WHAT IS CHEMICAL PATHOLOGY?

Chemical Pathology is the study of the changes in the chemical constitution and biochemical mechanisms of the body as a result of disease. In effect it plays an important role in the clarification of the abnormal metabolic processes which underlie the symptoms of an illness. It is a discipline that requires the application of chemistry, physiology and biochemistry. Thus it is variously termed clinical biochemistry, clinical chemistry, physiological chemistry, etc. by various institutions. These technologies differ slightly from each other but they tend to overlap and most people use them interchangeably. Biochemical tests are extensively used for diagnosis, management in an individual patient i.e. monitoring! eg. "iabetes mellitus patients#! for prognosis e.g. cholesterol concentration can predict the ris$ of coronary artery disease# and also screening of an apparently healthy population for pre% clinical disease e.g. phenyl$etonuria, haemoglobinopathies etc#. Objectives of the course- Chemic ! P tho!o"# &' To describe the biochemical and pathophysiological mechanisms of diseases and the biochemical principle underlying the treatment. (' To select the appropriate lab test and interpret the results to confirm or refute provisional clinical diagnosis and to monitor progress during treatment. )' To collect the right type of specimens for laboratory investigations under the right conditions *' To perform specified side%room tests and interpret the results S$ecime% co!!ectio% The most frequently used material for quantitative and chemical investigations are the blood specimen. This is followed by urine and faeces. +ther materials submitted include C,-, saliva, ,weat, pathological fluids obtained by parenthesis, aspirate# calculi, and samples of dietary inta$e. Currently interest is also being focused on the chemical analysis of cellular constituents. ad biopsy materials. /any factors associated with specimen collection can influence the validity and interpretation. These include the collection of the specimen from the wrong patient, incorrect identification of specimens after collection and subsequent handling of specimen prior to analysis.

SI&E 'OOM TESTS Point of care testing P+CT# % 0ear Patient Testing 0PT# % Bedside Testing There is a considerable range of chemical investigations which can be carried out in a doctor.s consulting room, or in a ward side%room, or a in some cases by patients themselves! all that is required is a small wor$ing area provided with a bench%top and a sin$.

Lecture notes for Intro to Chemical Pathology by Max E. Annani-Akollor 2

Chemical investigations that can be performed by clinicians, medical students, nursing staff and other non%laboratory personnel can be subdivided into the following categories1 &# ,imple side%room tests, qualitative or semi%quantitative, mostly performed on urine or faecal specimens (# ,imple side%room test, semi%quantitative or quantitative, mostly performed on blood specimens )# 2uantitative tests performed with equipment that in some cases have microprocessor% controlled operations and which can be quic$ly and reliably operated after very little instructions. There is currently available P+CT glucometers for monitoring blood glucose concentration, P+CT analy3ers for cardiac mar$ers e.g. C4 5 /B isoen3ymes, cardiac troponins the 6eflotron system is now being used for on%site screening diagnosis and therapy monitoring of lipid disorders being the most relevant ris$ factors for atherosclerosis and myocardial infarction. (ACTO'S TO CO)SI&E' P'IO' TO SPECIME) COLLECTIO)

1) &iet*
,ome chemical investigations are liable to interference from dietary constituents e.g. a patient on whom calcium analysis is to be performed should not ta$e dairy products. 7lso sub8ects for 9lucose tolerance test 9TT# should be on their normal glucose diet of about &:;g of carbohydrate a day for at least three days prior to the test. Patients for lipid analysis should be on their normal lipid diet for at a wee$ prior to the test. +, Curre%t me-ic tio% or -ru"s* /any drugs are $nown to interfere with chemical laboratory determinations e.g. +ral contraceptives, cough mixtures. Patients on cephalosporins and <itamin C are most li$ely to present with false 9lycosuria if the method for determining urine glucose is Benedict.s test. It is therefore important that doctors indicate or discuss the details of current medication of patients with lab staff.

3) Time of - #*
,everal Blood constituents show variation with the time of the day e.g. Corticosteroids and iron. -or such blood constituents is imperative that the time at which the sample is ta$en be indicated. (ACTO'S TO CO)SI&E' AT THE TIME O( COLLECTI)G THE LA. SPECIME)/ The $osture of the $ tie%t* ,everal patients with protein bound blood constituents show significant differences in the concentrations between samples collected from ambulatory =upright and reclining patients e.g. albumin, calcium, cholesterol, cortisol, and protein bound iodine. -or example when the posture is changed, from lying to standing, there may be an increase in as much as &)> in the concentration of non?diffusible components of blood within &: minutes due to redistribution of the @C-. It is very important for every health institution to standardi3e procedure for collecting blood specimen.

1) 0e%ost sis*

Lecture notes for Intro to Chemical Pathology by Max E. Annani-Akollor 2

@very effort must be made at avoiding prolonged stasis during venipuncture since it can mar$edly change the concentration of plasma proteins haemoglobin, hormones, calcium and lipids. The combined effect of raised intravenous pressure with its associated local anorexia from sustained occlusion results in the passage of water and small molecules from the lumen of the vein into the surrounding @C-. Intracellular constituents such as potassium and lactic acid lea$ intro the plasma causing falsely elevated results. The best procedure is to remove the tourniquets soon after puncturing. 2) Site of ve%i$u%cture* Ander no circumstances should blood be drawn from receiving an I< infusion since the fluid may not have mixed with the whole blood volume. Blood from the opposite arm usually provides valid results. If there are no available veins, on the arms, then the femoral blood should be ta$en. If this is not possible then blood should be ta$en from a site that is distant from the infusion site or preferably specimens should be obtained from a site that is distant from the infusion site or preferably about &;% (;mls of blood should be aspirated through the needle. If the patient is very sic$ avoid that.# 1, H emo!#sis This must be avoided at all costs because in certain invalidates certain determination owing to the release of erythrocytic contents e.g. 4B, Cactate dehydrogenase, acid Phosphatase, or through colour changes which interfere with photometric determination using shorter wavelengths of the visible spectrum *;;%:;;nm#. Daemoglobin also interferes with specific chemical reactions such as the diacetlyation of bilirubin. The specimen should be collected with only moderate suction. The plunger of the syringe should not be drawn too fast and there should be an easy flow of blood. The needle should be removed from the syringe before the specimen is expelled gently into an appropriate container, leaving behind any froth that may be in the syringe. Tubes into which the blood s expelled should be cleaned and dried. 7ny anticoagulant in the tube should be mixed with the blood by gentle rotation. If the blood is ta$en on the ward, and allowed to stand for more than an hour or two, the effect on plasma constituents is synonymous to haemolysis. 2, H %-!i%" of s$ecime% Identification of specimen is crucial. @ach specimen collected from a patient must be identified in such a way that the report issued from the lab can be matched with the correct set of patients. records. The minimum information on a label should include the patients name, location, ward, and an identifying number as well as the date and the time of specimen collection. The method used for identifying each patient must be unique and reproducible. CHA)GES THAT OCC3' I) THE .LOO& A(TE' COLLECTIO) "iffusion of potassium and some en3ymes such as Cactate "ehydrogenase C"# and 7spartate Transaminase 7,T# through the red cell membrane into the serum of plasma. "ecrease in the concentration of glucose by erythrocytic glycolysis. This can be prevented by the use of fluoride oxalate. Coss of C+(% Chloride ,hift Increase in plasma phosphate due to hydrolysis of organic esters. Coss of activity of liable en3ymes such as Prostatic acid phosphatase Photo degradation of bilirubin by light. This could be prevented by $eeping the sample in aluminum foil.

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Lecture notes for Intro to Chemical Pathology by Max E. Annani-Akollor 2

P'ESE'0ATIO) O( SPECIME) I) T'A)SIT. ,pecimen must be handled properly treated during its transport to the lab and from the time the specimen has been separated until it is analy3ed. -or some tests such as blood gas determination e.g. PC+( P+(, and blood pD, the sample must be $ept at *oC in a refrigerator# from the time the sample is drawn till serum or plasma is separated from cells. Transfer of specimen to the lab must be done by placing the specimen in a container of ice= ice cold water. ,pecimen for hormonal assays such as gastrin, rennin and the parathyroid hormone must and should be separated from the cells in a refrigerated centrifuge. ,pecimen for bilirubin and carotene must be protected from both sunlight and fluorescent light to avoid photo degradation. SEPA'ATIO) A)& STO'AGE O( SPECIME) ,pecimen should be centrifuged with stoppers in place. Closure of the tubes reduces evaporation which occurs in warm centrifuges as a result of the air current set up by centrifugation. ,toppers also prevent aerosol formation from infectious particles and the also maintain anaerobic conditions required for certain determinations such as carbon dioxide and ioni3ed calcium CHA)GES THAT OCC3' &3'I)G 3'I)E SEPA'ATIO) -resh clearly collected urine is required for urine analysis. The container should be dry, lea$ proof, and sufficiently wide nec$ to facilitate the collection of the specimen. The preferred specimen is that passed by the patient first in the morning some of the changes that may occur in the specimen at room temperature are1 &# Brea$down of urea to ammonia by urea splitting en3ymes leading to an increase in the pD of urine. This may lead to the precipitation of calcium and phosphate. (# +xidation of urobilinogen to urobilin )# Atili3ation of glucose by bacteria *# Precipitation of urea crystals in the acidic medium. These changes may held in chec$ by the use of preservatives e.g. E/ DCC or refrigeration. LO)G-TE'M .IOLOGICAL CHA)GES 4, .io v i! bi!it#* the i%f!ue%ce of r ce There is a genetic difference in the plasma concentration of cholesterol, glucose, urea, urate, and bilirubin. Blac$s are $nown to have a higher serum protein than whites. This is probably due to the higher level of gamma globulin in blac$s. The Ig9 is often *;> higher and Ig7 (;> higher in blac$s as compared to whites. The serum albumin of Blac$s is typically lower. The activities of creatinine $inase and lactate dehydrogenase are usually higher in blac$s than whites due to higher s$eletal muscle mass. Blac$s generally have serum al$aline phosphate activity at puberty than white children. 7fter *; years serum cholesterol and triglycerides are higher in whites than in blac$s. 9lucose tolerance is less in Blac$s, 7merican%Indians and @s$imos than in comparable age and sex matched Fhites. +, Ge%-er* Antil puberty there are a few differences in lab data between boys and girls. 7fter puberty, the serum activity of a' al$aline phosphatase, b' aminotranferase, c' creatine $inase d' aldolase are higher in men than women.

Lecture notes for Intro to Chemical Pathology by Max E. Annani-Akollor 2

7fter menopause, the activity of al$aline phosphatase increases until it is higher in women than men. The concentration of albumin, calcium and /g(B are higher in men than in women. But the concentration gamma globulin is less in men than in women. Db and serum bilirubin are less in females. ,erum iron is low during a woman.s reproductive life and her plasma ferritin is usually one ?third that of males. Cholesterol is typically higher in men than women. Plasma concentration of amino acids creatinine, urea, uric acid, are higher in men than women. Dyperuricaemic diseases li$e gout affect more men than women.

3) A"e * 9enerally individuals can be divided into the ff groups a# 0eonates, b# Childhood%Puberty, c# The Goung 7dult 5 d# @lderly 7dult
Neonates1 The serum activity of several en3ymes including Creatine 4inase C4#, 9ammaglutamyl transferase 99T# and 7spartate transaminase are high at birth. But the rise in 7lanine Transaminase is less. The serum bilirubin rises after birth and pea$s after about the )rd to :th day of life. Plasma urea decreases after birth because the infant synthesi3es new proteins. The plasma amino acids are also low as a result of the same reason. Arinary excretion of amino acids may be high because of the immaturity of the tubular reabsorptive mechanisms. The plasma urate is high at birth but the large clearance of urate soon reduces the plasma concentration below the adult value. The serum thyroxine of the healthy newborn is appreciably high. This physiological hyperthyroidism declines over the first year of life. The blood glucose in neonates is low because of small glycogen reserves. Blood lipids are low but reach H;> of adult values two wee$s post%partum. 7t &( hrs post%partum it decreases below the adult value before rising again to a value slightly higher than in an adult. This change is largely due to fluid transfer in and out from the capillaries. Plasma potassium may be as high as I mmol=l at birth 0ormalJ ).:%:.;mmol=l# but rapidly falls thereafter. Plasma Calcium is also initially high but it falls rapidly to ;.):mmol=l 0ormal J(.&% (.Emmol=l# during the first day of life. Childhood- puberty: The serum activity of most en3ymes is low during childhood and rises to adult values by puberty or earlier. 7CP levels in infants rise rapidly within a few days after birth and (%) times the adult value from age & year through puberty. ,erum al$aline phosphatase correlates with s$eletal growth and sexual maturity 7dditional increases occur during puberty, after which values fall to adult range. 7lthough the activity of 7CT at least in males until the middle age KKKKKKK.,.

Lecture notes for Intro to Chemical Pathology by Max E. Annani-Akollor 2

The serum creatinine rises steadily from infancy to puberty prior to the development of s$eletal muscle. The serum urate uric acid# concentration falls from the high level at birth until about I%&; years of sage at which time it begins to rise, especially boys until at about &E years of age. The young adult: The concentration of most constituents remains quite constant between puberty and the middle age in men and puberty to menopause in women. The serum total cholesterol and triglycerides concentration increases in both males and females at a rate of ;.;(mmol per annum to a maximum between :; to E; years. The elderly adult: ,ignificant increase in the plasma concentration of blood constituents occur in women after menopause. Area concentration rises with age. Dormone concentrations are affected by age e.g. T) concentration falls by up to *;> in people over *; years. ,ecretion and concentration of testosterone are decreased in males after :; years. In women, serum concentration of oestrogen decreases by about I;> or more after menopause. SI&E-'OOM TESTS O) 3'I)E 7 wide range of simple tests and observations can be carried out on urine. The most suitable specimens for analy3ers, in general, are fresh specimens that have been passed early in the morning, when the specific gravity is usually at its highest. In performing the tests, careful technique is required with precise adherence to simple instructions if reliable results are to be obtained cleanliness is particularly important. -resh specimens are usually required because of the changes which may occur in urine on standing. These include1 &# "estruction of glucose by bacteria (# Conversion of urea to ammonia by bacteria, with consequent rise in pD and tendency for phosphates to precipitate, and )# +xidation of urobilinogen to urobilin. These changes are slowed down by refrigeration. )O'MAL APPEA'A)CE O( 3'I)E 0ormal freshly passed urine is clear and straw or amber# described as straw Gellow# in colour. "ilute urine however, appears pale in colour and a concentrated one has a dar$ yellow appearance. The yellow colour is due to the pigments urochrome, urobilin, and porphyrine. Fhen normal urine is allowed to stand for some time, white phosphate desist may form if the urine is al$aline dissolved by adding acetic acid or a pin$ uric acid deposit may form if the urine is highly acidic or concentrated disappears on warning#. 7 Lmucus. cloud may also form if normal urine is left to stand. )O'MAL COMPOSITIO) O( 3'I)E

Lecture notes for Intro to Chemical Pathology by Max E. Annani-Akollor 2

The composition of urine usually depends on diet and the metabolic activities of the body.s cells. In health urine usually contains the following1 &. Fater ma$ing up about M;> @lectrolytes, including 0a, 4, /g, Cl 5 DC+) Faste products of metabolism, including urea, uric acid and creatinine.

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3'I)E CHA)GES I) &ISEASE ,ome of the changes which can occur in the volume, appearance, odour, constituents and mass density specific gravity# of urine are as follows1 4/ 0OL3ME CHA)GES 7n increase in the <ol. of urine is called $o!#uri . It occurs in diabetes mellitus due to an increase in the osmolality of the filtrate preventing the normal reabsorption of water +smotic diuresis#. Polyuria also occurs when the secretion of the antidiuretic hormone 7"D# is reduced e.g. in diabetes insipidus. 7 decrease in the vol. of urine excreted is called o!i"uri / It occurs when the renal blood flow and=or glomerular filtration rate are reduced. +ne of the causes of reduced blood flow is low blood pressure hypotension# caused e.g. by severe dehydration or cardiac failure. 7 fall in glomerular filteration rate occurs in acute glomerulonephritis inflammation of the $idney glomeruli# and also in the early stages of acute tubular necrosis. If severe oliguria progresses to a complete cessation of urine flow, this is called %uri and is usually due to severe damage to the renal tubules acute tubular necrosis#. 7cute tubular necrosis may follow any of the conditions which cause severe hypotension or may be due to a direct toxic effect on the tubules by drugs or following an incompatible blood transfusion. +/ APPEA'A)CE CHA)GES The appearance of urine may alter in many conditions including1 Arinary tract infections in which the urine appears cloudy because it contains pus cells and bacteria Arinary schistosomiasis in which the urine often appears red and cloudy because it contains blood haematuria# Blac$water fever and other conditions causing intravascular haemolysis in which the urine appears brown and cloudy because it contains free haemoglobin Naundice in which the urine may appear yellow%brown or green%brown because it contains bile pigments or increased amounts of uroblin oxidi3ed urobilinogen# Bancroftian filariasis in which the urine may appear mil$y%white because it contains chyle digested fats ? mil$y fluid#

1/ COMPOSITIO) CHA)GES 7bnormal chemical constituents in urine include1

Lecture notes for Intro to Chemical Pathology by Max E. Annani-Akollor 2

9lucose glycosuria#, which may be found in the urine of diabetes and occasionally in some healthy individuals 4etones 4etonuria#, which can be found in the urine of untreated diabetes or persons suffering from starvation Protein proteinuria#, which can be found in the urine of persons with urinary schistosomiasis, urinary tract infections, nephrotic syndrome, renal diseases such as pyelonephritis, and renal tuberculosis. It may also be found in urine from pregnant women and sometimes from healthy young adults Bilirubin bilirubinuria# found in hepatic and post hepatic 8aundiced patients Arobilinogen ? conditions causing abnormal haemolysis 9alactose ? persons with the rare congenital metabolic disorder called 9alactosaemia.

'ELATI0E MASS &E)SITY 5CHA)GES I) SPECI(IC G'A0ITY, The normal mass density specific gravity# of urine varies from 4/66+ 7 4/6+2 depending on the state of hydration of the person and the time of day. 0ormal mass density is proportional to the urea and sodium concentrations in the urine /ethods of measuring the concentration of urine include using a urinometer, weighing technique, or specific gravity strip. (L3I&S COMPA'TME)TS8 WATE' 9 ELECT'OLYTES &istributio% of .o-# W ter The total body D(+ of an adult constitutes :;%E;> of the TBF 5 comprises of the IC @.C. fluids. The lymph and transcellular fluids which are the alimentary secretions, cerebrospinal fluid, aqueous humour and synovial fluid are considered part of the extra%cellular fluid. The total volume of water in a I;$g man is about *:l, for a E;$g man is about )El. of this approximately EE> is found in the intracellular compartment, i.e. (:%);l of body water#. This constitutes about );%*;> of the body weight, and ))> is in the extra%cellular compartment, i.e. &)%&El that constitutes about &:%(;> of the body weight. The plasma constitutes )%).:l i.e. :> of the body weight. The lymph and transcellular fluid are each &.:l. *;%:;> of the body weight is made up of solids. +f this &=) is made up fat. In infants, water constitutes about I:%H;> of the body weight i.e. about )l# of which );> is found in the intracellular compartment, and water in extra%cellular fluid constitutes *:> of the body weight. ,olids constitute (:> of body weight.

Lecture notes for Intro to Chemical Pathology by Max E. Annani-Akollor 2