Blood Safety and Clinical Technology

Guidelines on Standard Operating Procedures for CLINICAL CHE IST!"

Contents
Foreword Acknowledgements Preface

SECTION A# GENE!AL INT!O$%CTION

Introduction Introduction to SOP

SECTION B# BIOCHE ICAL PLAS A&SE!%

EAS%!E ENTS IN

Glucose Glucose Oxidase Method Urea Diacet l Monoxime Method !reatinine "affe#s Method !holesterol !holesterol Oxidase Method $iliru%in "endrassik and Grof Method &otal Protein $iuret Method Al%umin ' $!G D e $inding Method &ransaminases ' !olorimetric (nd')oint Method Alkaline Phos)hatase ' P'nitro)henol Method !alcium'O'!resol)hthalein !om)lexone Method Phos)horus ' Stannous !hloride *eduction Method Sodium and Potassium ' Flame )hotometr

SECTION C# BIOCHE ICAL

EAS%!E ENTS IN CS'

!ere%ros)inal Fluid +!SF, !SF glucose Glucose Oxidase Method !SF Protein ' P rogallol D e $inding Method !SF )rotein &ur%idimetr Method

SECTION $# %!INAL"SIS
Introduction -ualitati.e &ests Semi'-uantitati.e &ests Di)stick &echnolog

Glucose ( Glucose O)idase

Introduction

ethod

Glucose is a reducing monosaccharide that ser.es as the )rinci)al fuel of all the tissues/ It enters the cell through the influence of insulin and undergoes a series of chemical reactions to )roduce energ / 0ack of insulin or resistance to its action at the cellular le.el causes dia%etes/ &herefore1 in dia%etes mellitus the %lood glucose le.els are .er high/ Some )atients with .er high %lood glucose le.els ma de.elo) meta%olic acidosis and ketosis caused % the increased fat meta%olism1 the alternate source for energ / 2 )ergl caemia is also noted in gestational dia%etes of )regnanc and ma %e found in )ancreatic disease1 )ituitar and adrenal disorders/ A decreased le.el of %lood glucose1 h )ogl caemia is often associated with star.ation1 h )er insulinaemia and in those who are taking high insulin dose for thera) / Principle of the *ethod

Glucose )resent in the )lasma is oxidi3ed % the en3 me glucose oxidase +GOD, to gluconic acid with the li%eration of h drogen )eroxide1 which is con.erted to water and ox gen % the en3 me )eroxidase +POD,/ 4 amino)hena3one1 an ox gen acce)tor1 takes u) the ox gen and together with )henol forms a )ink coloured chromogen which can %e measured at 565mm/ Speci*en type+ collection and storage Plasma is the s)ecimen of choice for glucose estimation/ Plasma glucose le.els ha.e %een checked to %e 7uite sta%le for 8 hours at room tem)erature +95 ':5 ;!, in the author#s la%orator / It is im)ortant that )lasma should %e se)arated from the cells soon after collection1 )refera%l within 6 hour/ A%out 9 ml of the )atient#s %lood should %e collected % .eni)uncture into a tu%e containing a mixture of )otassium eth lene diaminetetraacetate +(D&A, sodium fluoride at a ratio 6<9 +=>=,/ Fi.e mg of the mixture is ade7uate for 9 ml of %lood/ &he tu%e should %e gentl %ut thoroughl shaken for com)lete mixing/ Preparation of the anitcoagulant *i)ture# 6;; g of )otassium (D&A and 9;; g of sodium fluoride should %e mixed and ground into a fine )owder using a %lender/ &his should )refera%l %e done in a fume cu)%oard/ &he mixture should %e stored in a clean container/ A thin1 long s)atula that can scoo) 5 mg when le.elled1 can %e used for deli.ering the mixture into the tu%e/ !eagents All chemicals must %e Analar grade Phosphate Buffer # ,-- **ol&L. pH /.&o ?;; ml of distilled water add the following in the order< Disodium h drogen )hos)hate dih drate @Aa92PO4 929OB 69/C5 g Anh drous )otassium dih drogen )hos)hate @D29PO4B 4/C5 g Sodium a3ide @AaA:B ;/5 g Add one % one1 dissol.e and finall make u) to 6 litre with distilled water/ Sta%le for :'4 months1 at 9'?;!/ !heck that the final )2 is E/; F ;/;5 with a )2 meter/ Colour !eagent &o 6;;ml of the a%o.e )hos)hate %uffer add the following in the order and then mix to dissol.e< 4 amino )hena3one 68 mg GOD @Sigma G E;68B 6?;; units POD @Sigma P ?95; B 6;; units Phenol 6;5 mg &ween 9; @Sigma P 6:5CB 5;m l *econstitute the )urchased GOD G POD )owder with )hos)hate %uffer/ Dis)ense se)aratel into .ials so that each .ial re)resents the re7uisite num%er of units/ Store the .ials fro3en/ Sta%le for 9 weeks at 9'?;!/ Store in a %rown %ottle/ Ben0oic acid ,g&l. Dissol.e 6/;g of %en3oic acid in water and make u) to 6 litre with water/ &his solution is sta%le indefinitel at room tem)erature/

Stoc1 glucose solution+ , g&l. $efore weighing1 dr the glucose at 8;'?; ;! for 4 hours/ Allow to cool in a dessicator/ Dissol.e 6g of glucose in %en3oic acid solution and make u) to 6;; ml in a .olumetric flask/ Sta%le for six months at room tem)erature +95':5;!,/ $O NOT '!EE2E THE STAN$A!$ 3or1ing glucose standard ,-- *g&dl. Dilute 6; ml of stock glucose +use either a .olumetric )i)ette or a %urette, to 6;; ml with %en3oic acid in a 6;; ml .olumetric flask/ Mix well/ Sta%le for 8 months at room tem)erature +95':5 ;!,/ E4uip*ent+ glass5are and other accessories *efer to Section A +9,1 Introduction to SOP Procedure &he )rotocol of the )rocedure is descri%ed %elow/ $ilution of standards 6S,7S89+ Test : ;C Pi)ette the following into a))ro)riatel la%elled 6: x 6;; mm tu%es

S,
Distilled =ater +ml, 6;; mg>dl glucose +ml, &est sam)le >-! +ml, Mix well 6/C ;/6 '

S<
6/? ;/9 '

S=
6/E ;/: '

S>
6/8 ;/4 '

S8
6/5 ;/5 '

Test
6/C ' ;/6

;C
6/C ' ;/6

Colour de?elop*ent Pi)ette the following into another set of a))ro)riatel la%elled tu%es/

Blan1
!olour reagent +ml, 6/9 Distilled water +ml, ;/6 Diluted Standards +ml,

S,
6/9

S<
6/9 '

S=
6/9 ' ;/6 '

S>
6/9 ' ;/6 '

S8
6/9 ' ;/6 '

Test
6/9 ' ' ;/6

;C
6/9 ' ' ;/6

;/6 Diluted &est Sam)le>-! +ml, ' '

;/6 '

Mix all tu%es well/ Incu%ate at :E;! in a water%ath for 65 minutes/ *emo.e from water%ath and cool to room tem)erature/ Set the s)ectro)hotometer> filter )hotometer to 3ero using %lank at 56; nm> green filter and measure the a%sor%ance of Standards1 &est and -!/ &his )rotocol is designed for s)ectro)hotometers > filter )hotometer that re7uire a minimum .olume of reaction mixture in the cu.ette of 6 ml or less/ (conomical use of reagents is )ossi%le with this )rotocol1 thus the cost )er test can %e ke)t to the minimum/ 2owe.er1 if a la%orator em)lo s a )hotometer re7uiring a large .olume of the reaction mixture for measurement1 .i3/ 5 ml1 it is ad.isa%le to increase the .olume of all reagents mentioned under &a%ulation H+%, !olour de.elo)mentH )ro)ortionatel / Calculation and cali@ration graph Since the )rotocol for standard tu%e S6 and test is identical1 the standard S6 will re)resent a concentration of 6;; mg>dl/ &he glucose concentrations re)resented % other standard tu%es are S9 I9;;J S: I :;;J S4 I4;; G S5 I 5;; +mg>dl,/ Plot the a%sor%ance .alues of standards against their res)ecti.e concentrations/ &he measura%le range with this gra)h is from 6; to 5;; mg>dl/ Plot a%sor%ance .alues of &est>-! on the cali%ration gra)h and read off the concentrations/ Once linearit is )ro.ed1 it is not necessar to )re)are the standard gra)h e.er time that )atients# sam)les are anal sed/ It will %e ade7uate if standard S9 is taken e.er time and )atients# results are calculated using the formula < &est a%sor%ance '''''''''''''''''''''' x 9;; mg>dl Standard a%sor%ance

Analytical relia@ilities *efer to pages /7A of section , 6General Introduction9 on the use of internal -! and

inter)retation of dail -! data +for releasing )atients# results,/ Since glucose is the most common anal te measured in a la%orator 1 it is ad.isa%le to include an internal -! +normal -! )ool, with e.er %atch of sam)les anal sed in the da 1 irres)ecti.e of the num%er of sam)les in a %atch/ Further1 e.en when a single sam)le is anal sed as an Hemergenc H sam)le at an time of the da or night1 it is essential to include an internal -!/ From the -! results o%tained for the da 1 mean1 standard de.iation and K!L can %e calculated to ensure that within-day precision is well within the acce)ta%le limit1 i/e1 5K/ &he mean .alue of internal -! for the da can %e )ooled with the )receding 6; or 9; mean .alues o%tained in the )re.ious da s1 and betweenday precision can %e calculated and ex)ressed as K !L/ (nsure that this is well within the acce)ta%le limit1 i/e1 ?K/ At least once a da anal se another -! serum from either a low -! or high -! )ool/ HAssa edH -! sera with stated .alues +ranges, are a.aila%le from se.eral commercial sources1 .i3/ $oehringer Mannheim1 $io*ad G *andox/ If a laboratory uses QC sera from a commercial source, it is important that the company certifies that their QC materials are traceable to international reference materials. Ha0ardous *aterials This procedure uses sodium azide and phenol, which are poisonous and caustic. Do not swallow, and avoid contact with skin and mucous membranes !eference range and clinical interpretation Plasma glucose< Fasting< E; 66; mg>dl Post')randial< ?; 64; mg>dl *andom< 8; 64; mg>dl (le.ated )lasma glucose le.els are ex)ected in a .ariet of clinical conditions1 es)eciall dia%etes mellitus1 !ushing#s s ndrome and h )eradrenalism/ Decreased )lasma glucose le.els are o%ser.ed in h )er'insulinism1 anti'dia%etic treatment and h )oadrenalism/ Li*itations An sam)le that gi.es aglucose .alue M 5;; mg>dl should %e diluted 6<9 with ;/CgK sodium chloride solution and the correct .alue o%tained % multi)l ing the result % :/ At high )lasma le.els1 uric acid1 glutathione and %iliru%in ma interfere with the assa % causing a decrease in glucose .alues/ Ascor%ic acid will decrease glucose .alues % retarding colour de.elo)ment/ Do not re)ort results from s)ecimens with sus)ected interference/ Inform the re7uesting )h sician of the )ro%lem/ !eferences 6/ 9/ &rinder1 P/ +6C8C,/ Annals of !lin/ $iochem/ 8< 94 9E/ $arham D and &rinder P/ +6CE9,/ Anal st CE< 649 645

%rea ( $iacetyl *ono)i*e *ethod

Introduction Urea contri%utes most of the %od #s non')rotein nitrogen1 accounting for a%out 45K of the total/ It is the maNor end')roduct of )rotein cata%olism in humans/ It is s nthesi3ed in the li.er1 released into %lood circulation and excreted % the kidne s/ Measurement of urea in %lood is a useful indicator of renal and he)atic integrit / Principle of the *ethod Urea reacts directl with diacet l monoxime under strong acidic conditions to gi.e a ellow condensation )roduct/ &he reaction is intensified % the )resence of ferric ions and thiosemicar%a3ide/ &he intense red colour formed is measured at 54;nm> ellow green filter/ Speci*en types+ collection and storage Serum is the s)ecimen of choice/ Store sam)les for no longer than ? hours at room tem)erature +95' :5;!, and E da s at 9'?;!/ For a longer duration1 store in the free3er/ If the sam)les show e.idence of %acterial contamination1 do not use these for urea estimation/ Plasma could also %e used for urea estimation/ !eagents All chemicals must %e Analar grade/ Stoc1 acid reagent Dissol.e 6/;g of ferric chloride hexah drate in :; ml of distilled water/ Add 9; ml ortho)hos)horic acid and mix/ Store in a %rown %ottle at room tem)erature +95':5 ;!,/Sta%le for 8 months/ i)ed acid reagent Add slowl 6;; ml of !onc/ 29S;4 to 4;; ml distilled water taken in a 6'litre flat'%ottom conical flask ke)t in an icecold water%ath/ Mix well and add ;/:ml of stock acid reagent/ Mix and store in a %rown %ottle at room tem)erature +95':5;!,/ Sta%le for 8 months/ Stoc1 colour reagent ( A Dissol.e 9g diacet l monoxime in distilled water and make the .olume u) to 6;; ml in a .olumetric flask/ Store in a %rown %ottle at room tem)erature +95':5;!,/ Sta%le for 8 months/ Stoc1 colour reagent 7 B Dissol.e ;/5 g thiosemicar%a3ide in distilled water and make u) to 6;; ml in a .olumetric flask/ Store in a %rown %ottle at room tem)erature +95':5;!,/ Sta%le for 8 months/ i)ed colour reagent Mix :5 ml of stock colour reagent A with :5 ml of stock colour reagent $ and make u) to 5;; ml with distilled water/ Store in a %rown %ottle at room tem)erature +95':5 ;!,/ Sta%le for 8 months/ Stoc1 urea standard =eigh 6/;g of anal tical'grade urea and dissol.e in 6;;ml of %en3oic acid +6g>dl,/ Use a 6;;ml of .olumetric flask for )re)aring this/ Store at room tem)erature +95':5 ;!,/ Sta%le for 8 months/ 3or1ing standard 8-*g&dl

Dilute 5/;ml of stock urea standard to 6;; ml with %en3oic acid/ Store at room tem)erature +95' :5;!,/ Sta%le for 8 months/ E4uip*ent+ glass5are and other accessories *efer to Section A +9,1 Introduction to SOP Procedure &he )rotocol of the )rocedure is descri%ed %elow/ $ilution of Standards 6S,7S=9+ Test : ;C Pi)ette the following into a))ro)riatel la%elled 6: x 6;; mm tu%es

S,
Distilled =ater +ml, 6/C

S<
6/?

S=
6/E

Test
6/C

;C
6/C

5; mg>dl Urea +ml,

;/6

;/9

;/:

'

'

&est sam)le >-! +ml, Mix Well Colour $e?elop*ent

'

'

'

;/6

;/6

&he colour reagent is )re)ared fresh at the time of anal sis % reagent and mixed colour reagent in the ratio 6<6<6/

mixing distilled water1 mixed acid

Pi)ette the following into another set of a))ro)riatel la%elled 6? x 65; mm tu%es/

Blan1
!olour reagent +ml, :/6

S,
:/;

S<
:/;

S=
:/;

Test
:/;

;C
:/;

*es)ecti.e diluted standard ml,

'

;/6

;/6

;/6

'

'

Diluted test >-! +ml,

'

'

'

'

;/6

;/6

Mix all tu%es well/ Dee) them in a %oiling water%ath for 65 minutes/ *emo.e from water%ath and cool the tu%es for 5 minutes/ Set the s)ectro)hometer>filter )hotometer to 3ero with %lank at 54;nm> ellow green filter and measure the a%sor%ance of the other tu%es/ Calculation and cali@ration graph !oncentration of standards< S6 I 5; mg>dl

S9 I 6;; mg>dl S: I 65; mg>dl Plot the a%sor%ance .alues of standards against their res)ecti.e concentrations/ &he measura%le range with this gra)h is from 6; to 65; mg>dl/ A cali%ration gra)h should %e constructed whene.er a new set of reagents is )re)ared/ Plot a%sor%ance .alues of test>-! on the cali%ration gra)h and read off the concentrations/ Once linearit is )ro.ed1 it will %e enough if S: is set u) e.er anal sed and the results calculated using the formula< A%sor%ance of test Urea in test sam)le I ''''''''''''''''''''''''''''' x 65; mg>dl A%sor%ance of Standard time that )atients# sam)les are

Analytical relia@ilities *efer to pages /7A of section , 6General Introduction9 for the use of internal -! and inter)retation of dail -! data +for releasing )atients# results,/ Since urea is one of the most common anal tes measured in a la%orator after glucose1 it is recommended that an internal -! +normal -! )ool, %e included with e.er %atch of sam)les anal sed in the da 1 irres)ecti.e of the num%er of sam)les in a %atch/ Further1 e.en when a single sam)le is anal sed as an Hemergenc H sam)le at an time of the da or night1 it is essential to include an internal -!/ From the -! results o%tained for the da 1 mean1 standard de.iation and K!L can %e calculated to ensure that within-day precision is well within the acce)ta%le limit1 i/e/ 4K/ &he mean .alue of internal -! for the da can %e )ooled with the )receding 6; or 9; mean .alues o%tained in the )re.ious da s and %etweenda )recision can %e calculated and ex)ressed as K !L/ (nsure that this is well within the acce)ta%le limit1 i/e/ ?K/ At least once a da anal se another -! serum from either a low -! or high -! )ool/ HAssa edH -! sera with stated .alues +ranges, are a.aila%le from se.eral commercial sources1 .i3/ $oehringer Mannheim1 $io*ad G *andox/ If a laboratory uses QC sera from a commercial source, it is important that the company certifies that

their QC materials are traceable to international reference materials. Ha0ardous *aterials Most of the chemicals used in this method are acids. Care should therefore be taken to avoid mouth pipettin and contact with skin. !eference range and clinical interpretation Serum> Plasma Urea OO//65 4; mg>dl (le.ated serum urea le.els ma %e due to )re'renal1 renal or )ost'renal etiolog / Pre'renal causes could %e cardiac related or due to increased )rotein cata%olism1 and deh dration/ *enal causes include glomerulone)hritis1 chronic ne)hritis1 ne)hrotic s ndrome and other kidne disease/ Post'renal causes include o%struction of the urinar tract/ Decreased serum urea le.els could %e due to )regnanc 1 intra.enous infusion1 low antidiuretic hormone secretion1 low )rotein intake1 se.ere li.er diseases1 in%orn errors of urea c cle and SIAD2 +S ndrome of ina))ro)riate AD2 secretion,/ Li*itations S)ecimens with gross icterus cannot %e assa ed as it will cause falsel ele.ated urea .alues/ Do not re)ort results from s)ecimen with sus)ected interference/ Inform the re7uesting )h sician of the )ro%lem/ !eferences 1. 2. = %enga1 D/*/1 Di Glorgio1 "/G Pileggi1 L/"/ +6CE6,/ !linical !hem/1 6E1 ?C6'?C5/ Seaton1 $ G Ali/ A +6C?4, Med/ 0a%/ Sciences 461 :9E ::8/

Creatinine ( BaffeCs *ethod

Introduction !reatinine is a waste )roduct formed in muscle from a high'energ storage com)ound1 creatine )hos)hate/ !reatine )hos)hate can %e stored in muscle at a))roximatel four times the concentration of adenosine tri)hos)hate/ In muscles it s)ontaneousl undergoes degradation to form a c clic anh ride'creatinine/ &he %lood concentration of creatinine and its excretion in urine are remarka%l constant in normal indi.iduals/ &herefore serum creatinine le.el is used as an indicator for assessing kidne function/ Principle of the *ethod !reatinine )resent in serum or )lasma directl reacts with alkaline )icrate resulting in the formation of a red colour1 the intensit of which is measured at 5;5nm>green filter/ Protein interference is eliminated using sodium laur l sul)hate/ A second a%sor%ance reading after acidif ing with :;K acetic acid corrects for non's)ecific chromogens in the sam)les/ Speci*en type+ collection and storage Serum or )lasma can %e used/ A.oid using haemol sed or li)aemic sam)les/ Sta%le for 69 hours at room tem)erature +95':5;!,1 one week at 9'?;! and for : months at 9;;!/ !eagents

All chemicals must %e Analar grade !eagent A Into 4;;ml of distilled water taken in a 5;; ml %eaker add 4/4g of AaO2/ Mix to dissol.e1 then add C/5g trisodium )hos)hate @Aa:PO46929OB1 dissol.e and then add C/5g of sodium tetra%orate @Aa9$4OE6;29OB/ After dissol.ing check that the )2 is a%o.e 6;1 adNust if necessar % the dro)wise addition of 6M AaO2/ &ransfer to a 5;; ml .olume flask and make u) to 5;;ml with distilled water/ Mix well/ Sta%le for : months at 9'?;!/ !eagent B Dissol.e 9;g sodium laur l sulfate in a final .olume of 5;;ml distilled water/ Sta%le for 8 months at room tem)erature +95':5;!,/ !eagent C Picric acid su))lied commerciall contains 5;K % weight of water to ensure safet in transit/ &herefore the amount of )icric acid weighed out should %e )ro)ortionall more than the amount of the re7uired anh drous )icric acid/ For reagent !1 4/8g of anh drous )icric acid is re7uired/ &herefore weigh a))roximatel E/;g %ut not less than 8/;g moist )icric acid and add to 5;;ml of distilled water taken in a .olumetric flask1 mix and lea.e o.ernight at :E; !/ &hen filter and store in %rown glass %ottle at room tem)erature +95':5;!,/ Sta%le for 6 ear/ 3or1ing reagent At the time of anal sis freshl mix e7ual .olumes of the a%o.e three reagents/ After use discard an lefto.er working reagent/ Stoc1 creatinine standard ,--*g&dl Dissol.e 6;; mg of )ure creatinine in ;/6 M 2!l and make u) to 6;; ml with ;/6 M 2!l in a .olumetric flask/ Sta%le for 8 months at 9'?; !/ 3or1ing creatinine standard Dilute 91 41 8 and ? ml of stock creatinine standard each to 6;; ml with ;/6 M 2!l to get creatinine concentrations of 91 41 8 and ? mg>dl1 res)ecti.el / Sta%le for 8 months at 9'?;!/ =-D 6E&E9 Acetic acid Dilute :;ml of glacial acetic acid to 6;;ml with distilled water/ Sta%le for : months at room tem)erature +95':5; !,/ E4uip*ent+ glass5are and other accessories *efer to Section A +9,1 Introduction to SOP/ Procedure &he )rotocol of the )rocedure is descri%ed %elow/

Pi)ette the following into a))ro)riatel la%elled 6? x 65; mm tu%es +Standards<S9 I9mg>dl1 S4I4mg>dl1 S8I8mg>dl G S?I?mg>dl,

Blan1
=orking reagent +ml, Distilled =ater +ml, Standard +ml, &est sam)le >-! +ml, :/; ;/9 ' '

S,
:/; ' ;/9 '

S<
:/; ' ;/9 '

S=
:/; ' ;/9 '

Test
:/; ' ' ;/9

;C
:/; ' ' ;/9

Mix Well 0ea.e at room tem)erature +95':5;!, for :; minutes/ Set the s)ectro)hotometer> filter )hotometer to 3ero with %lank at 5;5 nm>green filter and measure the a%sor%ance of the other tu%es/ After measuring the a%sor%ance )our the solutions %ack into the res)ecti.e tu%es/ &hen add ;/9 ml of :;K acetic acid to the test and -! tu%es1 mix well and lea.e at room tem)erature +95':5 ;!, for 5 minutes/ Again set the s)ectro)hotometer>filter )hotometer to 3ero with %lank at 5;5nm>green filter and measure the a%sor%ance of test and -!/ Calculation and cali@ration graph Su%tract the second a%sor%ance .alues of test and -! from the first set of .alues/ Draw a cali%ration gra)h % )lotting the a%sor%ance .alues of standards against their res)ecti.e concentrations/ &he measura%le range with this gra)h is from ;/9 to ?/; mg>dl/ Plot the corrected a%sor%ance of test and -! and read off the .alues of creatinine/ Once linearit is )ro.ed1 it is enough if a single standard such as S8 is taken each time when )atients# sam)les are anal sed and the results are calculated using the following formula

Serum !reatinine I

&est a%sor%ance '''''''''''''''''''''''''' x 8 mg>dl Standard a%sor%ance

Analytical relia@ilities *efer to pages /7A of section , 6General Introduction9 on the use of internal -! and inter)retation of dail -! data +for releasing )atients# results,/ Since creatinine is one of the most common anal tes measured in a la%orator 1 inclusion of an internal -! +normal -! )ool, with e.er %atch of sam)les anal sed in the da 1 is recommended1 irres)ecti.e of the num%er of sam)les in a %atch/ Further1 e.en when a single sam)le is anal sed as an Hemergenc H sam)le at an time of the da or night1 it is essential to include an internal -!/ From the -! results o%tained for the da 1 mean1 standard de.iation and K!L can %e calculated to ensure that within-day precision is well within the acce)ta%le limit1 i/e1 5K/ &he mean .alue of internal -! for the da can %e )ooled with the )receding 6; or 9; mean .alues o%tained in the )re.ious da s and betweenday precision can %e calculated and ex)ressed as K !L/ (nsure that this is well within the acce)ta%le limit1 i/e1 ?K/ At least once a da anal se another -! serum from either a low -! or high -! )ool/ HAssa edH -! sera with stated .alues +ranges, are a.aila%le from se.eral commercial sources1 .i3/ $oehringer Mannheim1 $io*ad G *andox/ If a laboratory uses QC sera from a commercial source, it is important that the company certifies that their QC materials are traceable to international reference materials. Ha0ardous *aterials Picric acid is poisonous and sodiu* hydro)ide is caustic ( a?oid contact 5ith s1in and *ucous *e*@ranes. !eference range and clinical interpretation Serum>Plasma !reatinine< Male ;/E 6/4 mgK Female ;/4 6/9 mgK Serum creatinine concentration is related to muscle mass and the .alues are lower in children/ Increased serum creatinine is associated with decrease in glomerular filtration rate +GF*,1 whether the cause is )re'renal1 renal or )ost' renal/ Pre'renal factors include conditions such as congesti.e heart

failure1 shock1 diarrhoea1 uncontrolled dia%etes mellitus1 use of diuretics1 etc/ *enal factors in.ol.e mainl damage to the glomeruli/ Post'renal factors ma %e )rostatic h )ertro)h 1 calculi %locking the ureters or neo)lasms com)ressing the ureters/ &he serum creatinine concentration is monitored closel after a renal trans)lantation %ecause a rising concentration1 e.en though small1 ma %e an indication of graft reNection/ Li*itations Ascor%ic acid1 uric acid1 glucose1 ketones and ce)halos)orin anti%iotics1 if )resent at high concentrations1 ma interfere in the assa causing falsel high .alues/ Do not re)ort the result from s)ecimens with sus)ected interference/ Inform the re7uesting )h sician of the )ro%lem/ !eferences 6/ 9/ Slot !/ +6C85, Scand "/ !lin/ 0a% In.est/ 6E1 :?6 :?E/ Seation $/ G Ali A +6C?4, Med/ 0a% Sci/1 461 :9E' ::8/

Cholesterol ( Cholesterol o)idase *ethod

Introduction &he maNor constituents of )lasma li)ids are cholesterol and trigl cerides/ !holesterol is an im)ortant com)ound of cell mem%rane and )recursor for the s nthesis of %ile salts and steroid hormones/ !holesterol is s nthesi3ed in the li.er and trans)orted in the %lood mainl in the form of 0D0 and 2D0/ In %lood1 cholesterol is )resent in free as well as esterified form/ O.er the decades serum cholesterol has %een measured % methods em)lo ing 0ie%ermann'$urchard reaction/ &he en3 matic method has %ecome )o)ular in recent ears/ &he )ercentage of )artici)ants in the (xternal -ualit Assessment Scheme conducted from the author#s la%orator 1 em)lo ing the en3 matic method1 has increased significantl from 6; to ?5 in the last decade/ Principle of the *ethod !holesterol esters in serum are h drol sed % cholesterol esterase/ &he free cholesterol is then oxidi3ed % cholesterol oxidase to the corres)onding ketone li%erating h drogen )eroxide1 which is then con.erted to water and ox gen % the en3 me )eroxidase/ Para amino)hena3one +4 amino)hena3one, takes u) the ox gen and together with )henol forms a )ink coloured 7uinoneimine d e1 which can %e measured at 565nm> ellow green filter/ Speci*en type+ collection and storage Serum or )lasma can %e used/ A fasting %lood sam)le is )referred for li)id )rofile test/ 2owe.er1 if cholesterol alone has to %e anal sed1 a random sam)le can also %e used/ &he s)ecimen is sta%le for a week at 9 ' ?;! and at least for : months at '9;;!/ !eagents All chemicals must %e Analar grade !eneral note" Se.eral com)anies )ro.ide com)act kits for the measurement of cholesterol % the en3 matic method/ &hese kits are most economical and readil a.aila%le and therefore )racticall in most of the la%oratories cholesterol is measured % using kits/ 0a%oratories using kits are ad.ised to follow carefull the instructions gi.en in the leaflet/ !ommercial com)anies generall )ro.ide a single reagent consisting of the following chemicals<

4 amino)hena3one !holesterol esterase Phenol !holesterol oxidase Peroxidase Sodium a3ide &he reagent is )ro.ided in the l o)hil sed form and )ro)er instructions are gi.en for reconstitution and use in the assa / &he reagent is generall sta%le for one week when stored at 65 to 95 ;! and one month at 9'?;!/ E4uip*ent+ glass5are and other accessories *efer to Section A +9,1 Introduction to SOP/ Procedure &he )rotocol of the )rocedure is descri%ed %elow/ Pi)ette the following into a))ro)riatel la%elled 6: x 6;; mm tu%es

Blan1
*eagent solution +ml, 9/;

Standard
9/;

Test
9/;

;C
9/;

Standard +ml,

'

;/;9

'

'

&est sam)le >-! +ml,

'

'

;/;9

;/;9

Mix well/ Incu%ate at :E;! in a water%ath for 5 minutes or at room tem)erature +95':5 ;!, for 65 minutes/ *emo.e from water%ath and cool to room tem)erature/ Set s)ectro)hotometer > filter )hotometer to 3ero using %lank at 56; nm > ellow green filter and measure the a%sor%ance of standard1 test and -!/ &his )rotocol is designed for s)ectro)hotometers > filter )hotometers that re7uire a minimum .olume of reaction mixture in the cu.ette of 6ml or less/ Since economical use of the reagent is )ossi%le with this )rotocol1 the cost )er test can %e ke)t to the minimum/ 2owe.er1 if a la%orator em)lo s a )hotometer re7uiring a large .olume of reaction mixture for measurement1 .i3/ 5 ml1 it is ad.ised that the .olumes of reagent1 standard1 and test sam)le>-! mentioned under P8 %e increased )ro)ortionatel / Calculation and cali@ration graph 0inearit for cali%ration gra)h has %een well documented % la%orator it is from 9; to 5;; mg>dl/ se.eral kit com)anies/ In the author#s

&herefore a single standard +.i3/ 9;; mg>dl, can %e used and cholesterol in )atients# sam)les can %e calculated using the formula/ A%sor%ance of test '''''''''''''''''''''''''''' x !onc/ of Std OOOOO/ mg>dl A%sor%ance of standard +9;;, Anaytical relia@ilities *efer to pages /7A of section , 6General Introduction9 on the use of internal -! and inter)retation of dail -! data +for releasing )atients# results,/ Include one internal -! in e.er %atch of sam)les anal sed e.er da irres)ecti.e of the num%er of sam)les in a %atch/ Since cholesterol is anal sed in a single %atch in a da in an intermediate la%orator 1 it will not %e )ossi%le to anal se se.eral -! sam)les and calculate within'da )recision/

2owe.er1 e.en if a single -! sam)le is anal sed in a da 1 this .alue can %e )ooled with the )receding 6; or 9; .alues o%tained in the )re.ious da s and between-day precision can %e calculated and ex)ressed as K!L/ (nsure that this is well within the acce)ta%le limit1 i/e/ ?K/ Once a week it will %e good to anal se another -! serum from either a low -! or a high -! )ool/ HAssa edH -! sera with stated .alues +ranges, are a.aila%le from se.eral commercial sources1 .i3/ $oehringer Mannheim1 $io*ad G *andox/ If a laboratory uses QC sera from a commercial source, it is important that the company certifies that their QC materials are traceable to international reference materials. Ha0ardous *aterials This procedure uses phenol, which is caustic. Do not swallow, and avoid contact with skin and mucous membranes. !eference range and clinical interpretation Serum !holesterol ' 65;'95; mg>dl Serum .arious serious seen in cholesterol is increased in h )oth roidism1 dia%etes mellitus1 ne)hrotic s ndrome and in h )erli)idaemias es)eciall those causing xanthomatosis/ (le.ated serum cholesterol is a risk factor for the de.elo)ment of coronar arter disease/ Decreased serum cholesterol is se.ere he)atoclellular disease1 h )erth roidism and anaemia/

Li*itations 2aemol sis and li)aemia cause ele.ated cholesterol le.els/ Serum %iliru%in M5mg>dl and ascor%ic acid M 6; mg>dl also cause ele.ated cholesterol le.els/ Do not re)ort results from s)ecimens with sus)ected interference/ Inform the re7uesting )h sician of the )ro%lem/ !eference 6/ Allain !!1 Poon 0S1 !han !SG et al/ +6CE4, !lin !hem 9; < 4E;

Biliru@in ( Bendrassi1 : Grof *ethod

Introduction $iliru%in is formed from the haem fragment of haemoglo%in released % aged or damaged red %lood cells/ 0i.er1 s)leen and %one marrow are the sites of %iliru%in )roduction/ $iliru%in formed in s)leen and %one marrow is trans)orted to the li.er/ In the li.er it is con.erted into %iliru%in conNugates %iliru%in mono and diglucuronides/ An li.er disease affects the a%o.e s stems1 and hence %iliru%in accumulates in serum leading to Naundice/ Principle of the *ethod !onNugated +direct, %iliru%in in serum is cou)led with dia3otised sul)hanilic acid to form a red coloured com)ound/ Ascor%ic acid is used to sto) the cou)ling reaction1 and to eliminate interference % haemoglo%in/ !affeine %en3oate solution is used to s)lit the unconNugated %iliru%in )rotein com)lex releasing the %iliru%in so that it can react with dia3otised sul)hanilic acid/ &he tartrate %uffer makes the mixture alkaline and con.erts the red acid %iliru%in to a green coloured com)ound which shows )eak a%sor%ance at 8;E nm/ At this wa.elength the a%sor%ance due to haemoglo%in or carotene is minimal/ Speci*en type+ collection and storage Use onl clear1 non'haemol sed sam)les of serum/ $iliru%in is unsta%le and light sensiti.e and therefore the assa should %e carried out within 9 hours of sam)le collection/ If a longer dela is una.oida%le1 refrigerate the sam)le/ Sam)les can %e fro3en at 9; ;!1 to kee) %iliru%in sta%le for 9 months/ !eagents All chemicals must %e Analar grade Caffeine7@en0oate Dissol.e 6;;g caffeine sodium %en3oate and 95 g sodium %en3oate together in ?;; ml of distilled water/ 2eat the solution to 8;;! and then add 695g of h drated sodium acetate and 6g of (D&A/ Mix to dissol.e and then make u) to 6 litre with distilled water/ Filter the solution/ Store at room tem)erature +95':;;!,/ Sta%le for 8 months/ Sulphanilic acid Dissol.e 5 g sul)hanilic acid in 5;;ml distilled water with heating/ Add 65ml of conc/ 2!l and when cool make u) to 6 litre/ Store at room tem)erature +95':;;!,/ Sta%le for 8 months/ Sodiu* nitrite Dissol.e 5;; mg sodium nitrite in a%out ?;ml distilled water and then make u) to 6;; ml/ Store at 9'?;!/ Pre)are fresh once a month/ $ia0o reagent Mix 6;ml sul)hanilic acid with ;/95ml sodium nitrite/ &he solution is sta%le for a))roximatel hours at room tem)erature +95':;;!, and 94 hours at 9'?;!/ Al1aline tartrate Dissol.e 6;;g AaO2 and :5; g sodium )otassium tartrate in distilled water and make u) to 6 litre/ Store at room tem)erature +95':;;!,/ Sta%le for 8 months/ :

Ascor@ic acid 6> g&dl9 Dissol.e 9;; mg of ascor%ic acid in 5ml of distilled water/ &his solution must %e freshl each da / E4uip*ent+ glass5are and other accessories *efer to Section A +9,1 Introduction to SOP/ Procedure &he )rotocol of the )rocedure is descri%ed %elow/ Add reagents1 standards and test sam)les>-! in the order indicated into a))ro)riatel +6? x 65;mm, la%elled tu%es )re)ared

Standard Blan1
Distilled water +ml, ;/?

Std

Test&;C Blan1
;/?

Test&;C Test&;C $irect Bil. Total Bil.

;/? ;/?

;/?

$iliru%in Std +ml,

;/9

;/9

'

'

'

&est sam)le >-! +ml,

'

'

;/9

;/9

;/9

Dia3o reagent +ml,

'

;/5

'

;/5

;/5

!affeine sodium %en3oate +ml,

'

9/;

'

'

9/;

Mix and wait for 10 minutes at room temperature ( !"#0 0C$ Ascor%ic +ml,

;/6

;/6

;/6

;/6

;/6

Dia3o reagent +ml,

;/5

'

;/5

'

'

!affeine sodium %en3oate +ml,

9/;

'

9/;

9/;

'

Alkaline &artrate +ml,

6/5

6/5

6/5

6/5

6/5

Mix all tu%es/ Set the s)ectro)hotometer> filter )hotometer to 3ero with distilled water at 8;Enm> orange filter and read the a%sor%ance in the order of assa tu%es mentioned in the &a%le/ Preparation of @iliru@in standard !ommerciall a.aila%le %iliru%in is water insolu%le %ut the addition of a small amount of dimeth l sul)hoxide +DMSO, and AaO2 will dissol.e it/ As diluent1 non'icteric and non'li)aemic human )ooled serum tested negati.e for 2IL anti%odies and 2%s antigen could %e used/ 2owe.er1 there is alwa s a

risk of infection from this material/ Pool dail lefto.er normal human sera until a%out 69; ml are collected/ &ake an ali7uot for screening for the )resence of 2IL anti%odies and 2%s antigen and ensure that %oth are negati.e/ !entrifuge the )ooled serum twice at :5;; r)m for 6; minutes and collect the serum in a clean container/ Measure the total %iliru%in of the )ooled serum to ensure that it is Q ;/5 mg>dl/ =eigh accuratel 6;mg %iliru%in in a small sto))ered glass tu%e/ Add 9/; ml DMSO and ;/5 ml of ;/4M AaO2 and shake the tu%e in dim light until the %iliru%in has dissol.ed +It ma %e necessar to warm the tu%e in a :E;! water'%ath to s)eed u) this ste),/ &ransfer a%out E5ml of the )ooled serum into a 6;; ml .olumetric flask/ Add the %iliru%in solution slowl with continuous mixing to the serum/ *inse the tu%e with a few dro)s of DMSO and then with )ooled serum and add to the flask/ Make u) to the mark with the )ooled serum/ Froth can %e dis)ersed % touching with a glass rod minimall smeared with silicone or % adding a trace of ca)r l alcohol +octan 9'ol,/ =ra) the flask with car%on )a)er and store it at 9'? ;! until the standari3ation )rocedure is com)lete/ &his %iliru%in standard will ha.e a concentration of a%out 6;'66 mg>dl/ &he exact .alue will %e determined after carr ing out the )rocedure outlined in :/5/E Calculation and cali@ration graph E)trapolation *ethod Pre)are standards +S6'S5, as shown %elow

S,
$iliru%in standard +ml, Pooled serum +ml, &heoretical !oncentration %iliru%in +mg>dl, of 6/; ;/;9 ;/6?

S<
;/;5 ;/65 9/5

S=
;/6 ;/6 5/;

S>
;/65 ;/;5 E/5

S8
;/9 ' 6;

Proceed with %iliru%in estimation in the usual wa as for total %iliru%in/ Measure the a%sor%ance of all standards against distilled water in a s)ectro)hotometer at 8;E nm> orange filter in a filter )hotometer/ !onstruct a cali%ration gra)h % )lotting the theoretical %iliru%in concentrations against the corres)onding a%sor%ance .alues/ &his gra)h will not )ass through the origin1 instead it will interce)t the R # axis at a s)ecific )oint1 indicating that the %lank tu%e contains a certain amount of %iliru%in corres)onding to the R # interce)t a%sor%ance .alue/ U)on extra)olation of the cali%ration gra)h1 this will read a s)ecific .alue of %iliru%in on the negati.e side of the x axis +concentration axis,/ In the sam)le gra)h gi.en %elow +gra)h I,1 the %iliru%in content of the %lank is shown as ;/5 mg>dl/ &his .alue must %e added to the amount of %iliru%in dissol.ed in 6;; ml of )ooled serum/ &herefore1 in this exam)le the actual %iliru%in content in the standard S5 will %e 6;F;/5 I 6;/5 mg>dl/ 2ence the le.els of %iliru%in in the diluted standards from S4 down to S6 will .ar accordingl / &his is ex)lained in the &a%le gi.en %elow/

Standard
S5

Theoretical @iliru@in ?alue 6*g&dl9

6;/;

Actual @iliru@in ?alue after correcting for @lan1 6*g&dl9

6;/5;

S8 E/5 E/?5

S: 5/; S9 9/5 S6 6/; 6/;5 9/8; 5/95

&he cali%ration gra)h with corrected %iliru%in .alues should then %e )lotted against a%sor%ance .alues as shown in gra)h II/ &he test> -! a%sor%ance .alues should %e )lotted on this gra)h and concentrations read off/ &he measura%le range with this gra)h is from ;/9 to 9;/; mg>dl/ Once linearit is )ro.ed1 it will %e enough if a single standard is set u) e.er sam)les are anal sed and the results are calculated using the formula< &est a%sor%ance ' &est %lank a%sor%ance +&$D !oncentration '''''''''''''''''''''''''''''''''''''''''''''''''''''''''' x of $iliru%in Std/ A%sor%ance Std %lank a%sor%ance +S$D, standard &est +total, a%sor%ance'&$D !oncentration 6/ &otal $iliru%in mg>dl I ''''''''''''''''''''''''''''''''''' x of $iliru%in Standard a%sor%ance S$D standard &est +direct, a%sor%ance '&$D !oncentration 9/ Direct $iliru%in mg>dl I '''''''''''''''''''''''''''''''''' x of $iliru%in Standard a%sor%ance S$D standard time that )atients#

Storage of standard Ali7uot small .olumes of standard into screw'ca))ed .ials and store in the free3er on the same da it is )re)ared/ Sta%le for 9 months at '9;;!/ Do not refreeze leftover standard after use. Analytical relia@ilities *efer to pages /7A of section , 6General Introduction9 on the use of internal -! and inter)retation of dail -! data +for releasing )atients# results,/ Include one internal -! in e.er %atch of sam)les anal sed e.er da irres)ecti.e of the num%er of

sam)les in a %atch/ Since %iliru%in is anal sed in a single %atch in a da in an intermediate la%orator 1 it will not %e )ossi%le to anal se se.eral -! sam)les and calculate within'da )recision/ 2owe.er1 e.en if a single -! sam)le is anal sed in a da 1 this .alue can %e )ooled with the )receding 6; or 9; .alues o%tained in the )re.ious da s and between-day precision can %e calculated and ex)ressed as K!L/ (nsure that this is well within the acce)ta%le limit1 i/e1 6;K/ Once a week it is good to anal se another -! serum from either a low -! or high -! )ool/ HAssa edH -! sera with stated .alues +ranges, are a.aila%le from se.eral commercial sources1 .i3/ $oehringer Mannheim1 $io*ad G *andox/ If a laboratory uses QC sera from a commercial source, it is important that the company certifies that their QC materials are traceable to international reference materials / Ha0ardous *aterials This method uses sulphanilic acid and sodium hydro#ide. $void contact with eyes, skin and mucous membranes. !eference range and clinical interpretation Serum Direct $iliru%in ' u) to ;/5 mg>dl Serum &otal $iliru%in ' ;/9 6/; mg>dl 2 )er%iliru%inaemia is characteristic of Naundice/ Increase in unconNugated %iliru%in is o%ser.ed in haemol tic and neonatal Naundice/ In .iral and toxic he)atitis there is im)aired he)atocellular conNugation and excretion of %iliru%in with a maNor rise in conNugated and a lesser rise in unconNugated %iliru%in in serum/ In cirrhosis there is o.erall damage to li.er cells and hence the a%ilit of the li.er to form conNugated %iliru%in1 resulting in an increase in unconNugated %iliru%in in serum/ In o%structi.e Naundice there is an increase in )redominantl conNugated %iliru%in in serum/ Li*itations Sam)les with %iliru%in concentrations higher than 9;mg>dl should %e diluted with an e7ual .olume of distilled water and the result o%tained should %e multi)lied % 9/ &here is no interference in the assa % haemoglo%in u) to a concentration of 6/;g>dlJ howe.er1 strong haemol sis will interfere negati.el with measurement/ Do not re)ort results for s)ecimens with sus)ected interference/ Inform the re7uesting )h sician of the )ro%lem/ !eference 6/ Doumas $&1 Dwok'!heung PP1 Perr $=1 et al/ !lin/ !hem/ 9/ +6C?5, :6 < 6EEC'?C/ :/ &iet3/ A=/ Fundamentals of !linical !hemistr / 4/ Pu%lished % =$ Saunders !om)an / 6C?8/ )age 6:??'6:C;/ &otal Protein $iuret method Introduction &he serum'total )rotein1 as its name im)lies1 re)resents the sum total of numerous different )roteins1 man of which .ar inde)endentl of each other/ Proteins are )resent in all %od fluids %ut the )rotein concentration is normall high +M :g>dl, onl in )lasma1 l m)hatic fluids and some exudates/ Protein concentration in the cere%ro's)inal fluid of normal su%Nects is Q 45 mg>dl1 whereas the urine contains onl a trace/ Measurement of serum'total )rotein is useful in conditions relating to changes in )lasma or fluid .olumes1 such as shock and deh dration/ In these conditions concentration of serum'total )rotein is ele.ated indicating hemoconcentration/ 2aemodilution is reflected as relati.e h )o)roteinemia1 which

occurs with water intoxication or salt retention s ndrome1 during massi.e intra.enous infusions/ Princi)le of the method Proteins form a )ur)le coloured com)lex with cu)ric ions in alkaline solution/ &he reaction takes its name from the sim)le com)ound %iuret which reacts in the same wa / &he intensit of the )ur)le colour is measured at 54; nm > ellow green filter and com)ared with a standard serum of known )rotein concentration/ S)ecimen t )e1 collection and storage (ither serum or )lasma ma %e used1 %ut serum is )referred/ A fasting s)ecimen is not re7uired %ut ma %e desired to decrease li)aemia/ A.oid hamol sis/ &ightl sto))ered sam)les are sta%le for 94 hours at room tem)erature +95':5;!,1 one week at 9'?;! and for : months at '9;;!/ *eagents All !hemicals must %e Analar grade Sodium chloride diluent ;/CK =>L Dissol.e 4/5g sodium chloride in a%out 4;;ml of distilled water and then make u) to 5;;ml with distilled water/ Sta%le at room tem)erature +95':5;!,/ Make a fresh solution once in 8 months/ $iuret reagent Dissol.e 4/;g sodium h droxide in a%out 4;; ml of distilled water/ Add 4/5 g sodium )otassium tartrate/ Mix to dissol.e/ &hen add 6/5g co))er sul)hate followed % 4/5g )otassium iodide/ &ransfer the solution into a 5;; ml .olumetric flask and make u) to the mark with distilled water/ Store in a tightl sto))ered )ol eth lene %ottle at room tem)erature +95':5;!,/ Sta%le for 8 months/ Standard In man %iochemical estimations direct standardi3ation is em)lo edJ for exam)le1 glucose1 urea and creatinine are a.aila%le in )ure forms1 and can %e directl weighed and used/ In the case of serum total )roteins this is not )ossi%le since serum )roteins consist of se.eral )rotein fractions/ 2owe.er1 an acce)ta%le )rocedure is to make use of %o.ine al%umin to )re)are the standard/ Alternati.el 1 the la%orator can o%tain )rotein standard from commercial firms or )re)are standard in'house/ &he method of in'house )re)aration of standard is descri%ed %elow < &he la%orator can use either lefto.er )atients# sera or %o.ine serum to )re)are the standard/ Aote that there is a risk of infection from )ooled )atients# s)ecimens/ Pre)are )ooled serum as descri%ed in Section 6 General Introduction under RPre)aration of -! )ool# After ali7uoting and free3ing the )ooled serum1 anal se total )rotein in the ade7uots dail for a )eriod of 9; da s using a relia%le )rotein standard +commercial source or non'commercial source such as =2O,/ !alculate the mean .alue and assign this as the standard .alue/ (7ui)ment1 glassware and other accessories *efer to Section A +9,1Introduction to SOP/ Procedure &he )rotocol of the )rocedure is descri%ed %elow/ Pi)ette the following into a))ro)riatel la%elled 6? x 65; mm tu%es +Standard IS61 S9 G S:,

Blan1
Sodium +ml, !hloride diluent 9/5

S,
9/45

S<
9/4

S=
9/:5

Test
9/4 '

;C
9/4 '

Standard +ml,

'

;/;5

;/6

;/65

&est Sam)le >-! +ml,

'

'

'

'

;/6

;/6

Mix well $iuret reagent +ml, :/;

:/;

:/;

:/;

:/;

:/;

Mix well Incu%ate at room tem)erature +95':5;!, for 65 minutes/ Set the s)ectro)hotometer > filter )hotometer to 3ero using %lank at 54; nm> ellow green filter and measure the a%sor%ance of standards1 test G -!/ !alculation and cali%ration gra)h Since the )rotocol for standard tu%e S9 and the test is identical1 standard S9 will re)resent the actual concentration of the )ooled serum used as standard/ Standard S6 contains half the .olume of S9 and S: has 6S times the .olume of S9/ &herefore )rotein concentrations re)resented % S6 and S: will %e S

and 6S times of S9 concentration1 res)ecti.el / For exam)le1 if S9 concentration is 8g>dl1 S6 will %e :g>dl and S: will %e Cg>dl/ Plot the a%sor%ance .alues of standards against their res)ecti.e concentrations/ &he measura%le range with this gra)h is from ;/5 to6;/;g>dl/ Plot the a%sor%ance .alues of test>-! on the cali%ration gra)h and read off the concentrations/ Once linearit is )ro.ed1 it is not necessar to )re)are standard gra)h e.er time when )atients# sam)les are anal sed/ It will %e ade7uate if standard S9 is taken e.er time and )atients# results are calculated using the formula< &est ''''''''''''''''''''''''''' Standard a%sor%ance x !oncentration of S9 OOOO// a%sor%ance g>dl

Anal tical relia%ilities *efer to )ages E'C of section 6 +General Introduction, on the use of internal -! and inter)retation of dail -! data +for releasing )atients# results,/ Include one internal -! in e.er %atch of sam)les anal sed e.er da irres)ecti.e of the num%er of sam)les in a %atch/ Since total )rotein is anal sed in a single %atch in a da in an intermediate la%orator 1 it will not %e )ossi%le to anal se se.eral -! sam)les and calculate within'da )recision/ 2owe.er1 e.en if a single -! sam)le is anal sed in a da 1 this .alue can %e )ooled with the )receding 6; ;r 9; .alues o%tained in the )re.ious da s and %etween'da )recision can %e calculated and ex)ressed as K!L/ (nsure that this is well within the acce)ta%le limit1 i/e1 8K/ Once a week it will %e good to anal se another -! serum from either a low -! or high -! )ool/ HAssa edH -! sera with stated .alues +ranges, are a.aila%le from se.eral commercial sources1 .i3/ $oehringer Mannheim1 $io*ad G *andox/ If a la%orator uses -! sera from a commercial source1 it is im)ortant that the com)an certifies that their -! materials are tracea%le to international reference materials/ 2a3ardous materials Sodiu* hydro)ide used in this procedure is a strong al1ali and is caustic. $o not s5allo5+ and a?oid contact 5ith s1in and *ucous *e*@ranes. *eference range and clinical inter)retation Serum &otal Protein OOOOOO 8/5 ?/5 g>dl In addition to deh dration and diarrhoea1 increased serum'total )rotein le.els are o%ser.ed in multi)le m eloma/ 2 )o)roteinemia is generall seen in conditions associated with h )oal%uminemia/ 0imitations 2aemol sed and li)aemic sera interfere strongl with the measurement of )roteins/ Set u) a))ro)riate %lank for such sam)les % adding ;6/ml of serum to 5/5ml of sodium chloride diluent/ &he a%sor%ance o%tained against distilled water is then su%tracted from the test a%sor%ance %efore the test result is calculated/ *eference 6/ *einhold/ "/G/ +6C5:,/ Standard methods of clinical chemistr 6<) <??

Al@u*in 7 BCG $ye Binding

ethod

Introduction Serum al%umin consists of a single s)ecies of )roteins re)resenting a))roximatel 8;K of the total )rotein/ It is s nthesi3ed exclusi.el in the li.er and functions as a regulator of %lood oncotic )ressure1 as a carrier for man cations and water insolu%le su%stances1 and as a )ool of aminoacids for caloric or s nthetic )ur)oses/ Principle of the *ethod Al%umin %inds 7uantitati.el with %romocresol green at )2 4/65 resulting in the formation of a green colour which can %e measured at 8:;nm>red filter/ Speci*en type+ collection and storage (ither serum or )lasma ma %e used1 %ut serum is )referred/ A fasting s)ecimen is not re7uired %ut ma %e desired to decrease li)aemia/ A.oid haemol sis/ &ightl sto))ered sam)les are sta%le for 94 hours at room tem)erature +95':5;!,1 one week at 9'?;! and for : months at '9;;!/ !eagents All chemicals must %e Analar grade Sodiu* hydro)ide , #

=eigh out 4/; g of sodium h droxide +AaO2,1 dissol.e and make u) to 6;;ml with distilled water/ &his solution is sta%le for se.eral months at room tem)erature +95':5 ;!, in a )ol )ro) lene container/ BriF 7 =8........... =-g&dl *eadil a.aila%le at the a%o.e concentration from S/D Fine chemicals or 0o%a !hemical !om)an 1 in India/ Solid $riN can also %e o%tained from Sigma !o/ In this case1 warm :;g solid $riN in a %eaker in a small .olume of distilled water to dissol.e and make u) to 6;;ml with distilled water/ Bro*o Cresol Green 6BCG9 dye solution &ransfer 95ml of I M AaO2 into a one'litre .olumetric flask containing 8;;ml distilled water/ Add 5/8g succinic acid and then add 58 mg of $!G )owder/ Mix and then make u) to 6 litre with distilled water/ !heck the )2/ If it is less than 4/651 adNust to 4/65 F ;/;5 % the dro)wise addition of 6 M AaO2/ Add 6;; mg sodium a3ide and :/5ml :; g>dl $riN':5 to the reagent/ !heck the a%sor%ance of the reagent at 8:; nm> red filter against distilled water/ It should %e less than ;/9/ If it is greater than ;/91 add some more $riN to %ring down the a%sor%ance/ Store ina )ol ethl ene container/ Sta%le for 8 months at room tem)erature +95':5;!,/ Standard In man %iochemical estimations direct standardi3ation is em)lo edJ for exam)le1 glucose urea and creatinine are a.aila%le in )ure forms1 and can %e directl weighed and used1 whereas1 in the case of human al%umin1 this is not readil a.aila%le/ &he la%orator can o%tain al%umin standard from commercial firms or )re)are the standard in'house/ &he method of in'house )re)aration of the standard is descri%ed %elow<

Pre)are )ooled serum as descri%ed in Section , General Introduction under GPreparation of ;C PoolC/ &he la%orator should )ool dail lefto.er )atientsT sera/ For al%umin standard1 onl the use of human serum is recommended1 since it has %een documented that the affinit of $!G to %o.ine al%umin is different from its affinit to human al%umin %e ond :g>dI/ After ali7uoting and free3ing the )ooled serum1 anal se al%umin in the ali7uots dail for a )eriod of 9; da s using a relia%le al%umin standard +commercial source or non'commercial source such as =2O,/ !alculate the mean .alue and assign this as the standard .alue/ Aote< &here is alwa s a risk of infection from this material/ E4uip*ent+ glass5are and other accessories *efer Section A +9,1 Introduction to SOP/ Procedure &he )rotocol of the )rocedure is descri%ed %elow/ $ilution of Standards 6S, 7S>9+ Test : ;C Pi)ette the following into a))ro)riatel la%elled 6: x 6;;mm tu%es

S,
Distilled water +ml, Standard +ml, &est sam)le>-! +ml, 6/C ;/6 '

S<
6/? ;/9 '

S=
6/E ;/: '

S>
6/8 ;/4 '

Test
6/? ' ;/9

;C
6/? ' ;/9

Mix well Colour $e?elop*ent Pi)ette the following into another set of a))ro)riatel la%elled 6? x 5;mm tu%es

Blan1
Distilled =ater +ml, Diluted Standard +ml, Diluted Sam)le >-! +ml, $!G Solution +ml, &est ' 9/5 ;/6 '

S,
' ;/6 ' 9/5

S<
' ;/6 ' 9/5

S=
' ;/6 ' 9/5

Test
' ' ;/6 9/5

;C
' ' ;/6 9/5

Mix all tu%es well/ Incu%ate at room tem)erature +95':5 ;!, for 6; minutes/ Set the s)ectro)hotometer >filter )hotometer to 3ero using %lank at 8:; nm> red filter and measure the a%sor%ance of standards1 test G -!/

Calculation and cali@ration graph Since the )rotocol for standard tu%e S9 and the test is identical1 standard S9 will re)resent the actual concentration of the )ooled serum used as standard/ Standard S6 contains half the .olume of S9 1 S: has 6Stimes and S4 has twice the .olume of S9/ &herefore al%umin concentrations re)resented % S61 S: and S4 will %e S1 6 S and 9 times of S9 concentration res)ecti.el / For exam)le1 if S9 concentration is :g>dl1 SI will %e 6/5g>dl1 S: will %e 4/5g>dl and S4 will %e 8g>dI/ Plot the a%sor%ance .alues of standards against their res)ecti.e concentrations/ &he measura%le range with this gra)h is from ;/5 to 8/; g>dl/ Plot the a%sor%ance .alues of test>-! on the cali%ration gra)h and read off the concentrations/ Once linearit is )ro.ed1 it isnot necessar to )re)are the standard gra)h e.er time when )atientsT sam)les are anal sed/ It will %e ade7uate if standard S9 is taken e.er time and )atientsT results are calculated using the formula <

&est a%sor%ance '''''''''''''''''''' x !oncentration of S9 g>dl Standard a%sor%ance Analytical relia@ilities *efer to pages /7A of section , 6General Introduction9 on the use of internal -! and inter)retation of dail -! data +for releasing )atientsT results,/ Include one internal -! in e.er %atch of sam)les anal sed e.er da irres)ecti.e of the num%er of sam)les in a %atch/ Since al%umin is anal sed in a single %atch once a da in an intermediate la%orator 1 it will not %e )ossi%le to anal se se.eral -! sam)les and calculate within'da )recision/ 2owe.er1 e.en if onl a single -! sam)le is anal sed in a da 1 this .alue can %e )ooled with the )receding 6; or 9; .alues o%tained in the )re.ious da s and between-day precision can %e calculated and ex)ressed as K!L/ (nsure that this is well within the acce)ta%le limit1 i/e1 8K/ At least once a week anal se another -! serum from either a low -! or high -! )ool/ HAssa edH -! sera with stated .alues +ranges, are a.aila%le from se.eral commercial sources1 .i3/ $oehringer Mannheim1 $io*ad G *andox/ If a la%orator uses -! sera from a commercial source1 it is im)ortant that the com)an certifies that their -! materials are tracea%le to international reference materials/ Ha0ardous *aterials

%odium hydro#ide used in this procedure is a stron alkali and is caustic. Do not swallow, and avoid contact with skin and mucous membranes. !eference range and clinical interpretation Serum al%umin ' :/5'5/; g>dl Serum le.els of al%umin are used to assess nutritional status and ha.e im)ortant influences on the meta%olism of endogenous su%stances such as calcium1 %iliru%in and fatt acids and on the effect of drugs and hormones/ 2 )eral%uminemia has little diagnostic significance exce)t in deh dration/ 2 )oal%uminemia is .er common in man illnesses like im)aired s nthesis +li.er disease,1 increased cata%olism1 reduced aminoacid a%sor)tion1 )rotein loss in urine1 malnutrition and )rotein losing entero)ath 1 and in hos)ital )atients with acute illness/ Li*itations 2aemol sed and li)aemic sera interfere strongl with the measurement of al%umin/ Set u) a))ro)riate %lank for such sam)les % adding ;/6 ml of serum to 4/5 ml of sodium chloride diluent +*efer &otal Protein,/&he a%sor%ance o%tained is then su%tracted from its test a%sor%ance %efore calculating the test result/ !eference 6/ S)encer D and Price !/P/ +6CEE,/ Ann/ !lin/ $iochem 641 6;5'665/

Transa*inases 7 Colori*etric End7Point

Introduction

ethod

&he two transaminases of diagnostic im)ortance are< serum glutamic oxaloacetate transaminase +SGO&, or as)artate amino transferase +AS&,1 and serum glutamic ) ru.ate transaminase +SGP&, or alanine amino transferase +A0&,/ =hile AS& is found in e.er tissue of the %od 1 including red %lood cells1 and is )articularl high in the cardiac muscle1 A0& is )resent in moderatel high concentration in li.er and low in cardiac1 skeletal muscle and other tissues/ $oth AS& and A0& measurements are useful in the diagnosis and monitoring of )atients with he)atocellular disease/ Principle of the *ethod &ransamination is the )rocess in which an amino grou) is transferred from amino acid to an a 'keto acid/ &he en3 mes res)onsi%le for transamination are called transaminases/ &he su%strates in the reaction are a 'ketoglutaric acid +a DG, )lus 0'as)artate for AS&1 and a DG )lus 0'alanine for A0&/ &he )roducts formed % en3 me action are glutamate and oxaloacetate for AS& and glutamate and ) ru.ate for A0&/ Addition of 9141 dinitro)hen l h dra3ine results in the formation of h dra3one com)lex with the ketoacids/ A red colour is )roduced on the addition of sodium h droxide/ &he intensit of colour is related to en3 mic acti.it / Speci*en type+ collection and storage Serum or (D&A>he)arini3ed )lasma can %e used in this assa / &ransaminases are sta%le in serum for 8 hours at 95':5 ;!1 E da s at 9'?;! and for one month when stored at '9;;!/ !eagents

All chemicals must %e Analar grade Phosphate @uffer+ pH /.> # Dissol.e 64/C g disodium h drogen )hos)hate deh drate +Aa 92PO4 929;, and 9/9g anh drous )otassium dih drogen )hos)hate +D29PO2, in distilled water and make u) to one litre/ !heck the )21 and1 if necessar 1 adNust to E/4 using small amounts of either D2 9PO4 or Aa92PO4' Sta%le for : months when stored at 9'?U !/ AST Su@strate Dissol.e 9/88 g D0' as)artic acid and :; mg a 'keto glutarate in 9;/5 ml of 6 M AAO2/ AdNust the )2 to E/4 % adding I M AAO2 dro) wise while stirring/ Make u) to 6;; ml with )hos)hate %uffer/ Add 6 ml of chloroform as )reser.ati.e/ Sta%le for 9 months when stored at 9'?U !/ Discard if it %ecomes tur%id/ ALT Su@strate Dissol.e 6/E? g D0'alanine and :; mg a 'keto glutarate in 9; ml of )hos)hate %uffer containing 6/95 ml of ;/4 M AAO2/ Make u) to 6;; ml with %uffer and adNust to )2 E/4 if necessar / Add 6 ml chloroform as )reser.ati.e/ Sta%le for 9 months when stored at 9'?U !/ Discard if it %ecomes tur%id/ Pyru?ate standard < * *ol&*l Dissol.e 99; mg sodium ) ru.ate in)hos)hate %uffer and make u) to 6;; ml/ Dilute 6; ml of this solution to 6;; ml with )hos)hate %uffer to o%tain the working standard containing 9 m mol ) ru.ate )er ml/ &he remaining C; ml of the first solution should %e discarded/ &he working standard should %e stored in small ali7uots of 9 ml in the free3er/ One ali7uot of working standard should %e used for )re)aring a cali%ration gra)h/ Discard the lefto.er standard in the .ial/ Colour reagent Dissol.e 9;; mg 914 dinitro')hen lh dra3ine +914 DAP2, in hot 6M 2!I and make u) to 6 litre with 6M 2!I/ Sta%le for 8 months when stored at 9'?;!/ -.> Sodiu* hydro)ide

Dissol.e 68 g sodium h droxide in a%out ?;; ml of distilled water and make u) to 6 litre with distilled water/ Store in a )ol eth lene container at 95':5 ;!/ Sta%le for 8 months/ E4uip*ent+ glass5are and other accessories *efer to Section A +9,1 Introduction to SOP/ Procedure AST &he )rotocol of the )rocedure is descri%ed %elow/ Pi)ette the following into a))ro)riatel la%elled 6? x 65;mm tu%es &$D I &est $lank G -!$D I -! $lank

TBH
AS& Su%strate +ml, ;/5 &est sam)le>-! +ml, '

;CBH
;/5

TEST
;/5

;C
;/5

'

;/6

;/6

Mix and incubate at #% 0Cin a waterbath for 1 hour 914 DAP2 +ml, ;/5 ;/5 ;/5 ;/5

Mix and remo&e the tubes from the waterbath &est sam)le>-! +ml, ;/6 ;/6 ' '

Mix and lea&e the tubes for 0 minutes at room temperature ( !"#! 0C$ ;/4M AaO2 +ml, 5/; 5/; 5/; 5/;

Mix and lea&e the tubes for ! minutes at room temperature ( !"#! 0C$ Set the s)ectro)hotometer>filter )hotometer to 3ero using distilled water at 5 6 ; nm> ellow green filter and measure the a%sor%ance of &$D1 -!$D1 &est and -! in the order/ ALT Pi)ette the following into a))ro)riatel la%elled 6? x 65; mm tu%es/

TBH
A0& Su%strate +ml, ;/5

;CBH
;/5

TEST
;/5

;C
;/5

&est sam)le>-! +ml,

'

'

;/6

;/6

Mix and incubate at #% 0 C in a waterbath for #0 minutes 914 DAP2 +ml, ;/5

;/5

;/5

;/5

Mix and remo&e the tubes from the waterbath &est sam)le>-! +ml,

;/6

;/6

'

'

Mix and lea&e the tubes for 0 minutes at room temperature ( !"#! 0C$

;/4M AaO2 +ml,

5/;

5/;

5/;

5/;

Mix and lea&e the tubes for ! minutes at room temperature ( !"#! 0C$ Set the s)ectro)hotometer>filter )hotometer to 3ero using distilled water at 5 6 ; nm> ellow green filter and measure the a%sor%ance of &$D1 -!$D1 &est and -! in the order/ Calculation and cali@ration graph In the measurement of %oth serum AS& G A0&1 onl ) ru.ate is used as the standard/ &heoreticall s)eaking1 oxaloacetate should %e used as the standard for AS& assa and ) ru.ate as the standard for A0& assa / Oxaloacetate formed in the AS& assa is unsta%le and immediatel gets con.erted into ) ru.ateJ hence the use of ) ru.ate standard for AS& assa / One unit>0 of AS& or A0& is defined as the li%eration of 6m mol of ) ru.ate )er minute at :E;! incu%ation )er litre of serum/ As a 'keto glutarate'cosu%strate in the assa contri%utes to the final a%sor%ance1 the change in a%sor%ance is not linearl related to the theoretical .alue of ) ru.ate )roduced and hence the en3 me acti.it / &his is e.ident from the sam)le cali%ration gra)h shown/

Blan1
P ru.ate Standard +ml, ' A0& +or AS&, Su%strate +ml, 6/; Distilled water +ml, ;/9 941 DAP2 +ml, 6/;

S,
;/6

S<
;/9

S=
;/:

S>
;/4

;/C

;/?

;/E

;/8

;/9

;/9

;/9

;/9

6/;

6/;

6/;

6/;

Mix and lea&e the tubes for 0 min at room temperature( !"#! 0C$ ;/4 M AaO2 +ml, 6;/; 6;/; 6;/; 6;/; 6;/;

Mix and lea&e the tubes for 0 minutes at room temperature ( !"#! 0C$ (7ui.alent AS& in U>0 serum ' (7ui.alent A0& in U>0 serum ' 9? 5E CE 65; 94 86 664 6C;

!onstruct a cali%ration cur.e % )lotting the corres)onding a%sor%ance of standards against their res)ecti.e AS&>A0& acti.ities/ &he measura%le ranges with these gra)hs are from 5/; to 65; U>0 for A0& and 5/; to 6C; U>0 for AS&/ Plot the difference in the a%sor%ance %etween test and &$D as well as -! and -!$D for AS& G A0& and read off the en3 me acti.ities on the cali%ration gra)hsJ otherwise refer to the &a%le recommended % standard clinical chemistr text%ooks like &iet3 or Larle relating ) .urate .alues to AS&>A0& acti.ities +if it is a.aila%le,/ ALT

AST

Analytical relia@ilities *efer to )ages E'C of section 6 +General Introduction, on the use of internal -! and inter)retation of dail -! data +for releasing )atientsT results,/ Include one internal -! in e.er %atch of sam)les anal sed each da irres)ecti.e of the num%er of sam)les in a %atch/ Since AS& or A0& is anal sed in a single %atch in a da in an intermediate la%orator 1 it will not %e )ossi%le to anal se se.eral -! sam)les and calculate within'da )recision/ 2owe.er1 e.en if onl a single -! sam)le is anal sed in a da 1 this .alue can %e )ooled with the )receding 6; or 9; .alues o%tained in the )re.ious da s and between"day precision can %e calculated and ex)ressed as K!L/ (nsure that this is well within the acce)ta%le limit1 i/e1 6;K/

At least once a week anal se another -! serum from either a low -! or high -! )ool/ HAssa edH -! sera with stated .alues +ranges, are a.aila%le from se.eral commercial sources1 .i3/ $oehringer Mannheim1 $io*ad G *andox/ If a la%orator uses -! sera from a commercial source1 it is im)ortant that the com)an certifies that their -! materials are tracea%le to international reference materials/ Ha0ardous *aterials This procedure uses &a'(, which is caustic. Do not swallow, and avoid contact with the skin and mucous membranes. !eference range and clinical interpretation &he reference ranges % this method are< AS& ? ' 4; U>0 A0& ! ' :5 U>0 2igh le.els of serum AS& acti.it are seen in the heart1 li.er1 skeletal muscle and kidne tissues/ Increased acti.it of AS& in serum is o%ser.ed in m ocardial infarction after 9;':8 hours of onset and hence used as a su))orting e.idence in the diagnosis of m ocardial infarction/ Lalues are usuall less than 6; times the u))er limit of normal +U0A,/ Increased acti.ities are also o%ser.ed in .iral>toxic he)atitis1 muscular d stro)h and in )ulmonar em%olism/ A0& is distri%uted mainl in the li.er and to a lesser extent in the kidne and muscles/ Increased A0& acti.it is o%ser.ed in he)atitis and cirrhosis/ Lalues ma %e increased to M6; times 6;; times U0A in he)atitis/ Li*itations For sam)les with en3 me acti.it greater than 65; U>0 for A0& and greater than 6C; U>0 for AS&1 dilute the s)ecimen 6 in 6; with ;/C K saline/ Some s)ecimens ma re7uire a further 6 in 6; dilution to gi.e a final dilution of 6 in 6;;/ Multi)l the final result % the dilution factor/ A.oid using the haemol sed sam)le as this will cause falsel re7uesting )h sician and ask for another s)ecimen/ !eference 6/ *eitman1 S G Frankel1 S/ +6C5E, Am " !lin Pathol/1 9?1 58'8:/ ele.ated .alues/ In this case inform the

Al1aline Phosphatase 7 p7nitrophenol

ethod

Introduction Phos)hatases are en3 mes which catal se the s)litting of a )hos)hate from mono')hos)horic esters/ Alkaline )hos)hatase +A0P,1 a mixture of isoen3 mes from li.er1 %one1 intestine and )lacenta1 has maximum en3 me acti.it at a%out )2 6;/5/ Serum A0P measurements are of )articular interest in the in.estigation of he)ato%illar and %one diseases/ Principle of the *ethod Paranitro)hen l )hos)hate1 which is colourless1 is h drol sed % alkaline )hos)hatase at )2 6;/5 G :E;! to form free )aranitro)henol1 which is coloured ellow/ &he addition of AaO2 sto)s the en3 me acti.it and the final colour shows maximum a%sor%ance at 46; nm/ Speci*en type+ collection and storage Serum or he)arini3ed )lasma can %e used/ Sta%le for E da s at 9'?;!/ &he acti.it sam)les are left at room tem)erature +95':5;!, for se.eral hours/ !eagents All chemicals must %e Analar grade/ <7a*ino <7 *ethyl ,7propanol 6A P9 @uffer pH ,-.8 Add 668 ml of AMP to 8;; ml of distilled water/ Mix and adNust the )2 to 6;/5 with 8 M 2!I and then make u) to 6 litre with distilled water/ Sta%le for 8 months at 9'?;!/ agnesiu* chloride 6,.8 **ol&,9. Dissol.e :;; mg of magnesium chloride hexah drate in distilled water and make u) to 6 litre/ Sta%le for 8 months at room tem)erature +95':5; !, Su@strate Dissol.e ?:/5 mg of disodium )aranitro)hen l )hos)hate in 6/;ml magnesium chloride solution/ Sta%le for 94 hours at 9'? ;!/ &his solution should %e colourlessJ do not use it if the OD at 46;nm M ;/?;;/ Sodiu* hydro)ide -.<8 Dissol.e 6; g of AaO2 in a%out ?;; ml of distilled water and then make u) to 6 litre with distilled water/ Store in a )ol thene %ottle at room tem)erature +95':5U !,/ Sta%le for 8 months/ Stoc1 paranitrophenol 6PNP9 ,-.I **ol&l. =eigh out 65; mg of PAP and dissol.e in a%out ?;ml of AaO2 +;/95M, and then make u) to 6;; ml with the same AaO2 solution/ Store ina %rown glass %ottle at room tem)erature +95':5 ;!,/ Sta%le for : months/ 3or1ing PNP 8> * *ol&l Pi)ette ;/5 ml of the PAP stock solution into a 6;;ml .olumetric flask and make u) to the mark with AaO2 solution +;/95 M,/ Pre)are fresh %efore use/ E4uip*ent+ glass5are and other accessories increases if

*efer to Section A +9,1 Introduction to SOP/ Procedure &he )rotocol of the )rocedure is descri%ed %elow/ Preparation of standards 6SI 7SJ9 Pi)ette the following into a))ro)riatel la%elled 6? x 65;mm tu%es

S,
=orking PAP solution +ml, ;/5

S<
6/;

S=
9/;

S>
:/;

S8
4/;

SJ
5/;

AaO2 solution +ml,

4/5

4/;

:/;

9/;

6/;

'

Acti.it U>0

4;

?;

68;

94;

:9;

4;;

Mix Well Set the s)ectro)hotometer > filter )hotometer to 3ero a%sor%ance at 46; nm > .iolet filter against ;/95M AaO2 and measure the a%sor%ance of the a%o.e standards/ En0y*e *easure*ent in test & ;C Pi)ette the following into another set of a))ro)riatel la%elled 6? x 65; mm tu%es/

Blan1
AMP %uffer +ml, 6/4

Test
6/4

;C
6/4

Mix and Incubate at #%0C for ! minutes

&est Sam)le>-! +ml,

'

;/;5

;/;5

Su%strate +ml,

;/6

;/6

;/6

Mix and Incubate at #%0C for 1! minutes

AaO2 +ml,

4/;

4/;

4/;

+Aote< AaO2 should %e added to each tu%e inse7uence maintaining timed inter.als, Mix and cool the tu%es to room tem)erature +95':5 ;!,/ Measure the a%sor%ance of test > -! at 46;nm >.iolet filter1 setting the s)ectro)hotometer >filter )hotometer to 3ero with the %lank/ Calculation and cali@ration graph &he working PAP concentration is 54 m mol>0/ Standard SI contains ;/5ml PAP

54 !oncentration of PAP in SI I ''''''''''''' x ;/5 I ;/;9Em mol/ 6;;; A0P acti.it in U>0 I 0i%eration of 6 m mol of PAP )er minute at :E ;! incu%ation )er litre serum/ In the assa )rotocol1 ;/;5 ml serum is mixed with reagent and incu%ated for 65 minutes and the total .olume is made u) to 5/55ml/ $ut the total .olume in the case of each standard +SI to S8, is 5/; ml/ PAP in umol>0 or A0P acti.it in U>0 in the test sam)le I &est a%sor%ance ;/;9E 5/55 6;;; '''''''''''''''''''' x '''''' x '''''' x '''''' Std a%sor%ance 65 5/; ;/;5 &est a%sor%ance I ''''''''''''''''''''''' x 4; Std a%sor%ance i/e1 A0P acti.it e7ui.alent for S I I 4; U>0/ Similarl the A0P acti.ities re)resented % standards are < S9 I ?;1 S: I 68;1 S4 I 94;1 S5 I :9; and S8 I 4;; U>0/ other

!onstruct a cali%ration gra)h % )lotting the e7ui.alent acti.it of A0P of the standards against their corres)onding a%sor%ance .alues/ &he measura%le range with this gra)h is from 6; to 4;; U>0/ Plot the a%sor%ance .alues of test >-! on the cali%ration gra)h and read off the concentrations/ Once linearit is )ro.ed1 it will %e enough if a single standard is set u) e.er time that )atientsT sam)les are anal sed/ Use standard S8 in the assa and calculate the results using the formula < &est A%sor%ance ''''''''''''''''''''''' x 4;;///// U>0/ Std A%sor%ance

Analytical relia@ilities *efer to pages /7A of section , 6General Introduction9 on the use of internal -! and

inter)retation of dail -! data +for releasing )atientsT results,/ Include one internal -! in e.er %atch of sam)les anal sed e.er da 1 irres)ecti.e of the num%er of sam)les in a %atch/ Since alkaline )hos)hatase isanal sed in a single %atch in a da in an intermediate la%orator 1 it will not %e )ossi%le to anal se se.eral -! sam)les and calculate within'da )recision/ 2owe.er1 e.en if a single -! sam)le is anal sed in a da 1 this .alue can %e )ooled with the )receding 9; .alues o%tained in the )re.ious da s and between day precision can %e calculated and ex)ressed as K!L/ (nsure that this is well within the acce)ta%le limit1 i/e/ 6;K/ Once a week it is good to anal se another -! serum from either a low -! or high -! )ool/ HAssa edH -! sera with stated .alues +ranges, are a.aila%le from se.eral commercial sources1 .i3/ $oehringer Mannheim1 $io*ad G *andox/ If a laboratory uses QC sera from a commercial source, it is important that the company certifies that their QC materials are traceable to international reference materials. Ha0ardous *aterials This procedure uses NaOH+ 5hich is caustic. $o not s5allo5+ and a?oid contact 5ith the s1in and *ucous *e*@ranes. > nitro phenol is to)ic. $o not pipette @y *outh. %se a dispenser. !eference range and clinical interpretation Serum alkaline )hos)hatase +adults, ' 4;'695 U>0/ +0e.els u) to : times this ma %e normal in children, 0i.er1 %one and )lacenta contain .er high concentrations of A0P/ &herefore1 increase in A0P acti.it is usuall related to he)ato%iliar and %one disorders/ Increased A0P le.els are o%ser.ed in li.er diseases1 osteomalacia1 rickets and %one disorders/ Moderate ele.ations are sometimes noted in congesti.e heart failure1 intestinal disease and intra'a%dominal %acterial infections/ Li*itations A.oid ex)osure of the freshl dissol.ed su%strate to strong sunlight1 since the reagent is light sensiti.e/ &he change in a%sor%ance will increase with an increase in tem)erature1 since the )2 of the reagent will %e different at different tem)eratures/ 2aemol sed s)ecimens are not suita%le for anal sis/ Inform the re7uesting )h sician of the )ro%lem and ask for another s)ecimen/ An sam)le that gi.es a .alue M 4;; U>0 should %e diluted either 6<9 or 6<: with ;/C gK sodium chloride solution and the correct .alue o%tained % multi)l ing it % the a))ro)riate dilution factor/ !eference 6/ $omers GA1 Mc!om% *$ +6CE5,/ !lin/!hem/ 96< 6C??'6CC5/

Calciu*7O7Cresolphthalein Co*ple)one
Introduction

ethod

!alcium is the mineral )resent in the largest amount in the %od +665;g,/ A))roximatel CCK of total %od calcium is de)osited in the skeleton/ A higher )ro)ortion of non'skeletal calcium is )resent within cells than in extracellular fluids1 and most of this intracellular calcium is %ound to )roteins in the cell mem%rane/ Intracellular ioni3ed calcium is )h siologicall acti.e and functions as an intracellular messenger % %inding to or %eing released from s)ecific intracellular )roteins1 a )rocess that changes )rotein conformations and hence its acti.it or function/ Principle of the *ethod

!alcium forms a )ur)le'coloured com)lex with ortho'cresol)hthalein com)lexone in an alkaline medium/ &he inclusion of 2!l hel)s to release calcium %ound to )roteins and ? h drox '7uinoline eliminates the interference % magnesium/ 9'amino1 9'meth l1 6')ro)anol +AMP, )ro.ides the )ro)er alkaline medium for the colour reaction/ &he intensit of the colour is measured at 54;nm> ellow green filter/ Speci*en type+ collection and storage Serum is the )referred s)ecimen/ 2aemol sed and he)arinised sam)les are unsuita%le for this method/ Similarl 1 )lasma )re)ared using (D&A1 oxalate or citrate must not %e used as these )reser.ati.es cause remo.al of calcium % chelation/ !alcium in serum is sta%le for 69 hours at room tem)erature +95':5 ;!,1 one week at 9'?;! and for a longer )eriod u) to : months at '9;;!/ !eagents All chemicals must %e Analar grade A P Buffer pH ,-. / Measure :E/? ml of AMP reagent and add 65; ml of distilled water and mix/ AdNust the )2 to 6;/E with 8A2!I and make u) to 95;ml with distilled water/ Store in the refrigerator ina %rown coloured glass %ottle/ Sta%le for : weeks/ Colour reagent Add 65 ml conc/ 2!I to a 95; ml .olumetric flask containing a%out 95ml of distilled water/ &ransfer with washing 95 mg ;'cresol)hthalein com)lexone )owder into it1 mix to dissol.e/ &hen add 95; mg of ? h drox '7uinoline and dissol.e and then make u) to 95; ml with distilled water/ Store in a %rown coloured glass %ottle at room tem)erature +95':5;!,/ Sta%le for a%out one month/ Stoc1 calciu* standard 8- *g&dl $efore weighing1 dr calcium car%onate at 6;; ;! for 9 hours/ Allow to cool in a dessicator/ Dissol.e 895 mg of dried calcium car%onate in 5; ml of distilled water taken in a 5;; ml .olumetric flask and add :/5ml conc/ 2!l/ Mix to dissol.e and make u) to 5;; ml with distilled water/ Store in a %rown %ottle at room tem)erature +95':5;!,/ Sta%le for a%out 8 months/ 3or1ing standards Into four 6;; ml .olumetric flasks transfer 6;1 651 9; and 95 ml of stock calcium standard and dilute each to 6;; ml with %en3oic acid to get working standards containing 51 E/51 6;1 and 69/5 mg>dl calcium1 res)ecti.el / Store in %rown %ottles at room tem)erature +95':5 ;!,/ Sta%le for 9 months/ E4uip*ent+ glass5are and other accessories *efer to Section A +9,1 Introduction to SOP/ All glassware used in this method must %e scru)ulousl cleaned1 soaked o.ernight in :K +L>L, 2!l to remo.e traces of calcium1 thoroughl rinsed with distilled water and dried %efore use/ Procedure

&he )rotocol of the )rocedure is descri%ed %elow/ Add reagents1 standards +S5I 5mg>dl1 SE/5 I E/5mg>dl1 S6;I 6; mg>dl1 S69/5 I 69/5mg>dl, and test sam)le >-! in the order indicated into a))ro)riatel la%elled tu%es +6? x 65;mm,

Blan1
Distilled water +ml, ;/6

S8
'

S/.8
'

S,'

S,<.8
'

Test
'

;C
'

Standard +ml,

'

;/6

;/6

;/6

;/6

'

'

&est sam)les>-! +ml,

'

'

'

'

'

;/6

;/6

!olour reagent +ml,

9/;

9/;

9/;

9/;

9/;

9/;

9/;

Mix Well
$uffer 9/; 9/; 9/; 9/; 9/; 9/; 9/;

Mix Well
Incu%ate at room tem)erature +95':5 ;!, for 65 minutes/ Set the s)ectro)hotometer >filter )hotometer to 3ero using %lank at 54; nm> ellow green filter and measure the a%sor%ance of standards1 test G -!/ Calculation and cali@ration graph !onstruct a cali%ration gra)h % )lotting the a%sor%ance of the standards against their res)ecti.e concentrations/ Plot the a%sor%ance .alues of test>-! on the cali%ration gra)h and read off the concentrations/ &he measura%le range with this gra)h is from 6/; to 69/; mg>dl/ It is ad.isa%le to )lot a cali%ration gra)h whene.er the reagents are freshl )re)ared/ Once linearit is )ro.ed1 it is Nust enough if a single standard such as S 6; +6; mg>dl, is used and the conentration in the )atientTs sam)le is calculated using the formula < &est a%sor%ance '''''''''''''''''''''' x 6;///////////// mg>dl/ Standard a%sor%ance

Analytical relia@ilities *efer to pages /7A of section , 6General Introduction9 onthe use of internal -! and inter)retation of dail -! data +for releasing )atientsT results,/ Include one internal -! in e.er %atch of sam)les anal sed each da irres)ecti.e of the num%er of sam)les in a %atch/ Since calcium is anal sed single %atch in a da in an intermediate la%orator 1 it will not %e )ossi%le to anal se se.eral -! sam)les and calculate within'da )recision/ 2owe.er1 e.en if onl a single -! sam)le is anal sed in a da 1 this .alue can %e )ooled with the )receding 6; or 9; .alues o%tained in the )re.ious da s and between-day precisioncan %e calculated and ex)ressed as K!L/ (nsure that this is well within the acce)ta%le limit1 i/e1 ?K/ At least once a week anal se another -! serum from either a low -! or high -! )ool/ HAssa edH -! sera with stated .alues +ranges, are a.aila%le from se.eral commercial sources1 .i3 $oehringer Mannheim1 $io*ad G *andox/ If a laboratory uses QC sera from a commercial source, it is important that the company certifies that their QC materials are traceable to international reference materials. Ha0ardous *aterials Concentrated hydrochloric acid, which is corrosive, is used in this procedure. Do not mouth pipette or swallow, and avoid contact with skin and mucous membranes. !eference range and clinical interpretation Serum !alcium///O/ ?/5 ' 6;/4 mg>dl !alcium measurements are used in the diagnosis and treatment of )arath roid diseases1 a .ariet of %one diseases1 chronic renal failure and tetan / Increased serum calcium le.els are associated with )rimar h )er)arath roidism1 multi)le m eloma1 metastatic %one lesions and h )er.itaminosis D/ 2 )ocalcaemia is associated with h )o)arath roidism1 ne)hrotic s ndrome1 and rickets and renal failure/ Li*itations 2aemol sed and li)aemic sera interfere with the measurement of calcium/ Set u) an a))ro)riate %lank for such sam)les % adding ;/6ml of serum to 4ml of distilled water/ &he a%sor%ance o%tained against

distilled water is then su%tracted from the test a%sor%ance %efore the test result is calculated/ !eference 6/ Gitelman 2/+6C8E, Anal/$iochem 9; < 596/

Phosphorus7Stannous Chloride !eduction

Introduction

ethod

(ight )er cent of %od )hos)horus is laid down in %one matrix as insolu%le salts/ &he organic )hos)hate esters are )rimaril confined within cells1 associated with nucleo)roteins1 hexoses and )urines/ Phos)hate forms high energ %onds in A&P1 G&P and creatine )hos)hate/ Inorganic )hos)hate ions are mostl confined to the extracellular fluid where the are )art of %uffer s stems/ &he )lasma )hos)hate concentration is regulated % )arath roid hormone +P&2, and .itamin D:/ P&2 stimulates the kidne to excrete )hos)hate while conser.ing calcium/ In chronic renal disease1 )hos)hate retention occurs %ecause of im)aired glomerular filtration/ Principle of the *ethod Phos)horus in serum reacts with ammonium mol %date to form )hos)homol %date1 which is then reduced % stannous chloride and h dra3ine sul)hate to mol %denum %lue/ &he intensit of the colour is measured at 84; nm/ Speci*en type+ collection and storage Use onl clear1 non'haemol sed serum se)arated from er throc te as soon as )ossi%le1 as cells contain organic )hos)hate which can %e en3 maticall clea.ed there% increasing the serum concentration of )hos)horus/ Phos)horus in serum is sta%le for 69 hours at room tem)erature +95':5 ;!,1 one week at 9'?;! and for a longer )eriod u) to : months at 9;!/ !eagent All chemicals must %e Analar grade Trichloroacetic acid = g&dl Dissol.e : g trichloroacetic acid +&!A, in distilled water and make u) to a final .olume of 6;; ml/ Store in a %rown glass %ottle at room tem)erature +95':5;!,/ Sta%le for : months/ A**oniu* *oly@date solution Into a 6;; ml .olumetric flask add E; ml of distilled water and then add slowl :/5 ml of conc/ 29SO4/ Add 6g of ammonium mol %date/ Mix well and make u) to the mark with distilled water/ Sta%le for one month at 9'?;!/ Hydra0ine sulphate solution Into a 6;; ml .olumetric flask add E; ml of distilled water and add slowl 9/? ml of conc/ 29SO4 followed % ;/9 g h dra3ine sul)hate and ;/;9g stannous chloride/ Mix well and make u) to the mark with distilled water/ Store in a %rown glass %ottle/ Sta%le for one month at 9'? ;!/ Stoc1 phosphorus standard ,-- *g&dl

=eigh 9/6C g anh drous D29PO4 and dissol.e in distilled water and then make u) to 5;; ml with distilled water/ Sta%le for one month at 9 '?;!/ 3or1ing phosphorus standard ,- *g&dl Dilute 6; ml of stock )hos)horus standard to 6;; ml with distilled water/ Sta%le for one month at 9' ?;!/ (7ui)ment1 glassware and other accessories *efer to Section A +9,1 Introduction to SOP/ Procedure &he )rotocol of the )rocedure is descri%ed %elow/ Pi)ette the following into a))ro)riatel la%elled 6: x 6;; mm tu%es/ $ilution of Standards S,7 S>K Test : ;C.

S,
:gK &!A + ml, 6/C5

S<
6/C

S=
6/?5

S>
6/?

Test
6/?

6;mg>dl standard +ml,

;/;5

;/6

;/65

;/9

'

&est sam)le > -! +ml,

'

'

'

'

;/9 for 6;

Mix well/ 0ea.e for 5 minutes at room tem)erature +95 ':5 ;!, !entrifuge test G -! onl minutes at :;;; r)m/ Colour de?elop*ent Pi)ette the following into a))ro)riatel la%elled 6? x 65; mm tu%es

Blan1
:g>dl &!A +ml, 6/; Diluted standard +ml, ' Su)ernatant +ml, ' Amm/Mol %date +ml, 6/; 2 dra3ine +ml, Sul)hate 6/;

S,
'

S<
'

S=
'

S>
'

Test
'

;C
'

6/;

6/;

6/;

6/;

'

'

'

'

'

'

6/;

6/;

6/;

6/;

6/;

6/;

6/;

6/;

6/;

6/;

6/;

6/;

6/;

6/;

Mix and lea.e for 5 minutes at room tem)erature +95':5 U !,/ Set the s)ectro)hotometer>filter )hotometer to 3ero using %lank at 84; nm>red filter and measure

the a%sor%ance of standards1 test and -!/ Calculation and cali@ration graph Since the )rotocol for standard tu%e S4 and the test is identical1 the Standard S4 will re)resent a concentration of 6; mg>dl/ &he )hos)horus concentrations re)resented % other standards are SII9/51 S9I5/; G S: I E/5 mg>dl/ Plot the a%sor%ance .alues of standards against their res)ecti.e concentrations/ &he measura%le range with this gra)h is from ;/5 to 6;/; mg>dl/ Plot the a%sor%ance .alues of test>-! on the cali%ration gra)h and read off to the concentrations/ Once linearit is )ro.ed1 it isnot necessar to )re)are the standard gra)h e.er time that )atientsT sam)les are anal sed/ It will %e ade7uate if standard S9 is taken e.er time and )atientsT results are calculated using the formula<

&est a%sor%ance '''''''''''''''''''''' x 5O/////mg>dl Standard a%sor%ance

Analytical relia@ilities *efer to pages /7A of section , 6General Introduction9 on the use of internal -! and inter)retation of dail -! data +for releasing )atientsT results,/ Include one internal -! in e.er %atch of sam)les anal sed each da irres)ecti.e of the num%er of sam)les in a %atch/ Since )hos)horus is anal sed ina single %atch in a da in an intermediate la%orator 1 it will not %e )ossi%le to anal se se.eral -! sam)les and calculate within'da )recision/ 2owe.er1 e.en ifonl a single -! sam)le is anal sed in a da 1 this .alue can %e )ooled with the )receding 6; or 9; .alues o%tained in the )re.ious da s and between-day precision can %e calculated and ex)ressed as K!L/ (nsure that this is well within the acce)ta%le limit1 i/e1 ?K/ At least once a week anal se another -! serum from either a low -! or high -! )ool/ HAssa edH -! sera with stated .alues +ranges, are a.aila%le from se.eral commercial sources1 .i3/ $oehringer Mannheim1 $io*ad G *andox/

If a laboratory uses QC sera from a commercial source, it is important that the company certifies that their QC materials are traceable to international reference materials. Ha0ardous reagents The rea ents include conc. ()%'*, which is corrosive. $void contact with skin and eyes. +ash with copious amounts of water if it comes into contact with skin or eyes. !eference range and clinical interpretation Serum )hos)horous :/5 ' 5/; mg>dl An increase in serum )hos)horous is found in chronic ne)hritis )rogressing with increased renal failure/ A moderate increase is o%ser.ed in h )o)arath roidism and .itamin D excess/ A decrease in serum )hos)horous is o%ser.ed in rickets or osteomalacia and also in h )er)arath roidism/ 2 )o)hos)hatemia ma result due to disorders of renal tu%ular rea%sor)tion/ Li*itations !ontaminated glassware is the greatest source of error/ &herefore all glassware used in this method must %e scru)ulousl cleaned1 soaked o.ernight in :K +L>L, 2!l1 thoroughl rinsed with distilled water and dried %efore use/ 2aemol sed and li)aemic sera interfere with the measurement of )hos)horous/ Set u) an a))ro)riate %lank for such sam)les % adding ;/9ml serum to :ml of distilled water/ &he a%sor%ance o%tained against distilled water is then su%tracted from the test a%sor%ance %efore the test result is calculated Serum must %e se)arated % centrifugation as soon as )ossi%le after collection of the )atientTs %lood sam)le1 )refera%l Q9 hours1 otherwise1 )hos)hate )resent in er throc tes will %e released into the serum causing falsel ele.ated .alues/ !eference 6/ 2arold Larle HPractical !linical $iochemistr 8th (dn 6C?? )age 44?'44C/

Sodiu* and Potassiu* 7'la*e Photo*etry

Introduction Sodium1 the maNor extracellular cation1 )la s a role in fluid distri%ution among %od com)artments/ &he ingested sodium is filtered in the renal glomerulus and a))roximatel E;K is rea%sor%ed in the )roximal tu%ule/ Further rea%sor)tion occurs in the loo) of 2enle and Q5K is rea%sor%ed distall under the influence of aldosterone/ A%out 85'E;K of the total %od sodium is in its exchangea%le form/ &he exchangea%le sodium is made u) of extracellular and intracellular sodium/ &he intracellular sodium concentration isa%out 6; mmol>0 and the extracellular1 i/e/ the )lasma sodium concentration1 isa%out 64; mmol>0/ Sodium maintains the osmotic )ressure of the extracellular fluid and hel)s in retaining water in the extracellular com)artment/ Along with other cations it is also in.ol.ed in neuromuscular irrita%ilit 1 acid %ase %alance1 maintenance of %lood .iscosit and resting mem%rane )otential/ A high )lasma sodium concentration of more than 645 mmol>0 is referred to as h )ernatremia/ &his can occur due to sim)le deh dration1 excess sodium intake1 steroid thera) as well as in dia%etic insi)idus/ 2 )onatremia1 with )lasma sodium concentration less than 6:; mmol>01 can occur due to diuretic medication1 kidne disease1 excessi.e sweating1 congesti.e heart failure or gastrointestinal

disorder/ Potassium is the maNor intracellular cation/ It is widel distri%uted in the %od in muscle tissue1 ner.e tissue1 %lood cells and )lasma/ It is filtered in the glomerulus1 a%sor%ed in the )roximal tu%ule and finall excreted % exchange for sodium in the distal tu%ule/ Potassium influences muscular acti.it 1 cardiac function and ner.e conduction )rocess/ In h )erkalemia the )lasma )otassium concentration exceeds 5/5 mmol>0/ Acute h )erkalemia is a medical emergenc / In h )okalemia the )lasma )otassium le.el will %e less than :/5 mmol>0/ &his can occur due to excessi.e loss in gastrointestinal secretions and urine1 and also in renal tu%ular acidosis/ Principle of the *ethod =hen a solution of an inorganic salt such as sodium chloride is s)ra ed into the flame1 the elements in the com)ound are )artl con.erted into the atomic state/ Due to the heat energ of the flame a .er small )ro)ortion of these atoms is excited and the electrons mo.e to a higher energ le.el/ &he )ro)ortion of the atoms that are excited de)ends u)on the concentration of the )articular element and on the tem)erature of the flame/ In the excited state the electrons are unsta%le and the ra)idl re.ert %ack to their former lower energ le.el/ As the change from the excited state or higher energ le.el %ack to the lower energ le.el1 the emit the light in the form of a fixed wa.elength1 to )roduce a s)ectrum/ Under carefull controlled conditions the amount of light emitted is directl )ro)ortional to the num%er of atoms that are excited1 which in turn is )ro)ortional to the concentration of the su%stance in the sam)le/ Speci*en type+ collection and storage $oth sodium and )otassium are sta%le in serum for se.eral hours at 95':5U ! and for : months at '9;U !/ Anticoagulants containing sodium or )otassium salts are not suita%le1 %ut lithium he)arin ma %e used as an anticoagulant/ If whole %lood is left unse)arated for M:hours or refrigerated1 )otassium will leak out of the red cells gi.ing falsel increased .alues/ !eagents All chemicals must %e Analar grade/ Sodium chloride +Aa!I, and )otassium chloride +D!I, should %e dried for 9': hours at a%out 6;;U ! %efore use/ $efore weighing1 the chemicals must %e allowed to cool to room tem)erature either in a desiccator or in a container with a tight'fitting lid with a small air s)ace/ Stoc1 Sodiu* ,--- **ol&L =eigh out 9C/95 g dried Aa!l1 dissol.e in a%out 4;; ml of distilled water taken in a 5;; ml .olumetric flask and then make u) to 5;;ml with distilled water/ Store in a ) rex glass %ottle at 95' :5U !/ Sta%le for one ear/ Stoc1 Potassiu* ,-- **ol&L =eigh out ;/E48 g dried D!I1 dissol.e in a%out ?; ml of distilled water taken in a 6;; ml .olumetric flask and then make u) to 6;; ml with distilled water/ Store in a ) rex glass %ottle at 95':5;!/ Sta%le for one ear/ 3or1ing Standards 0ow standard for Sodium 6;;mmol>0< Dilute 6; ml of stock sodium to 6;; ml with distilled water/ Sta%le for 8 months at 95':5;!/ !om%ined standard for sodium and )otassium 64;AaF>5DF mmol>0< Dilute 64 ml of stock

sodium and 5 ml of stock )otassium together to 6;; ml with distilled water/ Store in a ) rex %ottle at 95':5;!/ Sta%le for 8 months/ As)iration standard for sodium ' 6/; mmol>0< Dilute 6/; ml of working standard to 6;; ml with distilled water/ Pre)are fresh each time/ !om%ined as)iration standard for sodium 6/4 mmol>0 and )otassium ;/;5 mmol>0/ Dilute 6/; ml of working standard +com%ined standard for AaF>DF64;>5mmol>0 , to 6;;ml with distilled water/ Pre)are fresh each time/ E4uip*ent+ glass5are and other accessories *efer to Section A +9,1 Introduction to SOP/ Procedure &he )rotocol of the )rocedure is descri%ed %elow/ Sa*ple dilution Dilute each serum sam)le 6<6;; with distilled water % water/ mixing ;/ 6ml sam)le with C/C ml distilled

Procedure for si*ultaneous *easure*ent of NaL : HL in the fla*e photo*eter 6digital fla*e photo*eter9 Switch on the flame )hotometer/ Digital dis)la should turn on/ &urn the set R+full scale, F/S/ coarse and fine controls# into maximum clockwise )osition/ Select a))ro)riate filter with the hel) of filter selector wheel +AaF on the left side and DF on the right side,/ Switch on the com)ressor and check the air )ressure/ AdNust it to read %etween ;/4 and ;/8 k g>cm9/ O)en the gas c linder1 remo.e the tra))er at the rear of the flame )hotometer and ignite the flame/ AdNust the gas regulator to get a maximum height non'luminous %lue flame with 6; distinct cones +5 on each side of the %urner head,/ Feed distilled water to the atomi3er and wait for at least :; seconds/ AdNust the TSet *ef !oarseT and Fine controlsT to 3ero digital readout for DF onl / As)irate 6/; mmol>0 AaF solution/ =ait at least :; seconds and then adNust the Set *ef !oarse and Fine controlsT to a digital read out of I ;; for AaF onl / As)irate the com%ined standard solution +6/4>;/;51 AaF>DF, and wait at least for :; seconds/ AdNust TF/S controlT on AaF side for readout 64; and that on DF side for a digital readout of 5;/ *e)eat ste)s C and 6; once again/ &he flame )hotometer now stands cali%rated/ Aow feed diluted test sam)le > -! to the atomi3er for at least :; seconds %efore recording the readings for AaF and DF/ Calculation After as)irating the standard solution1 the digital reading for Aa F is adNusted to 64; and that of DF to 5;/ &his is done in order to re)resent Aa F and DF .alues in undiluted serum/ Since the test sam)le>-! is diluted initiall 6< 6;; and then as)irated1 the initial standard .alues for Aa F G DF +6/4 G ;/;5 mmol>0, must %e multi)lied % 6;; to re)resent 64; mmol>0 AaF and 5 mmol>0 DF/ In the case of D F1 in order to im)ro.e the sensiti.it of the assa the digital reading for the standard is further multi)lied % 6; to show a reading of 5;/ In essence1 the test sam)le>-! digital readings are com)ared with the standard readings for Aa F and DF/ &he digital reading a))earing for AaT of the test sam)le>-! is read as mmol>0 .alue straightawa / On the other hand1 the test sam)le >-! DF .alue re)resents 6>6;th of the digital reading/ For exam)le1 digital reading for H64; Aa F I 64; mmol>0 AaFJ digital reading for 45 DF I 4/5 mmol>0 DF Analytical relia@ilities

*efer to pages /7A of section , 6General Introduction9 on the use of internal -! and inter)retation of dail -! data +for releasing )atientsT results,/ Since AaF1 DF are .er commonl anal sed )arameters in a la%orator 1 it isrecommended that internal -! +normal -! )ool, %e included with e.er %atch of sam)les anal sed in a da 1 irres)ecti.e of the num%er of sam)les in a %atch/ Further1 e.en when a single sam)le is anal sed as an Hemergenc H sam)le at an time of the da or night1 it is essential to include an internal -!/ From the -! results o%tained for the da 1 mean1 standard de.iation and K !L can %e calculated to ensure that withinday precision is well within the acce)ta%le limit1 i/e/ 4K/ &he mean .alue of internal -! for the da can %e )ooled with the )receding 6; or 9; mean .alues o%tained in the )re.ious da s and between-day precision can %e calculated and ex)ressed as K !L/ (nsure that this is well within the acce)ta%le limit1 i/e1 ?K/ At least once a week anal se another -! serum from either a low -! or high -! )ool/ HAssa edH -! sera with stated .alues +ranges, are a.aila%le from se.eral commercial sources1 .i3/ $oehringer Mannheim1 $io*ad G *andox/ 2owe.er1 care should %e taken when o)erating the flame )hotometer as the technician will %e using li7uefied )etroleum gas/ 0eakage of either air or gas during o)eration will cause ex)losion/ A))l soa) solution at the connecting )oint to check such leakage/ Ha0ardous *aterials The rea ents used are made up of only sodium chloride and potassium chloride. Therefore no precautionary measures are re,uired. !eference range and clinical interpretation &he reference ranges % this method are< Serum sodium 6:; ' 645 mmol>0 Serum )otassium :/5 ' 5/; mmol>0 (le.ated le.els of serum sodium occur in conditions such as se.ere deh dration1 h )eradrenalism and %rain inNur / 0ow serum sodium .alues are noticed in meta%olic acidosis1 salt' losing ne)hritis1 AddisonTs disease1 etc/ Increased serum )otassium le.el is o%ser.ed inanoxia1 meta%olic renal tu%ular acidosis and shock or circulator failure/ 0ow serum )otassium .alues are o%ser.ed due to low intake of dietar )otassium o.er a )eriod of time or increased loss through kidne 1 .omiting or diarrhoea/ Increased secretion of adrenal steroids or some diuretics ma also )romote the loss of )otassium/ Li*itations A.oid using haemol sed serum/ &his will cause ele.ated DF le.el/ *elia%ilit of the results de)ends on the )ro)er maintenance of the flame )hotometer1 salient features of which are listed %elow/ !e4uire*ents Aon'luminous %lue flame Su))l of dr air at a controlled )ressure1 .i3/ 6;' 65 Dg >cm9

*egular a.aila%ilit of li7uid )etroleum gas aintenance Disconnect )ower and gas su))l %efore )roceeding to do maintenance/ &urn the control in %oth AaF>DF dis)la full anti'clockwise/ Disconnect the drain outlet and the gas and air inlets/ *emo.e the to) )anel and disconnect the )hotocell/ *emo.e the side )anel and take out the atomi3er/ Disconnect the air line at the )ressure gauge/ Disconnect the air and gas inlets to the mixing cham%er/ *emo.e the %urner head and remo.e mixing cham%er =ash the a%o.e well with ta) water and distilled water/ !lean the atomi3er with a thin wire and adNust its s)ra % )assing com)ressed air/ &his can %e done % screwing > unscrewing the two knurled nuts in the atomi3er/ *emo.e the air tu%e and flush out an water remaining in the tu%e due to the cooling of com)ressed air/ Do not use ox acet lene or highl ex)losi.e mixture as fuel/ After cleaning1 refix e.er thing carefull / Cleaning of ?arious units in a fla*e photo*eter Ato*i0er and capillary tu@e# Flushing with co)ious amount of distilled water is ade7uate/ If %lockage occurs1 remo.e the atomi3er from its seating and flush with dr air or clean it using a thin wire/ If cleaning of atomi3er is done with a wire %efore and after using it1 %lockage will rarel occur/ If all the a%o.e fails1 a new atomi3er is to %e fixed/ i)ing cha*@er# Flushing with distilled water is ade7uate/ Do not use detergent or soa) solution %ecause it will remain inside if washing is not done )ro)erl out and will gi.e erratic reading due to the )resence of AaF > DF in the soa) solution/ 'ault diagnosis

Sy*pto*
6/ Unsta%le reading

$iagnosis
(xcessi.e .i%ration Air su))l %locked

!e*edy
Pro.ide shock')roof %ase +e/g/, glass )late on foam ru%%er/ !heck air su))l and clear %lockage

Atomi3er low gas )ressure *emo.e1 wash and dr %urner Filter dirt 9/ Intermittent reading $locked atomi3er Fault )hotocell Dirt )hotocell :/ 0ow sensiti.it $locked atomi3er 0ow gas )ressure Fault )hotocell !lean with iso)ro)anol/ !lean atomi3er using a thin wire !hange )hotocell !lean )hotocell !lean %lockage !heck gas )ressure !hange )hotocell/

Cere@rospinal 'luid 6CS'9

Introduction !SF originates from the %lood/ &he choroid )lexes in the 6st 1 9nd and :rd .entricles of the %rain are the sites of !SF )roduction/ !SF is formed from )lasma % the filtering and secretar acti.ities of the choroid )lexus and lateral .entricles/ !SF circulates around the %rain and the s)inal cord/

!SF nourishes the tissues of the central ner.ous s stem +!AS, and hel)s to )rotect the %rain and the s)inal cord from inNur / It )rimaril acts as a water shock a%sor%er/ It totall surrounds the %rain and the s)inal cord and thus a%sor%s an %low to the %rain/ !SF also acts as a carrier of nutrients and waste )roducts %etween the %lood and the !AS/ S)ecimen t )e1 collection and storage/ Onl a )h sician or a s)eciall trained nurse must collect the s)ecimen/ 0a%orator )ersonnel should %e )resent1 howe.er1 so that the s)ecimen is deli.ered to the la%orator immediatel after collection/ S)inal )uncture isa )rocess % which a long needle is inserted into the su%arachnoid s)ace %etween 0I and 05 and a%out 9ml of !SF is withdrawn/ &he s)ecimen is then transferred into a clean )enicillin .ial containing a%out ?mg of a mixture of (D&A and sodium fluoride in the ratio of 6<9/ Protein estimation can also %e carried out on this s)ecimen/ Glucose and )rotein estimations should %e )erformed as soon as )ossi%le after drawing the !SF s)ecimen/ If testing is dela ed1 the s)ecimen ma %e fro3en at '9; ;! u) to : da s/ !entrifuge s)ecimens containing red %lood cells or )articulate matter/ Do not dela testing the !SF %ecause cells and tr )anosomes are ra)idl l sed once the !SF is remo.ed from the %od / Glucose will %e ra)idl destro ed inthe a%sence of )reser.ati.es/ !e*e*@er# !SF is the most )recious %iological material/ Often1 onl small .olumes of !SF are a.aila%le for anal sis due to difficult in collection/ 2ence handle this with care/ &he s)ecimen ma contain .irulent organisms/ A.oid mouth )i)etting/

CS' Glucose ( Glucose O)idase

Principle of the *ethod

ethod

Glucose )resent in the !SF is oxidi3ed % the en3 me glucose oxidase +GOD, to gluconic acid with the li%eration of h drogen )eroxide1 which is con.erted into water and ox gen % the en3 me )eroxidase +POD,/ 4 amino)hena3one1 an ox gen acce)tor1 takes u) the ox gen and together with )henol forms a )ink coloured chromogen which can %e measured at 565nm/ GOD Glucose 4 Gluconic acid F 29O9 POD 29;9''''''''''''''M 4 29; F @;B @;B F 4 amino)hena3one F )henol ''''''M chromogen +)ink,
'''''''''''''''''''M 4

Gluconic acid F 29O9

!eagents All chemicals must %e Analar grade Phosphate @uffer # ,-- **ol&L. pH /.&o ?;; ml of distilled water add the following in the order< Disodium h drogen )hos)hate dih drate @Aa92PO4 929OBO//69/C5 gJ Anh drous )otassium dih drogen )hos)hate @D29PO4BO//4/C5 gJ Sodium a3ide @AaA:BO//;/5 g Add one % one1 dissol.e and finall make u) to one litre with distilled water/ Sta%le for :'4

months1 at 9'?;!/ !heck final )2 with a )2 meter/ Colour reagent &o 6;;ml of the a%o.e )hos)hate %uffer add the following in the order and then mix to dissol.e < 4 amino )hena3one 68 mg GOD @Sigma G E;68B 6?;; units POD @Sigma P ?95; B 6;; units Phenol 6;5 mg &ween 9; @Sigma P 6:5CB 5;m l *econstitute the GOD and POD )owder with )hos)hate %uffer/ Dis)ense se)aratel into .ials so that each .ials re)resents the re7uisite num%er of units/ Store the .ials fro3en/ Sta%le for 9 weeks at 9'?;!/ Store in a %rown %ottle/ Ben0oic acid ,g&l. Dissol.e 6/;g of %en3oic acid in water and make u) to one litre with water/ &his solution is sta%le indefinitel at room tem)erature/ Stoc1 Glucose solution+ , g&l. $efore weighing1 dr the glucose at 8;'?; ;! for 4 hours/ Allow to cool in a dessicator/ Dissol.e 6g of glucose in %en3oic acid solution and make u) to 6;; ml in a .olumetric flask/ Sta%le for six months at room tem)erature +95':5;!,/ $o not free0e the standard 3or1ing glucose standard ,-- *g&dl. Dilute 6; ml of stock glucose +use either a .olumetric )i)ette or a %urette, to 6;; ml with %en3oic acid in a 6;; ml .olumetric flask/ Mix well/ Sta%le for 8 months at room tem)erature +95':5 ;!,/ E4uip*ent+ glass5are and other accessories *efer to Section A +9,1Introduction to SOP/ Procedure &he )rotocol of the )rocedure is descri%ed %elow/ $ilution of standards 6S,7S89+ test : ;C Pi)ette the following into a))ro)riatel la%elled 6: x 6;; mm tu%es

S,
Distilled =ater +ml, 6/C

S<
6/?

S=
6/E

S>
6/8

S8
6/5

Test
6/C

;C
6/C

6;; mg>dl glucose +ml,

;/6

;/9

;/:

;/4

;/5

'

'

&est sam)le >-! +ml,

'

'

'

'

'

;/6

;/6

Mix well Colour de?elop*ent Pi)ette the following into another set of a))ro)riatel la%elled tu%es/

Blan1 !olour reagent +ml,

S,

S<

S=

S>

S8

Test

;C

6/9

6/9

6/9

6/9

6/9

6/9

6/9

6/9

Distilled water +ml,

;/6

;/6

'

'

'

'

'

'

Diluted Standards +ml, Diluted +ml, &est sam)le>-!

'

'

;/6

;/6

;/6

;/6

'

'

'

'

'

'

'

'

;/6

;/6

Mix all tu%es well/ Incu%ate at :E ;! in a water%ath for 65 minutes/ *emo.e from water%ath and cool to room tem)erature/ Set the s)ectro)hotometer> filter )hotometer to 3ero using %lank at 56; nm> green filter and measure the a%sor%ance of standards1 test and -!/ &his )rotocol is designed for s)ectro)hotometers > filter )hotometers that re7uire a minimum .olume of reaction mixture in the cu.ette of one ml/ or less/ Since economical use of reagents is )ossi%le with this )rotocol1 the cost )er test can %e ke)t to the minimum/ 2owe.er1 if a la%orator em)lo s a )hotometer re7uiring a large .olume of the reaction mixture for measurement1 .i3/ 5 ml1 it is ad.ised that the .olume of all reagents mentioned under &a%ulation H+%, !olour de.elo)mentH1 %e increased )ro)ortionatel / Calculation and cali@ration graph Since the )rotocol for standard tu%e S6 and test is identical1 the standard S6 will re)resent a concentration of 6;; mg>dl/ &he glucose concentrations re)resented % other standard tu%es are S9 I9;;J S: I :;;J S4 I4;; and S5 I 5;; mg>dl/ Plot the a%sor%ance .alues of standards against their res)ecti.e concentrations/ &he measura%le range with this gra)h is from 6; to 5;; mg>dl/ Plot a%sor%ance .alues of test>-! on the cali%ration gra)h and read off the concentrations/ Once linearit is )ro.ed1 it is not necessar to )re)are the standard gra)h e.er time that )atients# sam)les are anal sed/ It will %e ade7uate if standard S9 is taken e.er time and )atients# results are calculated using the formula < &est a%sor%ance ''''''''''''''''''''''''''' x 9;; OOOO mg>dl Standard a%sor%ance

Analytical relia@ilities *efer to pages /7A of section , 6General Introduction9 on the use of internal -! and inter)retation of dail -! data +for releasing )atients# results,/ Since !SF anal sis is carried out infre7uentl in intermediate la%oratories1 one -! for glucose should %e included as and when !SF glucose is anal sed/ 2ence it will not %e )ossi%le to anal se se.eral -! sam)les and calculate within'da )recision/ 2owe.er1 e.en if onl a single -! sam)le is anal sed in a da 1 this .alue can %e )ooled with the )receding 6; or 9; .alues o%tained in the )re.ious da s and between-day precision can %e calculated and ex)ressed as K !L/ (nsure that this is well within the acce)ta%le limit1 i/e1 ?K/ At least once a da anal se another -! serum from either a low -! or high -! )ool/ HAssa edH -! sera with stated .alues +ranges, are a.aila%le from se.eral commercial sources1 .i3/ $oehringer Mannheim1 $io*ad G *andox/ If a la%orator uses -! sera from a commercial source1 it is im)ortant that the com)an certifies that their -! materials are tracea%le to international reference materials/ Ha0ardous *aterials This procedure uses sodium azide and phenol, which are poisonous and caustic. Do not swallow, and avoid contact with skin and mucous membranes !eference range and clinical interpretation !oncentrations of anal tes in the !SF should alwa s %e com)ared with those in )lasma/ Aormal !SF glucose is a%out 8;K of the )lasma .alue/ Aormal range for !SF glucose 5;'?; mg>dl Decreased !SF glucose le.els are o%ser.ed in tu%erculosis1 %enign l m)hoc tic chronic meningitis and in h )ogl cemia/ Increased le.els are o%ser.ed in ence)halitis1 )oliom elitis and in cere%ral a%scess/ Li*itations Grossl %lood !SF ma gi.e s)uriousl ele.ated .alues for glucose/ Undue dela in anal sis ma gi.e

low .alues/ &he re)ort to the re7uesting )h sician should include the a))earance of the !SF %efore and after centrifugation/ !eferences 6/ 9/ &rinder1 P/ +I C8C,/ Annals of !lin/ $iochem/ 8< 94 ' 9E/ $arham D and &rinder P/ +6CE9,/ Anal st CE< 649'645/

CS' Protein 7 Pyrogallol $ye Binding

Principle of the *ethod

ethod

Protein molecules )resent in !SF %ind 7uantitati.el with ) rogallol red'mol %date com)lex at )2 9/; to form a .iolet com)lex1 the intensit of which is measured at 8;;nm and com)ared with the colour gi.en % a set of human )rotein standards/ !eagents All chemicals must %e Analar grade Pyragallol red dye Dissol.e 6; mg of disodium mol %date1 5/Cg of succinic acid1 6:4mg of sodium oxalate and 4:;mg of sodium %en3oate in a%out ?;; ml of distilled water taken in a one'litre .olumetric flask/ &o this add 95 mg of ) rogallol red d e and mix well till it is com)letel dissol.ed/ Make u) to the mark with distilled water/ Store in an am%er %ottle/ Sta%le at 9'?;! for : months/ Standards *efer &otal Protein+)age 4; +c, Standard, for the )re)aration of )rotein standard from )ooled serum/ Aote that there is alwa s a risk of infection from )ooled serum/ After determining the concentration of the )rotein standard1 dilute this to se.eral le.els as descri%ed %elow/ For exam)le1 if the total )rotein .alue of the )ooled serum is8/Eg>dl1 then the .olume of )ooled serum to %e diluted to 6;;ml to get a )rotein concentration of 9; mg>dl is found out using the dilution formula/ IL x I! I FL x F! IL I Initial Lolume of )ooled serum/ IC I Initial !oncentration of total )rotein +mg>dl,/ FL I Final Lolume/ F! I Final !oncentration IL x 8E;; I 6;; x 9; IL I 6;; x 9; 9; ''''''''''' ''''' I ;/: ml 8E;; 8E i/e/ Dilute ;/: ml of the )ooled serum to 6;;ml with ;/C g>dl sodium chloride containing ;/6 g>dl sodium a3ide/ Similarl 1 4;1 ?; and 69; mg>dl standards are )re)ared % diluting ;/81 6/9 and 6/? ml of the )ooled serum each to 6;; ml with the diluent/ &he standards are sta%le at 9'?U ! for one

month/ &he following ta%le summari3es the )re)aration of diluted standards/ Concentration of protein in pooled seru* is ta1en as J./g&dl.

Standard

Pooled seru* 6*l9

'inal diluted ?olu*e 6*l9

6;;

Concentration of Standard 6*g&dl9

9;

S6

;/:

S9

;/8

6;;

4;

S:

6/9

6;;

?;

S4

6/?

6;;

69;

E4uip*ent+ glass5are and other accessories *efer to Section A +9,1 Introduction to SOP/ Procedure &he )rotocol of the )rocedure is descri%ed %elow/ Pi)ette the following into a))ro)riatel la%elled 6: x 6;; mm tu%es

Blan1
P rogallol red +ml, :/;

S,
:/;

S<
:/;

S=
:/;

S>
:/;

Test
:/;

;C
:/;

Distilled water +ml,

;/;5

'

'

'

'

'

'

Standard +ml,

'

;/;5

;/;5

;/;5

;/;5

'

'

&est sam)le +ml,

'

'

'

'

'

;/;5

'

-! serum+6<6;;, +ml,

'

'

'

'

'

;/;5

Mix all tu%es well/ 0ea.e at 95':5 ;! for 65 minutes/ Set the s)ectro)hotometer >filter )hotometer to 3ero using %lank at 8;; nm> red filter and measure the a%sor%ance of standards1 test and -!/ Calculation and cali@ration graph Since standards and test> -! )rocedures are identical1 the a%sor%ance .alues of standards are )lotted

against their res)ecti.e concentrations/ &he cali%ration cur.e should %e linear u) to 69; mg>dl1 with a lower limit of 4'5mg>dl/ Plot the a%sor%ance .alues of test on the cali%ration gra)h and read off )rotein concentrations in )atients# !SF/ As 6< 6;; diluted -! serum is anal sed1 read off the )rotein concentration in -! on the cali%ration gra)h and multi)l the .alue % 6;; to get the correct )rotein .alue in -! serum/ Once linearit is )ro.ed1 it is not necessar to )re)are the standard gra)h e.er time that )atientsT sam)les are anal sed/ It will %e ade7uate if standard S4 is taken e.er time and )atientsT results are calculated using the formula/ &est a%sor%ance '''''''''''''''''''''''''''' x 69; mg>dl/ Standard a%sor%ance

Analytical relia@ilities *efer to pages /7A of section , 6General Introduction9 on the use of internal -! and inter)retation of dail -! data +for releasing )atients# results,/ Include one internal -! e.er time a )atient s)ecimen is measured1 irres)ecti.e of the num%er of sam)les in a %atch/ 2owe.er1 e.en if onl a single -! sam)le is anal sed in a da 1 this .alue can %e )ooled with the )receding 6; or 9; .alues o%tained in the )re.ious da s and the between-day precision can %e calculated and ex)ressed as K!L/ (nsure that this is well within the acce)ta%le limit1 i/e1 ?K/ At least once a week anal se another -! serum from either a low -! or high -! )ool/ HAssa edH -! sera with stated .alues +ranges, are a.aila%le from se.eral commercial sources1 .i3/ $oehringer Mannheim1 $io*ad G *andox/ If a laboratory uses QC sera from a commercial source, it is important that the company certifies that their QC materials are traceable to international reference materials. Ha0ardous *aterials This procedure uses sodium azide. Do not swallow, and avoid contact with skin and mucous

membranes. !eference range and clinical interpretation !SF )rotein ' 65'45 mg>dl Aormall the )rotein )resent in!SF is entirel al%umin1 %ut in man disease states1 !SF contains a mixture of al%umin and glo%ulins/ Increase in )roteins u) to 4;; mg>dl is o%ser.ed in meningitis and u) to se.eral grams in s)inal tumour/ In inflammator lesion1 increase in )rotein is associated with increase in cells/ A marked increase is also o%ser.ed in )aral sis and in disseminated sclerosis/ A%normall increased total !SF )rotein ma %e found in conditions where there is an increased )ermea%ilit of the ca)illar endothelial %arrier through which ultrafiltration occurs/ (xam)les of such conditions includeJ %acterial1 .iral and fungal meningitis1 traumatic ta)1 multi)le sclerosis1 o%struction1 neo)lasm and cere%ral infarction/ Li*itations An !SF )rotein .alue M 69; mg>dl should %e diluted with ;/CK saline either 6<9 or 6<: and reassa ed1 and the .alue o%tained is multi)lied % the a))ro)riate dilution factor to get the correct .alue/ &he )resence of %lood and )us will increase !SF )rotein/ &he re)ort to the re7uesting )h sician should include the a))earance of !SF %efore and after centrifugation/ !eference 6/ =atana%e et al +6C?8, !lin/ !hem :9 < 6556'6554

CS' Protein ( Tur@idi*etry

Principle of the *ethod

ethod

Protein )resent in !SF is measured % )reci)itating the )roteins with :gK sul)hosalic lic acid and com)aring the a%sor%ance of the tur%idit at 84; nm with that of )rotein standards/ !eagents All chemicals must %e Analar grade Sodiu* chloride diluent Dissol.e Cg of sodium chloride and ;/5 g of sodium a3ide together in a final .olume of one litre of distilled water in a .olumetric flask/ Store the solution in an am%er coloured %ottle/ Sta%le for one ear at 95':5;!/ Sulphosalicylic acid =g&dl Dissol.e :; g sul)hosalic lic acid in a final .olume of one litre distilled water/ Store in an am%er coloured %ottle at 95':5;!/ Sta%le for 8 months/ Standards *efer &otal Protein +)age 4;1 +c, Standard for the )re)aration of )rotein standard from )ooled serum/ Aote that there is alwa s a risk of infection from )ooled serum/ After determining the concentration of the )rotein standard1 dilute this to se.eral le.els as

descri%ed %elow/ For exam)le1 if the total )rotein .alue of the )ooled serum is8/Eg>dl1 then the .olume of )ooled serum to %e diluted to 6;;ml to get a )rotein concentration of 9; mg>dl is found out using the dilution formula/ IL x I! I FL x F! IL I Initial Lolume of )ooled serum/ I!I Initial !oncentration of total )rotein +mg>dl,/ FL I Final Lolume/ F! I Final !oncentration/ IL x 8E;; I 6;; x 9; IL I 6;; x 9; 9; ''''''''''' I ''''' I ;/: ml 8E;; 8E i/e/ Dilute ;/: ml of the )ooled serum to 6;;ml with ;/C g>dl sodium chloride containing ;/6g>dl sodium a3ide/ Similarl 1 4;1 ?; and 69; mg>dl standards are )re)ared % diluting ;/81 6/9 and 6/? ml of the )ooled serum each to 6;; ml with the diluent/ &he standards are sta%le at 9'?;! for one month/ &he following ta%le summari3es the )re)aration of diluted standards/ Concentration of protein in pooled seru* is ta1en as J./g&dl.

Standard

Pooled seru* 6*l9

'inal diluted ?olu*e 6*l9

6;;

Concentration of Standard 6*g&dl9

9;

S6

;/:

S9

;/8

6;;

4;

S:

6/9

6;;

?;

S4

6/?

6;;

69;

E4uip*ent+ glass5are and other accessories *efer to Section A +9,1 Introduction to SOP/ Procedure &he )rotocol of the )rocedure is descri%ed %elow/

Pi)ette the following into a))ro)riatel la%elled 6: x 6;; mm tu%es

Blan1
Protein standard +ml, '

S,

S<
6/;

S=
6/;

S>
6/;

Test
'

;C
'

6/;

&est sam)le +ml,

'

'

'

'

'

6/;

'

-! serum+6<6;;, +ml,

'

'

'

'

'

'

6/;

Sodium chloride +ml,

6/;

'

'

'

'

'

'

Sul)hosalic lic acid +ml,

:/;

:/;

:/;

:/;

:/;

:/;

:/;

Mix all tu%es well/ 0ea.e at 95':5 ;! for 5 minutes/ Set the s)ectro)hotometer >filter )hotometer to 3ero using %lank at 84; nm> red filter and measure the a%sor%ance of standards1 test and -!/ Calculation and cali@ration graph Since standards and test> -! )rocedures are identical1 the a%sor%ance .alues of standards are )lotted against their res)ecti.e concentrations/ &he cali%ration cur.e should %e linear u) to 69; mg>dl with a lower limit of 6; mg>dl/ Plot the a%sor%ance .alues of test on the cali%ration gra)h and read off )rotein concentrations in )atients# !SF/ As 6< 6;; diluted -! serum Ts anal sed1 read off )rotein concentration in -! on the cali%ration gra)h and multi)l the .alue % I ;; to get the correct )rotein .alue in -! serum/ Once linearit is )ro.ed1 it is not necessar to )re)are the standard gra)h e.er time when )atients# sam)les are anal sed/ It will %e ade7uate if onl standard S4 is taken e.er time and )atientsT results are calculated using the formula/ &est a%sor%ance ''''''''''''''''''''''''''''' x 69;////// mg>dl/ Standard a%sor%ance

Analytical relia@ilities *efer to pages /7A of section , 6General Introduction9 on the use of internal -! and inter)retation of dail -! data +for releasing )atients# results,/ Include one internal -! e.er time a )atient s)ecimen is measured1 irres)ecti.e of the num%er of sam)les in a %atch/ 2owe.er1 e.en if onl a single -! sam)le is anal sed in a da 1 this .alue can %e )ooled with the )receding 6; or 9; .alues o%tained in the )re.ious da s and between-day precisioncan %e calculated and ex)ressed as K!L/ (nsure that this is well within the acce)ta%le limit1 i/e1 ?K/ At least once a week anal se another -! serum from either a low -! or high -! )ool/ HAssa edH -! sera with stated .alues +ranges, are a.aila%le from se.eral commercial sources1 .i3/ $oehringer Mannheim1 $io*ad G *andox/ If a laboratory uses QC sera rom a commercial source, it is important that the company certifies that their QC materials are traceable to international reference materials. Ha0ardous *aterials This procedure uses sodium azide and sulphosalicylic acid. Do not swallow, and avoid contact with skin and mucous membranes. !eference range and clinical interpretation !SF )rotein ' 65'45 mg>dl Aormall the )rotein )resent in!SF is entirel al%umin1 %ut in man disease states !SF contains a mixture of al%umin and glo%ulins/ Increase in )roteins u) to 4;; mg>dl is o%ser.ed in meningitis and u) to se.eral grams in s)inal tumour/ In inflammator lesion1 increase in )rotein is associated with increase in cells/ A marked increase is also o%ser.ed in )aral sis and in disseminated sclerosis/ A%normall increased total !SF )rotein ma %e found in conditions where there is an increased )ermea%ilit of the ca)illar endothelical %arrier through which ultrafiltration occurs/ (xam)les of such conditions includeJ %acterial1 .iral and fungal meningitis1 traumatic ta)1 multi)le sclerosis1 o%struction1

neo)lasm and cere%ral infarction/ Li*itations An !SF )rotein .alue M 69; mg>dl should %e diluted with ;/CK saline either 6<9 or 6<: and re' assa ed1 and the .alue o%tained is multi)lied % the a))ro)riate dilution factor to get the correct .alue/ &he )resence of %lood and )us increases !SF total )rotein/ &he re)ort to the re7uesting )h sician should include the a))earance of !SF %efore and after centrifugation/ !eference 6/ Larle #s Practical !linical $iochemistr / 8 th edition1 )u%lished % +6C??, )age 44E'44?/ 2einemann Medical $ooks1 0ondon

Introduction
Urine is one of the most easil o%tained s)ecimens examined in the la%orator 1 and examination of the urine not onl )ro.ides information a%out the functioning of the kidne s and )ossi%le a%normalities of the urinar tract1 %ut ma also lead to the diagnosis of .arious s stemic diseases of the human %od which are reflected % the )resence of se.eral su%stances in the urine/ Collection of speci*en Early *orning urine &his is the %est urine s)ecimen for routine anal sis and is collected soon after the )atient awakens/ It is usuall concentrated and has an acid )2/ !asts and cells are )oorl )reser.ed in dilute or alkaline urine and traces of dissol.ed su%stances such as )rotein and sugar can %e missed if the urine is .er dilute/ !ando* urine &his s)ecimen is collected at an screening )ur)oses/ Preser?ati?e used For routine anal sis1 no )reser.ati.e is re7uired %ut the urine is %est examined fresh/ $acterial growth will ruin a s)ecimen if anal sis is dela ed for more than : hours/ *efrigeration is the %est wa to )reser.e it if anal sis is dela ed/ *efrigeration for more than 94 hours is not recommended/ Container for urine collection &he container used must %e thoroughl clean and free from an detergent or disinfectant residue since the oxidants contained in such cleaning agents ma cause the test areas for glucose and %lood to indicate false )ositi.e results/ After the urine is collected1 the container should )refera%l %e sealed/ time and is con.enient for the )atient and is suita%le for most

;ualitati?e Tests
Appearance Aormal urine colour .aries from light ellow to dee) am%er/ Urine colour sometimes ma .ar de)ending u)on the drink1 if an 1 consumed % the )atient/ &he colour of urine is sometimes related to a )igment called HurochromeH/ &he degree of colour also de)ends on whether the s)ecimen is concentrated or dilute/ Aormal urine is usuall clear/ If the )2 is alkaline1 tur%idit ma %e o%ser.ed due to the )reci)itation

of )hos)hates/ Such urine should %e centrifuged %efore anal sis/ &ur%idit due to the )resence of ch le +ch lomicrons, cannot %e centrifuged1 %ut re7uires filtration using a s)ecial cellulose filter ha.ing Q;/6 m m diameter/ pH Use a narrow range )2 )a)er or a )2 meter/ In some clinical situations1 measurements of a))roximate )2 within F ;/5 )2 units using a narrow' range )2 )a)er or exact )2 using a )2 meter ma %e .er hel)ful/ Procedure %sing pH paper Put a dro) of urine on a )ortion of )2 indicator )a)er/ &he colour o%tained is com)ared with a standard chart/ For checking the relia%ilit of the )2 )a)er cross check the )2 of %uffer solutions of known )2 .alues ha.ing acidic and alkaline )2 ranges/ %sing a pH *eter !ali%rate the )2 meter using standard %uffers1 one ha.ing an acidic )2 and the other an alkaline )21 )refera%l less than C/;/ Pour a%out 4;'5;ml of urine into a 6;;ml %eaker1 cali%rate the )2 meter once again using the %uffer of )2 E/; and measure the )2 of the urine/ !esult Aormal urine )2 ranges from 4/5 to ?/;/ &he urine )2 .alues are re)orted for exam)le as 8/; if )2 )a)er is used or as 8/6 if a )2 meter is used/ Interpretation and 4uality control Urine )2 is usuall .egetarians/ acidic in normal )eo)le1 es)eciall non'.egetarians1 and is usuall alkaline in

An earl morning urine )2 Q5/5 indicates that renal tu%ular acidification mechanism is intact/ As a 7ualit control measure1 use certified reference %uffers +commercial source,1 one in acidic range1 sa 1 )2 4/; and the other in alkaline range1 )refera%l )2 C/9 to check the relia%ilit of the )2 )a)er used or to assess the )erformance of the )2 meter/ Alwa s use a )2 indicator )a)er %efore the date of ex)ir / Do not use outdated )2 )a)ers/ Alwa s close the %ottle containing the )2 )a)er tightl / &he )2 meter must %e cali%rated against a correct cali%ration %uffer/ Hetone Bodies 7 !otheraMs test &he three main ketone %odies are acetone 1 acetoacetic acid +diacetic acid, and %eta'h drox %ut ric acid/ &esting for ketone %odies should %e done on fresh urine or the s)ecimen ke)t at 4 ;!/ Principle Acetone and acetoacetic acid react with sodium nitro)ruside in the )resence of alkali to )roduce a

)ur)le colour/ !eagents !otheraMs !eagent # $ry *i)ture Pul.eri3e E/5g sodium nitro)ruside with 9;;g ammonium sul)hate/ Store in a clean am%er %ottle at 95;':5;!/ Sta%le for 8 months/ A**onia concentrated+ specific gra?ity -.A, Procedure &o a%out 5ml of urine taken in an 6? x 65;mm glass tu%e1 add a%out one teas)oon of the mixture1 mix well1 then add ;/5 to 6/; ml of concentrated ammonia down to the side of the tu%e so that it la ers on to) of the urine/ O%ser.e for an colour change within :;'8; seconds/ !esult If acetone and diacetic acid are )resent1 then a )ur)le +)ermanganate calomel red, colour will form at the Nunction of the two la ers within :;'8; seconds/ &he result can %e graded from trace to :F %ased on the intensit of the colour formed1 as detailed %elow/ Ao change in colour ' Aegati.e Pinkish ring ' F *ed ring ' FF Dee) )ur)le ring ' FFF Interpretation and 4uality control Detone %odies are intermediar )roducts of fat meta%olism and their )resence in the %lood and then in the urine are indications that the meta%olism is disordered or incom)lete/ &his is associated with meta%olic acidosis/ &his occurs in )oorl controlled dia%etes mellitus and also in star.ation/ Aormal urine does not contain meth l ketone/ =eak false )ositi.e reactions ma contains 0'do)a and )hen l ) ru.ic acid/ occur if the urine

If there is sus)icion of a false )ositi.e test1 heat the urine in a test tu%e in a $unsen %urner flame for one minute1 allow to cool and re)eat the *otheraTs test/ 2eated urine will not gi.e a )ositi.e *otheraTs due to ketone %odies/ As a 7ualit control measure1 the reagent should %e checked fre7uentl using a )ositi.e control +6'9 dro)s of acetone is added to 5ml of urine,/ &he use of distilled water in )lace of urine for negati.e control is recommended/ %ro@ilinogen ( ErhlichCs test Principle (rhlichTs reagent in conc 2!I/ reacts with uro%ilinogen to form a )ink coloured aldeh de com)lex in chloroform/ !eagents ErhlichMs reagent Dissol.e 9 g of P'dimeth l amino%en3aldeh de in 6;;ml of 9;K 2!I/ Store at 95':5 ;! in an

am%er coloured %ottle/ Sta%le for : months/ ,-g&dl Bariu* chloride Dissol.e 6;g %arium chloride in 6;;ml of distilled water/ Store at 95':5 ;!/ Sta%le for 8 months/ Saturated a**oniu* sulphate Chlorofor* A! or G! 4uality Procedure &o 69 ml of urine taken in a 95ml or 5; ml measuring c linder add : ml of %arium chloride followed % : dro)s of saturated ammonium sul)hate/ Mix well/ &ransfer a )ortion of it into a 65 x 69;mm glass tu%e/ !entrifuge for 5 minutes at :5;; r)m/ &ransfer a%out 5ml of the su)ernatant into an 6? x 65; mm glass tu%e and add ;/5 ml of (rhlichTs reagent/ Mix well/ &hen add :ml chloroform and shake well/ Allow to stand one minute/ O%ser.e for an colour change in the chloroform la er +%ottom la er,/ !esult %ro@ilinogen Colourless # Not detected 'aint red colour # Nor*al !ed or @right red # Positi?e or highly positi?e depending upon the intensity of colour. Interpretation and 4uality control Uro%ilinogen is normall excreted in trace and a normal urine will alwa s show a faint red colour in the chloroform la er/ It is alwa s a good )ractice to run a normal urine as control whene.er an uro%ilinogen test is done/ (xcess uro%ilinogen is seen in urine in haemol tic Naundice1 .iral he)atitis and cirrhosis and is a%sent in o%structi.e Naundice/ Biliru@in 7 6Harison spot test9 'ouchetMs test Principle =hen ferric chloride in acid solution is added to a )reci)itate +*ef < Uro%ilinogen )rocedure, of urine containing %iliru%in1 a green colour is )roduced as the %iliru%in in the urine is oxidi3ed to %ili.erdin/ !eagent 'ouchetMs reagent. Dissol.e 95g of trichloroacetic acid in a%out 5;ml of distilled water1 then add 6g ferric chloride1 mix to dissol.e and then make u) to 6;;ml with distilled water/ Store at 95 :5 ;!/ Sta%le for 8 months/ Procedure &o 69 ml of urine taken in a 95 ml or 5; ml measuring c linder add : ml of %arium chloride followed % : dro)s of saturated ammonium sul)hate/ Mix well/ &ransfer a )ortion of it into a 65 x 69;mm glass tu%e/ !entrifuge for 5 minutes at :5;; r)m/ Decant the su)ernatant and add 6or 9 dro)s of FouchetTs reagent to the )reci)itate/ (xamine for an colour change/ !esult

No colour change in the precipitate # Negati?e Appearance of a green or @lue colour # Positi?e Interpretation and 4uality control $iliru%in is not )resent in normal urine/ For a )ositi.e control1 a few dro)s of either a %iliru%in standard or an icteric serum are added to a normal urine sam)le and the s)ecimen is anal sed for the )resence of %iliru%in/ An %iliru%in )resent in the urine is conNugated and indicates excess in the serum due to cholestasis/

Se*i7;uantitati?e Tests
Specific Gra?ity 6 ass $ensity9 %rino*eter *ethod Principle S)ecific gra.it is a function of the num%er1 densit and weight of the solute )articles )resent and is used as a measure of the concentrating )ower of the kidne / &he s)ecific gra.it of urine is its densit com)ared with the densit of distilled water that is con.enientl fixed as 6/;;; at 9;;!/ It ismeasured using a weighted c linder called HurinometerH1 which floats in the urine and which is cali%rated against distilled water at 9;U !/ !heck the working of the urinometer % floating it in distilled water to see if the reading is 6/;;;/ As the s)ecific gra.it .aries with tem)erature1 a))l tem)erature correction %efore re)orting/ Procedure Pour a%out 4; ' 5;ml of urine into a 6;;ml glass measuring c linder/ lower the urinometer gentl into the urine1 rotate and release +a.oid frothing,/ =ait for the urinometer to settle +make sure that the urinometer does not come into contact with the sides or %ottom of the c linder,/ *ead the s)ecific gra.it gi.en on the scale at the surface of the urine +use the lower )oint of the meniscus for reading,/ O%ser.e the tem)erature of the urine/ !esult !heck the tem)erature at which the urinometer is cali%rated/ It is usuall at 9; ;!/ For e.er :;! that the urine tem)erature is a%o.e the cali%ration tem)erature1 add ;/;;6 to the measured s)ecific gra.it and for e.er : ;! that the urine tem)erature is %elow the cali%ration tem)erature1 su%tract ;/;;6 from the measured s)ecific gra.it / E)a*ple Urinometer is cali%rated at 9;;! Urine tem)erature 9:;! &he measured s)ecific gra.it 6/;9: &he tem)erature of urine is :;! higher than the cali%ration tem)erature/ V S)ecific gra.it to %e added to the measured s)ecific gra.it I :>: x ;/;;6 I ;/;;:>: I ;/;;6/ V &he actual s)ecific gra.it I 6/;9:F;/;;6I6/;94/ Interpretation and 4uality control

&he normal urine's)ecific gra.it is 6/;6; 6/;:; &he )resence of an increased amount of )rotein affects the s)ecific gra.it % ;/;;6 for e.er ;/4g>dl )rotein le.el in urine/ As a 7ualit control measure1 the functioning of the urinometer must also %e checked % floating in other li7uids whose densities are greater than distilled water/ &here is also an adNustment for glucose Su%tract ;/;;6 for e.er 9E; mg>dl glucose in the urine/ Proteins ( Heat and acetic acid *ethod Principle Proteins in urine are coagulated % heat and the degree of coagulation is directl )ro)ortional to the amount of )roteins )resent/ !oagulation can %e further enhanced when dro)s of acetic acid are added/ Procedure Pour 9': ml of urine into a 6: x 6;;mm glass tu%e and hold it using a tu%e holder/ !heck the urine )2J if it is M)2 E or Q:1 adNust to %etween 4'5 using :K acetic acid/ 2eat the u))er half of the column of urine in a flame until it %oils/ 0ook for the a))earance of cloudiness in the heated )ortion and contrast it with the lower )ortion of the tu%e/ A))earance of cloudiness in the u))er )ortion indicates the )resence of )roteins/ Add 9': dro)s of :K acetic acid to the )reci)itate and o%ser.e/ If the )reci)itate disa))ears1 it indicates the )resence of )hos)hates and car%onate +later )roduces effer.escence when the )reci)itate disa))ears,/ Persistence of the )reci)itate shows the )resence of al%umin/ On adding 9': dro)s of conc/ 2A; : ifthe )reci)itate disa))ears1 the )resence of mucin or nucleo)rotein is suggested/ !esult &his test ma %e used as semi'7uantitati.e1 as followsJ

Colour change
Ao cloudiness

!esult
Aegati.e

Faint cloudiness +ma %e o%ser.ed onl if the tu%e is held against a %lack %ackground,/ Definite nongranular cloud without flocculation

&race

6F 2ea. and granular cloud without flocculation 9F Dense cloud with marked flocculation :F &hick curd flocculation G coagulation 4F Interpretation and 4uality control &his test is sensiti.e enough to detect )rotein down to a concentration of 9': mgK/ For 7ualit control1 dilute 99gK of human al%umin solution to get a concentration of 5 mg>dl/ Use this as a test and check the relia%ilit and sensiti.it of this method/

Note# If an alkaline urine is %oiled1 the )rotein ma %e con.erted into the so' called Halkaline meta)roteinH1 which is not coagulated % heat/ &herefore it is alwa s %etter to acidif the urine %efore doing this test/ If too much acetic acid is added1 the )rotein ma %e con.erted to the so'called Hacid meta)roteinH1 which is also not coagulated % heat/ &herefore the urine should %e onl mildl acidic/ Protein ( Sulphosalicylic acid *ethod Principle Urine )roteins are )reci)itated % sul)hosalic lic acid1 which gi.es a white )reci)itate1 and the degree of the )reci)itate is )ro)ortional to the )rotein le.el/ !eagent :g K sul)hosalic lic acid +SSA, =eigh E/5 g of sul)hosalic lic acid and dissol.e it in a%out 9;;ml of distilled water and then make u) to 95; ml with distilled water/ Store at 95 ' :5;!/ Sta%le for 8 months/ Procedure &o 9ml of urine taken in a 6: x 6;;mm glass tu%e1 add 9 ml of :gK SSA/ Mix gentl / 0ea.e for 5 minutes at room tem)erature/ !om)are the degree of the )reci)itate with 4ml of SSA taken in a similar test tu%e/ !esult Same as gi.en on )age CC H+c, *esultH Interpretation and 4uality control &he sul)hosalic lic acid method will not detect )rotein in a normal urine1 %ut will %e sensiti.e enough to detect )rotein )resent down to 9;mgK/ As a 7ualit control measure1 a 99g>dl al%umin solution can %e diluted a))ro)riatel with ;/C g>dl sodium chloride to get standards containing 9;1 5;1 9;;1 5;; and 95;; mg>dl )roteins/ &hese standards are sta%le for one month when stored at 9' ?;!/ =hen the are su%Nected to the same )rocedure as urine1 the results can %e inter)reted as follows<

Concentration of proteins
9; mg>dl 5; mg>dl 9;; mg>dl 5;; mg>dl 95;; mg>dl Sugar# BenedictMs test

!eported as
&race 6F 9F :F 4F

Principle Urinar sugars when %oiled in $enedictTs reagent reduce co))er sul)hate to a reddish cu)rous oxide )reci)itate in hot alkaline medium1 the intensit of which is )ro)ortional to the amount of sugar )resent in the urine/ &he results are re)orted as IF19F1 etc/ de)ending u)on the colour and intensit of the cu)rous oxide )reci)itate/ !eagent Dissol.e 6E/:g of cr stalline co))er sul)hate in a%out ?;;ml of distilled water1 then add 6;; g of sodium car%onate1 mix to dissol.e and finall add 6E5g of sodium citrate/ Mix well to dissol.e and then make u) to one litre with distilled water/ Store in an am%er coloured %ottle at 95':5;!/ Sta%le for one ear/ Procedure &o 5 ml of $enedictTs reagent taken in an 6? x 65;mm glass tu%e1 add ? dro)s +;/5 ml, of urine1 mix well and %oil for 9/: minutes1 )refera%l in a %oiling water%ath/ !ool the tu%e and o%ser.e for an colour change/ !esult &he results are re)orted as follows<

O@ser?ation
Ao change in the original colour of $enedict#s solution Solution a))ears and slightl cloud Definite cloud green )ale green

Inference
Aegati.e &race 6F

Wellow to orange )reci)itate 9F +6 g>dl, Orange to red )reci)itate :F +9 g>dl, $rick red )reci)itate G clear su)ernatant 4F +M9 g>dl, Interpretation and 4uality control Aormal urine does not contain an reducing sugar/ If )rotein is )resent in large amounts1 it ma interfere with the )reci)itation of the cu)rous oxide/ &o o.ercome this )ro%lem1 )reci)itate the )roteins using :K SSA filter using a =hatman filter )a)er and use the filtrate to test the amount of sugar )resent/ As a 7ualit control measure1 standards containing known amounts of glucose are )re)ared in saturated %en3oic acid and one of the standards is used e.er da to check the relia%ilit of the )atient#s results/ &he standard results ma %e transformed in the following semi'7uantitati.e wa /

6;;mg>dl

&race

95;mg>dl 5;;mg>dl E5;mg>dl 9 g>dl

6F 9F :F 4F

False )ositi.e reactions are known to occur due to the )resence of non' car%oh drate su%stances like ascor%ic acid1 homogentisic acid1 creatinine and uric acid/ *educing sugars like lactose1 galactose1 fructose and )entoses will also gi.e a )ositi.e reaction/ &he di)stick techni7ue is s)ecific for glucose and eliminates the false )ositi.e reaction due to the su%stances mentioned a%o.e/