INTRODUCTION

1. xxxxxxxxxNEW Liquid CK - NAC is a reagent set for determination ofcreatine kinase activity in serum and plasma based on UV - Kineticmethod.

2. xxxxxxxxxNEW Liquid CK - NAC is a ready-to-use, two liquid reagentsystem.

3. xxxxxxxxxNEW Liquid CK - NAC estimates creatine kinase activity in just5 minutes.

4. xxxxxxxxxNEW Liquid CK - NAC is linear upto 1800 IU/l.

5. xxxxxxxxxNEW Liquid CK - NAC can be used on anySpectrophotometer, Discrete semiautomated and Automatedanalyzers. Programme can be designed for any specific analyzer uponrequest.

6. xxxxxxxxxNEW Liquid CK - NAC is stable till expiry at 2 - 80C.

PRINCIPLE

Creatine kinase catalyzes the conversion of creatine phosphate and ADP tocreatine and ATP. ATP phosphorylates glucose to glucose-6-phosphate inthe presence of hexokinase. Glucose-6-phosphate is oxidized to6-phosphogluconate, reducing NADP to NADPH in presence of G-6-PDH.The rate of increase in NADPH absorbance at 340 nm. is directly proportionalto the activity of creatine kinase in serum/plasma.

Creatine phosphate + ADP creatine + ATP

ATP + glucose glucose-6-phosphate + ADP

Glucose-6-phosphate + NADP+ 6-phosphogluconate + NADPH + H+

AbbreviationsADP = Adenosine - 5’ - diphosphateATP = Adenosine - 5’ - triphosphateG-6-PDH = Glucose-6-phosphate dehydrogenaseCK = Creatine kinaseHK = Hexokinase

PREPARATION OF

WORKING SOLUTION

R1 : Ready-to-use reagent.

R2 : Ready-to-use reagent.

Prepare working solution by mixing Reagent R1 and Reagent R2 in theratio 5 : 1 as per requirement

REAGENT STORAGE & STABILITY

The reagent kit should be stored at 2 - 8° C and is stable till the expiry dateindicated on the label.

R1 & R2 reagents are stable till expiry at 2 - 8° C.

The working solution (5 R1 + 1 R2) is stable for 10 days at 2 - 8° C.

COMPONENTS & CONCENTRATION

OF WORKING SOLUTION

Component Concentration Component Concentration

• Imidazole buffer; pH 6.7 100 mmol/l • EDTA 2 mmol/l

• Phosphocreatine 30 mmol/l • ADP 2 mmol/l

• N-acetylcysteine (NAC) 20 mmol/l • NADP 2 mmol/l

• Magnesium acetate 10 mmol/l • AMP 5 mmol/l

• Glucose 20 mmol/l

• Di-(adenosine-5’)-pentaphosphate ≥ 10 μmol/l

• Glucose-6-phosphate dehydrogenase ≥ 1.5 KU/I

• Hexokinase ≥ 2.5 KU/I

PROCEDURE

� Reaction type ........................................................UV - Kinetic� Reaction direction ................................................ Increasing� Wavelength ................................................................. 340 nm.� Flowcell temperature ...................................................... 37oC� Zero setting with ........................................... Distilled water� Delay time ........................................................... 120 seconds� No. of readings ........................................................................ 4� Interval .................................................................. 60 seconds� Sample volume ................................................0.04 ml (40 μl)� Working solution volume (5R1 : 1R2) ......... 1.0 ml (1000 μl)� Factor .................................................................................. 4127

� Linearity ....................................................................... 1800 IU/I

Manual assay procedurePrewarm at 37oC the required amount of working solution be-fore use. Perform the assay as given below :

1.0 ml procedureSerum / plasma .................................................................... 0.04 ml

Working solution ................................................................... 1.0 ml

Mix thoroughly and transfer the assay mixture immediately tothe thermostated cuvette and start the stop watch simulta-neously. Record the first reading at 120th second and subse-quently four more readings with 60 seconds interval at340 nm.

Calculation:

Calculate the change in absorbance per minute.Activity of CK - NAC in IU/I = Δ Abs./min x 4127

Conversion factors :

Following factors can be used for conversion of IU/I from one temperatureto another :

Temperature Conversion

Assay temperature Desired temperature250C 300C 370C

250C 1.00 1.53 2.38

300C 0.65 1.00 1.56

370C 0.42 0.64 1.00

Note : Since temperature conversion factors are given only as an approximateconversion, it is suggested that values be reported at the temperature ofmeasurement.

EXPECTED VALUES

Temperature at 250C at 300C at 370C

MEN < 80 IU/I < 130 IU/I < 190 IU/I

WOMEN < 70 IU/I < 110 IU/I < 165 IU/I

BABIES

PROCEDURE LIMITATIONS1. Avoid using haemolysed serum since red blood cells may release enzymes

and intermediates such as ATP and G-6-P which may interfere in theassay.

2. CK activity in serum may be elevated in patients receiving intramuscularinjections upto one week prior to sample collection. Strenuous and / orunusual physical exercise may also cause elevated CK activity.

3. The working solution is considered unsatisfactory and should not beused if its absorbance exceeds 0.700 at 340 nm. against distilled water.

4. If the CK activity exceeds 1800 IU/I then dilute the specimen suitably withnormal saline and repeat the assay. In such case, the results obtainedshould be multiplied with the dilution factor to obtain correct CK activity.

CK - NAC

UV - K

inetic

CK - NAC • CK - NAC

UV - KINETIC • UV - KINETIC

CK - NAC • CK - NAC

UV - KINETIC • UV - KINETIC

CK - NAC • CK - NAC

UV - KINETIC • UV - KINETIC

CK - NAC • CK - NAC

UV - KINETIC • UV - KINETIC

CK - NAC • CK - NAC

UV - KINETIC • UV - KINETIC

CK - NAC • CK - NAC

UV - KINETIC • UV - KINETIC

CK - NAC • CK - NAC

UV - KINETIC • UV - KINETIC

CK - NAC • CK - NAC

UV - KINETIC • UV - KINETIC

CK - NAC • CK - NAC

UV - KINETIC • UV - KINETIC

CK - NAC • CK - NAC

UV - KINETIC • UV - KINETIC

CK - NAC • CK - NAC

UV - KINETIC • UV - KINETIC

CK - NAC • CK - NAC

UV - KINETIC • UV - KINETIC

CK - NAC • CK - NAC

UV - KINETIC • UV - KINETIC

CK - NAC • CK - NAC

UV - KINETIC • UV - KINETIC

CK - NAC • CK - NAC

UV - KINETIC • UV - KINETIC

CK - NAC • CK - NAC

UV - KINETIC • UV - KINETIC

AR. No.: I 68 LN-2009-09-00I

Perform total CK on all suspected casesof MI. It is essential to draw a minimum of2 samples of blood at least 4-6 hours apartand between 10 and 20 hours after theonset of chest pain. Upper limit of normalfor total CK [U/I]:

130 at 300C190 at 370C

MI INTERPRETATION CHART*A diagram for distinguishing myocardial infarction

Is the total CK value greaterthan the upper limit of normal?

Perform CK-MB assay at sametemperature as total CK test.Upper limit of normal for CK-MB [U/L]:

15 at 300C24 at 370C

Is CK-MB [U/L] value greaterthan upper limit of normal?

Calculate CK-MB (%) = x 100CK-MB [U/L]

Total CK [U/L]

Is CK-MB [%] less than 5.5%?

Is CK-MB [%] between 5.5% & 20%?

Is CK-MB [%] greater than 20%?

* (Chart modelled after that reported by Wu, AHB and Bowers, Jr, GN, Clin. Chem. 28 : 2017, 1982)

Most Probable Cause: No muscle damageOther Possibilities: MI occurred less than4 - 6 hours prior to blood collection;no MI occurred.

Most Probable Cause: No MI occurredOther Possibilities: MI occurred lessthan 4 - 6 hours prior to blood collection; MIoccurred more than 48 hours beforeblood collection; skeletal muscle damage;atypical CK; elevated CK-BB.

Most Probable Cause: No MI occurredOther Possibilities: MI occurred less than4 - 6 hours prior to blood collection; MIoccurred more than 48 hours before bloodcollection; skeletal muscle damage;atypical CK; elevated CK-BB.

Most Probable Cause: MI occurredOther Possibilities: Diverse myocardialdamage [e.g. cardiac catheterization];various muscle disorders [e.g. Duchenne’smuscular dystrophy] atypical CK; elevatedCK-BB.

Most Probable Cause: Atypical CKOther Possibilities: Elevated CK-BB.Persistent elevation of CK-MB for 48 hoursis a characteristic of atypical CK. Continuedelevation of CK-MB value after serumincubation at 450C for 20 minutes alsosuggests atypical CK. Electrophoresisshould be used for clarifying ambiguouscases.

NO

YES

NO

YES

YES

NO

YES

NO

YES

QUALITY CONTROLTo ensure adequate quality control, it is recommended to use a normal andan abnormal control serum to check instrument and reagent functions. Factorswhich might affect the performance of this test include proper instrumentfunction, temperature control, cleanliness of glassware and accuracy of

pipetting.

REFERENCES1. Ann. Biol. Clin. 40 (1982) 99.

2. Stein W. Med Weit. 1985, 36:572 & Burtis CA , Ashwood ER, Tietz Fund. ofClin. Chem. 5th ed. 30-54, 352-390 and 974-975.

3. Szasz G, Busch EW, Third European Congress of Clinical Chemistry,Brighton, England, 3-8 June 1979 (abstract).

67446755

NEW

/ColorImageDict > /JPEG2000ColorACSImageDict > /JPEG2000ColorImageDict > /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 300 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages false /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages false /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict > /GrayImageDict > /JPEG2000GrayACSImageDict > /JPEG2000GrayImageDict > /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages false /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages false /MonoImageFilter /CCITTFaxEncode /MonoImageDict > /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile (None) /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False

/Description > /Namespace [ (Adobe) (Common) (1.0) ] /OtherNamespaces [ > /FormElements false /GenerateStructure false /IncludeBookmarks false /IncludeHyperlinks false /IncludeInteractive false /IncludeLayers false /IncludeProfiles false /MultimediaHandling /UseObjectSettings /Namespace [ (Adobe) (CreativeSuite) (2.0) ] /PDFXOutputIntentProfileSelector /DocumentCMYK /PreserveEditing true /UntaggedCMYKHandling /LeaveUntagged /UntaggedRGBHandling /UseDocumentProfile /UseDocumentBleed false >> ]>> setdistillerparams> setpagedevice