Basic Concepts in Clinical Biochemistry: A Practical Guide

174 Pages·2018·4.19 MB·English

by  Vijay Kumar

Vijay Kumar
Kiran Dip Gill
Basic Concepts
in Clinical
Biochemistry:
A Practical Guide
Basic Concepts in Clinical Biochemistry:
A Practical Guide
Vijay Kumar (cid:129) Kiran Dip Gill
Basic Concepts in Clinical
Biochemistry:
A Practical Guide
VijayKumar KiranDipGill
DepartmentofBiochemistry DepartmentofBiochemistry
MaharshiDayanandUniversity PostgraduateInstituteofMedicalEducation
Rohtak,Haryana,India &Research
Chandigarh,India
ISBN978-981-10-8185-9 ISBN978-981-10-8186-6 (eBook)
https://doi.org/10.1007/978-981-10-8186-6
LibraryofCongressControlNumber:2018935904
#SpringerNatureSingaporePteLtd.2018
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Preface
Biochemistryisoneofthefundamentalsubjectsoflifescience,andknowledgeofits
practical aspects is absolutely essential for students and researchers. Performing a
practical in biochemistry requires adequate facilities and training laboratories for
organizing meaningful practicals both at undergraduate and postgraduate levels.
Clinicalbiochemistryisadiagnosticssubject,whichaimstousestandardmethods,
tomonitordiseasedevelopmentandtreatmentbybiochemicalmethods.Laboratory
investigations pertaining to clinical biochemistry provide useful information to
clinicians, both in the diagnosis of illness and the monitoring of treatment. The
necessity for a practical manual on orientation toward various aspects of clinical
biochemistry is always desired. The present manual is written in simple language
covering various experimental aspects related with clinical biochemistry such as
urinary analysis of bioorganic constituents and estimation of proteins, urea, sugar,
uricacid,bilirubin,etc.,inbloodwhichisessentialforearlydiagnosisofadisease
andalsoassessmentofitstherapyefficacy.
The book is specifically helpful to BSc, MLT, MSc, and MSc biochemistry
students. Each chapter begins with theoretical aspects of the practical, and the
experiments are further well supported by self-explanatory tables and simple
calculations that will make the students understand protocols very easily. The
manual is also helpful to teachers who are taking clinical biochemistry practicals
toguidestudentsinasimpleway.
Theauthorshopethatthemanualwillmeettherequirementofundergraduateand
postgraduatestudentsstudyingbiochemistryandwillbegladtoacceptconstructive
criticisms and suggestions from the faculty, students, and readers to make this
manualabetteroneinthefuture.
Rohtak,Haryana,India VijayKumar
Chandigarh,India KiranDipGill
v
Reference Biochemical Values in Serum/Plasma
Acidphosphatase 1–4KAU/dl Albumin 3.5–5g/dl
Totalproteins 3.5–5.5g/dl Globulins 2.5–3.5g/dl
Alaninine 3–40IU/L Alkaline 3–13KAU/dl
transaminase phosphatase
(ALT,SGPT) Amylase 5–45SU/dl
Aspartate 5–45IU/L Calcium 9–11mg/dl
transaminase
(AST,SGOT) Calcium(U) 100–200mg/
day
Bilirubin(total) 0.2–1mg/dl Ceruloplasmin 25–50mg/dl
Bilirubindirect <0.4mg/dl Cholesterol 150–200mg/dl
Bilirubinindirect <0.6mg/dl HDL 30–60mg/dl
Creatinine 0.5–1.5mg/dl LDL 60–130mg/dl
Creatinine(U) 0.8–2g/day VLDL 20–40mg/dl
Chloride 103–105mEq/L Inorganic 3.0–4.5mg/dl
phosphorus
Creatinekinase, 24–195U/L Phosphorus(U) 0.5–1.5g/day
CK-MB
Creatinekinase,total 10–400U/L LDH 100–190U/L
Fibrinogen 0.2–0.4g/dl Urea 15–40mg/dl
Glucose,fasting 70–100mg/dl Urea(U) 15–40g/day
Lipase 0.5–160U/L Uricacid 3.5–8mg/dl
Transferrin 200–300mg/dl Uricacid(U) 200–500mg/
day
Triglyceride,fasting 50–175mg/dl Urobilinogen(U) 1–3.5mg/day
Sodium 135–145mEq/L
Potassium 3.5–5mEq/L
Chloride 103–105mEq/L
U-Urine
vii
Contents
1 CommonClinicalLaboratoryHazardsandWasteDisposal. . . . . . 1
1.1 WasteDisposalinLaboratory. . . . . . . . . . . . . . . . . . . . . . . . 2
2 BloodCollectionandPreservation. . . . . . . . . . . . . . . . . . . . . . . . . 5
2.1 BloodCollection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3 QualityControlinLaboratory. . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3.1 TypesofLaboratoryErrors. . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.2 MethodstoMinimizetheLaboratoryErrors. . . . . . . . . . . . . . 11
4 AutomationinClinicalLaboratory. . . . . . . . . . . . . . . . . . . . . . . . . 13
4.1 TypesofAutoanalyzers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
5 Photometry:ColorimeterandSpectrophotometer. . . . . . . . . . . . . . 17
5.1 ColorimeterandSpectrophotometer. . . . . . . . . . . . . . . . . . . . 18
6 PreparationofGeneralLaboratorySolutionsandBuffers. . . . . . . 21
6.1 MolarSolutions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
6.1.1 Molarity(M). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
6.2 NormalSolutions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
6.2.1 Normality(N). . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
6.3 Percent(%)Solutions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
6.4 BufferSolutions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
6.5 PhysiologicalBuffersintheHumanBody. . . . . . . . . . . . . . .. 25
6.6 PreparationofCommonLaboratoryBuffers. . . . . . . . . . . . . . 25
6.6.1 0.2MAcetateBuffer(pKa4.86). . . . . . . . . . . . . . . . 25
6.6.2 0.2MSodiumPhosphateBuffer(pKa6.86). . . . . . . . 26
6.6.3 0.2MTris-HClBuffer(pKa8.1). . . . . . . . . . . . . . . . 26
7 ExaminationofUrineforNormalConstituents. . . . . . . . . . . . . . . . 29
7.1 PreservativesUsedforUrineCollection. . . . . . . . . . . . . . . . . 29
7.2 PhysicalExaminationofUrine. . . . . . . . . . . . . . . . . . . . . . . . 30
7.2.1 ColorandOdor. . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
7.2.2 Appearance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
ix
x Contents
7.2.3 SpecificGravity. . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
7.2.4 Volume. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
7.2.5 pH. . . .. . . . . . . . . . .. . . . . . . . . .. . . . . . . . . . .. . 31
7.3 ChemicalExaminationofUrine. . . . . . . . . . . . . . . . . . . . . . . 31
7.4 TestsforInorganicConstituentsofUrine. . . . . . . . . . . . . . . . 31
7.5 TestsforOrganicConstituents. . . . . . . . . . . . . . . . . . . . . . . . 32
7.6 DetectionofUrea. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
8 ToPerformQualitativeTestsforUrinaryProteins. . . . . . . . . . . . . 33
8.1 Theory. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
8.2 TestsforUrinaryProteins. . . . . . . . . . . . . . . . . . . . . . . . . . . 34
8.2.1 DipstickTest. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
8.2.2 TheBoilingTestforCoagulableProteins. . . . . . . . . . 34
8.2.3 SulphosalicylicAcidTest. . . . . . . . . . . . . . . . . . . . . 35
8.2.4 NitricAcidRingTest(Heller’sTest). . . . . . . . . . . . . 35
8.2.5 Bence-JonesProteins. . . . . . . . . . . . . . . . . . . . . . . . 35
9 ToDeterminetheQuantityofProteinsinUrineSample
UsingBiuretReaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
9.1 Theory. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
9.2 SpecimenRequirements.. . . . .. . . . .. . . . . .. . . . .. . . . . .. 39
9.3 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
9.4 Reagents. . . . .. . . . . .. . . . .. . . . . .. . . . . .. . . . .. . . . . .. 40
9.5 Calculations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
9.6 ClinicalSignificance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
10 ToEstimatetheAmountofTotalProteinandAlbumininSerum
andtoFindA/GRatio. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
10.1 Theory. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
10.2 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
10.3 SpecimenRequirements. .. . . . .. . . . .. . . . .. . . . . .. . . . .. 44
10.4 Reagent. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
10.5 Procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
10.6 Calculations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
10.7 EstimationofAlbumininSerum. . . . . . . . . . . . . . . . . . . . . . 46
10.8 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
10.9 Reagents. . . . . .. . . . .. . . . . .. . . . .. . . . . .. . . . .. . . . . .. 46
10.10 Procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
10.11 Calculations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
10.12 ClinicalSignificance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
10.13 Precautions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Contents xi
11 ToPerformQualitativeTestforReducingSubstancesinUrine. . . 49
11.1 Theory. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
11.2 QualitativeTestforReducingSugars. . . . . . . . . . . . . . . . . . . 49
11.2.1 Benedict’sTest. . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
11.2.2 Benedict’sQualitativeReagent. . . . . . . . . . . . . . . . . 50
11.2.3 Procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
11.3 CLINISTIX/Uristix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
11.3.1 Procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
11.4 Precautions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
11.5 ClinicalSignificance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
12 QuantitativeAnalysisofReducingSugarsinUrine. . . . . . . . . . . . . 53
12.1 Theory. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
12.2 SpecimenRequirement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
12.3 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
12.4 Reaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
12.5 Reagents. . . . . .. . . . .. . . . . .. . . . .. . . . . .. . . . .. . . . . .. 54
12.6 Procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
12.7 Precautions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
12.8 Calculation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
12.9 ClinicalSignificance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
13 EstimationofBloodGlucoseLevelsbyGlucoseOxidaseMethod. . 57
13.1 Theory. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
13.2 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
13.3 SpecimenRequirements. .. . . . .. . . . .. . . . .. . . . . .. . . . .. 58
13.4 Reagents. . . . . .. . . . .. . . . . .. . . . .. . . . . .. . . . .. . . . . .. 58
13.5 Procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
13.6 Calculations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
13.7 ClinicalSignificance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
14 ToDeterminetheBloodGlucoseLevelsbyFolin
andWuMethod. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
14.1 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
14.2 Reagents. . . . . .. . . . .. . . . . .. . . . .. . . . . .. . . . .. . . . . .. 61
14.3 Procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
14.4 Calculations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
15 ToPerformGlucoseToleranceTest. . . . . . . . . . . . . . . . . . . . . . . . 63
15.1 MethodofCarryingGTT. . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
15.2 SpecimenRequirements. .. . . . .. . . . .. . . . .. . . . . .. . . . .. 63
15.3 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
15.4 Reagents. . . . . .. . . . .. . . . . .. . . . .. . . . . .. . . . .. . . . . .. 64
15.5 Procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
15.6 Calculations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
15.7 ClinicalSignificance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
xii Contents
16 EstimationofUreainSerumandUrine. . . . . . . . . . . . . . . . . . . . . 67
16.1 Theory. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
16.2 SpecimenRequirements. .. . . . .. . . . .. . . . .. . . . . .. . . . .. 67
16.3 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
16.4 Reagents. . . . . .. . . . .. . . . . .. . . . .. . . . . .. . . . .. . . . . .. 68
16.5 Procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
16.6 Calculations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
16.7 ClinicalSignificance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
17 ToDetermineUreaClearance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
17.1 Theory. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
17.2 MaximumUreaClearance. . . . . . . . . . . . . . . . . . . . . . . . . . . 71
17.3 StandardUreaClearance. . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
17.4 Procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
17.5 Precautions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
17.6 ClinicalSignificance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
18 ToEstimateCreatinineLevelinSerumandUrine
byJaffe’sReaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
18.1 Theory. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
18.2 SpecimenRequirements. .. . . . .. . . . .. . . . .. . . . . .. . . . .. 75
18.3 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
18.4 Reagents. . . . . .. . . . .. . . . . .. . . . .. . . . . .. . . . .. . . . . .. 77
18.5 PreparationofProtein-FreeFiltrate. . . . . . . . . . . . . . . . . . . . . 77
18.6 Procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
18.7 Calculations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
18.8 ClinicalSignificance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
19 ToDetermineCreatinineClearance. . . . . . . . . . . . . . . . . . . . . . . . 79
19.1 Theory. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
19.2 SpecimenRequirementandProcedure. . . . . . . . . . . . . . . . . . 79
19.3 ClinicalSignificance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
20 ToDeterminetheUricAcidConcentrationinSerumandUrine. . . 81
20.1 Theory. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
20.2 SpecimenRequirements. .. . . . .. . . . .. . . . .. . . . . .. . . . .. 81
20.3 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
20.4 Reagents. . . . . .. . . . .. . . . . .. . . . .. . . . . .. . . . .. . . . . .. 82
20.5 Procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
20.6 Calculations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
20.7 ClinicalSignificance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
20.8 Precautions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
21 EstimationofTotalCalciuminSerumandUrine. . . . . . . . . . . . . . 85
21.1 Theory. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
21.2 SpecimenRequirements. .. . . . .. . . . .. . . . .. . . . . .. . . . .. 85