SAFETY IN THE LABORATORY Health hazards come from a variety of sources: 1 - Chemical hazards: Examples include strong acids and alkalis 2 - Biological hazards: Examples include human body fluids that may carry infections such as HIV, 3 - Electrical and mechanical hazards: All electrical apparatus should be used and maintained in accordance with the manufacturers’ instructions. 4 - General laboratory hazards: Common examples include syringe needles, broken glassware and liquid nitrogen flasks. "Specimen Collection and Processing" Q/Many errors can occur during these steps? Proper collection, identification, processing, storage, and transport Q/Enumeate Types Of Specimens? Types of biological specimens that are analyzed in clinical laboratories include (1) whole blood; (2) serum; (3) plasma; (4) urine; (5) feces; (6) saliva; (7) spinal, synovial, amniotic, pleural, pericardial, and ascitic fluids; and (8) various types of solid tissue. Q/Enumeate Preliminary Steps Before any specimen is collected? the phlebotomist must confirm the identity of the patient. Two or three items of identification should be used? -(e.g., [1] name, [2] medical record number, [3] date of birth, [4] address if the patient is an outpatient). *Before collection of a specimen, a phlebotomist should dress in personal protective equipment (PPE), such as an impervious gown and gloves applied immediately before approaching the patient, to adhere to standard precautions against potentially infectious material and to limit the spread of infectious disease from one patient to another. 1-Blood Blood for analysis may be obtained from veins, arteries, or capillaries. * venipuncture is the method for obtaining this specimen. *Arterial puncture is used mainly for blood gas analyses. Q/define Timing? The time at which a specimen is obtained is important for those blood constituents that undergo marked diurnal variation (e.g., corticosteroids, iron) and for those used to monitor drug therapy. Q/Explain Venous Occlusionاحتباس الدم with example? define Venous Occlusion? After the skin is cleaned, a blood pressure cuff or a tourniquet is applied 4 to 6 inches (10 to 15 cm) above the intended puncture site (distance for adults). Example *Total protein increase and potassium decrease Q/Give the reason for ?The increase in activity of creatine kinase CK and aspartate aminotransferase AST in serum seen after venipuncture ? may be caused by 1-hemoconcentration, 2- slight trauma to tissue as the needle pierces the skin, 3-stasis of blood in the tissue. Q/Pumping اول قطرة of the fist before venipuncture should be avoided? -because it causes an increase in plasma potassium, phosphate, and lactate concentrations. Q/Stress associated with blood collection can have effects on patients at any age.? As a consequence, plasma concentrations of cortisol and growth hormone may increase. Give the reason for ? blood collection under stress condition in young children give false result for glucose concentration and skeletal muscle enzyme ? - adrenal stimulation leading to an increased plasma glucose concentration or may create increases in the serum activities of enzymes that originate in skeletal muscle. Anticoagulants and Preservatives for Blood Serum is defined as : the watery portion of blood that remains after coagulation has occurred and is the specimen of choice for many analyses, including viral screening and protein الجزء المائي المتبقي بعد عملية الجمع electrophoresis. *Samples are collected into tubes with no additive or with a clot activator and must be allowed to complete the coagulation process before further processing. النفي يشمل الكلوت Plasma is defined as : the noncellular component of anticoagulated whole blood and is increasingly being used for routine chemistry testing to decrease turnaround time. *A number of anticoagulants are available, including: 1- heparin, 2-ethylenediaminetetraacetic acid (EDTA), 3-sodium fluoride, 4-citrate, 5-acid citrate dextrose (ACD, oxalate, and iodoacetate. 1-Heparin(Defined) Heparin is the most widely used anticoagulant for chemistry and hematology testing. It is a mucoitin polysulfuric acid حامض الكبريتيك المتعدد and is available موجود على شكلas sodium, potassium, lithium, and ammonium salts, all of which adequately prevent coagulation. *Mechanism Of Action ? This anticoagulant accelerates تعمل على زيادة فاعلية the action of antithrombin III, which neutralizes thrombin and thus prevents the formation of fibrin from fibrinogen. يعتمد على عوامل مهمة الكالسيوم والفايبرين *The heparin is usually present as a dry powder that is hygroscopic and dissolves rapidly. Heparin has the disadvantages ? 1-high cost 2-more temporary action of anticoagulation than is attained by chemical means, such as those discussed below. 3- It produces a blue background in blood smears that are stained with Wright’s stain. 4-In addition, heparin is said to inhibit acid phosphatase activity and to interfere with the binding of calcium to EDTA in analytical methods for calcium involving complexing with EDTA. 5- unacceptable for most tests performed using the polymerase chain reaction (PCR)? -because of inhibition of the polymerase enzyme by this large molecule. 2-Ethylenediaminetetraacetic Acid EDTA is a chelating agent of divalent cations such as Ca2+ and Mg2+ that is particularly useful for(advantage) (1) hematologic examinations, (2) isolation of genomic DNA, and (3) qualitative and quantitative virus determinations by molecular techniques, because it preserves the cellular components of blood. * It is used as the disodium, dipotassium, or tripotassium salt, the last two being more soluble. *It is effective at a final concentration of 1 to 2 g/L of blood. مهم*Higher concentrations hypertonically shrink the red cells. *EDTA prevents coagulation by binding calcium, which is essential for the clotting mechanism. Disadvantage of EDTA? 1- probably by chelation of metallic cofactors, inhibits alkaline phosphatase, creatine kinase, and leucine aminopeptidase activities. Because it chelates calcium and iron, 2-EDTA is unsuitable for specimens for calcium and iron analyses using photometric or titrimetric techniques. 3-Sodium Fluoride (defined ) Sodium fluoride is a weak anticoagulant that is often added as a preservative for blood glucose. As a preservative, together with another anticoagulant such as potassium oxalate, it is effective at a concentration of approximately 2 g/L blood. Why used ? It exerts its preservative action by inhibiting the enzyme systems involved in glycolysis, although such inhibition is not immediate and a certain amount of degradation occurs during the first hour after collection. على الرغم من انه مانع ضعيف الا انه يستخدم لحفظ عينة الدم للسكر لانه يعمل على تثبيط الانزيمات المشاركة في عملية التحلل السكري 4-Citrate Sodium citrate solution, Because citrate chelates calcium, (disadvantage)? 1-it is unsuitable as an anticoagulant for specimens for measurement of this element. 2-It also inhibits aminotransferases and alkaline phosphatase but 3-stimulates acid phosphatase when phenylphosphate is used as a substrate. 4-Because citrate complexes molybdate, it decreases the color yield in phosphate measurements that involve molybdate ions and produces low results. 5-Oxalates Sodium, potassium, ammonium, and lithium oxalates inhibit blood coagulation by forming rather insoluble complexes with calcium ions. Combined ammonium and/or potassium oxalate does not cause shrinkage of erythrocytes. However, 1-other oxalates have been known to cause shrinkage by drawing water into the plasma. 2-Reduction in hematocrit may be as much as 10%, causing a reduction in the concentration of plasma constituents of 5%. *As fluid is lost from the cells, an exchange of electrolytes and other constituents across the cell membrane occurs. 3-Oxalate inhibits several enzymes, including acid and alkaline phosphatases, amylase, and lactate dehydrogenase, and may cause precipitation of calcium as the oxalate salt. B-Urine *A clean, early morning, fasting specimen is usually the most concentrated specimen, and *thus is preferred for microscopic examinations and for the detection of abnormal amounts of constituents, such as proteins, or of unusual compounds, such as chorionic gonadotropin. Urine Preservatives The most common preservatives and the tests for which preservatives? preservatives Concentration /volumes 1-HCL 6 mol/l 30ml per 24 hour collection 2-Boric acid 10g per 24 hour collection Preservatives have different roles but usually are added ? 1-to reduce bacterial action or chemical decomposition, 2- to solubilize constituents that otherwise might precipitate out of solution. 3-to decrease atmospheric oxidation of unstable compounds. *Some specimens should not have any preservatives added because of the possibility of interference with analytical methods. Investigation Of Renal Function Function of the kidney: The function unit in the kidney is the nephron, shown in Figure 1. The kidney regulate تنظيم Extra Cellular Fluid (ECF) volume and electrolyte composition to compensate for wide daily variations in water and electrolyte intake. تنظيم حجم السوائل خارج الخلوية ومكونات الاليكترولايت داخل جسم الانسان والتغيرات التي تحدث نتيجة لتناول الماء والايلكترولايت They form urine in which the potentially toxic waste products of metabolism are excreted. هو المسؤول عن تكوين الادرار )النواتج الايضية الغير مرغوب بها ( ?What are The functions of the kidneys • Regulation of water, electrolytes and acid-base balance. • Excretion of the products of protein and nucleic acid metabolism: urea, creatinine, creatine, uric acid, sulphate and phosphate. Q/ why The kidneys are consider endocrine organs? -because producing a number of hormones, and are subject to control by other. 1-Arginine vasopressin (AVP) acts to influence water balance. موازنة المحتوى المائي 2- aldosterone affects sodium reabsorption in the nephron. اعادة امتصاص الصوديوم 3-Parathyroid hormone promotes tubular A-reabsorption of calcium, phosphate excretion and B-synthesis of 1,25-dihydroxycholecalciferol, which regulates calcium absorption by the gut. تنظيم امتصاص الكالسيوم بواسطة القناة الهضمية Erythropietin : is glycoprotein consist 165 a.a act the receptor on RBC act to reduce apoptosis on RBC in bone marrow يفرز عندما تكون كمية انسيايبة الدم قليلة الواصل للكلية ويعمل على تحويل الانجو الى ال1والى 2 والذي يحفز : Rennin افراز الاديستيرون والباراثارايد والارجنين Tests Of Glomerular Function: Define the (GFR): The glomerlar filtration(GFR): الترشيح الكلوي is an ultra filtrate فلترة of plasma, and has the same composition as plasma without most of the proteins. Plasma is filtered by the glomeruli at a rate of approximaltely 140 ml/minute. A normal glomerular filtration rate (GFR) will depend on there being normal renal blood flow and الدم يعتمد بشكل اساي على سرعة الدم المتدفق والضغط pressure. What are the most factor effect to the GFR is ? a-directly related to body size يتاثر بحجم الجسم b-consequently is higher in men than women. c- affected by age, declining in the elderly. Q/Give the reasons for the GFR falls تنخفض ? 1-restriction of the renal blood supply 2-or as a result of destruction ضرر of nephrons by renal disease, there نتيجة is retention of the waste products of metabolism in the blood. what happen dut to this fall ? In chronic disease. A new ‘steady state حالة استقرار’ is reached with a constant elevation in the serum concentration of substances such as urea and creatinine. بشكل ثابت اعلى من الحالة الطبيعية As the renal disease progresses, urea and creatinine concentration may increase slowly over many كل ما قل كفائة النظام الكلوي كل ما زاد اليوريا والكرياتنين months. Fig. 1: Endocrine links in the kidney. Define Creatinine clearance:? An estimate of the GFR can be calculated from the creatinine content of a 24-hour from urine collection,The ‘clearance’ of creatinine from plasma is directly related to the GFR. كمية الكرياتنين الموجود في البلازما خلال فترة 22 ساعة من جمع عينة الادرار The GFR is calculated as follows: كيفية حساب GFR = U × V/P U = urine concentration of creatinine P= serum or plasma concentration of creatinine V= urine flow in ml/min Note: that these be in the same units. A common mistake is to consider V انسيابية الادرار as urine volume, which it is not. It is the urine volume collected in 24 hours, and this figure is divided by 24 x 60 to give the volume produced per minute. V= urine volume collected in 24 hours/24 x 60 Q/ Why Serum urea concentration is less useful as s measure of GFR?. لماذا لا يتم الاعتماد على اليوريا في سرعة الترشيح الكلوي؟ويتم الاعتماد على الكرياتنين؟ Dietary protein intake affects serum urea concentration.-1 2-Gastrointestinal bleeding will cause serum urea to be elevated this does not indicate that glomerular filtration is compromised. 3-Urea is reabsorbed in the tubules. This reabsorption increases at low urine flow rates. creatinine which is not so reabsorbed. • Renal Stones: Renal stones produce severe pain and discomfortعدم راحة, and are common causes of obstruction in the urinary tract (Fig. 2). Chemical analysis of renal stones is important in the investigation of why they have formed. Types of stone include: Stone Casus Calcium phosphate 1-hyperparathyroidism 2-renal tubular acidosis. Magnesium, urinary tract infections Ammonium and phosphate Oxalate hyperoxaluria Uric acid Increase the acidity of urine Acute Renal Failure: Renal failure is the cessation توقف of kidney function. What deferent between ? In acute renal failure (ARF), Chronic renal failure (CRF) the kidneys fail over a period of hours or days. لفترات زمنية قصيرة develops over months or years and leads eventually to end stage renal failure (ESRF). reversed and normal renal function regained irreversible. • Aetiology: (etiology: the cause of disease) ARF arises from a variety of problem affecting the kidneys and/or their circulation. It usually presents as a sudden deterioration حدوث تدهور مفاجئ للوظيفة الكلويةof renal function undicated مصاحب by rapidly rising ارتفاع serum urea and creatinine concentrations. Kidney failure or uraemia ارتفاع اليوريا بالدمcan be classified as (Fig. 3): • Pre-renal: the kidney fails to receive a proper blood supply كمية الدم الواصلة للكلية قليلة وسرعة انسياب الدم تكون بطيئة • Post-renal: the urinary drainage مخرجات التدررof the kidneys is impairedقليلة because of an obstruction.(stone ,tumor) • Renal: intrinsic damage ضرر حقيقيto the kidney tissue. This may be due to a variety مختلفة of disease, or the renal damage may be a consequence of prolonged pre-renal or post-renal problems. • Diagnosis: In nearly all cases the clinical history and presentation will indicate that a patient has, or may develop, ARF. The first step in assessing the patient with ARF is to identify any pre-or post-renal factors which could be readily corrected and allow recovery of renal function. تقييم جميع المشاكل ما قبل او بعد المرحلة الكلوية لتصحيح المشكلة واعادة الوضيفة الكلوية الى عهدها السابق The second step The history and examination of the patient, including the presence of other severe illness امراض اخرى, drug history and time course of the onsetمعرفة مدة بداية حدوثof the ARF, may well provide important clues ادلة مهمة. -It is important to note that in the first 24 hrs of ARF the serum and urine tests may not reveal any في اليوم الاول لانستطيع اكتشاف المرض abnormality. *Factors which precipitate pre-renal uremiaمرتبطة بكمية السوائل المجهزة للنظام الكلوي are usually associated with a reduced effective ECF volume and include: • Decrease plasma volume because of blood loss, burn, prolonged vomiting, or diarrhea. • Local factors such as an occlusion انسداد of the renal arteryالشريان الرئوي. Therefore, biochemical finding in pre-renal uraemia include the following:ماذا يحدث • Serum urea and creatinine are increased: urea is increased proportionally more than creatinine? because of its reabsorption by the tubular cells, particularly at low urine flow rates. This leads to a relatively higher serum urea concentration than creatinine which is not so reabsorbed. • Metabolic acidosis: because of the inability of the kidney to excrete hydrogen ions. • Hyperkalaemia: because of the decreased glomerular filtration rate and acidosis. ارتفاع احد الاسباب السابقة مؤشر على المشاكل قبل الكلوية Post-renal factors cause decreased renal function, because the effective filtration pressure الضغط التناضحي at the glomeruli محفضة بومانis reduced due to the back pressure accused by the blockage. Caused include: • Renal stones. • Carcinoma of cervix, prostate, or occasionally bladder. If these pre- or post-renal factors are not corrected اذا لم يتم العلاج?, patients will develop intrinsic renal damage (acute tubular necrosis).  CHRONIC RENAL FAILURE: Chronic renal failure (CRF) is the progressive تدريجي irreversible destruction of kidney tissue by disease which, if not treated لارجعة فيه by dialysis or transplant, will result in the death of the patient. the aetiology of CRF encompasses the spectrum of known of kidney disease. The end result of progressive renal damage is the same no matter what the cause of the disease may have been. *The major effects of renal failure all occur? because of the loss of functioning nephrons. It is a feature of CRF that patients may have few if any symptoms until the glomerular filtration rate falls below 15 ml/min (i.e. to 10% of normal function % 11 مميزات المزمن يقلل وضيفة الكلية الى), and the disease is far مراحل متقدمة من الفشل advanced. • Erythropoietin synthesis: Erythropoietin : glycoprotein release from renal system act as to stimulate red blood cell production from boon marrow Anemia is often associated with chronic renal disease. The normochromic normocytic anemia is due primarily to failure of erythropoietin production. نوع من انواع فقر الدم يحدث عندما يكون حجمها وشكلها طبيعي لكن اعدادها قليلة Biosynthesized human erythropoietin may be used to treat the anemia of CRF. اي سبب بالكلة يسبب فقر دم MEASUREMENT OF SERUM POTASSIUM AND SODIUM The role of electrolytes is extensive, particularly that of the cations (positively charged ions) sodium and potassium which exist in the body fluids largely as free ions. Function of cations ? 1-maintaining cellular tonicity 2- fluid balance between the various cellular components, 3-they are involved in most metabolic processes, 4-maintenance of pH, 5- regulation of neural and muscular function. *Abnormal levels can be either the cause or result of a wide range of disorders. Sodium is the main extracellular cation. The plasma sodium level is a major factor in the control of water homeostasis and extracellular fluid volume. An increase in plasma sodium normally results in three compensatory mechanisms coming into 1-thirst prompts oral fluid intake. العطش الحاد 2-anti-diuretic hormone (ADH) secretion from the pituitary is increased, leading to renal water retention. 3-there is a shift of water from intracellular to extracellular. *As the total intake of sodium chloride is almost completely absorbed from the gastrointestinal tract with no active control, regulation of the retained body sodium is maintained by the kidneys, with the excess excreted in the urine and fine control carried out by tubular reabsorption. After initial glomerular filtration : *some 60% of the filtered sodium is recovered in the proximal tubules together with bicarbonate *25% is reabsorbed in the Loop of Henle of the renal tubule with chloride. *the remainder is reabsorbed in the distal tubules where, with aldosterone governing its reabsorption, it competes with potassium and hydrogen ions. Potassium is the principal intracellular cation, 98% of which is maintained within the cells by the ATP dependent mechanism known as the sodium pump. اغلب البوتاسيوم موجود داخل الخلية وينتقل الى داخل بواسطة ميكانيكية الطاقة والتي تعرف بالبوابة Here, any sodium which diffuses into cells is actively excreted in exchange for potassium. هذه الميكانيكية تعتبر تبادلية ((Insulin also accelerates the cellular uptake of potassium and elevated levels of plasma potassium encourage secretion of insulin.)) تقليل نسبة البوتاسيوم يحفز البوتاسيوم الى الدخول في الخلية In addition function ? 1- to its role in intracellular osmolality, 2-potassium is essential for many enzymatic reactions, 3-the regulation of muscles (in particular heart muscle), 4- the transmission of nerve impulses. *An important factor in the control of potassium cellular transport is the acid/base status. In acidosis the flow of hydrogen ions into cells causes the outflow of an equivalent number of في حالة الحامضية يؤدي الى خروج جزيئات البوتاسيوم من الداخل الى الخارج وبالتالي يرتفع potassium ions. Dietary potassium intake is normally in excess of requirement and the surplus is excreted via the kidneys. Following potassium ingestion or who remove potassium or hyperkilimia ? 1-aldosterone secretion is increased to enhance renal clearance and 2-insulin levels rise to increase cellular absorption. Osmolality: Number of osmoles (Osm) of solute per Kg of solvent (Osm/Kg). Osmolarity : Number of osmoles (Osm) of solute per Liter of solvent (Osm/L). Interpretation Of Sodium And Potassium Test Results: Sodium(135-145)mmol/l Hormone control on sodium =aldesteron Increases An elevated sodium level is known as hypernatraemia. It is nearly always due to dehydration with the rise in sodium (also chloride and urea) being due to a concentrating effect. It is usually brought about by a reduction of body water content by fluid loss without a compensatory reduction in sodium content, rather than a dietary overload, although excessive IV saline is a potential factor. Typical causes of hypernatraemia are: 1-Severe vomiting 2-Prolonged diarrhoea 3-Profuse sweating, fever 4-Polyuria, as in diabetes 5-Hyperaldosteroidism 6-Cushing’s syndrome 7-Inadequate water intake 8-Accidental ingestion of sea water. Note: A high sodium level must be reported as soon as possible.? Severe hypernatraemia (sodium level that has reached 155 mmol/l) is a serious finding which requires immediate correction to prevent coma and death. Decreases A low sodium level is known as hyponatraemia. It is a commoner finding than hypernatraemia. A greatly reduced level (as low as 125 mmol/l) indicates a dangerous condition and must be reported as soon as possible. A low sodium level may accompany any severe illness including viral and bacterial infections, malaria, heart attacks, heart failure, strokes, and tumours of the brain and lung. Other causes of hyponatraemia include: 1-Surgery or severe accident. 2-Treatment with diuretics. 3-Side effect of some drugs. 4-When loss of salt and water (e.g. by vomiting, diarrhea or excessive sweating) is replaced by water only. 5-Loss of sodium in the urine as in severe renal impairment and salt-losing nephritis. 6-Hypoadrenalism (Addison’s disease). In tropical countries hypoadrenalism can be caused by tuberculosis of the adrenal glands. Potassium (3.5-5.5) mmol/l Increases A raised potassium level is known as hyperkalaemia. Levels above 6.5 mmol/l are particularly dangerous and must be reported immediately? -because fatal disorders of heart rhythm can occur suddenly. Typical causes of hyperkalaemia are: 1-Excessive IV infusion, or increased ingestion of potassium. 2-Reduced renal excretion, renal failure with oliguria, anuria, acidosis. 3-Addison’s disease. 4-Hypoaldosteronaemia. 5-Leakage of cellular potassium following: acute starvation, gross haemolysis, diabetic ketoacidosis, dehydration, severe tissue injury. Falsely high potassium result: ? 1-This can occur if a blood sample is haemolyzed due to poor venepuncture technique, a 2-sample is left for a long time (e.g. overnight) without the plasma or serum being removed or 3- if whole blood is refrigerated before it is centrifuged. 4- Red cells contain a high concentration of potassium. Decreases A low potassium level is called hypokalaemia. The depletion of potassium can be masked by the topping up of the plasma levels from intracellular sources and clinical symptoms may present in the face of apparently normal values. These include weakness, tetany, polyuria and ECG changes. Causes of hypokalaemia include: 1-Inadequate intake of potassium in the diet and long term starvation. 2-Increased loss of potassium due to prolonged vomiting or diarrhoea, renal tubular failure, diuretics, hyperaldosteroidism. 3-Redistribution from plasma into cells; insulin therapy, metabolic or respiratory alkalosis. لماذا ينخفظ عند الحامضية Note: In the management of patients with salt and water depletion a simple test for urine chloride may be of value when facilities are not available for measuring serum or plasma electrolytes. NOTE: The number 1.73 m2 refer to the surface area for human body by weight 70 Kg. Cases of chemistry CASE: 1 A 56-year-old man attended the renal out-patient clinic because of polycystic kidneys, which had been dlagnosed 20 years previously. He was hypertensive and the following blood results were returned: Plasma Sodium 136 mmol/L (135-145) Potassium 6.2 mmol/L (3.5-5.0) Urea 23.7 mmol/L (2.5-7.0) Creatinine 360 umolL (70-110) Estimated glomerular filtration rate (EGFR) 14 mL/ min per 1.73 m2 Albumin-adjusted calcium 1.80 mmol/L (2.15-2.55) Phosphate 2.6 mmol/L (0.80-1.35) Bicarbonate 13 mmol/L (24-32) Answer Case 1 DISCUSSION These results are typical of a patient with chronic kidney disease (CKD) with raised plasma urea and creatinine concentrations. The patient has hyperkalaemia and a low plasma bicarbonate concentration, suggestive of a metabolic acidosis. The hypocalcemia and hyperphosphataemia are also in keeping with CKD stage 5. NOTE: The number 1.73 m2 refer to the surface area for human body by weight 70 Kg. Case 2 A 21-year-old man presented to the urology outpatient clinic because of renal calculi. There was also a family history of renal calculi. Plasma Sodium 137 mmoL (135-145) Potassium 4.2 mmol/L (3.5-5.0) Urea 5.9 mmol/L (2.5-7.0) Creatinine 108 umol/L (70-110) Estimated glomerular filtration rate (EGFR)90 mL/ min per1.73m2 Albumin-adjusted calcium 2.43 mmolL (2.15-2.55) Phosphate 1.1 mmol/L (0.80-1.35) Bicarbonate 27 mmol/L (24-32) Urate 0.33 mmol/L (0.20-0.43) Urinary excretion of both calcium and oxalate fell within the laboratory reference ranges. However, cystine was detected in the urine Answer Case 2 DISCUSSION In conjunction with the family history and relatively young age of presentation, the results are suggestive of cystinuria manifesting cystine stones. This is one of the most common amino acidurias, although a rare cause of renal calculi, and is treated by increasing fluid intake and alkalinizing the urine. Note: The types of kidney stones: 1-Cystine stone. 2-Callcum stone. 3-Uric acid stone. 4-Struvite stone. result from analyses urea to ammonia which combined with phosphate or magnesium . CASE: 3 A 5-year old girl was admitted to hospital because of a 4-day history of diarrhea and vomiting on examination she was found to be clinically dehydrated with loss of skin turgor ,her pulse was 120 beats/min and her blood pressure74/50mmHg.Her admission results were as follow: Plasma Sodium Na+=167 mmol/L (135-146) Potassium K+3.0 mmol/L (3.5-5.0) urea=19 mmol/L (2.5-7.0) Creatinine110 umol/L (70-110) Answers Case 3 DISCUSSION the patient have hypernatremia because of hypotonic fluid loss due to the diarrhea and vomiting .loss of skin turgor, tachycardia and hypotention because of hypovolaemia the patient have mild alkalosis becanse of hypernatremia. Case 4 A 53-year-old man saw his general practitioner because of bone pain and constipation. A number laboratory tests were requested, the results for the most relevant of which were as follows: Plasma Albumin-adjusted calcium 2.96 mmol/L(2.15-2.55) Phosphate 0.62 mmol/L. (0.80-1.35) Parathyroid hormone 157 ng /L (20-65) Answer Case 4 DISCUSSION The patient has hypercalcemia. Note also the hypophosphatemia and inappropriately raised PTH concentration. Due to the primary hyperparathyroidism the PTH will act on renal tubule to excrete phosphate out the body. “LIVER FUNCTION TEST” Introduction: function 1-The liver plays a major role in protein, carbohydrate and lipid homeostasis استتباب(Fig. 1). 2-The metabolic pathways of glycolysis, the Krebs cycle, amino acid synthesis and degradation, 3-the processes of oxidative Phosphorylation are all carried out in the hepatocytes which are well endowed with mitochondria. 4-The liver contains an extensive reticuloendothelial شبكي داخليsystem for the synthesis and breakdown of blood cells. 5-Liver cells metabolize, detoxify ازالة السموم and excrete فرز both endogenous and exogenous compounds. 6-Excretion of water-soluble end products from the metabolism of both nutrients and toxin, and of digestive aids such as bile acids, occur into the biliary tree. Liver Function Tests: What are usually called liver function tests (LFTs) do not assess quantitatively the capacity of that tissue to carry out any of the functions . ‘LFTs’ :are measurements of blood components which simply provide a lead to the existence, the extent and the type of liver damage. *Usually, a request for LFTs will provide results for bilirubin, the aminotransferases and alkaline phosphatase in a serum specimen. Knowledge of the serum albumin concentration may also be of some value in the investigation of liver disease. These biochemical investigations can assist تساعدin differentiatingتحديدthe following: Obstruction to the biliary tract(cholestasis) TSB ,ALP, ɣGT High Acute hepatocellular damage(the integrity of liver cells.) AST,ALT High Chronic liver disease(liver’s synthetic capacity) Albumin High ɣGT High only =liver disease ALP High only=bone disease 1-THE AMINOTRANSFERASES (AST and ALT):20) Alanine aminotransferase 111 U/L (>42) ( 250 )>Alkaline phosphatase (ALP) 826 U/L Albumin 34 g/L (35-45) ϒ- Glutamyl transferase (GGT) 764 U / L (>55) She had a positive test result for anti-mitochondrial antibodies. Subsequent studies, including liver biopsy, showed the patient to have primary biliary cirrhosis. Note the predominant cholestatic biochemical picture with raised plasma ALP and GGT activities. This condition is associated with hyperlipidaemia, hence the xanthelasma, and is more common in middle- aged women with other autoimmune disorders. There may also be raised plasma IgM concentration and osteoporosis and osteomalacia. Case: 2 A middle aged chronic alcoholic male was brought to the casualty with complaints of hematemesis. On examination he had icterus and hepatomegaly. Biochemical investigations showed the following: Serum albumin 2.5 gm % (3.5-5 g/dl) Serum bilirubin 12 mg %(0.2-1 mg/dl) Alkaline phosphatase 350 IU/L (40-125 IU/L) AST 134 IU/L (8-20 IU/L) ALT 360 IU/L (13-35 IU/L) Diagnosis :alcoholic cirrhosis The hematemesis occur as a result of digestive tract bleeding . Albumin level decteased due to the damge (cirrhosis )in liver where its synthesized . Cholestasis occur due to cirrhosis as aresult of elevated bilirubin level . The enzymes (ALP,ALT,AST )elevated also due to liver damage 1. Kidney function: Glomerular and tubular function evolve to mature levels between 12-18 months, يكون التطور من سنة الى سنة ونص but the glomerular functions(GFR) develops more rapidly than tubular(reabsorbtion), عملية الطرح اكثر تطور من عملية اعادة الامتصاص No new nephrons are made during childhood but the existing ones grow in size and mature in function. نفس العدد للكبار والصغار The kidney regulates homeostasis by two fundamental functions, the glomerular and the tubular. In neonates both functions are deficient, they are severely limited in their response to stress. Glomerular filtration rate (GFR) increase within the first month of life, and the velocity of this increase is lower in preterm neonates طفل خدج, اطفال الخدج اقل ترشيح من الاطفال الطبيعيين It increases after birth and reaches approximately 20 ml/min/1.73 m2 at 1 month of age in term and preterm neonates. Immature tubular function, neonates responsible for reduced concentrating capacity, negative sodium equilibrium, reduced bicarbonate levels, thus, prone to dehydration and hyponatremia, water, electrolytes and acid-base balance. 2. Water: During fetal life water is abundant and is exchanged freely between mother and fetus without any concentrative mechanisms. عملية تبادل الماء بين الام والطفل تكون حرة At birth the total body water (TBW) accounts for 75% of the weight of the newborn, most of which is extracellular fluid (ECF). حجم الماء يمثل 57 من حجم الطفل Within days the total amount of water starts to decrease(ECF)? ((physiological weight loss)) 1- due to very little fluid intake and 2-the increasing GFR and3- at the same time a shift of fluids between compartments commences. The ECF space contracts and water inter the cells. These changes result in the so called ((physiological weight loss)) of 5-10% of birth weight which that occurs within the first week of life. After this period the kidneys concentrating capacity increases and water loss minimized. 3. Sodium Management:(NA neonatal low –adult high) Sodium is essential for growth and a positive sodium balance that is a necessary for adequate growth and development. Cases naterimia in neonatal ? * The increased water loss right after birth is accompanied by sodium loss, which is more prominent in premature neonates اطفال الخدج. Fractional excretion of sodium (FENa) immediately after birth can be as high as 5% compared with 1% in the adult, but it falls within days as the mechanisms for concentration and saving sodium develop quickly to compensate for the very low concentration in salt of the human milk, at least the development of renal function. 4. Hyperkalemia:( k neonatal high –adult low) Normal serum and plasma potassium concentrations in children and adolescents are similar to levels in adults. However, infants have a higher normal range of potassium ? -because of their reduced urinary potassium excretion caused by their relatively increased aldosterone insensitivity and decreases glomerular filtration rate, Thus , potassium values of 6 mEq/L or even 6.2 mEq/L in premature babies are considered normal in early infancy. 5. Phosphorous:( p neonatal high ?–adult low) Is an essential element not only for growth but also for metabolism and its mechanisms of *reabsorption are well developed at birth and work more efficiently than they do in adult life. Phosphate values are higher during neonatal period and infancy, especially in breast-fed infants. 6. Acid-base balance and other substances: The regulation of acid-base homeostasis is achieved through buffer systems and appropriate respiratory and renal adaptions. At birth, respiratory adaptive responses are adequate and work immediately in a spontaneously breathing and neurologically intact neonate. The renal compensatory mechanisms are slower and limited due to low neonatal GFR and the not yet developed tubular transport systems of bicarbonate and hydrogen ions. Thus neonate are in a physiological acidotic state, with healthy term newborns to have bicarbonate levels of 18-20 mEq/L compared with 24-26 mEq/L in the adult, a level which is reached at about لان عملية الترشيح قليلة وعملية التطور غير كافية في امتصاص الهايدوجين وغيره 1 year of age. Premature infants may have bicarbonate levels as low as 14 mEq/L. 7. The blood urea:( neonatal low –adult high?) *Is low in newborn infants compared with that adult despite relatively low GFR. ? *The high anabolic rate results in more nitrogen being incorporated into protein rather than into urea. 8. Serum creatinine:( at birth high – low after two weeks) Is a better index of renal function in infancy and childhood . Its concentration is highest at birth, reflecting maternal creatinine levels. During the first two weeks of the life serum creatinine decreases rapidly to reach the stable neonatal levels of 0.5 mg/dl and continues to drop, but at a low rate, in the following month to be stabilized at 0.3-0.4 mg/dl during infancy. In premature neonates creatinine levels increase transiently with the peak at day 4, and a more progressive decline towards normal neonatale levels at about 4 weeks of age. Although creatinine concentration reflects muscle mass and would be expected to rise with age, during the first 2 years of age little change occurs. *This is because of the dramatic rise of GFR and therefore creatinine clearance during the first 23 years of age. Beyond that time increase in muscle mass is reflected in creatinine concentration. Premature infant Term infant 1st week 1st week 2nd week 8 weeks 1-2 years Daily excretion of urine (ml/kg/24h) 15-75 20-75 25-120 80-130 40-100 GFR (mL/min/1.73m2) 10-15 15-20 35-45 60-75 90-110 Serum creatinine (mg/dl) 0.9 0.7 0.5 0.3-0.4 0.3-0.4 Max urine Osmolality (mOsm/kg H2O) 400-500 500-600 700-800 1000-1200 1200-1400 9. Hypoglycemia :( at birth low –high after two or three days) Hypoglycemia can occur in the newborn, particularly in preterm infants also called neonatal hypoglycemia: It refers to low blood sugar (glucose) in the first few days after birth. Babies need blood sugar (glucose) for energy. Most of that glucose is used by the brain. The baby gets glucose from the mother through the placenta before birth. After birth, the baby get glucose from the mother through her milk of from formula, and the baby also produces it in the liver. * Insulin helps his body to store sugar (blood glucose) and release it when he need it. When everything is working well, your baby’s hormones keep his blood sugar levels balanced. When the balance is out, hypoglycemia can happen. Glucose level can drop if: When babies are just 1 hour to 2 hours old, the normal level is just under 2 mmol/L, but it will rise to adult levels (over 3 mmol/L) within two to three days. In babies who need treatment for low blood glucose or are at risk for low blood glucose, a level over 2.5 mmol/L is preferred. Causes of Hypoglycemia: 1-There is too much insulin in the blood after birth if the mother have diabetes. Insulin is a hormone that pulls glucose from the blood. 2-In adequate glycogen stores and an inability to perform gluconeogenesis, especially preterm. 3-Baby born under significant stress used their glycogen stores during intrapartum asphyxia. ( the baby may be cold, or might cry too much after birth). 4-The baby is not able to feed enough to keep the glucose level up. 10. Phenylketonuria (PKU): PKU is an inherited condition caused by a defect in the PAH gene. The PAH gene helps create phenylalanine hydroxylase, this enzyme responsible for convert phenylalanine into tyrosine, which your body needs to create neurotransmitters such as epinephrine, norepinephrine and dopamine. When this enzyme is missing, your body can not break down phenylalanine. This causes a buildup of phenylalanine in your body. A dangerous buildup of phenylalanine can occur when someone eats high-protein foods, such as eggs and meat. Early diagnoses and treatment after birth can help relieve symptoms of PKU and prevent brain damage. Case 1 A 4_year old boy was seen in the paediatric out-patient department because of hepatomegaly, metabolic acidosis and growth retardation some of his abnormal fasting blood results were as follows: Plasma (fasting) Glucose 2.0mmol/L (3.0-5.5) Urate 0.61mmol/L (0.20-0.43) Lactic acid 3.7mmol/L (0.5-1.5) Cholesterol 5.4mmol/L (3.0-5.0) Triglycerides 6.7mmol/L (0.5-1.5) Discussion The child has hyperlactataemia , hypoglycaemia , hyperuricaemia and hyperlipidaemia .he was later found to have von gierkes disease (or type I glycogen storage disease) do to glucose-6-phosphatase deficiency . this enzyme deficiency lead to abnormalities of glycolysis and gluconeogenesis ,resulting in the hypoglycaemia and lactic acidosis .the raised plasma lactic acid concentration may interfere with uric acid renal excretion, leading to hyperuricaemia Quality Management Q/What do you mean TQM (Defined): provides both a management philosophy for organizational development and a management process for improving the quality of all aspects of work. ادارة فسلجية لتحسين العمل من عدة نواحي quality is defined as :conformance with the requirements of users or customers. More directly, quality refers to satisfaction of the needs and expectations of users or customers. Who can improvement Quality? Quality improvement occurs when problems are eliminated permanently. Industrial experience الخبرات الصناعية has shown that 85% of all problems are process problems that are solvable only by managers; المدير مسؤول عن تصحيح الاخطاء the remaining 15% are problems that require the action and improvement in performance of individual workers. What are the activities of Quality assessment? *Quality assessment QA, as currently applied, is primarily concerned with broader measures and monitors of laboratory performance, such as (1) turnaround time, (2) specimen identification, (3) patient identification, (4) test utility. What are the Errors Made in the Clinical Laboratory? Incidents included those in which: (1) physicians orders for laboratory tests were missed or incorrectly Interpreted. (2) patients were not properly prepared for testing or were incorrectly identified. (3) specimens were collected in the wrong containers or were mislabeled or mishandled. (4) the analysis was incorrect. (5) data were entered improperly. (6) results were delayed, not available, or incomplete, or they conflicted with clinical expectations. Q/Compare between quality control and Quality assurance? quality control Quality assurance is often used to represent those techniques and procedures that monitor performance parameters. is used here to represent practices that are generally recommended for ensuring that desired quality goals are achieved Generally, these are quantitative techniques that monitor particular sources of errors, estimate the magnitude of the errors, and alert laboratory personnel when indications suggest that quality has deteriorated. It is a broad spectrum of plans, policies, and procedures that together provide an administrative structure for a laboratory’s efforts to achieve quality goals. What are the causes of falling quality goals in lab ? A quality assurance program involves virtually everything and everybody in the clinical laboratory. An error in any one step during the (1) acquisition, (2) processing, (3) analysis of a specimen, and (4) reporting of a laboratory test result affects the quality of the analysis and causes the laboratory to fall short of its quality goals. Q/What are Facilities and Resources to TQM? التسهيلات Laboratories must have the administrative support necessary to provide the quality of services desired. This means having : (1) adequate space, (2) equipment, (3) materials, (4) supplies, (5) staffing, (6) supervision, (7) budgetary resources. These resources provide the basis upon which quality services are developed and maintained. Q/Who are a chief quality improvement throat technical Procedures? Technical procedures necessary for laboratory services include the following: 1. Control of preanalytical conditions or variables, 2. Control of analytical variables, 3. Monitoring of analytical quality through the use of statistical methods and control charts. 4. Control of postanalytical conditions or variables. 1-Control Of Preanalytical Variables Q/what are potential error between the time of the physiciain initial request and final interpretation resalt ?مهم جدا Process Potential error Test ordering Cost or delayed order Wrong patient identification Specimen acquisition Incorrect tube or container Incorrect patient identification Analytical measurement Specimen mixup Incorrect volume of specimen Test reporting Report delayed Wrong patient identification Test interpretation Specificity of test not understood Analytic sensitivity not appropriate Types of Preanalytical Variables Turnaround Time تقليل الوقت major problems for laboratories. ? Delayed and lost test requisitions, specimens, and reports have been What do you mean flag “out-of-range” specimens.? مهم-Lists of delayed specimens also provide a powerful mechanism for detecting lost specimens or reports. -Centrifuge Performance Who can calipration of ? by checking the speed, timer, and temperature. Control Of Analytical Variables Reliable analytical methods are obtained by a careful process of selection, evaluation, implementation, maintenance, and control. Certain variables: 1-water quality, 2-calibration of analytical balances, 3-calibration of volumetric glassware and pipettes, 4-stability of electrical power, and 5-the temperature of heating baths, refrigerators, freezers, and 6-centrifuges—should be monitored on a laboratory-wide basis because they will affect many of the methods used in the laboratory. In addition, certain variables will relate more directly to individual analytical methods, and these require that procedures be developed to deal specifically with the characteristics of those methods. • Choice of Analytical Method • Reference Materials and Methods Role of International Organization for Standardization (ISO) ISO is a worldwide federation of national standards bodies from more than 150 countries accessed March 22, 2011. The work of the ISO results in international agreements, which are published as international standards. Control Materials Specimens that are analyzed for QC purposes are called control materials. what are required for control materials.? (1) stable, (2) available in aliquots or vials, and (3) amenable to analysis periodically over a long time. *The control material preferably should have the same matrix as the test specimens of interest. General Principles of Control Charts The most common method of comparing the values observed for control materials with their known values is the use of control charts. لمقارنة اكثر من قيمة للمواد المرجعية *Control charts are simple graphical displays in which the observed values are plotted versus the time when the observations were made. External Quality Assessment And Proficiency Testing Programs internal quality control external quality assessment. procedures described earlier in the chapter have focused on monitoring a single laboratory. procedures used to compare the performance of different laboratories for the daily monitoring of the precision and accuracy of the analytical method for maintaining long-term accuracy of the analytical methods The two are complementary activities, Quality Assurance in the biochemistry laboratory is intended to ensure the reliability of the laboratory tests. The objective of quality assurance is to achieve reliable test results by • Accuracy • Precision Accuracy Precision This refers to the closeness of the estimated value to that considered to be true. Accuracy can, as a rule, be checked only by the use of reference materials which have been assayed by reference methods. لدقة النتائج This refers to the responsibility of the result, but a test can be precise without being accurate. Precision can be controlled by replicate tests and by repeated tests on previously measured specimens. And the test result or value which we get should be closer to the previous one. DIAGNO STIC ACCURACY OF TESTS The extent of agreement of test results with accurate patient diagnosis is represented in several ways, including (1) sensitivity and specificity, (2) predictive values, (3) receiver operating characteristic (ROC) curves, and (4) likelihood ratios. Sensitivity and Specificity The sensitivity specificity of a test reflects the fraction of those with a specified disease that the test correctly predicts. The is the fraction of those without the disease that the test correctly predicts. high sensitivity (few FN) high specificity (few FP) Table 3-1 shows the classification of unaffected and diseased individuals by test result. True positives (TP) are those diseased individuals who are correctly classified by the test. False positives (FP) are nondiseased individuals misclassified by the test. False negatives (FN) are those diseased patients misclassified by the test. True negatives (TN) are nondiseased patients correctly classified by the test.