University of Baghdad / College of Pharmacy 

Clinical Laboratory Science Department 

Clinical Biochemistry Manual 

Fifth Stage 

2009-2010
INTRODUCTION

Laboratory and diagnostic tests are tools to gain additional information about the patient. By themselves, these tests are not therapeutic, however, when used in conjunction with patients history and physical examination, these tests may confirm a diagnosis provide valuable information about a patients status and response to therapy that may not be apparent from the history and physical examination alone. 
Generally, several approaches had been considered to request specific laboratory  tests such as:
1-	Basic screening.
2-	Establishing (initial) diagnosis.
3-	Different diagnosis.
4-	Evaluation of current medical case management and outcomes.
5-	Evaluating diseases severity.
6-	Monitoring course of illness and response to treatment.
7-	Group and panel testing.
8-	Testing related to specific events, certain signs and symptoms, or other exceptional situations. e.g. sexual assault, pheochromocytoma, drug screening .
These investigations could involve blood, urine, stool, x-ray, nuclear scanning, ultrasound and endoscopy.

Diagnostic Test Basics:

Some important factors that could increase a patient’s risk for complications (during sampling) that may affect testing outcomes should be considered , such as :
1.	Age > 70 years
2.	History of illness.
3.	History of allergies (e.g. latex, contrast iodine, radio pharmaceuticals and other medications).
4.	Infection or increased risk for infection e.g. human immunodeficiency virus (HIV) , organ transplant chemotherapy, radiation therapy.
5.	Aggressive or antisocial behavior
6.	Uncontrolled pain
7.	Neuromuscular conditions
8.	Weakness, fatigability
9.	Parasthesias
10. Sever visual problems
11. Hearing impairment
12.Use of diuretics, sedatives, analgesics
13.Alcohol or illegal drug use or addiction.

The diagnostic testing process is passing through three major phases:
I – Pre- test Phase:
This phase emphasis of pretest care including appropriate test selection, proper patient preparation, individualized patient education and emotional support.
II - Intra test Phase:
This phase include the performance of procedure, collection and transport of specimens, observation of standard ,precautions, documenting and keeping proper records.

III- Post- test phase:
Include interpretation of test results and informing the patient, identification of critical value, and following infection control guidelines.

History and Assessment:
Obtain a relevant, current health history , perform a physical assessment if indicated. Identify conditions that could influence the acute testing process or test out comes (e.g. pregnancy, diabetes mellitus, cultural diversity, physical impairment, altered mental state).
Record all drugs the patient is taking because these may influence test outcomes.

Methodology of Testing:
1-	Ensure that specimens are correctly obtained, preserved, handled, labeled and delivered to the appropriate department. Follow testing producer accurately. e.g., it is not generally acceptable to draw blood samples when an intravenous line is infusing proximal to the intended puncture site.
2-	Observe precautions for patients in isolation.
3-	As much as possible, coordinate patient activities with testing schedules to avoid conflicts with meal times , administration of medications, treatments, or other diagnostic tests.

Interfering Factors:
1- Sampling Errors:
Minimize test outcome deviations by following proper test protocols.
-	Make certain the patient know what is expected of them. Written instructions are very helpful.
-	Deviations may include the following:
-	Incorrect specimen collection, handling, storage, or labeling.
-	Wrong preservation or lack of preservative.
-	Delayed specimen delivery.
-	Incorrect or incomplete patient preparation.
-	Hemolyzed blood samples
-	Old or deteriorating specimens.
2- Patient Factors:
Patient factors that can alter test results may include the following:
	Incorrect pretest diet.
	Current drug therapy.
	Patient’s illness
	Dehydration
	Position or activity at time of specimen collection.
	Postprandial status (i.e, time patient last  ate).
	Time of day
	Pregnancy
	Age and gender
	Level of patient knowledge and understanding of testing.
	Stress.
	Noncompliance with instructions and pretest preparation.
	Alcohol use.

     Patient posture is important when plasma volume is measured because this value is 12%-15% greater in a person who has been supine for several hours. Changing from a supine to standing position can alter values as follows:
Increased hemoglobin (Hb), red blood cell count (RBC) , hematocrit (Hct), calcium (Ca), potassium (K), phosphorus (P), asparate aminotransferase (AST), phosphatases (ALP&ACP), total protein (T.ptn), albumin (Alb.), cholesterol (chol.) and triglyceride (TG).
     Going from an upright to a supine position results in decreased Hct, Ca, T.ptn and chol.
     Tourniquet applied for > 1 minute produces laboratory value increase in protein 5%, iron (6-7%), AST (9.3%), and cholesterol 5% and decreases in K+ (6%) and creatinine (2-3%)

Test results:

Knowledge of normal or reference values is vital. Theoretically (normal) can refer to the ideal health state, to average references values .The majority of normal blood test values are determined by measuring ˝Fasting˝ specimens.
Laboratory Reports:
Scientific publications and many professional organizations are cLinging clinical laboratory data values from conventional units (mg/dl) to system international (SI) units. The SI concentrations are written as amount per volume (moles or millimoles per liter) rather than as mass per volume (grams or milliequivalents per deciliter (100 milliliters), or liter).

Collecting And Transporting Specimens
Biological specimens that are utilized for laboratory investigations include: blood (whole, plasma, serum), urine, stool, sputum, biological fluids (peritoneal, synovial, cerebrospinal, amniotic (aminiocentesis), hair, skin, tissue, semen) and bone marrow.
Initially you should determine specimen type needed and method of sample handling. Examples may include urine collection, venipuncture, arterial puncture, or bone marrow aspirate. Additionally, special requirement and supplies may be necessary (e.g. sterile containers, special kits).
Some specimens collection requires a clinician to perform the entire collection e.g. CSF specimen.Time of collection is also important, e.g. results from a fasting blood glucose test versus results from a 2-hr postprandial blood glucose test have a entirely different significance from each other as a diagnostic parameter.

Blood Specimen Collection:
	Patient preparation by considering whether fasting is required [e.g. fasting blood sugar (EBS), cholesterol (Chol.), triglycerides (TG)], or withholding medications until blood is drawn, otherwise indicate any drug that the patient is taking.
	Record the time of day that the blood is drawn (some analytes such as cortisol vary according to diurnal pattern.
	Note the patient position for certain tests (e.g. renin level is affected by body position).
	Collect the blood in the proper color-coded test tube.

Blood Collection Tubes :
Blood collection tubes have color-coded stoppers to indicate the presence or absence of different types of additives (preservatives and anticoagulants). A preservative prevents change in the specimen and an anticoagulant inhibits clot formation or coagulation. 
1-	White-top (‘plane,) tubes contain no additive, usually to obtain serum from a blood sample.
2-	Lavender-top tubes contain ethylenediaminetetraacetic acid -K3 (EDTA-K-3) used for hematological studies.
3-	Yellow-top tubes contain Sod. Fluoride for blood glucose measurement because fluoride inhibits blood glucose conversion to lactic acid.
4-	Blood culture tubes (sterile tubes to maintain sterility).
5-	Green-top tubes these contain heparin used for hematological tests.

Important Guidelines:
To obtain valid results do not fasten the tourniquet for longer than 1 min. (prolonged tourniquet application can cause stasis and hemoconcentration).
Do not shack blood specimen, hemolysis may result from vigorous shaking and invalidate test results.
	Blood specimens could be obtained by venipuncture, arterial puncture or skin punctures. Skin punctures can be used for blood tests on capillary blood. Common puncture sites include the finger tips earlobes and heel surfaces.
	Ensure that the blood tubes are correctly labeled and delivered to the laboratory.
After the specimen is drawn, apply pressure or a pressure dressing to the vein puncture site. Assess the site for bleeding.
If the patient fasted before the blood test, reinstitute appropriate diet.

Veni puncture:

Venipuncture allows collection of larger quantities of blood for testing usually, the antecubital veins the veins of choice because of ease of access.

1-	Place the arm in a fully extended position with palmar surface facing upward (for antecubital access).

2-Position and tighten tourniquet on the upper arm to produce venous congestion.
3-	Ask the patient to close the fist in the designated arm. Select an accessible vein.
4-	Cleanse the puncture site and dry it properly with sterile gauze. Using povidone-iodine which must dry thoroughly.
5- Lincttire the vein Lising a syringe. Usually, for an adult, anything smaller than a number 21-gauge needle might make blood withdrawal somewhat more difficult.
6-	Once the vein has been entered by the collecting needle, blood will fill the attached syringe automatically because of negative pressure within the collection syringe.
7-	Remove the tourniquet before removing the needle from the puncture site otherwise bursing will occur.
8-	Remove needle. Apply pressure and sterile dressing strip to site.
9-	The preservative or anticoagulant added to the collection tube depends on the test ordered.

URINE SPECIMENS
Urine tests are easy to obtain and provide valuable information about many body system functions such as kidney function , glucose metabolism and various hormone levels.
Guide lines:
Use the first morning specimen for routine urinalysis because it is more concentrated. To collect a first morning specimen, have the patient void before going to bed and collect the first urine specimen immediately on rising.
-	Random urine specimens can be collected at any time. They are usually obtained during day time hours and without any prior patient preparation.
-	If a culture and sensitivity ( C & S ) study is required or if the specimen is likely to be contaminated by vaginal discharge or bleeding , collect a clean-catch or mid stream specimen. After capping the container, allow the patient to finish voiding.
-	One- time composite urine specimens are collected over a period that may range anywhere from 2-24 hrs.  to collect a timed specimen, instruct the patient to void and discard the first specimen. This is noted as the (start time) of test. Instruct the patient to save all subsequent urine in a special container for the designated period of time. A preservative is usually used in the collection container.
- For 24 hrs urine specimens should hold 3 to 4 L of urine and have tight-fitting lids. They should be labeled with patient’s name, the starting collection date and time ,the ending collection data and time , the name of test , the preservative and storage requirement during collection (mostly by keeping specimen in ice or under refrigeration)
- Indicate on the laboratory slip any medications that may affect test results.

STOOL SPECIMENS:
The examination of feaces provides important information that aids in the diagnosis of various gastrointestinal disorders. Fecal tests may also include microbiologic, chemical and parasitic examinations.

Guidelines
- Collect stool specimens in a clean container that has a fitted cover.
- Do not mix urine and toilet paper with the stool specimen.
-Fecal analysis for occult blood, white blood cells, or qualitative fecal fat requires only a small amount of a randomly collected specimen.
-Some fecal collections require dietary restrictions before the collection (e,g tests for occult blood).
-Consider various factors, medications taken by the patient (e.g. tetracyclines and antiadiarrheal preparations ) affect the detection of intestinal parasites.
-Correctly label and deliver stool specimens to the lab. within 30 minutes after collection, otherwise , the specimen should be refrigerated for up to 2hr.

Anticoagulants And Preservatives For Blood Specimens
Additive 	Concentration	Disadvantages	
Heparin 
as Sod. Pot.,Lethium salts	20U/ml Blood
0.2 mg/ml Blood	High coast,temporary action,
Interfere with acid phosphatase activity,Affect binding of T35T4to their carrier protein .	
EDTA
di Na.diK,or Tetra K Salt	1-2 mg/ml Blood	
Causes hypertonically shrinking of RBC 	
Sodium Flouride 
A preservative for blood glucose	2 mg/ml Blood	
Inhibit enzymes of glycolysis	
Citrate 
Sod, Citrate Salt	3.4-3.8 g/dl
or 1:9 parts of blood	Inhibit aminotransrerases and alkaline phosphatase,increase phosphates estimation because of it’s complexing  with molybdate,	
Oxalate
Sod.Pot.Ammonium and Lethium Salts	1-2 mg/dl Blood	
Complexing with Ca ions	

BIOCHEMICAL TESTS
    Biochemical studies identifies many chemical constituents mainly in blood. It is often necessary to measure several blood chemicals to establish a pattern of abnormalities. Selected tests serve as screening devices to identify target organ damage. 

GENERAL BIOCHEMICAL PROFILES

Profiles are group of selected tests that screen for certain conditions. Some of the more common profiles are listed below:

	       Test suggested	
Basic Metabolic Screen:	Chloride, sodium, potassium, carbon dioxide, glucose. Blood urea nitrogen (BUN), creatinine.	
Diabetes Mellitus Diagnosis:	Fasting blood glucose, postprandial blood glucose, glucose tolerance test
	
Kidney Functions:	BUN, phosphorus, LDH, creatinine, creatinine clearance, uric acid, total protein, A/G ratio (Albumine/Globulin ratio), albumin, globulins, calcium, glucose, cholesterol.	
Liver Function:	Total bilirubin, alkaline phosphatase, cholesterol, gamaglutamyl transferase (GGT), AST, ALT, total protein, A/G ratio LDH, viral hepatitis panel, PT.	
Lipids:	Cholesterol, triglycerides, HDL. LDL, lipoprotein electrophoresis	
Cardiac Enzymes:	CPK, AST, LDH.	

BLOOD GLUCOSE

Fasting Blood Glucose ( FBG), Fasting Blood Sugar ( FBS).

Normal values:

Adult:3.6 —6.1 mmol/l (65-110 mg/dl)

Child (6-18 yr.): 3.9-6.0 mmol /l ( 70-108 mg/dI)

Critical Values:

Adult:	<2.8 and > 22 mmol/l (<50 and > 400 mg /dl)

Newborn : < 1 .66 and > 16.7 mmol/l (<30 and> 300 mg /dl)

     During carbohydrate metabolism glycogen is formed, the conversion of glycogen to glucose by the liver is controlled by two hormones that directly regulate blood glucose are glucagon and insulin. Glucagon accelerates glycogen breakdown in the liver and causes blood glucose level to rise. Insulin increase cell membrane permeability to glucose, transports glucose into cells (for metabolism), stimulates glycogen, formation, and reduces blood glucose levels. As cellular glucose metabolism occurs blood glucose levels fall. Adrenocorticotrophic hormone (ACTH), adrenocorticosteroids, epinephrine and thyroxine also play key roles in glucose metabolism.
Serum glucose levels must be evaluated according to the time of day they are preformed. eg, a glucose level of 135 mg/dl may be abnormal if the patient is in the fasting state, but this level would be within normal limits if the patient had eaten a meal within the last hour.
Fasting blood glucose is a vital component of diabetes management. Abnormal glucose metabolism may be caused by inability of pancreatic islet β-cells to produce insulin, reduced numbers of insulin receptors, glucose absorption, inability of the liver to metabolize glycogen or altered level of hormones that play a role in glucose metabolism.
In most cases, significantly elevated fasting blood sugar levels 7.0mmol/L (> 125 mg/dl) , hyperglycemia are usually diagnostic for diabetes mellitus. However , mild, borderline cases may present with normal fasting glucose value. If diabetes is suspected a postprandial or  oral glucose tolerance test or glycosylated hemoglubin (Hb A1C) can confirm the diagnosis.

Specimen:
-  5 ml venous blood sample is drawn from an overnight fasting person. In known cases of diabetes blood drawing should precede insulin or oral hypoglycemic administration. Serum could be used.
- Glucose levels can be also evaluated by performing a finger stick and using either visually read or a reflectance meter eg, glucotestmeter.
Abnormal Findings:
Increased levels 		Decreased levels  
(hyperglycemia)		(hypoglycemia)
	Diabetes mellitus		Insulinoma
	Acute stress response		Hypothyroidism
	Cushing’s syndrome		Hypopituitrasim
  Pheochromocytoma		Addison’s disease
	Chronic renal failure		Extensive liver disease
	Glucagonoma		Insulin overdose
	Acute pancreatitis		Starvation
	Diuretic therapy
	Corticosteroid therapy
	Acromegally

PLASMA GLUCOSE ESTIMATION

Principle:
Glucose is oxidized by glucose oxidase to gluconate and hydrogen peroxide according to the following equation :-
                 
                                  GOD
Glucose + 02+H20	             H202 + Gluconate
                                                           Peroxidase
2H202 + Phenol + 4 Amino-antipyrine          	 H20 + Quinonimine

Samples:

    Serum ( not hemolyzed)

Plasma collected on heparin fluoride or heparin iodoacetate (not hemolyzed)

Spinal fluid.

Procedure:
	Blank	Standard	Sample
Standard	  _	  10 µl                _
Sample	  _	    _	 10 µl 
Working reagent	1 ml	  1 ml	 1 ml

Mix. incubate 10 min. at 37 0C or 30 min. at room temperature (20 0C- 25 0C )
Read at 505 nm.The colour is stable for 30 min.

Calculations:

                   O.D. Sample
Glucose Conc.=	                      x n
                  O.D. Standard

mg/dl: n = 100

g/l: n = 1

mmol/l : n = 5.56

Glucose Post Prandial (2-Hour Post Parandial Glucose) (2h PPBG),

[1-Hour Glucose Screen For Gestational Diabetes Mellitus ]

(O’Sullivan Test)

Normal Values:

2-hppG

1-50 yrs: <7.8 mmol /l (< 140 mg /dl)

50-60 yrs : <8.3- mmol/l (<150 mg /dl)

60 years and older : <8.9 mmol I l (<160 mg /dl)

1 hour glucose screen for GD : <7.8 m mol/l (<140 mg/dl)

     A post prandial test is performed 2 hours after a meal. Normally, insulin is secreted immediately after a meal in response to the elevated blood glucose level causing the level to return to the pre-meal range within 2 hours. In patients with diabetes, the glucose level usually is still elevated 2 hours after the meal. The PPG is an easily performed screening test for diabetes mellitus. If the results are (> 140 mg/dl and < 200 mg/dl), a glucose tolerance test may be performed to confirm the diagnosis. If the 2-hr PPG is (11 mmol/l > 200 mg/dl), diagnosis of diabetes mellitus is confirmed.
    The 1- hour glucose screen is used to detect gestational diabetes mellitus (GDM). GDM affects 3% to 8% of pregnant women with up to half of these women developing overt diabetes later in life. The detection and treatment of GDM may reduce the risk for several adverse prenatal outcomes (such as excessive fetal growth and birth trauma, fetal death or neonatal morbidity). Screening for GDM is performed with a 50-g oral glucose followed by a glucose level determination 1 hour later. Screening is done between 24 and 28 weeks of gestation. Patients whose serum glucose level equals or exceeds 7.8 mmol/l (140 mg/dl) should be evaluated by glucose tolerance  (GTT).

Interfering Factors:

-	-Stress can increase blood glucose level.
-Vomiting, gum chewing and snacking during the 2- hour test interval invalidates the test result.

Patient preparation for 2hr-PPG:
1-The patient should be on a high carbohydrate diet (at least 75 gm) 2-3 days before testing.
2-The patient should eat a high - carbohydrate meal, the test is timed from the beginning of the meal.
3-The patient should rest during the 2-hour interval. Smoking is not permitted during this time. Smoking may increase glucose levels.

	Abnormal Findings: 
Increased levels▲		 Decreased levels▼
	Diabetes mellitus	 Insulinoma
	Gestational diabetes mellitus	 Hypothyroidism
	Malnutrition                       Hypopituitaism
	Hyperthyroidism	 Addison’s disease
	Acute stress response	 Malabsorption or maldigestion
	Cushining’s syndrom
	Pheochromocytoma
	Chronic renal failure
	Glucagonoma
	Diuretic therapy
	Corticosteroid therapy
	Acromegaly
	Extensive liver disease

ORAL GLUCOSE TOLERANCE TEST (OGTT)

Normal Values:

*Fasting:
Adult:	3.9-6.1 mmol/l or 70-110 mg/dL

Child : <7.2 mmol/l or < 130 mg /dl

*30 min.:
Adult:	6.1-9.4 mmol/l or 110- 170 mg/dl.

*60 min. (1 hour): 
Adult:  6.7-9.4 mmol /l or 120-170 mg/dl
Child : <7.8 mmol/l or < 140 mg /dl

*120 min (2 hours):
   Adult:	3.9-6.7 mmol/l or 70-120 mgldl

   Child:	<7.8 mmol /l or < 140 mg /dl

*3 hours:
   Adult:	3.9-6.7 mmol/l or 70-120 mg/dl

Note: All four blood values must be within normal limits to be considered normal GGT.

Specimen:

-Collect fasting blood and urine specimens.
-Collect approximately 5 ml of venous blood at 30 mm. and at hourly periods.
-Collect urine specimens at hourly periods.
-Mark the tubes with the time that the specimens are collected.

Explanation of GTT:
If fasting and postprandial glucose test results are borderline, the GTT can support or rule out a diagnosis of diabetes mellitus , it can also be part of workup for un explained hypertriglyceridemia, neuropathy, renal diseases, or retinopathy. This test may be ordered when there is glucose in urine or when the fasting blood glucose level or 2-hour postprandial blood sugar level is significantly elevated. GTT is also suggested for the following conditions:
-	Patients with a family history of diabetes.
-	Patient who are massively obese.
-	Patients with a history of recurrent infections.
-	Patient with delayed healing of wounds (especially on the lower legs or feet).
-	Women who have a history of delivering large babies.
-	Patients who have transient glucosuria or hyperglycemia during pregnancy or following myocardial infarction, surgery, or stress.

Glucose Tolerance Test Curve For A Diabetic And Prediabetic Patient.

Potential Complications:

A.	  Dizziness, tremors, anxiety, sweating, fainting during testing.
B.  If these symptoms occur, a blood specimen is obtained, if the glucose levels too high, stop the test and start the administration of insulin. 
C.  Patients with concurrent infections or endocrine disorders, because glucose intolerance will be observed even though these patients may not have diabetes.

Interfering Factors:

1-	1-Smoking during test stimulates glucose production because of nicotine.
2-Stress (eg, from surgery , infection) can increase glucose levels.
3-Exercise during the rest can affect glucose levels.
4-Reduced caloric intake before GTT can cause glucose intolerance.
5-Drugs that may cause glucose intolerance include antihypertensive, antiintlamrnatory drugs; aspirin , β- blockers, furosemide, nicotine, oral contraceptives, psychiatric drugs, steroids and thiazide diuretics.
6-If the patient vomit the glucose solution ,the test is declared invalid it can be repeated in 3 days.

Patients Preparation for GTT

1-Patient should have a high carbohydrates diet (150 gm) for 3 days preceding the test. Instruct the patient to abstain from alcohol.
2-Patient should fast at least 12 hours but not more than 16 hours before the test. Only water may be ingested during the fasting time and test time. (smoking is not permitted during testing).
3-Patient should rest or walk quietly during the test period. They may feel weak, faint or nauseated during the test. Vigorous exercise should be avoided during testing.

4-Collect blood specimens at the prescribed times (at 30 mm. intervals) and record exact times collected and urine specimens at hourly periods.
5-Record the patient’s weight.
6-This is a timed test. A 2-hour test done for detecting diabetes in individuals other than pregnant women, the 3-hour test done for pregnant women and the 5-hour test evaluates possible hypoglycemia.
7-For IV-GTT administer glucose load intravenously over 3-4 minutes.

Glucose Load:
a)	Pediatric doses of glucose are based on body weight calculate as 1.75 gm/kg of body weight up to 75 gm.
b)	Pregnant women:100 gm glucose.
c)	Non- pregnant adult: 75 gm glucose.

Abnormal Findings:
	 Diabetes mellitus
 Acute stress response
 Cushing’s syndrome
 Pheochromocytoma
 Chronic renal failure
 Glucagonoma
Acute pancreatitis
 Corticosteroid therapy
 Acromegaly
 Myoxedema
 Somogyi response to hypoglycemia
 Post gastrectomy
 Diuretic therapy

BLOOD UREA
Blood Urea Nitrogen (BUN), Serum Urea Nitrogen (SUN)

Normal Values:

Adult:2.5-6.4 mmol/l or 7-18 mg/dl

Elderly (>60 yr.) 2.9- 7.5 mmol/l or 8- 20 mg/dI

Child :1.8-6.4 mmol/l or 5-18 mg/ dl

Critical values : > 36 mmol/l ( > 100 mg/dl ) indicates serious impairment of renal function.

   The BUN measures the amount of urea nitrogen in the blood. Urea is formed in liver as the end product of protein metabolism. After the breakdown of protein into amino acids, these are catabolized in the liver to form ammonia. Urea is formed from the combination of ammonia with CO2 to be transported by the blood to kidneys for excretion.
    Therefore, BUN is directly related to the metabolic function of the liver and the excretory function of the kidneys. It serves as an index for the function of these organs. Elevated level of BUN is referred to as azotemia. A markedly increased BUN is conclusive evidence of severe impairment of glomerular function. In chronic renal disease the BUN level correlates better with symptoms of uremia than does the serum creatinine.

Specimen: 
Serum is preferred (no fasting is required). Avoid hemolysis.

Interfering Factors:
-	Changes in protein intake may affect BUN levels.
-	Advanced pregnancy may cause increased levels.
-	Overhydration and underhydration will affect levels.

-	Drugs that may cause increased B UN levels include:

Allopurinol, aminoglycosides, cephalosporines, chloralhydrate, furosemide, guanethidine, indomethacin, methotrexate, methyldopa, nephrotoxic drugs (eg, amphotericin B, Bacitracin, carbamazepine, gentamicin, neomycine, pencillamine, polymyxin B, aspirin, probencid, vancomycin), propranolol, rifampin, spiranolactone, tetracyclines, thiazide diuretics and triametrene.
-	Drugs that may cause decrease BUN levels include chloramphenicol and streptomycin.

Abnormal Findings:
Increased levels▲	Decreased levels ▼
   Prerenal causes	 Liver failure
Hypovolemia                       Overhydration caused by fluid
 Shock	 overload
 Burns	 Negative nitrogen balance (eg,
 Dehydration	 malnutrition or malabsorption).
 Congestive heart failure	 Pregnancy
 Myocardial infarction	 Nephroic syndrme
GI bleeding
 Excessive protein ingestion
 Starvation
 Sepsis
	 Renal causes
	Renal disease ( e.g. glomerulonephritis, 
pyelonephritis, acute tubular necrosis)
	Renal failure
	Nephrotoxic drugs
    Postrenal azotemia
	Ureteral obstruction
	Bladder outlet obstruction

In clinical laboratories blood urea estimation can be performed by either:

1-	Nessler’s method.

2- Urea kit (Brethelot test).

3-	Diacetyl monoxime method.

Most techniques have used the conversion of urea into ammonium carbonate by action of the enzyme urease found in soya and Jack beans. The ammonia formed has been determined by reaction into alkaline media (with salicylate and hypochlorite to form green colored indophenol-Brethelot method) and colorimetrically by nesslerization while the direct colorimetric method include reaction of urea with diacetyl in hot acid solution at nearly 100 0C to give a colored product by forming the chromogen diazine.

UREA KIT

Procedure:
	Blank	Std.	Sample
Sample	 -	 -	l0µl
Std 98.33 mmol/l	 -	10 µ1	 -
Working solution	1 ml	1 ml	1 ml
     (R2+R3)

Shake and incubate for at least 3 min. at 370C or 5 min. at 20-25 0C.
Reagent4	200µl	200µl	200µl

Sake and incubate for 5 min. at 37 0C or 10 min. at 20-25 0C then read at 580 nm the color intensity is stable 2 hours away from direct light.

Calculations:

OD sample
Conc. of urea =	x  n
                  OD Std.

Where n = concentration of Std.

Reagents:

Reagent 1 Std.: urea 8.33 mmol/L

Reagent 2 enzyme : urease≥ 5000 u/L
Reagent 3 colour reagent: phosphate buffer pH 8	40 mmol/l
	Sod. Salicylate	52 mmol/l
	Sod. Nitroprusside	2.3 mmol/l
	EDTA	1 mmol/l
Reagent 4	Sod. Carbonate	83 mmol/l
(Alkaline reagent)	Sod. Hypochlorite	3.75 mmol/l

BLOOD CRAETININE (SERUM CREATININE)

Normal Values:

Adult:62- 125 µmol/l or 0.6- 1.5 mg/dl

Child (3-18 yr.) : 44- 88 µmol/l or 0.5-1.0 mg /dl

Young child (0-3 yr.) : 27-62 µmol/l or 0.3- 0.7 mg/dl

BUN/ creatinin ratio:  10: 1 to 20: 1

Creatinine is a byproduct in the breakdown of muscle creatine phosphate from energy metabolism. It is produced at a constant rate depending on the muscle mass of the person and is removed from body by the kidneys. Production of creatinine is constant as long as muscle mass remains constant. Disorder of kidney function reduces excretion of creatinine, resulting in increased blood creatinine level.
Serum creatinine test aid in diagnosis of impaired renal function. It is more specific and sensitive indicator of kidney disease than BUN, although in chronic renal disease, both BUN and creatinine are ordered to evaluate renal problems, because the BUN / CREATININE provides more information.

Specimen: Collect 5 ml venous blood sample. Serum is preferred, but heparinized blood can be used.

Interfering Factors:
1-	High levels of ascorbic acid and cephalosporin antibiotics can cause a falsely increased creatinine level. These agents also interfere with BUN/ creatinine ratio.
2-A diet rich in meat can cause increased creatinine levels.

3-Creatinine is falsely decreased by bilirubin, glucose , histidine and quinidine compounds.
4-Ketoacidosis may increase serum creatinine substantially.

5-	Drugs that may increase creatinine values include ; aminoglycosides (eg, gentamicin), cimet~dine, heavy- metal chemotheraprutic agents (eg, cisplatin) , and other nephrotoxic drugs such as cephalosporins.

Abnormal Findings:
Increased levels▲		Decreased levels▼	
 Glomerulonephritis                   Debilitation
 Pyelonephritis	 Decreased muscle mass(eg,
	 muscular dystrophy,
	 myasthenia gravis)
 Acute tubular necrosis
 Urinary tract obstruction
 Reduced renal blood flow
 (eg, shock, dehydration, CHF,
 artherosclerosis)
 Diabetic nephropathy
 Nephritis
 Rhabdomyolsis
 Acromegaly
 Gigantism

ESTIMATION OF CREATININE

Colorimetric method (with Deproteinzation):

Principle:
Creatinine in an alkaline solution reacts with picrate to form a coloured complex. Deproteinization  procedure: mix equal volumes of Trichloroacetic acid (TCA) and  serum sample:
1 ml(serum or heparinized plasma)+ 1 ml TCA
   Mix well with a glass rod, centrifuge at 2500 rpm for 10 min. then pour off the supernatant, which can be stored up to 7 days at 2 to 8 0C.
   Reagent	Blank	Std.	Sample	Urine
				sample
D.W	0.5 ml	  -            -               -
Solution 1	  -	0.5 ml         -               -
TCA(1.2 mol/l)	0.5 ml	0.5 ml	  -	0.5 ml
Supernatant	  -	  -	 1 ml            -
Urine (1+49)	  -	  -	  -	0.5 ml
(R2+R3)	lml	lml	 lml	 lml

Mix and stand for 20 min. at 25 0C measure the absorbance of sample and Std. against blank.
A sample
Calculations: creatinine conc.:	X  n
A std.
n = conc. of Std.
*For urine multiply result by 50

Reagent 1 (Std.): 177µmol/l (2 mg/dl) of creatinine

Reagent 2: picric acid 35 mmol/l

Reagent 3: Sod. Hydroxide 1.6 mol/l

CREATININE CLEARANCE

Normal Values:
Adult (> 40 yr.):
Male:	107-139 ml/min.
Female:	87-107 ml/min.
Values decrease 6.5 ml/min / decade of life because of ↓GFR.

Explanation of test:
The creatinine clearance test is a specific measurement of kidney function , primarily for glomerular filtration. It measures the rate at which the kidneys clear creatinine from the blood. Generally, clearance of a substance may be defined as the volume (milliliters) of plasma from which the substance would have to be completely cleared by kidney in 1 minute. In addition to estimating the GFR, this test is used to evaluate renal function in patients to monitor the progression of renal disease.

Specimen:
-	Collect a serum sample to estimate creatinine level.               
-	  Mark the patient’s age, weight and height on the requisition sheet.

Interfering Factors:
•	Exercise may cause increase creatinine values.
•	Pregnancy increases creatinine clearance.
•	A diet high in meat content can transiently cause elevation of the          creatinine clearance.
•	Incomplete urine collection may give a falsely lowered value.
Drugs that may cause increased levels include aminoglycosides (eg, gentamicin) , cimetidine, heavy- metal chemotherapeutic agents (eg, cisplatin) , and nephrotoxic drugs such as cephalosporins (eg, cefoxitin)

Abnormal Findings:

Increased levels▲                             Decreased levels▼

	Exercise                                  Impaired kidney function
                              (eg, renal artery                   
                                                atherosclerosis,                                     
                                                glomerulonephritis and
                                                acute tubular necrosis)
	Pregnancy                                Conditions causing ↓ GFR               
                             (eg, GHF, cirrhosis with 
                                        ascites, shock, dehydreation)
 High cardiac output syndrome

Calculation:
                            Cockcroft-Galt equation allows creatinine clearance estimation for patients with a stable serum creatinine. 

	  (140 - age) x LBW [kg] 		
  CCr (in men) = 	----------------------------- =	 mL/min	
	  PCr [mg/dL] x 72 		

  CCr (in women) = 0.85 x above value =  mL/min 

 Where :
 CCr   = Creatinine Clearance 
 LBW =Lean Body Weight 
 PCr   = Plasma Creatinine 
         The correction factor of 0.85 reflects the lower muscle mass and therefore ,the lower rate of creatinine production in women when compared to men .  
 { LBW= lean body weight which is calculated from the following equation for males :LBW ( kg) = [ 0.73 x   Hight cm  ] -   59.42  
 whereas for females  :LBW ( kg) = [ 0.65 x   Hight cm  ] -   50.74   } 

CHOLESTEROL
Normal Values:
Adult:	<5.20 mmol/l or 200 mg/dl
             Child and Adolescent (12-18 yr.): 3.12-5.20 mmol/l or 120- 200 mg/dl
             Normal values vary with age, diet and geographical region.

     Cholesterol is a steroid alcohol formed in animal fats. It is widely distributed throughout the body, especially in the blood, brain, liver, kidneys and nerve fiber myelin sheaths and it is an essential component of cell membrane development and production of bile acids , adrenal steroids and sex hormones. Most of the cholesterol we eat comes from foods of animal origin. The liver metabolizes the cholesterol to its free from and cholesterol is transported in blood stream by lipoproteins. Nearly 75% of the cholesterol is bound to low-density’ lipoproteins (LDLs), and 25% is bound to high -density lipoproteins (HDLs). LDL is the most directly associated with increased risk of coronary heart disease (CHD). Cholesterol testing is usually done as a part of lipid profile testing, which also evaluates lipoprotein.
Specimen: Obtain 5 ml venous blood sample. Fasting is required. Serum is used for analysis.
•	Instruct the patient to fast 12-14 hr after eating a low fat diet before testing. Only water is permitted also no alcohol should be taken 24 hr before the test.

Interfering Factors;
1-Estrogen decreases plasma cholesterol levels, pregnancy increases these levels.
2-	Positional variations occur; levels are lower when sitting, versus standing and lower when recumbent versus sitting.
3-	Seasonal variations in cholesterol levels, higher levels were observed in winter and lower in spring and summer.
4-	Drugs that may cause increased levels include adrenocorticotropic hormone, anabolic steroids, beta-adrenergic blocking agents, corticosteroids, epinephrine, oral contraceptives, phenytion, sulfonamides, thiazide diuretics, cyclosporine and vitamin D.
     Other drugs that may causes decreased levels including allopurinol, bile salt binding agents, captopril , chlorpropamide, clofibrate, colchicine, erythromycin, isoniazide, lovastatin, monoaminoxidase inhibitors, neomycin (oral), niacin and nitrates.
Abnormal Findings:
Increased levels ▲                                       Decreased level▼
	Hypercholesterolemia                             Malabsorption
	Hyperlipidemia                                   Malnutrition
	Hypothyroidism                                  Hyperthyroidism
	Uncontrolled diabetes                            Cholesterol lowering                        mellitus                                           medications                
	Nephrotic syndrome                              Pernicious anemia
	Pregnancy                                        Sepsis
	High cholesterol diet                              Stress
	Hypertension                                     Liver disease
	Myocardial infarction                             Acute myocardial infraction
	Atherosclerosis
	Biliary cirrhosis
	Nephrosis
CHOLESTEROL ESTIMATION

(ENZYMATIC METHOD)
Principle:
Cholesterol is determined according to the following reaction:
                    Cholesterol esterase
Cholesterol ester                     cholesterol + fatty acids
                Cholesterol oxidase
Cholesterol                      cholest-4-en-3one + H2O2
                                       peroxidase
2H2O + phenol +4-aminoantipyrine               Quinoneimine +4H2O
Procedure:
                  Reagent blank               Std.                Sample
Std.(2g/l            -                           10µl                     -
cholesterol)                                                
Sample              -                           -                      10µl
Working solution  1ml                        1ml                    1ml
(R1+R2)  
Mix and measure absorbance after incubation at 37 0C for 5 min. or incubate for 10 min. at 20-25 0C. The color intensity is stable for 30 min.

Reagents:
Reagent 1 (buffer) phosphate buffer      0.1mol/l
	Phenol                15 mmol/l
	sod.cholate            3.74 mmol/l
	surfactant

Reagent 2       4-aminoantipyrine       0.5 mmol/l
      (enzymes)	peroxidase               ≥1000 U/l
	cholesterol oxidase	≥200 U/l
	cholesterol esterase	≥124 U/l

Calculation:

Where:
   n=5.17 mmol/l    or   200 mg/dl

LIPOPROTEINS (HIGH- DENSITY LIPOPROTEIN -
CHOLESTEROL ( HDL), LOW DENSITY LIPOPROTEIN-
              CHOLESTEROL (LDL-C) , VERY LOW DENSITY
LIPOPROTEIN (VLDL).

Normal Values:
HDL:
Male : > 0.75 mmol/l or > 45 mgldl

Female:	> 0.91 mmol/l or > 55 mg/dl

       LDL : <3.3 mmol/l  or 60-180 mgldl

VLDL: 25-50%

Lipoprotiens are considered to be an accurate predictor of coronary heart disease. As part of the lipid profile these tests are performed to indicate persons at risk for developing heart disease and to monitor therapy if abnormalities are found.
Lipoproteins are proteins in blood whose main purpose is to transport
cholesterol, triglycerides and other insoluble fats. With the use of electro- phoresis these lipoproteins can be grouped into:
1-	Chylomicrons (origin ) ,which are primarily triglycerides.
2-	LDLs (beta-lipoproteins), which are primarily cholesterol.
3-VLDLs (prebeta-lipoproteins), which are mainly triglycerides.
4-	HDLs (alpha-lipoproteins), which are predominalty protein with small amount of cholesterol.
The lipid profile usually includes total cholesterol, triglycerides, HDL, LDL, and VLDL.
       HDLs are carriers of cholesterol, that are produced in liver. It is main function is to remove cholesterol for excretion. Also HDLs prevent cellular uptake of cholesterol and lipids. These potential actions may be the cause of their protective cardiovascular characteristics associated with HDLs (good cholesterol) within the blood.
     LDLs are cholesterol rich. Cholesterol carried by LDLs can be deposited into the peripheral tissues and is associated with increased risk of arteriosclerotic heart and vascular diseases. Therefore, high levels of LDL (bad cholesterol) are atherogenic. LDL is very difficult to isolate and measure. Hence, LDL is most usually derived by the Friedwald formula as follows:
 
LDL = TOTAL CHOLESTEROLT- (HDL + TG/5)

VLDLs although carrying a small amount of cholesterol these are the predominant carriers of blood triglycerides. The VLDL value is usually expressed as a percentage of total cholesterol levels in excess of 25-50% are associated with increased risk coronary heart disease.

Interfering Factors:
1-	Smoking and alcohol ingestion decrease HDL levels.
2-HDL values are age and sex dependent.
3-	HDL values, like cholesterol, tend to significantly decreased for as long as 3 months following MI
4-	HDL level is evaluated in hypothyroidism and diminished in hyperthyroidism.
5-	Increased LDL levels are associated with pregnancy while women
taking oral estrogen therapy show decreased LDL and increased
HDL levels.
6-Drugs may cause increased lipoprotein levels include , steroids, progesterone (oral contraceptive) , androgens, phenothiazines and sulfonamides.
7-	Some drugs decrease HDL levels such as : steroids, diuretics, β-blockers.
Specimen:
Collect 5-10 ml of venous blood.
•	Instruct the patient to fast for 12-14 hours before sampling only water is permitted.
•	No alcohol should be consumed for at least 24 hr.
•	If possible, all medications should be withheld for at least 24 hr.

Abnormal Findings:
Increased HDL levels▲                    Decreased HDL levels▼
  Familial HDL lipoproteinemia            Familial low HDL
  Excessive exercise                      Hepatocellular disease (eg,                                                                
                                           hepatitis or cirrhosis)
                                             Hypoproteinemia (eg, nephrotic                                   
                                              syndrome or malnutrition)

Increased LDL and VLDL                 Decreased LDL and VLDL
	Familial LDL lipoproteinemia              Familial hypolipoproteinemia
  Glucagon storage diseases m               Hypoproteinemia
 (eg, Von Gierke’s diseade)                    (eg, malabsorption         
                                                 sever burnes, or malnutrition)                                                     
	Hypothyroidism                             Hyperthyroidism
	Alcohol consumption
	Chronic liver disease 
(eg, hepatitis, cirrhosis)
	Hepatoma
	Multiple myeloma
  Cushing’s syndrome

ESTIMATION OF HDL-CHOLESTEROL

Principle:
The chylomicrons VLDL and LDL — lipoproteins that are contained in the sample are precipitated by the addition of phosphotungestic acid in the presence of Mg +2 The supernatant obtained after centrifugation contains high density lipoproteins (HDL) from which the cholesterol can be determined by using the cholesterol enzymatic kit.

Procedure:
Serum 500 µl + 50 µl R1
Mix and stand for 10 min. centrifuge for 15 min. at 500 rpm.
For the determination of HDL-cholesterol use the same procedure used for cholesterol estimation using the resultant supernatant from the above mixture.
	Reagent	Std	Sample
	 blank
D.W	 50µl	  -                    -
HDL- cholesterol calibrating	  -	50µl                 -
sol. 1.3 mmol/l (50 mg/dl)
Supernatant	  -	50µl                 -
Working sol.	                          1 ml            1 ml               l ml (Reagent 1 + Reagent 2 of
cholesterol kit)
Mix and incubate for 5 min. at 37 0C, measure at 500 nm, the color is stable for 30 min.
Reagents:
Reagent 1             phosphotungestic acid                  40 g/l
                         Mg Cl2.6H20                          100g/l
                         PH 6.2
Calculations:

Where :
n =1.42 mmol/l   or    55 mg/dl

TRIGLYCERIDES (TG)

Normal Values:
Adult:
  Male  0.45-1.81 mmol/l or 40-160 mg/dl
  Female  0.40-1.52 mmol /l or 35-135 mg/dl
Child (6-11 yr.): 33-112 mg/dl
Triglycerides (TGs) are transported in blood by very low density lipoproreins VLDL and chylomicrons. TGs are produced in liver by using glycerol and fatty acids as building blocks. TGs are a part of lipid profile that also evaluates lipoproteins.

Interfering Factors:
1- Ingestion of fatty meals may cause elevated TG levels.
2-Ingestion of alcohol may cause elevated TG levels.
3-Pregnancy may cause increased levels.
4-Drugs that may cause increased TG levels include cholesytramine, estrogens and oral contraceptives.
5-Drugs that may cause decreased levels include ascorbic acid, clofibrate and colestipol.

Specimen:

Collect 5 to 10 ml of venous blood. Instruct the patient as with lipoproteins estimation to obtain serum or plasma (collected in heparin, or oxalate or citrate salts or EDTA) . Hemolysis will interfere.

Abnormal Findings:

Increased TG levels▲                            Decreased TG levels▼
	Glycogen storage diseases                   Malabsorption system
	Hyperlipidemias                             Malnutrition
	Hypothyroidism                            Hyperthyroidism
	High-carbohydrate diet
	Poorly controlled diabetes
	Occlusive coronary disease 
and peripheral vascular disease
	Nephrotic syndrome
	Hypertension
	Alcoholic cirrhosis
	Pregnancy
	Myocardial infarction

ESTIMATION OF TRIGLYCERIDES (ENZYMATIC METHOD)

Principle:
                             Lipase
Triglycerides             Glycerol + Fatty Acids
                          Glycerokinase
Glycerol + ATP               Glycerol -3 phosphate + ADP
                             Glycerol -3phosphate oxidase	
Glycerol 3-phosphate 	           Dihydroxy Acetone Phosphate +H2O2                                                                                                                                                                                                                                                         
                                                Peroxidase
2H2O + P- chlorophenol +4-Aminoantipyrine         Quinoneimine + 4H2O          
Procedure:                                                          
                                   Reagent blank         Std.          Sample
Standard R1	    -	    10 µl	     -
Sample	    -	   10 µl                 -
Working solution (R2 + R3)         l ml	    1ml	   l ml

 Mix, incubate for 5 min at 37 0C or 10min. at 20-25 0C measure absorbance at 505 nm, the color intensity is stable for 30 min.

 Calculation:                                           

Where: 
n= 2.29 mmol/l  or  200 mg/dl
Reagents: 
Reagent I(Std. Glycerol)                       2.29 mmol/l or 200 mg/dl
Reagent 2             tris buffer pH 7.6                  100 mmol/l 
                        Parachlorophenol                  2.7 mmol/l
                        Magnesium                          4 mmol/l
Reagent 3 (enzymes)    Amino- antipyrine                > 0.4 mmol/l
                        Lipase	                               1000 u/l
                        glycerokinase	                        ≥ 200 u/l
                        Glycerol 3-phosphate oxidase         ≥2000u/l
                        Peroxidase                            ≥ 200u/l
                        ATP                                 0.8mmol/l

BLOOD PROTEINS

( Albumin, Total Protein, Protein Electrophoresis)

Normal Values:
Adult:		Children:
Total protein	64-83 g/l	Total protein	62-80 g/l
Albumin	35-50 g/l	Albumin	40-59 g/l
Globulin	23-34 g/l
α1 globulin:	1-3 g/l
α2  globulin:	6- l0g/l
β1globulin :	7-11g/l

Albumin and globulin constitute most of the protein within the body and are measured in the blood total protein. Albumin is a protein that is formed within the liver. It makes up approximately 60% of the total protein. The major effect of albumin within the blood is to maintain colloid osmotic pressure. Albumin transport important blood constituents such as drugs, hormones and enzymes. When disease affects the liver cell, the hepatocytes lose their ability to synthesize albumin. Serum albumin level is greatly decrease, because the half-time of albumin is 12-18 clays, however, sever impairment of hepatic albumin synthesis may not be recognized until after that period
Globulins are the building block of antibodies. The various fractions sever a wide variety functions from enzymatic activity as α1 antitrypsin to transporting function e.g. thyroxine- binding globulin.
Serum albumin and globulin are measure of nutrition. Malnourished patients , especially after surgery, have a greatly decreased level of serum proteins. 
Other causes of hypoproteinemia include: Burns, protein — losing enteropathies and uropathies. Those patients have normal protein synthesis but excessive protein loss.

In some diseases , albumin is selectively diminshed and globulins are normal or increased to maintain a normal total protein level eg, lupus erythrematosus.
     Another group diseases similarly associated with low albumin, high globulin and normal total protein levels as in chronic liver diseases.
Serum protein electrophoresis can separate various components of blood protein into bands or zones according to their electrical charge and molecular weight.
Interfering Factors:

-	Prolonged application of a tourniquet can increase both albumin and globulin.
-	Sampling of peripheral venous blood proximal to an IV fluid site can result in low protein levels and massive IV infusion of crystalloid fluid can cause acute hypoproteinmia.
-	Drugs that may increase protein levels include anabolic steroids, androgens, corticosteroids , dextran , growth hormone, insulin, phenazopyridine and progesterone.
-	Drugs may cause decreased protein levels include ammonium ions, estrogens, hepatotoxic drugs and oral contraceptives.
Specimen:
Collect approximately 5-7 ml of blood use serum or heparinized plasma for analysis. Serum (only) is applied for protein electrophoresis. No fasting is required.

Abnormal Findings:
Increased Albumin Levels▲                  Decreased Albumin Levels▼
  Dehydration                              Malnutrition
                                              Pregnancy
                                              Liver disease
                                              Protein losing enteropatheis
                                              Overhydration
                                              Increased capillary permeability
                                                                    Inflammatory disease

Increased α1- globulin ▲                     Decreased α1- globulin▼

	Inflammatory disease                      Juvenile pulmonary emphysemia

Increased α2 -globulin ▲                     Decreased α2 -globulin▼
	Nephrotic syndrome                       Hemolysis
	Inflammatory disease                      Wilson’s disease
                                               Hyperthyroidism
                                               Sever liver dyfunction
Increased β-globulin ▲                      Decreased β-globulin▼
	Hypercholesterolemia                      Malnutrition
   Iron deficiency anemia
Increased γ- globulin levels ▲             Decreased γ- globulin levels ▼
	Multiple myeloma                          Genetic immune disorders
   Chronic inflammatory disease             Secondary immune deficiency
	Malignancy
   Hyperimmunization
   Acute and chronic infection

ESTIMATION OF SERUM TOTAL PROTEIN

(BIURET METHOD)

Principle:
Cupric ions, in an alkaline medium, interact with protein peptide bonds resulting in the formation of a coloured complex.

Procedure:       
                              Reagent blank      Std.        Sample

D.W                              0.02 ml	-	-
Standard protein (6.0 g/l)	-                                    0.02 ml           -
Serum plasma	-	-	                          0.02 ml
Biutet reagent	1 ml         l ml	           1ml

Mix and incubate for 30 mm. at 20-25 0C. Measure absorbance of the sample and standard against reagent blank.

Calculation:

                       A Sample
Total protein conc. =               X  Std.conc.
    ( g/l )               A Std

 Biuret reagent: 
            Sod. Hydroxide               100mmol/l
	Na-K tartarate                16 mmol/l
	Potassium iodide             15 mmol/l
	Cupric sulphate               6 mmol/l

	

ALBUMIN

(BROMOCRESOL GREEN)

Principle:

The measurement of serum albumin is based on its quantitative
binding to the indicator 3,3؍ ,5,5.؍ - tetrabromo-m-cresol sulphanphthalein
(bromocresol green, BCG).
Procedure:
	Reagent	Std.	Sample
	Blank
D.W	0.01 ml		      -	    -
Std. Human Serum                 -  	                                0.01 ml  	    -
Albumin (45 g/L)
Serum or Plasma                   -		           -	0.01 ml
BCG reagent                     3 ml 		          3 ml 	   3 ml
 Mix and incubate for 5 min. at 20-25 0C, 	measure the absorbance of sample and of Std. against reagent blank.؍

Procedure:

                       A Sample
Albumin conc. =                   X  Std.conc.
    ( g/l )               A Std

BILIRUBIN

Normal values:

Adul:
Total bilirubin: 5.1 - 17 µmol/l           or  0.3 – 1.0 mg/dl
Indirect bilirubin: 3.4 – 12.0 µmol/l      or  0.2 – 0.8 mg/dl
Direct bilirubin: 1.7 – 5.1 µmol/l         or  0.1 – 0.3 mg/dl

Newborn (0 – 7 days):
Total bilirubin: 17 – 170 µmol/l         or  1.0 – 10.0 mg/dl
Indirect bilirubin: 0 – 170 µmol/l        or  0 – 10.0 mg/dl
Direct bilirubin: 0 – 1.36 µmol/l         or  0 – 0.8 mg/dl

Panic volume for neonatal bilirubin: > 15 mg/dl (mental retardation can occur).
     
     Bilirubin metabolism begins with breakdown of red blood cells in the reticulo-endothelial system. Hemoglobin released from RBCs and broken down to heme and globin molecules. Heme is then catabolized to form biliverdin, which is transformed to bilirubin. This form of bilirubin is called unconjugated (indirect) bilirubin. In the liver, indirect bilirubin is conjucated with glucuronide resulting in conjucated (direct) bilirubin. The conjucated bilirubin is then excreted from the liver cells into the intrahepatic canaliculi, which eventually lead to the hepatic ducts, the common bile duct and the bowel.  
     Jaundice is the discoloration of body tissues by abnormally high blood levels of bilirubin. This yellow discoloration is recognized when the total serum bilirubin exceeds 2.5 mg/dl. Physiologic jaundice of the newborn occurs if the newborn’s liver is immature and does not have enough conjugating enzymes. This results in a high circulating blood levels of unconjugated bilirubin , which can pass through the blood-brain barrier and deposit in the brain cells of the newborn. This can cause encephalopathy (kernicterus).

The jaundice cases caused by hepatocellular dysfunction (eg, hepatitis) results in elevated levels of unconjugated bilirubin. Jaundice resulting from extrahepatic obstruction of the bile ducts (eg, gallstones or tumors bloking the bile ducts) results in elevated conjugated bilirubin levels , this type of jaundice usually can be resolved surgically or endoscopically.
Total serum bilirubin level is the sum of the conjugated (direct) and unconjugated (indirect) bilirubin.
     Normally the unconjugated bilirubin makes up 70% to 85% of the total bilirubin. Conjugated hyperbilirubinemia could result from gallstones, tumor, inflammation, scarring or obstruction of the extrahepatic ducts. Unconjugated hyperbilirubinemia exists when less than 15% to 20% of the total bilirubin is conjugated this mostly result from accelerated erythrocyte hemolysis or hepatitis. 
Specimen:
-	Collect 5-7 ml venous blood.
-	Serum is used for analysis. Fasting is preferred. Avoid hemolysis during sample collection.
-	Use a heel puncture for blood collection in infants.

Interfering Factors:
-	Blood hemolysis and lipemia can produce false results.
-	Avoid the exposure of the specimen to sunlight or high intensity artificial light at room temperature because this will decrease bilirubin content.
-	Air bubbles and shaking of specimen may cause decreased bilirubin levels.
-	Certain food (eg. carrots, yams) may increase the yellow hue in serum thus causing falsely increased bilirubin levels by spectrophotometric methods.
-	Prolonged fasting raises bilirubin level as does anorexia.
-	Drugs that may increase levels of total bilirubin include:
Allopurinol, anabolic steroids, antibiotics, antimalarials, ascorbic acid, chlorpropamide, diuretics, methyldopa, monoamine oxidase inhibitors, 

morphine, nicotinic acid (large dose), oral contraceptives, refampicin, salicylates , theophlylline and vitamin A.
  -   Drugs that may decrease levels of total bilirubin include barbiturates, caffeine , penicillin and high doses of salicylates.

ESTIMATION OF BILIRUBIN

Principle:
Sulfanilic acid reacts with sodium nitrite to form diazotized sulfanilic acid. In the presence of dimethyl sulfoxide, total bilirubin reacts with diazotized sulfanilic acid to form azobilirubin. In the absence of dimethyl-sulfoxide, only direct bilirubin reacts with diazotized sulfanilic acid to form azobilirubin.
Procedure:

Total Bilirubin

                   Std. blank     Std.      Test Blank      Test 
Reagent 4            50µl        50µl	     -	  -
Sample	-	-	50µl	50µl
Reagent R1           1 ml          -	          1 ml	   -
Working sol.	-                                1 ml	-	1ml
Mix well and incubate exactly for S mm. at 37 0C read the absorbance of Std. and testing against their blanks at 555 nm.
Direct Bilirubin
                   Std. blank     Std.      Test Blank      Test 
Reagent 4            50µl        50µl	     -	  -
Sample	-	-	50µl	50µl
Reagent R2           1 ml          -	          1 ml	   -
Working sol.	-                                1 ml	-	1 ml

Mix well and incubate exactly for 5 mm. at 37 0C read the absorbance of Std. and Test against their blanks at 555 nm stable in the dark for 6h at  20-25	0C.

	
 Serum Calcium

Normal Values:
Children:	3-9 yr. : 2.2-2.7 mmol/l (8.8-10.1 mg/dl) 
          13-15 yr. : 2.10-2.55 mmol/l (9.2-10.7 mg/dl)
Adult:	2.25-2.75 mmol/l (8.4-10.2 mg/dl)

The bulk of body calcium (98-99%) is found in the skeleton and teeth. About 50% of blood calcium in ionized, the rest is protein-bound. Only ionized calcium can be used by the body in vital processes an muscular contraction, cardiac function, transmission of nerve impulses and blood clotting.
The amount of protein in blood also affects calcium levels, because 50% of blood calcium is protein- bound, however, ionized form of calcium is not affected. Parathyroid hormone calcitonin, vitamin D, estrogens, androgens, carbohydrates and lactose all are factors that could influence calcium levels.
Specimen:
Obtain 5 ml venous blood sample to get serum for calcium estimation. Heparinized plasma is preferred for ionized calcium studies. (EDTA or oxalate cause decreased calcium levels).
-	Avoid prolonged tourniquet use (otherwise falsely high calcium levels).
Interfering Factors:
-	Vitamin D intoxication may cause increased serum calcium levels.
-	Excessive ingestion of milk may cause increased level.
-	Serum pH can effect calcium levels. A decrease in pH causes increased calcium levels.
-	Hypoalbuminimia artifactually associated with decreased levels of total calcium.

-	Drugs that may cause increased serum levels include calcium salts, hydralazine, thiazide diuretics parathyroid hormone, thyroid hormone, alkaline antacids ergocalciferol , androgens and vitamin D.
-	Drugs that may cause decreased serum levels include: acetazolamide, anticonvulsants, aspirin, corticosteroids , heparin, laxatives, loop diuretics , Mg salts , estrogen and oral contraceptives.

Abnormal Findings:
Increased levels (hypercalcemia)▲
	Hyperparathyroidism
	Metastatic tumor to the bone
	Paget’s disease of bone
	Prolonged immobilization
	Milk-alkali syndrome
	Vitamin-D intoxication
	Lymphoma
	Addison’s disease
	Acromegaly
	Hyperthyroidism	Decreased levels (hyocalcemia)▼
	Hypoparathyroidism
	Renal failure
  Hyperphosphatemia secondary to renal failure
	Rickets
	Vit-D deficiency
	Osteomalacia
	Malabsorption
  Pancreatitis
	Fat embolism
  Alkalosis	

ESTIMATION OF CALCIUM

Principle:
Colorimetric determination of calcium without deproteinization using 0-cresol phthalein complex. Interference due to Mg +2 is eliminated by β-hydroxyquinoline (up to 4 mmol/l 0.10 mg/l).

Procedure:

	Reagent blank	Std.	Sample	
Sample	-	-	20µl	
Reagent 1 (std)	-	20 µl	-	
Working solution
(R2+R3)	         1ml	lml	lml	
Mix , read absorbance after 5 mm. at 572 nm against blank. The color intensity is stable for 1 hour.	

INORGANIC PHOSPHATE
Normal Values:
Adult:	0.87-1.45	mmol/l	or	2.5-4.5 mg/dl
Child:	1.45-1.78	mmol/l	or	4.5-5.5 mg/dl
Newborn:	1.45-2.91	mmol/l	or	4.5-9.0 mg/dl
Of the human body’s total phosphprus content, 85% is combined with calcium in the bone, and the remainder resides within the cells. Most of the phosphorus in the blood exists as phosphates or esters. Phosphate is required for the generation of bony tissue and functions in the metabolism of glucose and lipids, in the maintenance of acid-base balance, and in the storage and transfer of energy from one site in the body to another. Phosphorus enters the red blood cells with glucose and therefore is lowered in the plasma after carbohydrate ingestion or infusion.

Interfering Factors:

-	Laxatives or enemas containing sod. Phosphate can increase phosphorus levels.
-	Recent carbohydrate ingestion, including IV glucose administration, causes decreased phosphorus levels because phosphorus enters the cell with glucose.
-	Drugs that may cause increased levels included methacillin and vitamin D (excessive).
-	Drugs that may cause decreased levels include antacids and mannitol.
-	Phosphorus levels are normally high in children.
-	Seasonal variation could exist in phosphorus levels (maximum in May and June, lowest in winter).
-	Note the time day sample is drawn; levels are highest in the morning and lowest in the evening.

-	Specimen:
-	Collect venous blood to separate serum as soon as possible.
-	Handle the sample carefully , avoid hemolysis.
-	Use a heel stick to draw blood from infants.
-Take the specimen to the lab. Immediately.

Abnormal Findings:

Increased levels (Hyperphosphatemia)▲
	Renal failure
	Increased dietary or IV intake of P
	Acrornegaly
	Hypoparathyroidism
	Acidosis
	Rhbdomyolysis
	Hemolytic anemia
	Advanced lymphorna or myeloma
ingestion	Decreased levels 
(hypophosphatemia)▼
	inadequate dietary ingestion of phosphorus
	Chronic antacid
	Hypercalcemia
  Chronic alcoholism
	Vit. D deficiency
	Diabetic acidosis
	Rickets
	Malnutrition
	Alkalosis, Sepsis
	
DETERMINATION INORGANIC PHOSPHORUS

Principle:
    Colorimetric determination, without deprotrinization , of serum phosphorus using a single reagent which forms a phosphomlybdate complex in the presence of a reducing agent (ferrous sulphate).
Procedure:
	Reagent Blank	Std.	Sample

Sample                          -                      -                   100 µl            

Reagent (std) (5mg/dl,            -                      100 µl            -

1.61	mmol/l)

D.W	100 µl            -                -
Working solution 
   (R2+R3)	2.5 ml	2.5 ml	      2.5 ml
Mix and wait 10 min. measure the absorbance of Sample and Standard at 690 nm 

ALKALINE PHOSPHATASE (ALP)

Normal Values:
Adult: 30-120 U/L or 3-13 kind and king U/100 ml 
Children (>2yr) : 85-235 U/l
        (2-8 yr.) : 62-210 U/l
Adolescent:	(16-21) : 30-200 U/l

Although ALP is found in many tissues, the highest concentrations are found in the liver, biliary tract epithelium , and bone. The intestinal mucosa and placenta also contain ALP. Measurements of this enzyme is important for determining liver and bone disorders.
ALP levels are greatly increased in both extrahepatic and intrahepatic obstructive biliary disease and cirrhosis. Other liver abnormalities such as hepatic tumors , hepatotoxic drugs, and hepatitis causes lesser elevations in ALP levels.
Bone is the most frequent extrahepatic source of ALP, new bone growth is associated with elevated ALP levels , which explains why ALP levels are high in adolescents. Pathologic new bone growth occurs with osteoblastic metastate eg, breast, prostate tumors. Paget’s disease healing fractures, rheumatoid arthritis , hyperparathyroidism and normal-growing bones are sources of elevated ALP levels as well.
Isoenzyrnes of ALP are sometimes used to distinguish between liver and bone diseases. The detection of isoenzymes can help in differentiation of the source of the pathology associated with elevated total ALP.
AlP1 is from the liver. ALP2 is from the bone.

Specimen:
Collect venous blood fasting is preferred. Serum is used for this test. Perform the test as soon as possible otherwise refrigerate the sample. Hemohysis will interfere.Record age and gender in test requisition.
Interfering Factors:
-	Recent ingesting of a meal increase ALP levels.
-	Drugs that may cause elevated ALP levels include allopurinol, antibiotics, azothioprine, colchicine, fluoride, indomethacin, isoniazide (INH) , methyldopa, nicotinic acid, phenothiazine , tetracycline and verapamil.
-	Drugs that may cause decreased levels include nitrofurantion, oxalates and zinc salts.

Abnormal Findings:
Increased levels▲ 
 Cirrhosis
	Intrahepatic or extrahepatic 
   biliary obstruction
	Primary or metastatic liver tumor
	Normal pregnancy (trimester, early post partum)
	Metastatic tumor to bone
	Healing fracture
	Hyperparathyroidism
	Rheumatoid arthritis
	Normal bones of growing children
	Decreased levels▼
	Hypoparathyroidism
	Malnutrition
	Pernicious anemia
	Hypophosphatemia
	Scurvy (vit. C deficiency)
	Celiac disease
	Excessive vit. B ingestion	

CLOROMETRIC DETERMINATION OF ALKALINE

PHOSPHATES ACTIVITY

Principle:
Clorimetric determination of alkaline phosphates activity according to the following reaction: 
                            Alkaline phosphatase
Phenylphosphate                    phenol + phosphate
                                     PH10
The phenol liberate is measured in the presence of amino-4-antipyrine and potassium ferriciyanide. The addition of sodium arsenate to the reagents stop the enzymatic reaction of the end of the incubation period.

Procedure:
	Sample	Sample Blank	Std.	Reagent Blank	
Reagent 1	2ml	2ml	2ml	2ml	
Incubate for 5 min. at 37 0C	
Serum	50 µl	-	-	-	
Reagent 2	-	-	50 µl	-	
Incubate for exactly 15 min. at 37 0C	
Reagent 3	0.5 ml	0.5 ml	0.5 ml	0.5 ml	
Mix well then add:	
Reagent 4	0.5 ml	0.5 ml	0.5 ml	0.5 ml	
Serum	-	50 µl	-	-	
D.W.	-	-	-	50 µl	
     Mix and stand for 10 min. in dark, measure absorbance at 510 nm against reagent blank, the color intensity is stable for 45 min.	

Calculation:
                                      OD Serum Sample - OD Serum Blank
Alk. Phosph. Activity =                                     X  n
                                                        OD Std.

n= 142 u/l   or  n= 20 Kind and King unit/100ml

Reagents:
Reagent 1
(Substrate buffer)	Disodium phenyl phosphate
Carbonate-bicarbonate
buffer PH=10	5 mmol/l
50 mm0l/l	
Reagent 2
(Std.)	Phenol 	Equal to 20 kind and king unite/100 ml	
Reagent 3
(Blocking reagent)	4-Aminoantipyrine	60 mmol/l	
Reagent 4
(Color reagent)	Sod. Arsenate
Potassium ferricyanide	75g/l
150 mmol/l	

Note: reagent 3 is a toxic reagent (by inhalation or swallowing), handle with proper care.

ACID PHOSPHATAES(ACP)

Normal Values:
Total acid phosphatase up to 11 u/l
Prostatic phosphatase up to 4 u/l

Acid phosphatase is found in many tissues, including liver, red blood cells and platelets. Highest levels are found in the prostate gland. Determination of acid phosphatase level is primarily used to diagnose and stage prostatic carcinoma and t monitor the efficiency treatment.
The most clinically significant isozyme is the prostatic acid phosphatase (PAP). It is more accurate than total acid phosphatase in the prostatic cancer patient but less accurate than the prostate specific antigen (PSA).
The identification of tartarate-resistant acid phosphatase (TRAP) is very helpful in the diagnosis of hairy-cell leukemia.
Interfering Factors:
-	Alkaline and acid phosphatase are very similar enzymes that differ in the
       PH at which they are identified.
-	Falsely high levels of acid may occur in males after prostatic stimulation.
-	Drugs that may cause elevated levels include androgens (in females) and clofibrate.
-	Drugs that may cause decreased levels include: fluorides, phosphates, oxalates and alcohol.
Specimen:
Collect venous blood. Fasting is not indicated. Avoid hemolysis, because red blood cells contain acid phosphatase.
-Note on the lab. slip if the patient has had a prostatic examination within the last 24 hr.

-	Do not leave the specimen at room temperature for 1 hr. or longer, because the enzyme is heat and pH sensitive and its activity will decrease.
-	Have the test performed without delay or freeze the serum.

Abnormal Findings:
Increased levels▲ 
	Prostatic carcinoma
	Multiple mycloma
	Paget’s disease
	Sickle cell crisis
	Renal impairment
	Recent prostste manipulation
	Benign prostatic hypertrophy
	Prostatitis
	Cancer of the breast and bone
	Cirrhosis
	Hyperparathyroidism
	Thrombocytosis
  Cancer metastasis to bone

ESTIMATION OF ACID PHOSPHATASE

Colorimetric Method:
Acid phosphatase
P- Nitrophenyl Phosphate + H20	P-nitrophenol + Phosphate

Procedure:
	Reagent blank	Sample 1	Sample 2
Solution2	1.0ml	1.0ml	1.0ml

Solution 3	-	-	0.1 ml

Incubate for exactly 5 min. at 37 0C (add at 30 sec. intervals)
Sample		-	0.2 ml	0.2m1

Incubate for exactly 5 min. at 37 0C add at 30 sec. intervals
Dilute NaOH	10.0 ml	10.0 ml	10.0 ml
Sample	0.2 ml               -                    -
Mix, read the absorbance of the sample against the reagent blank at 405
nm.

Calculation:

To calculate acid phosphatase activity use the following formula.
Total Acid Phosphatase: 101 x  A Sample 1
Postatic acid phosphatase: 101 x A Sample 1- A Sample 2

Reagents:

Reagent 1	citrate buffer	55 mmol/ pH 4.8
(Used for the preparation of reagent 2)

Reagent 2
p-nitrophyenyl phosph.	   (substrate)	       5.5 mmol/l
Reagent 3                Sod. Tartarate           200 mmol/l
Reagent 4	Sod. Hydroxide          200 mmol/l 
                     (used after with DW 1+9)

Alanine Aminotransferase (ALT)

Serum Glutamic-Pyruvic Transaminase (SGPT)

Normal Values:
Adult /child : up to 12 U/l .
Elderly: may be slightly higher than adult.
Infant: 	may be twice as high as adult.

ALT is found predominantly in liver, kidneys (to a lesser extent), heart and skeletal muscles. Injury or disease affecting liver cells will cause a release of this hepatocellular enzyme into the blood stream, thus elevating serum ALT levels.
Generally most ALT elevation are caused by liver diseases. Therefore, this enzyme is not only sensitive but also quite specific in indicating hepatocellular disease. In hepatocellular disease other than viral hepatitis the ALT/AST ratio (DeRitis ratio) is less than1. In viral hepatitis, the ratio is greater than 1. This is helpful in the diagnosis of viral hepatitis.

Interfering Factors:
-	Previous intramuscular injections may cause elevated levels.
-	Drugs that may cause increased ALT levels include: acetaminophen, allopurinol, aminosalicylic acid, ampicillin, carbamazepine,
cephalosporins, chlordiazepoxide, chlorpropamide, clofibrate, cloxacillin, codien, indomethacin, isoniazid, methyldopa.
Specimen:
Obtain  venous blood to obtain serum.

Abnormal Findings:
Increased levels ▲
	Hepatitis
	Hepatic necrosis
	Hepatic ischemia
	Cirrhosis
	Cholestasis
	Hepatic tumor
	Hepatoxic drugs
	Obstructive jaundice
	Sever burns
	Trauma to striated muscle
	Myositis
  Pancreatitis
  MI.
	Infectious mononucleosis
	shock

MEASUREMENT OF GLUTAMIC -
PYRUVIC TRANSAMINASE (ALT)

(Colorimetric Method)

Principle:
                                    ALT
α-Oxoglutarate + L- Alanine	  L-glutamate + Pyruvate

Gltamic pyruvic transaminase is measured by monitoring the concentration of pyruvate. Hydrazone formed with 2,4- dinitrophenylhydrazine.

Procedure:
	Reagent blank	Sample

Sample	-	0.1 ml
Buffer	0.5 ml	0.5 ml

DW	0.1 ml	-

Mix, incubate for exactly 30 min at 37 0C.
2,4-DNP	0.5m1	0.5m1
Mix, allow to stand for exactly 20 min. at 20-25 0C.
Sod. Hydroxide	5 ml	5 ml
Mix, read the absorbance of sample at 550 nm, using reagent blank to adjust the spectrophotometer to read zero absorbance to estimate the activity of ALT in serum, see the following table.

Absorbance	U/l	Absorbance	U/l	
0.025	4	0.275	48	
0.050	8	0.300	52	
0.075	12	0.350	62	
0.100	17	0.375	67	
0.125	21	0.400	72	
0.150	25	0.425	77	
0.175	29	0.450	83	
0.200	34	0.475	88	
0.225	39	0.500	94	
0.250	43			

Reagents:

•	Buffer (phosphate buffer)
L- alanine
α - oxoglutarate	100 mmol/l, PH 7.4
200 mmol/l
2.0 mmol/l	
•	2,4- dinitrophenyl hydrazine	2.0 mmol/l	
•	Sod. Hydroxide	4.0 mmol/l	
•	Pyruvvate Std.	2.0 mmol/l	

ASPARTATE AMINOTRANSFERASE (AST)

Glutamic-Oxaloacetate Transaminase (SGOT)

Normal Values:

Adult:	up to 12 U/l

Elderly:	values slightly higher than adult.

Child:	values similar to adult.

Aspartate aminotransferase is measured in the evaluation of suspected coronary occlusive heart disease or suspected hepatocellular disease. When disease or injury affects the cells of these tissues causing cells lyses.
AST is released and picked up by the blood and the serum level rises. The amount of AST elevation is directly related to the number of cells affected by the disease or injury. AST is cleared from the blood in a few days. Serum AST levels become elevated 8 hours after cell injury, peak at 24 to 36 hours, and retum to normal in 3 to 7 days. If the cellular injury is chronic, levels will be persistently elevated.
     AST (SGOT) enzyme is one of the enzymes tested in the cardiac enzyme profile. Although not specific for myocardial injury, when observed with the enzymes creatine phosphokinase (CPK) and lactate dehydrogenase (LDH), it is usedful in diagnosis of a recent MI and the extent of MI.. In the case of an acute MI, the AST level rises within 6-10 hours after MI, peaks at 12-48 hrs and returns to normal in 3-4 days. A second rise in AST would indicate extension or progression of myocardial injury despite therapy. Myocardial injuries such as angina, pericarditis or rheumatic carditis do not increase AST levels.
Diseases that affect the hepatocyte cause elevated levels of this enzyme. In acute hepatitis AST levels can raise 20 times the normal value.
In acute extrahepatic obstruction (eg, gall stone) AST levels quikly rise to
10 times the normal and swiftly fall. In cirrhotic patien ts the level of AST depends on the amount of active inflammation.
The AST/ALT ratio is usually greater than 1.0 in patients with alcoholic cirrhosis and metastatic tumor of the liver. Ratios less than 1.0 could be seen in patients with acute hepatitis, viral hepatitis or infections mononucleois.

Interfering Factors:

1-Exercise may cause increased levels.
2-	Drugs that may cause increased levels include: anihypertensive, cholinergic agents, coumarin-type anticoagulants, digitalis preparation, erythromycin, methyldopa, oral contaceptives, salicylates hepatotoxic medications.
3-	Decreased levels could be seen in pyridoxine deficiency (beriberi or pregnancy), sever long standing liver disease, uremia or diabetic ketoacidosis.

Specimen:
Collect a venous sample of blood. This is usually done daily for three days and then again after 1 week. (in case of suspected MI for emergency room patients) It is preferred to instruct the patient to hold drugs that could interfere with test results for 12 hrs before the test. Avoid hemolysis.

Abnormal Findings:
Increased levels▲
•	  Heart disease:
 Myocardial infarction
 Cardiac catheterization
•	Liver diseases
	Hepatitis
	Hepatic cirrhosis
	Drug- induced liver injury
	Hepatic metastasis
	Hepatic surgery
•	Skeletal muscle diseases:
  Skeletal muscle trauma
	Multiple traumas
	Sever, deep burns
	Progressive muscular dystrophy
•	Other diseases
	Acute hemolytic anemia
	Acute pancreatitis
	Decreased levels▼
 Acute renal diseases
 Beriberi
 Diabetic ketoacidosis
 Pregnancy
 Chronic renal dialysis

Estimation of Glutamic-Oxaloacetatic Transaminase 
(SGOT) (AST)Activity
Colorimetric Method 
Principle: 
                                                    GOT
α-oxoglutarate + L-aspartate         L- glutamate + oxaloacetate

Glutamic- oxaloacetic transaminase is measured by monitoring the concentration of oxaloacetate by hydrazone formed with 2,4- dinitrophenylhydrazine.

Procedure:
	Reagent blank	Sample
Sample		0.1 ml
GOT buffer	0.5 ml	0.5 ml
D.W	0.1 ml
Mix, incubate for exactly 30 min. at 37 0C.
2,4- dinitrophneyl hydrazine	0.5 ml	0.5 ml
Mix, allow to stand for exactly 20 mm at 20 to 25 0C.
Sod. Hydroxide	5.0 ml	5.0 ml

Mix, read the absorbance of sample at 530 nm against the reagent blank after 5 min.
Obtain the activity of GOT in the serum from the table:

Absorbance	U/l	Absorbance	U/l	
0.020	7	0.100	38	
0.030	10	0.110	41	
0.040	13	0.120	47	
0.050	16	0.130	52	
0.060	19	0.140	59	
0.070	23	0.150	67	
0.080	27	0.160	76	
0.090	31	0.170	89	

LACTATE DEHYDROGENASE (LDH)

Normal Values:
Adult:	80-190 U/l
Child:	60-170 U/l
Newborn:	160-450 U/l
LDH is found in the cells of many body tissues especially the heart, liver, red blood cells, kidneys, skeletal muscle, brain and lungs.
When a diseases or injury effects the cells that contain LDH, the cell lyse, and LDH is spilled into the blood stream, where it is identified in higher than normal levels. The LDH is a measure of total LDH (which is not specific indicator of any one disease affecting any one organ).
Actually five separate fractions (isoenzymes) that make up the total LDH. In general , isoenzyme LDH-1 comes mainly from the heart, LDH-2 comes primarily from the reticuloendothelial system, LDH-3 comes from the lungs and other tissues, LDH-4 comes from the kidney, placenta and pancreas , and LDH-5 comes mainly from the liver and striated muscle.
Normally, LDH-2 makes up the greatest percentage of total LDH. With myocardial injury, the serum LDH level rise within 24-48hr. after a myocardial infarction , peaks in 2 to 3 days and return to normal in 5-10 days (i,e LDH level is useful for delayed diagnosis of patient with Ml).
Normally the LDH-1 /LDH-2 ratio is less than 1. The reversal of the normal ratio (i.e, the ratio is greater than 1) is referred to as a (flipped  LDH)). In acute MI, the flipped LDH ratio usually appears in 12-24 hours and is present within 48 hours in approximately 80% of patients.

Interfering Factors:

1-	Strenous exercise may cause elevated levels of total LDH and specifically LDH-1, -2 and -5
2-	Hemolysis of blood will cause false-elevated LDH levels.
3-	Drugs that may cause increased LDH levels include: alcohol, anesthetics, aspirin, clofibrate, narcotics and procainamide.
4-	Drugs that may cause decreased levels include ascorbic acid.

Specimen:

Collect venous blood, no fasting is required. Serum is separated for test.

Abnormal Finding s:
Increased levels ▲
	Myocardial infraction
	Pulmonary disease (eg, embolism, pneumonia, CHF).
	Hepatic disease (eg, hepatitis, active cirrhosis, neoplasm).
	Red blood cells diseases (eg, hemolytic or megaloplastic anemia).
	Skeletal muscle disease and injury (e.g. muscle dystrophy, recent very strenous exercise, or muscular trauma).
	Renal paranchymal disease (e.g, infraction, glomerulonephritis, acute tubular necrosis, kidney transplantation rejection).
	Intestinal ischemia and infraction.
	Advanced solid tumor malignancies pancreatitis.

Colorimetric Method For The Estimation Of Lactate
Dehydrogenase

Principle:
This method is based on the reduction of pyruvate to lactate in the presence of NADHI by the action of lactate dehydrogenase.
                                    LDH
Pyruvate + NADH + H+                Lactate + NAD+
The pyruvate that remains unchanged reacts with 2,4- dinitrophenyhydrazone, which is determined colorimetrically in an alkaline medium.
Procedure:
Pipette into cuvette
NADH             0.9ml (place in water bath at 37 0C for 2-3 min.) 
Sample             0.1 ml (mix, incubate in water bath at 37 0C for
                    exactly 30 min.)
Colour reagent	1.0 ml (mix; allow to stand at room temperature for 20 min.)
Sod. Hydroxide    (1.6%) 10 ml (mix, allow to stand for 10 min.. read absorbance of samples against D.W)
Enzyme activity is determined and by reference to the calibration curve.
Calibration curve:

Test tubeNo.	1	2	3	4	5	6	
NADH (ml)	0.9	0.7	0.5	0.3	0.2	0.1	
D.W (ml)	0.1	0.3	0.5	0.7	0.8	0.9	
Color reagent(ml)	1.0	1.0	1.0	1.0	1.0	1.0	
Mix and allow to stand for 20 min.	
NaOH (1.6%)	10	10	10	10	10	10	
Mix and allow to stand at room temperature for 10 mm. read absorbance of samples against D.W.	
The corresponding values in U/l are given in a specific table
CRAETINE PHOSPHOKINASE(CPK)

(CREATINE KINASE CPK)

Normal Values:
Adult:	male : 55-170 U/l
Female: 30-135 U/l
Newborn: 68-580 U/l
Isomers:	CPK- MM:100% 
          CPK-MB : 0 %
CPK-BB : 0 %
CPK is found predominately in the in the heart muscle, skeletal muscles and brain. Serum CPK levels are elevated whenever injury occurs to these muscle or nerve cells. CPK levels in plasma can rise within 6 hours after damage.
To test specifically for myocardial muscle injury electrophoresis is performed to detect three CPK iso-enzymes: CPK-BB (CPK1), CPK-MB (CPK2) and (CPK-MY) (CPK3). The CPK-MB isozyme part appears to be specific for myocardial cells. CPK-MB levels rise 3-6 hrs after infraction occurs, wit peak levels at 12-24 hrs and returns to normal 12-48 hrs after infarction.
Sever injury to skeletal muscle can be significant enough to raise the CPK-MB isozyme above normal. To avoid misdiagnosis of myocardial injury, a relative index is calculated to determine whether myocardial injury has occurred. The ratio of CPK-MB / total CPK is calculated to determine this index. An index of > 2.5 is highly suggestive of myocardial injury.
A CPK-MB of a relative index of < 2.5 is indeterminant for myocardial injury.
Because CPK-BB isoenzyme is predominantly found in brain and lung , injury to either of these organs(eg, cerebreovascular accident, pulmonary infarction) is associated with elevated levels of this isozyme.

The CPK-MM isozyme normally comprises almost all of the circulatory total CPK enzymes in healthy people. So when the total CPK level is elevated as a result of increased CPK MM, this would indicate injury or disease of skeletal muscle is present eg, myopathies, vigorous exercise, and multiple IM injection.

Interfering Factors:
1-	IM injections can cause elevated CPK levels.
2-	Strenous exercise and recent surgery may cause increased levels.
3-	Early pregnency may produce decreased levels.
4-	Drugs that may cause increased levels include amphotericin B, ampicillin, some anesthetics-fanticoagulants, aspirin, clofibrate, dexamethasone, furosemide, captopril, alcohol, propanol and morphine.

Specimen:
Collect venous blood sample to separate serum, avoid hemolysis. This is usually done daily for 3 days and then at 1 week when cardiac disease is suspected. Record the exact time and date of venipuncture on laboratory slip, to aid in the interpretation of pattern of enzyme elevation.
Abnormal Findings:
Increased levels of CPK ▲
	Diseases or injury effecting the heart muscle, skeletal muscle and brain.

Increased levels of CPK-BB isozyme 
	Disease effecting the central nervous system.
	Carcinoma of breast and lung.
	Pulmonary infraction.

Increased levels of CPK-MB isozyme 
	Acute myocardial infraction
	Myocarditis
	Ventrical arrhythmias
	Cardiac ischemia.

Increased levels of CPK-MM isozyme 
  Rhadbomyolysis
	Muscular dystrophy
	Myositis
	Recent surgery
	IM injections
	Crush injury
	Shock
	Hypokalemia
  Hypothyroidism

Creatine Kinase Activity Measurement
(Kinetic Method)

Principle:
Kinetic determination of creatinine kinase:
                                CPK
Creatine phosphate + ADP           creatine + ATP
                     HK
Glucose + ATP         D-glucose -6- phosphate + ADP
                                       G-6-P-DH
D-Glucose —6- phosphate + NADP+	  D-gluconate 6-phosphate +
	NADPH+ H+
The catalytic activity of CPK is determined by the measurement of NADPH formation.

Procedure:

Pipette into cuvettes:
Working solution	1000 µl
Incubate for 5 min. at 37 0C. Then add 40 µl of sample mix and incubate at 37 0C for 2 min. read the initial absorbance at 365 nm and started timer immediately. Read again at constant intervals for 3 min.

Calculation:

Calculate the average value of variations of absorbance per mm OD/min then apply the following formula:

   OD nm/min  x 7429 =  U/l

Reagents:
Reagent 1
Imidazol acetate buffer pH 6.5 Magnesium acetate
Reagent 2 (substrate)
D-	glucose
Creatin phosphate
EDTA
N-acetyl cystein
ADP
AMP
Diadenosine pentaphosphate
NADP
Hexokinase (HK)
G-6-	PDH
	
100 mmol/l
10 mmol/l

20 mmol/l
30 mmol/l
2 mmol/l
20 mmol/l
2 mmol/l
5 mmol/l
10 µ mol/l
2 mmol/l
3500 u/l
2000 u/l