Diagnostic Biochemistry

8/12/2019 Diagnostic Biochemistry

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Practicals

Lab. Practical 1 Blood Glucose Estimation

Lab. Practical 2 Oral Glucose Tolerance

Lab. Practical 3 Glycated Hemoglobin Estimation

Lab. Practical 4 easurment o! Triglycerides

Lab. Practical " easurement o! Total #$olesterol % H&L and L&L

Lab. Practical ' (enal )unction Tests* B+, and #reatinine estimation

Lab. Practical - #reatinine #learance Estimation

Lab. Practical Li/er )unction Tests 1* Bilirubin 0 Total and &irect

Lab. Practical Li/er )unction Tests 2* Enymes0 LT% T and GGT

Lab. Practical 15 lbumin and Total Protein Estimation

Lab. Practical 11 Bone 6ro!ile Testes

Lab. Practical 12 #ardiac 6ro!ile testes

Lab. Practical 13 1" Tutorials


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Introduction To Applied Biochemistry

General Comments about testing

T$ere are so many di!!erent met$ods used to analye di!!erent c$emical com6ounds t$at to state onemet$od o/er anot$er is un!air. not$er issue is t$at your bodyc$emistry c$anges t$roug$out t$e day inres6onse to e7ternal conditions suc$ as e7ercise and internal conditions suc$ as 8idney !unction. T$isma8es com6arisons among /arious tests di!!icult to do. One met$od to lessen t$ese /ariables is to try to$a/e your tests done by t$e same laboratory so t$at com6arisons o! test /alues are 6ossible. 9t is alsobene!icial t$en to $a/e your tests dra:n under t$e same conditions ;!asting


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Practices of Clinical Biochemistry Part II:

Estimation of Blood Glucose

Introduction:

T$e im6ortance o! testing t$e blood glucose le/el comes !rom t$e !act t$at t$e brain cells are/ery de6endent on t$e e7tracellular glucose concentration !or t$eir energy su66ly0 $y6oglycemia is li8elyto im6air cerebral !unctions as :ell as do t$e $y6erglycemia es6ecially o! ra6id onset% :$ic$ can causecerebral dys!unction by a!!ecting e7tracellular osmolarity.

Obecti!es:

>To 8no: t$e di!!erent met$ods !or estimation o! blood glucose>To 8no: t$e 6recautions needed to get accurate results and better inter6retation o!glycemic status in relation to disease condition.

"ethods:any met$ods :ere de/elo6ed to estimate t$e glucose le/el in body !luids among :$ic$ t$e

commonly used no:adays% t$e enymatic met$ods. T$ese met$ods can be summaried andcategoried into

= (eduction met$ods:T$ese met$ods de6end on t$e reducti/e 6ro6erty o! glucose;aldose=

1>Ferriccyanide( Hoffmans) method:using !erricyanide :$ic$ is reduced by t$eglucose .

)e??? )e??;color c$ange !rom yello: to colorless solution t$at :illdiminis$ t$e absorbance measured 6$otometerically =

2#Copper sulfate methods:

Benedict* T$e reagent contains ,a>citrate @,a carbonate :it$ #uO4. 9t gi/escolor acc. To conc. o! glucose ;green>>>>>yello:>>>>>bro:n>>>>>red=.)e$ling * using AOH @,a u * l8aline #u O4?P$os6$omolybdic acid molybdenum blue

by reducing #u2O #uO2

3>Smogi-Nelson method: using rsenomolybdate

,.B. T$e reduction met$ods need al8aline medium @$eatT$ese met$ods are Cualitati/e @ semi>Cuantitati/e.

B= romatic amines met$od:O>toludine ?glucose ;ald$yde= $eat @acidity glucosamine ;colored =


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#= Enymatic met$ods*1-Hexokinase methods(The reference method=.it$ 6re>de6roteiniation o! sam6le or :it$out.Glucose ?TP ?HA&P?G'P

G'P ?,& ?G'P&' P>gluconolactone ?$A%&'&;measured at 345=

2- Glucose oxidase methods*

>TrinderDs ;En.>&ye #olorimetric = met$od* :$ic$ is colorimetric eit$er by s6ectro6$otometer or re!ractrometer

;re!ractrometeric met$ods eit$er in a !ilm !orm 8oda8 Ectac$emF or a stri6 !orm&ry c$emistryF =.

>Ainetic met$od*by measuring t$e increase in absorbance t$roug$ increase in ,&H?H

>Polarigra6$ic met$od* using O2 electrode to detect O2 utiliation.

,.B. GO&


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or8ing reagent ;(=*&issol/e t$e contents o! one /ial ( 2 Enymes in one bottle o! ( 1 Bu!!er.#a6 and mi7 gently to dissol/e contents.T$e reagent is stable 1 mont$ a!ter reconstitution in t$e re!rigerator ;2>I#= or - days at room

tem6erature ;1">2"I#=.

0igns of reagent deterioration:1> Presence o! 6articles and turbidity.2> Blan8 absorbance ;= at "5" nm 5.15.

(eCuirements:

Jam6les*>Blood sam6les

$ole blood

erumPlasma ;:it$ #a.o7alates)res$ urine by double /oid collection tec$niCueKK.>#) collected in sterile clean container and to be done immediately or centri!uged to get cell!ree !luid.

9nstrumentation*>P$otometer adusted on :a/elengt$ "45 nm>#u/ette ;lig$t 6at$= 1 cm>ater bat$ at 3- I#>utomatic 6i6ettes% dis6osable test tubes % rac8s and dis6osable ti6s !or t$e

dis6ensers.

P-OCE%1-E11. ssay conditions*a/elengt$* . . . . . . . . . . . . . .. . "5" nm ;45>""5=#u/ette* . . . . . . . . . . . . . . . . . . . . .. 1 cm lig$t 6at$Tem6erature. . . . . . . . . . . . . . . . . . . 3-I# < 1">2"I#12. dust t$e instrument to ero :it$ distilled :ater.23. Pi6ette into a cu/ette*

8( ;mL=

tandard ;L=am6le ;L=

14. i7 and incubate !or 15 min at 3-I# or 1">25 min at room tem6erature ;1">2"I#=.

1". (ead t$e absorbance ;= o! t$e sam6les and standard% against t$e Blan8.T$e colour is stable !or at least 35 minutes.

CA.C1.ATIO$0


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;= am6le 7 155 ;tandard conc.= M mg


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I #&ypoglycemia * T$e 6atient considered critically $y6oglycemic i!*

$ole Blood glucose le/el 45mg


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- &iabetes ellitus- Hemoc$romatosis- Hy6o8alemia- tress

- P$eoc$romocytoma- nest$esia- Pregnancy- Hy6ert$yroidism- #us$ing disease- Hy6er6ituitarism ;gigantism=

%iscussion:

JPhysiological 2 Biochemical Bac3ground* Glucose metabolism% 9nsulin action and ot$er $ormonal e!!ects on glucose int$e $uman body.

4Pathological 2 %isease Correlation: &iabetes ellitus% #us$ingdisease %Hy6ert$yroidism K..etc

5uestions:

1> $at is t$e basis o! reduction met$ods !or glucose estimation 2> Gi/e s$ort notes on TrinderDs met$od !or glucose estimation.3> $en does a 6erson considered $y6oglycemic4> $at are t$e ty6es o! $y6oglycemia "> Gi/e an account on t$e 6rinci6le o! glucose o7idase met$od !or glucose estimation.

O-A. G.1CO0E TO.E-A$CE TE0T

Introduction:


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On standard oral glucose dose% t$e res6onse o! t$e body regarding t$e absor6tion andmetabolism o! glucose said to be tolerant on meeting t$e normal ele/ation and return. $ereasabnormal and im6ro6er glucose metabolism is termed glucose intolerance. T$is used to diagnosediseases :$ere t$e glucose metabolism is im6aired as in &iabetes mellitus. Oral glucose tolerance test

;OGTT= $as been :idely used as t$e golden standard !or diagnosing diabetes mellitus in clinicallydoubt!ul cases. Lately% t$oug$t% t$e use o! OGTT in 6rimary care $as been Cuestioned !or se/eralreasons. 9t $as lo: re6roducibility and is /ery e76ensi/e. Ho:e/er% !or t$e detection o! diabetes in6regnant :omen% it is still recommended.

Obecti!es:

9t is to 6ractice t$e OGTT and 8no:ing t$e uses and inter6retation regarding t$e diagnosticbene!its o! t$is laboratory test.

Indications:

1> Borderline !asting blood sugar !or Q2 times ;N 115 12"mg &iagnosis o! Gestational &iabetes ;G&= at 24 2 :ee8s o! gestation es6ecially !or t$ose

$a/e a !amily $istory o! diabetes.3> !ter deli/ery !or t$ose :as su!!ering !rom G&.

OGTT )E(periment * 6,:

4Patient preparation )Per7uisites,0

cti/ity>>&onRt smo8e or e7ercise strenuously !or $ours be!ore t$e test or during t$etest.&iet>>Eat a $ig$>carbo$ydrate diet ;Q 1"5 g9n!orm t$e 6erson 6er!orming t$e test to omit any medications listed%as under ta8ing t$ese drugs t$e test results may di!!er ;contrace6ti/es to be sto66ed onecycle be!ore t$e 6er!ormance o! OGTT=.

T$e test must be 6er!ormed at daytime ;morning=.

4 General description of test

Test usually ta8es 3 $ours but can last as long as ' $ours ;e7tended OGTT=.

&rin8 :ater !reCuently during t$e test ;t$e only allo:ed !luid to drin8=.T$e !irst blood sam6le and t$e !irst urine sam6le are collected bet:een - .. and ..% a!ter you $a/e !asted !or 12 $ours.

O6erator gi/es a test load o! glucose% usually -" 155 gram de7trose < 355 ml :ater%lemon !la/ored . &rin8 t$e entire solution in " minutes.Blood and urine sam6les are collected at 35 min.% '5 min.% 5 min.%125 min. and 3 $oursand sometimes immediately a!ter drin8ing oral glucose solution.

%ose of Oral Glucose*&e7trose* 1 1.-" g


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J0amples:Blood sam6les 0 !asting;basal= sam6le% 35min. a!ter oral glucose load% '5min% 5min%125min. ;in e7tended OGTT anot$er 2 sam6les :ill be ta8en at 2S$our and 3 $ours=.

+rine sam6les 0 !irst !asting urine and t$e $ourly collected urine sam6les.

Calculation: t$ere are di!!erent met$ods to calculate and inter6ret t$e glucose le/els ;mgconn criteria (e/ised ummation

)asting Q 1351 6oint >

J9! o! results;) ? '5min. ? 5 min. ? 125min.= Q '55 mg Q 1'"?1

125 min. Q145S 6oint Q 145?1

2 S $our Q135 1 6oint >

#alculation o! (esults

2 3 6oint&iabetic

S > 1 S 6ointus6ect

Uero,on diabetic

3&iabetic

1 2us6ect

Uero,on diabetic

-esults and %iagnosis: Glucose tolerance tests may lead to one of the follo8ing diagnoses:

$ormal -esponse

6erson is said to $a/e a normal res6onse :$en t$e 2>$our glucose le/elis less t$an or eCual to 115 mg


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6erson is said to $a/e im6aired glucose tolerance :$en t$e 2>$ourglucose results !rom t$e oral glucose tolerance test are greater t$an oreCual to 145 but less t$an 255 mg


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%iscussion:

%rugs may affect OGTT resultsm6$etamines.rginine.Benodiae6ines.Beta>adrenergic bloc8ers.#$lort$alidone.#lo!ibrate.

#orticosteroids.&e7trot$yro7ine.&iao7ide.E6ine6$rine.)urosemide.Glucose 9.V.9nsulin.Lit$ium.O in$ibitors.,icotinic acid ;large doses=.Oral contrace6ti/es ;estrogen>6rogestogen combination=.

Oral $y6oglycemics.P$enol6$t$alein.


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P$enot$iaines.P$enytoin.T$iaide diuretics.Triamterene.

Other factors that may affect test resultsEt$anol.

#a!!eine.(ecent in!ection.)e/er.Pregnancy.cute illness.Peo6le o/er age "5 tend to:ard decreasing carbo$ydrate tolerance% :$ic$ may causecon!licting results.#us$ingRs disease% $emoc$romo>cytosis% 6$eoc$romocytoma% inury to central ner/ous system%tumor o! 6ancreas islet cells% malabsor6tion% ddisonRs disease% $y6ot$yroidism% $y6o6ituitarism.

(educed carbo$ydrate inta8e !or se/eral days be!ore t$e test.)ailure to !ollo: dietary and e7ercise restrictions.

51E0TIO$0:

a. $at are t$e indications o! OGTT b. $at are t$e 6rereCuisites o! OGTT c. &ra: a gra6$ o! a normal glucose tolerance.


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Glycated &emoglobins

Introduction:

Glyco$emoglobin ;GHb% glycated $emoglobin% glycosylated $emoglobin= is a generic term !or$emoglobin bound irre/ersibly ;8etoamine !orm= to glucose. O!ten% t$e term is used to mean totalglycated $emoglobin% and sometimes to mean $emoglobin 1c.

Total glycated $emoglobin ;Total GHb= re!ers to all t$e glycated $emoglobins% including glycated$emoglobin /ariants. Total glycated $emoglobin is usually determined by a!!inity c$romatogra6$y orimmunassays.

Hemoglobin 1c ;Hb1c= is t$e maor sub!raction o! t$e glycated normal $emoglobin ;Hb1=.

&etermination o! Hb1c is usually ac$ie/ed by ion>e7c$ange HPL# or gel electro6$oresis.

Obecti!es:

9t is to 8no: t$e im6ortance o! glycated $emoglobin as a long term monitoring test :$ic$ may be usedto $el6 controlling t$e treatment o! diabetes mellitus.

Types of Glycated hemoglobins*

Hb1a1 M Hb ? )ructose>1%'>bis6$os6$ate ;)BP=

Hb1a2 M Hb ? Glucose>'>6$os6$ate

Hb1b M Hb ? Pyru/ic acid

Hb1c M Hb ? Glucose ;,>terminal o! W>c$ain=

Hb1d M Hb ? Glucose ; internal a.a. o! X c$ain=

1sing G&b :

onitoring blood glucose is a 8ey com6onent o! success!ul diabetes management. it$ t$e a/ailability

o! sel!>monitoring and Hb1#testing% laboratory testing !or !asting glucose and 2>$our 6ost>-"g glucoseload s$ould no longer be used routinely to assess glucose control. Laboratory measurement o! glucose%$o:e/er% may be use!ul to /eri!y t$e accuracy o! $ome glucose monitoring eCui6ment or :$en t$ere $asbeen a loss o! diabetic control.

Hb1#measurement 6ro/ides a Cuantitati/e and reliable measure o! glycemic status and control o/er ane7tended 6eriod o! time% t$ereby com6lementing day>to>day monitoring. Hb 1#le/els are a better ;andless e76ensi/e= measure o! long>term glucose control t$an re6eated !asting and 6.c. glucose le/els.O/er t$e li!e o! a red blood cell ;:$ic$ a/erages 125 days=% a !raction o! $emoglobin :ill becomeco/alently bound to glucose and ot$er sugar molecules. T$is reaction occurs non>enymatically and at a

rate :$ic$ is 6ro6ortional to t$e concentration o! glucose in t$e blood. Hb 1#is t$e largest singlecom6onent o! t$ese glycated $emoglobins.


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,.B. Blood Glucose le/el re!lects t$e 6re/ious !e: $ours glycemic state% glycated lbumin re!lects 15 14 days glycemic state% :$ile Hb1c re!lects t$e longest ;2>3 mont$s= glycemic state.

"ethods:

T$ere are currently !our main tec$niCues !or determining glycated $emoglobins*

1. #ation>e7c$ange c$romatogra6$y > se6arates $emoglobins using HPL# based on net c$argeas a result o! glycation0

2. Gel electro6$oresis03. !!inity c$romatogra6$y > se6arates total glycated $emoglobins by binding to solid>6$ase

di$ydro7yborate04. 9mmunoassay > based on binding to s6eci!ic antibodies.

E(periment * 9: Estimation of &bA+c by using affinity chromatography column

Principle:

9n a c$romatogra6$y column% t$e $emoglobins in a $emolysed sam6le is bound by di!!erent a!!inity todi$ydro7yborate. Elution o! Hb1c is carried out by 6$os6$ate bu!!er% :$ile t$e ot$er $emoglobinsse6arate ;elute= a!ter by sodium c$loride solution.

Procedure:

Calculation: Y Hb1c M A+ +;;


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nnual Hb1c Q 1.- times t$e u66er limit o! normal ;13."Y=% suggesting more li8ely occurringcom6lications.

Correlation 8ith "ean Blood Glucose .e!els

single !asting blood glucose measurement only gi/es an indication o! t$e 6atientRs immediate 6ast;last 1 to 2 $ours= condition% and may not re6resent t$e true status o! blood glucose regulation. 9ncontrast% t$e le/el o! glycated $emoglobin is directly related to t$e a/erage glucose concentration o/ert$e li!e>s6an o! t$e $emoglobin in t$e circulation.

Various !ormulae $a/e been 6ro6osed to demonstrate t$e correlation bet:een t$e mean blood glucose;BG= and Hemoglobin 1c ;Hb1c=.

BG mg '

Or% BG mg


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.ipid Profile

Introduction:

ome bene!icial as6ects o! li6ids include t$e !ollo:ing* energy course% !unction and structuralcom6onents o! cell membranes% and 6recursor com6ound to many im6ortant substances suc$as /itamin & and steroid ;se7= $ormones.

it$ e/idence o! a lin8 bet:een ele/ated li6ids and at$erosclerosis ;also 8no:n asarteriosclerosis or at$erot$rombosis=% t$ere is increase interest !rom bot$ t$e medical and laycommunity in t$e battery o! tests commonly ordered as a li6id 6ro!ile. Pre6aration !or $a/ingblood collected !or li6id testing s$ould include a 12>14 $our o/ernig$t !ast.

Obecti!es:

> T$e contribution o! $y6erc$olesterolemia to coronary $eart disease ;#H&= ris8% including t$eim6ortance o! ele/ations in total c$olesterol% L&L c$olesterol% H&L c$olesterol% ratio o! total toH&L c$olesterol.

> T$e classi!ication o! dysli6idemias% including :$o to screen% and $o: o!ten

> T$e a/ailable diagnostic studies and t$eir use% 6articularly determinations o! H&L% L&L andtotal c$olesterol% as :ell as t$e need to test !or ot$er cardio/ascular ris8 !actors .

E(periment * =:Glycerol#Phosphate O(idase method for Triglycerides

P-I$CIP.E O/ T&E "ET&O%

am6le triglycerides incubated :it$ li6o6roteinli6ase ;LPL=% liberate glycerol and !ree !atty acids.Glycerol is con/erted to glycerol>3>6$os6$ate ;G3P= and adenosine>">di6$os6$ate ;&P= by glycerol8inase and TP. Glycerol>3>6$os6$ate ;G3P= is t$en con/erted by glycerol 6$os6$ate de$ydrogenase;GPO= to di$ydro7yacetone 6$os6$ate ;&P= and $ydrogen 6ero7ide ;H2O2=. 9n t$e last reaction%$ydrogen 6ero7ide ;H2O2= reacts :it$ 4>amino6$enaone ;4>P= and 6>c$loro6$enol in 6resence o!6ero7idase ;PO&= to gi/e a red colored dye*

Principle:

Triglycerides ? H2O li6ase Glycerol ? ))

Glycerol ? TP glycerol 8inase Glycerol>3>6$os6$ate ? &P

Glycerol>3>6$os6$ate ? O2 Glycerol>P$os6$ate O7idase &HP ? H2O2

H2O2 ? 4> minoanti6yrine ? #$loro6$enol 6ero7idase uinoneimine

T$e intensity o! t$e color !ormed is 6ro6ortional to t$e triglycerides concentration in t$e sam6le.


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C.I$ICA. 0IG$I/ICA$CETriglycerides are !ats t$at 6ro/ide energy !or t$e cell. Li8e c$olesterol% t$ey are deli/ered to t$e bodyDs

cells by li6o6roteins in t$e blood. diet :it$ a lot o! saturated !ats or carbo$ydrates :ill raise t$etriglyceride le/els. T$e increases in serum triglycerides are relati/ely non>s6eci!ic. )or e7am6le li/erdys!unction resulting !rom $e6atitis% e7tra $e6atic biliary obstruction or cirr$osis% diabetes mellitus isassociated :it$ t$e increase #linical diagnosis s$ould not be made on a single test result0 it s$ouldintegrate clinical and ot$er laboratory data.

P-EPA-ATIO$or8ing reagent ;(=* &issol/e ;= t$e contents o! one /ial ( 2 Enymes into one bottle o! ( 1 Bu!!er.or8ing reagent ;(=* &issol/e ;= t$e contents o! one /ial ( 2 Enymes in 15 mL o! ( 1 Bu!!er.#a6 and mi7 gently to dissol/e contents.( stability* ' :ee8s at 2>I# or 1 :ee8 at room tem6erature ;1">2"I#=.

0A"P.E0erum or $e6arinied or E&T 6lasma1.tability o! t$e sam6le* " days at 2>I# .

P-OCE%1-E

1. ssay conditions*a/elengt$* . . . . . . . . . . . . . .. . . . . "5" nm ;45>""5=#u/ette* . . . . . . . . . . . . . . . . . . . . . . . . 1 cm lig$t 6at$Tem6erature . . . . . . . . . . . . . . . . . . . . 3-I# < 1">2"I#2. dust t$e instrument to ero :it$ distilled :ater.3. Pi6ette into a cu/ette*

Blan8andard( ;mL=1.00tandard ;L=--0

am6le ;L=--

4. i7 and incubate !or " min. at 3-I# or 15 min. at room tem6erature.". (ead t$e absorbance ;= o! t$e sam6les and tandard% against t$e Blan8. T$e colour is stable !or atleast 35 minutes.

CA.C1.ATIO$0A Sample x 200 (Standard conc.) = mg/dL triglycerides in the sampleA StandardConversion factor: mg/dL x 0.0113= mmol/L.

-eference ranges:

,ormal )asting blood triglycerides M '5 1'5 mg


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9t is considered normal as long as it is belo: 255 mgTy6es o! $y6erli6idaemias

51E0TIO$0:

a. Gi/e t$e di!!erent met$ods !or t$e determination o! triglycerides.b. rite a s$ort note on $y6ertriglyceridemia.c. Gi/e t$e u66er cut o!! /alue o! triglyceride !or a diagnosis o!

$y6ertriglyceridemia.


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%ETE-"I$ATIO$ O/ C&O.E0TE-O.:

I$T-O%1CTIO$:

#$olesterol is a :a7y substances used in e/ery cell membrane you $a/e and as a base !orse/eral $ormones. T$e recommended daily allo:ance !or dietary c$olesterol inta8e is 355 milligrams.ost cells $a/e some ca6acity to synt$esie c$olesterol. T$e largest 6ercentage o! synt$esiedc$olesterol is made in t$e li/er. #$olesterol lo:ering medications 6rescribed by 6$ysicians in$ibit t$esynt$esis o! c$olesterol by t$e li/er% t$ereby reducing t$e le/el in t$e blood stream.

OBECTIE0:

T$e estimation o! c$olesterol along :it$ ot$er 6arameters o! li6id 6ro!ile is necessary !or t$eclassi!ication and diagnosis o! li6emias

P-I$CIP.E O/ T&E "ET&O%T$e c$olesterol 6resent in t$e sam6le originates a coloured com6le7% according to t$e !ollo:ingreaction* T$e intensity o! t$e color !ormed is 6ro6ortional to t$e c$olesterol concentration in t$e sam6le

Principle )E(periment *@,:

#$olesterol esters ? H2O#$olesterol esterase #$olesterol ? )

#$olesterol ? O2

#$olesterol O7idase

#$olesterol>3>one ? H2O2

H2O2? 4>P ? P$enolPero7idase uinonimine

C.I$ICA. 0IG$I/ICA$CE#$olesterol is a !at>li8e substance t$at is !ound in all body cells. T$e li/er ma8es all o! t$e c$olesterolt$e body needs to !orm cell membranes and to ma8e certain $ormones. T$e determination o! serumc$olesterol is one o! t$e im6ortant tools in t$e diagnosis an classi!ication o! li6emia. Hig$ bloodc$olesterol is one o! t$e maor ris8 !actors !or $eart disease"%'. #linical diagnosis s$ould not be madeon a single test result0 it s$ould integrate clinical and ot$er laboratory data.

P-EPA-ATIO$or8ing reagent ;(=* &issol/e ;= t$e contents o! one /ial ( 2 Enymes in one bottle o! ( 1 Bu!!er.#a6 and mi7 gently to dissol/e contents.;(= is stable* 4 mont$s at 2>I# or 45 days at 1">2"I#. /oid direct sunlig$t.0A"P.E0erum or 6lasma1%2* tability o! t$e sam6le !or - days at 2>I# or !reeing at 25I# :ill 8ee6 sam6lesstable !or a !e: mont$s.P-OCE%1-E1. ssay conditions*a/elengt$* . . . . . . . . . . . . . .. . . "5" nm ;"55>""5=#u/ette* . . . . . . . . . . . . . . . . . . . . .. 1 cm lig$t 6at$

Tem6erature . . . . . . . . . . . . . . . .. . . . .3-I#


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13. Pi6ette into a cu/ette*

Blan8tandardam6le( ;mL=1.01.01.0

tandard ;L=--10--am6le ;L=----10

14. i7 and incubate !or " min. at 3-I# or 15 min. at room tem6erature.2". (ead t$e absorbance ;= o! t$e sam6les and tandard% against t$e Blan8. T$e colour is stable

!or at least '5 minutes.

CA.C1.ATIO$0 ;am6le= 7 255 ;tandard conc.= M mg


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.%. Cholesterol

P-I$CIP.E O/ T&E "ET&O%&irect determination o! serum L&Lc ;lo:>density li6o6rotein c$olesterol= le/els :it$out t$e need !orany 6re>treatment or centri!ugation ste6s. T$e assay ta8es 6lace in t:o ste6s.

1I Elimination o! li6o6rotein no>L&L#$olesterol esters ? H2O

#$olesterol esterase #$olesterol ? )

#$olesterol ? O2#$olesterol O7idase #$olesterol>3>one ? H2O2

H2O2catalase 2 H2O ? O2

2I easurement o! L&Lc

#$olesterol esters ? H2O#$olesterol esterase #$olesterol ? )

#$olesterol ? O2#$olesterol O7idase #$olesterol>3>one ? H2O2

H2O2? 4>P ? P$enolPero7idase uinonimine ? 4 H2O2

T$e intensity o! t$e color !ormed is 6ro6ortional to t$e L&Lc concentration in t$e sam6le.

C.I$ICA. 0IG$I/ICA$CET$e L&Lc 6article is li6o6roteins t$at trans6ort c$olesterol to t$e cells. O!ten called ]bad c$olesterol^because $ig$ le/els are ris8 !actor !or coronary $eart disease and are associated :it$ obesity% diabetesand ne6$rosis 1%"%'. #linical diagnosis s$ould not be made on a single test result0 it s$ould integrateclinical and ot$er laboratory data.

P-EPA-ATIO$> - + and - 6* re ready to use. >&%.c-55= nm#u/ette* . . . . . . . . . . . . . . . . . . . . .. . .1 cm. lig$t 6at$Tem6erature . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-I#2. dust t$e instrument to ero :it$ distilled :ater.



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Blan8 tandard am6le(1;L! 355 355 355

tandard ;L= >>>>>>> 4 >>>>>>

am6le ;L! >>>>>>> >>>>>>>> 4

4. i7 and incubate !or " min. at 3-I#.". dd*

(2 ;L! 155 155 155

'. i7 and incubate !or " min. at 3-I#. -. (ead t$e absorbance ;=% against t$e Blan8.

CA.C1.ATIO$0;= am6le 7 tandard.conc. M mg


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&%. cholesterol

P-I$CIP.E O/ T&E "ET&O%T$e /ery lo: density ;VL&L= and lo: density ;L&L= li6o6roteins !rom serum or 6lasma are 6reci6itatedby 6$os6$otungstate in t$e 6resence o! magnesium ions. !ter remo/ed by centri!ugation t$e clearsu6ernatant containing $ig$ density li6o6roteins ;H&L= is used !or t$e determination o! H&L c$olesterolC.I$ICA. 0IG$I/ICA$CEH&L 6articles carry c$olesterol !rom t$e cells bac8 to t$e li/er. H&L is 8no:n as ]good c$olesterol^because $ig$ le/els are t$oug$t to lo:er t$e ris8 o! $eart disease. lo: H&L c$olesterol le/els% isconsidered a greater $eart disease ris8. #linical diagnosis s$ould not be made on a single test result0 its$ould integrate clinical and ot$er laboratory data.

Procedure :

PT ? B. sam6le

(T incubation !or 15 min

#entri!ugation !or 15 min. at 4555 r6m

u6ernatant c$olesterol ? #$ol.O7idase reagent >H&L> #$ol. #onc.

0A"P.E0erum or 6lasma1* )ree o! $emolysis. (emo/ed !rom t$e blood clot as soon as 6ossible.

tability * H&L #$olesterol is stable !or - days at 2>I# .

P-OCE%1-E

Precipitation11. Pi6ette into a centri!uge tube*

12. i7 :ell0 allo: to stand !or 15 min at room tem6erature.

23. #entri!uge at 4555 r.6.m. !or 25 min or 2 min at 12555 r.6.m..34. #ollect t$e su6ernatant and test H&Lc.

( ;L=155am6le ;mL=1.5


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Test)ollo:ing t$e #$olesterol reagent instructions.

CA.C1.ATIO$0> it$ )actor*"5" nm am6le 7 325 M mgH&L c$olesterol

5uestions:a. rite a s$ort note on Hy6erli6idemia.b. $ic$ one o! t$e t:o H&L#$ol

rite the e7uation for &%.#cholesterol calculated from TGD Total Chol>2 .%.>

.aboratory -enal /unction Tests


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I# 1rea Estimation 2 Blood 1rea $itrogen )B1$,

Introduction:

Aidney 6roblems are /ery common in clinical medicine. Essentially all seriously sic8 6atients :illneed t$eir 8idney !unction e/aluated during t$e course o! t$eir illnesses.

!ter $istory and 6$ysical e7am are com6lete% t$e initial ste6s in c$ec8ing 6atientsR 8idneys are6er!orming t$e !ollo:ing tests* ;1= urinalysis ;2= serum creatinine ;3= serum urea )bloodurea nitrogenD B1$,. ,e7t% you may c$ec8 ;4= ability to concentrate urine.

Bot$ creatinine and B+, are included on t$e common c$emical 6ro!iles. _ou can c$ec8 t$eability to concentrate urine using a $ygrometer% re!ractrometer% or di6stic8.

Obecti!es:

"ethods:

+# Chemical )direct, method:

+rea ? &iacetyl mono7ime;&= &iacetyl>+rea

&iacetyl>+rea ? )e3??acidic 6H _ello: &iaine ;measured at "45=

6#EnFymatic )indirect, method:

+rea ? H2O+rease #O2? $&9

_ello: Orange

;at "45 nm=

E(periment * )"odified BerthlotHs -eaction,:

NH3

" indi#ator

d$e %dr$

#hemistr$!

&mmonia

dete#ting

ele#trode'ond(#tivit$

di))eren#e bet*een

non-ioni+ed (rea and

ioni+ed ammonia

,ineti#

(lti-

en+$mati#

methodBerthlots

% g2


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P-I$CIP.E O/ T&E "ET&O%+rea in t$e sam6le is $ydrolied enymatically into ammonia ;,H4?= and carbon dio7ide ;#O2=.mmonia ions !ormed reacts :it$ salicylate and $y6oc$lorite ;,a#lO=% in 6resence o! t$e catalystnitro6russide% to !orm a green indo6$enol*

T$e intensity o! t$e color !ormed is 6ro6ortional to t$e urea concentration in t$e sam6le

C.I$ICA. 0IG$I/ICA$CE+rea is t$e !inal result o! t$e metabolism o! 6roteins0 it is !ormed in t$e li/er !rom its destruction.Ele/ated urea can a66ear in blood ;uremia= in* diets :it$ e7cess o! 6roteins% renal diseases% $eart!ailure% gastrointestinal $emorr$age% de$ydration or renal obstruction1%'%-. #linical diagnosis s$ould notbe made on a single test result0 it s$ould integrate clinical and ot$er laboratory data.

Principle*

+rea ? H2O +rease #O2? ,H3

,H3 ? ,a>salicylate ? ,a>$y6oc$lorite ?,a>nito6russide 9ndo6$enol

P-EPA-ATIO$> or8ing reagent ;(=* &issol/e one tablet ( 3 Enymes in one bottle o! ( 1 Bu!!er. #a6 and mi7gently to dissol/e contents.tability* 4 :ee8s in t$e re!rigerator ;2>I#= or - days at room tem6erature ;1">2"I#=.> ( 2 ,a#lO is ready to use*0A"P.E0

1> erum or $e6arinied 6lasma* &o not use ammonium salts or !luoride as anticoagulants.2> +rine* &ilute sam6le 1I# !or " days0

P-OCE%1-E11. ssay conditions*

a/elengt$* . . . . . . . . . . . . . .. . . . .. . . . . . "5 nm#u/ette* . . . . . . . . . . . . . . . . . . . . .. 1 cm lig$t 6at$Tem6erature. . . . . . . . . . . . . . . . . .. . . 3-I# < 1">2"I#

12. dust t$e instrument to ero :it$ distilled :ater.13. Pi6ette into a cu/ette*

8( ;mL=tandard ;L=am6le ;L=

14. i7 and incubate " min at 3-I# or 15 min at room tem6erature ;1">2"I#=.2". Pi6ette*


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( 2 ;mL=

1'. i7 and incubate " min at 3-I# or 15 min at room tem6erature ;1">2"I#=.2-. (ead t$e absorbance ;= o! t$e sam6les and calibrator% against t$e Blan8. T$e colour is

stable !or at least 35 minutes at 1">2"I#.

CA.C1.ATIO$0;= am6le 7 "5 ;tandard conc.= M mg


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%$erhydration;iatrogenic% 6syc$ogenic :ater>drin8ing=

%iscussion*

>P$ysiological @ Bioc$emical Bac8ground*>Pat$ological @ &isease #orrelation*

5uestions:

- rite t$e di!!erent met$ods !or estimation o! blood urea- #alculate B+, on estimation o! blood urea.> ention causes o! $y6eraotemia.


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II# Plasma Creatinine Estimation

Introduction:

#reatinine is t$e end 6roduct o! muscle metabolism. 9t is e7creted t$roug$ t$e 8idneys and c$anges increatinine are an early indicator o! 8idney disease as :ell as being seen in se/ere muscle damage or

:asting diseases or :it$ many medications suc$ as antibiotics. t$is test can be 6er!ormed ons6ecimens dra:n !rom 6atients in eit$er t$e !asting or non !asting state.

"ethods:

1> %irect Chemical methods*a= &affe method* ee t$e 6rinci6le and 6rocedure ;E76eriment =

) 'N method (used in dry chemistry:

#reatinine ?&initrobenoic acid ?al8aline 6H 6ur6lis$ rose

6# Indirect EnFymatic methods:a= &eaminase met$od ;One enyme ste6 met$od=*

#reatinine

&eaminase

met$yl $ydantoin ? ,H3

;detected by di!!erent tec$niCues=

b= mido$ydrolase met$od ; multi>enymatic met$od=*

> #reatinine #reatinine. mido$ydrolase #reatine

> #reatine #reatine 8inase #reatine >6

> #reatine>6 ?TP #reatine ?&P

> &P ? P>enol 6yro/ate ;PEP= TP ?Pyru/ate

> Pyru/ate ? ,&H?H? L&H Lactate ? $A%;:it$ diminis$ed absorbance at 345 nm=

P-I$CIP.E O/ T&E "ET&O%T$e assay is based on t$e reaction o! creatinine :it$ sodium 6icrate as described by a!!. #reatininereacts :it$ al8aline 6icrate !orming a red com6le7. T$e time inter/al c$osen !or measurements a/oidsinter!erences !rom ot$er serum constituents. T$e intensity o! t$e color !ormed is 6ro6ortional to t$ecreatinine concentration in t$e sam6le.


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Principle )affeH "ethod,:

#reatinine ? Picric acid ? al8aline 6H 2%4%' trinitro6$enol

;ano/s8iDs com6le7=measured at "25 nm

C.I$ICA. 0IG$I/ICA$CE#reatinine is t$e result o! t$e degradation o! t$e creatine% com6onent o! muscles% it can be trans!ormedinto TP% t$at is a source o! $ig$ energy !or t$e cells. T$e creatinine 6roduction de6ends on t$emodi!ication o! t$e muscular mass% and it /aries little and t$e le/els usually are /ery stable. 9s e7cretedby t$e 8idneys. it$ 6rogressi/e renal insu!!iciency t$ere is retention in blood o! urea% creatinine and

uric acid. Ele/ate creatinine le/el may be indicati/e o! renal insu!!iciency1%4%". #linical diagnosis s$ouldnot be made on a single test result0 it s$ould integrate clinical and ot$er laboratory data.

P-EPA-ATIO$or8ing reagent ;(=*i7 eCual /olumes o! ( 1 Picric (eagent and ( 2 l8aline reagent.T$e :or8ing reagent is stable !or 15 days at 1">2"I#.

0igns of reagent deterioration:1> Presence o! 6articles and turbidity.2> Blan8 absorbance ;= at 42 nm 1.5.

0A"P.E01> erum or $e6arinied 6lasma.

#reatinine stability* 24 $ours at 2>I#.1> +rine* &ilute sam6le 1I#.

P-OCE%1-E11. ssay conditions*a/elengt$* . . . . . . . . . . . . . . . . . 42 nm ;45>"15=

#u/ette* . . . . . . . . . . . . . . . . . . . . . . . . 1 cm. lig$t 6at$Tem6erature. . . . . . . . . . . . . . . . . . . . . 3-I# < 1">2"I#12. dust t$e instrument to ero :it$ distilled :ater.23. Pi6ette into a cu/ette*

Blan8( ;mL=1.0

tandard ;L=--

am6le ;L=--

1 4. i7 and start sto6:atc$.


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2". (ead t$e absorbance ;1= a!ter 35 seconds and a!ter 5 seconds ;2= o! t$e sam6le addition.3'. #alculate* M 2 1.

CA.C1.ATIO$0

am6le Blan8 7 2 ;tandard conc.= M mg


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Tutorial

Creatinine clearance* is :idely used to a66ro7imate glomerular !iltration. _ou need a timed urinesam6le and a blood sam6le.

T$e clearance o! a substance is t$e /olume o! 6lasma `cleared` o! t$at substance 6er unit time.

Clearance )conc> in urine, ( )urine !olume,

)conc> in plasma, time of urine collection )min>,>

9n deciding $o: to `time` your collection% remember t$at you donRt really need to collect urine !ora !ull 24 $ours. One grou6 got more reliable results by a controlled collection o/er 4 $ours%

monitoring body 6osition ;8e6t t$em lying do:n= and $ydration :it$ body sur!ace areameasurement.

#reatinine clearance is not a 6er!ect measure o! G)(% because some is not !iltered and some issecreted into t$e 6ro7imal tubule. T$ese !ractions tend to cancel eac$ ot$er out in $ealt$% but

:$en G)( dro6s belo: 35 mLric$= can lead to o/erestimates ;maybe35Y= in G)( in renal !ailure 6atients.

(e!erence range !or creatinine clearance is 5>125 mL


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%ETE-"I$ATIO$ O/ 1-IC ACI%

I$T-O%1CTIO$:

+ric acid is a6urine com6ound t$at circulates in 6lasma as sodium urate and is e7creted by8idney. 9t is deri/ed !rom t$e brea8 do:n o! nucleic acids t$at are ingested or come !rom t$e destructiono! tissue cells0 it is also synt$esied in t$e body !rom sim6le com6ounds as s$o:n in !igure.

OBECTIE0:

To 8no: t$e uric acid le/el in t$e body To diagnose a case o! Hy6eruricemia ;Gouts,

"ET&O%0:

Chemical "ethod )Phosphotungestic acid "ethodJ,

EnFymatic "ethod

P-I$CIP.E EnFymatic Colorimetric )1ricase "ethod,K:+ric acid is o7idied by uricase to allantoine and $ydrogen 6ero7ide ;2H2O2=% :$ic$ under t$e in!luenceo! PO&% 4amino6$enaone ;4>P= and 2>4 &ic$loro6$enol sul!onate ;P= !orms a red Cuinoneiminecom6ound*+ric acid ? 2H2O ? O2 +ricase llantoine ? #O2 ? 2H2O2

2H2O2 ? 4>P ? P PO& uinoneimine? 4H2O

T$e intensity o! t$e red color !ormed is 6ro6ortional to t$e uric acid concentration in t$e sam6le.

C.I$ICA. 0IG$I/ICA$CE+ric acid and its salts are end 6roducts o! t$e 6urine metabolism. it$ 6rogressi/e renal insu!!iciency%t$ere is retention in blood o! urea% creatinine and uric acid. Ele/ate uric acid le/el may be indicati/e o!

renal insu!!iciency and is commonly associated :it$ gout #linical diagnosis s$ould not be made on asingle test result0 it s$ould integrate clinical and ot$er laboratory data.

P-EPA-ATIO$or8ing reagent ;(=* &issol/e t$e contents o! one /ial ( 2 Enymes in one bottle ( 1 Bu!!er. #a6and mi7 gently to dissol/e contents. ;(= is stable a!ter reconstitution 1 mont$ at 2>I# or 15 days atroom tem6erature.0A"P.E0 >erum or 6lasma* tability 3>" days at 2>I# or ' mont$s at 25I#.> +rine ;24 $=1* tability 4 days at 1">2"I#% 6H Q. &ilute sam6le 1


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P-OCE%1-E1. ssay conditions*a/elengt$* . . . . . . . . . . . . . . . . . . . ."25 nm ;45>""5=

#u/ette* . . . . . . . . . . . . . . . . . . . . .. . . . . 1 cm lig$t 6at$Tem6erature . . . . . . . . . . . . . . . . . . . . . . 3-I# < 1">2"I#2. dust t$e instrument to ero :it$ distilled :ater.3. Pi6ette into a cu/ette*

Blan8 tandard am6le

( ;ml= 1.5 1.5 1.5

tandard ;L= >>>>>>> 2" >>>>>>>>

am6le ;L= >>>>>>> >>>>>>>>> 2"

4. i7 and incubate !or " min at 3-I# or 15 min at 1">2"I#.". (ead t$e absorbance ;= o! t$e sam6les and tandard% against t$e Blan8.T$e colour is stable !or at least 35 minutes.

CA.C1.ATIO$0erum or 6lasma;= am6le7 ' ;tandard conc.=M mg


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3. E76lain t$e $y6eruricemia.PRERENAL VS. RENAL AZOTEMA !

/ery common clinical 6roblem is to distinguis$ 6rerenal aotemia ;due to s$oc8% de$ydration%

#H) >> also `$e6atorenal syndrome`= !rom renal aotemia ;acute tubular necrosis% `renals$utdo:n`.=

Eit$er could be t$e cause :$en a 6atient $as been $y6otensi/e and no: is aotemic andoliguric. T$e management is di!!erent.

One older tec$niCue is to calculate t$e B1$

9n 6rerenal aotemia% urine sodium is lo: ;t$e 8idney res6onds to lo: blood !lo: by `trying toretain all t$e sodium it can.`=

9n acute tubular necrosis% urine sodium is $ig$er ;t$e renal tubules are unable to concentrate ordilute t$e glomerular !iltrate e!!ecti/ely.=

+rinary sodium under 25 mEC


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JTi6* 9! you obtain urine by sCueeing a dia6er or t$e absor6ti/e balls you 6laced into t$e dia6er% yourestimate o! urine creatinine :ill be lo: because t$ese t$ings absorb creatinine .

1rine Protein Plasma protein

Generally t$e concentration o! 6lasma 6rotein is ele/ated due to de$ydration but can bereduced in 6rimary glomerular diseases suc$ as glomerulone6$ritis and renal amyloidosis.

=> Amylase and lipase

Ele/ated 6lasma li6ase and amylase le/els can be obser/ed in dogs :it$ renal disease%

because t$ese t:o enymes are eliminated by t$e 8idneys.

@> Total -ed blood cell

9n c$ronic renal disease% non>regenerati/e anemia is commonly obser/ed. 9t is mainly due to areduced eryt$ro6oietin le/el secondary to t$e loss o! renal 6arenc$yma.Ot$er causes o! anemiain renal disease include $aemorr$age% s$orter t$e li!e s6an o! eryt$rocytes and bone marro:de6ression.

> $#acetyl#beta#%#glucosaminidase )glucosaminidaseD $AG = is a lysosomal enyme ;

145%555= !ound in serum and urine. +rinary ,G is a 6ro6osed mar8er !or tubular disease%


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es6ecially subtle industrial 6oisoning% acute 6yelone6$ritis% early acute tubular necrosis% andearly trans6lant reection. ;T$ese !unctions are no: largely ta8en o/er by beta>2 microglobulin=.

?> Adenosine %eaminase Binding Proteinis an enyme !rom t$e brus$ borders o! t$e

6ro7imal tubule. Li8e ,G% its 6resence in urine indicates tubular disease.

L> Al3aline phosphatasein urine comes !rom t$e 6ro7imal tubular brus$ border .

M> Beta#6 microglobulin )beta#6#m,is t$e s$ort c$ain o! t$e HL class 9 6roteins. 9n $ealt$% it is!reely !iltered by t$e glomerulus% and !ully reabsorbed by t$e 6ro7imal tubule.

erum beta>2>m $as been suggested as a measure o! glomerular !iltration rate% similarto creatinine. Ob/iously t$is isnRt a good idea !or 6atients :it$ tissue necrosis%lym6$omas% etc.

+rine beta>2>m $as !ound :ides6read acce6tance as an researc$ tool. 9t a66ears i!le/els in t$e serum and glomerular !iltrate e7ceed :$at t$e 6ro7imal tubule canreabsorb ;more t$an 4." mg Tubular functions* 1rinary amino acids andma(imum concentrating abilityaresensiti/e screens !or tubular damage. .ithium clearanceis a researc$erRs :ay o! estimatingdeli/ery to t$e distal tubule.

++> Isotope scanse7ist to com6are t$e !unction o! t$e 8idneys. T$ese may 6ro/e a /aluablesu66lement to t$e intra/enous 6yelogram. ore recently% t$e +6>color %oppler sonogram%

:$ic$ is c$ea6 and 6ortable% $as 6ro/ed e/en more use!ul t$an t$ese scans in trans6lant6atients. ost recent o! all% t$ereRs a Tc scanner t$at monitors glomerular !iltration minute byminute% suitable !or t$e intensi/e care .

+9> Positron emission tomographyis t$e latest :ay o! measuring renal blood !lo:.

0PECI/IC G-AITN O/ 1-I$E

$ile not a `blood test`% c$ec8ing urine s6eci!ic gra/ity 6ro/ides /ery im6ortant in!ormationabout tubular !unction and $ydration.

Peo6le in our culture drin8 relati/ely little !luid. T$us `normal` 6eo6le $a/e !airly concentratedurine ;G greater t$an 1.515=. O! course% t$e same is true o! 6atients in 6rerenal aotemia ;$ig$urinary s6eci!ic gra/ity% lo: or ero urinary sodium=.

Patients :it$ tubular disease ;`renal aotemia`% i.e.% acute tubular necrosis% really bad bilateral6yelone6$ritis or interstitia ne6$ritis% or on diuretics% or :it$ end>stage 8idney= :ill $a/eisost$enuria.

Patients getting lots o! !luid by 9V% or :it$ diabetes insi6idus% or ent$usiastic :ater>drin8ers;ast$matics% craies= :ill $a/e lo: urine s6eci!ic gra/ity.


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0erum and 1rine Osmolality

T$e term osmolality re!ers to t$e osmotic concentration o! a !luid. T$e osmolality o! serum% urine% or anyot$er body !luid de6ends on t$e number o! acti/e ions or molecules in a solution. 9n laboratory re6orts%osmolality is e76ressed as `so many` milliosmoles 6er 8ilogram o! :ater ;mOsm 2" mOsm 1455 mOsm


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.i!er /unction Tests )./T,

Albumin estimation

Introduction:

lbumin is made in t$e li/er and is res6onsible !or maintaining 6ro6er !luid balances. Too little albuminmay result in !luids `lea8ing` out o! t$e blood /essels into surrounding s6aces suc$ as t$e abdomen.&ecreased amounts o! albumin can occur :$en t$e li/er is not ma8ing enoug$ or i! albumin is being lostt$roug$ t$e 8idneys. 9ncreases in albumin do not occur naturally but can be seen in 6atients :$o $adrecei/ed albumin sus6ensions.

"ethods

1>Preci6itation met$od2>Electro6$oresis3>Globulin Try6to6$an content met$od4>9mmunoc$emical met$ods.">&ye binding met$ods

I, Precipitation method

J+se serum onlyJ,ot a66lied !or automationJ+sed no: !or se6aration @ manu!acturing albumin . JPreci6itation is done by salting out of globulins @t$en albumin in t$e su6ernatant is

measured using a 6rotein estimation met$od.

II, ElectrophoresisMMMMMMMMMMM

Je6aration o! lbumin !rom t$e maor classes o! 6rotein in an electrical !ield @ t$e staining Yis

obtained .Calculation of Albumin Albumin Total Protein

J&i!!icult met$od !or utomation.J9t is a Cuantitati/e met$od but tends to o$er estimate luminbecause albumin is t$e best binder o!

staining dyes @t$e band density o! alb. scanned by densitometer.

III, Globulin Tryptophan content method

J9n t$is met$od Try6to6$an content o! t$e globulin is !ist estimated as !ollo:ing0

Glyco7ylic acid ?try6to6$an ;Globulin=Pur6le c$romogen ;measured at "45= #alculation o! lbumin M T.Protein > Globulin


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I, Immune chemical methods

A=>Electro #immune#diffusion )EI%=* #onsidered t$e Reference method%uantitati!e @Manual"Jigration o! 6rotein !ractions in an electrical !ield t$roug$ a medium contains s6eci!ic antibodies toalbumin. T$e $eig$t o! t$e Rocket #reci#itin lineis correlated to albumin conc.J+sed !or serum only.

B=>-adial immune diffusion )-I%=*By measuring t$e diameter o! 6reci6itin ring bet:een albumin @itsantibodies incor6orated in agarose gel.J+sed !or serum @#). Ta8es long time.

C=>Turbidimetry* T$e reaction bet:een albumin and its s6eci!ic antibodies!ormcom6le7es %t$at :illdecrease t$e lig$t transmission t$roug$ t$e reaction 6$ase more t$an !ree albumin ;antigen=.

%=>$ephelometry:>

E,>(adio immune assay ;(9=.

/,>Enyme immune assay ;EL9=

P-I$CIP.E O/ T&E "ET&O%lbumin in t$e 6resence o! bromcresol green at a slig$tly acid 6H% 6roduces a colour c$ange o! t$eindicator !rom yello:>green to green>blue. T$e intensity o! t$e color !ormed is 6ro6ortional to t$ealbumin concentration in t$e sam6le.C.I$ICA. 0IG$I/ICA$CEOne o! t$e most im6ortant serum 6roteins 6roduced in t$e li/er is albumin. T$is molecule $as ane7traordinarily :ide rage o! !unctions% including nutrition% maintenance o! oncotic 6ressure and trans6orto! #a??% bilirubin% !ree !atty acid% drugs and steroids. Variation in albumin le/els indicate li/er diseases%malnutrition% s8in lesions suc$ as dermatitis and burns or de$ydration. #linical diagnosis s$ould not bemade on a single test result0 it s$ould integrate clinical and ot$er laboratory data.

P-EPA-ATIO$

(eagent and calibrator are ready to use

0igns of reagent deterioration:1> Presence o! 6articles and turbidity.2> Blan8 absorbance ;= at '35 nm 5.45.

0A"P.E0erum or 6lasma% !ree o! $emolysis* tability 1 mont$ at 2>I# or 1 :ee8 at 1">2"I#.



43/84

a/elengt$* . . . . . . . . . . . . . .. . '35 nm ;'55>'"5=#u/ette* . . . . . . . . . . . . . . . . . . . . . . 1 cm lig$t 6at$Tem6erature . . . . . . . . . . . . . . . . . . . .. . . . 1">2"I#12. dust t$e instrument to ero :it$ distilled :ater.


an8( ;mL=0

tandard ;L=am6le ;L=

14. i7 and incubate !or 15 min at room tem6erature ;1">2"I#=.2". (ead t$e absorbance ;= o! t$e sam6les and tandard% against t$e Blan8.T$e colour is stable 1 $our at room tem6erature.

CA.C1.ATIO$0;= am6le 7 " ;tandard conc.= M g


44/84

Total protein estimation

Introduction:

Total Protein can be done on eit$er a !asting or non !asting s6ecimen. 9t is usually done as a generalscreening assay since it is com6osed o! t:o maor !ractions ;albumin and globulin=. Ele/ations ordecreases in a total 6rotein must be in/estigated to !ind out :$ic$ o! t$e t:o com6onents is causing t$e6roblem. ince many o! t$e ne7t le/el tests may be re6orted as 6ercentages or ratios% it is necessary to

$a/e t$e total 6rotein rerun at t$e time t$ese tests are 6er!ormed. O/erall% a general re!erence range is".5 > .5 gram#uO4 ;#u>Pr com6le7= et$ods a=Lo:ry or b= Biuret

Normal (,eference),anges:> mmonia ;Plasma on He6arin=M 1">"1 ug T.P Premature babies M begin !rom 3.' g .5 g E7ercise @mbulatory 5." g


45/84

2-5 25 nm

255 >22" nm

J+sed !or olutions rat$er t$an serum.J+sing uart #u/ette ;:it$ no scratc$es=

On using serum % &ilute 1*1555 :it$ ,a#l 5.1" mol


46/84

2-34eldahl +ethod:

JT$e re!erence met$odJusing 6rotein !ree !iltrate.J&e6end on estimation o! 6rotein nitrogen.

Protein

H2O4?#atalyst ?,a>olybdate

$&='

l8aline PH

$&9 H#l;standard ol.= $A%P& '2o7oglutarate ; eit$er=

;Titration=

,eutral PH $A%P ' glutamate;,isseleriation= ;onitor abs.c$ange at 345 nm=

#oncentration o! total 6roteinM detected nitrogen 155


47/84

5-l!aline CS%2 soln0+ethods:

0ample

,aOH ? #uO4>

#o66er '>6e6tide bond 6rotein com6le7

)olin;)enol=? A@,aTartarate;Mcolor stabilier=

#io>#alteau;PT?P$>olbdic a.=

olybdinum blue ? Violet color o! #u>Pr.#om6le7Tungesten blue;at '"5> -"5nm= ;at "4'nm=

#%6,7Hs "ethod I,.Hs "ethod

0ensiti!ity: +;; times BiuretHs good for Pr> 6#+6 g

0pecificity: .ess specific specific

$o> of reagents: 6 -eagents One reagent

%rug Interference %ependence on Tryptophan2Tyrosine)salicylatesDsulfa2tetracyclines, e>g Alb>;>6 Tryptophan by 8t>

Glob>6 Tryptophan by 8t>


48/84


Proteins gi/e an intensi/e /iolet>blue com6le7 :it$ co66er salts in an al8aline medium. 9odide isincluded as an antio7idant. T$e intensity o! t$e color !ormed is 6ro6ortional to t$e total 6roteinconcentration in t$e sam6leC.I$ICA. 0IG$I/ICA$CET$e 6roteins are macromolecular organic com6ounds% :idely distributed in t$e organism. T$ey act li8estructural and trans6ort elements. T$e 6roteins o! t$e serum are di/ide in t:o !ractions% albumin andglobulins T$e determination o! total 6roteins is use!ul in t$e detection o!* > Hig$ 6rotein le/els caused by$emoconcentration li8e in t$e de$ydrations or increase in t$e concentration o! s6eci!ic 6roteins. > Lo:6rotein le/el caused by $emodilution by an im6ared synt$esis or loss ;as by $emorr$age= or e7cessi/e6rotein catabolism. #linical diagnosis s$ould not be made on a single test result0 it s$ould integrate

clinical and ot$er laboratory data

P-EPA-ATIO$T$e reagents are ready to use

0igns of reagent deterioration:1> Presence o! 6articles and turbidity.2> Blan8 absorbance ;= at "45 nm 5.22.

0A"P.E0erum or $e6arinied 6lasma* tability o! t$e sam6le* 1 mont$ at re!rigerator ;2>I#=.

P-OCE%1-E11. ssay conditions*a/elengt$* . . . . . . . . . . . . . . . . . . . . . . . "45 ;"35>""5= nm#u/ette* . . . . . . . . . . . . . . . . . . . . .. . . . . . . . 1 cm. lig$t 6at$Tem6erature . . . . . . . . . . . . . . . . . . . . . . . . . .3-I# < 1">2"I#12. dust t$e instrument to ero :it$ distilled :ater.23. Pi6ette into a cu/ette*

Blan8 tandard am6le

( ;mL= 1.5 1.5 1.5

tandard ;L= >>>>>> 2" >>>>>>>

am6le;L= >>>>>> >>>>>>>>> 2"

14. i7 and incubate " min at 3-I# or 15 min at room tem6erature.2". (ead t$e absorbance ;= o! t$e sam6les and tandard% against t$e Blan8.T$e colour is stable !or at least 35 minutes.

CA.C1.ATIO$0;= am6le7 - ;tandard conc.=M g


49/84

Ad!lts" #.# $ %.3 g/dL&e'orn" .2 $ .1 g/dL

Bilirubin estimation

Introduction:

Bilir(bin is the end "rod(#t o) red #ell l$sis and re#$#ling o) hemoglobin *hi#h is "er)ormed

in the liver. 7he test 8(anti)ies t*o di))erent )orms o) bilir(bin one is the )inal "rod(#t *hile

the other is an intermediate )orm.

7he b(ild (" o) bilir(bin in the blood stream is #alled a(ndi#e and is a general sign o) liver

disease. an$ medi#ations gall bladder disease as *ell as vir(ses s(#h as in)e#tio(s

monon(#leosis and he"atitis *ill have a(ndi#e. an$ in)ants are born *ith less than )(ll$

mat(re livers. &s a #onse8(en#e )or the )irst several da$s the$ ma$ sho* neonatal a(ndi#e

*hi#h is a b(ild (" o) bilir(bin in the blood stream. 7his sho(ld go a*a$ as the liver mat(res.

Bilir(bin determinations are (sed to st(d$ liver )(n#tion and red #ell metabolism

"LN"AL S#N$"AN"E

Bilirubin is a brea8do:n 6roduct o! $emoglobin. 9t is trans6orted !rom t$e s6leen to t$e li/er ande7creted into bile. Hy6erbilirubinemia results !rom t$e increase o! bilirubin concentrations in 6lasma.#auses o! $y6erbilirubinemia* Total bilirubin ;T=* 9ncrease $emolysis% genetic errors% neonatal aundice%ine!!ecti/e eryt$r6oiesis% and drugs. &irect bilirubin ;&=* He6atic c$olestasis% genetic errors%$e6atocellular damage1%"%'. #linical diagnosis s$ould not be made on a single test result0 it s$ould

integrate clinical andother laborator$ data.

"ethods for Bilirubin estimation

+# %irect 0pectrophotometry:

4-estricted to ne8born)Q 6L days, up to 9 monthsD because their serum contains noCarotenes>

Jeasuring absorbency o! Bilirubin in serum at 2 :a/e lengt$s 4"5 @ "45 nm The difference in the absorbance represents bilirubin absorbance )A=@; # A@=; ,

4That is because &emoglobin reads the same at both >. 8hile bilirubin reads at =@; nm>

6# %irect 03in Bilirubinometer:

J(estricted also to ne:borns u6 to 3 mont$s.J,eeds calibration using*>et$yl Orange solution.

Or >)ilter ultilayered color glass. 4"5 > "45 M bsorbance o! bilirubin

9# 0pectral shift change method:J6ectral s$i!t t$roug$ adding $ydro6$obic cationic 6olymer.


50/84

J+sed by AO&A E#T# only.

=#&P.C)&igh Purity .i7uid Chromatography,:

+sing ,ormal ilica #$romatogra6$y .

T$e (e!erence met$od.

@#Colometric methods )E(periment * L,:JT$e most commonly used met$ods.

&e6end on reaction o! bilirubin@ &iaotied ul!anilic acid ;&=

"E..ON 2EE.N$ "> E$%-A00I.#G-O/ ">

4Accelerator: "ethanol or 1rea Caffeine 2$a>BenFoate4P& : $eutral Al3aline)P&+6,4>. : ; @; nm4Color : -ed purple red

Coloumetric -eaction: 4Bil.Glucuronides' %0A AFobilirubin 'H2O?#o2?Hcl

)'irect iliruin)4Bil.Glucuronides'%0A 'ccelerator AFobilirubin

'H2O?#o2?Hcl).otal iliruin)

#Bilirubin O(idase "ethod :

J6eci!ic !or Bilirubin only.JBil. ?Bil. O7idaseBili/erdin ;measured at 45" nm=

PRN"PLE O$ THE METHO%

Bilir(bin is #onverted to #olored a+obilir(bin b$ dia+oti+ed s(l)anili# a#id and meas(red

"hotometri#all$. ;) the t*o )ra#tions "resents in ser(m bilir(bin-gl(#(romide and )ree

bilir(bin loosel$ bo(nd to alb(min onl$ the )ormer rea#ts dire#tl$ in a8(eo(s sol(tion

%bilir(bin dire#t! *hile )ree bilir(bin re8(ires sol(bili+ation *ith dimeth$ls(l)o;!

to rea#t %bilir(bin indire#t!. n the determination o) indire#t bilir(bin the dire#t is also

determined the res(lts #orres"ond to total bilir(bin. 7he intensit$ o) the #olor )ormed is

"ro"ortional to the bilirr(bin #on#entration in the sam"le

P-EPA-ATIO$


51/84

ll t$e reagents are ready to use

0igns of reagent deterioration:1> Presence o! 6articles and turbidity.

2> #olor de/elo6ment in ( 2.

0pecimen Precautions:+# 0erum or Plasma6# A!oid &emolysis9# A!oid light e(posure=# 0torage for 9 days in dar3 2 refrigerator )for months if freeFed at ?;R C,@# 1rine sample either -andom or 6= hrs> not stored for 6= hrs>

P-OCE%1-E

11. ssay conditions*a/elengt$* . . . . . . . . . . . . . K . . . . """ nm ;"35>"5=#u/ette* . . . . . . . . . . . . . . . ... . . K K . . .1 cm lig$t 6at$Tem6erature . . . . . . . . . . . . . . . . . . . . . . . . . . . 1">2"I#

12. dust t$e instrument to ero :it$ distilled :ater.23. Pi6ette into a cu/ette*

Blan8.

Total BBlan8

( 1 ;&= ;mL=----1.?

( 2 ;T= ;mL=1.?1.?--( 3 ;L =--?0--am6le100100100

14. i7 and incubate e7actly !or @ minutes at 1">2"I#.

2". (ead t$e absorbance ;=.

CA.C1.ATIO$0ith /actor:

;;= am6le > ;= am6le Blan8= 7 /actor4 M mg


52/84

- rite causes o! aundice- $at are t$e commonest met$ods o! estimating serum bilirubin in neonates- ention t$e di!!erent causes o! ele/ated direct and indirect bilirubins.

Catalytic )EnFymatic, acti!ities of .i!er )E./T,

+#Gamma Glutamyl Transferase )GGT,

Introduction:

T$is enyme used to metabolie materials in t$e 8idney% li/er% gall bladder% and 6ancreas. 9t is ane7ce6tionally sensiti/e indicator o! stress in t$ese sites. s a conseCuence% /ariations in results may beCuite common. lco$ol consum6tion ;e/en a little= and many medications are t$e c$ie! causes o! t$eses:ings. T$is test is used to !ollo: 8idney% li/er or 6ancreatic !unction

P-I$CIP.E O/ T&E "ET&O% ;Ainetic test ;as=

Gamma>glutamyl trans!erase ;h>GT= catalyses t$e trans!er o! h>glutamyl grou6 !rom h>glutamyl>6>nitroanilide to acce6tor glycylglycine% according to t$e !ollo:ing reaction*

h>>L>Glutamyl>3>carbo7y>4>nitroanilide ? Glycylglycine h>GTh>L>Glutamyl>glycylglycine ? 2>,itro>">aminobenoic acid

T$e rate o! 2>nitro>">aminobenoic acid !ormation% measured 6$otometrically% is 6ro6ortional to t$ecatalytic concentration o! h>GT 6resent in t$e sam6le

C.I$ICA. 0IG$I/ICA$CEGamma>glutamyl trans!erase ;h>GT= is a cellular enyme :it$ :ide tissue distribution in t$e body%6rimarily in t$e 8idney% 6ancreas% li/er and 6rostate.easurements o! gamma>glutamyl trans!erase ;h>GT= acti/ity are used in t$e diagnosis and treatmento! $e6atobiliary diseases suc$ biliary obstruction% cirr$osis or li/er tumours#linical diagnosis s$ould not be made on a single test result0 it s$ould integrate clinical and ot$erlaboratory data.

P-EPA-ATIO$or8ing reagent ;(=*&issol/e one tablet o! ( 2 ubstrate in one /ial o! ( 1 Bu!!er.

#a6 and mi7 gently to dissol/e contents.tability* 21 days at 2>I# or " days at room tem6erature ;1">2"I#=.

0igns of reagent deterioration:1> Presence o! 6articles and turbidity.2> Blan8 absorbance ;= at 45" nm 1.25.

0A"P.E0erum. hjGT is stable !or at least 3 days at 2>I#% $ours at 1">2"I# and 1 mont$ at 25I#.


a/elengt$* . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45" nm


53/84

#u/ette* . . . . . . . . . . . . . . . . . . . . .. . . . . . . 1 cm lig$t 6at$#onstant tem6erature . . . . . . . . . . . . . . .2"I# 1 +


54/84

6# Alanine Transaminase )A.T,

P-I$CIP.E O/ T&E "ET&O%lanine aminotran!erase ;LT= o Glutamate 6yru/ate transaminase ;GPT= catalyses t$e re/ersibletrans!er o! an amino grou6 !rom alanine to X>8etoglutarate !orming glutamate and 6iru/ate.T$e 6iru/ate 6roduced is reduced to lactate by lactate de$ydrogenase ;L&H= and ,&H*

X>8etoglutarate ? L>lanine LT ;GPT= L>Glutamate ? Pyru/ate

Pyru/ate ? ,&H?H? Lactate de$ydrogenase ;L&H= L>Lactate ? ,&

T$e rate o! decrease in concentration o! ,&H% measured 6$otometrically% is 6ro6ortional to t$ecatalytic concentration o! LT 6resent in t$e sam6le

CLINICAL SIGNIFICANCE

T$e LT is a cellular enyme% !ound in $ig$est concentration in li/er and 8idney. Hig$ le/els areobser/ed in $e6atic disease li8e $e6atitis% diseases o! muscles and traumatisms% its better a66lication is

in t$e diagnosis o! t$e diseases o! t$e li/er.$en t$ey are used in conunction :it$ T aid in t$e diagnosis o! in!arcts in t$e myocardium% since t$e/alue o! t$e LT stays :it$in t$e normal limits in t$e 6resence o! ele/ated le/els o! T#linical diagnosis s$ould not be made on a single test result0 it s$ould integrate clinical and ot$erlaboratory data.

P-EPA-ATIO$or8ing reagent ;(=*&issol/e one tablet o! (2 ubstrate in one /ial o! (1.

#a6 and mi7 gently to dissol/e contents.tability* 21 days at 2>I# or -2 $ours at room tem6erature ;1">2"I#=.

0igns of reagent deterioration:1> Presence o! 6articles and turbidity.2> Blan8 absorbance ;= at 345 nm 1.55.

0A"P.E0erum or 6lasma* tability - days at 2>I#..


a/elengt$* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 nm

#u/ette* . . . . . . . . . . . . . . . . . . . . .. . . . . . . . 1 cm lig$t 6at$#onstant tem6erature . . . . . . . . .. . . . . .2"I# < 35I# < 3-I#


55/84

2. dust t$e instrument to ero :it$ distilled :ater or air.3. Pi6ette into a cu/ette*

14. i7% incubate !or 1 minute.2". (ead initial absorbance ;= o! t$e sam6le% start t$e sto6:atc$ and read absorbances at 1>

minute inter/als t$erea!ter !or 3 minutes.3'. #alculate t$e di!!erence bet:een absorbances and t$e a/erage absorbance di!!erences 6er

minute ;k


56/84

9# Aspartate Transaminase )A0T,

Introduction:

s6artate Transaminase ;T= is also 8no:n by its older name% GOT% t$is enyme is needed in t$eutiliation o! energy sources. 9t is !ound in $ig$ concentrations in muscle ;cardiac and ot$ers=% li/er% and

ot$er organs. T$is test usually is ordered to !ollo: cardiac and muscle disease .

T$is test can be 6er!ormed on s6ecimens !rom 6atients :$o are eit$er in a !asting or non !asting. dultre!erence ranges /ary :idely :it$ di!!erent instruments.

P-I$CIP.E O/ T&E "ET&O%s6artate aminotrans!erase ;T= !ormerly called glutamate o7aloacetate ;GOT= catalyses t$ere/ersible trans!er o! an amino grou6 !rom as6artate to X>8etoglutarate !orming glutamate ando7alacetate. T$e o7alacetate 6roduced is reduced to malate by malate de$ydrogenase ;&H= and,&H*

X>8etoglutarate ? L>s6artate T ;GOT= L>Glutamate ? O7aloacetate

O7aloacetate ? ,&H?H? alate de$ydrogenase ;&H= alate? ,&T$e rate o! decrease in concentration o! ,&H% measured 6$otometrically% is 6ro6ortional to t$ecatalytic concentration o! T 6resent in t$e sam6le.

C.I$ICA. 0IG$I/ICA$CET$e T is a cellular enyme% is !ound in $ig$est concentration in $eart muscle% t$e cells o! t$e li/er% t$ecells o! t$e s8eletal muscle and in smaller amounts in ot$er :ea/es.lt$oug$ an ele/ated le/el o! T in t$e serum is not s6eci!ic o! t$e $e6atic disease% is used mainly todiagnostic and to /eri!y t$e course o! t$is disease :it$ ot$er enymes li8e LT and LP.

lso it is used to control t$e 6atients a!ter myocardial in!arction% in s8eletal muscle disease and ot$er#linical diagnosis s$ould not be made on a single test result0 it s$ould integrate clinical and ot$erlaboratory data.

P-EPA-ATIO$or8ing reagent ;(=*&issol/e one tablet o! (.2 ubstrate :it$ one /ial o! (1 Bu!!er.#a6 and mi7 gently to dissol/e contents.tability* 21 days at 2>I# or -2 $ours at room tem6erature ;1">2"I#=.

0igns of reagent deterioration:

1> Presence o! 6articles and turbidity.2> Blan8 absorbance ;= at 345 nm 1.55.


57/84

0A"P.E0erum or 6lasma* tability - days at 2>I#..


a/elengt$* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 nm#u/ette* . . . . . . . . . . . . . . . . . . . . .. . . . . . . .1 cm. lig$t 6at$#onstant tem6erature . . . . . . . . . . . . . . .2"I#


58/84

Bone Profile Testes

Calcium %etermination

Introduction:

#alcium is reCuired !or cell !unction o/erall and !or bone metabolism. Too little calcium gets you eit$er aloss o! tissue !unction or so!t bones ;osteo6orosis= :$ile too muc$ gi/es you tetni ; cardiac arrest and% W> and h>globulins.

>To 8no: t$e status body calcium ;Tetany=

"ET&O%0:

i. #$elation :it$ o>#resol6$t$alein #om6le7one;#olorimetric=ii. tomic absor6tion 6ectro6$otometry ;=iii. )lame 6$otometeri/. 9E

P-I$CIP.E O/ T&E "ET&O%T$e measurement o! calcium in t$e sam6le is based on !ormation o! color com6le7 bet:een calcium

and o>cresol6$talein in al8aline medium*

#a?? ?o>#resol6$talein OH #olored com6le7O>#resol6$t$alein #om6le7 one gi/es /iolet color in al8aline medium.T$e intensity o! t$e colour !ormed is 6ro6ortional to t$e calcium concentration in t$e sam6le

C.I$ICA. 0IG$I/ICA$CE#alcium is t$e most abundant and one o! t$e most im6ortant minerals in t$e $uman body.66ro7imately Y o! body calcium is !ound in bones. decrease in albumin le/el causes a decreasein serum calcium. Lo: le/els o! calcium are !ound in $y6o6arat$yroidism% 6seudo$y6o6arat$yroidism%/itamin & de!iciency% malnutrition and intestinal malabsortion. mong causes o! $y6ercalcemia are

cancers% large inta8e o! /itamin &% en$aced renal retention% osteo6orosis% sarcosidosis% t$yroto7icosis%


59/84

$y6er6arat$yroidism. #linical diagnosis s$ould not be made on a single test result0 it s$ould integrateclinical and ot$er laboratory data.

P-EPA-ATIO$ll t$e reagents are ready to use. To 6re6are monoreagent% mi7 according to t$is 6ro6ortion* "5 /ol. o!(1 and 1 /ol. o! (2.

0A"P.E0 >erum or 6lasma* e6arated !rom cells as ra6idly as 6ossible. Blood anticoagulants :it$ o7alate orE&T are not acce6table since t$ese c$emicals :ill strongly c$elate calcium.> +rine* #ollect 24 $our urine s6ecimen in calcium !ree containers. T$e collecting bottles s$ould contain15 ml o! diluted ,itric acid ;"5Y />>>> 25 >>>>>

am6le ;L= >>>>> >>>>>> 25

4. i7 and incubate !or " min at 3-I# < 1">2"I#. ". (ead t$e absorbance ;= o! t$e sam6les andcalibrator% against t$e Blan8.T$e color is stable !or at least 45 minutes.

CA.C1.ATIO$0

erum and 6lasma ;= am6le 7 15 ;tandard conc.= M mg15." mg 2.' mmol12 mg


60/84

-E01.T0:

C.I$ICA. 0IG$I/ICA$CE:

4 &NPOCA.CE"IA1. Vitamin & de!iciency2. Hy6o6arat$yroidism3. l8alosis ;l8alemia=

4 &NPE-CA.CE"IA1. Hy6er6arat$yroidism2. alignancy o! bone3. T$yroto7icosis4. Vitamin & into7ication". 9dio6at$ic

%I0C100IO$:

TETA$N

P-ECA1TIO$0:

+> /oid /enous stasis ;9ncrease 6rotein @ calcium=6> &o not use contaminated glass :are ;9ncrease calcium=9> Li6emic am6les ;Pre6are blan8 5.5" ml sam6le ? 2." &.,

51E0TIO$0:

1. $at is H_PO#L#E9 rite a s$ort note on Tetany.2. Gi/e t$e 6rinci6le !or t$e determination o! serum calcium by colorimetric met$od.3. Enumerate di!!erent met$ods !or t$e determination o! serum calcium.


61/84

5uantitati!e determination of phosphorus


9norganic 6$os6$orus reacts :it$ molybdic acid !orming a 6$os6$omolybdic com6le7. 9ts subseCuentreduction in al8aline medium originates a blue molybdenum colour.

T$e intensity o! t$e color !ormed is 6ro6ortional to t$e inorganic 6$os6$orus concentration in t$e sam6le

C.I$ICA. 0IG$I/ICA$CE

P$os6$orus is an essential mineral !or tissue bone !ormation and is reCuired by e/ery cell in t$e body

!or normal !unction. 66ro7imately "Y o! t$e body 6$os6$orus is !ound in bone and in teet$.

Lo: le/els o! 6$os6$orus% can be caused by $y6er/itaminosis 5% 6rimary $y6er6arat$yroidism% renal

tubular disorders% antacids or malabsortion.Hig$ le/els o! 6$os6$orus can be caused by diet% bone metastases% li/er disease% alco$ol ingestion%

diarr$ea and /omiting

#linical diagnosis s$ould not be made on a single test result0 it s$ould integrate clinical and ot$er

laboratory data.

P-EPA-ATIO$

or8ing reagent ;(=*

i7 eCual /olumes o! ( 1 ;olybdic= and ( 2 ;#atalyer=

tability* 24 $ at 2>#% 6rotected !rom lig$t.

0A"P.E0

> erum*

)ree o! $emolysis. erum s$ould be remo/ed !rom t$e clot as Cuic8ly as 6ossible to a/oid ele/ation o!

serum 6$os6$orus !rom $ydrolysis or lea8age o! 6$os6$ate 6resent in eryt$rocytes.

tabilitr - days at 2>#.

> +rine ;24 $=*

#ollect t$e s6ecimen into a bottle containing 15 mL o! 15Y !$! $ydroc$loric acid ;H#9= to a/oid

6$os6$ate 6reci6itations. dust to 6H 2. &ilute t$e sam6le %$%&:it$ distilled :ater. i7. ulti6ly t$e

result by 15 ;dilution !actor=. tability* 15 days at 2>Bo#.

P-OCE%1-E

1> ssay conditions*

a/elengt$* .................-15 nm ;'25>-"5=

#u/ette* ............................1 cm. lig$t 6at$

Tem6erature ....................3-# % 1">2"#


62/84

2> dust t$e instrument to ero :it$ distilled :ater.

3> Pi6ette into a cu/ette*

4. i7 and incubate !or 15 min at 3-# or 35 min at room tem6erature ;1">35#=.

"> (ead t$e absorbance ;= o! t$e sam6les and calibrator% against t$e Blan8.

T$e colour is stable !or at least 2 $ours.

CA.C1.ATIO$0

erum

;= am6le 7 " ;#alibrator cone.= M mg


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5uantitati!e determination of al3aline phosphatase )A.P,


l8aline 6$os6$atase ;LP= catalyses t$e $ydrolysis o! 6>nitro6$enyl 6$os6$ate at 6H 15.4% liberating 6>

nitro6$enol and 6$os6$ate% according to t$e !ollo:ing reaction*

6>,itro6$enyl6$os6$ate ? H25 6>,itro6$enol ? P$os6$ate

T$e rate o! 6>,itro6$enol !ormation% measured 6$otometrically% is 6ro6ortional to t$e catalytic

concentration o! al8aline 6$os6$atase 6resent in t$e sam6le

C.I$ICA. 0IG$I/ICA$CE

l8aline 6$os6$atase is an enyme 6resent in almost all :ea/es o! t$e organism% being 6articularly

$ig$ in bone% li/er% 6lacenta% intestine and 8idney.

Bot$ increases and decreases o! 6lasma LP are o! im6ortance

clinically. #auses o! increased 6lasma LP* PagetRs disease o!

bone% obstructi/e li/er disease% $e6atitis% $e6atoto7icity caused by

drugs or osteomalacia. #auses o! decreased 6lasma LP*

#retinism and /itamin # de!iciency1%"%'. #linical diagnosis s$ould

not be made on a single test result0 it s$ould integrate clinical and

ot$er laboratory data.

P-EPA-ATIO$

8or3ing reagent )-,:

&issol/e one tablet o! ( 2 ubstrate in one /ial o! ( 1 Bu!!er.

0A"P.E0

erum or $e6arined. 6lasma +se un$emolyed .serum% se6arated !rom t$e clot as soon as 6ossible.

tability* 3 days at 2>#.

P-OCE%1-E

1>ssay conditions*

a/elengt$* ................. .. . ...................45" nm

#u/ette* . . . . . . . . . . . . . . . . . . . . .. . .. . 1 cm lig$t 6at$

#onstant tem6erature ...................2"# 3&) 3*)


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2>dust t$e instrument to ero :it$ distilled :ater or air.

3>Pi6ette into a cu/ette*

( ;mL= 1.2

;L=am6le 25

4. i7% incubate !or 1 minute.

">(ead initial absorbance ;= o! t$e sam6le% start t$e sto6:atc$ and read absorbances at 1 minute

inter/als t$erea!ter !or 3 minutes.

'>#alculate t$e di!!erence bet:een absorbances and t$e a/erage absorbance di!!erences 6er minute

;


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Cardiac profile Testes

5uantitati!e determination of creatin 3inase )CS,

C.I$ICA. 0IG$I/ICA$CE#reatine 8inase is a cellular enyme :it$ :ide tissue distribution in t$e body. 9ts 6$ysiological role isassociated :it$ adenosine tri6$os6$ate ;TP= generation !or contractile or trans6ort systems.Ele/ated #A /alues are obser/ed in diseases o! s8eletal muscle and a!ter myocardial in!arction#linical diagnosis s$ould not be made on a single test result0 it s$ould integrate clinical and ot$er

laboratory data.

P-I$CIP.E O/ T&E "ET&O%#reatine 8inase ;#A= catalyses t$e re/ersible trans!er o! a 6$os6$ate grou6 !rom 6$os6$ocreatine to&P. T$is reaction is cou6led to t$ose catalysed by $e7o8inase ;HA= and glucose>'>6$os6$atede$ydrogenase ;G'P>&H=*

P$os6$ocreatine ? &P #A #reatine ? TPTP ? Glucose HA &P ? Glucose>'>6$os6$ateG'P ? ,&P? G'P>&H '>P$os6$ogluconate ? ,&PH ? H ?

T$e rate o! ,&PH !ormation% measured 6$otometrically% is 6ro6ortional to t$e catalytic concentration o!#A 6resent in t$e sam6le

P-EPA-ATIO$or8ing reagent ;(=*&issol/e 1 tablet o! ( 2 ubstrate :it$ one /ial o! ( 1.

#a6 /ial and mi7 gently to dissol/e contents.tability* " days at 2>I# or 24 $ours at room tem6erature ;1">2"I#=.

0TO-AGE A$% 0TABI.ITNll t$e com6onents o! t$e 8it are stable until t$e e76iration date on t$e label :$en stored tig$tly closedat 2>I#% 6rotected !rom lig$t and contaminations 6re/ented during t$eir use.&o not use t$e tablets i! a66ears bro8en.

&o not use reagents o/er t$e e76iration date.

0igns of reagent deterioration:1> Presence o! 6articles and turbidity.2> Blan8 absorbance ;= at 345 nm 1.'5.

0A"P.E0erum or 6lasma* tability - days at 2>I#% 6rotected !rom lig$t.T$e creatin 8inase acti/ity decreases 15Y a!ter 1 day at 2>"I# or a!ter 1 $our at 1">2"I#.

P-OCE%1-E1. ssay conditions*a/elengt$* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 nm


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#u/ette* . . . . . . . . . . . . . . . . . . . . .. . . . . . . .1 cm lig$t 6at$#onstant tem6erature . . . . . . . . .. . . . . .2"I# < 35I# < 3-I#

2. dust t$e instrument to ero :it$ distilled :ater or air.3. Pi6ette into a cu/ette*

( ;mL=am6le ;L=

14. i7% incubate !or 2 minutes.2". (ead initial absorbance ;= o! t$e sam6le% start t$e sto6:atc$ and read absorbances at 1

minute inter/als t$erea!ter !or 3 minutes.

3'. #alculate t$e di!!erence bet:een absorbances and t$e a/erage absorbance di!!erences 6erminute ;k


67/84


68/84

4. i7. 9ncubate !or 15 minute.". (ead initial absorbance ;= o! t$e sam6le% start t$e sto6:atc$ and read again a!ter " minutes ;2=.

'. #alculate t$e di!!erence bet:een absorbances * M 2 1.

CA.C1.ATIO$0

7 2" M +B1'"1 M +B

1nits: One international unit ;9+= is t$e amount o! enyme t$at trans!orms 1 mol o! substrate 6erminute% in standard conditions. T$e concentration is e76ressed in units 6er litre o! sam6le ;+B Q 15 +


69/84

Appendi( )+,

Collecti$e 3no"ledge of +ost Common #a0.ests

lood .ests

Glucose:Glucose is the primary blood sugar test and indicates blood sugar le!el at the timeblood 8as dra8n> &igh !alues are seen in diabetics> In addition to pancreatic functionsD Glucosemay be altered by diet and medication> $ormal fasting !alue is ?;#++;>

/ructosamine:Indicates blood sugar le!els o!er the past one to three 8ee3s>

&GB A+C )Glycohemoglobin,:Indicates blood sugar acti!ity for the past three months>

B1$:B1$ stands for Blood 1rea $itrogen and is a 8aste product 8hich should be remo!edfrom the blood by the 3idneys> This test measures 3idney function> $ormal range is #6;>

Creatinine:Creatinine is a 8aste product 8hich should be remo!ed from the blood by the3idneys> This test measures 3idney function> $ormal range is ;>@#+>6>

A0AT


70/84

Total Protein:This is a combination of albumin and globulinD 8hich are proteins> Abnormal!alues occur in li!er disease and poor nutrition> $ormal range is >?#L>;>

Globulin:Globulin helps to combat infection on a normal le!el> It is the total protein !alue minus

albumin !alue> $ormal range is 6>9#=>;>

A $ormal range is ;>L#6>=>

Calcium:The most abundant mineral found in the human body> Abnormalities are found in lossof boneD 3idney disease and lac3 of itamin %> $ormal range is L>@#+;>@>

Phosphorous:-elated to bone acti!ity and usually follo8s e(act opposite of calcium> $ormalrange is 6>@#=>>

1ric Acid:A material 8hichD if in e(cessD can deposit stones in the 3idney or in the oints and

cause gout> $ormal range for males is =>;#?>; normal range for females is 6>;#>;>

Cholesterol:A blood fat related in part to eating animal fats such as eggsD cheeseD creamD li!erDpor3D beefD etc> Increased !alues may indicate a tendency to ha!e hardening of the arteries>alues of +L; or less are associated 8ith least ris3 of heart disease in addition to diet ande(ercise>

.ipoproteins:Proteins combined 8ith lipids that ser!e as carriers of cholesterol> .%. )BadCholesterol, &%. )Good Cholesterol,> The higher the !alueD the less li3ely that cholesteroldeposits are in the blood stream and the less li3ely the chance of coronary heart disease>Cholesterol

Triglycerides:A blood fat related to calories and starch )s8eets, in the diet> &igh le!els canimpair circulation and lead to hardening of the arteries> Alcohol also 8ill increase the !alue> /asto!ernight test for accurate test results> $ormal range for males is =;#+; normal range forfeales is 9@#+9@>

"agnesium:An element absorbed in the intestine> Abnormal le!els are found in pancreatitisDalcoholism and AddisonUs disease> $ormal range is +>L#6>=>

0ocium:A body saltD also termed electrolyte> Sidney disease and some diseases of the adrenal

gland and dehydration can cause abnormal results> $ormal range is +9@#+=@>

Potassium:A body salt or electrolyte found mostly inside of cells> ater pills may lo8erpotassium and increase 3idney damage> $ormal range is 9>#@>;>

Chloride:A body salt $ormal range is+;+#+++>

Co6:Buffer system 8hich assists in the transport of carbon dio(ide from the tissue to the lungs>$ormal range is 6+#9+>

&I antibody:Presence of antibody is associated 8ith ha!ing been infected by the !irus 3no8nto cause AI%0 )Ac7uired Immune %eficiency 0yndrome,>


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P0A:Abnormal le!els in the serum are associated 8ith clinical abnormalities of the prostateDincluding prostate cancer> Because P0A is found in normalD malignant and benign prostatictissueD clinical discrimination is based upon its serum le!el>

Complete lood Count

BC )hite Blood Cells,:hite blood count is the number of 8hite blood cells> It helps combatinfection> $ormal range is =>L#+;>L>

-BC )-ed Blood Cells,:-ed blood count is the number of red blood cells> It relates to anemiaand o(ygen transport> $ormal range for males is =>?#>+ normal range for females is =>6#@>=>

&GB

$ormal range for males is +=#+L normal range for females is +6#+>

"C&


72/84

Appendi( )6,

Common Blood Profiles

(e!erence /alues !or t$e more commonly em6loyed laboratory tests are gi/en in t$e !ollo:ing table. T$ere!erence /alues are in t$e units currently o!ten used and in t$e 9nternational ystem ;9= o! +nits.

Test Current units /actor 0I units

%iabetic 0creen

Glucose% !asting'">115 mg


73/84

lbumin3.">4. gm 5.-4mmol3. g3 gGlutamy trans6e6tidase;GGT= >ale

11>"5 9+

5.-">2.1 15>

Aatal .4 >'- 15>Aatal. mmol


74/84

Other Common 0erum Chemistries3" mol3" mol 6H

-.3">-.4" 1 -.3">-.4"

Blood gases ;arterial% :$oleblood= > PO2

5>15" mmHg 5.133 15.'>14.5 8Pa

Blood gases ;arterial% :$oleblood= > P#O2

3" > 4" mmHg 5.133 4.->'.5 8Pa

Blood gases ;arterial% :$oleblood= >#arbon dio7idecontent

22>31 mmol31 mmol#arotene"5>355 g3. mol145 g1"" g


75/84


76/84

Protein electro6$oresis >Gamma globulin

5.->1.- gm1- gm" g3.32 mol55 6g"1 6mol13." 15>Aatal +6 to 3515>Aatal )emale+6 to 1"5 9+ 1"5>41- 15>Aatal24 9+245 9+


77/84

)H )emale > Luteal+6 to 1" 9+35 9+-5 ngHydro7y6rogesterone >)emale > Luteal

3">25 ng idcycle "5>1"5 9+Postmeno6ausal

Q35 9+ ale +6 to 155 ng


78/84

Progesterone > )emale>)ollicular

+6 to 1"5 ng )emale >Luteal

2"5>255 ng )emale >1sttrimester

1355>"555 ng ale 2>12> ng12 ug )emale 2>25 ng25 ug3.3 ng1555 ng1.5 nmol )emale 1.1>'.3 6g22 15>q nmol4"Y 5.51 5.3">5.4" arb units

Triiodot$yronine ;T3= 155>21' ng3.23 nmol"" ng


79/84

Common 1rinary Chemistries


>aminole/ulinic acid 1.3>-.5 mg 24 nmol ale 1.5>2.5 gm5.51 mmol )emale 5.'>1." gm' mg31. mol- mg


80/84

Common &ematologic 0tudies


#oagulation studies > Bleedingtime

2.">." min '5 1"5>"-5 sec

#oagulation studies >Partialt$rombo6lastin time

2">41 sec 1 2">41 sec

#oagulation studies >Prot$rombin time

15.>13.5 sec 1 15.>13.5 sec

#oagulation studies >T$rombin time

11.3>1." sec 1 11.3>1." sec

Hematocrit > ale 45.->"5.3Y 5.51 5.4>5."53 arb units

Hematocrit > )emale 3'.1>44.3Y 5.3'>5.44 arb units

Hemoglobin > ale 13.>1-.2 gm15.- mmol )emale 12.1>1".1 gm".- 15'< l 15' 4.">".- 15)emale

3.> ".515'< l

3.>".5 15. 15q< l 15'

3.>. 15

Lym6$ocytes

1.2>3.3 15q< l

Leu8ocyte 6ro!ile >ononuclear cells

5.1>5.-15q< l

Leu8ocyte 6ro!ile >Granulocytes

1.>'.' 15q< l


81/84

Platelet count 15 >45" 15q< l 15' 15>45" 15 eancor6uscular $emoglobin 2'.->33.- 6g22.5 mmol eancor6uscular /olume

5.5>-.' cu 5.5>-.' !l

Eryt$rocyte indices > (ed celldistribution :idt$

11.>14.'Y

Eryt$rocyte edimentationrate

+6 to 35 mm


82/84

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-esults*

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CO""E$T0 2 51E0TIO$0

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Diagnostic Biochemistry

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Transcript of Diagnostic Biochemistry