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FINALS: PRINCIPLES OF MEDICAL LABORATORY SCIENCE PRACTICE 2 HISTOPATHOLOGY Bronchial brushing/washings Sputum Histol...

FINALS: PRINCIPLES OF MEDICAL LABORATORY SCIENCE PRACTICE 2 HISTOPATHOLOGY Bronchial brushing/washings Sputum Histology - study of normal tissues Gastric washings Histopathology - study of abnormal tissues Urine sediment Prostatic secretions/fluid Tests Commonly Offered in the Histopathology Section Cervicovaginal (paps) smear Biopsy Autopsy Cell Cytology Cell Block Pap’s Smear Personnel in Histopathology Section Pathologist Histotechnologist Histotechnician Gross Examiner Histopath Quality Assurance and Documentation A. Histopath Reports Histopathologic Techniques a. Surgical Pathology - Involves the different procedures that have been adopted for b. Cytopathology Report the preparation of materials and tissues for microscopic c. Autopsy Report investigation, whether they are normal or abnormal B. Signatories - Includes examination of smears, preservation and a. Request Forms - patient’s physician processing of tissues sections prior to actual evaluation of b. 2. Result Forms - Pathologist tissue details C. Specimen Handling Importance a. FIX FIRST!!! - A well processed tissue can help in the confirmation and b. 2. Proper Label proper evaluation of disease entity leading to a proper mode D. Routine Turn-Over Results of treatment a. Surgical pathology and cytology= 24 hours Main Concern of Medtechs b. 2. Frozen Section = within 5 to 15 minutes - See to it that the tissues collected have been properly c. 3.Autopsy Report = 1 week preserved and adequately prepared for microscopic study E. Storage of Specimens, Tissue Blocks, Slides a. Specimen = 1 month to 1 year EXFOLIATIVE CYTOLOGY b. Tissue Blocks= 3-10 years - Branch of general cytology which deals with microscopic c. Slides= indefinitely study that have been desquamated from the epithelial surfaces. Forensic and autopsy results should be kept permanently! - synonyms : Papanicolaou’s method or Pap’s method Recommended for: - Detection of malignant cells or cancerous conditions in the RECORDS DURATION body - Detection of asymptomatic or precancerous cervical lesions Requisitions 2 years in women - Assessment of female hormonal status in case of sterility Quality Control 2 years and endocrine disorders - Determination of genetic sex Instrument Maintenance 2 years - Detection of presence of infectious agents Blood Bank Donor/ recipient Indefinitely DIVISIONS: records 1. Cytopathology - study of abnormal cells as Cancer cells. 2. Cytotechnique - study of the methods preparing the cells for Blood bank employee signatures 10 years microscopic examinations Specimens for Examination for Cytology Blood bank quality control 5 years Peritoneal, pericardial and pleural fluids CSF Nipple discharge  FINALS: PRINCIPLES OF MEDICAL LABORATORY SCIENCE PRACTICE 2 1.streaking REPORTS ko DURATION - used for preparing mucoid secretions Clinical pathology reports 2 years vaginal secretions, sputum and gastric content) Autopsy forensic reports Indefinitely - use a spatula, dissecting needle or applicator stick and streak in a zigzag Surgical Pathology and bone 10 years fashion marrow reports 2.spreading - used for thick mucoid secretions Cytogenetics Reports 20 years (smears of fresh sputum and bronchial aspirates) 3.pull apart SPECIMENS DURATIONS - for serous fluids, concentrated sputum, and enzymatic lavage form the GIT, Serum/others body fluids 48 hrs. smears of urinary sediment, vaginal pool and breast secretions Blood smears 7 days 4.touch or impression smear - PREPARATION: for preparation of direct Pathology / bone marrow 10 years impression from the cut surface of tissue like the lymph nodes and other Pathology blocks 10 years surgical or autopsy secretions. Microbiology smears 7 days FROZEN SECTION Blood bank donor/ recipient 7 days post transfusion - Utilized when rapid diagnosis of tissue is required - Recommended for lipids and nervous tissue Cytogenetics slides 3 years - Thickness: 10-15 micra - Temperature: -10 to -20°C Cytogenetics diagnostic images 20 years - Normally used when a rapid diagnosis of a tissue is required. EXAMINATION OF TISSUES - Applications: 1. Rapid pathologic diagnosis during Methods of Tissue Examination surgery Fresh Tissue Examination 2. Enzyme histochemistry Preserved/Fixed Tissue Examination 3. 3. Demonstration of soluble substances such as lipids and carbohydrates Methods of Fresh Tissue Examination 4. Immunofluorescence and Teasing or Dissociation immunocytochemical staining Squash Preparation (Crushing) 5. Some specialized silver Smear Preparation stains,particularly in neuropathology Frozen Section Two methods of Preparing Frozen Sections Teasing or Dissociation A. Cold Knife Procedure - A selected tissue specimen is immersed in a watch glass Almost any microtome can be used. It uses carbon dioxide. containing NSS, carefully dissected or separated and Optimum condition for sectioning: examined Knife= -40 to -60 degrees celsius Squash Preparation Tissue= 5 to -10 degrees celsius - Small pieces of tissues are placed in a microscopic slide and Environment= 0 to -10 degrees celsius forcibly compressed with another slide or coverslip Smear Preparation B. Cryostat procedure(Cold Microtome) - smears should be from fresh material Optimum working temp = -18 to -20 degrees celsius - see requisition form (patient’s ID: name, age; date and type CRYOSTAT- a refrigerated cabinet in which a modified microtome is of specimen requested housed. - label the slide Methods of Smear Preparation: All the controls to the microtome are operated from outside the cabinet. Presently, the rotary microtome is the type of choice.  FINALS: PRINCIPLES OF MEDICAL LABORATORY SCIENCE PRACTICE 2 Mounting media for cryostat sections: Instruments used for the aspiration of secretions: - Water - Rubber bulb with glass pipette – for aspiration of fluid. - 20-30% bovine albumin - Ayres spatula – for obtaining mucoid specimens for - von apathy’s gum syrup hormonal studies - O.C.T. = best synthetic water- soluble glycols and resins Examination of Fixed Tissues Histopathologic Techniques/ Steps - Curette – spoon, curved or blunt types – commonly used for many biopsies to obtain pieces of the tissues from the TISSUE PROCESSING vagina and cervix or placental tissues from the - Tissue Processing is designed to remove all extractable endometrium. water from the tissue, replacing it with a support medium that provides sufficient rigidity to enable sectioning of the - Pap smear, also called a Pap test, is a procedure to test for tissue without parenchymal damage or distortion. cervical cancer in women. Types of Tissue Processing - A Pap smear involves collecting cells from the A. Manual Tissue Processing cervix — the lower, narrow end of the uterus that's - In this process the tissue is changed from one at the top of the vagina. container of reagent to another by hand. - Named after George Nicholas Papanicolau who B. Mechanical Tissue Processing created this test. - In this the tissue is moved from one jar to another by mechanical device. Timings are controlled by a 2. Prostatic secretions – done by massage through an timer which can be adjusted in respect to hours intra-rectal route using the middle and index index finger and minutes. aseptically. 3 samples collected (urine before massage, prostatic secretion by massage, urine after massage) HISTOPATHOLOGIC TECHNIQUES 3. Bronchial secretions or sputum – “deep cough” specimen is Numbering the best study - first and most important step in histopathology. Three consecutive morning sputum sample - Identify properly all the specimens received without the need Sputum – collected in wide-mouth container containing of writing the patient’s name to the accompanying specimen SACCOMANO FLUID (50% ETOH and 2% Carbowax) tag. 4. Gastric Contents and duodenal fluid – specimens obtained - Entering details in the log book by the incubation technique using rubber tubings of - In numbering the specimen number is preceded by either S specimen diameter and length known as nasogastric tube surgical, A autopsy, or C cytology. Year must be indicated. (Levin tube and Rehfuss tube). - Unique accession number or code should be assigned 5. CSF (Cerebrospinal fluid) – obtained by lumbar puncture or lumbar tap down in the intervertebral spaces between the GROSS EXAMINATION OF SPECIMEN second the third lumbar vertebrae or between the third and - Surgical cut –up fourth lumbar vertebrae. The fluid is a secretion of the - Specimen Dissection choroidal plexus of the brain circulating within the ventricles - Grossing and central canal of the spinal cord. Medtech will assist pathologists in doing the gross examination, he/she 6. Serous Fluids – specimens from different body cavities will write down descriptions of the specimen received. such as: - Specimen size 3 x 2 cm and 3- 5 mm thick. Pleural cavity – fluids are obtained by - Use pencil in writing descriptions thoracentesis, done commonly on the left lateral thoracic wall. Procedures/methods done in boiling specimens for exfoliative Pericardial cavity – fluids are obtained by cytology pericardiocentesis, done commonly on the left sternal 1. Scrapings – specimens come from lining epithelium and border. mucosa or secretions of the female reproductive tract as Peritoneal cavity – fluids are obtained by various vagina, cervix and endometrium of the uterus. Paracentesis abdominis, done on the midline of the abdomen below the naval (at the linea alba)  FINALS: PRINCIPLES OF MEDICAL LABORATORY SCIENCE PRACTICE 2 7. Bone marrow substances (blood) - specimens from the bone INSTRUMENTATION IN HISTOPATHOLOGY marrow cavities (red bone marrow or myeloid tissue) obtained by puncture using a bone marrow borer at the Tissue Cassette sternum or iliac crest. Microtomy 8. Urine - A microtome is a basic instrument used in microtomy in - Voided urine of male is usually sufficient for which it is used to cut thin sections of tissue. cytological evaluation - 3 essential parts - But for female patients, catheterized specimen is - Block Holder “ chuck” recommended - Knife Carrier and knife 9. Nipple Discharge - Pawl, Ratchet Feed Wheel and Adjustment screws - Spontaneous nipple discharge is collected with a (line up the tissue block) cotton swab Kinds of Microtome Rocking microtome ○ Paldwell Trefall BIOPSY ○ Simplets microtome - Process of taking pieces of tissues from a living patient for ○ Can cut 10-12 um tissue sections the purpose of microscopic examination and diagnosis. ○ For large paraffin embedded blocks - This is commonly used in histopathology for laboratory ○ Not for serial sections interpretation of tumors, especially in differentiation Rotary Microtome between benign and malignant tumors. ○ Routinely used microtome - The main purpose of biopsy is attained if the tissue under ○ Cutting of paraffin embedded blocks examination turns out to be cancer (CA) or malignant ○ Not for serial sections - Greek origin: (bios - life and opsia - see/look/appearance) ○ This is the most commonly used microtome for - The excision (removal) and examination of tissue from a routines and research purposes. living subject for diagnostic purposes ○ So called because of the rotary action of the hand - It could be therapeutic wheel actuates and the basic mechanism requires - Careful handling of the tissue is mandatory for sarcoma the rotation of a fine advance handwheel through (general term associated to cancer in connective tissue) , and 360º moving the specimen vertically past the immediate touch preparation for lymphoma cutting surface and returning to the starting Historical Perspective position - 1870, Ruge and Joham Vert in Berlin introduced surgical ○ Sometimes called minot microtome after their biopsy as an essential tool for diagnosis inventor Prof.George Richards Minot. - 1889n, Emarch put forward and argument that confirmation should be made before surgeries for malignancies - William halsted 1st introduced this principle in United States - 1941, study of exfoliated cells from female genital tract by Papanicolaou. - This was adapted to study cells from other body systems - Along with this were innovations in various kinds of tissue tissue preparations and staining techniques Indications for Biopsy in Surgery I. Any lesion that persists for more than 2 weeks with no apparent etiologic basis II. Any inflammatory lesion that does not respond to local Sliding Microtome treatment after 10-14 days ○ Adams III. Persistent hyperkeratotic changes in surface tissues For celloidin sections and hard rough IV. Any persistent tumescence , either visible or palpable tissue block beneath relatively normal tissue 2 types V. Evaluation and monitoring of tissue rejection after Basic Sledge transplantation - kidney and liver Standard Sliding : more dangerous Knife is moving Freezing Microtome ○ Quekett ○ For rush frozen sections ○ CO2 is used as a propellant  FINALS: PRINCIPLES OF MEDICAL LABORATORY SCIENCE PRACTICE 2 ○ Cryostat or Cold Microtome - then add distilled water until the solution reaches For rush frozen section, now commonly the 95-ml line. used - This gives you 95 ml of 70% ETOH. Refrigerated ( -5 to -30 C) average of -20 - If you are making a 50% solution of ETOH , C - pour 95% ETOH up to the 50-ml line, Can cut sections of 4 um - add distilled water up to the 95-ml line. Inside is a rotary microtome - To make larger amounts, just use quantities that are Ultrathin Microtome multiples of the numbers above ○ Cutting sections as thin as 0.5 um Electron Microscopy TYPES OF BIOPSY Tissues are usually embedded in Plastic Special Knife: Diamond Knife- not easily Aspiration Biopsy dulled - Use of a needle and syringe to penetrate a lesion for aspiration if its contents Microtome Knives Indications: (3 basic types) - To determine the presents of fluid within a lesion Plane Concave Knife - To ascertain the type of fluid within a lesion - Usually 25mm in length - When exploration of an intraosseous lesion is - Less concave side: for celloidin indicated - More concave side: for paraffin - Outpatient procedure - For base sledge, rotary pr rocking microtome - Infiltrate the site with LA Biconcave - 22G needle attached to a 10ml syringe (syringe holder) - Usually 120mm in length - Place the needle in the mass - Both sides are concave - Apply suction while the needle is move back and forth within the - Paraffin Sections mass - Rotary Microtome - Release the suction and withdraw needle once cellular aspirate is Plane - Wedge Knife seen - Usually 100 mm in length - The cellular material is then expressed unto the microscope slide - for frozen section or very hard tissues - air dry of fixed with 95% ethanol - Base sledge or sliding microtome Other equipment used in sectioning Flotation Water bath Temperature 10C below Melting Point of Wax Colored Enamel Diameter 2 inch Height= 4 inch 2 Liter capacity Drying Oven Core needle Biopsy ○ Temperature - Skin cleansing +La 5 C Higher than Melting Point of Wax - Small skin incision - Lesion approach at an angle 45 degree Forceps - Stabilize the lesion and introduce the needle via the skin Clean slides 76x 25 mm 1- 1.2 mm thick Frosted until it abuts against the lesion - Fully mechanical biopsy gun is then fired Reagent Preparation - Tissue fixed in formalin - Making a graded series of alcohol: - Bleeding usually not a problem,, apply pressure - Always use 95% ethanol (ETOH) to dilute from, rather than - Incision covered by an occlusive dressing 100% ETOH.(Ethanol) - Sensitivity of 80-90% - Absolute (or 100%) ethanol is exceedingly expensive compared to 95%. Here is an easy method for making any Exfoliative Cytology percentage of alcohol below 95% that you wish. - Is now widely used, especially for the diagnosis of malignant - To make a 70% solution of ETOH, conditions and sites as the cervix, uterus, vagina, and the - pour 95% ETOH into a graduated cylinder up to the bronchial but also in urine and CSF and in fluid aspirated 70 ml line, from the pleural and peritoneal cavities. - Smears made from suspected metastasis  FINALS: PRINCIPLES OF MEDICAL LABORATORY SCIENCE PRACTICE 2 Excisional Biopsy ADVANTAGES: - Complete removal of lesion provides the most reliable - Its quick and surgeons can decide the further biopsy steps to follow - Permits the pathologist to examine all of the suspicious tissue Stereotatic Biopsy - Performed under local anesthesia before treatment is - This uses images intensifiers to enhance the accuracy of the planned, but may be done using frozen section site of the biopsy - Removal of lesion in its entirety - Radiological images of the site of the lesion, the location the - For discrete lesions size and the shape the dept and other characteristics are - Include 2-3mm perimeter of normal tissue employed in order to increase the accuracy of the procedure - Certainly benign lesions this involves ultrasound CT-scan MRI and mammography Incisional Biopsy AUTOPSY - Removal of part of a lesion for histological examination - “See for yourself” - Preferred for large situated tumors that cannot be excised - A post mortem examination performance to determine the - Removal of small piece of tumor cause of death - Significant tumor remains - Cause of death deemed suspicious, or involves criminal action. Bite Biopsy - Biopsy in which small pieces of tumor are removed with Classification special forceps - In forensic autopsy a medical examiner or coroner declares - Endoscopic biopsy is example of this type the death to be: Natural Cutaneous Biopsy - Caused by a known disease: cancer, heart disease, stroke, - Cutaneous biopsy are removed from the skin lesion genetic disorders, etc. - Often just simply “old age” Punch biopsy - It is larger than 2 millimeter Accidental Death - Stained with H and E. - Caused by mistake or freak occurrence - Gross lesion assessment is very limited - Death not planned, but can be explained by circumstances Shave Biopsy Homicidal Death - Shave biopsy always present orientation problems and - Killing another person should be reserve for secretions requiring only - Close to you? histopathologic diagnostic - infanticide , fratricide, sororicide, parricide, patricide, matricide, mariticide, uxoricide Wedge Biopsy - Lots of people - Size should be thicker than 3 to 4 mm - genocide - killing a national, ethnic, racial or - If EM is used, it should be less than 1mm thin religious group - Homicide is the most investigated death, therefore the most Marginal Excision autopsied - It refers to excisional or shell out and is a procedure in which the lesion is not entered but removed. Suicidal Death - Killing of self Endoscopic Biopsy - Often the easiest to identify wrt cause. - Gastroscopic or colonoscopic or through ERCP or - Can be elaborated further in the report cystoscopic, arthroscopic - Toxic, firearm, blunt force trauma, asphyxiation, etc. Frozen Section Biopsy - Done whenever a report is needed at the earliest time. Here Unknown death an unfixed fresh tissue is frozen (using CO2) in a metal and - At sea sections are made and stained - Badly decayed bodies - PIT FALLS - Technically difficult Experts who perform forensic autopsies - Difficult to get accurate result - Forensic pathologist, aka a medical examiner  FINALS: PRINCIPLES OF MEDICAL LABORATORY SCIENCE PRACTICE 2 - 4 year undergrad CLINICAL CHEMISTRY - 4 year medical school - 4 year pathology “residency” CLINICAL – comes from the greek word kline meaning bed. External Examination CHEMISTRY - the science that deals with the elements, their Steps: compounds and the chemical structures and interactions of matter. 1. Photographed 2. Samples taken: hari, nails CLINICAL CHEMISTRY - is a basic science that utilizes the specialty of 3. Undressed , examined wounds chemistry to study human beings in various stages of health and 4. Measured, weight, cleaned disease. It is also an applied science when analyses are performed on body fluids or tissue specimens to provide important information for the diagnosis or treatment of disease. Clinical Chemistry - Clinical chemistry is the branch of laboratory medicine that uses chemical analysis to study the levels of various body constituents during health and disease - These chemical tests are usually performed on blood samples, but urine and other body fluids are also analyzed. - The test results are used by the physician to diagnose disease, institute treatment, and follow the disease’s progress. - The physician also uses the results to counsel the patient in preventive medicine - Clinical chemistry is a science, a service, and an industry. As a science, clinical chemistry links the knowledge of general chemistry, organic chemistry, and biochemistry with an understanding of human physiology. - As a service, the clinical chemistry laboratory produces objective evidence from which medical decisions may be made. - As an industry, clinical laboratories are businesses, which operate under the regulations and practices that guide commerce in the world Patient Preparation - The pre examination stage of laboratory testing involves processes that occur before testing of the specimen. - Errors that occur during this stage often happen during blood collection and are primarily controlled by the phlebotomist. Numerous variables in patient preparation can affect sample quality, and the phlebotomist cannot be expected to control and monitor all variables. - However, phlebotomists should be aware of the critical variables that can affect sample quality and consequently laboratory results and report them to the nursing staff or phlebotomy supervisor. - The phlebotomist should also be able to recognize various patient conditions and complications that may occur during or after blood collection - Numerous preexamination variables associated with the patient’s activities before sample collection can affect the quality of the sample.  FINALS: PRINCIPLES OF MEDICAL LABORATORY SCIENCE PRACTICE 2 - These variables can include diet, posture, exercise, stress, - Physical activity can have different effects on analyte alcohol, smoking, time of day, and medications. concentrations – volume shifts between the vascular and - Physiological variables, such as age, altitude, and gender interstitial compartments, volume loss by sweating and affect normal values for test results. changes in hormone concentrations. - Other patient conditions that may influence laboratory test - Increased in lactate, fatty acid, ammonia, ALT, AST, CPK, LD, results are dehydration, fever, and pregnancy. Growth hormone, Prolactin, testosterone, luteinizing hormones - Elevated levels of proteins in the urine (proteinuria) - Vigorous hand exercise (fist clenching) increases potassium, 5/1/24lactate and phosphate Fasting - Absolute Fasting: FBS, Uric acid , Lipid profile - Relative Fasting: BUN, Creatinine,SGPT, Phosphorous FBS: 8 hours of fasting Lipid Profile: 12-14 hours fasting Reminders: Strictly avoid smoking, eating, drinking after the last meal on the night before the examination. Avoid physical and strenuous exercises or activity the night before the examination. - 8 – 12 hours is required for glucose, lipids and lipoproteins - Serum bilirubin may increase after 48 hours of fasting - Plasma Triglycerides increases after 72 hours of fasting - Basal State - The ideal time to collect blood from a patient is when the patient is in a basal state (has refrained from strenuous exercise and has not ingested food or beverages except water for 12 hours [fasting]). - The best comparison of a patient’s results with the normal values can be made while the patient is in the basal state. This explains why phlebotomists begin blood collection in the hospital very early in the morning while the patient is in a basal state and why the majority of outpatients arrive in the laboratory as soon as early in the morning. - BASAL STATE COLLECTION – glucose, cholesterol, triglycerides and electrolytes. Diet - Metabolic products of food can increase in venous blood (high –Protein can increase urea) - Serotonin – will increase the urinary 5HIAA - Caffeine – increases glucose - Increases turbidity or latescence – Triglyceride levels exceed 400 mg/dL - Contributes to the degree of Icteric (bilirubin) - Icteric samples interfere with albumin, Total Cholesterol, FACTORS CONTRIBUTING TO THE VARIATION OF RESULTS glucose and Total Protein. Exercise - The tests most affected are glucose and triglycerides. - Moderate or strenuous exercise affects laboratory test Serum or plasma collected from patients shortly after a meal results by increasing the blood levels of creatinine, fatty may appear cloudy or turbid (lipemic) due to the presence of acids, lactic acid, aspartate aminotransferase (AST), fatty compounds such as meat, cheese, butter, and cream. creatine kinase (CK), lactic dehydrogenase (LD), aldolase, - Lipemia will interfere with many test procedures hormones (antidiuretic hormone, catecholamines, growth - Caffeine has been found to affect hormone levels, whereas hormone, cortisol, aldosterone, renin, angiotensin), bilirubin, hemoglobin levels and electrolyte balance can be altered by uric acid, high-density lipoprotein (HDL), and white blood cell drinking too much liquid. (WBC) count and decreasing arterial pH and PCO2.  FINALS: PRINCIPLES OF MEDICAL LABORATORY SCIENCE PRACTICE 2 Posture or Position - Alcohol consumption can cause a transient elevation in - Changes in patient posture from a supine to an erect glucose levels, and chronic alcohol consumption affects position cause variations in some blood constituents, such tests associated with the liver and increases triglycerides as cellular elements, plasma proteins, compounds bound to plasma proteins, and high molecular weight substances. Stress (Anxiety) - Upright position or supine (lying) – preferred - It affects adrenal hormone secretion - The concentration of analytes can increase 4 percent to 15 - It is associated with increase levels of albumin, glucose, percent within 10 minutes after changing from a supine insulin, lactate and cholesterol position to standing. After returning to the supine position - It also results in hypoventilation which in turn, affects acid – from the standing, it takes about 30 minutes for the analytes base balance to decrease back to the original level. - Plasma renin, serum aldosterone, and catecholamines can Stress double in 1 hour; therefore, patients are required to be lying - Failure to calm a frightened, nervous patient before sample down for 30 minutes before blood collection. collection may increase levels of adrenal hormones (cortisol - The National Institutes of Health recommends that patients and catecholamines), increase WBC counts, decrease serum be lying or sitting for 5 minutes prior to blood collection for iron, and markedly affect arterial blood gas (ABG) results. It lipid profiles to minimize the effects caused by posture. The has been shown that WBC counts collected from a violently increase is most noticeable in patients with disorders such crying newborn may be markedly elevated. This is caused by as congestive heart failure and liver diseases that cause the release of WBCs attached to the blood vessel walls into increased fluid to remain in the tissue. the circulation. In contrast, WBC counts on early morning - Patient should be supine / seated or at least 30 minutes samples collected from patients in a basal state will be before blood collection to prevent hemodilution or decreased until normal activity is resumed. hemoconcentration - Elevated WBC counts return to normal within 1 hour - Prolonged bed rest results to decreased plasma albumin due to fluid retention Medications - Supine to sitting – increases levels of albumin , enzymes and - Administration of medication prior to sample collection may calcium affect test results, either by changing a metabolic process - Sitting to supine – increases the levels of proteins, lipids, within the patient or by producing interference with the BUN, iron and calcium testing procedure. IV administration of dyes used in diagnostic procedures, including radiographic contrast Tourniquet Application media for kidney disorders and fluorescein used to evaluate - One – minute is recommended cardiac blood vessels, can interfere with testing procedures. - In general, understanding the effect of medications and Tobacco/ Cigarette Smoking (nicotine) diagnostic procedures on laboratory test results is the - The immediate effects of nicotine include increases in responsibility of the health-care provider, pathologist, or plasma catecholamines, cortisol, glucose, blood urea clinical laboratory testing personnel. Phlebotomists, nitrogen (BUN), cholesterol, and triglycerides. however, should be aware of any procedures being - The extent of the effect depends on the type and the number performed at the time they are collecting a sample and note of cigarettes smoked and the amount of smoke inhaled. this on the requisition form Glucose and BUN can increase by 10 percent and triglycerides by 20 percent. Chronic smoking increases Drugs hemoglobin, red blood cell (RBC) counts, the mean - Therapeutic Drug Monitoring specimen collection should be corpuscular volume (MCV), and immunoglobulin (Ig) E. scheduled according to the time of the last dose Immunoglobulins IgA, IgG, and IgM are decreased, lowering - Medications affecting plasma volume can affect protein, the effectiveness of the immune system BUN, iron and calcium concentrations. - Increased in plasma non esterified fatty acid (NEFA) - Hepatotoxic drugs can elevate liver function test concentration - Diuretics can decreased plasma Na and K - Increase in plasma catecholamines and serum cortisol - Increase in glucose, growth hormone, TC, TG and urea Alcohol Ingestion - Increases the plasma concentration of urate and TG - Increases GGT concentration - It causes hypoglycemia (chronic alcoholism)  FINALS: PRINCIPLES OF MEDICAL LABORATORY SCIENCE PRACTICE 2 stoppers. Tests are also performed on plasma, urine, and other body fluids. Serum and plasma are obtained by centrifugation, which should be performed within 1 to 2 hours of collection. - Fasting Samples drawn from patients who have not eaten for 8 to 12 hours are preferred. Serum separator tubes contain an inert gel that prevents contamination of the specimen by RBCs or their metabolites. - Samples must be allowed to clot fully before centrifugation to ensure complete separation of the cells and serum. Many chemistry tests require special collection and handling procedures, such as chilling and protection from light, and these tests Centrifugation - In the accessioning area, there are a number of types of centrifuges available, including table models, floor models, and refrigerated models. Altitude - The relative centrifugal force (RCF) of a centrifuge is - RBC counts and hemoglobin (Hgb) and hematocrit (Hct) expressed as gravity (g) and is determined by the radius of levels are increased in high-altitude areas such as the the rotor head and the speed of rotation (revolutions per mountains where there are reduced oxygen levels. minute [rpm]). - The body produces increased numbers of RBCs to transport - Most laboratory samples are centrifuged at 850 to 1000 g or oxygen throughout the body. Normal ranges for RBC 3500g for 10 minutes. parameters must be established for populations living at Rules for Centrifugation of Samples 5,000 to 10,000 feet above sea level. - Improper use of the centrifuge can be dangerous, and the - It is important to note this information if when speaking with following rules of operation must be observed: the patient you realize that he or she has just traveled from 1. Tubes placed in the cups of the rotor head must another geographical area be equally balanced. This is accomplished by placing tubes of equal size and volume directly Age and Gender across from each other. Failure to follow this - Laboratory results vary between infancy, childhood, practice will cause the centrifuge to vibrate and adulthood, and the elderly because of the gradual possibly break the tubes. A final check for - change in the composition of body fluids. Hormone levels balancing should be made just before closing the vary with age and gender. RBC, Hgb, and Hct values are centrifuge lid higher for males than for females. Normal reference ranges 2. A centrifuge should never be operated until the are established for the different patient top has been firmly fastened down, and the top - age and gender groups; therefore, the age and gender of the should never be opened until the rotor head has patient should be present on the requisition come to a complete stop. Should a tube break during centrifugation, pieces of glass and bio Pregnancy hazardous aerosols will be sprayed from a - Pregnancy-related differences in laboratory test results are centrifuge that is not covered. caused by the physiological changes in the body including 3. Do not walk away from a centrifuge until it has increases in plasma volume. The increased plasma volume reached its designated rotational speed and no may cause a dilutional effect evidence of excessive vibration is observed. - and cause lower RBC counts, protein, alkaline phosphatase, 4. When a tube breaks in the centrifuge, estradiol, free fatty acids, and iron values. immediately stop the centrifuge and unplug it - The erythrocyte sedimentation rate and coagulation factors before opening the cover. Don puncture-resistant II, V, VII, VIII, IX, and X may be increased gloves before beginning the cleanup. The cup containing the broken glass must be completely SPECIMEN COLLECTION AND HANDLING emptied into a puncture-resistant container and Sample Collection and handling disinfected. The inside of the centrifuge must also - Clinical chemistry tests are performed primarily on serum be cleaned of broken glass and disinfected. collected in gel barrier tubes, but the serum may also be Deposit any cleaning materials that may contain collected in tubes with red, green, gray, or royal blue broken glass into a puncture- resistant container.  FINALS: PRINCIPLES OF MEDICAL LABORATORY SCIENCE PRACTICE 2 5. Samples should not be re-centrifuged. This can 2. Sleeping patients cause hemolysis and erroneous test results. They must be awakened When the serum or plasma has been removed 3. Unconscious, Mentally Incompetent Patients from the tube, the volume ratio of plasma to red identify by asking the attending nurse/ relative blood cells has been altered. Substances from 4. Infants and Children the cellular leakage or exchange, caused by the identify by asking the attending nurse/ relative blood clotting, will then be centrifuged into the 5. Outpatient/ Ambulatory patients serum or plasma and alter test results verbally ask full names, countercheck with their ID card Requisitions - All phlebotomy procedures begin with the receipt of a test Labeling requisition form that is generated by or at the request of a - Always sa container, wag na wag sa lid health-care provider. The requisition becomes part of the patient’s medical record and is essential to provide the ★ Because many tests are performed on instruments that take phlebotomist with the information needed to correctly photometric readings, differences in the appearance or color identify the patient, organize the necessary equipment, of a specimen may adversely affect the test results. collect the appropriate samples, and provide legal ★ Specimens of concern include hemolyzed specimens that protection. Phlebotomists should not collect a sample appear red because of the release of hemoglobin from RBCs, without a requisition form, and this form must accompany icteric specimens that are yellow because of the presence of the sample to the laboratory. excess bilirubin, and lipemic specimens that are cloudy - The method by which a phlebotomist receives a requisition because of increased lipids varies with the setting. Requisitions from outpatients may be ★ The clinical chemistry section is the most automated area of hand carried by the patient, or requests may be telephoned the laboratory. Instruments are computerized and designed or faxed to the central processing or accessioning area by to perform single and multiple tests from small amounts of the health-care provider’s office staff, where the laboratory specimen staff generates a requisition form. In some health-care providers’ offices, the healthcare provider will use a prescription pad to write the names of lab tests ordered. - The patient then takes the prescription to a clinical lab where the requisition is then prepared following the health-care providers’ orders. Inpatient requisitions may be delivered to the laboratory, sent by pneumatic tube system, or entered into the hospital computer at the nursing station and printed out by the laboratory computer. - Phlebotomists should carefully examine all requisitions for which they are responsible before leaving the laboratory. The requisition should be reviewed to verify the tests to be collected and the time and date of collection, and to determine whether any special conditions such as fasting or patient preparation requirements must be met before the venipuncture. - They should check to be sure that all requisitions for a particular patient are together so that all tests are collected with one venipuncture. They must be sure they have all the necessary equipment. Patient Identification - Proper identification is the first step in sample collection. - Prime factor in attaining accurate results. - Procedures: 1. Conscious Inpatients/ hospitalized patients. verbally ask their full names to verify the name using identification bracelet  FINALS: PRINCIPLES OF MEDICAL LABORATORY SCIENCE PRACTICE 2 Specimen Rejection - Misidentification of patient - Mislabeling of specimen - Short draws/wrong anticoagulant to blood ratio - Mixing problems/clots - Wrong tubes/anticoagulant - Hemolysis/lipemia - Hemoconcentration - Exposure to light - Improperly timed specimens - Processing errors: incomplete centrifugation, incorrect log-in, improper storage Transporting Sample to the Laboratory - Deliver each sample to the laboratory as soon as possible. - Follow procedures for samples requiring special handling, which is covered in the following chapters, and in stat situations. When possible, the phlebotomist should try to schedule patients so that - a sample requiring special handling is collected last. - Use designated biohazard containers for transport, and securely attach the requisitions with the sample when using Cardiac Tests the pneumatic tube system. Verify that the pneumatic tube - Cardiac Enzymes- AST, CKMB, LDH has been sent before leaving. - Cardiac Panel - Blood samples should be transported to the laboratory for - Troponin I, Myoglobin, CKMB, D'DIMER, BNP –To processing in a timely manner. The stability of analytes assess cardiac diseases like Acute Myocardial varies greatly, as do the accepted methods of preservation. - Infarction, Coronary Heart This is why delivery to the laboratory or following laboratory Diseases,Atherosclerosis prescribed sample-handling protocols is essential. - CLSI recommends centrifugation of clotted tubes and anticoagulated tubes and separation of the serum or plasma from the cells within 2 hours. Ideally, the sample should reach the laboratory within 45 minutes and be centrifuged on arrival. Tests most frequently affected by improper processing include glucose, potassium, and coagulation tests. Glycolysis caused by the use of glucose in cellular metabolism causes falsely lower glucose values. Hemolysis and leakage of intracellular potassium into the serum or plasma falsely elevated potassium result Specimen Storage - Based on the tests requested, separated serum or plasma Non Protein Nitrogenous Compounds may remain at room temperature (24 °C ) for 8 hours except - Creatinine for glucose testing. - Blood urea nitrogen - If testing has not been completed in 8 hours, the specimen - Uric Acid should be refrigerated (2°C to 8°C). - Ammonia - If testing is not complete in 48 hours, the serum or plasma should be frozen at or below –20°C. Lipid Profile - Refer to the procedure manual for specific analyte - Total Cholesterol instructions. - High Density Lipoprotein - Low Density Lipoprotein - Triglycerides  FINALS: PRINCIPLES OF MEDICAL LABORATORY SCIENCE PRACTICE 2 OVERVIEW OF IMMUNOLOGY Laboratory Applications and Immunologic Assessment - (study of how our immune system works) Immune System - The immune system is structured to recognize, respond to, and destroy a wide variety of invading organisms - Immunologic function can be summarized as searching for nonself antigens that our body cannot recognize and then destroying them. Cellular and Humoral Immunity A. Humoral Immunity or Antibody-mediated Immunity - Cell produce chemicals for defense B. Cellular Immunity or Cell-mediated Immunity - Cells directly targets virus infected cells Serology - Study of constituents of the serum - Tests in serology section detects the presence of antibodies Storage Requirement of Serum Samples against certain bacteria, fungi, parasite, and viruses and Delayed testing - stored in refrigerator autoantibodies. Delated testing for 3 days - freeze the serum sample @ - 20 degree celsius Immunology - Study of molecules, cells, organs, and systems responsible In Vitro & In Vivo for the recognition and disposal of nonself materials and In vivo - means research done on a living organism how they work, or can be manipulated In vitro - means research done in a laboratory dish or test - All aspects of body defense, such as antigens and tube antibodies, allergy and hypersensitivity are included Inactivation of Complement - It is a process that destroys complement activity to prevent interference with some immunologic assays such as syphilis testing Process of Inactivation: Heat serum @56 degree Celsius for 30 mins. Hepatitis Screening - Viral hepatitis is the most common liver disease worldwide - The viral agents of acute hepatitis can be divided into two major groups, as follows:  FINALS: PRINCIPLES OF MEDICAL LABORATORY SCIENCE PRACTICE 2 A. Primary hepatitis viruses: A, B, C, D, E, and GB Immunochemistry may be used to detect HBsAg in liver virus C tissue samples. B. Secondary hepatitis viruses: Epstein-Barr virus Serologic Reporting (EBV), Cytomegalovirus (CMV), herpesvirus, and Positive test - Reactive others Negative test - non-reactive Hepatitis B - (HBV) is the classic example of a virus acquired through blood transfusion - Viral proteins of importance include the following: a. Hepatitis B surface Antigen (HbsAg) - the envelope of the protein b. Hepatitis B core Antigen (HBcAg) - Nucleocapsid core protein c. Hepatitis B e Antigen (HBeAg) - soluble nucleocapsid protein Persons at risk of exposure to HBV, including those mentioned earlier, include members of the following groups: Human Immunodeficiency virus (HIV) Heterosexual men and women - HIV is the predominant virus responsible for acquired Homosexual men with multiple partners immunodeficiency syndrome (AIDS) Household contacts and sexual partners of HBV carriers - Older names Lymphadenopathy- associated virus (LAV), Infants born to HBV-infected mothers Human T-lymphotropic retrovirus (HTLV) type III. Patients and staff in custodial institutions for HIV- 1 - is responsible for the main AIDS epidemic developmentally disabled persons HIV - 2 - is endemic in parts of West Africa Recipients of certain plasma- derived products, including patients with congenital coagulation defects Retrovirus Health care and public safety workers who may be in contact - Contain a single, positive-stranded ribonucleic acid (RNA) with infected blood. with the genetic information of the virus and a special Persons born HBV- endemic areas and their children enzyme called reverse transcriptase in their core. Reverse transcriptase enables the virus to convert viral RNA into Serological Examination deoxyribonucleic acid (DNA) - Serum testing procedures may be performed by qualitative - This reverses the normal process of transcription in which chemiluminescent immunoassay, qualitative EIA, DNA is converted to RNA - thus, the term retrovirus. quantitative real-time PCR, quantitative real-time PCR-nucleic acid sequencing, or real-time PCR with reflex to genotype.  FINALS: PRINCIPLES OF MEDICAL LABORATORY SCIENCE PRACTICE 2 Confirmatory Testing Western Blot Quantitative RNA Assay Immunofluorescence Assay Immunohistochemical testing Viral Load Testing - Testing of the viral load should be performed as soon as patient treatments begin. Subsequent viral load testing can be used as a marker for HIV viremia and should be carried out every 3 to 6 months for patients undergoing treatment. Laboratory Criteria for HIV infection - New guidelines recommend testing of viral load every 2 to 8 - Positive result from an HIV antibody screening test (e.g., weeks after the initiation of HAART (Highly Active reactive enzyme immunoassay (EIA*)) confirmed by a Antiretroviral Therapy) to determine early response to positive result from a supplemental HIV antibody test (e.g., therapy. Western blot or indirect immunofluorescence assay test) Or - Positive result or report of a detectable quantity (i.e., within Syphilis the established limits of the laboratory test) from any of the following HIV virologic (i.e., non-antibody) tests: - HIV nucleic acid (DNA or RNA) detection test (e.g., polymerase chain reaction (PCR) - HIV p24 antigen test, including neutralization assay - HIV isolation (viral culture) - Caused by a bacteria called Treponema pallidum - A sexually transmitted disease HIV TESTING METHODS 1. Detection of HIV antibodies Antibodies 2. Detection of antigens, particularly p24 A. Anti Treponemal antibodies - produced in response against 3. Detection of quantification of viral nucleic acids T. pallidum. B. Non treponemal antibodies or reagin antibodies - produced Enzyme Immunoassay by the infected person against components of their own. Can - Detection of HIV antibodies by EIA was the first technology be seen in patients with other infectious diseases. developed for HIV diagnosis in 1985. - Antibody testing by EIA remains the standard method for screening potential blood donors.  FINALS: PRINCIPLES OF MEDICAL LABORATORY SCIENCE PRACTICE 2 BLOOD DONORS, BLOOD COLLECTION & STORAGE Dengue Virus Primary Vector: Aedes aegypti mosquito It has Serotypes 1 to 4 After an incubation period 3 to 7 days, signs and symptoms start suddenly and follow three phases. 1. Initial Febrile Phase 2. Critical phase at about the time that the fever subsides (defervescence), 3. Final spontaneous recovery phase BLOOD BANKING WAS BORN OF WAR - Attempts to find a nontoxic anticoagulant and preservative Laboratory Examination (boosted during world wars) Viral component testing - To prevent clotting of blood ○ Detection of viral nucleic acid in serum by reverse - To maintain cell viability and function during storage so that transcriptase polymerase-chain reaction (RT-PCR) post transfusion survival is maximum Or Introduction of indirect transfusions ○ Detection of soluble nonstructural protein 1 (NS1) by enzyme-linked immunosorbent assay (ELISA) or Donations must be accomplished in such a way that the safety of both lateral flow rapid testing the donor and the potential recipient is assured. Serologic testing ○ Detection of IgM seroconversion by ELISA Or ○ Lateral flow methods Or ○ Detection of IgG in secondary infections by ELISA or lateral flow methods.  FINALS: PRINCIPLES OF MEDICAL LABORATORY SCIENCE PRACTICE 2 Criteria for Pre Deposit Donation can be made at weekly interval (1-5 units) Hb (>11.0 g/dl) HCT (> 33%) Last donation should be 72 hrs before surgery Cross match is required before each transfusion Blood not required during or after the patient surgery normally is discarded Screening test are performed as per normal donation Label should clearly state “For Autologous use Only” Double Red Cell Donation - An automated process where the red blood cells are separated by a machine and reserved while the remaining blood components are returned to the donor. Blood Donation Sites Walk in donation DEFINITIONS ○ Blood donors coming to the blood bank for Autologous donations ○ Derived from the organisms of the self; same ○ Usually regular blood donors individual; “autologous blood donation” Mobile blood donations Heterologous ○ Major part of blood donations ○ Derived from organisms of a different but related ○ Blood donations outside the campus for species; “a heterologous blood donation” Targeted population group Apheresis Untargeted population group ○ Greek word meaning “take out” Cytapheresis Donor Declaration ○ To harvest specific cellular components such as - Two crucial factors for safe blood products platelets, granulocytes or red cells - Accurate donor screening or selection Plasmapheresis - Accurate laboratory testing on each unit collected ○ To harvest plasma only and return back the cellular components to the donor/patient Why does one donate? - Volunteer donation Criteria for Apheresis Platelet Donation - Donation to replace products used Preferably regular donor - Donation to cover family and friends Weight (>55 kgs) Good venous access Purpose of Donor Screening Prior investigation required FIRST ○ FBC ○ Healthy enough to donate ○ VDRL SECOND ○ HbsAG ○ Recipient is protected ○ Anti HIV ○ Laboratory staff is protected ○ Anti HCV OUTCOMES OF DONOR SCREENING ○ Serum lipid profile ○ Acceptance ○ Temporary deferral Autologous Donation ○ Permanent deferral - A donation by the intended recipient of his or her own blood or component for a possible subsequent transfusion Donor Screening - Classification Registration of the donor - Preoperative or pre deposit Medical history - Perioperative hemodilution Physical examination - Intraoperative salvage ○ Upon successful completion of these the donor - Postoperative salvage proceeds to phlebotomy  FINALS: PRINCIPLES OF MEDICAL LABORATORY SCIENCE PRACTICE 2 ○ Donors must be assured of a private and confidential interview process for the medical history and the physical examination Demographic Data - Donors full name as in identification card and - Id card number - Permanent address with telephone number - Gender - Age - 18-55 years - Date of birth - Donors occupation - Date of last donation - Whole blood should be 3 months - Apheresis 2 weeks - Autologous depends on the equipment - Weight - Should be > 45 kgs Physical Examination - General appearance of donor - Determination of hemoglobin - Males Hb: >12.5 g/dl - Females Hb: >12.0 g/dl - Pulse - 50-100 beats/min - Blood pressure - Maximum 140/90 mmHg - Temperature - Maximum 37.5 degree celsius - Donor weight - Maximum 45kgs - Amount of blood to be drawn - Donor wt.in kg /50)x 450 - Venipuncture site - Inspection for scar marks