Specimen Variables (MLS 11A) - University of San Agustin-Iloilo PDF

MODULE 1: SPECIMEN VARIABLES CLINICAL MLS 11A || September 2021 CHEMISTRY Transcribers:...

MODULE 1: SPECIMEN VARIABLES CLINICAL MLS 11A || September 2021 CHEMISTRY Transcribers: Altura, Jovero, Saradolla Editors: Last Name, Last Name, Last Name, Last Name, Last Name, Last Name University of San Agustin-Iloilo Legend: INTRODUCTION Remember Previous Trans (Exams) Lecturer Book Trans Comm ACCURACY VS. PRECISION      Accuracy - Near the target value OUTLINE Precision INTRODUCTION - The nearness of the results to each other PRE-ANALYTICAL PHASE Our results may not be accurate, but it must be precise. Pre-Analytical Variables That Can Affect Laboratory We need to be both accurate and precise and use quality Testing and Results control. - Exercise & Physical Training Why do we need to maintain quality control? - Fasting and Food Intake - Expensive equipment and reagents - Alcohol Ingestion - We are dealing with lives - Tobacco Smoking - Treat each sample or lab as your relative’s - Drugs - Doctors rely on us - Oral Contraceptives Example: Potassium - Posture - You used a hemolyzed sample - Stress It resulted to the increased sample level of K - Circadian/Cyclical Variation - Travel = Hyperkalemia - Age: Newborn, Children to Puberty, and Adult But the true value is actually low. - Sex Loving our work and maintaining quality. - Temperature - Obesity What is Clinical Chemistry? - Other Variables CLINICAL Pseudohyperkalemia - Clinical comes from a Greek word “kline” which means ANALYTICAL PHASE “bed”. Analytical factors CHEMISTRY POST-ANALYTICAL PHASE - is the science which deals with the elements, their Post analytical factors compounds and the chemical structures and interactions of Specimen collection, handling, and processing matter. Pre-analytical variables Basic science Pre-analytical variables - utilizes the specialty of chemistry to study human beings - Specimen transport different human body fluids and different samples - Turnaround time from human body - Patient preparation - various stages of health and disease. Control of analytical variables Applied science Post analytical variables - performed on body fluids or tissue specimens Anticoagulant Interference - - provides important information for the diagnosis or Specimen handling and processing treatment of disease. - Specimen transport - Specimen interference The laboratory diagnostic process to obtain a result can be divided Reasons for Specimen Rejection into three phases: the pre-analytical, analytical and post-analytical Samples phases. Specimen Storage - evaporation induced errors - Stability of analytes on storage PRE-ANALYTICAL PHASE - Glucose 1. Patient Preparation and Identification - Ammonia This phase involves an individual’s preparation for collection - Storage at 4c of the biological material, the collection itself, storage of the collected - Permeability changes sample and its transport to the laboratory, and preparation of the - Light exposure sample for the assay. Factors affecting the pre-analytical phase before - Specimen required chilling before transport the biological material collection can be further divided into controllable and non-controllable variables. Controllable Variables LEARNING OBJECTIVES Physiological Variable At the end of this topic in Specimen Variables, the student - Posture/position shall be able to: - Prolonged bed rest 1. Apply concepts of pre-analytical, analytical, and post analytical - Fasting analysis. - Diet - Exercise - Physical training - Tourniquet application - Circadian variation - Menstrual cycle - Others: lifestyle, stimulants, drugs, herbal preparations, recreational drug ingestion Non-Controllable Variables Biological influences - Age - Sex - Environmental factors ✓altitude ✓temperature) ✓ Geographical location ✓ Seasonal influences Page 1 of 10 [MLS 11] Module 1 SPECIMEN VARIABLES Underlying medical condition Why will HDL concentration increase? Obesity HDL is a good cholesterol (carriers of Blindness cholesterol) Pregnancy A lot of HDL is not bound to cholesterol if Stress cholesterol lowers. Fever A lot of HDL becomes free HDL Shock & trauma Transfusion & infusions Shock or Trauma Situation: Hypobulimic shock Massive blood loss Hematology effects: Decreased Hemoglobin, RBC count, and Hematocrit Pre-Analytical Variables That Can Affect Laboratory Testing and Results 1. Exercise & Physical Training During physical activity the following happens: ✓ shift of fluid between intravascular & interstitial compartments ✓ change in hormone concentration particularly changes in hormonal activity ✓ loss of fluid through sweating The following are the effects of exercise and physical activities: ✓ Generally, the following increases: Hormones: prolactin, testosterone, and LH Proteins in urine- increase in albumin ✓ Transient effects (temporary) Immediate fall & subsequent increase in FFA, Increased alanine by 180%, increased lactate by 300% Increase in blood gases (Ammonia) due to breathing Why we have immediate (initial) decrease in FA? It is because of the utilization of FFA 2. Fasting and Food Intake during physical exercise but after this, it 8-14 hours also immediately returns to high levels. Glucose fasting time: 6-8 hours Why we have increased levels of Alanine? Lipid profile: 12-14 hours Alanine is the major Amino acid for If the patient cannot tolerate 12-14 hours of fasting, gluconeogenesis. Glucose is taken from 9 hours is accepted, but note it. proteins. Due to glucose being utilized, Tests: Total cholesterol, TAGs, HDL, LDL there will be more production of Alanine. Why Lactate increases? TAGs: cleared out 8 hours after eating 9 hours of fasting is enough if this is the only test It is produced from exercising muscles. required. Lactate is later converted into glucose in Greatly affected by diet the Live. In short, because the glucose reserve was PROLONGED FASTING: already utilized, glucose will be taken from alanine and lactate to compensate. This explains why their Drinking of water is allowed. Half glass only. levels increase. Excess drinking of water affects analyte and makes it diluted, decreasing analyte cells. ✓ Long-term effects: (Chronic)- gradual CPK (creatinine kinase), AST (aspartate amino 48 hours fasting: transferase), LD (lactate dehydrogenase), and aldolase Increase bilirubin by 240% 74 hours fasting: ✓ For long distance athletes: ✓ Increase plasma TAG, glycerol & FFA decreased FSH and LH (plasma) ✓ No change in cholesterol ✓ Vigorous hand exercise: ✓ Decrease plasma glucose to 45 mg/dL increased potassium, lactate and phosphate What if the patient did not fast but they must Phlebotomy: make a fist= good picture of vein undergo cholesterol determination, is it okay for but vigorous hand exercise is for blood donation them not to fast? only. Yes, it is okay because cholesterol is not affected by diet. Potassium is a major intracellular cation. Vigorous hand exercise causes the shifting of K What if the patient did not fast but they must outside of the cell (extracellular fluid) towards the undergo lipid profile, is it okay for them not to plasma fast? No, because the patient must fast. Aside from cholesterol, lipid profile also has ✓ Walking for about 4 hours each week: TAGs. Cholesterol concentration 5% lower HDL concentration 3.4% higher Immediately after eating: Page 2 of 10 [MLS 11] Module 1 SPECIMEN VARIABLES All analytes increase except for inorganic phosphorus After eating: ✓ Increase K & TAG (chylomicrons will also increase) Why will chylomicrons increase if TAG levels are high? Chylomicrons are carriers of TAGs ✓ Increased ALP (alkaline phosphatase) intestinal isoenzyme in Lewis positive secretors, type B & O patients ✓ Hypertriglyceridemia: accumulation of chylomicrons until cleared by lipoprotein lipase  Anticoagulant EDTA (Ethylenediaminetetraacetic acid) Inversion of 3-4 times light blue top Inversion of 8-10 times EDTA Substances that prevent the blood from Anticoagulants clotting What tube to use for serum? Red top Can we use SST to acquire serum? Yes (Serum Separator Tube) What is the difference between SST and SST has a gel that separates Red top tube? serum from formed elements while red top has no gel. Normally, plasma is clear yellow Why is it turbid after eating cheeseburger? Because of the presence of chylomicrons (transporters of TAGs) Cheeseburger is rich in TAGs FOOD OR DIETARY REGIMENS FOR THE PAST FEW WEEKS: High-meat protein diet: 3. Alcohol Ingestion ✓ Increase urea, ammonia, urate (beef, red meat) Increase lactate, urate, acetaldehyde, acetate due to the Non-protein nitrogens, used to evaluate kidney accumulation of NADH function Acetaldehyde is a metabolite of alcohol ✓ Increase urinary urea excretion Increase TAG, HDL due to impaired lipase ✓ Serum creatinine not affected Lipoprotein lipase: enzyme necessary for the Not affected by meat diet breaking down of Tags and HDL. Increase GGT, MCV Specific marker for kidney function ✓ Indicators of alcohol consumption: 1. GGT (Gamma-Glutamyl Transferase) Purine-rich food: Increase urate 2. HDL All alcoholic beverages 3. MCV (Mean Corpuscular Volume) Fish, seafoods, sardines, scallops, bacon, turkey, liver 4. Tobacco Smoking ✓ High fat diet: Acute effects: Increase TAG, depletes nitrogen pool ✓ Increase catecholamines & cortisol Chocolates ✓ Increase glucose, impaired glucose tolerance ✓ High ratio of unsaturated to saturated FA: Decrease CHOL & LDL May develop to diabetes over time ✓ Increase FFA ✓ Beverages with caffeine: Chronic effects: Increased FFA (3x); Release catecholamines from adrenal medulla & brain ✓ Increased carboxyhemoglobin (blood gases), MCV, WBC count Increases neural excitation ✓ Lower immune response, presence of ANA (Antinuclear Pituitary gland receives signal that there’s an Antibodies) & CEA (Carcino-Embryonic Antigen) emergency -> Signals adrenal medulla -> release of catecholamines -> Increased heart rate, bp, CEA is a tumor marker breathing rate, muscle strength, and mental ✓ Reduced sperm count, Vitamin B12 alertness. Effects of Nicotine ✓ Triggers the production of steroid hormones Bananas, pineapple, tomatoes & avocados: ✓ Results to the migration of leukocytes from marginal to Increase blood serotonin (happy hormone) circulating pool Increase 5-HIAA in urine Leukocytes that adhere to the endothelium of blood vessels free themselves to the circulation in the presence of nicotine. Will result to increased WBC count Page 3 of 10 [MLS 11] Module 1 SPECIMEN VARIABLES 6. Oral Contraceptives Affects concentration of transport proteins & certain enzymes Increase ceruloplasmin, transcortin, TBG (Thyroxine-binding globulin), plasminogen, transferrin, iron & TAG *transport proteins Increase ALT (Alanine Aminotransferase) and GGT (Gamma- Glutamyl Transferase) Decrease albumin, orosomucoid, zinc Of all the proteins mentioned, only albumin decreased. 7. Posture One’s posture affects redistribution of water leading to the following: Increase albumin, enzymes, calcium, bilirubin, TAG, 5. Drugs cholesterol, certain drugs Directed at the liver Albumin-bound substances: Induce hepatic microsomal enzymes (barbiturates, Unaffected freely diffusible substance phenytoin, tolbutamide) Sudden change in posture may decrease blood volume about Hepatocellular damage (methotrexate as anti-neoplastic) 10% and increase plasma proteins 8— 10% Cholestasis (steroids, tolbutamide, chlorpromazine) ✓ These happens 10 minutes from lying to Cholestasis means blocked bile flow standing except for prolonged bed rest or 30 Liver function tests: minutes from standing to lying ✓ Increase urine bilirubin ✓ Greatly affected by these changes: 1. Hypertensive patients ✓ Increase serum ALP, bilirubin, BSP, ALT, AST 2. Individuals with low plasma protein ✓ Decrease glucose 3. Elderly Drink many water if you took a drug ✓ Least affected: 1. Patients with abnormally high concentrations of protein 2. Patients on prolonged bed rest results to: 3. Fluid retention 4. Dilution effects: decreased plasma proteins and albumin - Albumin is responsible for oncotic pressure maintenance  Page 4 of 10 [MLS 11] Module 1 SPECIMEN VARIABLES 13. Age: Adult Analyte concentrations remain constant between puberty & 8. Stress menopause in women & middle age in men (e.g. Hyperventilation) ALP activity higher in women at menopause Alteration in acid-base balance Pregnant women are expected to have high Increase FFA levels of ALP Increase lactate Serum uric acid: first to increase in men at 20s Generally, increase enzyme activity due to greater physiological activities 9. Circadian/Cyclical Variation 14. Sex Activity of muscle enzymes is higher in men owing to increase muscle mass Cholesterol and LDL- varies ALP activity higher in women 15. Temperature Acute exposure to heat results to: ✓ expansion of plasma volume and ✓ Influx of interstitial fluid to intravascular space Hormones are mostly affected ✓ reduced glomerular filtration ✓ 10% decrease plasma protein 10. Travel Sweating results decrease potassium as these are taken up by the cells Differences in time zones affects normal circadian rhythm Alters pituitary & adrenal function Sweat is made up by water and Na. When we sweat, we are losing more Na. That would mean Sample effects: that there is an electrolyte imbalance. ✓ 5 days; required to have stable rhythm after travel across 10 time zones Na is major extracellular cation. ✓ During a flight: increase glucose & TAGs, stimulated It means there is higher levels of K than Na. glucocorticoid secretion This is not normal High altitude: production of stress-related So in response, the K will shift back inside the hormones= increase glucose cell and the Na will go outside to maintain electrolyte balance. Then, there would be ✓ Prolonged flight: fluid & sodium retention, normal urinary decreased K level in blood. excretion after 2 days Lessened movement 16. Obesity Increase cholesterol, TAGs, beta-lipoprotein 11. Age: Newborn Increase LDL Fluid passes from blood vessels to extravascular spaces: Growth hormone reduced: increase plasma protein (albumin) responds poorly to normal challenges Increase enzyme activity of CK (Creatinine kinase), GGT & Impaired glucose tolerance AST (Aspartate Amino Transferase) High risk to develop diabetes. Increase bilirubin due to enhanced RBC destruction RBC lysis: release of heme and globin Low blood glucose due to small glycogen reserve 12. Age: Children to Puberty Increased ALP activity due to skeletal growth; ALP is necessary for maximum osteoblastic activity with bone growth 9-19 years old Normal in increased ALP ALP cannot be used as sole indicator of liver damage Serum creatinine increases due to skeletal muscle development Page 5 of 10 [MLS 11] Module 1 SPECIMEN VARIABLES All ase from the table: enzymes found inside ->Analytes can be changed if prolonged period the RBC Calcium, K, Na, Phosphate Pre-analytical Variables electrolytes, they are greatly affected by 1. Test usage and practice guidelines hemolysis 2. Patient Identification 3. Turnaround time 18. Pseudohyperkalemia 4. Laboratory logs 5. Transcription errors - Falsely elevated K level in the blood 6. Patient preparation extremely high blast counts in acute or accelerated phase 7. Specimen collection leukemias 8. Specimen transport blasts are extremely fragile and are easily 9. Specimen separation and distribution of aliquots hemolyzed fragile and may lyse during standard phlebotomy Disadvantages: extremely elevated platelet count o Difficult to monitor and control ▪ We can’t monitor how the patients Analytical Factors collect their samples Factors that can lead to an increase or decrease of o Requires the coordinated effort of many individuals results which can happen during the analysis of the sample. and departments Involves the Medical Technologists, and Laboratory ▪  (e.g) if in-patient, you can check with Scientist because we are the one who will analyze the the nurse why a certain test became sample abnormal when it was on normal level on previous days 1. Reagents o May require support from outside the laboratory 2. Instrumentation & equipment ▪ Institution’s clinical practice committee - The problem is on the machine and on the equipment used Control of Pre-analytical Variables 3. Testing procedure 1. Patient identification 4. Quality control Handwritten labels and request forms 5. Preventive maintenance o Use of bar coding technology (US); LIS -  (e.g) monitoring of temperature of refrigerator o Use of wrist bands, ID, folks confirmation of identity 6. Access to procedural manuals 2. Test ordering - If not sure of what to do… Second hand test ordering by nurses or clerks exhibits higher 1. Get ask for help to your seniors error rates 2. Check the manuals o Direct ordering by clinicians 7. Competency of personnel performing the test o Avoiding abbreviations 3. Specimen collection Post-analytical Factors Improper container, incorrect preservatives, These are factors or conditions leading to an erroneous anticoagulants results after the sample had been analyzed. Involves the medical technologists Pre-analytical Variables o if in-patient or admitted, interns bring the results to the nurses’ station Specimen Transport o if no intern available, phlebotomists bring the Ideally within 45 minutes after collection specimen must be transported results to the laboratory o  (floating) going to one patient to another for 1. Delays in transport and mishandling of specimen blood extraction - The authority to reject specimen that arrive in the laboratory is Reporting of results an obviously unsatisfactory condition, comes from MT. (e.g) if the patient need to test for FBS and did not fast, we have the o For medical technologists authority to reject. Explain first. But if the patient insists to be Interpretation of results tested, just take note in the result. o For physicians o For MT, never interpret results to patients 2. Specimen Separation and Distribution of Aliquots - Centrifuges should be calibrated and maintained Specimen Collection, Handling & Processing o Calibration is a requirement for every 6 months. Because this can affect the quality of the sample - Collection tubes, pipettes, stoppers, and aliquot tubes should be tested for contamination by calcium and other elements Turnaround Time Record actual time of specimen collection, receipt in the laboratory, and reporting of the test results. All during the blood collection, you need to put your name as phlebotomist, time collected the sample. In urine, note the time received. Patient Preparation Lab must provide well-written, understandable procedures for proper patient preparation and specimen acquisition → must be made available Before using the tubes, it must be checked for any evidence of vacuum loss and for the expiration date. Control of Analytical Variabels Ideally, after collection: 1. Schedule daily and monthly maintenance 45 minutes to 1 hour: analysis should be performed - There are variables that we need to have preventive maintenance within 1 hour: serum or plasma separated from cells everyday, or monthly -  (e.g) Everyday: Running a Quality Control Page 6 of 10 [MLS 11] Module 1 SPECIMEN VARIABLES 2. Routine instruments function/system checks should be detailed and their performance should be documented 3. Reagents and kits should be dated when received and opened 4. New lots of reagents should be run in parallel with old reagent lots before being used for analysis Post-Analytical Variables 1. Variables involve report generation, delivery, and interpretation of results and subsequent actions 2. Generally experience fewer and less severe quality problems but errors can be serious and significant. A lost report or delay in reporting a panic or critical value can jeopardize patient care. Anticoagulant Interference - Prevent clotting of blood Light blue top - Stimulate ACP. It can increase the level of ACP. - Citrate is the most ideal for coagulation test. Because it preserves labile coagulation factors  Two types of EDTA - Versene - Sequestrene - Increase T3, T4 (thyroid hormones) - Not for hematology, esp for differential count (WBC). It - Best used in hematology section. Because it preserves causes blue background. It is difficult to identify nucleus and the morphology of blood. platelets. - In Clinical Chemistry, we cannot use for ALP, CK and ALP (enzyme). Because if it inhibits the activity of the enzyme, it lowers the level of the enzyme. - Increase in protime (PT), Partial Thromboplastin (PTT), Sodium (Na), and Potassium (K). These are coagulation tests. - In Clin. Chemistry, most of the analytes requires serum as sample because of its small or less interference. - Decrease in Calcium and Iron. Because the action of EDTA is to chelate or chelation of calcium. - Prevent platelet aggregation. It preserves RBCs and at the same time prevents platelet clumping. Specimen handling and processing Clotting time 20-30 mins: make sure to stand by the blood first before centrifugation Importance: allows the complete clot retraction If did not set up: attained inadequate serum (only 1 ml obtained instead of ideal 2.5) due to blood did not clot in the ideal time span (20-30 mins), can result to re-centrifugation and it can induce the lysis. Preferred clinical chem sample: serum preferred over plasma Note: some of the color as are different depending on the analytes that - Inhibit. Because they are enzyme are elevated in the sample - Oxalate is not for Hematology because it destroys the morphology of the cells. Difficult to identify RBC, WBC, and Reasons: platelets. Lipemia precipitated during clotting Optically clearer -> clear yellow Potential interference with the use of anticoagulants ->Enzyme inhibitors - Na & K: cause of loss of water (where sodium goes water follows) - NH4 salts: cause influx if water in the cells (Hypotonic solution) - Anticoagulants lower the calcium by chelation (EDTA), Ionization(citrate), formation of Oxalate salts (Oxalate) etc. except for heparin which is a natural anticoagulant Page 7 of 10 [MLS 11] Module 1 SPECIMEN VARIABLES INCREASE Album concentration in Specimen transport BCG Ideally within 45 minutes Bilirubin in Dependent on the stability spectrophotometry Avoid agitation -> can cause lysis to the red blood cells Bilirubin in Prevent direct exposure to light diazotization (van den Keep at specified temperature Bergh reaction) Note: Avoid exposure to sunlight since analytes particularly bilirubin Any form of hemolysis present in the serum sample can be rejected if it will be affected. When exposed, the amount of bilirubin in the blood will is induced by controllable variables decrease as it is broken down by the light. A similar example would be an infant with jaundice being exposed to sunlight to reduce bilirubin Remember: levels in their body. How to check for susceptible hemolytic sample? Remember: some samples are transported with ice ex. Blood gas In tube-> place on a white background Specimen interference THROMBOLYSIS Result of clotting Clot retraction can cause platelet disintegration INCREASE Acid phosphate (ACP) Mg K Aldolase (Correlate values between plasma and serum specimen) Note: K is inside the cell-> cell rupture-> increase k GRANULOCYTOLYSIS INCREASE Muramidase(lysozyme) Phosphohexose LYSIS of CELLS - leakage of intracellular substances isomerase (PHI) a. In vivo hemolysis: Possibility of hemolytic crises Arginase Note: possibility of a hemolytic disease Glucose-6- phosphate Recent blood transfusion dehydrogenase (CPD) Glutamate Note: In vivo hemolysis through blood transfusion is caused by dehydrogenase mismatch of blood type by the donor and the recipient. Given a person has a blood type O, this blood type possesses Both a and B antibodies. When the blood type O receives a blood from blood donor AB, which ICTERIC SERUM harbors both A and B antigen, upon contact, the antibody of blood type It has increased bilirubin >25 mg/dL and interferes with antibodies in O will destroy the antigen of blood type AB causing RBC lysis. colorimetric assay Scenario: We check the hemolysis on patient before doing the INCREASE Alb by HABA centrifugation, take a small sample and check. CHOL by FeCl3 Glucose by O- b. in vitro hemolysis: toluidine >Hb concentration of 200 mg/L (20 mg/dL) TP by biuret Improper collection & handling Refer in Torrent pg. 22-23 Use of vacuum tubes: expansion of cells -Note: the force of the close system evacuated tube can LACTESCENT SERUM cause lysis It has physiologic increase in chylomicrons carrying TAGs Vigorous mixing TAG level is 4.6 mmol/L (4 g/L). -Note: Mixing causes lysis of cells. Instead, inversion is used. Too narrow or too wide needle bores -Note: Narrow needle bores and forcing to extract the blood when there is no backflow during phlebotomy causes hemolysis. Too narrow->slow movement of blood to the syringe->can cause the delay of extraction->lysis Too wide-> easier to pull the plunger-> Excreted great pressure on blood extraction-> lysis Alcohol applied on skin -Note: Alcohol must be air-dried before extraction to avoid pain and RBC lysis. Indicated by a stinging sensation Centrifugation & separation steps -Note: refer to the clotting time Effects of hemolysis if not detected: Scatters light and visible turbidity (due to the increase of 1.) Release of RBC constituents including water chylomicrons carrying trigycerides) INCREASE Acid phosphate (ACP) Zn Interference in test associated with lactescent Mg Albumin in BCG K Ca2+ in O-cresolphthalein CK Inorganic phosphate in acid ammonium molybdate Adenylate kinase 2.) Interference of Hb in color reactions at wavelength 400 to 600 nm Page 8 of 10 [MLS 11] Module 1 SPECIMEN VARIABLES DECREASE Amylase Uricase Serum with hemolysis Urease Prominent red color in the serum Creatinine kinase Cell lysis in specimen collection refers to lysis of RBC (CK) wherein the breakdown of RBC caused the serum’s color to Bilirubin turn red. Rupturing of RBC causes the hemoglobin to TP breakdown into heme and globin. Globin in particular is responsible for the redness of the serum. During this time, Note: microscopic hemolysis can be detected through a machine with several analytes for laboratory testing will be affected. grade: 1,2,3 Lactescent serum with hemolysis It has physiologic increase in chylomicrons carrying TAGs - TAG level is 4.6 mmol/L (4 g/L). Reasons for Specimen Rejection ✓ Hemolysis/lipemia Can we reject this sample? Note: Lipemic specimen, high concentration of lipids in We can reject this specimen if the patient did not follow the pre- blood specimen, is only rejected when the patient did not collection procedure particularly fasting for 14 hours. However, when follow the pre-collection procedure particularly fasting for 14 the patient followed the pre-collection procedures, recollection can be hours. However, when the patient followed the pre-collection done for the confirmation of result and if the results are similar, the procedures, recollection can be done to compare the results specimen is not rejected. and if the results are similar, the specimen is not rejected ✓ Clots present in an anticoagulated tube Specimen storage Note: Don’t reject specimen in a following condition: it there is presence of a micro clot of a sample in the tube with Evaporation-induced Errors anticoagulant Plasma CO2: evaporates easily ✓ non-fasting specimen when test requires fasting Decrease of 5 mmol/L in 60 minutes at RT ✓ Improper blood collection tube Cause increase in pH (8.5) Note: Don’t reject specimen in a following condition: if the Destroy ACP: add buffer to maintain pH 6.2 test tube has no anticoagulant ex. Red and SST. It is okay to get an inadequate blood sample but just follow how many ml Stability of Analytes on Storage of blood needed for a certain test. Lecithin over sphingomyelin (L/S) ratio Reject if: test tube has anticoagulant, must follow the Assess fetal lung maturity recommended C:blood ratio 2:1 fetal maturity ✓ Short draws, wrong volume ✓ Improper transport condition (e.g., samples for blood gases should ->Use refrigerated centrifugation in amniotic fluid be transported with ice) Why are we doing this? Related to blood gasses: carbon dioxide, oxygen, ammonia Inhibit phospholipase etc. ✓ Discrepancies between requisition and specimen label Phospholipase is the enzyme that breaks down phospholipids. If serum is transported or centrifuged ina higher temperature, the enzyme ✓ Unlabeled or mislabeled specimen would still function. It would affect the Lecithin over sphingomyelin ✓ Contaminated specimen/leaking container because sphingomyelin is a lipid that has a phospholipid property. Prevent phospholipid degradation Important reminder: Refer to specimen interference Glucose Separated from clot ASAP SAMPLES: Prevent utilization of glucose by RBCs - Decrease by 10% in contact with clot for 2 hours Note: - Prolonged contact of the blood and serum will cause the RBCs to utilize the glucose decreasing the blood sugar present in the blood. All the color top tube is applicable to this except for SST because when it was centrifuged, the serum is already separated using the gel (polymer barrier) thus less interaction of blood and serum. Ammonia on Increase on storage Must proceed with the testing process immediately RBC proteolytic & hydrolytic processes Permeability changes Specimen from left to right: INCREASE P K Normal serum Mg Clear yellow plasma Note: - Longer storage, higher levels of ammonia. Same with K, P, Mg Icteric serum due to electrolyte shifting. Dark yellow plasma -> can be cause by the red blood cell lysis thus presence of bilirubin Important: K is the most abundant intracellular cation. It has increased bilirubin >25 mg/dL and interferes with antibodies Na is the most abundant extracellular cation. in colorimetric assay because of its own light absorbance An exchange between them in the RBC, K goes out of the RBC; Na goes in the RBC interchangeably, is called electrolyte balance. The color of serum depends on the level of bilirubin in the blood. When the level of bilirubin is high, the serum is dark yellow. On the SEXPIN other hand, the serum is pale yellow when the bilirubin level is low. Sodium- most abundant EXtracellular cation. Normally, the serum is pale yellow when cell lysis does not exhibit. Potassium- most abundant INtracellular cation. Can we reject this sample? -Prolonged storage of RBC causes lysis. No, we can’t reject the serum because this is a cause of an Potassium from the RBC goes to serum, which will be extracted for uncontrollable variable. The in vivo hemolysis is the is the destruction of laboratory testing. Hence, high K level in lab results. RBC inside that can cause jaundice in which there is an increase of bilirubin. It is not the fault of patient, but it can indicate an underlying condition. Page 9 of 10 [MLS 11] Module 1 SPECIMEN VARIABLES Storage in 4 degree C Permeability changes also noted in this condition loss of CK from red blood cells; K goes to the plasma increase inorganic phosphate (plasma) loss of activity: Lactate dehydrogenase isoenzymes (LD4 and LD5) = low level Rapid freezing by liquid nitrogen @ 80° C Short-term Storage (6 weeks) @ -20° C For creatinine kinase (CK): -70° C Light Exposure (protect them from light) Note: Bilirubin, D-ALA, porphyrins, and porphobilinogen are analytes that get affected once exposed to light. They will DECREASE because the tendency is these analytes, since they are very sensitive to light, will break down once they are exposed to light. Bilirubin D-ALA Porphyrins Porphobilinogen Note: Porphyrins and Porphobilinogen are part of the metabolism of the Bilirubin Specimens requiring chilling during transport: Ammonia BGA Lactic acid PTH Pyruvate renin Note: Arterial blood must be stored and chilled properly compared to the venous blood. Specimens that require chilling for transport are usually used for blood gas analysis. The tube used is green top. Usually, this specimen is arterial blood. The choice of the collection system is also very important. Options include a closed or an open sample collection system. Anticoagulants (heparin, citrate, oxalate, etc.) can be chosen depending on the required test. Order of draw for multiple tube collections and the number of inversions when mixing the sample must be strictly followed. Page 10 of 10  MLS11: Clinical Chemistry 1: Lecture Module 2: Analytic Technique and Instrumentation Trans team: Almario, Altura, Andrade, Berja, Dichoson, Gallego, Jovero, Noble, Palencia, Saradolla Legend: Remember Previous 🔊 Lecturer Book Trans Comm (Exams) Trans ☝ 🕮 🗐 🖉 fdsg TOPIC OUTLINE 🔊 When for example there is energy absorbed the valence electrons will moved to an orbital with a higher energy level, following once there is an energy absorption the electron which is on the excited state it will now fall again into its ground state after that they will emit a little 🔊 amount of energy in the form of the wavelength. Which means there is a production of wavelength because of energy. PLANCK’S FORMULA Where: E = hv E = energy h = Plank’s constant (6.626 x 10 -34 erg sec) v = frequency If the H is a Planck’s constant meaning it does not vary while our v or your frequency it becomes v because it is variable, so your frequency is the only one changing Lesson 1. FREQUENCY COLORIMETRY AND SPECTROMETRY - Number of vibrations of wave motion per second Energy - Lower wave frequency: longer wavelength wavelength - Transmitted via electromagnetic waves - Wavelength inversely related to frequency and energy 🔊 - Characterized by their frequency and wavelength The principle of spectrometry is based on the principle of colorimetry meaning the basis of our read of analyte is the colored - Shorter wavelength: higher frequency and energy and vice versa 🔊 reaction. Energy is very necessary when we measure analyte particularly for 🔊 spectrometry and colorimetry 🔊 Manually operated machine- we record wavelength. Fully automated- does not produce wave length but the 🔊 concentration. Visible spectrum that’s the wavelength where we can observe by our naked eye the colored reaction (it means when we measure an 🔊 We have to remember even if there is a small change on the wave analyte, we can see visible results) length it could have a greater effect on the absorbance reading in return, because the absorbance is necessary for us to solve the Wavelength concentration of the analyte that will affect the concentration (e.gThe - Distance between two successive peaks concentration of creatinine is 115 mmol/liter, we can solve this by - Expressed in terms of nanometer (nm) basing on the results for the wavelength of the absorbance. 400-700 nm Visible spectrum < 400 nm UV region Notes to remember >700 nm IR Region Nominal wavelength - Represents the wavelength in nanometers at peak transmittance Slight error in wavelength adjustments: introduce a significant error in absorbance readings Wavelength accuracy: [MLS 11] Module 2 ANALYTICAL TECHNIQUE AND INSTRUMENTATION - Indicated on the control dial the concentration of your substance is also high (the darker the color is - Actual wavelength of light passed by the monochromator the higher is the concentration of the analyte) 🔊 🔊 Control dial- it is found in the machine - COLORIMETRY Measurement of the concentration of an analyte in a colored 🔊 Monochromator-select the amount of light that will pass through solution the solution/ cuvette. It totally eliminate the stray light This refers to the visible spectrum (lighter solution, lower concentration of the substance) Didymium or holmium oxide filter - Used to check wavelength - Concentration of an analyte in a solution is directly 🔊 proportional to the amount of light it absorbs o Beer’s law Didymium or holmium oxide- to calibrate the wave length 🔊 Colorimeter - Instrument used in Colorimetry To calibrate that as for to prevent some type of error in our - Uses ordinary filters to screen light which will strike the wavelength readings we need to have filters solution o Measure the amount of light intensity in multiple Neutral density filters and dichromate wavelength - Verify absorbance accuracy on linearity 🔊 Neutral density filters and dichromates (to confirm the wavelength accuracy) for some this is also known as dichromium filter Types of Colorimetry: 1. Visual colorimetry 2. Photoelectric colorimetry Visual Colorimetry COLORIMETRY - Color intensity of the solution is matched - Used to quantify and describe physically the human color against a standard solution perception o Dubowski (Dubosq) Colorimeter 🔊 - Similar to spectrophotometry but is distinguished by its interest in reducing spectra to the physical correlates of color perception Standard solution- basis (e.g. we dip the - Utilized extensively for identification and determination of reagent strip in the urine after that we compare to the concentrations of substances that absorb light color to the bottle outside the reagent kit to read the 🔊 pH, specific gravity, and glucose) Colorimetry- color we are basing in the visible results. Somehow similar to spectrophotometry but the difference is spectrophotometry has now the ability to remove substances that can hinder our reading Photoelectric Colorimetry - Uses a photoelectric device or detector Photoelectric colorimetry - Primary clinical utility of spectrophotometry or filter photometry: isolation of discrete portions of the spectrum for the purpose of measurement Types: - Spectrophotometric measurement FACTORS AFFECTING THE INSTRUMENTATION - Photometric measurement 🔊 Wavelength - To get the sensitivity and selectivity, it is important to try to Spectrophotometric measurement- we measure the light intensity get as close as possible to an absorbance peak 🔊 🔊 in a narrower wave length. Photometric- the measurement of light intensity. Wavelength - What is the importance of having a correct choice or correct wavelength, because we need to get accurate and precise results in order for us to make sure it is sensitive or selective, sensitive Principle of Spectrophotometric means it can measure specific analyte) e.g., the required glucose - A substance absorbs light at unique wavelength wavelength is 405 then your machine is not on the 405 wavelength so - The amount of light absorbed is proportional to the amount if our machine does not have the capacity at least we are near on 405 of substance that is present either we choose 400 or 410 in order to make sure that our machine is - To shine light on a sample and to analyze how the sample sensitive or selective affects the lights 🔊 For example, sodium has a different wavelength for potassium it's also different it means that no all analytes have the same wavelength. Sample blanks and zeros - It is good practice to run a “zero” (untreated sample) - To correct for stray light, sample color or turbidity or 🔊 The amount of light absorbed is directly proportional to the concentration of the analyte meaning if you have high light absorption, - absorption of the measured wavelength by some constituents of the sample To correct for internal reflection of light by container walls 2 🔊 [MLS 11] Module 2 ANALYTICAL TECHNIQUE AND INSTRUMENTATION Zero’s actually means we are reading the concentration of water, solvent? When we are measuring glucose, the solute is glucose, the solvent is the reagent that we use. remember water is pure especially distilled water so it has 0 🔊 absorbance reading Purpose why we run zero’s or sometime called blanking? It is in Wavelength Accuracy - Wavelength indicated on the control dial is the actual 🔊 order to eliminate errors 🔊 If manual operation on machine we do zero’s If fully automated no need to run zero wavelength of light passed by the monochromator Stray light - Refers to any wavelength outside the band transmitted by the monochromator The difference Spectrophotometry Colorimetry - Not come from the light source In non-technical and Single wavelength is A particular band is simple terms selected selected Linearity Can be manipulated by Able to transmit light of - Demonstrated when a change in concentration results in a the experimenter only a few wavelength straight-line calibration curve Monochromator or The filter is used % Transmittance The wavelengths prisms is used in here - Ratio of the radiant energy transmitted (T) divided by the It is usually used in a Used in High school radiant energy incident on the sample (I) professional research setting laboratories 🔊 Transmittance- is the ratio of the transmitted light to the incident light (emergent light) in short ratio of the radiant energy transmitted Spectrophotometry Colorimetry divided by the radiant energy incident on the sample Colored and colorless solution Colored solution only PHOTOMETRY 𝐼𝑡 Where: 𝑥 100 - Measurement of light in the form of wavelength 𝐼𝑜 It : transmitted light thru - Selection of a range of the incident wavelength using Io : intensity of light striking the sample o Filters: photometers o Prism / gratings: spectrophotometers Spectrophotometer - Radiant energy is partially reflected, absorbed and - Enhances the sensitivity and precision of colorimetric assays transmitted - Uses monochromators instead of low-resolution filters - Analysis is suitable for both colored and colorless solutions - A photometer that can measure intensity as a function of the light source wavelength 6 BASIC COMPONENTS OF A SPECTROPHOTOMETER 1. stable source of radiant energy 2. Filter 3. Sample holder 4. Radiation detector 5. Signal processor 6. Read-out device PARTS OF A SPECTROPHOTOMETER SPECTROPHOTOMETRY - Quantitative measurement of the reflecting of transmission properties of a material as a function of wavelength - To determine the concentration of the light-absorbing substances in the solution, the amount of the light 🔊 transmitted by a solution is being measured. Spectrophotometry gives us the ability to measure the 🔊 concentration of the solution either it is colored or colorless. 🔊 Spectrophotometry- we are measuring quantitatively e.g. analytes that are present in the serum of an individual they have the ability to absorb light meaning glucose in which is on our serum if we put a reagent has the ability to absorb light, creatine- it is an analyte it is a substance also has the ability to absorb light. 1. Light Source 5. Cuvette Absorbance 2. Entrance Slit 6. Photodetector - Amount of light absorbed / blocked by the solution 3. Monochromator 7. Read-out device - Proportional to the inverse log of transmittance 🔊 4. Exit Slit 8. Power source - Mathematically derived from %T Absorbance- amount of light absorb by the solution, solution is a mixture of solute and solvent, if we are measuring glucose what is the 1. LIGHT SOURCE - Provides polychromatic light 3 [MLS 11] Module 2 ANALYTICAL TECHNIQUE AND INSTRUMENTATION - Intense beam of light is directed through the monochromator (to avoid further entry of particles that can affect the absorbance and the sample reading. - Response to change in light must be linear Kinds of Monochromator Light Source a. PRISMS Visible 1. Tungsten bulb - Triangular or wedged piece transparent material 2. Tungsten iodine lamp - Allows only desired wavelength to pass through an exit slit 3. Tungsten halogen lamp UV 1. Hg arc lamp 1. Visible: glass 2. Deuterium lamp 2. UV: Quartz 3. H vapor lamp 3. IR: Sodium chloride and Potassium bromide 🔊 4. Xenon arc lamp 5. Tungsten Iodide lamp A narrow light is focused on a prism and is refracted as it enters a 🔊 IV 1. Tungsten halogen lamp 🔊 more dense glass. Why the light is narrow? Because it is specific for a particular One example of a line source that is used in the laboratory are analyte. mercury lamps and hollow cathode lamps (used for atomic 🔊 absorption spectrophotometry). Another example of a line source is the laser. - b. DIFFRACTION GRATING Grooved piece transparent material (3000 or more per mm) that function as prism 🔊 TWO TYPES OF LIGHT SOURCE What it is made up of? It is placed in a semi transparent, silver a. CONTINUUM SOURCE film on both sides. Both sides of the material are silver films while in - Emits radiation that changes in intensity between them are Magnesium fluoride. b. LINE SOURCE c. FILTERS - Emits limited radiation and wavelength - Produced monochromatic light 🔊 Light source or the bulb, or the radiant energy source, or the - Light waves enter one side of the filter and reflected on the second surface exciter lamp. 🔊 The prisms have narrower wavelengths, while in the filters, it has Mercury arc ALTERNATIVES FOR TUNGSTEN BULB Merst glower 🔊 wide bands of wavelengths. Prisms are still the BEST kind of monochromator. - Visible and UV - IR 4. EXIT SLIT Deuterium lamp Globar (silicone carbide) - controls the amount of emergent light that passes through - 165 nm - IR the cuvette - UV 5. CUVETTE Hydrogen lamp - UV 2. ENTRANCE SLIT - Minimizes stray light emitted by the lamp - Prevents scattered light from entering the monochromator 🔊 Entrance slit prevents the entry of unwanted/stray lights to the 🔊 monochromator. Stray lights didn’t come from the light source but from the external - analytical cell, sample cell, sample holder or absorption cell 🔊 - holds the solution whose concentration is to be assayed environment. - sample holder (serum + reagent→ depends on the test eg. If there’s no entrance slit, there will be a limitations in the reagent for glucose) absorbance reading of the sample because stray lights can affect the absorbance reading, which can further affect the concentration of the analyte. 🔊 REMEMBER, we are not placing serum in the cuvettes because serum is the solute. So it needs a diluent (solute + solvent or solute + - 3. MONOCHROMATOR used to eliminate unwanted wavelengths (stray light) of 🔊 diluent). Emergent light passed thru cuvette= transmitted light KINDS OF CUVETTE - 🔊 lights Total elimination of stray light for light source will only come from the light bulb 🔊 REMEMBER, the choice of cuvette depends on the type of - Isolate sharply specific wavelengths of light solution. 🔊 - Selects amount of light The most important function of the monochromator is the isolation of individual wavelengths of light. Which means the monochromator a. a. GLASS Aluminosilicate glass: Acidic solutions allows measurement of light intensity in a much narrower wavelength b. Borosilicate glass: Alkaline solutions 4 [MLS 11] Module 2 ANALYTICAL TECHNIQUE AND INSTRUMENTATION b. SOFT GLASS - In short, the function of a photodetector is to detect the amount of light that passes through the sample in a cuvette c. QUARTZ or PLASTICS or the solution that is in the cuvette itself and means that it - Visible and UV spectra measures the amount of light that passes through the 🔊 This is the most IDEAL cuvette that is being used in the 🔊 cuvette holding the sample or the solution. What can we use as a photodetector/photocell? 🔊 laboratory. - Any photosensitive device can be used as detector of the radiant energy REMEMBER, the clear side of the cuvette should be faced on the - Remember, the function of photodetector is to convert the direction of the light source. If it is done incorrectly, the absorbance reading will be affected. transmitted light into photoelectric energy or the radiant energy CONSIDERATIONS AND PRECAUTIONS - So, which means that if one device is photosentive already, it 🔊 1. Free from scratches and dirt (fingerprints) can be used as a detector of the radiant energy. While holding the cuvettes, we should hold it on the frosted side. - Several types of photodetector that is being used in a If it is done on the clear side of the cuvette, fingerprints will be left and spectrophotometers: can now affect the absorbance reading. 1. Photocell 2. Phototubes 🔊 2. Avoid prolonged standing of alkaline solution in cuvettes Should not be exposed in alkaline solutions or higher pH because our cuvettes are made up of glass.Alkaline solutions tend to dissolves - Some of the simplest photodetector that is being used in the laboratory the glass that will lead to etching or scratches. Kinds of Photodetector a. BARRIER LAYER CELL 🔊 3. Position properly in photometer The clear side of the cuvette should face the light source (see 🔊