt please contact the Hematology Lab at (816) 234-3831 for processing and transport instructions. Plasma based assays will require the specimen be spun down ASAP following collection, plasma removed and frozen. Ship with dry ice; specimens must be frozen when received at the CMH lab CMH Lab Manual Last updated: 4/2012 Page 9 of 23 Flow Cytometry – Special Instructions Lab Hours/Phone • • Monday – Friday; 0800 – 1630 (816) 234-3983 General Information • • • • Samples to be processed the same day should be received in the laboratory no later than 1200 (noon) Results are reported in percents, absolute number per mm3, and may include a brief interpretive report Custom panels may be requested using available antibodies. Consultation with our Medical Director is required Research Flow Cytometry services are available Peripheral Blood Panels – Collection Guidelines • • • Specimens should be collected during regular Flow Cytometry Laboratory hours Friday afternoon after 14 and weekend collections should be avoided if possible A CBC with differential should accompany all peripheral blood panels to allow calculation of absolute number results Weekday Specimen Requirements • • • • • EDTA (purple top) tube Specimen volume: optimal 3-mL, minimum 1.5-mL Optimal to have the Flow Cytometry EDTA be a separate tube from a CBC tube; however, if a single tube is collected and must be shared for testing the minimum is 3.0 mL Do not refrigerate or spin May be held at room temperature up to 24-hours Weekend & After Hours Specimen Requirements • • • • 1 ACD-B (2.5 mL) must fill completely 1 EDTA (purple top); can be a microtainer Do not refrigerate or spin ACD may be held at room temperature up to 48-hours Specimen requirements and handling expectations can not be modified. Issues with collections or handling will be cause for rejection and request for recollection. CMH Lab Manual Last updated: 4/2012 Page 10 of 23 Test Name Information/ Instructions Immune Status Assessment Peripheral Blood Panels See Peripheral Blood Collection Guidelines on previous page • • T Cell subset analysis (T-Helper & T-Suppressor and ratio) Immune deficiencies, DiGeorge Syndrome, monitor transplant rejection therapy B & T CELL IMMUNE DEFICIENCY PANEL • • Lymphocyte subset analysis (T Cells and CD19+ B Cells) Immune deficiencies, B Cell therapy monitor EXTENEDED IMMUNE DEFICIENCY PANEL • • Lymphocyte subset analysis (T, B, NK, and activated T Cells) Immune deficiencies, post BMT monitoring T CELL IMMUNE DEFICIENCY PANEL Characterization of thymic production of T Cells; specific for T Cell that can respond to new antigen • Flow Extended Immune Def Panel analysis is required • NAÏVE /MEMORY T CELL Characterization of naïve and memory B cells including immunoglobulin class-switched B cells • Common variable and other immunodeficiencies, and B cell humoral immunity post transplant • Recommend test is not done as a standalone order. Best when done in conjunction with an immune deficiency panel. • NAÏVE /MEMORY B CELL Hematology/Oncology Immunophenotyping of neoplastic cells for diagnosis and assessment of lineage • Monitor of disease progression (minimal residual disease) and chemoterapy effectiveness • Various specimens: bone marrow, peripheral blood, cell suspensions of solid tumors • LEUKEMIA/LYMPHOMA DNA PLOIDY/CELL CYCLE ANALYSIS • • • Determination of DNA ploidy (chromosome content) of specimen Estimation of the proliferative activity (cell cycle analysis) Various specimens: bone marrow, peripheral blood, cell suspensions of solid tumors GRANULOCYTE OXIDATIVE BURST IMMUNE DEFFICIENCY PANEL • • • Useful for diagnosis of Chronic Granulomatous Disease Requires 3-mL blood less than 24-hours old at room temperature Only collected Monday-Friday (must be in Lab by 1400 Friday) GRANULOCYTE ADHESION MOLECULE IMMUNE DIFFICIENCY PANEL • • • Useful for diagnosis of Leukocyte Adhesion Deficiency Requires 3-mL blood less than 24-hours old at room temperature Only collected Monday-Friday (must be in Lab by 1400 Friday) • Enumerates CD34 (HPCA-2) positive progenitor cells • • Viability assessment done if specimen is a leukapheresis product Specimens: peripheral blood (EDTA), leukapheresis products, thawed products • • Determines the proportion of live cells in a sample Various specimens: peripheral blood, bone marrow, cell suspensions, and leukapheresis products Bone Marrow Transplant STEM CELL (CD34 ONLY) STEM CELL WITH VIABILITY Specimen Assessment CELL VIABILITY CMH Lab Manual Last updated: 4/2012 Page 11 of 23 Histology – Special Instructions Lab Hours/Phone • • Monday – Friday: 700 – 1700 (816) 234-3827 please ask to speak to the supervisor or charge tech • Histology staff and a Pathologist are on-call outside normal hours. Call the Main Lab at (816) 234-3230 General Information • • • Specimens must arrive in Histology by 1530 for next day results STAT/ same day results must be approved by the Pathologist Notify the Histology lab in advance of collection when the specimen will require frozen section or special handling Labeling Expectations • • • • Containers labels must include a minimum of 2-patient identifiers and the specimen source Specimens without source information will require follow-up before processing Specimens without sufficient patient identifiers require the collecting individual to come to Histology to complete proper labeling Please utilize shared specimen labels when appropriate Collection Guidelines • • All routine specimens must be placed in formalin (1:15 – 1:20 volume) Fresh specimens or specimens in normal saline must be labeled with a “fresh” label CMH Lab Manual Last updated: 4/2012 Page 12 of 23 Microbiology – Special Instructions Test Name CLOSTRIDIUM DIFFICILE TOXIN Collection & Other Instructions 2-3 mL of liquid stool in a poly cup. Deliver to the lab immediately or refrigerate up to 24 hours. Stool samples from infants <1year will not be routinely tested due to high incidence of C difficile colonization in the age group. CULTURE ACID FAST Sputum, urine or gastric washing: collect one specimen per day, a first morning is recommended. Collection containers for gastric aspirates can be obtained from the Microbiology Lab. Tissue: Transport to the lab immediately or cover with a small amount of non-bacteriostatic saline. Blood and bone marrow: collect a Wampole isolator tube. Feces: collect at least 1 gram of stool in a poly cup. Swabs are not optimal for the recovery of acid fast organisms. Specimens will be sent to St. Luke's Hospital for AFB smear & culture. CULTURE-ANAEROBIC Aspirate or body fluid transport in the collection syringe with a luer lock top or a sterile vial. For small volumes, collect pus on a transport swab with charcoal. Tissue transport in a sterile container and deliver to the lab immediately or cover with a small amount of non-bacteriostatic saline. Respiratory tract submit transtrachael aspirate, pleural or empyema fluid only. Transport samples for anaerobic culture to the laboratory immediately. Do not refrigerate. The following are not acceptable for anaerobic culture: superficial material collected on a swab, sputum, nasotracheal aspirate, throat or np aspirate, bronchoscopy specimen inappropriately collected, vaginal/cervical specimens or clean catch urine. CULTURE-BLOOD Use Persist or Chlroprep to disinfect skin. Apply disinfectant by beginning at venipuncture site, work in circular motion, cover 23 inches diameter. Air dry; do not touch or palpate. Wt in Kg Peds Plus Aerobic Plus 1-3 0.5mL 0.5mL 3.1-6 1 mL 1 mL 6.1-9 2 mL 2 mL 9.1-12 3 mL 3 mL 12.1-20 4 mL 4 mL 20.1-25 5 mL 5 mL 25.1-40 5 mL 5 mL >40 n/a 10 mL x 2 bottles Contact Microbiology at 234-3386 for Anaerobic, Fungal & Mycobacterial blood culture media when needed. CULTURE-BODY FLUID CULTURE-BONE MARROW CMH Lab Manual Last updated: 4/2012 Disinfect overlying skin with 2% iodine. Obtain specimen via percutaneous needle aspiration or surgery. Transport in a sterile container. If the specimen might clot use a sterile tube with heparin. EDTA tubes are not acceptable for culture. Prepare puncture site as for surgical incision. Inoculate bone marrow into blood culture bottles. Transport immediately. Page 13 of 23 Test Name Collection & Other Instructions CULTURE-CATHETER TIP Aseptically remove and clip 5 cm of the distal end of the catheter directly into a sterile tube. Acceptable IV catheters for semi-quantitative cultures: central line, CVP, Hickman, Broviac, peripheral, arterial, umbilical, hyperalimentation, Swan-Ganz. Catheter tip cultures can not be evaluated without a concurrent blood culture CULTURE-CSF Transport immediately in sterile screw cap tube. Never refrigerate. CULTURE-DERMATOPHYTES Skin scrapings, nail or hair clippings in a sterile petri dish or poly cup. Samples from the scalp may be obtained using a sterile toothbrush. CULTURE-DIPHTHERIA Respiratory illness: throat or np swab. Cutaneous Diphtheria: skin, throat or np swab. Specimens will be sent to a reference laboratory CULTURE-EAR Outer ear: take a representative sample with a swab and transport using charcoal transport medium. Inner ear: collect aspirate from tympanocentesis in a sterile tube. CULTURE-EYE Conjunctiva: Swab conjunctiva use charcoal swab for transport. Corneal scraping: Blood agar plate, chocolate agar plate and eugonic broth tube to be inoculated by the physician at the bedside. Add an inhibitory mold agar plate if fungus is suspected. Fluids of aspirates: sterile screw cap tube or direct inoculation. CULTURE-FECES ROUTINE Feces: Screw cap Cary-Blair vial. Do not exceed the fill line. Routine culture will screen for E. coli 0157 Salmonella, Shigella, Yersinia, Campylobacter sp. and Shiga Toxin for enterohemorrhagic E. coli. A rectal swab may be submitted if it is not possible to obtain feces but Shiga Toxin testing will not be performed from swabs. CULTURE -FECES AEROMONAS PLESIOMONAS Collection and transport-see culture-feces routine. Not included in routine culture must be ordered separately. CULTURE-FECES VANCOMYCIN RESISTANT ENTEROCOCCUS Collection and transport see culture-feces routine. Not included in routine culture must be ordered separately. CULTURE-GENITAL Cervical and vaginal: collect with a swab inserted through a speculum. Avoid touching swab to uninfected mucosal surfaces. Urethra: collect exudate with a swab. If no discharge can be obtained, cleanse the external urethra and insert a urethral swab. 2-4 cm into the urethra. Directly inoculate a Jembec plate and send a transport swab in charcoal medium. Send a slide for gram stain if requested. CULTURE-FUNGUS Body fluid: collect in a sterile container. If specimen may clot use a tube with heparin. Wound or abscess: send aspirate or drainage in a sterile container or on a charcoal swab. Tissue: transport to lab immediately or cover with a small amount of non-bacteriostatic saline. CMH Lab Manual Last updated: 4/2012 Page 14 of 23 Test Name CULTURE-BLOOD FOR FUNGUS CULTURE – MALASSEZIA FURFUR CULTURE MYCOPLASMA/UREAPLASMA Collection & Other Instructions Decontaminate the puncture site as for blood culture. Decontaminate the top of the Wampole Isolator tube. Collect a 0.5-1.5 mL of blood using a vacutainer needle or a syringe. Butterfly draws are not recommended as blood may clot prior to inoculating the Isolator tube. Transport to the lab immediately. Special culturing techniques required. Indicate on requisition or computer if M furfur is suspected. Order as a Culture Fungal or Blood Culture Fungal.. Collect at least 1mL sputum or tracheal aspirate. Transport in a sterile cup or trach tube. Deliver to lab immediately Samples are sent to St. Luke’s Hospital for culture. CULTURE-MRSA Acceptable sources for MRSA screening include axilla, groin, nose, throat,,and rectum. Swabs of dry skin surfaces should be premoistened with nonbacteriostatic saline. Both nares can be sampled using the same swab. Transport swabs in charcoal transmort media. CULTURE-NOSE Swabs for the nares are accepted for surveillance purposes only (MRSA colonization, ICN admission and dialysis patients) and are not appropriate for evaluation of sinusitis. CULTURE - PERITONEAL DIALYSIS FLUID 20-50 CC of fluid in a sterile screw cap tube or poly cup. Deliver to the lab immediately. If delayed, refrigerate but do not freeze. BORDETELL PERTUSSIS PCR/ CULTURE Contact the microbiology lab (816) 234-3386 for a transport kit and collection instructions. CULTURE - RECTAL NEISSERIA GONORRHOEAE Carefully insert a swab≈ 1 inch beyond the anal sphincter. Directly inoculate a Jembec plate and send a transport swab in charcoal medium. CULTURE-RECTAL BETA STREP GROUP A Carefully insert a swab≈ 1 inch beyond the anal sphincter. Transport the swab in charcoal medium. Order as an Aerobic Culture and choose Rectal for Group A Strep as the specimen source. CULTURE - RESPIRATORY Sputum: Instruct the patient to cough deeply, not just spit. A first morning specimen is best. Collect sample in a sterile container. Culture processing must be preceded by Gram stain demonstrating >25 PMN and <10 epithelial cells/lpf. Tracheal aspirate: Collect sample in a sterile container. Refrigerate. Note if patient has cystic fibrosis. CULTURE - RESPIRATORY quant Specimen: Bronchia alveolar lavage. Transport immediately or on ice. Note if patient has cystic fibrosis. CMH Lab Manual Last updated: 4/2012 Page 15 of 23 Test Name Collection & Other Instructions CULTURE - SURGICAL Tissue: place in a sterile tube or poly cup and cover with a small amount of non-bacteriostatic saline. Fluid or pus: aspirate specimen and transport in a sterile tube or poly cup. If swabs must be used, collect one charcoal swab for each type of culture ordered. If anaerobic, fungal or AFB cultures are required, separate orders are required. CULTURE - THROAT Swab the back of the throat between and around the tonsils, being careful not to touch the tongue. Use a charcoal transport swab. Routine cultures will screen fro Group A Beta Streptococcus and upon special request Arcanobacter haemolyticum. For patients with cystic fibrosis, throat cultures will be evaluated for Staphylococcus aureus, Pseudomonas aeruginosa and Burkholderia cepacia. CULTURE – THROAT NEISSERIA GONORRHOEAE Collect sample as described for routine culture. Directly inoculate a Jembec plate and send a transport swab in charcoal. CULTURE - URINE If possible, collect an early morning specimen. Collect voided midstream urine into sterile container; do not use a bedpan or urinal. Straight catheter collection is better for avoiding skin contamination, but the risk of introducing organisms into the bladder is increased. Suprapubic aspiration of the bladder is occasionally necessary in infants. Transport immediately or refrigerate. Specimens may be refrigerated up to 24 hours. Refrigeration is not required if a transport tube with appropriate preservative is used. Foley catheter tips are unacceptable for culture. CULTURE - WOUND Decontaminate surrounding skin, then open the lesion and express pus onto a charcoal transport swab. Sample the advancing margin or the lesion. Label the specimen as to the source of the wound (i.e. incision, burn, dog bite etc.) CMH Lab Manual Last updated: 4/2012 Page 16 of 23 Test Name Collection & Other Instructions HANGING DROP FOR TRICHOMONAS Vaginal or urethral swab transported in 1 cc of nonbacteriostatic saline or an InPouch Trichomonas transport. Send to the laboratory immediately as specimen must be fresh. If the InPouch is used, inoculate by rinsing a vaginal or urethral swab in the fluid of the upper chamber, or by transferring a small amount of the saline suspension from a hanging drop tube to the pouch. Roll down the top and seal with the side closures. Transport to the LAB immediately. If there is a delay in transport beyond 15 minutes, move the fluid from the top chamber to the bottom chamber. A negatiave hanging drop will reflex to Trichomonas culture or PCR depending on patient age. H. PYLORI SCREEN Place biopsy sample in Remel rapid urease tube. KOH PREP Skin scrapings, hair or nail clippings. Transport in a sterile Petri dish or poly cup. O&P GIARDIA/ CRYPTOSPORIDIUM ANTIGEN Feces must be preserved within 30 minutes. Add feces to PVA vial and 10% formalin vial according to package directions. Do not overfill. Do not submit samples contaminated with urine or water. Samples containing bismuth, barium or magnesium are unacceptable. Allow 7 to 10 days for these substances to clear before collecting feces. OVA AND PARASITES COMPLETE EXAM See O&P Giardia/Cryptosporidium PINWORM EXAM A clear scotch tape prep or a pinworm collection paddle obtained first thing in the morning before the patient has bathed or used the bathroom. Press the adhesive side of the scotch tape or pinworm paddle firmly against the anus, flattening the peri-anal skin folds. If using scotch tape, affix the tape to a class slide for transport to the laboratory. Shiga Toxin Assay Shiga Toxin –Producing E. coli THROAT - RAPID STREP GROUP A WORM IDENTIFICATION - ROUND WORMS OR TAPE WORMS CMH Lab Manual Last updated: 4/2012 Feces: Screw cap Cary-Blair vial. Do not exceed the fill line This is included in Routine stool culture but may be ordered separately. Dual Dacron swabs without tramsport media. Sample only the back of the throat between and around the tonsils. Place both swabs back into the plastic sleeve. Negative rapid Strep tests are automatically confirmed with a throat culture for Group A Strep. Submit whole worm or tapeworm proglottids. Transport in 10% formalin. Page 17 of 23 Stool Specimens Test Name Collection & Other Instructions Routine culture for enterics – Salmonella, Shigella, Campylobacter, Yersinia and E Coli 0157 and Shiga Toxin Feces: Screw cap Cary-Blair vial. Do not exceed the fill line. Routine culture will screen for E. coli 0157 Salmonella, Shigella, and Shiga Toxin for Yersinia, Campylobacter sp. enterohemorrhagic E. coli. A rectal swab may be submitted if it is not possible to obtain feces but Shiga Toxin testing will not be performed from swabs. Shiga Toxin Assay Shiga Toxin –Producing E. coli Cary Blair vial Rectal swabs are not acceptable Aeromonas/Plesiomonas Same as routine culture Rectal for GC (Neisseriaeae) Jembec plate and charcoal swab VRE -Vancomycin resistant Enterococcus Same as routine culture Yeast or Fungus Cary Blair vial or tube or charcoal swab C. difficile toxin Stool in poly cup AFB/TB Stool in poly cup Giardia / Cryptosporidium Antigen Stool in 10% formalin and zinc PVA See Ova and parasites Stool in 10% formalin and zinc PVA. 1 to 3 samples collected over 7-10 days but no more than one sample per day will be accepted. Sample should be free of barium & mineral oil. Ova and parasites Pinworm Clear scotch tape or pinworm collection paddle take from perirectal area. Collection must be done in the morning upon awakening. Rotavirus Rectal swab or fecal material in a poly cup Virus culture Rectal swab or fecal material in viral transport media. Stool Specimens – other testing Test Name Collection & Other Instructions Fecal Fat Stool in poly cup, no preservatives, pea size Fecal WBCs Stool in poly cup, no preservatives, pea size Non-reducing substances Stool in poly cup, no preservatives, pea size Reducing substances Stool in poly cup, no preservatives, pea size Occult blood Stool in poly cup or Hemocult card Stool pH Stool in poly cup, no preservatives, pea size Stool electrolytes- NA, K, CL, CO2 Liquid stool in poly cup CMH Lab Manual Last updated: 4/2012 Page 18 of 23 Molecular Microbiology/Virology – Special Instructions Test Name Collection and Other Instructions PCR Chlamydia & N gonorrhoeae, PCR Chlamydia trachomatis, PCR Neisseria gonorrhoeae ER or SCAN Patient:: Urogenital or rectal swab specimens – collected and transported in M4 Culture Transport Media. Urine collection – patient should not have urinated for 1 hour. Patient collects first 20-30 mL of initial urine stream into a specimen cup. Urine cup transported to the lab. Adolescent Clinic Patient: Vaginal swab specimens collected in GenProbe vaginal swab specimen collection kit. Urine specimens collected in a primary urine collection cup and then transferred into Gen-Probe urine transport tubes by Adolescent Clinic staff or Virology lab staff. PCR Trichomonas reflex Adolescent Clinic Patient only: Vaginal swab specimens collected in Gen-Probe vaginal swab specimen collection kit. This is the same specimen collected for the PCR Chlamydia and N gonorrhoeae test. NOTE: Reflex PCR is performed on patient specimens that are negative for Hanging Drop for Trichomonas. PCR Enterovirus CSF, PCR Enteroviurs and Parechovirus, PCR Parechovirus, PCR Parechovirus Reflex CSF collected using standard lumbar puncture techniques. 200 µL volume is needed for testing. Volumes between 50 – 200 µL are tested with a low-volume specimen disclaimer. Volumes < 50 µL are not tested. NOTE: Parechovirus PCR reflex testing is performed on patients ≤ 6 months old. PCR Respiratory Virus Panel PCR HSV 1/2 CSF Nasopharyngeal swabs, nasal aspirates, and tracheal aspirates collected in viral transport media. CSF collected using standard lumbar puncture techniques. 200 µL volume is needed for testing. Volumes between 50 – 200 µL are tested with a low-volume specimen disclaimer. Volumes < 50 µL are not tested. PCR EBV Quant Blood Whole blood should be collected in lavender top vacutainer using EDTA as the anticoagulant, (heparin as the anticoagulant is unacceptable). Minimum volume is 0.5 mL. PCR CMV Quant Plasma Whole blood should be collected in lavender top vacutainer using EDTA as the anticoagulant, (heparin as the anticoagulant is unacceptable). Plasma separation is performed by the laboratory. Minimum volume is 1 mL. PCR HHV-6 Quant WB Whole blood should be collected in lavender top vacutainer using EDTA as the anticoagulant, (heparin as the anticoagulant is unacceptable). Minimum volume is 0.5 mL. PCR BKV Quant PCR Influenza A/B/H1N1 CMH Lab Manual Last updated: 4/2012 Plasma: Whole blood should be collected in lavender top vacutainer using EDTA as the anticoagulant, (heparin as the anticoagulant is unacceptable). Plasma separation is performed by the laboratory. Minimum volume is 1 mL. Urine: Specimen collected in a polycup. Nasopharyngeal swabs and nasal aspirates collected in viral transport media. Note: Tracheal aspirate, nasal swab, throat swab, and BAL specimens will be run with a disclaimer. Page 19 of 23 Virology – Special Instructions Specimens • Use rayon-, Dacron-, or cotton-tipped swabs • Do NOT use CULTURETTES or CALGI-SWAB o CULTURETTES are NOT an unacceptable means of isolating viruses o CALGI-SWAB will inhibit growth Media • Use viral transport media unless otherwise specified by test • Specimens placed in inappropriate media will be rejected Transport • • • • after collection, place the swab in viral transport media leave the swab in the tube secure the screw-capped lid tightly Transport to the lab ASAP Containers must be properly sealed and labeled, including specimen source. Specimens without source may require additional follow-up and testing will be delayed. Test Name Collection and Other Instructions Rapid Ag Flu A/B Rapid Ag RSV Nasal wash, Nasal aspirate or Nasopharyngeal swab in Viral Transport Media. Refrigerate if there will be a delay in getting sample to the lab. Swabs and Viral Transport Media can be obtained by going to the Virology Dept. (53060) Culture Viral Respiratory Nasal wash, Nasal aspirate, Nasopharyngeal swab, throat, Nose, Sputum, Tracheal aspirate, Bronch wash place in Viral Transport Media and deliver to lab ASAP. Refrigerate if there is a delay. Culture Viral Stool Collect a fresh stool specimen. Place a small pea size amount in Viral Transport Media. Refrigerate if there is a delay of more than 30 minutes in transporting. Culture Viral Urine Collect in a sterile cup. Transport on ice to lab. Culture Viral CSF Transport immediately in sterile screw cap tube. 0.5mls needed for culture. Culture Viral Blood Collect in 3ml Green top tube. Transport on ice to lab. Culture Viral Misc. Transport in Viral Transport Media. Write accurate description of the specimen on the tube. Deliver to lab. If there is a delay refrigerate. CMH Lab Manual Last updated: 4/2012 Page 20 of 23 Cytogenetics – Special Instructions Lab Hours/Phone/Fax • • • Monday – Friday; 0800 – 1630 & Saturday; 0800 – 1530 Phone: (816) 802-1220 Fax: (816) 802-1204 General Information • • Testing methods include: conventional chromosome analysis, florescence in-situ hybridization (FISH), post-natal microarray (aCGH) and cell culture. Prenatal, postnatal and oncologic testing is performed using most tissue types and fluids, includes: amniotic fluid, bone marrow, chorionic villus, CSF, lymph node, skin, solid tumor, peripheral blood, POCs, pleural fluid and other. Specimen Handling and Transport • • • • • • • • Label specimens with two patient identifiers as well as date/time of collection A completed Cytogenetics Requisition must accompany all specimens Contact the Lab for transport kits and medium Specimens should be collected using aseptic techniques, stored and transported at room temperature Special arrangements are required for extremes in ambient temperature or with extended transport times Notify the laboratory in advance of specimen arrival Specimens should be transported ASAP and arrive within 24 hours of collection; tumors must arrive the date of collection NEVER FREEZE OR PLACE SPECIMEN IN FIXATIVE (example- formalin) CMH Lab Manual Last updated: 4/2012 Page 21 of 23 Specimen Type Instructions Prenatal AMNIOTIC FLUID CHORIONIC VILLI • • • • TISSUE: PRODUCTS OF CONCEPTION • Gestational age at least 14 weeks (preferably 15-18 weeks) 15-20 mL fluid (≈10mL in 2 sterile clear plastic-capped centrifuge tubes) >30mg of tissue in sterile medium Products of conception at ≤9 weeks gestational age, send entire specimen. Specimens >9 weeks gestational age, send chorion or chorionic villi. For identifiable fetal parts, send each tissue type separately in sterile culture medium or balanced salt solution. Send connective tissues such as cartilage, cornea, pericardium, dura mater, fascia, lung and skin Postnatal PERIPHERAL BLOOD TISSUE: SKIN MICROARRAY aCGH • • • • • Collect 2-5 mL whole blood in sodium heparin (green top tube) Mix well by inverting tube several times Aseptically acquire a skin biopsy (including dermis); place in sterile tissue culture medium; keep at room temperature Collect 1-3 mL whole blood in EDTA (lavender top tube) Mix well by inverting tube several times Oncology Collect 2-5 mL bone marrow in sodium heparin (green top tube) Mix well by inverting tube several times Collect 7-10 mL whole blood in sodium heparin (green top tube). LEUKEMIC BLOOD Mix well by inverting tube several times Send WBC count and differential with the sample Aseptically collect specimen 0.5-1.0cm3 (500-1000mg); place in sterile SOLID TUMOR transport medium LYMPH NODE • Never freeze or add fixative to tumor samples FISH (fluorescence in-situ hybridization) • To detect subtle chromosomes abnormalities, e.g., microdeletions, to characterize structural abnormalities, e.g., translocations and inversions; to detect aneuploidy in interphase cells; and other. Contact CONSTITUTIONAL the Cytogenetics Lab at (816)802-1220 regarding probe availability. • Prenatal Screening to detect aneusomy of chromosomes X, Y, 13, 18, and 21 • Hemato-oncology disorders, including CML, ALL, CLL, AML, MDS, Multiple Myeloma, LPDs, NHLs • Solid tumors • To detect common gene rearrangements, aneuploidy, gene ONCOLOGY amplification • FISH probe panels are available for ALL, CLL/LPDs, AML/MDS, and Multiple Myeloma • Contact the Cytogenetics Lab at (816) 802-1220 regarding probe availability • Unstained tissue sections (≈ 3-4 microns thick) on sialinized slides accompanied by a sequentially cut H&E stained slide FORMALIN-FIXED • Indicate area of tumor on H&E slide PARAFFIN EMBEDDED • Include a copy of the pathology report TUMOR • Contact the Cytogenetics Lab at (816) 802-1220 regarding probe availability BONE MARROW CMH Lab Manual Last updated: 4/2012 • • • • • • Page 22 of 23 Molecular Genetics – Special Instructions Lab Hours/Phone/Fax • • • • Monday – Friday; 0800 – 1630 Phone: (816) 234-3588 Genetic Counselor phone: (816) 983-6984 Fax: (816) 983-6696 • After hours/weekends: page the Genetic Counselor at (816) 458-5011 General Information • • • Test requests should include the reason for the test Pedigrees are requested for family studies Requisition Constitutional Studies (Inherited Disease) Specimens • Peripheral blood in an EDTA vacutainer o Adults: 3-5 mL o Infants: 2 mL • Tissues (such as muscle or skin) must be placed immediately on dry ice unless cultured • Urine- minimum 1 mL volume, random colletion • Please contact the lab for instruction regarding prenatal specimens Neoplastic Studies (Cancer) Specimens • Bone marrow should be drawn with no anticoagulant and rapidly placed into an EDTA vacutainer • Peripheral blood in an EDTA vacutainer • Tumors should be immediately placed on dry ice. An adjacent section should be analyzed by pathology so we know the histology of the specimen. Tumor Specimen Handling • • • Place on dry ice immediately Transport with sufficient quantity of dry ice to maintain frozen state A -700C or colder freezer is acceptable for storage CMH Lab Manual Last updated: 4/2012 Page 23 of 23