Lab sample collection techniques pathology
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1. The document outlines various protocols for collecting and handling specimens for diagnostic testing in pathology. It discusses appropriate samples, containers, transport conditions, and preservation methods for urine, CSF, fluids, sputum, stool, semen, and blood samples. 2. Guidelines are provided for venous blood collection including equipment, anticoagulants used, and proper labelling and transport of samples. 3. An overview is also given of histopathology specimen handling, blood banking techniques including blood donation and components, and cross matching procedures.
Lab sample collection techniques pathology
- 1. Protocols in diagnostic services in Pathology Dr E Kiran Kumar Prof and Head Dept of Pathology GVP Medical college Visakhapatnam
- 2. Urine analysis • 1. A single specimen – first morning voiding sample, random sample, post-prandial sample. • First morning sample – routine examn, fasting glucose, proteins, microscopic analysis for cellular elements, pregnancy test, bacteriological analysis, etc • Random sample – for routine urine examn.
- 3. • Post prandial urine sample – for estimation of glucose, to monitor insulin therapy in DM or for urobilinogen estimation. 2. 24 hour specimen – First urine in morning is discarded. Then all the urine for 24 hours is collected along with next day first morning urine, in a clean bottle of 2 litre capacity with a cap. Preserved at 4-6 C. – For quantitative estimation of proteins and hormones.
- 4. • Midstream specimen – initial half of urine is voided and then a part of urine is collected in a bottle. Initial half flushes out contaminating cells and microbes from urethra and perineum. Rest of the stream is from urinary bladder. • Clean-catch specimen – For bacteriological culture. • Urine sample must be tested in lab within 2hrs of collection to get the correct results.
- 5. • Refrigeration at 4-6 C is the best general method of preservation upto 8 hours. • Chemical preservatives are HCl, Toluene, Boric acid, thymol and formalin – for 24hr urine sample.
- 6. CSF examination • Spinal or Lumbar puncture is done between L3/L4 or L4/L5 lumbar vertebrae, and CSF obtained from subarachnoid space. To prevent injury to spinal cord which ends at about T12. • Site is disinfected thoroughly with povidone iodine and sterile LP or spinal needle is used of 22G size. CSF is collected in sterile plain tubes- for chemistry, microbiology, hematology, cytology. Total 3-5 ml collected.
- 7. • Sample transported to lab immediately and examined without delay.
- 8. Pleural/peritoneal fluids • Pleural fluid collected by inserting needle in chest wall – thoracocentesis. • Heparinised tubes can be used to prevent clotting . EDTA anticoagulant is used for cell counts, and thoroughly mixed. • Peritoneal fluid – by puncture of peritoneal cavity per abdomen – abdominal paracentesis. Hollow needle inserted through abdominal wall, usually left lower quadrant of abdomen below the border of shifting dullness – into peritoneal cavity and 20-50ml fluid drawn under aseptic conditions. • 100ml collected for cytology studies to obtain sufficient cell yield. EDTA used for cell counts.
- 9. Sputum examination • Collected in the morning, soon after awakening, and before taking any mouthwash or food. • Collected in clean, dry, wide-mouthed 25ml capacity container with securely fitting screw cap. Deep breath 2-3times , coughs deeply, and then spits into container. 2-5ml sputum collected. Only saliva is not acceptable.
- 10. Stool examination • Random sample of 4ml collected in clean, dry container with tightly fitting lid and transported soon to lab. Parasites detected in immediate examn. 10% formalin fixative used for preservation of eggs, larvae or cysts, if examn is delayed. •
- 11. Semen analysis • Collected after about 3 days of sexual abstinence. Obtained by masturbation, collected in clean, dry, sterile, leak proof and wide-mouthed plastic container. • Brought to lab within 1 hour of collection. Entire ejaculate is collected. During transport, kept close to body temp or ideally, collected near the testing site. Two samples examined , 2-3 weeks apart for better interpretation of results.
- 12. Hematological samples • Skin puncture – commonly used in infants and small children, if the reqd amount of blood is small – for cell counts, Hb estmn, pcv, blood films, etc. – It is called capillary blood – actually, a mixture of blood from capillaries, arterioles, venules, with some tissue fluid. • In adults – blood obtained from side of ring or middle finger distal digit or from ear lobe. In infants – from lateral or medial aspect of plantar surface of heel or great toe.
- 13. • Skin puncture site cleansed with 70% ethanol. After dryting, puncture is made with sterile dry disposable lancet free flow of blood. • First drop of blood is wiped away with sterile, dry cotton since it has tissue fluid. Then few drops are collected. Excess squeezing is avoided since it will dilute the blood with tissue fluid. • After collection, piece of sterile cotton is pressed over puncture site till bleeding stops.
- 14. • Compared to venipuncture blood, skin puncture blood has slightly higher Hb, PCV and RBC counts. Platelets counts are lower, since they adhere to puncture site. • Blood not collected from cold, cyanosed skin, since false elevation of Hb, RBC/WBC counts occurs.
- 15. Venous blood collection • Ideal for many tests, and when more blood reqd. Anticoagulated venous blood is obtained. • Best obtained from veins of antecubital fossa. A rubber torniquet is applied to upper arm, not too tight and kept for maxm of 2mins. Patient asked to make a fist, to make the veins more prominent and palpable. • Venepuncture site is cleansed with 70% ethanol and allowed to dry.
- 16. • Selected vein is anchored by compressing and pulling the soft tissues below the puncture site with left hand. • Sterile, disposable needles and syringes are used. 19-21G needle in adults and 23G needle in children. • While puncturing, bevel of needle is kept up along the direction of vein, and blood withdrawn slowly with syringe, since fast withdrawl can cause hemolysis and collapse of the vein.
- 17. • Torniquet is released as soon as the blood begins to flow into the syringe. After collection, patient asked to open his fist. Needle withdrawn from the vein and sterile cotton guaze is pressed over the puncture site. • Patient is asked to press the guaze over the site till bleeding stops. Needle is detached from syringe and blood discharged into tube containing reqd anticoagulant, since whole blood is reqd for hematological tests.
- 18. • If blood is discharged through needle, hemolysis can occur. Containers are usually disposable plastic bottles with caps and flat bottom. • Blood is thoroughly mixed with anticoagulant in container by gently inverting the container serveral times. Vigorous shaking is not done since it causes frothing and hemolysis, and sample becomes unfit for testing. • Disposable syringes are placed in puncture-proof containers for proper disposal. Recapping of needle by hand may cause needle stick injury.
- 19. • Container is labelled. Time of collection should be noted on the label, and sent to lab soon with properly filled requistion form with all clinical details and name of tests and patient details. • Precautions – Blood never collected from intravenous line since it will dilute the blood sample.
- 20. • EDTA blood – for Hb, pcv, cell counts, blood films, sickling test, reticulocyte count, Hb electrophoresis. – 1.5mg/ml of blood is used. Excess EDTA causes damages to red/white cells, decr pcv, incr mchc, etc. • Heparinised blood – 15U/ml - used for osmotic fragility test, immunophenotyping. • Sodium citrate/trisodium citrate - 0.109mg/ml(1:9) - used for coagulation studies, ESR estimation by westergren method (1:4).
- 21. • Double oxalate or wintrobe mixture – for routine tests and esp for ESR by wintrobe method. - 3:2 ratio of ammonium oxalate and potassium oxalate.
- 22. Blood banking techniques • Whole blood donation – 350ml of whole blood in anticoagulant solution. • Blood donor – 18-60yrs. • 3 months interval between two successive donations • Wt 45kgs. Preg and lactating mothers , upto 1yr postpartum cannot be donors • HIV patients, viral hepatitis(HBV, HCV) not accepted.
- 23. Cross matching/ compatibility testing To avoid hemolytic transfusion reactions in the recipient. Major crossmatch – testing recipient’s serum against donor’s red cells. Crossmatching test not reqd for transfusion of platelets or FFP. But FFP should be ABO compatible.
- 24. Blood components • Whole blood with CPDA anticoagulant – shelf life is 35days. – platelets are not effective, factor V and VIII are not present. Transfusion of whole blood – within 30mins of removal from refrigerator, and should be complete within 4hrs of starting. One unit rises Hb by 1gm% or haematocrit by 3%. • Packed red cells – in chronic severe anemias, severe anemia with CCF, anemia in elderly.
- 25. • Platelet concentrate – stored at 20-24C with continuous agitation, in a device called platelet agitator. Shelf life is 5days. • One unit of platelet concentrate raises the platelet count in recipient by 5000microlitre. • Fresh frozen plasma – prepared from whole blood within 6hrs of collection, since labile coagulation factors are lost after this time. • After plasma is separated, it is rapidly frozen at - 20C. FFP has all coagulation factors.
- 26. • FFP can be stored for 1yr at -25C – Thawed between 30-37C and stored at 2-6C , when reqd for transfusion. Should be transfused within 2hrs of thawing, since labile coagulation factors will rapidly deteriorate. • Cryoprecipitate – prepared from plasma which is freshly separated, by rapidly freezing at -20C and thawing slowly at 4-6C. Again refrozen at -20C for 1yr shelflife. Contains FVIII, vWF, fibrinogen.
- 27. Histopathology • Tissue samples or surgical specimens are kept immediately in 10% formalin, which should completely immerse the tissue, which acts as a fixative, orelse, autolysis of tissue, and unfit for tissue processing and examination.