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1 Welcome to the online QML Pathology Reference Manual For ease of use please download this manual to your desktop. Viewing via your web browser may take time to reload depending on internet connection speeds. This manual is best viewed in Full Screen mode in Adobe Acrobat Reader version 7 or higher. Press Escape key to exit Full Screen Mode. Use the buttons below to navigate this manual. Search Manual Contents Page Launch Full Screen Mode Reference Manual

2 CONTENTS Home Full Screen CONTENTS The content of the QML Pathology Reference Manual is provided as current information as at July Information in this manual may change over time. For the latest information, please refer to the QML Pathology website or contact your local QML Pathology laboratory. Click on the links below to jump to the required section Introduction 1.0 Preface 5.0 Appendices 12.0 CONTENTS Mission Statement 1.1 Company History 1.2 Collection Facilities 2.0 Collection Centres A-Z 2.1 Special Tests 2.13 Additional Services 3.0 Vaccine Service 3.1 Travel Health Service 3.1 Warfarin Service 3.2 Occupational Pathology 3.4 Vetnostics 3.5 Departmental Directions on Specimen Collection, Storage and Transport 5.1 Blood Bank 5.9 Cytology 5.11 Endocrinology 5.21 Genetics 5.25 Haematology 5.31 Histology 5.35 Immunology 5.47 Microbiology 5.51 C C Test Listing A-Z Common Causes of Abnormal Biochemical Results 12.1 Serum Tumour Markers 12.4 Common Reference Ranges 12.7 Glucose Tolerance Test 12.9 Gestational Diabetes 12.9 Dietary Restrictions and Special Diets Qualitative Urine Drug Screen Quantitative Drug Assays for Therapeutic Monitoring Poisons and Toxic Substances Used in Pest Control Acid Base Analysis Lipids Endocrinology Pregnancy Timeline Investigation of Hirsutism Recommended Age Guidelines for Men s Health Testing Immunology Antibodies to Tissue Antigens (Autoantibodies) Antibodies to Microbial and Parasitic Agents Arbovirus Screen Skin Tests for Allergy RAST Allergen List Skin Allergen List Microbiology Infection Control in Medical Consulting Rooms Blood Collection, Waste Management, Handling Sharps Guidelines for Gloves, Handwashing Protocol Clean Up Procedure for Blood and Body Fluids Body Fluid Exposure Procedure Validation of your Steriliser Symbols for Hazardous Categories Collection Materials 4.0 Specimen Storage 4.1 Order of Draw 4.2 Blood Collection Tubes 4.3 Specimen Containers 4.6 Swabs 4.14 Skin Devices 4.16 Test Selection Guide 6.1 Test Listing A-F 7.0 Test Listing G-L 8.0 Test Listing M-R 9.0 Test Listing S-Z 10.0 Contact Details 11.0 Rule 3 Exemption 11.1 Genetics Cytogenetic Tests Molecular Genetic Tests Haematology Basic Haematology Parameters Leucocyte Reference Ranges Initiating Warfarin Therapy Range of Target INRs Duration of Warfarin Therapy Drugs that Interact with Warfarin 12.34

3 COLLECTION MATERIALS COLLECTION MATERIALS 4.1 SPECIMEN STORAGE All EDTA blood and blood films in Haematology are stored refrigerated for 1 week. Blood films showing significant pathology are archived for 1 year. All Bone Marrow blocks and slides are archived for 14 years. All sera in, Endocrinology and Haematology are stored refrigerated for 7 days after collection Note: Some analytes may deteriorate in this time. Serum collected for viral, bacterial or parasitic antibody testing is kept frozen for 12 months to follow the course of the illness or to make a diagnosis retrospectively [Immunology (07) or Branch Laboratory]. Gram-stained slides and culture plates are kept in Microbiology for 1 week should further sensitivity testing or identification be required. Histology tissue specimens are stored for 4 weeks before disposal. Blocks and slides are archived for 14 years. All cytology smears and preparations (normal and abnormal) are archived for 14 years. Specimen Labelling Requirements Please ensure all request forms and specimens have correct patient details Our minimum requirements are: Surname Given Names Date of Birth Date and time of collection Please understand that incorrect or insufficient labelling can necessitate a recollection All tubes MUST be signed by patient or collector to confirm patient identity. ORDER OF DRAW Vacutainer & Syringe Method 1. Blood culture bottles 2. Pale blue top (Sodium citrate)** 3. Tubes without chemical additive (SST, Red top, Navy top) 4. Green/Orange top (Lithium heparin) 5. Pink/Lavender top (EDTA) 6. ESR (if required) 7. Grey top (Fluoride oxalate) 8. Yellow top (ACD - Acid citrate dextrose) Aerobic bottle Anaerobic bottle Paediatric micro container Paediatric mini container Paediatric mini container Paediatric mini container Paediatric micro container Paediatric bottle Yellow rubber top Tube MUST be filled to indicated level Paediatric micro container Paediatric micro container **If the citrate tube is the only tube to be drawn (or if it is the first tube to be drawn), this tube is acceptable for routine coagulation testing (APTT and PT/INR). For special coagulation testing (e.g. Factor VIII and Heparin Therapy) the citrate tube should not be the first tube drawn. Use of a plain discard tube may be considered in this situation. 4.2 COLLECTION MATERIALS

4 COLLECTION MATERIALS COLLECTION MATERIALS 4.3 Blood Collection Tubes Serum separation tube (SS tube) (Yellow plastic top) After clotting, the tube should be centrifuged for 10 minutes and may then be left refrigerated overnight. Endocrinology tests (Thyroid function tests, FSH, LH, etc.) E/LFT: including urea, glucose, electrolytes, liver function tests, cholesterol, triglycerides (lipids) Autoantibodies (Including antisperm antibodies) Microbial, parasitic and viral serology (hepatitis serology, rubella antibodies, etc.) Pregnancy tests Paul-Bunnell test Tumour markers Iron studies/b12 Plain tube (Red top) All drug assays Vitamin D Paediatric micro container Paediatric micro container Fluoride oxalate tube (Grey top) Blood alcohol Lactate studies Blood glucose (if a delay in cell separation is unavoidable) Paediatric micro container Blood Bank EDTA tube (Pink top) Blood group Rh antibodies Crossmatch (+ EDTA lavender top) Group & hold serum (+ EDTA lavender top) HLA B27 testing Genetics (some) Sodium citrate tube (Pale blue top) Coagulation studies: Prothrombin time Thrombophilia tests Factor assays etc. INR APTT D-Dimer Fibrinogen. Acid citrate dextrose - ACD tube (Yellow rubber top) HLA tissue typing Leukaemia marker studies Lymphocyte studies Lymphocyte subset analysis HIV viral load EDTA tube (Lavender top) Full blood count: including haemoglobin, white cell count, platelet count Red cell folate Hb EPP ACTH Hb A1C ESR Paediatric mini container Paediatric micro container Tube MUST be filled to indicated level Paediatric mini container 4.4 COLLECTION MATERIALS

5 COLLECTION MATERIALS COLLECTION MATERIALS 4.5 Lithium heparin tube (Green top, orange top) Used for a wide variety of tests covering biochemistry, haematology and genetics Heavy metals screens Chromosome analysis Paediatric Paediatric micro container mini container Trace metal tube (Navy top) Must be centrifuged immediately for 10 minutes. Zinc Selenium Aluminium. ESR tube ESR SPECIMEN CONTAINERS Urine for microbiology (MUC M/C/S) After collection of the urine into a sterile container, aspirate the specimen into the Monovette. Transport to the laboratory immediately. If transport to the laboratory is likely to be delayed for more than 12 hours, refrigerate until transport is available. Suprapubic aspirates should be collected into a sterile container and refrigerated if transport to the laboratory is delayed. Urine collection bottles Used for timed urine collections. Patient instructions are written on the bottle. The bottle should be refrigerated between collections. Certain collections may need preservative (Check A-Z test listing of this Reference Manual). Funnel 8hr urine collection bottle Sterile container 24hr urine collection bottle Yellow monovette 4.6 COLLECTION MATERIALS

6 COLLECTION MATERIALS COLLECTION MATERIALS 4.7 Aptima urine tube Chlamydia trachomatis Neisseria gonorrhoeae Urine drug screen collection kit For details of use and Chain-of-Custody documentation requirements, see Drug Screening section (5.4) in Preface of this Reference Manual. Blood culture bottles Adults - take 16-20mL of blood on each occasion and divide evenly into 2 adult culture bottles (aerobic and anaerobic). Children - take 1-3mL of blood on each occasion and place in a paediatric blood culture bottle. If difficulty is experienced in obtaining blood from some patients, the paediatric blood culture bottle will suffice for adults. Faeces container Liquid stools should be examined promptly - please contact the laboratory to arrange pickup. Formed and semi-formed stools should be received by the laboratory within two hours of collection. Faeces container Aerobic bottle Anaerobic bottle Paediatric bottle 4.8 COLLECTION MATERIALS

7 COLLECTION MATERIALS COLLECTION MATERIALS Seminal fluid collection Collect specimen into a sterile container. This sample needs to reach the laboratory within two hours. Sterile container Skin scrapings containers Superficial mycoses may infect skin, hair and nails. Skin scrapings from the active edge of the lesion and scrapings from nails, together with clippings of nails and hair, may be placed in a sterile container. The paper envelope may also be used for collecting skin scrapings. If the lesion is exuding material and may be painful to scrape, a swab may be collected as an alternative. Use a dry swab previously moistened with saline to swab the lesion. Place the swab in a container without transport medium. COLLECTION MATERIALS Antibiotic transport medium Suitable for the transport of all viable tissues. Genetics (POC, etc.) Tissue culture Tissue tumour markers (lymph nodes, etc.) Nasopharyngeal tubing Nasopharyngeal aspirate RSV (Respiratory Syncytial Virus) Influenza A & B, Parainfluenza 1, 2, 3, Adenovirus Bordetella Pertussis PCR Histology specimen container Routine histology tissue and biopsy specimens are placed into 10% formalin for fixation and submitted to the laboratory for paraffin processing

8 COLLECTION MATERIALS COLLECTION MATERIALS 4.11 Synovial fluid collection kit 1. Crystals, rheumatoid factor latex, gram stain and culture. Several ml in a sterile screw top (urine) container. 2. Cell count and differential. 1-2mL in lithium heparin (green top) tube to prevent specimen clotting. 3. Protein, albumin and glucose. 1-2mL in a EDTA (lavender top) tube. Sterile container Cytology kits Monolayer cytology kit After preparing a conventional Pap smear, rinse the Cervex brush or preferred collection device thoroughly in the cell preserving solution. Transport the Pap smear in slide carrier and the labelled cell preserving solution to the laboratory. EDTA tube Lithium Heparin tube Single-use Pap smear kits Three separate kits available: Cervex brush Cytobrush Combination. Cervex brush Pap smear test kit Cytobrush Pap smear test kit Combination Pap smear test kit 4.12 COLLECTION MATERIALS

9 COLLECTION MATERIALS COLLECTION MATERIALS 4.13 Fine needle aspiration kit The fine needle aspiration kit contains all materials necessary to perform a fine needle aspiration of a lesion by any preferred technique. It comes packaged in a handy rigid transport cylinder which can be utilised to transport the specimen back to the laboratory. Swabs Bacteriology transport swab May be left overnight at room temperature except where gonorrhoea or anaerobic infection is suspected. In these cases, please contact the laboratory to arrange prompt pick up. Viral culture transport swab May be left overnight refrigerated. Use for routine virology COLLECTION MATERIALS

10 COLLECTION MATERIALS COLLECTION MATERIALS 4.15 Aptima Chlamydia trachomatis/neisseria gonorrhoeae swab for molecular testing Store at room temperature before and after collection. Flocked swab (Dry flexible swab) Used for PCR and respiratory viruses. Store at room temperature before and after collection. Nasopharyngeal swab (Dry swab) Store at room temperature before and after collection. May be left overnight refrigerated. Skin Devices Skin punch biopsy devices The punch biopsy with internal plunger system allows the lodged skin specimen inside the metal lumen of the punch to be easily ejected. Available in several sizes: Punch Biopsy with internal plunger available in 2, 3 and 4mm Punch Biopsy without plunger available in 2, 3, 4, 5, 6 and 8mm. 2mm Punch Biopsy with internal plunger 2mm Punch Biopsy without plunger BIOPBLADE The sterile, single-use BIOPBLADE is a flexible scalpel used for cutaneous surgery, including: shave biopsy, saucerisation of flat lesions and levelling of pedunculated lesions. The unique design of the BIOPBLADE incorporates a comfortable and protective Fingerguard in addition to the flexible super sharp blade. This flexibility allows the blade to be positioned at the correct angle for the intended procedure. The BIOPBLADE is utilised for removal of lesions, either elevated (shave biopsy) or flat (saucerisation). After the site is anaesthetised, the BIOPBLADE is held and bowed between the thumb and fingers. The lesion is removed at or just below the surface epithelium. Cosmetic results are normally good and the wound heals without the need for suturing. The Clinician will remove all of the lesion without overly deep penetration to avoid scarring COLLECTION MATERIALS

11 PREFACE - biochemistry PREFACE - BIOCHEMISTRY TESTS WITH SPECIAL COLLECTION REQUIREMENTS Many of the range of close to 1200 tests performed by or arranged through the Department have requirements which, if not recognised and met, may lead to misleading or delayed results. The section below refers only to requirements at the time of collection. Those tests that require pre-test preparation of the patient are listed subsequently. Test Requirements at Time of Collection BLOOD, SERUM OR PLASMA TESTS Arterial or Venous Blood Gases Blood remains living, actively metabolising tissue after collection and unless measures are taken to slow or halt metabolic activity, misleading results will be obtained. It is important that blood collected for oxygenation and ph studies should be cooled on ice or cold packs as soon as possible (within 20 minutes) after collection or an artefactual metabolic acidosis (low ph and bicarbonate, with raised negative base excess) may ensue from red cell generation of lactic acid from glucose. A slower process, white cell aerobic metabolism leading to a slow fall of po 2 and a rise of pco 2 with apparent respiratory acidosis will further complicate interpretation. Serum Calcium, Iron and Alkaline Phosphatase When using evacuated blood tubes (vacutainers) for sample collection, it is important to collect biochemistry samples before haematology. If a full vacutainer is collected, a small volume of blood usually refluxes back into the needle during withdrawal of the container. If haematology is collected first, this blood contains the EDTA anticoagulant and can pass into the next tube attached. If the latter is used for biochemical profile assay, we occasionally note a small but significant fall in calcium and iron. Rarely, a marked suppression of these as well as suppression of alkaline phosphatase may be noted. There is concern that milder artefacts go unrecognised Serum Therapeutic Drugs After administration of a drug, there is an interval between the absorption of the material and its uptake into the tissues within which it is active, during which the serum levels are misleadingly high (because they do not meaningfully reflect end-organ or tissue levels). This interval is known as the distribution phase. Clearly drug levels must be examined after this interval to give most useful information. The exception to this is seen with the antibiotics, in which the peak level itself conveys valuable information relating to bactericidal effect and to risk of toxicity. Blood Alcohol and other Medico-legal Collections When blood is collected for possible legal purposes (e.g. to challenge a police breathalyser finding), it is important to first of all obtain samples as close as possible to the time of the initial sampling and then to adequately seal those samples so that the pathologist can certify that no tampering has occurred between collection and testing. An appropriate method of sealing a blood tube is the placement of one QML Pathology bar code label, saddle-like across the top of the tube so that the ends reach approximately a centimetre down the glass, followed by the wrapping of a second label around the body of the tube so that it covers the ends of the first. The patient details are then completed and the collector and patient each sign across the joint of the two labels. The protocol also includes a Chain-of-Custody form which records the legally correct Chain-of-Custody of the specimen from collection to production of a report. This form is to be signed by both donor and collector. Chain-of-Custody forms detail the procedure and are available on request from QML Pathology. We also strongly recommend that saline or sterile water only be used to cleanse the skin before venepuncture, and not alcohol swabs. Although isopropanol does not crossreact as ethanol in the laboratory assay, the necessity to argue this point in the court setting can lead to the whole collection being discounted. PREFACE - BIOCHEMISTRY Serum Electrolytes, Glucose, Enzymes and Phosphate A living cell maintains a steep electrolyte gradient across the cell membrane with high extracellular sodium and chloride, and high intracellular potassium concentrations. In contrast, intracellular sodium and chloride and extracellular potassium concentrations are around 5% of the corresponding transmembrane levels. The maintenance of these gradients is an active process, requiring plentiful ATP. If blood is stored at room temperature, glucose is consumed (metabolised to lactic acid) to maintain the membrane gradients. This is accompanied by a fall of bicarbonate to <10 mmol/l. When the sample glucose falls to <2 mmol/l, cellular metabolism fails and electrolyte leakage occurs. Plasma sodium falls sequentially to as low as mmol/l, chloride to mmol/l, and potassium rises to as high as mmol/l. Lactate dehydrogenase (LD/LDH) and aspartate transaminase (AST) escape and may elevate the plasma level to 4-5 times the upper limit of normal. Intracellular phosphate also escapes with the shutdown of glycolysis and may elevate the plasma phosphate to 4-5 mmol/l. The only way to prevent this sequence of events is to centrifuge and separate the serum/plasma from the cell mass, preferably within 20 minutes of collection into a serum separation tube (SS tube), plain tube or other tube. The gel plug of the SS tube separates cells from serum. With other tubes it is advisable to decant the supernatant serum/plasma into a sterile plain tube(s) for storage. This should be stored refrigerated. Collection of the sample into fluoride oxalate preservative or refrigeration of the sample eliminates the loss of glucose and maintains a normal anion gap but other changes proceed. Plasma Lactate Lactic acid is the end product of anaerobic metabolism and is elevated in states associated with liver disease, ischaemia, shock and blockade of Kreb s Citric Acid Cycle. However, it is also the normal product of red cell glycolysis. Hence if blood is collected and allowed to stand at room temperature, red cells will convert glucose to lactate and produce a spurious lactic acidosis (with lowered glucose and bicarbonate, and a raised anion gap and lactate). Examination of the results reveals a pattern that is indistinguishable from a

12 PREFACE - biochemistry true pathological lactic acidosis. To guard against this, it is necessary to centrifuge the blood within 20 minutes of collection and separate the cells from the plasma/serum. A serum separation tube (SS tube) allows this without decanting the serum. Alternatively, collection into a fluoride oxalate preservative tube inhibits metabolism such that physical separation may be deferred until the sample reaches the laboratory. Cooling the sample to refrigerator temperature also partially achieves this end but at the expense of meaningful electrolytes (see previous page). Plasma Very Long Chain Fatty Acids and Phytanic Acid VLCFA are performed in the diagnosis of adreno-leucodystrophy, Refsum s Disease, Zellweger s Syndrome and related abnormalities of cellular peroxisomal function. Phytanic acid is relatively more specific to Zellweger s Syndrome. Both of these tests are referred to Royal Brisbane Hospital for analysis. In all laboratory analyses, some clinical details are valuable in case extra testing or additional tests are indicated. However, with these tests the referral laboratory will not commence the analyses unless adequate clinical details are supplied with the sample. So as to avoid undue delay, please write appropriate clinical details on the request form. Neonatal Screen (Heel Skin-Prick Blood) In Queensland, the routine neonatal screen includes tests for phenylketonuria, hypothyroidism, galactosaemia and cystic fibrosis. The tests are routinely performed on paper discs punched from a standard filter paper card. Assume even application of the infant s blood onto marked areas of the card. Uneven application, particularly reapplication onto areas previously dried may lead to falsely high results and hence potential risks of false alarms for all of the tests. If a card is not available, blood may be collected into an EDTA or Lithium heparin tube and the application to the card made in the laboratory before transfer to the screening laboratory. Urinary Catecholamines and VMA Epinephrine (adrenaline), norepinephrine (noradrenaline), dopamine and their metabolites vanillylmandelic acid (VMA, 4-hydroxy-3-methoxymandelic acid, HMMA), and homovanillic acid (HVA) are the key elements in the diagnosis of phaeochromocytoma and childhood neuroblastoma and ganglioneuroma. The metanephrines have declined in popularity with improvements in the former assays. Current assays are not susceptible to interference from dietary vanillin. However, mild pathophysiological elevation of excretion in response to illness, injury, psychiatric agitation and to fluctuations in blood pressure is common. Particularly difficult are the often marked elevations of excretion in response to commencement or dose increases of antihypertensive therapy; increases which may persist for 1-2 weeks while a new steady state is achieved. In the case of newly diagnosed or suddenly deteriorating hypertension, the ideal would be to collect a single 24 hour urine sample before changing therapy. Plasma catecholamines are available but their diagnostic value as a screen is not as clear (because of rapid elevation before or during venepuncture). Dihydroxyphenylglycol assay is also available but again this is not as attractive as a first line test because it has a turnaround time of several weeks. 24 hour urinary catecholamines must be collected into acid preservative (or if collected as a stat or random collection, the sample must be kept refrigerated until acidified in the laboratory). Please refer to the specific test in the A-Z listing for full collection details. Plasma Ammonia Ammonia cannot be meaningfully assayed on skin-prick blood because the high sweat ammonia level always leads to marked false elevation of the apparent blood level from contamination. Trace and Toxic Elements from Skin-Prick Blood Rigorous attention to skin cleansing is always essential before collection as contamination from material on the surface of the skin can produce marked elevation. URINE TESTS Urinary Porphobilinogen (PBG) A raised PBG excretion in a stat urine collection taken during a symptomatic episode is a key finding in the diagnosis of an acute porphyria (Acute Intermittent Porphyria, Hereditary Coproporphyria, or Variegate Porphyria). However, PBG is quite unstable and the sample must be refrigerated and protected from light (wrapped in foil or brown paper), as well as tested as soon as possible after collection. Urinary Drug Screen for Overdose Please notify the laboratory and seal samples as described under blood alcohol and other medico-legal collections (5.2) if foul play or potentially lethal toxicity is suspected. Clearly, there may be medico-legal implications. Urine Drug Screen for Industry, Occupational and Drugs of Abuse This test is probably the one most likely to give misleading results as a direct result of deliberate interference with the collection by the patient. Substitution with urine from another, dilution with tap or toilet water or saliva, oral water loading to dilute urine, consumption of other substances in an attempt to mask drug findings and addition of chemicals to the urine to attempt to breakdown urinary drug metabolites are common occurrences among the group of patients who find themselves required to undergo this testing. Supervision of the collection and sealing of the sample is essential. The QML Pathology protocol complies with Australian/New Zealand Standard AS/NZS4308. The protocol includes a specifically designed tamper-evident urine specimen bottle, a procedure designed to ensure collection of a truly representative sample of urine from an identified patient, and documentation that is signed by the donor and collector and that records the legally correct Chain-of-Custody of the specimen from collection to production of a report. Chain-of-Custody forms detail the procedure and are available on request from QML Pathology. Please refer to the Appendix (12.13) for a full list of drugs assayed PREFACE - BIOCHEMISTRY PREFACE - BIOCHEMISTRY

13 PREFACE - biochemistry FAECES TESTS NOTE: Faeces has a proportionally huge bacterial load and their continuing metabolism may significantly alter the faecal biochemical profile. WITH ALL biochemical faecal tests, it is essential that the sample be refrigerated or frozen as soon as possible after collection. Faecal Analysis for Reducing Substances and Sugar Chromatography When testing for sugar/lactose intolerance, it is advisable to ascertain that the child has not commenced a lactose-free diet. Parents have been known to commence treatment before firm diagnosis, and this will certainly produce a false negative (normal) result. NOTE: It is the fluid component of the faeces specimen that is required for testing. Use of a non-absorbing liner such as Glad Wrap to prevent absorption by the baby s nappy when collecting a specimen is advised. Faecal Fat Analysis Refrigerated sample storage is very important (see above). Fly larvae (maggots), an occasional finding in the laboratory, may both consume malabsorbed triglyceride and produce their own. Nappy liners must not be used during collection. The patient must be taking an adequate diet not excluding fat or a falsely normal test will result. CSF TESTS Cerebrospinal Fluid Protein Rarely, we receive CSF which has been contaminated with myelogram contrast material. This results in a false elevation of the assayed protein level which may be marked (e.g. up to 20 g/l {R.R 0.4 g/l}). If there is any suspicion of this, the situation can be rapidly clarified with CSF albumin assay. SWEAT TESTS Sweat Electrolytes Only under exceptional circumstances will the clinician collect sweat samples. We strongly support this - the collection is too difficult unless performed by trained and experienced staff. However, should a collection be unavoidable, it is essential that any evaporative loss must be avoided as it leads to false elevation of electrolytes and, potentially, misdiagnosis of Cystic Fibrosis. SALIVA TESTS Salivary Screen for Drugs of Abuse This test requires 10 ml of saliva in a sterile screw top (urine) container. Collection must be supervised as described for urine. Faecal Porphyrin Excretion In addition to standard sample-handling procedures, it is essential that the patient should avoid contamination of the collection with urine. The high urinary uroporphyrin and coproporphyrin will falsely elevate the total and mask the characteristic faecal pattern. Faecal Alpha-1-Antitrypsin Analysis Alpha-1-antitrypsin is used as the marker of choice for the detection of enteric proteinlosing states, not because of any unique handling of this protein but simply because it is relatively resistant to bacterial degradation. However, the sample must be refrigerated as soon as possible after collection. Faecal Pancreatic Elastase-1 (PE1) The faecal elastase-1 concentration reflects the secretory capacity of the pancreas. That is, the diagnosis or exclusion of pancreatic exocrine insufficiency. The concentration of PE1 may be lowered in very watery stool samples. Formed stool samples are the preferred sample. Samples should be frozen ASAP. HAIR AND/OR NAILS TESTS Hair or Nail Analysis for Toxic Elements Hair or nails must be thoroughly cleaned without shampoo, soap or detergents before collection. Because maximal deposition takes place in the keratin being laid down while the metal level is maximal in soft tissue, nail can detect exposure 4 to 8 months previously and hair 2 to 6 months previously (depending on the length). Quantity of material for analysis: Hair - A packed matchbox Nail - As much as possible from fingers and toes. Clearly, if exposure is very recent, i.e. of the order of weeks, blood or urine testing may be more appropriate. STOMACH CONTENTS/VOMITUS TESTS Vomitus Analysis for Drugs in Suspected Overdose In addition to standard requirements of refrigeration to stabilise the sample both chemically and microbiologically, it is essential that any suspicions of possible exposure should be noted on the request form. This is to expedite testing the more likely drug classes. If there is any possibility of medico-legal implications, this should be noted on the request form and the sample should be sealed as described for blood alcohol PREFACE - BIOCHEMISTRY PREFACE - BIOCHEMISTRY

14 PREFACE - biochemistry BREAST MILK TESTS Breast Milk Analysis for Nutritional Qualities In cases of maternal concern, we occasionally test breast milk for glucose, lactose, lipid and protein content. Because the sample is invariably contaminated during collection, it must be refrigerated promptly until arrival at the laboratory. DETECTION AND IDENTIFICATION OF SNAKE VENOM IN SUSPECTED SNAKE BITE The most appropriate and preferred sample for testing is snake venom from the site of the bite. Moisten a cotton swab or cotton bud with saline or tap water and swab the site of the puncture wound(s). A small piece of clothing cut from the bite site may also be taken if appropriate. The swab(s) and the cloth sample should be placed in a labelled dry sterile screw top container (microurine container) - one sample per jar. Please contact the laboratory and forward the specimen as soon as possible. Urine collected in a sterile screw top container and blood collected in a Lithium Heparin tube may also be tested, however, the swab and/or cloth from the bite site(s) are the preferred samples. The service is available on an urgent basis 24 hours per day. TESTS REQUIRING PRE-TEST PATIENT PREPARATION Breath hydrogen analysis (general fasting instructions and no smoking one hour prior to test) Cholesterol and triglycerides (may require fasting if directed by doctor) Glucose tolerance test 5-Hydroxyindoleacetic Acid (5-H.I.A.A.) Water deprivation test Details of preparation for these tests can be found by referring to the A-Z test listing and the Dietary Section (12.10) of the Appendix. Printed instruction forms for patient preparation for these tests are available on request from QML Pathology Collection Centres, QML Pathology Brisbane Liaison Services (07) or your local Branch Laboratory PREFACE - BIOCHEMISTRY PREFACE - BIOCHEMISTRY

15 PREFACE - blood bank PREFACE - BLOOD BANK HOMOLOGOUS BLOOD TRANSFUSION SERVICE QML Pathology provides a Cross Match Service for elective surgery using homologous random donor blood provided through the Australian Red Cross Blood Service. AUTOLOGOUS BLOOD TRANSFUSION SERVICE QML Pathology offers an Autologous Blood Donation Program for patients desiring an alternative to homologous random donor blood provided through the Australian Red Cross Blood Transfusion Service. There are many recognised advantages for autologous transfusion in selected patients undergoing elective surgical procedures, including the elimination of serological incompatibility and transfusion acquired HIV and Hepatitis infection. Patients who are excluded by the Red Cross guidelines may be acceptable for autologous blood collection by QML Pathology. The referring practitioner must indicate that in his/her opinion the patient s physical condition will permit venesections to be performed safely. It is emphasised that initiation of the collection request should allow sufficient time for clinical review, an achievable collection plan for the desired number of autologous donations and coordination of iron supplement therapy where directed by the QML Pathologist. If autologous blood collection is desired, the following important information should be noted: 1. Prior to venesection it is requested that basic testing (full blood count, blood group and antibody screen) be performed to ensure there is no haematological contraindication to the procedure. All autologous units collected will be screened for Syphilis, Hepatitis B and C, HTLV-1 and HIV by QML Pathology. This is in accordance with the recommendations by NATA/RCPA Accreditation Authority and the Australian New Zealand Society for Blood Transfusion. Referring doctors should advise their patients accordingly. 2. Blood donations are collected into CPD-Adenine anticoagulant and have a shelf life of approximately 35 days. Donations are collected at weekly intervals for a maximum of four donations, and preferably no venesections are performed in the week prior to surgery. The expected fall in haemoglobin for an average adult male is approximately 10 gm per litre per donation (the fall will be somewhat greater in females). A check haemoglobin is therefore performed prior to each venesection. Patients are usually placed on an oral iron supplementation (Ferrous Sulphate mg -1 tablet twice daily prior to their procedure) by the QML Pathologist. 3. On admission to hospital, a specimen is collected for Cross Match and Antibody Screening. Although the patient is to receive autologous blood it is essential for safety and medico-legal reasons (clerical errors, etc.) that compatibility tests be performed. Secondly, the autologous collection procedure is only performed on the proviso that if additional blood is required beyond the autologous reserve the patient will accept homologous (Red Cross) blood. Should this prove necessary the laboratory will have a stored cross match specimen available and there will be no additional fee applicable for cross matching the homologous blood Requests for autologous transfusion should be written on the special QML Pathology form and be accompanied by request forms for: 1. Serology for Syphilis, Hepatitis B and C, HTLV-1 and HIV 2. Hb, Antibody Screen and Cross Match. Pre-printed request forms for collection of autologous donations by QML Pathology and the patient information brochure Autologous Blood Donations are available on request from QML Pathology Collection Centres, QML Pathology Brisbane Liaison Services (07) or your local Branch Laboratory. For further details please contact QML Pathology Blood Bank (07) or your local Branch Laboratory. PREFACE - BLOOD BANK

16 PREFACE - cytology PREFACE - CYTOLOGY SPECIMEN COLLECTION FOR GYNAECOLOGICAL CYTOLOGY Most cervical cancers and precancers arise in the transformation zone that is, where everted endocervical epithelium comes to be replaced by metaplastic squamous epithelium. Therefore, it is crucial that this area is well visualised and adequately sampled. The location of the transformation zone may vary depending on a woman s age and menstrual history. The use of the modified Ayre s spatula or Cervex sampler is adequate in most instances. A cytobrush may be used when the transformation zone is located further up the endocervical canal, as is the case in post-menopausal women. Preparation Label frosted-ended glass slides with the patient s name and date of birth using a pencil. If collecting a biopsy specimen at the same time, it is important to keep histology specimens and cytology slides physically separated during storage and transport. Liquid formalin vapour has an adverse effect on Pap smears which can make morphological assessment difficult. Spatula & Cytobrush Cervex Sampler Complete the request form including the patient s name, date of birth, date of last menstrual period and other relevant clinical information e.g. pregnant, post natal, post menopausal, use of hormones, presence of IUD. Record any high risk factors including prior treatment for CIN, abnormal cervical appearance, presence of contact or post coital bleeding. Check whether the patient wishes her name to be withheld from the Pap Smear Register. If there is no indication on the form the result details will automatically be sent to the register. Collection After introduction of a vaginal speculum and proper visualisation of the cervix, gently rotate the spatula 360 about its axis to ensure sampling of the entire transformation zone. If a Cervex sampler is used, rotate through 360, three times in both clockwise and anticlockwise directions. Press the outer bristles of the brush firmly against the ectocervix. If using a cytobrush in conjunction with the spatula, perform this sampling after an ectocervix specimen has been collected to avoid contamination by blood. Avoid inserting the cytobrush too far into the os, in order to minimise sampling of the lower uterine segment. Some bristles should still be visible. Rotate the brush twice (180 ). It is advisable not to use the cytobrush if the patient is pregnant. Preparing and Fixing the Sample onto the Slide Transfer the sample onto the slide using a painting action, with just enough pressure to ensure cell transfer. This is best achieved by smearing the spatula or Cervex sampler, or by rolling the brush. Fix the smear quickly to prevent air drying by spraying with Cytospray at a distance of 15-20cm from the slide. Alternatively immerse the slide immediately in 95% ethanol for 15 minutes. Leave the slide to dry for 15 minutes and place into the plastic slide carrier provided. Place the carrier along with the patient request form into a plastic specimen bag PREFACE - CYTOLOGY

17 PREFACE - cytology PREFACE - CYTOLOGY ThinPrep Specimens and HPV Testing The ThinPrep Imaging System utilises cells harvested from the collection device by rinsing into a preservative fluid. HPV DNA Testing for high risk HPV types can also be performed from the ThinPrep vial. It is desirable when collecting a liquid-based sample to use either a cervex sampler or a plastic spatula. A wooden spatula is not recommended as cells tend to stick to the wood and are not easily released into solution. Pap Smear Collection Kits QML Pathology supplies various collection kits for cervical smears which are conveniently packaged to suit different clinical indications. Single Use Pap Smear Kits: May be left overnight at room temperature after fixing. These kits are suitable for conventional Pap smears and ThinPrep. PREFACE - CYTOLOGY Prepare the conventional slide and rinse the device(s) in the PreservCyt solution. Push the Cervex brush into the bottom of the vial and twirl vigorously to ensure as much cellular material as possible is released in the solution. Discard the collection device after rinsing. Replace the PreservCyt cap and tighten so that the small black mark passes the corresponding line on the vial. Label the vial with the patient name and date of birth, and place into the plastic specimen bag along with the conventional smear and completed request form. Cervex Pap smear test kit Cytobrush Pap smear test kit Combination Pap smear test kit Monolayer Cytology: After preparing a conventional Pap smear, rinse the cervex brush or preferred collection device thoroughly in the cell preserving solution. Transport the labelled Pap smear in slide carrier, and labelled cell preserving solution to the laboratory.

18 PREFACE - cytology PREFACE - CYTOLOGY Nipple Discharge Label slide/s with a pencil. Gently squeeze the nipple until fluid appears. Smear the fluid directly onto the glass slide. Fix the slide immediately. Allow slides to dry. Send to the laboratory - in a slide carrier with the request form. Effusions and Washings Collect in a sterile dry container of appropriate size. The entire effusion specimen should be submitted. Label the specimen container and send to the laboratory as soon as possible or Refrigerate if a delay in sending the specimen is anticipated (Do not add any fixative). If Lymphoma is suspected then immediate transportation to the laboratory should be arranged. Consider requesting fluid protein examination. Transudates have a protein content of <3.0 g/dl and a specific gravity of < Exudates have higher protein and specific gravity, and are more likely to be inflammatory or neoplastic. PREFACE - CYTOLOGY Fluids Sputum Collect deep cough early morning specimens preferably on three consecutive days. Instruct the patient to collect specimens before breakfast preferably after rinsing out the mouth. Collect each specimen in a sterile container. Use one container for each collection. Label the specimen container and send each one to the laboratory as soon as possible or Refrigerate if a delay in sending the specimen is anticipated. Note: Please specify X 3 sputum collection on consecutive days on request form. Urine Urine may be collected as a random specimen or as a series of three specimens preferably on consecutive days. Collect the first part, or all (not mid stream) of the second or later void of the day. The first void contains cells which have been sitting in urine for hours and will show degenerative changes. Collect the urine in a sterile container. Label the specimen container and send it to the laboratory as soon as possible or Refrigerate if a delay in sending the specimen is anticipated (Do not add any fixative). Note: Please specify X 3 urine collection on consecutive days on request form. Cerebrospinal Fluid - CSF Collect the CSF in a sterile dry container. Label the specimen container, mark the request form as urgent and send to the laboratory as soon as possible or Refrigerate if a delay in sending the specimen is anticipated (Do not add any fixative) FINE NEEDLE ASPIRATION CYTOLOGY Scope for Use Fine needle aspiration (FNA) is a branch of diagnostic cytology that interprets changes in cells extracted from within organs, tumours and non-neoplastic abnormal tissues. FNA contrasts with exfoliative cytology, which studies cells shed or scraped from surface epithelia or mesothelia. The diagnostic criteria of both branches have many common features as well as many important differences. Just as FNA is an extension of morphologic diagnosis within both diagnostic cytology and histology, it is also a useful tool for the oncologist who deals with undiagnosed palpable and non palpable masses and lesions. It is a short cut to direct diagnosis and can be carried out at the surgery, clinic or bedside. It may also obviate the need for radiographic and surgical procedures, and save time, expenses and morbidity, and allay anxiety. The common targets currently being aspirated are thyroid, liver, breast and lung. Multiple sites of cyst formation are easily aspirated. These include breast, thyroid gland, parotid gland, branchial cysts and cavitating squamous cell carcinoma. Aspiration of cysts can be both diagnostic and therapeutic. Basic Equipment Syringe pistol (now commercially available - but not essential). 10/20 ml disposable plastic syringe - can be used without a syringe pistol. Apply negative pressure by pulling on the piston in the usual way. Plastic extension tubing. Fine needles of gauge varying from 1cm to 20cm in length (23 gauge needles are used for most aspirations of palpable lumps). Alcohol prep sponges.

19 PREFACE - cytology PREFACE - CYTOLOGY Sterile gauze pads. Microscope glass slides with frosted ends (label with pencil). Suitable spray fixative (Cytofix) or a coplin jar of 95% ethyl alcohol, to hold glass slides for immediate fixation of wet smears. 2 ml jars of sterile normal saline. Transport cylinders for recapped, secured, labelled needles and syringes. NOTE: QML Pathology can supply the necessary equipment and materials contained in a handy rigid transport cylinder kit on request (illustrated in Collection Materials Section (4.13) of this Manual). A small plastic tray easily holds all the equipment as well as longer needles measuring 15cm and 20cm, employed for transthoracic and transabdominal aspirations. Local anaesthesia, 1% or 2% lignocaine (Xylocaine), may be required for needle aspiration of transthoracic or transabdominal masses, but it is rarely necessary for other clinically palpable lumps. Since FNA is virtually non-traumatic, it may be repeated frequently enough to procure adequate amounts of material for diagnostic purposes. Diagram A Diagram A Insert needle into mass Insert needle into mass Apply suction Syringe pistol Syringe pistol A A B Standard syringe Standard syringe PREFACE - CYTOLOGY D NOTE: If an immediate diagnosis is required with FNA biopsy, attendance by a pathologist at the procedure can be arranged with prior notification to the laboratory. Apply suction B Aspiration Techniques There are 3 routine methods of aspiration for palpable masses: 1. Apply negative pressure for aspiration using a 10 or 20 ml syringe attached to a 25 or 23G needle by a flexible plastic extension tube (Diagram A) 2. Apply negative Diagram pressure A for aspiration using a 20 ml syringe in a syringe pistol (Diagram B) 3. Using a needle alone (no syringe), substitute a negative pressure with capillary action only (Diagram C). Move needle back and forth in slightly different directions through mass Move needle back and forth in slightly different directions through mass Release suction, then withdraw slowly Release suction, then withdraw slowly C D C D Our preferred method is (1). Note that QML Pathology FNA kits contain flexible plastic extension tubing. This method has the advantage of Syringe allowing pistol greater sensitivity Standard and syringe accuracy in placement of the needle. A disadvantage is that an assistant is required to hold the syringe and to pull back on the syringe. This is, however, a simple procedure - once the needle is in position in the lump, indicate for the assistant to draw the syringe plunger back Insert (8 mls needle in a into 10 ml mass syringe and 15 mls in Aa 20 ml syringe); then, on completion, allow the plunger to return over 1-2 seconds before withdrawing the needle from the tissue. Diagram B Diagram C Diagram C 5.17 Method (2) using a syringe pistol has the advantage that only one operator is required, BUT we have found it to be clumsy. Apply suction B Method (3) (no syringe - using needle only - repeatedly rapidly inserted into and withdrawn from the lesion inducing cells into the bore of the needle by capillary action) is preferred in many centres but is not appropriate for cysts and has not, in our hands, produced consistently good material. Move needle back and forth in slightly C different directions through mass mass 5.18

20 PREFACE - cytology PREFACE - CYTOLOGY For all of these techniques, the basic procedure is as follows: Thoroughly palpate the target area and delineate the most suspicious, usually the most firm portion Fix the mass with the palpating hand Prepare the skin with an alcohol swab, alcoholic chlorhexidine or povidone iodine Introduce the needle into the mass. In the case of the vacuum assisted technique: Application of full vacuum to the syringe with needle in the mass Continuously apply full suction to the aspirating syringe while the needle is moved back and forth with short quick strokes and in slightly different directions. (The variation in needle direction has been greatly exaggerated in the illustration.) This alteration in direction should be fairly minimal in practice, and, coupled with the forward and backward motion, is carried out within the mass One smear should be immediately fixed (in 95% ethyl alcohol or spray fixed), the other allowed to air dry. NOTE: It is very important when using spray fixatives to avoid holding the can closer than 15cm from the slide. Place labelled smears in slide carriers for transport to laboratory. 2. Preservation of Aspirated Material Residual material always remains in the needle after smear preparation. On the other hand it may not always be possible or desirable to prepare a smear in the clinic or at the bedside. This material may be preserved and transported as outlined below Cease aspiration when material enters the transparent hub of the needle Release suction before withdrawing the needle, and then apply pressure to the puncture site with the patient s or nurse s assistance. In the case of the needle-only technique: Rapidly and repeatedly insert the needle into and withdraw it from the lesion inducing the cells into the bore of the needle by capillary action Cease when material is seen to enter the hub of the needle and slowly and gently withdraw To make smear preparations, attach a syringe - with the plunger drawn back a few ml - to the needle and proceed as outlined below. HANDLING OF SPECIMENS 1. Preparation of Smears Making the smear is critical because it determines the quality of the material the microscopist will examine. In practical terms, it may be the most important manoeuvre in the whole range of steps in the aspiration. Rapidly separate the needle containing the aspirated material from the syringe and draw a few ml of air into the syringe. Reattach the needle to the syringe. Express the material onto a glass slide, generally forming a drop or droplets with small particles of tissue. Care must be taken to place the bevel of the needle against the slide so there are no intervening air gaps allowing the material to splatter across the slide. Place a second glass slide on top and pull both slides rapidly apart (see diagram above right). Two slides (smears) are thus prepared from each drop. i. Syringes and Needles Syringes and needles used in preparation of smears should not be discarded following preparation of smears. Rinse material remaining in needle and syringe into a labelled 2 ml jar containing normal saline and send to laboratory where cells will be retrieved by centrifugation and/or filter techniques. The syringe and needle may now be discarded. ii. Fluid or Bloody Aspirates Where specimens are diluted by fluid or blood, or for specimens which are largely fluid e.g. cyst fluid obtained from breast or thyroid, immediate despatch of the carefully recapped, labelled needle and syringe with contents to the laboratory is recommended. The cap should be secured, syringe labelled and placed in a transport cylinder. This should be forwarded to the laboratory in an insulated container with ice brick as soon as possible. Standard cytologic filter and centrifuged preparations can then be prepared in the laboratory. Rinsings are preferable. Transport of FNA to the Laboratory Preparation of smears and rinsing of syringe and needle into saline as described above in handling of specimens. Labelled smears should be placed into slide carriers. Store the labelled saline specimens in a refrigerator (crisper bin area). Transport specimen to laboratory in a rigid transport cylinder, placed in a cooled (ice brick) insulated container as soon as possible. NOTE: QML Pathology can supply the necessary equipment and materials on request PREFACE - CYTOLOGY