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 Concise Book of Medical Laboratory Technology Methods and Interpretations Ramnik Sood MD (Path, Gold Medalist) Consultant Reem Medical and Diagnostic Center Healthcare Mena Limited Sharjah United Arab Emirates 2nd Edition




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Preface to the Second Edition I authored an exhaustive book entitled “Medical Laboratory Technology – Methods and Interpretations” that hit the stands in mid-eighties in the previous century and is now running in its 6th edition. This was the first such book in the subcontinent and was much appreciated and used by technologists and pathologists alike. The book has seen testimonies in courts and has been appreciated in the west too. However, in our subcontinent, I was requested by many technologists that they wanted a little younger sister of the book popularly known as MLT authored by me. And so was born the Concise Book of Medical Laboratory Technology: Methods and Interpretations. The book essentially covers everything presented in MLT-6 but in an abridged/shortened and easy-to-digest format. The book is presented in a flowing noninterrupted format and not in a cumbersome experiment-wise cascading flow. There is no break in the style that runs smoothly and is easier to absorb and assimilate. As experiments are a part of any course, more stress has been laid out in the book to understand the intricacies of relevant theories and even troubleshooting all experiments that you would conduct during the course of your study. As I have written multiple modules for many Universities in India, I did not have to think for too long to devise a style and format for this book. You will find everything from ESR to PCR and you will find foam test for bile pigments as also complete automation in urinalysis. You will find basic biochemistry as also detailed cytogenetics. So whatever be your course or query, you will find it within the covers of this short but sweet book now going into its second edition. The book is designed for you not to mug but to understand and elicit the answers from the book of all questions in your mind. I am aware that this book is used by most of your teachers and tutors too. Nothing wrong that you are holding now. It will help you all your life!


Preface to the First Edition

The first book on Medical Laboratory Technology from the house of Jaypee’s came out in 1985 and has been the best seller in its class till date. The title “Medical Laboratory Technology – Methods and Interpretations” has seen 6 editions. The latest one hit the stands in January this year and was released in two volumes. It is a four-color book and has over 1670 pages. The reader base of all editions of our vastly popular title “MLT” has been upcoming laboratorians, undergraduate and postgraduate medical students. It was requested by a few institutes to produce a little smaller version of the two-volume set that would be suitable for the upcoming Laboratory Technology students. So here it is! It is exhaustive yet precise and concise too. The book will help you to appreciate things as they appear in real life under the microscope and otherwise. All current technologies find a mention within the covers of this book. Gone are the days when we had to prepare reagents first thing in the morning (or the days usage); therefore, the current trend of consuming ready-to-use reagents/kits is followed here to make your job and understanding simpler. So, what is available in the market for all investigations, is what is presented here. The Tulip Group has very kindly given us the rights to reproduce the text related to all their kits and reagents in this book. Latest instruments are not forgotten too. Most important— Parasitology section is presented in ample detail as it is relevant to all the developing nations. Quality control/assurance is mentioned in appropriate details. Working is not enough. Working properly and producing nothing but the most accurate reports are the order of the day. This book will not fail you there. Follow the recommendations to the hilt and you would be running the most accurate laboratory available anywhere. Should problems arise! The book has “Troubleshooting” section for every possible test mentioned inside. If this happens – then what! If that happens – then what! You will find all answers. From ESR to PCR – you will find everything. The book is based on most syllabi as applicable to most institutes in India and elsewhere internationally. All necessary care has been taken to weed out any discrepancies/typographical errors at the time of going to press. However, if anything has remained inadvertently, the publishers/author do not take any responsibility for the same in any manner whatsoever. Learning can be enjoyable experience, flip a few pages to experience that




Chapter 1. Laboratory 1 Laboratory Set-up 3 Code of Conduct for Medical Laboratory Personnel 6 Accidents 6 Accidents in the Laboratory 10 Universal Work Precautions (Uwp) for Laboratory Personnel (Especially in Relation to Hiv Transmission) 15 Medicolegal Aspects of Clinical Practice 17 Laboratory Instruments 17 Chapter 2. Sterilization 29 Methods Commonly Used for Sterilization 29 Modern Day Disinfection 34 Chapter 3. SI Units 41 Liter 41 Gram 41 Mole (Mol) 41 International Unit (U) 42 Conversion Factors Between Conventional and System International Units (Siu) 42 Chapter 4. Fundamental Chemistry 52 Indicators 52 Solutes, Solvents and Solutions 52 Periodic Table of Elements 54 Chapter 5. Urine Analysis 56 Composition of Urine 56 Gross Examination of Urine 57 Chemical Examination of Urine 60 Multiple Reagent Strips for Urinalysis 71 Automation in Urinalysis 78 Special Urine Tests 82 Microscopy of the Urinary Sediment 92 Chapter 6. Renal Function and its Evaluation 104 Renal Physiology in Brief 104 Functions of the Kidney 104 Concentration: Dilution Tests 105 Phenol Red Test 105 Clearance Tests 106 Principles of Precise Tests of Renal Function 107 Maximal Tubular Capacity (Tm) 108 Contents Chapter 7. Stool Examination 112 Specimen Collection 112 Inspection of Feces 113 Chapter 8. Medical Parasitology 124 Medical Parasites 124 Intestinal Protozoa of Man 124 Laboratory Examination for Parasites 201 Chapter 9. Clinical Hematology 205 Ways of Obtaining Blood 205 Anticoagulants 206 Blood Collection System 208 Hemoglobin 210 Anemia 211 Hematocrit/Packed Cell Volume (Pcv) 212 Blood Cell Counts 213 Erythrocyte Indices 216 Complete Blood Count (Cbc) 217 Erythrocyte Sedimentation Rate (Esr) 220 Blood Film Examination 222 Rapid Diagnostics 225 Development of Blood Cells and Sites of Blood Formation 227 Morphological Types of Red Blood Cells 234 Qualitative Assessment of G6pd Deficiency 240 Examination of Fetal Hemoglobin 244 Laboratory Diagnosis of Disorders Related to Rbcs 248 Thalassemias (Reduced Synthesis Rate) 256 Normal White Cell Values and Physiological Variations 259 White Blood Cells 263 Quality Control in Hematology 270 Chapter 10. Clinical Hematology: Bleeding Disorders 272 Platelets, Coagulation and Bleeding Disorders: Laboratory Investigations 272 Laboratory Diagnosis of Platelet Disorders 272 Quality Assurance for Routine Hemostasis Laboratory 278 Buffered 3.2% Citrate Solution (Profact) 280 Prothrombin Time (Quick One-Stage Method) Liquiplastin® 283 Sensitive Thromboplastin Reagent for Prothrombin Time (Pt) Determination (Isi = 1.0) Uniplastin® 285 xii Concise Book of Medical Laboratory Technology: Methods and Interpretations Thromboplastin Reagent for Prothrombin Time (Pt) Determination, Lyoplastin® (Lyophilized Reagent, Isi = 1.0) 287 Aptt/Pttk Cephaloplastin Reagent for Partial Thromboplastin Time (Aptt) Determination Using Ellagic Acid as Activator Liquicelin-E® 293 Normal and Abnormal Control Plasmas for Coagulation Assays Plasmatrol H-I/Ii ® 296 Fibroscreen Thrombin Time Test for Qualitative Estimation of Fibrinogen Fibroscreen® 297 Fibrinogen Estimation-Quantitative Fibroquant, Reagent for Quantitative Estimation of Fibrinogen 299 Fibrinolytic Activity 301 Fdps A Qualitative and Semiquantitative Latex Slide Test for Detecting Cross Linked Fibrin Degradation Products in Human Plasma X-L Fdp 302 Laboratory Diagnosis of Coagulation Disorders 304 Automation in Coagulation Analysis 305 Troubleshooting 309 Prothrombin Time 310 Dptt/Pttk 312 Fibrinogen Estimation 314 Chapter 11. Blood Banking (Immunohematology) 317 Blood Group Antibodies 317 Anti-A, Anti-B, Anti-Ab Blood Grouping Antisera for Slide and Tube Tests 320 Anti-A, Anti-B, Anti-Ab Monoclonal }|Blood Grouping Antibodies for Slide and Tube Tests 321 Anti-A1 Lectin Dolichos Biflorus Lectin for Slide and Tube Tests 322 Anti-H Lectin Ulex Europaeus Lectin for Slide and Tube Tests 323 Physiological Saline Solution for Serological Applications (Sodium Chloride 0.9% W/V) 325 Bovine Serum Albumin 22% Solution for Serological Applications 325 Concentrated Iso-Osmotic Phosphate Buffered Saline for Serological Applications 327 Red Cell Preserving Solution for Serological Applications 328 Abo Grouping 329 Rh Blood Group System 331 Anti-D (Rho) Human (Igg) Polyclonal Blood Typing Antibodies for Slide and Modified Tube Tests 333 Anti-D (Rho) (Igm) Monoclonal Blood Typing Antibodies for Slide and Tube Tests 335 Anti-D (Rho) (Igg) Monoclonal Blood Typing Antibodies for Slide and Modified Tube Tests 336 Anti-D (Rho) (Igm + Igg) Monoclonal Blood Typing Antibodies for Slide and Tube Tests 338 Anti-human Igg Monospecific Coomb’s Reagent for Direct and Indirect Antiglobulin Test 353 Anti-human Globulin Reagent for Direct and Indirect Antiglobulin Tests 356 Preparing Coomb’s Control Cells Agtrol® 358 Low Ionic Salt Solution for Serological Applications 359 Stabilized, Activated Papain Enzyme Solution for Serological Applications 361 Blood Transfusion 363 Trouble Shooting 372 Chapter 12. Cerebrospinal and Other Body Fluids 382 Cerebrospinal Fluid 382 Synovial Fluid (Sf) 388 Pleural Fluid 389 Pericardial Fluid (Pf) 391 Peritoneal Fluid 392 Amniocentesis and Amniotic Fluid Analysis, Diagnostic 394 Chapter 13. Semen Analysis 398 Semen Analysis 398 Chapter 14. Sputum Examination 405 Sputum 405 Common Respiratory Disorders 406 Chapter 15. Pregnancy Tests 411 Bioassays 411 Immunologic Methods 412 Slide Test for Pregnancy 412 Slide Test for Pregnancy 414 Elisa Pregnancy Test 416 Dipstick Ict Pregnancy Test 416 Device Ict Pregnancy Test 417 Dipstick Ict, Urine/Serum Pregnancy Test 418 Device Ict Urine/Serum Pregnancy Test 419 Troubleshooting 421 Rapid Formats 423 Chapter 16. Examination of Gastrointestinal Contents 425 Normal Saliva—Constituents 425 Gastric Juice 425 Examination of Duodenal Contents 430 Composition of Bile 430 Pancreatic Function Tests 430 Sweat Electrolytes Pilocarpine Iontophoresis 432 Contents xiii Chapter 17. Diabetes Mellitus: Laboratory Diagnosis 434 Diabetes Mellitus 434 Glycosylated Hemoglobin Kit (Ion Exchange Resin Method) for the Quantitative Determination of Glycohemoglobin in Blood (for in Vitro Diagnostic use Only) 443 Rapid Diagnostics 448 Chapter 18. Liver Function Tests 454 Tests of Excretion by the Liver 454 Evaluation of Synthesis in Liver 457 Evaluation of Enzyme Activity 458 Suggested Liver Function Tests 458 Chapter 19. Clinical Chemistry 461 Colorimetry 461 Photometer 462 Clinical Chemistry 465 Total Proteins 478 Serum Albumin 479 Serum Cholesterol 481 Hdl Cholesterol 484 Blood Glucose 490 Uric Acid 492 Calcium 493 Phosphorus 497 Chloride 500 Serum Iron and Tibc 501 Trace Elements 503 Zinc 503 Zinc (Colorimetric Method) 503 Copper 504 Magnesium 506 Automation in Clinical Chemistry 508 Principles of Quality Assurance and Standards for Clinical Chemistry 514 Chapter 20. Enzymology 519 Alpha-amylase 519 Lipase 520 Phosphatases 522 Transaminases 530 Gamma-glutamyl Transpeptidase (Ggtp) Blood 536 Lactic Dehydrogenase 538 Automation in Clinical Chemistry: Random Access Autoanalyzer 546 Chapter 21. Blood Gases and Electrolytes 548 Blood Gases 548 Automation in Blood Gas Analysis 556 Avl Compact 2 Blood Gas Analyzer 557 Electrolyte Analysis by Flamephotometer 558 Rapid Diagnostics in Electrolyte Analysis 561 Chapter 22. Serology/Immunology 563 Basic Immunology 563 Technologies 568 Enzyme Immunoassay 572 Chemiluminescence: The Technology 585 Polymerase Chain Reaction 587 Ria 590 Liquid Handling Systems 591 Streptavidin-Biotin Systems 594 Representative Elisa/Clia Techniques 595 Examples of Detailed Elisa Methods 597 Tests for Syphilis 608 Modified Vdrl Reagent Trepolipin® 610 Toluidine Red Unheated Serum Test for Rapid Serodiagnosis of Syphilis Redgen® 613 Latex Slide Test for Vdrl Syphfinal 615 Rapid Plasma Reagin (Rpr) Card Test/Carbon Antigen for Syphilis Testing (Carbogen) 618 One-step Test for Syphilis: Dipstick Syphicheck® 621 One-step Test for Syphilis (Device) Syphicheck 622 Third Generation Double Antigen Sandwich Enzyme-linked Immunosorbent Assay (Elisa) for the Detection of Antibodies to Treponema Pallidum in Human Serum or Plasma Trepolisa 3.0 624 Tests for Typhoid/Enteric Fever Widal Antigen Set/Antigens for Tube Tests (Typhochek) 624 Widal Antigen Set/Antigens for Slide and Tube Tests (Tydal)® 627 Reduced Widal Antigen Set: O and H for Tube Tests (Vital Widal) 630 Positive Control for Widal Test 631 Rapid Test for Detection Igm Antibodies to S. typhi in Serum/Plasma/Whole Blood (Device) Enterocheck – Wb 632 Slide and Tube Test for Detection of Antibodies to Brucella Abortus/Melitensis Brucel A/M 635 Slide Screening Test for Brucella Antibodies (Brucel-Rb) ® 636 Brucellosis Positive Control 638 Rapid Test for Igm and Igg Antibodies to Dengue Virus: Dengue Fever (Denguecheck-Wb) (Device) 639 Test for Infectious Mononucleosis (Immutex) 643 Rapid Test for Igm Antibodies to Leptospira: Leptospirosis (Leptochek-Wb) (Device) 645 Rapid Test for Malaria Pan/Pv/Pf (Paramax-3®) (Device) 647 Slide Test for C-reactive Protein (Rhelax Crp) 650 Slide Test for Antistreptolysin O (Rhelax Aso®) 653 Slide Test for Rheumatoid Factors (Rhelax Rf) 656 Slide Test for Anti-deoxyribonucleoprotein (Rhelax Sle) 659 xiv Concise Book of Medical Laboratory Technology: Methods and Interpretations Australia Antigen Hbsag (Virutex Hbsag) 662 One-Step Test for Hbsag Virucheck Device 664 Hcv Flavicheck Device 665 Toxoplasma Infections 668 Rapid Immunoconcentration Test for Hiv-1 and Hiv-2 Antibodies Flow through Method Retroquick-Hiv 669 Rapid Test for Simultaneous/Differential Detection of total Antibodies to Hiv1 and Hiv-2 in Human Serum/Plasma Retroscreen 671 Tuberculosis 673 Rapid Test for Detection of Antibodies to Mycobacterium tuberculosis (Device) Serocheck-Mtb 676 Tb Igg, Iga, Igm Ab, Mfd Anda 678 Tumor Markers 679 Tumor Markers Standard Methodologies Available on Elisa and Clia, as on Ria too 683 Ca 15-3 (Carcinogenic Antigen 15-3) 683 Ca 19-9 (Carbohydrate Ag 19-9, Gicam Gastrointestinal Cancer Antigen) Blood Mfd: Can Ag, 683 Ca 242 Mfd: Can Ag, 684 Ca 125 (Cancer Antigen 125) Mfd: Monobind 684 Carcinoembryonic Antigen (Cea) Mfd: Monobind 685 Prostate-specific Antigen (Psa) Total Prostate Specific Antigen (Tpsa) Elisa, Mfd: Monobind 685 Prostatic Acid Phosphates (Pap), Blood Method: Biochemical Analysis 686 Elisa Troubleshooting Aspects 686 Technical Tips 690 Chapter 23. Diagnostic Immunology 693 Qualitative Determination of Plasma Proteins by Immunoprecipitation 693 Fundamental Quantitative Considerations 698 Turbidimetry 699 An Example of Turbidimetric Immunoassay 718 C-reactive Protein 720 Turbidimetric Immunoassay for Determination of C-reactive Protein 722 Turbidimetric Immunoassay for Ultrasensitive Determination of C-Reactive Protein 723 Turbidimetric Immunoassay for Determination of Antistreptolysin ‘O’ in Human Serum 724 Turbidimetric Immunoassay for Determination of Microalbuminuria 724 Immunoglobulins (Ig) 724 Turbidimetric Immunoassay for Estimation of Immunoglobulin Iga in Human Serum 725 Turbidimetric Immunoassay for Estimation of Immunoglobulin Igg in Human Serum 726 Turbidimetric Immunoassay for Estimation of Immunoglobulin Igm in Human Serum 726 Turbidimetric Immunoassay for Estimation of Complement C3 in Human Serum 726 Turbidimetric Immunoassay for Estimation of Complement C4 in Human Serum 727 Turbidimetric Immunoassay for Estimation of Antithrombin Iii in Human Serum 727 Quantitative Immunoturbidimetric Assay for Estimation of Fibrinogen 727 Turbidimetric Immunoassay for Estimation of Lipoprotein (A) in Human Serum 727 Quantitative Turbidimetric Immunoassay for Estimation of Apolipoprotein A-I 728 Quantitative Turbidimetric Immunoassay for Estimation of Apolipoprotein B 728 Automation in Turbidimetry 729 Chapter 24. The Endocrine System 731 Pituitary Gland 731 Anterior Lobe: Growth Hormone (Gh) 732 Method of Evaluation: StreptavidinBiotin Elisa 733 Corticotropin (Acth) 735 Other Anterior Pituitary Hormones 735 Intermediate Lobe (Pars Intermedia) 736 Posterior Pituitary (Neurohypophysis) 736 Disorders of the Pituitary System 738 Hypothalamus 738 Adrenal (Suprarenal) Gland 738 Mineralocorticoids 738 Glucocorticoids 739 Adrenal Medulla 742 Thyroid 742 Calcitonin 753 Parathyroid 754 Parathyroid Hormone (Intact) Elisa 756 Pancreas 757 Testes 758 Ovary 758 Pineal Gland 759 Hormones and Fertility 759 Male Fertility 760 Female Fertility 763 Algorithm for Evaluating Amenorrhea, Immunoassays for Lh, Fsh and Prl 768 Adrenal Cortex 775 Adrenal Medulla 777 Testes 779 Steroids 781 17-Β-Estradiol 781 Dheas (Dehydroepiandrosterone Sulfate) 782 ∆4-Androstenedione 782 Progesterone 782 17-Alpha-hydroxyprogesterone 783 Total Tri-iodothyronine (T3) 783 Cia™ Insulin (Chemiluminescence Immunoassay) 788 Contents xv Chapter 25. Histopathology 791 Preparation of Tissues 791 Routine Staining Procedures 794 Some Staining Techniques in Detail 799 Automation in Histopathology 805 Chapter 26. Cytology 808 Papanicolaou Method of Staining Smears (Modified) 808 Fnac (Fine-Needle Aspiration Cytology) 809 Smearing Techniques 812 Requirements for Laboratory Set Up 812 Immunoperoxidase Staining for Cyto and Histopathology 817 Automation in Cytology 818 Chapter 27. Microbiology and Bacteriology 819 Classification 819 Culture 825 Ready to Pour, Sterilized Pouched Media for Microbiological Applications Instaprep 828 Easybact 833 General Instructions for Microbiology 837 Gram-positive Cocci 837 Gram-negative Cocci 839 Anaerobic Spore Bearing Bacilli 840 Aerobic Spore Forming Bacilli 842 Gram-positive Bacilli 842 Mycobacteria 843 Overview of M. tuberculosis: Diagnostic Approach, Afb Staining, Culture and Sensitivity 845 Mucolytic, Disinfectant, Specimen Pretreatment and Buffering System for Afb Staining and Culture 848 Rapid Two Step Cold Afb Stain 851 Ready to use Lj Solid Medium for Mycobacterium tuberculosis Isolation 855 Combipack of Solid and Liquid Medium for Mycobacterium tuberculosis Isolation 857 Primary/Secondary Drug Containing Lowenstein-Jensen Media Panel Mtb Sensitivity Tests 861 In Determination of Adenosine Deaminase Activity in Serum, Plasma and Biological Fluids 863 Gram-negative Bacilli 865 Spirochetes 871 Gram Stainer 872 Quality Assurance in Bacteriology 872 Color Atlas—Media and Colonies 873 Chapter 28. Mycology 883 Intermediate Superficial Deep Mycoses 884 Deep or Systemic Mycoses 884 Fungi Usually Present as Contaminants but Which Rarely Cause Disease—Usually in Patients Chronically Ill from Other Diseases 885 Mycological Methods 885 Chapter 29. Diagnostic Skin Test 886 Technique of Skin Tests 886 Immunologic Basic for Skin Tests 886 Common Skin Tests 887 Immediate Reaction Type of Skin Tests 887 Delayed Reaction Type of Skin Tests 889 Chapter 30. Cytogenetics 891 Blood Lymphocyte Culture 891 Karyotyping 893 G and Q Bandings 894 Importance of Chromosomal Studies 894 Barr Body Analysis and Buccal Smear for Staining of Sex Chromatin Mass 895 Chapter 31. World’s Latest and Best Technologies by Roche 896 Business Areas 896 Revaluating Diagnostics 896 Testing Efficiency and Medical Value 896 Effective Management of Infectious Diseases 898 Cobas® Modular Platform 900 Your Benefit 900 Cobas® 8000 Modular Analyzer Series 901 Cobas® 8000 Modular Analyzer Series 902 Cobas® 6000 Analyzer Series 903 Cobas® 4000 Analyzer Series 906 Cobas C 311 Analyzer 906 Cobas E 411 Analyzer 906 Cobas C 111 Analyzer 907 Cobas Integra® 400 Plus 908 Cobas P 312 Pre-analytical System 910 Cobas P 512 Pre-analytical System 910 Cobas P 612 Pre-analytical System 911 Cobas P 501 and Cobas P 701 Modular® Pre-analytics Evo 912 Cobas® Connection Modules (Ccm) 913 Cobas® 8100 Automated Workflow Series 914 Cobas® It Solutions 916 Cobas® Middleware Solution 917 Cobas® Laboratory Information System 918 Cobas® Infinity It Solutions 920 Overview of Serum Work Area Tests 921 Ecl—Unique Immunoassay Technology 925 Technology for Homogeneous Immunoassay Detection 926 Elecsys® Hbsag Ii Quant 926 Elecsys® Hiv Combi Pt 4th Generation (Ag+Ab Test) 927 The Syphilis Assays 929 Elecsys® Syphilis Immunoassay 929 Elecsys® Torch Panel 930 Elecsys® Troponin T High Sensitive (Tnt Hs) 931 xvi Concise Book of Medical Laboratory Technology: Methods and Interpretations Key Benefit: Earlier Diagnosis of Ami 932 Elecsys® Nt-proPnB 932 Elecsys® Tumor Marker Portfolio 933 Elecsys® He4 935 Elecsys® Pro-grp 936 Elecsys® Calcitonin 937 Elecsys® Anti-Tshr 938 Elecsys® Tg Ii 939 Tina-Quant® Lipoprotein (A) Gen 2 Test 940 Tina-Quant® Immunoglobulin A and M Csf 941 Tina-Quant® Hemoglobin A1c 942 Tina-Quant® Cystatin C Gen 2 942 Elecsys® Preeclampsia 943 Elecsys® Vitamin D Total 945 Elecsys® Il-6, Pct and Tina-Quant® Crp 945 Elecsys® Tacrolimus and Cyclosporine 947 Hemostasis Testing 949 Multiplate® Analyzer 949 Urinalysis 951 Urinalysis From Roche 951 Combur-Test® Strip 951 Urisys 1100® Analyzer 952 Cobas U 411 Urine Analyzer 953 Cobas® 6500 Urine Analyzer Series* 954 Point-of-Care Testing 956 Cobas Poc it Solution 958 Cobas bge Link Software 960 Cobas B 121 System 961 Cobas B 221 System 962 Cobas B 123 Poc System 963 Accu-Chek® Inform Ii System 964 Accu-Chek® Safe-T-Pro Plus 965 Cobas H 232 Poc System 966 Roche Cardiac® Trop T Sensitive Test 967 Coaguchek® Xs System 968 Coaguchek Xs Plus System 969 Accutrend® Plus System 970 Reflotron® Plus System 971 Cobas B 101 System 972 Molecular Diagnostics 974 Solutions from Roche for Molecular Diagnostics 974 Cobas P 630 Instrument 977 Cobas® Ampliprep Instrument 978 Cobas® Taqman® Analyzer and Cobas® Taqman® 48 Analyzer 979 Cobas® Ampliprep/Cobas® Taqman® Hcv Qualitative and Quantitative Tests, V2.0 980 Cobas® Taqman® Mtb Test 981 Cobas P 480 Instrument 982 Cobas® 4800 System V2.0 983 The Cobas® Hpv Test 985 The Cobas® Oncology Tests 985 Cobas® Mrsa/Sa Test 987 Cobas® Cdiff Test 987 Cobas® Hsv 1 and 2 Test 988 Cobas S 201 System 988 Lightcycler® Systems 990 Lightcycler® 2.0 Instrument 991 Test Kits, Validated for Ivd 992 Lightcycler® Septifast Test 992 Lightcycler® Mrsa Advanced Test 993 Magna Pure Systems 993 Symphony System 996 Benchmark Special Stains 997 Primary Antibodies 999 Ihc Detection 1001 Breast Cancer Diagnostics 1002 Prostate Cancer Diagnostics 1003 Hematopathology 1004 Colorectal Diagnostics 1004 Lung Cancer Diagnostic Solutions 1005 Benchmark Ihc/Ish Platform 1007 Benchmark System Features 1007 Digital Pathology 1008 Vantage Workflow Solution 1009 Consultancy Services 1011 Sequencing Solutions 1013 Genome Sequencer Flx+ System 1013 Gs Junior System 1014 Nimblegen Sequence Capture 1015 Roche Dialog 1017 Appendix 1019 Index 1033

 


Biowavers

Biowavers are the exemptions that are granted by USFDA from conducting human

bioequivalence studies. Biowavers reduce the need for bioequivalence studies. They

are given when in vitro studies data provides sufficient estimate of a relative in vivo

performance of two products. These are given when API meets certain solubility and

permeability criteria.

BIOSTATISTICS IN PHARMACEUTICAL PRODUCT DEVELOPMENT

Statistics is the science which deals with collection, tabulation and classification of

numerical facts as the basis for explanation, description and comparison of the

phenomenon.

Biostatistics is the branch of statistics concerned with mathematical facts and data

related to biological events. Sir Francis Galton is the Father of biostatistics.

There are two types of biostatistics:

1. Inferential biostatistics: It is the methods of generalising a larger group. The

generalization is based on information about a sample. The sample is considered to

be similar to the population.

2. Descriptive biostatistics: These are used to explain the basic features of the data.

They also provide simple summaries of the data.

Application of Biostatistics

After the Kefauver Harris drug amendments, it is compulsory for the firms to prove

the safety of the drug with suitable statistical evidence. Statistics is involved in all

stages of drug development, i.e. from drug discovery to post-marketing processes.

DATA PRESENTATION FOR FDA SUBMISSIONS

The FDA accepts electronic submissions. The electronic submission's regulatory process

is divided into four steps:

1. Early planning and management of the project

2. Document Preparation

3. Dossier publishing

4. Dossier submission

During the planning stage applicants are recommended to use the following tools:

• Checklist • Glossary

• Template • Timeline table

MANAGEMENT OF CLINICAL STUDIES

The clinical trial management requires efficient management. Clinical trial consists of

a series of stages. Each of the stages requires effective management. The stages are as

follows:

1. Initiating 2. Planning 3. Executing

4. Monitoring and controlling 5. Analysis and reporting

Managing these five stages is the most effective way of managing clinical studies.

 


10. Quality and Related Concepts

11. Total Quality Management and Other Quality

Management Systems

12. ISO Series, NABL and GLP

Summary

4

Quality Management Systems

150 Industrial Pharmacy II

QUALITY

If we go with the dictionary meaning of quality, then quality means “standard of

something when compared with one that already exists, or degree of excellence of

something.”

Quality is something that motivates organisations not only to achieve the minimum

excellence mark but also to improve it continuously. Quality of any product depends

upon the stakeholders, i.e. their demands and expectations from the products. Here,

stakeholder means everyone who has an interest in the product directly or indirectly.

Customers comprise a large group of stakeholders. Other stakeholders are investors,

employees, suppliers, etc.

Quality of any product can be assured by three ways:

i. By setting guidelines which are compulsory for an organisation to follow.

ii. By setting up an effective quality control system in the organisation that will

maintain the ensured quality.

iii. By developing a culture in the organisation of improving the quality.

This unit deals with the same concept of quality and different quality management

systems developed for ensuring quality. Different ISO series have also been discussed

in this unit.

10.1 CONCEPT OF QUALITY

The quality in the pharmaceutical industry is a very important topic. The government

regulatory agencies, drug manufacturers, customers and other stakeholders all joined

together to give or to have a quality product. Every agency or stakeholders define

quality differently. Everyone has its meaning of quality.

According to ASQC (American Society for Quality Control) quality can be defined

as the totality of features and characteristics of a product that bears on its ability to

satisfy the stated or implied needs.

ISO, an international body for formulating standards, has defined quality as: “Degree

to which a set of inherent characteristics fulfils requirements”.

According to ICH quality is the degree to which a set of inherent properties of a

product, system or process fulfils requirements.

150

Quality and Related Concepts

10

Quality and Related Concepts 151

There are many ways to define quality like:

a. “It delivers the properties described on the label and is not contaminated”

b. “Fitness for intended use”

c. “Freedom from defects”

d. “Meeting or exceeding customer expectations”

We can summarise these definitions of quality as:

i. User-based: “In the eyes of beholder/what user expects.”

ii. Manufacturing-based: “Right at the first time”

iii. Product-based: “Precise as per specifications”

Dimensions of Quality

There are eight dimensions which decide either the product is a quality product or

not. These are as follows (Fig. 10.1):

i. Performance

ii. Features

iii. Reliability

iv. Conformance

v. Durability

vi. Serviceability

 


IND is not required:

• In case the drug is not intended for human subjects.

• In case the drug is intended for in vivo testing.

• When the drug is already approved and study is conducted within the approved

indication for use.

INVESTIGATOR'S BROCHURE (IB)

It is a compilation of the clinical and non-clinical data on the investigational product

that is relevant to the study of the product in human subjects. The main purpose of an

investigator's brochure is to provide information to the investigator. It also enables

the investigator to understand the trial and make benefit/risk assessment of the

investigational product by own. The investigational brochure is edited by a medically

qualified person.

NEW DRUG APPLICATION (NDA)

The application through which drug sponsors propose the approval of a new drug

product; for marketing in front of regulatory authority. It includes all clinical and

non-clinical data.

Review time frame: The FDA will review and issue an approval, approvable,

or non-approvable letter within 180 days of receipt of application. This period is

known as review-clock. In this period the application is reviewed by the FDA

thoroughly. During this period applicant may withdraw the application and resubmit

it later.

Filing time frame: After 60 days of receiving the application, the FDA will decide

whether the application may be filed or not. If the FDA filed the application, then

applicants will be notified otherwise the applicant will be allowed to discuss the reason

for non-filing of application with FDA.

CLINICAL RESEARCH/BE STUDIES

BE stands for bioequivalence. It is a relative term. It denotes that a drug substance in

two or more identical dosage forms reaches the systemic circulation at the same relative

rate and to the same relative extent.

Summary 147

 


Summary 145

NON-CLINICAL DRUG DEVELOPMENT

A targeted drug has to go through from various studies such as non-clinical and clinical

studies. Non-clinical studies are also known as pre-clinical studies. It is a stage of

research that happens before clinical studies. During non-clinical studies important

drug safety data is collected. These studies are necessary before filing Investigational

New Drug (IND) application.

Objectives of Non-clinical Drug Development

The main objective of non-clinical study is to confirm that either the target drug is safe

for use in human or not. This is done by studying animal pharmacology and toxicology

testing.

The need for non-clinical studies:

Non-clinical studies are essential:

• To determine the lethal dose, toxic dose (like LD50, ED50, etc.).

• To determine the pharmacological action of the target drug.

• It is necessary to submit the animal testing report during the IND application.

• To determine the pharmacokinetic properties of a drug.

• To determine the route of administration.

Types of non-clinical study

There are two types of non-clinical study. These are:

1. Pharmacodynamics (What drug does to the body or how the drug makes the body

to react?)

2. Pharmacokinetics (What the body does to the drug?)

TOXICOLOGY

This study helps in determine the toxicity of the compound.

DRUG METABOLISM

It is defined as the chemical alteration of a drug in the body. This chemical alteration

converts nonpolar compounds to polar and lipid-soluble compounds to lipid-insoluble

compounds. The products of drug metabolism are called metabolites. Drug metabolism

includes conversion of:

• Active drug to inactive or less active metabolite (pharmacological inactivation). For

example, phenobarbitone to p-hydroxyphenobarbitone.

• Active to a more active metabolite (bioactivation or toxicological activation). For

example, codeine to morphine.

• Inactive to more active toxic metabolite (lethal synthesis)

• Inactive drug (pro-drug) to an active metabolite (pharmacological activation). For

example, phenacetin to paracetamol.

• Active drug to an equally active metabolite (no change in pharmacological activity).

For example, digitoxin to digoxin.

• Active drug to an active metabolite which is having different pharmacological

activity (change in pharmacological activity). For example, iproniazid to isoniazid.

146 Industrial Pharmacy

The main and major site of drug metabolism is liver. Other sites of biotransformation

because a variety of metabolising enzymes are present in liver.

GENERAL CONSIDERATIONS OF INVESTIGATIONAL NEW DRUG (IND)

APPLICATION

INDA filed before conducting clinical trials. It is a submission to the regulatory

authority requesting permission to initiate a clinical study on the new drug product

by sponsor.

IND is required

At the time of the clinical trial of an unapproved drug.

 


INTRODUCTION

Medicines have their existence in parallel with the existence of mankind. It is perhaps

as old as a human being. The current pharmaceutical industry is more systematic,

well organised and in compliance with respective guidelines. This is because of effective

pre- and post-approval regulation of new drugs and the existing drugs. Regulatory

affairs play this role for pharmaceutical manufacturing companies and the government

of the country. It is a new profession which emerges after government and

manufacturing companies felt the need for a bridge in them which promotes

coordination and communication. The regulatory affairs act as an interface between

government and manufacturing companies.

Regulatory affairs is actively involved in every stage of drug development in case

of new drug and post-approval surveillance in case of an existing drug. All the

applications for getting a pharmaceutical product approved for entering into the market

has been prepared by the regulatory department of companies. The regulatory

professionals act as liaison with government regulatory agencies. These applications

are reviewed by government regulatory agencies to check the compliance with issued

guidelines. The regulatory department of the company internally also liaison with

QC, marketing, production, administration and other departments of company to keep

them updated with changes in guidelines and to ensure their compliance with the

guidelines.

HISTORICAL OVERVIEW OF REGULATORY AFFAIRS

The quality assurance and regulation of medicines evolved gradually over time. Many

unfortunate incidents have catalysed the development of regulations and guidelines

of assuring quality, safety and efficacy of medicines. In 1947, in United States over

100 died due to sulphanilamide elixir. This event led to the introduction of Federal

Food and Cosmetics Act for new drugs in 1948. In 1956, the introduction of Thalidomide

(a drug prescribed to pregnant women for morning sickness) in 46 different countries

resulted in an estimated 10000 babies born with phocomelia (Deformities related to

limbs). This event results in reshaping of the whole regulatory system. Due to these

types of incidents at different stage regulatory bodies introduce new laws and

guidelines which make norms related to drug approval or site approval stricter than

before. Due to more guidelines and laws, the need for regulatory affairs has been felt.

It led to the emergence of regulatory affairs as a profession.

 


REGULATORY AUTHORITIES

For every country, the health of its people is of utmost importance. To maintain the

health of the people country has to ensure the quality of medicines and other

142

Summary

Summary 143

pharmaceutical products as well as devices. So, every country has its regulatory

authority which regulates the following:

1. Import of drugs from another country

2. Export of drugs to another country

3. Manufacturing of drugs in the country

4. Establishment of manufacturing companies in the country

5. Dispensing of medicines

6. Approval of new drugs

7. Approval for clinical trials

8. Testing of drugs in the certified laboratories

9. The compliance with international guidelines

10. Regular inspections

11. Issuing various types of licence related to drugs

ROLE OF REGULATORY AFFAIRS DEPARTMENT

Drug regulatory affairs is a dynamic and challenging field in the pharmaceutical

industry. It has to deal with government authority on one hand and applicant/company

on another hand. Because of the crucial nature of the job regulatory affairs professionals

should be experts in managing the product life cycle. They should have the capability

to solve technical as well as administrative problems within the limits of laws and

regulations. Regulatory affairs department is a bridge between government regulatory

authority and pharmaceutical company. It also has a connection with all the

departments of the company internally.

Regulatory affairs department plays many important roles. These are as follows:

• Role in drug development

• Role in drug approval

• Keep other departments updated

• Acts as interface

• Acts as an adviser

• Maintenance of licences

• Preparation of documents

• Helps in the execution of clinical plan

• Data collection and storing

RESPONSIBILITY OF REGULATORY AFFAIRS PROFESSIONALS

There are some qualities which make efficient regulatory professionals. These are:

• Good communication skills

• Good computer knowledge

• Work independently

• Active listeners

• Good presenter

• Good data management skills

• Patient worker

• Effective negotiator

• Punctual

• Have sound knowledge

144 Industrial Pharmacy

Because of the critical nature of this job, there are several important responsibilities

of regulatory affairs professionals. These are as 

 


c. Conducting plant studies d. None of the above

3. IND is required:

a. In case the drug is not intended for human subjects.

b. In case the drug is intended for in vivo testing.

c. When the drug is already approved and study is conducted within the approved

indication for use.

d. At the time of the clinical trial of an unapproved drug

4. Which CTD module is not required while filing INDA?

a. Module 4 b. Module 2

c. Module 3 d. Module 5

5. How many types of INDA are there?

a. 2 b. 4

c. 6 d. 3

6. The FDA will review and issue an approval, approvable, or non-approvable letter

within 180 days of receipt of application. This time period is known as:

a. Filing time frame b. Patent time frame

c. Review time frame d. None of the above

7. The term that denotes that a drug substance in two or more identical dosage forms

reaches the systemic circulation at the same relative rate and to the same relative

extent is known as:

a. Bioequivalence b. Bioavailability

c. Therapeutic value d. Absorption

8. It is a measure of the extent of absorption:

a. Cmax b. Tmax

c. AUC d. None of the above

9. The exemptions that are granted by USFDA from conducting human

bioequivalence studies are known as:

a. Clinical trial waivers b. Clinical waivers

c. Geowaivers d. Biowavers

INDA and NDA 141

10. Bioequivalence studies are not required in case of:

a. Drugs are parenterally administered b. Drugs in solution form

c. Drugs in gaseous form d. All of the above

11. Biowavers are given in case of:

a. Drugs are highly soluble and highly permeable.

b. Highest dose strength of the drug is soluble in 250 ml aqueous solution over a

pH 1–7.5 at 37°C.

c. 90% of the drug is absorbed if administered orally.

d. All of the above

12. Match the following

Form no Required for filing

a. Form FDA-356h i. INDA

b. Form FDA-3397 ii. Application to market a new drug, biologic, or an

antibiotic drug for human use

c. Form FDA-3331 iii. User fee cover sheet

d. 21 CFR, Sec. 312.23 vi. New drug application field report

or Sec. 312.20

13. Fill in the blanks

a. _________ IND is submitted primarily by companies whose main goal is to obtain

the approval for new drug marketing.

b. _________ IND is for the experimental drugs which show promising results in clinical

testing of life-threatening conditions.

c. After _______ days of receiving the application, the FDA will decide whether the

application may be filed or not. It is known as _________.

d. The main and major site of drug metabolism is ______.

e. ______is the observed maximum concentration of a drug.

f. _______ is a measure of the rate of absorption.

g. Bioequivalence studies are of two types such as __________ and ________.

ANSWERS

1. b 2. a 3. d 4. d 5. b 6. c 7. a 8. c

9. d 10. d 11. d 12. a (ii), b(iii), c(iv), d(i)

13. (a) Commercial, (b) Treatment, (c) 60, Filing time frame, (d) Liver, (e) Cmax,

(f) Tmax, (g) In vivo and In vitro.

142 Industrial Pharmacy

 


i. To play a lead role in initiating, planning, executing, monitoring and controlling,

analysis and reporting a project

ii. To communicate and present trial effectively whenever needed

iii. To organise and motivate others

iv. To manage the trial budget.

Effective trial management includes the following:

i. Proper planning of the project

ii. Time management

iii. Timely approval of the trial

iv. Proper designing of the clinical trial protocol

v. Good collaboration between the trial staff and participants

vi. Good communication between each section of the trial pyramid

vii. Proper management of the budget

viii. Proper training to the staff

ix. Appropriate credit will be given to the responsible persons

x. Effective recruitment of staff and trial participants

xi. Efficient evidence and study in the support of the trial

xii. Genuine reporting to the competent authority

xiii. Compliance with the regulatory guidelines for clinical trials

xiv. Efficient management of the resources

xv. Good record keeping

Effective management of clinical studies will only be possible if all the stakeholders

come together and ‘own’ the project.

PRACTICE QUESTIONS

Long Answer Type Questions

1. Explain the general considerations of INDA.

2. Explain the procedure for NDA filing in detail.

140 Industrial Pharmacy II

Short Answer Type Questions

1. What are bioequivalence studies? Describe in detail.

2. Explain the importance of biostatistics in drug development process.

3. What are the contents of IB?

4. What are the general considerations for data submission in FDA?

5. Explain the following:

a. Biowavers

b. Management of clinical studies

Objective Type Questions

1. _____filed before conducting clinical trials

a. ANDA b. INDA

c. NDA d. All of the above

2. INDA filing is done for:

a. Conducting animal studies b. Conducting human studies

 


INDA and NDA 135

Fig. 9.7: Bioequivalence study is the process of estimation of closeness of the effects innovator drug

and its generic version.

Important pharmacokinetic parameters are:

AUC: Area under the concentration–time curve. It is a measure of the extent of

absorption.

Cmax: It is the observed maximum concentration of a drug. It is a measure of rate and

extent of absorption.

t

max: It is the time at which Cmax is observed. It is a measure of the rate of absorption.

Types of bioequivalence studies (Figs 9.8 and 9.9)

Fig. 9.8: Types of bioequivalence studies

Fig. 9.9: Types of in vivo bioequivalence studies

136 Industrial Pharmacy II

Biowavers

Biowavers are the exemptions that are granted by USFDA from conducting human

bioequivalence studies. Biowavers reduce the need for bioequivalence studies. They

are given when in vitro studies data provides sufficient estimate of a relative in vivo

performance of two products. These are given when API meets certain solubility and

permeability criteria. Biowavers are given in case if:

i. Drugs are highly soluble and highly permeable.

ii. Highest dose strength of the drug is soluble in 250 ml aqueous solution over a

pH 1–7.5 at 37°C.

iii. 90% of the drug is absorbed if administered orally.

iv. 80% of the drug is absorbed within 15 min of administration at 37°C.

v. Clinical research protocols

Bioequivalence studies are not required in case of:

i. Drugs are parenterally administered

ii. Drugs in solution form

iii. Drugs in gaseous form

iv. The new drug has a high therapeutic window.

v. Drugs with rapid and similar dissolution.

9.5 BIOSTATISTICS IN PHARMACEUTICAL PRODUCT DEVELOPMENT

Statistics is the science which deals with collection, tabulation and classification of

numerical facts as the basis for explanation, description and comparison of the

phenomenon.

Biostatistics is the branch of statistics concerned with mathematical facts and data

related to biological events. Sir Francis Galton is the Father of biostatistics.

There are two types of biostatistics

1. Inferential biostatistics: It is the methods of generalising a larger group. The

generalization is based on information about a sample. The sample is considered to

be similar to the population.

2. Descriptive biostatistics: These are used to explain the basic features of the data.

They also provide simple summary of the data (Fig. 9.10)

mcq general

 

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