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Concise Book of Medical Laboratory Technology: Methods and Interpretations
Ramnik Sood
1:
Laboratory
INTRODUCTION
PRIMARY LABORATORY
SECONDARY LABORATORY
TERTIARY LABORATORY
LABORATORY SET-UP
LABORATORY BUILDING AND SPACE
PHYSICAL ASPECTS OF A LABORATORY
PROVISIONS AND PRECAUTIONS
FIRE PREVENTION
ELECTRICAL INSTALLATIONS
LIQUIFIED AND COMPRESSED GASES
CHEMICALS AND RADIOACTIVE SUBSTANCES
STORES
STAFF SAFETY AND FACILITIES
BASIC LABORATORY SAFETY
CODE OF CONDUCT FOR MEDICAL LABORATORY PERSONNEL
ACCIDENTS
SAFETY MEASURES IN THE LABORATORY
HAZARDS IN THE CLINICAL LABORATORY
Physical Hazards
Laboratory Equipment
SAFETY WITH CHEMICALS/REAGENTS
LABELING OF HAZARDOUS REAGENTS/CHEMICALS
Incompatible Chemicals
Flammable Chemicals
SIGNS FOR MEDICAL LABORATORIES
GENERAL LABORATORY
LABORATORY CAUTIONARY
Corrosive Chemicals
Toxic, Harmful, and Irritating Chemicals
GENERAL PROHIBITION
GENERAL LABORATORY
Oxidising Chemicals
Explosive Chemicals
Carcinogens
ACCIDENTS IN THE LABORATORY
FIRST AID IN LABORATORY ACCIDENTS
Acid Burns
Swallowing Acids
Poisoning
Burns Caused by Heat
Injuries Caused by Broken Glass
Contamination by Infected Material
Bodily Damage by Electric Shock
Precautions for the Avoidance of Accidents
Suggested List of First Aid Equipment for Laboratory
Contamination from Infective Material
PRECAUTIONARY MEASURES
UNIVERSAL WORK PRECUATIONS (UWP) FOR LAB PERSONNEL (ESPECIALLY IN RELATION TO HIV TRANSMISSION)
INTRODUCTION
COMPONENTS OF UWP
GUIDELINES OF BASIC PRACTICES AND PROCEDURES
BIOSAFETY REGULATIONS FOR LABORATORY PROCEDURES
STERILIZATION (FOR NONDISPOSABLE ITEMS)
WASTE DISPOSAL
FINAL DISPOSAL
POST-EXPOSURE CARE
First Aid
Report
Initial Consultation
Laboratory Testing
Clinical Follow-up
Containing Spills
Collection of Specimen
Transport of Specimen
MEDICOLEGAL ASPECTS OF CLINICAL PRACTICE
LABORATORY INSTRUMENTS
MICROSCOPE
Parts of the Microscope
SPECIAL APPLICATIONS OF THE MICROSCOPE
Phase Contrast Illumination
Dark Ground Illumination
Fluorescence Microscopy
Electron Microscope
WEIGHING SCALES OR ANALYTICAL BALANCE
Use and Care
CENTRIFUGE
Types of Centrifuge
GLASSWARE (MANY ITEMS ARE NOW MADE OF PLASTIC)
OTHER NECESSARY EQUIPMENTS
Serological Water Bath
Incubator
Hot Air Oven
REPORTING LABORATORY TESTS AND KEEPING RECORDS
Standardization
Lab Reporter
2:
Sterilization
METHODS COMMONLY USED FOR STERILIZATION
STERILIZATION BY HEAT
DRY HEAT
Flaming
Red Heat
Hot Air Oven
MOIST HEAT
Temperature
Boiling
Steam
Inspissation
COLD
ULTRAVIOLET RADIATION
IONIZING RADIATIONS
FILTRATION
CHEMICAL STERILIZATION
Chemicals Used
GLASSWARE PREPARATION FOR USE
Dichromate Cleaning Solution
SYRINGES
Choice of Syringes and Needles
Disposable Sterile Syringes
MODERN DAY DISINFECTION
PREVENTION BEFORE CURE
Icons Used
HAND CARE
ANTISEPTICS
General Antisepsis
Specialized Antisepsis
Skin infections (including scalp face and neck)
Blood infections
SKIN PREPARATIVES
ENVIRONMENT AND SURFACES
INSTRUMENTS
3:
SI Units
LITER
GRAM
MOLE (MOL)
INTERNATIONAL UNIT (U)
CONVERSION FACTORS BETWEEN CONVENTIONAL AND SYSTEM INTERNATIONAL UNITS (SIU)
HEMATOLOGY
CHEMISTRY
ENZYME
THERAPEUTIC AND TOXIC DRUGS
4:
Fundamental Chemistry
INDICATORS
SOLUTES, SOLVENTS AND SOLUTIONS
Solute
Solvent
Solution
STRENGTH OF A SOLUTION
PERCENT SOLUTIONS
Weight Per Unit Weight (w/w)
Weight Per Unit Volume (w/v)
Volume Per Unit Volume (v/v)
PART DILUTIONS
MOLAR SOLUTIONS
NORMAL SOLUTIONS
Saturated Solution
Standard Solution
ABBREVIATIONS AND DEFINITION
Atomic Number
Atomic Mass
Isotope
Atomic Weight
Electron Configuration
5:
Urine Analysis
COMPOSITION OF URINE
PRESERVATION OF SPECIMEN
GROSS EXAMINATION OF URINE
COLOR AND APPEARANCE
Interfering Factors
REACTION
Urine pH
Interfering Factors
Be Careful
ODOR
SPECIFIC GRAVITY
Interfering Factors
URINARY VOLUME
Polyuria
Oliguria
TURBIDITY
Interfering Factors
CHEMICAL EXAMINATION OF URINE
TESTS FOR PROTEIN
Heat and Acetic Acid Test
Sulphosalicylic Acid Test
Paper Strip Method
Quantitative Estimation of Protein in Urine
Bence Jones Protein Tests
Interpretation of Proteinuria
Other Important Related Aspects
Collecting Specimen for Orthostatic Proteinuria
Interfering Factors
Mechanisms of Proteinuria
Microalbuminuria
MICROALBUMINURIA
Summary
Reagent
Reagent Storage and Stability
Principle
Note
Sample Collection and Preparation
Materials Provided with the Kit
Interpretation of Results
Remarks
TESTS FOR GLUCOSE
Benedict's Qualitative (Semiquantitative) Glucose Test
Benedict's Quantitative Glucose Test
Quantitative Method
Significance of Sugars in Urine
KETONE BODIES IN URINE
Tests for Ketone Bodies
Causes of Ketonuria
Clinical Implications
Clinical Relevance
BILE SALTS
Method
Interpretation
BILE PIGMENTS
1. Foam Test
2. Iodine Ring Test
3. Harrison Test
4. Diazo Test
Causes of Hyperbilirubinuria
UROBILINOGEN AND UROBILIN
Urobilinogen
Urobilin (Schlesinger's Test)
Estimated Urobilinogen
Urobilinogen (Quantitative)
Clinical Relevance
PORPHYRINS
Causes
Normal Values
Clinical Relevance
BLOOD IN URINE (HEMATURIA)
1. Guaic Test
2. Benzidine Test
Causes
Diffuse Renal Lesions
NITRITE/BACTERIA
Procedure
Clinical Implications
RAPID DIAGNOSTICS
MULTIPLE® REAGENT STRIPS FOR URINALYSIS
SUMMARY AND EXPLANATION/INTENDED USE
CHEMICAL PRINCIPLES OF THE PROCEDURE
Glucose
Bilirubin
Ketone
Specific Gravity
Blood
pH
Protein
Urobilinogen
REAGENTS
Glucose
Bilirubin
Ketone
Specific Gravity
Blood
pH
Protein
Urobilinogen
WARNING AND PRECAUTIONS
RECOMMENDED PROCEDURES FOR HANDLING BAYER REAGENT STRIPS
IMPORTANT
SPECIMEN COLLECTION AND PREPARATION
PROCEDURE
QUALITY CONTROL
RESULTS
LIMITATIONS OF PROCEDURES
Glucose
Bilirubin
Ketone
Specific Gravity
Blood
pH
Protein
Urobilinogen
EXPECTED VALUES
Glucose
Bilirubin
Ketone
Specific Gravity
Blood
pH
Protein
Urobilinogen
SPECIFIC PERFORMANCE CHARACTERISTICS
Glucose
Bilirubin
Ketone
Specific Gravity
Blood
pH
Protein
Urobilinogen
MULTISTIX® URINALYSIS STRIPS (FIG. 5.3)
Dependable Results When and Where You Need Them
Easy to Use (Figs 5.4 to 5.6)
Rapid Results (Fig. 5.6)
First Line Health Screen for a Variety of Settings
Improved Use of Resources
Multistix Configurations
AUTOMATION IN URINALYSIS
CLINITEK STATUS® URINE ANALYZER (FIG. 5.7)
Dependable Results in Any Patient Setting
A Wide Range of Test Parameters
CLINITEK® 50 URINE ANALYZER (FIG. 5.11)
Dependable Results in Any Patient Setting
Easy to use
Specifications
There's no Denying Easy Chemistry
A Helping Hand
User Friendly
Rapid Results
Improved Use of Resources
A Wide Range of Test Parameters
CLINITEK® 500 URINALYSIS INSTRUMENT
Efficiency in Processing
User Friendly
Intelligent Data Management
A Wide Range of Test Parameters
SPECIAL URINE TESTS
CALCIUM IN URINE (SULKOWITCH TEST)
Clinical Relevance
SEROTONIN (5-HYDROXYTRYPTAMINE)
Carcinoids
Test
Method
Clinical Relevance
CYSTINE
Clinical Relevance
FAT IN URINE
HEREDITARY METABOLIC DISORDERS
Errors of Carbohydrate Metabolism
Errors of Amino Acid Metabolism
Abnormal Porphyrin Metabolism
FERRIC CHLORIDE TESTING
URIC ACID
Normal Values
Clinical Relevance
Interfering Factors
VANILLYLMANDELIC ACID (VMA)
Method/Principle
Clinical Relevance
Interfering Factors
17-KETOSTEROIDS (17-KS)17-KETOGENIC STEROIDS (17-KGS)17-HYDROXYCORTICOSTEROIDS (17-OHCS)
Normal Values
Method/Principle 17 KS
Method/Principle 17-OHCS
Clinical Relevance
Interfering Factors
CHLORIDES
Normal Values
Method
Clinical Relevance
Interfering Factors
SODIUM
Normal Values
Method
Clinical Relevance
Interfering Factors
POTASSIUM
Normal Values
Method
Clinical Relevance
Interfering Factors
FOLLICLE STIMULATING HORMONE (FSH) LUTEINISING HORMONE (LH)
Normal Values
Method
Clinical Relevance
PREGNANEDIOL
Normal Values
Method
Clinical Relevance
PREGNANETRIOL
Normal Values
Method
Clinical Relevance
ESTROGEN FRACTIONS
Normal Values
Method
Clinical Relevance
Interfering Factors
HEAVY METALS AND TRACE ELEMENTS BLOOD/ URINE
Description
Toxic/Poisoning Symptoms and Treatment
MICROSCOPY OF THE URINARY SEDIMENT
DETAILED STUDY OF IMPORTANT URINARY MICROSCOPY CONSTITUENTS
Red Cells and Red Cell Casts
White Cells and White Cell Casts
Epithelial Cells and Epithelial Cell Casts
Hyaline Casts
Granular Casts
Waxy Cysts
Oval Fat Bodies and Fatty Casts (Fig. 5.30)
CRYSTALS
Crystals Seen in Normal Acid Urine (Fig. 5.31)
Crystals Seen in Normal Alkaline Urine (Fig. 5.32)
Crystals Seen in Abnormal Urine
BACTERIA, FUNGUS AND PARASITES
COMMON PATTERNS OF ABNORMAL URINE COMPOSITION IN DISEASE
PARASITES AND PARASITIC OVA
CASTS IN URINE—COMMON CAUSES
Hyaline
Red Cell
White Cell
Epithelial Cell
Granular
Waxy Casts
6:
Renal Function and Its Evaluation
RENAL PHYSIOLOGY IN BRIEF
FUNCTIONS OF THE KIDNEY
URINALYSIS
IMPAIRED RENAL FUNCTION AND BLOOD CHEMISTRY
CONCENTRATION: DILUTION TESTS
PRINCIPLE
Concentration Test
Dilution Test (Water Test)
Mosenthal Modification of Concentration—Dilution Tests
Vasopressin Concentration Test
Conditions that Impair Concentrating Ability
PHENOL RED TEST
PRINCIPLE
Intravenous Method
Interpretation
Ureteral Catheterization Method (Cystoscopy)
PHENOL RED TEST FOR RESIDUAL URINE
Interpretation
CLEARANCE TESTS
CREATININE CLEARANCE
Methods
Interpretation
Creatinine Clearance Test
PRINCIPLES OF PRECISE TESTS OF RENAL FUNCTION
GLOMERULAR FILTRATION RATE (GFR)
Renal Function Tests at a Glance
RENAL PLASMA FLOW (RPF)
FILTRATION FRACTION
MAXIMAL TUBULAR CAPACITY (TM)
7:
Stool Examination
MICROSCOPIC EXAMINATION OF STOOL SPECIMENS
STOOL CONCENTRATION METHODS
Flotation Concentration Methods
Sedimentation Concentration Methods
8:
Medical Parasitology
MEDICAL PARASITES
IMPORTANCE OF MORPHOLOGIC IDENTIFICATION
INTESTINAL PROTOZOA OF MAN
MALARIAL PARASITES OF MAN
BLOOD FLAGELLATES OF MAN
LEISHMANIASIS
VISCERAL LEISHMANIASIS (KALA AZAR) CAUSED BY LEISHMANIA DONAVANI
CUTANEOUS LEISHMANIASIS (ORIENTAL SORE, CHICLERO'S DISEASE, UTA, ETC.) CAUSED BY LEISHMANIA TROPICA
TRYPANOSOMIASES: AFRICAN TYPE: SLEEPING SICKNESS
AFRICAN TRYPANOSOMIASES (SLEEPING SICKNESS) (CONTD…)
TRYPANOSOMIASIS SOUTH AMERICAN TYPE: CHAGAS' DISEASE
SOUTH AMERICAN TRYPANOSOMIASIS (CHAGAS' DISEASE) (CONTD…)
COMMON INTESTINAL ROUNDWORMS OF MAN–PATHOLOGY
EXTRAINTESTINAL ROUNDWORM INFECTION OF MANLARVAL WORM PATHOLOGY
PLASMID NEMATODES (CONTD…)
Hookworms
Enterobius Vermicularis (Thread, Pin or Seat Worm)
PLASMID NEMATODES (CONTD…)
Ascaris Lumbricoides (The roundworm)
The Hookworms
TISSUE ROUNDWORMS OF MAN, CHIEFLY FILARIAE
PLASMID NEMATODES (CONTD…)
The Filarial Worms
Wuchereria bancrofti
TAPEWORMS OF MAN
FLUKES OF MAN
RECAPITULATION—PARASITOLOGY AT A GLANCE
LABORATORY EXAMINATION FOR PARASITES
PRESERVATION AND SHIPMENT OF SPECIMENS TO BE EXAMINED FOR TROPHIC AND ENCYSTED PROTOZOA
PVA (Polyvinyl Alcohol) Method of Brooke and Goldman
MIF (Merthiolate—Iodine-Formalin) Method of Sapero and Lawless
ROUTINE STOOL EXAMINATION AND CONCENTRATION METHODS HAVE BEEN DEALT WITH ELSEWHERE (PREVIOUS CHAPTER)
Negative Stain Direct Fecal Smear Examination
Kato Cellophane Thick Smear Technique (Kato and Miura, 1954)
Heidenhain's Iron Hematoxylin Staining Method for Intestinal Protozoa
Gomori's Trichrome Stain
Procedure
Cultivation of Intestinal Protozoa
URINE
SPUTUM
GASTRIC WASHINGS
DUODENAL ASPIRATES
SPINAL FLUID
Graham Cellulose Tape Technique for Diagnosis of Enterobiasis
BLOOD
Combined Thin and Thick Films
Concentration of Microfilariae
Bone Marrow Smear
Serological Tests
Intradermal Tests
Casoni's Test
Muscle Biopsy for Trichinella spiralis
9:
Clinical Hematology
WAYS OF OBTAINING BLOOD
CAPILLARY OR PERIPHERAL BLOOD
VENOUS BLOOD (VENIPUNCTURE)
Complications
Clinical Alert
ANTICOAGULANTS
EDTA
Advantages of EDTA
Disadvantages of EDTA
Making EDTA Bulbs
OXALATES
Making Double Oxalate Bulbs
TRISODIUM CITRATE
HEPARIN
SPECIAL ANTICOAGULANTS
WHEREVER POSSIBLE, THE NECESSARY TESTS, INVESTIGATIONS AND PREPARATION OF BLOOD FILMS SHOULD BE DONE IMMEDIATELY
ANTICOAGULATED BLOOD STORAGE AND BLOOD CELL MORPHOLOGY
Peripheral Smears
EDTA Blood
DIAGNOSTIC ALERTS
BLOOD COLLECTION SYSTEM
HEMOGLOBIN (HB)
HEMOGLOBIN ESTIMATION: SAHLI'S METHOD: (SAHLI'S HEMOGLOBINOMETER) (FIG. 9.3)
CYANMETHEMOGLOBIN METHOD
Drabkin's Reagent
SHEARD-SANFORD OXYHEMOGLOBIN METHOD
OTHER METHODS
Alkali Hematin Method
Gasometric Method
Specific Gravity Method
Chemical Methods
SODIUM LAURYL SULPHATE METHOD (Available from Coral Clinical Systems, Goa)
Normal Hemoglobin Values
ANEMIA
CAUSES OF ANEMIA
1. Blood Loss
2. Impaired Red Cell Formation
3. Increased Destruction of Red Cell (Hemolytic) Anemia
POLYCYTHEMIA
CAUSES
Primary
Secondary
HEMATOCRIT/PACKED CELL VOLUME (PCV)
DEFINITION
METHODS
Wintrobe's Tube
Microhematocrit
INTERPRETATION
BLOOD CELL COUNTS
WBC
Counting Chamber
Methods
Calculation
RBCs
Diluting Fluid
Method
Interpretation
PLATELETS
Method
Rees-Ecker Method for Platelet Count
Rough Estimation of Platelet Count from Stained thin Smear
ERYTHROCYTE INDICES
1. THE MEAN CELL VOLUME (MCV)
2. THE MEAN CELL HEMOGLOBIN (MCH)
3. THE MEAN CELL HEMOGLOBIN CONCENTRATION (MCHC)
COMPLETE BLOOD COUNT (CBC), BLOOD
NORMAL VALUES
COMPLETE BLOOD COUNT (CBC)
ERYTHROCYTE SEDIMENTATION RATE (ESR)
METHODS
WESTERGREN'S METHOD
Normal Values
WINTROBE'S METHOD
Normal Values
MICROSEDIMENTATION (LANDAU) METHOD
Procedure
Normal Values
ZETA SEDIMENTATION RATE (ZSR)
SOURCES OF ERROR FOR ANY ESR METHOD
INTERPRETATION OF ESR
Rapid ESR is Found in
Slow ESR is usually seen in
FACTORS THAT PLAY A ROLE IN ESR
1. Plasma Factors
2. Red Cell Factors
3. Anticoagulants
Stages in ESR
INTERFERING FACTORS
BLOOD FILM EXAMINATION
PREPARATION OF A THIN BLOOD FILM
MAKING OF SPREADERS (Fig. 9.9)
MAKING THICK SMEARS
FIXING OF BLOOD FILMS
STAINING OF BLOOD FILMS
Buffer Solution used in the Laboratory
Stain Preparation and Staining
Staining of Thick Films
Mounting and Preservation of Films
RAPID DIAGNOSTICS
AUTOMATION IN HEMATOLOGY
COULTER PRINCIPLE
THE BASICS OF HEMATOLOGY ANALYZERS IN A NUTSHELL
COULTER¨ MAXM AND MAXM AL HEMATOLOGY FLOW CYTOMETRY SYSTEMS (FIG. 9.11)
DEVELOPMENT OF BLOOD CELLS SITES OF BLOOD FORMATION
DEVELOPMENT OF BLOOD CELLS (CHART 9.1)
Erythropoiesis (Fig. 9.12)
Leucopoiesis (Fig. 9.13)
Thrombopoiesis (Fig. 9.13)
EXAMINATION OF A BLOOD FILM
Method
Always Note
BONE MARROW EXAMINATION
Bone Marrow Examination and Reporting
MORPHOLOGICAL TYPES OF RED BLOOD CELLS (Fig. 9.16)
RBC Morphology, Blood
RETICULOCYTE COUNT
Normal Values
Staining
Stain
Method
RED BLOOD CELL ABNORMALITIES SEEN ON STAINED SMEAR (IN BRIEF)
RED CELL FRAGILITY TEST
Quantitative Test
Clinical Implications
QUALITATIVE ASSESSMENT OF G6PD DEFICIENCY
Methemoglobin Reduction Test
COMMERCIALLY AVAILABLE KIT FOR G6PD ASSESSMENT (QUALITATIVE)
QUANTITATIVE ESTIMATION OF G6PD
EXAMINATION OF FETAL HEMOGLOBIN
Qualitative Method
Quantitative Method
TESTS FOR SICKLING
Methods
COMMERCIALLY AVAILABLE KIT
LABORATORY DIAGNOSIS OF DISORDERS RELATED TO RBCs
LABORATORY DIAGNOSIS OF IRON DEFICIENCY ANEMIA
Peripheral Blood
Peripheral Smear
Bone Marrow
Serum Biochemistry
Laboratory Findings in Iron Deficiency
Causes of Iron Deficiency Anemia
LABORATORY DIAGNOSIS OF MEGALOBLASTIC MACROCYTIC ANEMIAS
Peripheral Blood Findings in Vitamin B12 or Folic Acid Deficiency
Bone Marrow
SPECIAL TESTS FOR DIAGNOSING VITAMIN B12 DEFICIENCY
Causes of Vitamin B12 Deficiency
Special Tests for Diagnosing Folate Deficiency
Causes of Folic Acid Deficiency
Laboratory Findings in Megaloblastic Anemias
Causes of Bone Marrow Megaloblastosis
LABORATORY DIAGNOSIS OF HEMOLYTIC ANEMIAS
CAUSES AND CLASSIFICATION OF HEMOLYTIC ANEMIAS
Intracorpuscular Defects
Extracorpuscular Defects
Evidences of Hemolysis
LABORATORY DIAGNOSIS OF HEREDITARY SPHEROCYTOSIS
Blood Picture
LABORATORY DIAGNOSIS OF HEREDITARY ELLIPTOCYTOSIS
LABORATORY DIAGNOSIS OF ENZYME DEFICIENCY RELATED ANEMIAS
G6PD Deficiency
LABORATORY DIAGNOSIS OF AUTOIMMUNE HEMOLYTIC ANEMIA (AIHA)
Laboratory Diagnosis of Warm Antibody AIHA
Laboratory Diagnosis of Cold Antibody AIHA
LABORATORY DIAGNOSIS OF PAROXYSMAL NOCTURNAL HEMOGLOBINURIA
Blood Picture
LABORATORY DIAGNOSIS OF LEAD POISONING
Blood Picture
Bone Marrow
Urine
LABORATORY DIAGNOSIS OF HEMOGLOBIN STRUCTURE AND SYNTHESIS DISORDERS
SPECIAL TESTS
Tests Depending on Physiochemical Properties
Hemoglobin Electrophoresis
Autoscanning and Computing Densitometer 205
Power
LABORATORY DIAGNOSIS OF SICKLE CELLS TRAIT
LABORATORY DIAGNOSIS OF SICKLE CELL ANEMIA
Blood Picture
LABORATORY DIAGNOSIS OF UNSTABLE HB HEMOGLOBINOPATHY
Blood Picture
Special Tests
THALASSEMIAS (REDUCED SYNTHESIS RATE)
In β Thalassemia
In α Thalassemia
Laboratory Diagnosis of β Thalassemia Minor
Laboratory Diagnosis of β Thalassemia Major
Laboratory Diagnosis of α Thalassemia
Hb-H Disease
Hb Bart's—Hydrops Fetalis
LABORATORY DIAGNOSIS OF APLASTIC ANEMIA
Blood Picture
Classification and Causes of Aplastic Anemia
PANCYTOPENIA
Blood Picture
Causes
NORMAL WHITE CELL VALUES AND PHYSIOLOGICAL VARIATIONS
PATHOLOGICAL VARIATIONS IN WHITE CELL COUNTS NEUTROPHILIA
Metabolic Disorders—due to Varied Causes Leading to
Neoplasms
Conditions Causing Cell Necrosis or Destruction
Various Drugs/Chemicals Implicated are
Trauma and Hemorrhage
Cardiac Disorders
Collagen Diseases
Miscellaneous
EOSINOPHILIA
Allergic States
Parasitic Diseases
Neoplasms
Miscellaneous
LYMPHOCYTOSIS
Acute Infections
Chronic Infections
Endocrine Disorders
Neoplasms
MONOCYTOSIS
Infections
Neoplasms
Collagen Diseases
Miscellaneous
BASOPHILIA
MORPHOLOGIC FORMS OF LYMPHOCYTES
Virocyte
Transformed Lymphocytes
ARNETH COUNT
Arneth Index
NEUTROPENIA AND AGRANULOCYTOSIS
LYMPHOPENIA
EOSINOPENIA
Drug/Hormone Therapy
Response to Stress
Endocrine Diseases
Miscellaneous
BASOPHILOPENIA
LEUKEMOID REACTIONS
Neutrophilic
Lymphocytic
Eosinophilic
BONE MARROW PLASMACYTOSIS
Acute Infections
Chronic Infections
Allergic States
Collagen—Vascular Disorders
Neoplasms
Others
WHITE BLOOD CELLS
NEUTROPENIA AND AGRANULOCYTOSIS
Blood Picture of Drug-induced Neutropenia
Bone Marrow
Causes of Neutropenia
OTHER CAUSES OF NEUTROPENIA
LABORATORY DIAGNOSIS OF INFECTIOUS MONONUCLEOSIS
Blood Picture
Paul Bunnel Test for Heterophile Antibody
LUPUS ERYTHEMATOSUS (LE) CELL/PHENOMENON
Method
CLASSIFICATION OF ACUTE MYELOMONOCYTIC LEUKEMIAS
FAB Classification
FAB Classification of Lymphoblastic Leukemias
LABORATORY DIAGNOSIS OF LEUKEMIAS
Cytochemical Methods for Staining Leucocytes
Neutrophil Alkaline Phosphatase (Kaplow's Method)
Peroxidase (Myeloperoxidase, Kaplow's Method)
Periodic Acid-Schiff (PAS) Reaction
Sudan Black B Stain (Sheehan and Storey)
Nonspecific Esterase (Yam et al)
ACUTE LEUKEMIAS, LABORATORY DIAGNOSIS
Routine Hematologic Investigations
Differentiation of ALL from AML
CHRONIC MYELOID LEUKEMIA
Laboratory Investigations
CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)
Laboratory Findings
PARAPROTEINEMIAS
Causes
MULTIPLE MYELOMA
Laboratory Diagnosis
Other Laboratory Findings
POLYCYTHEMIA VERA
Laboratory Diagnosis
MYELOSCLEROSIS
Laboratory Diagnosis
HODGKIN'S DISEASE
Laboratory Findings in Hodgkin's Disease
QUALITY CONTROL IN HEMATOLOGY
INTERNAL QUALITY CONTROL
Testing Control Sample
Control Chart (Levy-Jennings or L-J Chart)
Cusum Analysis
Duplicate Tests
Inbuilt Quality Control
B. EXTERNAL QUALITY ASSESSMENT
C. STANDARDIZATION
D. PROFICIENCY SURVEILLANCE
10:
Clinical Hematology Bleeding Disorders
PLATELETS, COAGULATION AND BLEEDING DISORDERS: LABORATORY INVESTIGATIONS
CAPILLARY FRAGILITY TEST OF HESS (Rumpel-Leede Sign, Tourniquet Test)
Clinical Implications
LABORATORY DIAGNOSIS OF VASCULAR BLEEDING DISORDERS
Interfering Factors
LABORATORY DIAGNOSIS OF PLATELET DISORDERS
IDIOPATHIC THROMBOCYTOPENIC PURPURA (ITP)
DRUG INDUCED IMMUNE THROMBOCYTOPENIA
DISSEMINATED INTRAVASCULAR COAGULATION (DIC)
Causes of DIC
FUNCTIONAL PLATELET DISORDERS
Laboratory Diagnosis
Hereditary Disorders
Acquired Disorders
BLEEDING TIME
Duke's Method
Requirements
Method
Precautions
Ivys's Method
Method
Interpretation
Clinical Implications
Interfering Factors
Patient Preparation
COAGULATION TIME
Capillary Tube Method of Wright
Lee and White's Method
Requirements
Method
Normal Values
Precautions and Errors
Clinical Implications
Interfering Factors
CLOT RETRACTION
Principle
Requirements
Method
Interpretation
Errors
HEPARIN THERAPY
Protocols and Blood Coagulation Tests
QUALITY ASSURANCE FOR ROUTINE HEMOSTASIS LABORATORY
BUFFERED 3.2% CITRATE SOLUTION
(PROTHROMBIN TIME)
THROMBOPLASTIN REAGENT FOR PROTHROMBIN TIME (PT) DETERMINATION
Summary
Reagent
Reagent Storage and Stability
Principle
Note
Sample Collection and Preparation of PPP
Additional Material Required for Manual and Calibration Curve Methods
Test Procedure
Manual Method
Calculation of Results
Manual Method
Expected Values
Remarks
SENSITIVE THROMBOPLASTIN REAGENT FOR PROTHROMBIN TIME (PT) DETERMINATION (ISI=1.0)
THROMBOPLASTIN REAGENT FOR PROTHROMBIN TIME (PT) DETERMINATION, LYOPLASTIN®
MNPT
ISI Value of PT Used and Method of Clot Detection
Advantages of the INR system
Disadvantages of the INR System
Patient Variables in PT/INR Testing
Factors that Influence Coagulation Test Results
PROTHROMBIN DETERMINATION (TWO STAGE METHOD)
Principle
Results
APTT/PTTK CEPHALOPLASTIN REAGENT FOR PARTIAL THROMBOPLASTIN TIME (APTT) DETERMINATION USING ELLAGIC ACID AS ACTIVATOR
CIRCULATING ANTICOAGULANTS
NORMAL AND ABNORMAL CONTROL PLASMAS FOR COAGULATION ASSAYS
FIBROSCREEN THROMBIN TIME TEST FOR QUALITATIVE ESTIMATION OF FIBRINOGEN
FIBRINOGEN ESTIMATION-QUANTITATIVE
REQUIREMENTS
Method
Result
DILUTE BLOOD CLOT LYSIS TIME
FDPs
SCREENING TESTS FOR DIAGNOSIS OF PROCOAGULANT DEFICIENCY
Requirements
Method
Results
LABORATORY DIAGNOSIS OF COAGULATION DISORDERS
LABORATORY DIAGNOSIS OF HEMOPHILIA
CHRISTMAS DISEASE (HEMOPHILIA B)
VON WILLEBRAND'S DISEASE
HEMORRHAGIC DISEASE OF NEWBORN
AUTOMATION IN COAGULATION ANALYSIS
HEMOSTAR
Technical Features
HEMOSTAR XF
Technical Features
COALAB 6000
Compact
Versatile
User Friendly
Technical Specifications
TROUBLESHOOTING
GENERAL INSTRUCTIONS FOR COAGULATION TEST
Sample Collection Techniques
Sample Preparation
Sample Processing and Storage
Calibration of Instruments/Equipments
Storage and Procedure
Critical Requirement of MNPT in the Derivation of INR
ISI value of PT used and Method of Clot Detection
INR SYSTEM
PROTHROMBIN TIME
Uniplastin®/Liquiplastin®/Lyoplastin® Problem: Prolonged Clotting Time
Problem: Shortened Clotting Time
DPTT/PTTK
Liquicelin-E® Problem: Prolonged Clotting Time
Problem: Shortened Clotting Time
FIBRINOGEN ESTIMATION
Fibroscreen®/Fibroquant® Problem: Prolonged Clotting Time
Problem: Shortened Clotting Time
FIBRINOGEN DEGRADATION PRODUCTS (D-DIMER) ESTIMATION XL-FDP®
Problem: False Positive Results
Problem: Delayed Agglutination
Problem: False Negative Results
Problem: Positive Control Giving Negative Reaction
11:
Blood Banking (Immunohematology)
BLOOD GROUP ANTIBODIES
NATURALLY OCCURRING ANTIBODIES
GENETICS OF ABO SYSTEM
BIOCHEMISTRY
SUBGROUPS OF A AND AB
Subgroups of B
Antibodies of ABO System
Anti-H
ABO TESTING PROCEDURES
ABO Antibody Reagents
Red Cells Reagents
Group O Reagent Screen Cells
Preparation of Red Cell Suspensions
Method (for 2% suspension)
Blood grouping sera should meet the following requirements
ANTI-A, ANTI-B, ANTI-A,B
ANTI-A, ANTI-B, ANTI-A, B
ANTI-A1 LECTIN
ANTI-H LECTIN
PHYSIOLOGICAL SALINE SOLUTION FOR SEROLOGICAL APPLICATIONS
BOVINE SERUM ALBUMIN 22% SOLUTION FOR SEROLOGICAL APPLICATIONS
CONCENTRATED ISO-OSMOTIC PHOSPHATE BUFFERED SALINE FOR SEROLOGICAL APPLICATIONS
MEDIA FOR COLLECTION AND PRESERVATION OF REAGENT RED CELLS
Precautions
RED CELL PRESERVING SOLUTION FOR SEROLOGICAL APPLICATIONS
ABO GROUPING
SLIDE ABO GROUPING TEST
SIX-TUBE METHOD (FIG. 11.3)
Interpretation
Briefly the reasons can be described as:
This leads to false negative results
This may cause false positive reaction
Solving Problems of Discrepancies
Repeat Preliminary Procedures
RH BLOOD GROUP SYSTEM
RH(D) GROUPING PROCEDURES
Slide Testing
Method
Tube Method
Interpretation
Testing for Du
Method
Interpretation
ANTI-D (Rho)
ANTI-D (Rho) (IgM)
ANTI-D (Rho) (IgG)
ANTI-D (Rho) (IgM + IgG)
BLOOD GROUP TESTING IN MICROPLATES
DIRECT ANTI-HUMAN GLOBULIN TEST (DAT)
MAJOR APPLICATIONS OF DAT IN BLOOD GROUP SEROLOGY
Hemolytic Disease of the Newborn (HDN)
Transfusion Reactions
Other Immune Hemolytic Diseases
Classification of Autoimmune Hemolytic Anemia
Drug-induced Hemolytic Anemia
Importance of Serological Studies in DAT Positive Results (Table 11.5)
INDIRECT ANTI-HUMAN GLOBULIN TEST (IAT)
Applications of IAT
Probable Sources of Error in Anti-human Globulin Testing
False Negative Results
False Positive Results
COOMB'S CONTROL CELLS/COMPLEMENT COATED CELLS
Indirect Anti-human Globulin Test for the Detection of Red Blood Cell Antibodies
Saline Phase Indirect Anti-human Globulin Test
Albumin Phase Indirect Anti-human Globulin Test
LISS Phase Indirect Anti-human Globulin Test
PEG-enhanced Indirect Anti-human Globulin Test
LIM (Low Ionic Medium Polybrene) Indirect Anti-human Globulin Test
Interpretation of Results for Anti-human Globulin Tests
Controls
Notes
Papain—One-stage Enzyme technique / Two-stage Enzyme Technique
ANTIBODY TITRATION STUDIES
Antibody Titration for Characterizing Type of Antibody in Serum
Specimen
Reagents
Procedure
Interpretation
Notes
Antibody Titration Studies for Early Detection of Hemolytic Disease of the Newborn
Specimen
Materials
Quality Control
Procedure
Results
Notes
Use of Sulfhydryl Reagents to Distinguish between IgM and IgG Antibodies
Specimen
Reagents
Procedure
Notes
ELUTION TECHNIQUES
Citric Acid Elution Method
Specimen
Reagents
Procedure
Notes
Cold Acid Elution
Specimen
Reagents
Procedure
Notes
Glycine-HCl/EDTA Elution
Specimen
Reagents
Procedure
Notes
Heat Elution
Specimen
Reagents
Procedure
Donath-Landsteiner Test
Specimen
Reagents
Procedure
Interpretation
Notes
CHEQUER BOARD TITRATION FOR QUALITY CONTROL OF ANTI-IGG POTENCY IN POLYSPECIFIC AHG REAGENT AND EVALUATION OF COMPLEMENT POTENCY WITH COMPLEMENT-COATED CELLS
Reagents and Materials Required for Chequer Board Titration
Reagent Preparation Procedure
DILUTIONS OF ANTI-D (IGG) REAGENT
Cell Sensitization
Note
Dilutions of Anti-human Globulin Reagent
Preparation of Complement-Coated Cells
Preparation of 50% Cell Suspension of O Group Red Blood Cells
Collection of Inert O Group Serum
Sensitization of O Group Red Blood Cells
Preparation of Coomb's Control Cells
Procedure
ANTI-HUMAN IgG MONOSPECIFIC COOMB'S REAGENT FOR DIRECT AND INDIRECT ANTIGLOBULIN TEST
ANTIHUMAN GLOBULIN REAGENT
Summary
Reagents
Reagent Storage and Stability
Principle
Note
Sample Collection and Storage
For Direct Antiglobulin Test
For Indirect Antiglobulin Test
Preparation of Coomb's Control Cells
Additional Material Required
Procedure
Direct Antiglobulin Test
Indirect Antiglobulin Test
Interpretation of Results
Direct Antiglobulin Phase
Indirect Antiglobulin Phase
Remarks
PREPARING COOMB'S CONTROL CELLS
LOW IONIC SALT SOLUTION FOR SEROLOGICAL APPLICATIONS
STABILIZED, ACTIVATED PAPAIN ENZYME SOLUTION FOR SEROLOGICAL APPLICATIONS
BLOOD TRANSFUSION
BLOOD DONORS
Donor Screening
Drawing of Blood
ADVERSE DONOR REACTIONS
Problems with Blood Flow
Hematoma
Accidental Puncture of the Artery
Mild, Moderate or Severe Reactions
Hyperventilation
Accidents
COMPATIBILITY TESTING
The Cross-match
Saline Cross-match
Method of Cross-matching Universal Donor Blood
Factors Leading to False Results
If Still an Unexpected Incompatibility is Obtained
CHOICE OF MATERIAL FOR TRANSFUSION
BLOOD AND ITS PRODUCTS
Fresh Blood
Packed Cells
Plasma
BLOOD TRANSFUSION COMPLICATIONS
INVESTIGATIONS IN A CASE OF TRANSFUSION REACTION
Proceed as Mentioned below
Interpretation of Results
LABORATORY DIAGNOSIS OF HEMOLYTIC DISEASE OF THE NEWBORN
TROUBLE SHOOTING
GENERAL INSTRUCTIONS FOR BLOOD GROUPING
Sample Preparation
Sample Processing
Sample Storage
Equipments
Reagents
Common Causes of False Negative and False Positive Results in ABO Testing
False Negative Results
False Positive Results
Interpretation of Agglutination Reactions
ABO GROUPING
Problem: False Positive Results
Problem: False Negative Results
Problem: Hemolysis or Red Blood Cells
Problem: Weak Agglutination
Problem: Mixed Field Agglutination in ABO Grouping or Discrepancy Observed Between the Red Cell Group and the Reverse Group
Problem: Delayed Agglutination
RH TYPING
Problem: False Positive Results
Problem: False Negative Results
Problem: Hemolysis of Red Blood Cells
Problem: Delayed or Weak Agglutination
GENERAL INSTRUCTIONS FOR ANTIHUMAN GLOBULIN (COOMBS REAGENT)
Sources of Error in Antiglobulin Testing—Coombs Cells
False Negative Results
False Positive Results
ANTIHUMAN GLOBULIN (AHG OR COOMBS REAGENT)
Problem: False Positive Results
Problem: False Negative Results
ANTI-A1 LECTIN
Problem: False Positive Results
Problem: Hemolysis of Red Blood Cells
Problem: False Negative Results
ANTI-H LECTIN
Problem: False Positive Results
Problem: Hemolysis of Red Blood Cells
Problem: False Negative Results
12:
Cerebrospinal and Other Body Fluids
CEREBROSPINAL FLUID
NORMAL VALUES FOR LUMBAR CSF IN ADULTS
LUMBAR PUNCTURE
Indications
Complications of Lumbar Puncture
CSF RHINORRHEA AND OTORRHEA
CSF PRESSURE
CSF
GROSS EXAMINATION
CELL COUNTS
Diluting the Fluid
Count
Various Types of Cells in CSF
Mixed Reaction (Neutrophils, Lymphocytes and Monocytes) occurs in:
Monocytic and/or Lymphocytic Reaction is Seen in:
GLOBULIN TEST
Conditions that Elevate CSF Protein
Mild Elevation, to 300 mg%
Electrophoretic Evidence of IgG
Moderate or Pronounced Elevation
Protein Electrophoresis of CSF
Lange's Colloidal Gold Test
Colloidal Gold Test
Glucose
Conditions that Effect the CSF Glucose
Enzymes
LDH
SGOT
CPK
BACTERIOLOGIC EXAMINATION
Serologic Tests
SYNOVIAL FLUID (SF)
PLEURAL FLUID
PERICARDIAL FLUID (PF)
PERITONEAL FLUID
AMNIOCENTESIS AND AMNIOTIC FLUID ANALYSIS, DIAGNOSTIC
13:
Semen Analysis
SEMEN ANALYSIS
SEMEN ANALYSIS
Why it is done?
How to Prepare?
How it is done?
How it Feels?
Risks
Results
What Affects the Test?
What to Think about?
Collection
GROSS EXAMINATION
Physical Characteristics
MICROSCOPIC EXAMINATION
Sperm Counts
Safety Precautions
Sperm Counting Methods
When Count are towards lower limit use the method given as under
Motility
Sperm Morphology (Fig. 13.1)
CHEMICAL EXAMINATION OF SEMEN
Reagent
Method
Reporting
OTHER TESTS OF SEMEN
ANTIBODIES TO SPERMATOZOA
14:
Sputum Examination
SPUTUM EXAMINATION
COMMON RESPIRATORY DISORDERS
MYCOBACTERIA
MYCOTIC (FUNGAL) DISEASE
PATHOLOGICAL FUNGI
Actinomyces israelii
Nocardia asteroides
Cryptococcus neoformans
Histoplasma capsulatum
Coccidiodes immitis
Blastomyces dermatidis
Candida albicans
Aspergillus fumigatus
Phycomycetes
BRONCHIAL ASTHMA
BRONCHIECTASIS
CHRONIC BRONCHITIS
LUNG ABSCESS
PNEUMONIA
PNEUMOCONIOSIS
PULMONARY EMBOLISM
HEART DISEASE
VIRAL INFECTIONS
PULMONARY ALVEOLAR PROTEINOSIS
CYTOLOGIC EXAMINATION IN MALIGNANCY
15:
Pregnancy Tests
BIOASSAYS
HISTORICAL ASPECTS
Aschheium and Zondek Test (1928)
Friedman Test (1931)
Bellerby Test (1934)
Rat Ovarian Hyperemia Test of Frank and Berman
Tests Using Male Toad/Male Frog
NORMAL VALUES OF HCG (SERUM/PLASMA)
COMMENTS
IMMUNOLOGIC METHODS
IMMUNOLOGIC TESTS FOR PREGNANCY
SLIDE TEST FOR PREGNANCY
LATEX AGGLUTINATION INHIBITION METHOD
Summary
Reagents
Reagent Storage and Stability
Principle
Note
Sample Collection and Preparation
Qualitative Method
Semi-quantitative Method
Material Provided with the Kit
Reagent Pack
Accessories Pack
Additional Material Required
Test Procedure
Qualitative Method
Semi-quantitative Method
Interpretation of Results
Qualitative Method
Semi-quantitative Method
Remarks
SLIDE TEST FOR PREGNANCY
DIRECT LATAX AGGLUTINATION METHOD
Summary
Reagent
Reagent Storage and Stability
Principle
Note
Specimen Collection and Preparation
Qualitative Method
Semi-quantitative Method
Material Provided with the Kit
Additional Material Required
Test Procedure
Qualitative Method
Semi-quantitative Method
Interpretation of Test Results
Qualitative Method
Semi-quantitative Method
Remarks
ELISA PREGNANCY TEST
DIPSTICK ICT PREGNANCY TEST
DEVICE ICT PREGNANCY TEST
ICT TECHNIQUES FOR URINE/SERUM SAMPLE
ADVANTAGES OF SERUM TESTING
DIPSTICK ICT, URINE/SERUM PREGNANCY TEST
DEVICE ICT URINE/SERUM PREGNANCY TEST
ALTERED LABORATORY RESULTS IN NORMAL PREGNANCY
TROUBLESHOOTING
LATEX METHODS
INDIRECT/LATEX AGGLUTINATION INHIBITION METHOD
Foretel
Problem: False Positive Results
Problem: Delayed Agglutination
Problem: False Negative Results
LATEX AGGLUTINATION/DIRECT METHOD
Foresight®
Problem: False Positive Results
Problem: False Negative Results
RAPID FORMATS
DEVICE/DIPSTICK
Clue
Problem: False Positive Results
Problem: Delayed Positive Results
Problem: False Negative Results
Problem: Invalid Results
Gravicheck®
Problem: False Positive Results
Problem: Delayed Positive Results
Problem: False Negative Results
Problem: Invalid Results
16:
Examination of Gastrointestinal Contents
NORMAL SALIVA—CONSTITUENTS
GASTRIC JUICE
ROUTINE GASTRIC JUICE EXAMINATION
Gross Examination
Chemical Examination
1. Blood
2. Qualitative Test for Free HCI (Topfer's Test)
3. Titration for Acid
NORMAL GASTRIC CONSTITUENTS IN ADULTS
Lactic Acid (Kelling's Test)
Microscopic Examination
GASTRIC TEST MEALS
Procedures
Tubeless Gastric Analysis
Basal Gastric Secretion
Method
Augmented Histamine Test (AHT)
Histamine Infusion Test
Histalog Test
Insulin Hypoglycemia Test
Gastrin Secretory Test
Miscellaneous Investigations
EXAMINATION OF DUODENAL CONTENTS
DUODENAL DRAINAGE
Indications
Method for Diagnostic Drainage
Examination for Diagnosis
Interpretation
COMPOSITION OF BILE
PANCREATIC FUNCTION TESTS
COMPOSITION OF PANCREATIC JUICE
Gross and Chemical Characteristics of Pancreatic Juice
Digestive Enzymes
Proteolytic Enzymes
Peptidases
Nucleases
Amylolytic Enzymes
Lipolytic Enzymes
Acute Pancreatitis
Chronic Pancreatitis (Cirrhosis of Pancreas)
Carcinoma of Pancreas
Lipase
Secretin Test
Other Laboratory Tests in Acute Pancreatitis
Miscellaneous Tests for Chronic Pancreatitis
SWEAT ELECTROLYTES PILOCARPINE IONTOPHORESIS
DIAGNOSTIC APPLICATION OF SWEAT TESTING
17:
Diabetes Mellitus Laboratory Diagnosis
DIABETES MELLITUS
CLASSIFICATION AND CAUSES OF DIABETES
SCREENING TESTS
Urine Glucose (Methods mentioned elsewhere)
Fasting Blood Sugar
Two-hours Postprandial Blood Glucose
DIAGNOSIS AND CLASSIFICATION OF DIABETES MELLITUS NEW CRITERIA
Previous Classification
Changes in the Classification System
New Diagnostic Criteria of Diabetes Mellitus
Glycated Hemoglobin
Impact of the New Diagnostic Criteria
Screening Recommendations
Final Comment
CONVENTIONAL DIAGNOSTIC TESTS
Oral Glucose Tolerance Test (OGTT)
Patient Preparation
Interpretation
Interfering Factors
I/V GTT
Rapid I/V GTT
Cortisone Glucose Tolerance Test
Parenteral Administration of Glucagon or Epinephrine
I/V Tolbutamide Test
I/V Insulin Tolerance Test
GLYCOSYLATED HEMOGLOBIN (HBA1C);GLYCO-HEMOGLOBIN (G-HB); DIABETIC CONTROL INDEX
Kits Available Commercially
Increased
Decreased
Description
Factors that Affect Results
Other Data
Test Significance
Clinical Relevance
Interfering Factors
GLYCOSYLATED HEMOGLOBIN KIT
INSULIN
Normal values
Test Significance
Clinical Relevance
Interfering Factors
C-PEPTIDE
Normal Values
Test Significance
Clinical Relevance
GLUCAGON
Normal Values
TEST SIGNIFICANCE
Clinical Relevance
Interfering Factors
OTHER IMPORTANT TESTS IN DIABETICS
HYPOGLYCEMIA
Causes of Hypoglycemia
Spontaneous (fasting) Hypoglycemia
Induced Hypoglycemia
RAPID DIAGNOSTICS
ACCU-CHEK®
Accu-Chek Active System: Virtually Painfree Testing in 5 Seconds
Accu-Chek Softclix (Fig. 17.1)
Accu-Chek Active Meter
Active Glucose Test Strips
Running a Quality Control Test
Cleaning the Meter
Storing the Meter
Light Conditions
Atmospheric Humidity
Sources of Interference
DCA 2000 PLUS ANALYZER (FIG. 17.11)
Accuracy, Precision, and Reproducibility with the Convenience of In-office Results
Easy Procedure
DCA 2000 Plus Analyzer
Intensive Management Improves Glycemic Control
Reduce the Risk Monitor HbA1c Levels
Laboratory-Accurate Results Just Minutes After Testing
HbA1c Results… in Minutes
Microalbumin/Creatinine Ratio…in minutes
Detect Early Stages of Diabetic Nephropathy. Protect your Patient from Complications
Specifications
Size
Weight
Power
Ambient Operating Temperature Range
Ambient Operating Humidity Range
18:
Liver Function Tests
TESTS OF EXCRETION BY THE LIVER
BILE PIGMENT
Types of Bilirubin
Classification of the Causes of Jaundice
Unconjugated Bilirubin
NORMAL
Hemolytic jaundice
HEPATITIS
OBSTRUCTION
Conjugated Bilirubin
Hyperbilirubinemia
Urine Urobilinogen
Urine Urobilinogen Absent
Fecal Urobilinogen
Bromsulphalein (Sulphobromophthalein) Excretion Test
Method
Conditions Associated with Increased BSP Retention
Artefacts
EVALUATION OF SYNTHESIS IN LIVER
SERUM PROTEINS (ALBUMIN ESPECIALLY)
PROTHROMBIN CONCENTRATION
Low Prothrombin in Presence of Jaundice
Low Prothrombin in the Absence of Jaundice
CHOLESTEROL AND ITS ESTERS
Decrease of Both Substances
Increase of Total but Decrease of Esters
DETOXIFICATION
Hippuric Acid Test
EVALUATION OF ENZYME ACTIVITY
SERUM TRANSAMINASES
SERUM ALKALINE PHOSPHATASE
Serum Protein Changes in Selected Diseases
SUGGESTED LIVER FUNCTION TESTS
LIVER BATTERY (PROFILE), SERUM
Normal Values are Dependent Upon Methods/Kits/Manufacturers
Usage
Increased
Decreased
Description
19:
Clinical Chemistry
COLORIMETRY
PHOTOMETER
CLINICAL CHEMISTRY
SPECIMEN COLLECTION AND PROCESSING
PROPER SPECIMEN COLLECTION
Specimen Collection
Chemistry (plain tube)
Chemistry (Heparin)
Chemistry
Hematology (EDTA)
Hematology (EDTA)
Hematology (plain tube)
Hematology (Sodium citrate)
Blood bank (plain tube)
Serology (plain tube)
PROCESSING
CENTRIFUGE
DIFFICULTIES
BLOOD COLLECTION, PRECAUTIONS AND ERRORS
Clinical Chemistry and Drug Interference (See Appendix II)
Pharmacologic Interference
Chemical Interference
CONTROL SERA
NORMAL VALUES DIFFER WITH DIFFERENT KITS AND MANUFACTURES. ALWAYS CONSULT THE PRODUCT INSERT FOR EXACT METHOD FOR A PARTICULAR KIT, FOLLOW THE MANUFACTURER'S INSTRUCTIONS STRICTLY
Control Sera from Boehringer
BLOOD UREA NITROGEN (BUN)
UREA (DAM METHOD)
Summary
Principle
Normal Reference Values
Storage/Stability
Reagent Preparation
Sample Material
Procedure
Calculations
Linearity
Note
UREA (MOD. BERTHELOT METHOD)
Summary
Principle
Normal Reference Values
Storage/Stability
Reagent Preparation
Sample Material
Procedure
Calculations
Linearity
Note
UREA (GLDH KINETIC METHOD)
Summary
Principle
Normal Reference Values
Storage/stability
Reagent Preparation
Sample Material
Procedure
Substrate Start Assay
SAMPLE START ASSAY
Calculations
Linearity
Note
NORMAL VALUES (GENERAL REFERENCE)
Clinical Relevance
Prerenal
Renal
Post-renal
Interfering Factors
Comments
PLASMA OR SERUM CREATININE
CREATININE (ALKALINE PICRATE METHOD)
Summary
Principle
Reference Values
Storage/stability
Reagent Preparation
Sample Material
Procedure
Deproteinization of specimen
Color Development
Calculations
Linearity
Note
CREATININE (MOD JAFFA'S KINETIC METHOD)
Summary
Principle
Reference Values
Storage/stability
Reagent Preparation
Sample Material
Serum or Urine
Procedure
Calculations
Linearity
Note
Clinical Relevance
Decreased Creatinine levels occur in
Interfering Factors
SERUM BILIRUBIN
BILIRUBIN
Summary
Principle
Normal Reference Values
Storage/Stability
Reagent Preparation
Sample Material
Procedure
Direct Bilirubin Assay
Total Bilirubin Assay
Calculations
Linearity
Note
Causes of Hyperbilirubinemia
Conjugated (Direct) Hyperbilirubinemia
Interfering Factors
Comments
ICTERUS INDEX
Reagents
Method
Calibration Curve
TOTAL PROTEINS
BIURET METHOD
Summary
Principle
Normal Reference Values
Storage/Stability
Reagent Preparation
Sample Material
Procedure
Calculations
Linearity
Note
SERUM ALBUMIN
DETERMINATION OF SERUM ALBUMIN (BCG METHOD)
Summary
Principle
Normal Reference Values (Albumin)
Storage/stability
Reagent Preparation
Sample Material
Procedure
Calculations
Linearity
Note
Normal Values
Specimen Collection and storage
Clinical Relevance
Causes of Hypoalbuminemia
Disorders Associated with Polyclonal Gammopathies
Causes of Monoclonal Gammopathies
Interfering Factors
SERUM CHOLESTEROL
CHOLESTEROL (CHOD/PAP Method)
Summary
Principle
Normal Reference Values
Storage/stability
Reagent Preparation
Sample Material
Procedure
Calculations
Linearity
Note
Normal Values
Clinical Relevance
Interfering Factors
Patient Preparation
HDL CHOLESTEROL
PEG/CHOD-PAP METHOD
Summary
Principle
Normal Reference Values
Storage/stability
Reagent Preparation
Sample Material
Procedure
Precipitation of VLDL and LDL
Cholesterol Assay
Calculations
Calculation of LDL Cholesterol (mg/dl)
Linearity
Note
RISK FACTOR
HDL CHOLESTEROL PPT SET
Summary
Principle
Normal Reference Values
Storage/stability
Reagent Preparation
Sample Material
Procedure
Procedure for the cholesterol assay
Calculations
Linearity
Note
Clinical Relevance
Interfering Factors
Patient Preparation
PATIENT AFTERCARE
CHOLESTEROL: LDL AND VLDL
Normal Values
LDL-CHOLESTEROL FULLY ENZYMATIC, COLORIMETRIC TEST
Principle
Sample
Reagents
Preparation of Reagents
Procedure
Calculation
Clinical Interpretation
Note
Test Significance
TRIGLYCERIDES
Summary
Principle
Normal Reference Values
Storage/stability
Reagent Preparation
Sample Material
Procedure
Calculations
Linearity
Note
Normal Values
CLASSIFICATION OF TRIGLYCERIDE LEVELS
CLINICAL IMPLICATIONS
BLOOD GLUCOSE
GLUCOSE (GOD/POD Method)
Summary
Principle
Normal Reference Values
Storage/stability
Reagent Preparation
Sample material
Procedure
Calculations
Linearity
Note
BODY FLUID, GLUCOSE
Persistent Hyperglycemia
Transient Hyperglycemia
Persistent Hypoglycemia
Transient Hypoglycemia
URIC ACID
URICASE/PAP METHOD
Summary
Principle
Normal Reference Values
Storage/stability
Reagent Preparation
Sample Material
Procedure
Calculations
Linearity
NORMAL VALUES
Factors Affecting Serum Uric Acid levels
Increased Production, Raised Serum Levels
Decreased Excretion, Raised Serum Levels
Increased Excretion, Lowered Serum Levels
Decreased Production, Lowered Serum Levels
CALCIUM
CALCIUM
Summary
Principle
Normal Reference Values
Storage/stability
Reagent Preparation
Sample material
Procedure
Linearity
Note
NORMAL VALUES
Specimen Collection and Storage
Clinical Relevance
Hypercalcemia (increased total calcium)
Hypocalcemia (decreased total calcium levels)
Increased Ionized Calcium
Decreased Ionized Calcium
Be Careful
Interfering Factors
PHOSPHORUS
MOLYBDATE UV METHOD
Summary
Principle
Normal Reference Values
Storage/stability
Reagent Preparation
Sample Material
PROCEDURE
Calculations
Linearity
Note
PHOSPHORUS
Mod Gomorri's Method
Summary
Principle
NORMAL REFERENCE VALUES
Storage/stability
Reagent Preparation
Sample Material
Procedure
Calculations
Linearity
Notes
NORMAL VALUES
Hyperphosphatemia (increased phosphorus levels)
Hypophosphatemia (Decreased phosphorus levels)
Interfering Factors
CHLORIDE
THIOCYANATE METHOD
Principle
Normal Reference Values
Chloride Kit
Storage/stability
Reagent Preparation
Procedure
Chloride Assay
Linearity
Notes
Calculation
NORMAL VALUES
Interfering Factors
Be Careful
SERUM IRON AND TIBC
FERROZINE METHOD
Summary
Principle
Normal Reference Values
Storage/stability
Reagent Preparation
Sample Material
Procedure
Iron assay
TIBC assay
Calculations
Linearity
Notes
Interfering Factors
TRACE ELEMENTS
ZINC
ZINC
NORMAL VALUES
Toxic Level Symptoms
Deficiency Symptoms
Values are Increased in
Values are Decreased in
COPPER
COLORIMETRIC METHOD
Summary
Principle
Normal Reference Values
Storage/stability
Reagent Preparation
Working Reagent
Sample Material
Procedure
Calculations
Linearity
Notes
Toxic Level Symptoms
Deficiency Symptoms
Values are Increased in
COPPER URINE
Normal Values
Values are increased in
Values are decreased in
MAGNESIUM
CALMAGITE METHOD
Summary
Principle
Normal Reference Values
Storage/stability
Reagent Preparation
Sample Material
Procedure
Calculations
Linearity
Notes
NORMAL VALUES
Toxic Level Symptoms
Deficiency Symptoms
Values are Increased in
Values are Decreased in
MAGNESIUM IN URINE
Values are increased in
Values are Decreased
AUTOMATION IN CLINICAL CHEMISTRY
INSTRUMENTATION
PRESENT STATUS
BENEFITS OF A SEMIAUTOMATIC ANALYZER
Economy
Wide Spectrum of Tests
Speed
Accuracy and Reliability
Convenience
Non-dependence on Technicians
Business Growth
WHICH LAB NEEDS AUTOMATION?
SELECTION OF A MODEL
ANALYZER CLASSIFICATION
Semiauto Analyzers
Batch Analyzers
Random Access Auto Analyzer
CLIN CHECK PLUS
EP/KIN/FXT/MSD and ABS mode
Features
Technical Features CCP
SCREEN MASTER 3000
Features
Technical Features of Screenmaster 3000
MAP LAB PLUS
Features
Technical Features of Maplab Plus
FULLY
System Overview
Modern
Compact
Software
Technical Specifications of Fully Measuring System
PRINCIPLES OF QUALITY ASSURANCE AND STANDARDS FOR CLINICAL CHEMISTRY
I. PREANALYTICAL FACTORS IMPORTANT IN CLINICAL CHEMISTRY
A. Specimen Collection, Handling, and Transport to the Laboratory
B. Specimen Identification
C. Test Identification
D. Specimen Accessioning
E. Client Communication and Education
F. Personnel Safety
G. Laboratory Environment
H. Personnel Requirements
II. ANALYTICAL FACTORS IMPORTANT IN CLINICAL CHEMISTRY
A. Monitoring
B. Method Validation
C. Instrumentation
D. Quality Control
E. Procedures Manual
F. Comparison of Test Results
POSTANALYTICAL FACTORS IMPORTANT IN CLINICAL CHEMISTRY
A. Computer Entry of Data
B. Report Generation
C. Report Delivery
D. Specimen Storage
E. Specimen Disposal
F. Personnel Safety
G. Laboratory Environment
H. Personnel Requirements
20:
Enzymology
α AMYLASE
SERUM AND URINE—αAMYLASE
Summary
Principle
Normal Reference Values
Storage/stability
Reagent Preparation
Sample Material
Procedure
For Urine as Sample
Calculations
Linearity
Note
NORMAL VALUES
Clinical Relevance
Increased levels are found in
Pronounced elevation (5 or more times normal)
Moderate elevation (3 to 5 times normal)
Decreased Levels are found in
LIPASE
LIPASE SERUM (TURBIDIMETRIC METHOD)
Intended Use
Clinical Significance
Turbidimetric Method
Principle
Sample
Reagent Composition
Safety Precautions and Warnings
Stability and Preparation of Reagents
1. Buffer
2. Substrate
3. Standard
Materials Provided
Materials Required but not Provided
Procedure Notes
Manual—Lipase
Procedure
Calculation
Quality Control
Interference
Normal Values
Linearity
Normal Value
Clinical Relevance
Increased
Description
PHOSPHATASES
ALKALINE PHOSPHATASE (ALP)
ALKALINE PHOSPHATASE (MOD. KIND AND KING'S METHOD)
Summary
Principle
Normal Reference Values
Storage/stability
Reagent Preparation
Sample Material
Procedure
Calculations
Linearity
Note
ALKALINE PHOSPHATASE (DEA)
Summary
Principle
Normal Reference Values
Storage/stability
Reagent Preparation
Sample Material
Procedure
Calculations
Temperature Conversion Factors
Linearity
Note
Clinical Relevance
Elevated Levels
Reduced Levels
Interfering Factors
ALKALINE PHOSPHATASE ISOENZYMES
Normal Values
Clinical Relevance
ACID PHOSPH ATASE
MOD. KING'S METHOD
Summary
Principle
Normal Reference Values
Storage/stability
Reagent Preparation
Sample Material
Procedure
Assay
Calculations
Linearity
Notes
ACID PHOSPHATASE
Summary
Principle
Normal Reference Values
Storage/stability
Reagent Preparation
Sample Material
Procedure
Total ACP Assay
Non Prostatic ACP Assay : (Tartrate Inhibited)
Calculations
Linearity
Notes
Clinical Relevance
Interfering Factors
SERUM ALKALINE PHOSPHATASE AND ACID PHOSPHATASE
TRANSAMINASES
SGOT (AST)
Summary
Principle
Normal Reference Values
Storage/stability
Reagent Preparation
Sample Material
Procedure
Assay
Note
SGOT (AST)
Summary
Principle
Normal Reference Values
Storage/stability
Reagent Preparation
Sample Material
Procedure
Substrate Start Assay
Sample Start Assay
Calculations
Linearity
Note
SGPT (ALT)
Summary
Principle
Normal Reference Values
Storage/stability
Reagent Preparation
Sample Material
Procedure
Plotting of the Calibration Curve
Assay
Note
SGPT (ALT)
Summary
Principle
Normal Reference Values
Storage/stability
Reagent Preparation
Sample Material
Procedure
Substrate Start Assay
Sample Start Assay
Calculations
Substrate/Sample start
Linearity
Note
Clinical Relevance of SGPT/ALT/ALAT
A. Increased levels are found in
B. SGOT/SGPT Comparison
Clinical Relevance of SGOT/AST/ASAT
A. Increased levels occur in
B. Decreased levels occur in
C. Interfering factors
GAMMA-GLUTAMYL TRANSPEPTIDASE (GGTP) BLOOD
GLUTAMYL TRANSFERASE
Summary
Principle
Normal Reference Values
Storage/stability
Reagent Preparation
Sample material
Procedure
Calculations
Linearity
Note
Clinical Relevance
LACTIC DEHYDROGENASE
CK (NAC ACT)
Summary
Principle
Normal Reference Values
Storage/stability
Reagent Preparation
Sample Material
Procedure
Substrate Start Assay
Sample Start Assay
Calculations
Linearity
Note
CK MB (NAC ACT)
Summary
Principle
Normal Reference Values
Storage/stability
Reagent Preparation
Sample Material
Procedure
Substrate Start Assay
Substrate Start Assay
Calculations
Linearity
Note
CREATINE KINASE (CK)
Clinical Relevance
C K Isoenzymes
Normal Values (at 37°C)
Diagnostic Alert
6% Rule
Increased Total CK
Increased CK-BB
Increased CK-MB
Increased CK-MM
Decreased Total CK
Decreased CK-BB, CK-MB, CK-MM
Interfering Factors
LIVER DISEASE (SERUM ENZYME PATTERNS) VALUES ARE X TIMES THE UPPER NORMAL LIMITS
AUTOMATION IN CLINICAL CHEMISTRY RANDOM ACCESS AUTO ANALYZER
ROCHE HITACHI 911 CHEMISTRY ANALYZER
Assay Types
Sampling System
ISE System
Reagent System
21:
Blood Gases and Electrolytes
BLOOD GASES
BLOOD GASES, ARTERIAL (ABG), BLOOD
Normal Values
BLOOD GASES, CAPILLARY, BLOOD
Normal Values
BLOOD GASES, VENOUS, BLOOD
Normal Values
PARTIAL PRESSURE OF CARBON DIOXIDE (PCO2)
Normal Values
Explanation of Test
Procedure
Clinical Alert
OXYGEN SATURATION (SO2)
Normal Values
Explanation of Test
Procedure
OXYGEN (O2) CONTENT
Normal Values
Explanation of Test
Procedure
PARTIAL PRESSURE OF OXYGEN (PO2)
Normal Values
Background
Explanation of Test
Procedure
CARBON DIOXIDE (CO2) CONTENT OR TOTAL CARBON DIOXIDE (TCO2)
Normal Values
Background
Explanation of Test
Procedure
Clinical Alert
Interfering Factors
BLOOD pH
Normal Values
Background
Explanation of Test
Procedure
Clinical Alert
Interfering Factors
BASE EXCESS/DEFICIT
Normal Values (± 3 mEq/liter)
Explanation of Test
Procedure
AUTOMATION IN BLOOD GAS ANALYSIS
THE BASIS OF BLOOD GASES
Sample Collection
Blood Gas Analyzers
AVL COMPACT 2 BLOOD GAS ANALYZER (FIG. 21.1)
ELECTROLYTE ANALYSIS BY FLAMEPHOTOMETER
FLAME PHOTOMETER 129
Microprocessor-based Automation
Salient Features
Easy Menu Driven Operation
Technical Specifications
Normal Values
Alterations of Sodium and Extracellular Fluid (ECF)
Hyponatremia (Serum Sodium Concentration Lower than Normal)
Hypernatremia (serum sodium concentration higher than normal)
Abnormalities of Serum and Whole Body Potassium
Hyperkalemia (serum potassium concentration more than normal)
Hypokalemia (serum potassium concentration lower than normal)
RAPID DIAGNOSTICS IN ELECTROLYTE ANALYSIS
BAYER 614 NA+ K+ ELECTROLYTE ANALYZER (FIG. 21.5)
Ranges
22:
Serology/Immunology
BASIC IMMUNOLOGY
ANTIGEN-IMMUNOGEN
Antigenicity
EPITOPES (Fig. 22.1)
How big is an epitope?
What are the different kinds of epitopes?
Some Examples of Antigens
What is the structure of Antibody?
WHAT IS THE KINETICS OF ANTIGEN – ANTIBODY REACTION?
IMMUNOLOGICAL REACTIONS
How is binder – ligand assays classified?
What is the difference between all these reactions?
What form of Reaction Takes Place in Hla Typing?
What is the Principle of Hla Typing?
What are the different indicators used in Immunoassay?
INTERFERENCES IN IMMUNOASSAYS
Definition of Interference
Preanalytical Variables
Matrix Effects
Mechanical Interference
Non-specific Interference
Hook Effect
Assay Specificity
TECHNOLOGIES
RAPID IMMUNOCHROMATOGRAPHIC TECHNIQUES
Perspective on Membrane-based Rapid Diagnostic Tests
What are the Principles of Membrane-based Rapid Diagnostic Tests?
What are the Components of Membrane-based Rapid Diagnostic Tests and how are they Constructed?
What are the Limitations and Effects of Various Components on the Performance of Membrane Rapid Diagnostic Tests?
How does the Nitrocellulose Membrane affect the Sensitivity of Rapid Diagnostic Tests?
Pore Size and Capture Reagent Binding Properties
Pore Size and Lateral Flow Rate
Why are Colloidal Gold Sol Particles commonly employed in the Detector Reagent in Membrane-based Rapid Diagnostic Tests?
Colloidal Gold Sol Particles as Indicator
Effect of Shape of Colloidal Gold Sol Particles on Stability
Effect of Shape of Colloidal Gold Sol Particles on Sensitivity
Effect of Size on Color of Colloidal Gold Sol Particles
Why are Variations in Band Appearance Commonly Observed in Membrane-based Rapid Diagnostic Tests Employed for Antigen Detection?
What is the Role of Sample Pad in Membrane-based Rapid Diagnostic Tests?
What is the Role of Soak Pad in Membrane-based Rapid Diagnostic Test?
Why do “Faint Ghost Bands” Appear at the Test Region if the Device is left out on the Worktable?
How do we Interpret “Broken Bands” at the Test/Control Region?
ENZYME IMMUNOASSAY
CLASSIFICATION OF ELISA
1. Direct ELISA
2. Indirect ELISA
3. Capture ELISA
4. Competitive ELISA
5. Streptavidin-biotin ELISA
6. Immunocapture ELISA
7. Interference Corrected ELISA
8. Homogeneous ELISA
9. Heterogeneous ELISA
10. Quantitative ELISA
11. Semi-quantitative ELISA
12. Qualitative ELISA
ELISA: PRACTICAL ASPECTS
Solid Surface
Calibrators/Controls
The Features of an Ideal Calibrator
References
Conjugate
Most Commonly Used Enzymes in Immunoassays
Substrate
The Commonly Used Substrates
Stop Solution
STEPS IN ELISA
Dilution
Addition
Incubation
Wash
Estimation
INTERFERENCES IN IMMUNOASSAYS
Definition of Interference
PREANALYTICAL VARIABLES
Patient based
Specimen based
Nature of the sample
Hemolysis and Hyperbilirubinemia
Assay based
ANALYTICAL VARIABLES
Washing Errors
Pipetting error
Equipment error
Procedural errors
POSTANALYTICAL VARIABLES
Use of Wrong Reference Values
Use of Wrong Units
ELISA TROUBLESHOOTING
PRACTICAL TIPS ON ELISA
Normal Washing
Strip/Plate Washers
Washing Tips
Pipetting Tips
Microplates
Substrate Preparation
Conjugates
General Tips
MATRIX EFFECTS
1. The Effect of Reagents
Immunoassay Labels
Separation of the Antibody-bound and Free Fractions
2. Effect of Proteins
Albumin
Rheumatoid Factors
Complement
Lysozyme
Endogeneous Hormone-binding Proteins
Abnormal forms of Endogeneous binding Proteins
Heterophilic Antibodies
MECHANICAL INTERFERENCE
NONSPECIFIC INTERFERENCE
HOOK EFFECT
Reduction of Hook Effect
EDGE EFFECT
ASSAY SPECIFICITY
ASSAY SENSITIVITY
CHEMILUMINESCENCE: THE TECHNOLOGY
INTRODUCTION
COMPONENTS OF CHEMILUMINESCENT SYSTEM
The Signal
Signal Quantity
Signal Duration
Flash
Glow
Instrumentation
Comparison with Other Technologies
Drawbacks of Other Technologies
References
Advantages of Chemiluminescence Technology
POLYMERASE CHAIN REACTION (FIG. 22.22)
RIA
LIQUID HANDLING SYSTEMS
STREPTAVIDIN-BIOTIN SYSTEMS
STREPTAVIDIN-BIOTIN SYSTEMS, BETTER THAN TRADITIONAL ANTIBODY CAPTURE SYSTEMS
WHY STREPTAVIDIN-BIOTIN BASED IEMA SYSTEMS ARE A BETTER CHOICE FOR TROPICAL LABORATORIES?
SIGNAL NOISE RATIO
PRIMARY CALIBRATORS AND MATRIX EFFECT
ULTRASENSITIVE ASSAYS
EPITYPE CHARACTERIZATION
REPRESENTATIVE ELISA/CLIA TECHNIQUES
ELISA/CLIA ANALYTE DETERMINATION PRINCIPLES
EXAMPLES OF DETAILED ELISA METHODS
COMPETITIVE ELISA
Total Triiodothyronine (tT3)
IMMUNOENZYMOMETRIC/SANDWITCH (STREPTAVIDIN-BIOTIN) ELISA
Thyrotropin (TSH)
IMMUNOENZYMOMETRIC/SANDWICH SEQUENTIAL (STREPTAVIDIN-BIOTIN) ELISA
Prolacting Hormone (PRL) Sequential Method
“MU CAPTURE” IMMUNOCAPTURE ELISA
HAV-IgM
DIRECT ELISA
DNase Activity
CLIA—IMMUNOENZYMOMETRIC/SANDWITCH (STREPTAVIDIN-BIOTIN) ELISA
TESTS FOR SYPHILIS
VDRL TEST
CONVENTIONAL VDRL TEST
Principle
Preparation of Patient's Serum (Inactivation)
Preparation of Working Solutions
Note
Preliminary Testing of the Working Antigen Suspension
Storage and Stability
Procedure
Note
Interpretation of Test Results
Notes
Limitation of the Test
MODIFIED VDRL REAGENT TREPOLIPIN®
TROUBLESHOOTING
Problem: False Positive Results
Problem: False Negative Results
TOLUIDINE RED UNHEATED SERUM TEST FOR RAPID SERODIAGNOSIS OF SYPHILIS REDGEN®
TROUBLESHOOTING
Problem: False Positive Results
Problem: False Negative Results
Problem: Negative Control Giving False Positive Reaction
LATEX SLIDE TEST FOR VDRL SYPHFINAL®
TROUBLESHOOTING
Problem: False Positive Results
Problem: Negative Control Giving Positive Reaction
Problem: False Negative Results
RAPID PLASMA REAGIN (RPR) CARD TEST/CARBON ANTIGEN FOR SYPHILIS TESTING (CARBOGEN®)
TROUBLESHOOTING
Problem: False Positive Results
Problem: Negative Control Giving False Positive Reaction
Problem: False Negative Results
Problem: Negative Control Giving False Positive Reaction
ONE-STEP TEST FOR SYPHILIS: DIPSTICK Syphicheck®
ONE-STEP TEST FOR SYPHILIS (DEVICE) Syphicheck®
THIRD GENERATION DOUBLE ANTIGEN SANDWICH ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) FOR THE DETECTION OF ANTIBODIES TO TREPONEMA PALLIDUM IN HUMAN SERUM OR PLASMA
TESTS FOR TYPHOID/ENTERIC FEVER
WIDAL ANTIGEN SET/ANTIGENS FOR TUBE TESTS
TROUBLESHOOTING
Problem: False Positive Results
Problem: False Negative Results
WIDAL ANTIGEN SET/ANTIGENS FOR SLIDE AND TUBE TESTS
TROUBLESHOOTING
Problem: False Positive Results
Problem: False Negative Results
REDUCED WIDAL ANTIGEN SET: O AND H FOR TUBE TESTS
POSITIVE CONTROL FOR WIDAL TEST
Summary
Reagent
Reagent Storage and Stability
Principle
Note
Additional Material Required
Procedure
Interpretation of Results Slide Test Method
TROUBLESHOOTING
Problem: False Positive Results
Problem: False Negative Results
RAPID TEST FOR DETECTION IGM ANTIBODIES TO S.TYPHI IN SERUM/PLASMA/WHOLE BLOOD (DEVICE)
SLIDE AND TUBE TEST FOR DETECTION OF ANTIBODIES TO BRUCELLA ABORTUS/MELITENSIS
TOUBLESHOOTING
Problem: False Positive Results
Problem: False Negative Results
SLIDE SCREENING TEST FOR BRUCELLA ANTIBODIES
BRUCELLOSIS POSITIVE CONTROL
RAPID TEST FOR IgM AND IgG ANTIBODIES TO DENGUE VIRUS: DENGUE FEVER
TROUBLESHOOTING
DENGUECHECK WB AND LEPTOCHECK-WB
Problem: False Positive Results
Problem: Faint Lines Observed in Control and Test Region
Problem: Delayed Results and Altered Flow
Problem: False Negative Results
Problem: Invalid Results
TEST FOR INFECTIOUS MONONUCLEOSIS (IMMUTEX®)
TROUBLESHOOTING
IMMUTEX
Problem: False Positive Results
Problem: False Negative Results
RAPID TEST FOR IgM ANTIBODIES TO LEPTOSPIRA: LEPTOSPIROSIS
TROUBLESHOOTING
Problem: False Positive Results
Problem: Faint Lines Observed in Control and Test Region
Problem: Delayed Results and Altered Flow
Problem: False Negative Results
Problem: Invalid Results
RAPID TEST FOR MALARIA PAN/PV/PF
SPECIMEN COLLECTION AND PREPARATION
Test Procedure
Limitation of the Test
SLIDE TEST FOR C-REACTIVE PROTEIN
TROUBLESHOOTING
Problem: False Positive Results
Problem: False Negative Results
Problem: Positive Control giving Negative Reaction
Problem: Positive Result with our Kit and Negative with Another Kit or Vice Versa
SLIDE TEST FOR ANTI STREPTOLYSIN O
TROUBLESHOOTING
Problem: False Positive Results
Problem: Delayed Agglutination
Problem: False Negative Results
Problem: Positive Control Giving Negative Reaction
Problem: Positive Result with this Kit and Negative with Other Kit or Vice Versa
SLIDE TEST FOR RHEUMATOID FACTORS
TROUBLESHOOTING
Problem: False Positive Results
Problem: False Negative Results
Problem: Positive Control giving Negative Reaction
Problem: Delayed Agglutination
Serological diagnosis rheumatic disorders with latex agglutination tests RF, CRP, ASL
SLIDE TEST FOR ANTI DEOXYRIBONUCLEOPROTEIN
TROUBLESHOOTING
Problem: False Positive Results
Problem: Delayed Positive Results
Problem: False Negative Results
Problem: Positive Control giving Negative Reaction
Problem: Delayed Agglutination
AUSTRALIA ANTIGEN HBsAg
SLIDE TEST FOR HEPATITIS B SURFACE ANTIGEN
Summary
Reagent
Reagent Storage and Stability
Principle
Note
Sample Collection and Storage
Material Provided with the Kit
Reagent Pack
Accessories Pack
Additional Material Required
Procedure
Interpretation of Results
Remarks
ONE-STEP TEST FOR HBsAG
HCV
ONE-STEP IMMUNOCHROMATOGRAPHIC TEST FOR HCV ANTIBODIES
Introduction
Summary
Principle
Reagents and Materials Supplied
Kit Components
Storage and Stability
Note
Specimen Collection and Preparation
Test Procedure and Interpretation of Results
Remarks
TORCH INFECTIONS—INTRODUCTION
Toxoplasma Infection
Rubella Infection
Cytomegalovirus Infection
Herpes Simplex Virus (1 + 2) Infections
TOXOPLASMA INFECTIONS
SLIDE TEST FOR ANTIBODIES TO TOXOPLASMA GONDII, TOXOGEN®
Summary
Reagent
Reagent Storage and Stability
Principle
Note
Specimen Collection and Storage
Material Provided with the Kit
Reagent Pack
Accessories Pack
Additional Material Required
Test Procedure
Qualitative Method
Semiquantitative Method
Interpretations of Results
Qualitative Method
Semi Quantitative Method
Differentiation IgG - IgM
Significance of Test Results
Remarks
RAPID IMMUNOCONCENTRATION TEST FOR HIV I AND HIV 2 ANTIBODIES FLOW THROUGH METHOD
RAPID TEST FOR SIMULTANEOUS/DIFFERENTIAL DETECTION OF TOTAL ANTIBODIES TO HIV-1 AND HIV-2 IN HUMAN SERUM/PLASMA
TUBERCULOSIS
MYCOBACTERIUM TUBERCULOSIS
MICROBIOLOGY
Classification
Pathogenesis
Immune Response
Serological Response
Virulence Factors
Clinical Manifestations
Clinical Manifestation of Pulmonary TB
TB Infection and Disease –Differentiation
Diagnosis
Tuberculous Skin Testing
Pitfalls of Laborartory Diagnosis
Importance of Serological Diagnosis
Treatment
RAPID TEST FOR DETECTION OF ANTIBODIES TO MYCOBACTERIUM TUBERCULOSIS (DEVICE)
TB IgG, IgA, IgM Ab, Mfd. ANDA
TUMOR MARKERS
TUMOR MARKERS
STANDARD METHODOLOGIES AVAILABLE ON ELISA AND CLIA, AS ON RIA TOO.
ALPHA-FETOPROTEIN (AFP) ELISA
Summary and Explanation of the Test
Interpretation
Expected Ranges of Values
CA 15-3
CA 19-9 (CARBOHYDRATE AG 19-9, GICAM GASTROINTESTINAL CANCER ANTIGEN) BLOOD
CA242
CA 50 (CARBOHYDRATE ANTIGEN 50)
Increased
Decreased
CA 125 (CANCER ANTIGEN 125)
CARCINOEMBRYONIC ANTIGEN (CEA)
DECREASED
PROSTATE-SPECIFIC ANTIGEN (PSA) TOTAL PROSTATE SPECIFIC ANTIGEN (TPSA) ELISA,
PROSTATIC ACID PHOSPHATES (PAP), BLOOD
ELISA TOUBLE SHOOTING ASPECTS
INTRODUCTION
Problem: High Negative Control Value or High Background
Problem: Low Positive Control Value or Low Absorbance
Problem: Entire Plate gives Positive OD or Color All Over Plate
Problem: False Positive Reactions
Problem: Poor Reproducibility or Bad Duplication
Problem: Poor Sensitivity
Problem: High Absorbance of Calibrator
Problem: Specimen Absorbance Out of Range of Calibrators
Problem: Overall Low Absorbance
Problem: Controls Out of Range
Problem: Strips Slip from Holder
Problem: Strips do not Fit in Holder
Problem: Substrate A is Blue
Problem: Substrates A and B Turn Blue when Mixed
Problem: Stop Solution Yellow
Problem: Waited Over 30 minutes Before Measuring Plate
Problem: No Color Even After 30 minutes Incubation with Substrate
Problem: Color Develops Very Quickly
Problem: Color Develops too Slowly
TECHNICAL TIPS
Washing
Normal Washing
Strip/Plate Washers
Washing Tips
Pipetting Tips
Microplates
Substrate Preparation
Conjugates
Addition of Samples
Stopping Reagents
Temperature
Rotation of Plates While Incubating Reagents
Laboratory Conditions
23:
Diagnostic Immunology: Non Enzymatic, Quantitative Techniques (Immunodiffusion, Electrophoresis and Turbidimetry)
QUALITATIVE DETERMINATION OF PLASMA PROTEINS BY IMMUNOPRECIPITATION
IMMUNODIFFUSION METHOD OF OUDIN
DOUBLE DIFFUSION METHOD OF OUCHTERLONY
GRABAR AND WILLIAMS'S METHOD OF IMMUNOELECTROPHORESIS
METHODOLOGY
Double Diffusion of Ouchterlony
Macrotechnique
Gel Diffusion Procedure
Microtechnique
Recording of Results
Possible Sources of Error
IMMUNOELECTROPHORESIS
Reagents and Equipment
Agar
Buffer
Antisera
Preparation of Agar Gel Slides
Application of Antigens
Electrophoresis
Application of Antiserum
Possible Sources of Errors
Fixation of Precipitin Patterns
Protein Stains
Lipoprotein Stains
Peroxidase Reaction (for haptoglobin and hemopexin)
Ceruloplasmin Stain
Cholinesterase Stain
Lipid-Protein Double Staining
Identification and Interpretation of Precipitin Lines
PRACTICAL APPLICATION OF IMMUNOELECTROPHORESIS
QUANTITATIVE DETERMINATION OF PLASMA PROTEINS BY IMMUNOPRECIPITATION
FUNDAMENTAL QUANTITATIVE CONSIDERATIONS
PHOTOMETRIC METHOD
SINGLE LINEAR IMMUNODIFFUSION OF OUDIN
SINGLE RADIAL IMMUNODIFFUSION
ELECTROIMMUNODIFFUSION
SINGLE RADIAL IMMUNODIFFUSION
Equipment
Procedure
TURBIDIMETRY
INTRODUCTION
1. Detection
2. Quantification
3. Monitoring
4. Prognosis
IMMUNOASSAYS
QUALITATIVE IMMUNOASSAYS
QUANTITATIVE IMMUNOASSAYS
Heterogeneous Immunoassays
Homogeneous Immunoassays
SPECTROPHOTOMETRY
Beer's Law
Lambert's Law
Beer-Lambert Law
Measuring Principles in Biochemistry
Criteria for Wavelength Selection
Complementary Filters for Measuring Color Complexes
Reading Methods
Standardization of Time Interval for Rate Reactions
Measurement of Immune Complexes by Spectrophotometry
Selection of Wavelength for Measuring Immune Complexes
Turbidimetry vs Nephelometry
CONSIDERATIONS FOR MEASUREMENTS OF TURBIDIMETRIC IMMUNOASSAYS (TIA)
The Antibody Excess Zone
Zone of Equivalence
The Antigen Excess Zone
OPTIMIZATION, STANDARDIZATION AND QUALITY CONTROL OF TURBIDIMETRIC ASSAYS
Ionic Strength
pH
Temperature
Enhancers
Interference
Characteristics of Antibody Used as a Reagent
Standardization and Calibration
Quality Control
Reading Principles in Turbidimetry
Real Sample Blanking
Immediate Mixed Blanking
Reaction Kinetics and its Effect on Blanking
CONCEPTS OF ASSAY OPTIMIZATION
Detection Limit
Measuring Range
Security Range
Reagent Optimization
Standard Curve
INSTRUMENTATION FOR TURBIDIMETRY
Aspiration Mode
Cuvette Mode
Classification of Analyzers
Semiautomated Analyzers
Automated Analyzers
AN EXAMPLE OF TURBIDIMETRIC IMMUNOASSAY
TURBIDIMETRIC IMMUNOASSAY FOR DETERMINATION OF RHEUMATOID FACTORS
C–REACTIVE PROTEIN
TURBIDIMETRIC IMMUNOASSAY FOR DETERMINATION OF C-REACTIVE PROTEIN,
TURBIDIMETRIC IMMUNOASSAY'FOR ULTRASENSITIVE DETERMINATION OF C-REACTIVE PROTEIN
TURBIDIMETRIC IMMUNOASSAY FOR DETERMINATION OF ANTISTREPTOLYSIN ‘O’ IN HUMAN SERUM
TURBIDIMETRIC IMMUNOASSAY FOR DETERMINATION OF MICROALBUMINURIA
IMMUNOGLOBULINS (IG)
TURBIDIMETRIC IMMUNOASSAY FOR ESTIMATION OF IMMUNOGLOBULIN IgA IN HUMAN SERUM
TURBIDIMETRIC IMMUNOASSAY FOR ESTIMATION OF IMMUNOGLOBULIN IgG IN HUMAN SERUM
TURBIDIMETRIC IMMUNOASSAY FOR ESTIMATION OF IMMUNOGLOBULIN IgM IN HUMAN SERUM
TURBIDIMETRIC IMMUNOASSAY FOR ESTIMATION OF COMPLEMENT C3 IN HUMAN SERUM
TURBIDIMETRIC IMMUNOASSAY FOR ESTIMATION OF COMPLEMENT C4 IN HUMAN SERUM
TURBIDIMETRIC IMMUNOASSAY FOR ESTIMATION OF ANTITHROMBIN III IN HUMAN SERUM
QUANTITATIVE IMMUNOTURBIDIMETRIC ASSAY FOR ESTIMATION OF FIBRINOGEN
TURBIDIMETRIC IMMUNOASSAY FOR ESTIMATION OF LIPOPROTEIN (A) IN HUMAN SERUM
QUANTITATIVE TURBIDIMETRIC IMMUNOASSAY FOR ESTIMATION OF APOLIPOPROTEIN A-I
QUANTITATIVE TURBIDIMETRIC IMMUNOASSAY FOR ESTIMATION OF APOLIPOPROTEIN B
AUTOMATION IN TURBIDIMETRY
QUANTIMATE TURBIDIMETRY ANALYZER (FIG. 23.17)
Optimized Measuring System
Instrument Specifications
TROUBLESHOOTING
IMMUNOTURBIDIMETRY TESTS
For Product Range
Problem: Testing Errors
Problem: Errors in Real Sample Blanking and Immediate Mixed Blanking Methods
24:
The Endocrine System
INTRODUCTION
PITUITARY GLAND
ANTERIOR PITUITARY (ADENOHYPOPHYSIS)
Hormones Secreted by the Pituitary Gland
Causes of Hypopituitarism Resulting in Multiple or Single Deficiencies
Intrinsic Pituitary Disease
Extrinsic Pituitary Disorders
ANTERIOR LOBE
GROWTH HORMONE (GH)
METHOD OF EVALUATION: STREPTAVIDIN-BIOTIN ELISA
GROWTH HORMONE (hGH)
CORTICOTROPHIN (ACTH)
OTHER ANTERIOR PITUITARY HORMONES TSH
FSH
LH
LH, FSH—RECOMMENDATIONS FOR TESTING
PROLACTIN
PROLACTIN—RECOMMENDATIONS FOR TESTING
INTERMEDIATE LOBE (PARS INTERMEDIA)
POSTERIOR PITUITARY (NEUROHYPOPHYSIS)
ADH
OXYTOCIN
FEEDBACK MECHANISM
DISORDERS OF THE PITUITARY SYSTEM
HYPOTHALAMUS
ADRENAL (SUPRARENAL) GLAND
MINERALOCORTICOIDS
GLUCOCORTICOIDS
CORTISOL
Plasma Cortisol
Normal Values
Cortisol Levels are Increased in
Cortisol Levels are Decreased in
Interfering Factors
Cortisol Suppression (Dexamethasone Suppression)
Test Significance
Method
Clinical Relevance
Cortisol Stimulation (Cortrosyn Stimulation)
Test Significance
Method
Clinical Relevance
Water Excretion Test (Soffer)
Interpretation
Corticotrophin (ACTH) Response Test (Thorn test)
Method
Interpretation
Adrenocortical Inhibition Test
1. Test with dexamethasone
2. Rapid dexamethasone test
3. Tests with metyrapone (an endogenous ACTH test)
Method
Cortisol Abnormalities
Excess
Deficiency
ADRENAL MEDULLA
THYROID
TESTS FOR THYROID function
Generally recommended in
Different Markers of Thyroid Function are Tested in Combination for Assessment of Thyroid Status
Information to be Taken before Sample Collection
FT3, FT4—Recommendations for Testing
Free Thyroxine Index (FTI)
Thus: FT4 Index = T4 (total)× T3UR
T3 Uptake Ratio (T3UR)
Clinical Significance
Pregnancy
High or Low-T4 in a Euthyroid Individual
A Normal T3 in a Hypothyroid Patient
THYROID FUNCTION TESTS
Introduction
Tests for Thyroid Function
Binding Proteins
Importance of Free T4 and Free T3
Alterations in the Concentrations or Affinity of Thyroid Hormone-Binding-Proteins (Fig. 24.6)
Increases in
Decreases in
The TSH-Free T4 Relationship
Problems in the Interpretation of Thyroid Function Tests
Common Causes of TSH/FT4/FT3 Discrepancies
Effects of Drugs on Thyroid function
Drugs that Affect Results of Thyroid Function Tests
Relationship Between Serum Total T4 and Total T3 Concentrations in Various Disorders
Relationship Between Serum FT4, FT3 and TSH Concentrations in Various Disorders
Free T4 and Free T3 in Various Disease Conditions
1. Hyperthyroidism
2. Nonthyroidal Illness
3. Hypothyroidism
Assay Choice Application
Screening and Case Finding
Untreated Patients
Assessment of the Response to Treatment
Difficult Diagnostic Situations
Thyroid Diagnosis and Treatment
Recommendations for Thyroid Testing
Diagnostic Approach to Anomalous Serum T3, T4, and TSH Values
Typical Reference Ranges for Serum Thyroid Hormones and TSH in Humans*
CALCITONIN
PARATHYROID
PARATHYROID HORMONE (INTACT) ELISA
PANCREAS
INSULIN
Functions of Insulin
Clinical Relevance of C-peptide
Anti-insulin Antibodies
Against Endogenous Insulin
Against Exogenous Insulin
TESTES
OVARY
PINEAL GLAND
HORMONES AND FERTILITY
MALE FERTILITY
THE FOUR FACTORS OF MALE FERTILITY
Pretesticular Function (Hormones)
Testicular function
Post-testicular function
Ejaculatory Disturbance, Impotence, and Sexual Problems
PROBLEMS ASSOCIATED WITH MALE INFERTILITY
THE MALE HORMONE SYSTEM
The Hypothalamus and Pituitary Start the Action
Feedback Hormones from Testicles
WHERE CAN THINGS GO WRONG?
Fertility Factor: The Hormone Balancing Act
An Overview of Hormonal Treatment
DIAGNOSING AND BEATING SPECIFIC HORMONAL PROBLEMS
Hyperprolactinemia
Hypothyroidism
Congenital Adrenal Hyperplasia
Hypogonadotrophic Hypopituitarism
Panhypopituitarism
Kallmann's Syndrome
Delayed Puberty
Fertile Eunuch
What Causes Testicular Failure?
Varicocele
Cryptorchidism
Infection
Torsion
Klinefelter's Syndrome
Cushing's Syndrome
Germ Cell Aplasia (Sertoli Cell Only)
Testicular Enzyme Defects
FEMALE FERTILITY
THE FIVE FEMALE FERTILITY FACTORS
Fertility Factor—1: Ovulation
Fertility Factor—2: Sperm-Mucus Interaction
Fertility Factor—3: Fertilization
Fertility Factor—4: Tubal Factor
Fertility Factor—5: Embryo Implantation
FEMALE HORMONE SYSTEM
What is Ovulation?
What makes One have a Period?
CLUES FROM THE MENSTRUAL HISTORY
The Three Types of Menstrual Patterns
The Regular Menstrual Period
Irregular Menstrual Periods or Amenorrhea for Six or More Months
Nonexistence of the Menstrual Period
CLUES FROM PHYSICAL EXAMINATION
The Physical Examination
Diagnostic Approaches for Irregular Menstrual Periods or Amenorrhea
Pregnancy Test
Progesterone Withdrawal Test
Positive Response to Progesterone Withdrawal
Negative Response to Progesterone Withdrawal
Positive Withdrawal to Estrogen/Progesterone Stimulation
Detecting Ovarian Failure
Diagnosing Anovulation
Symptoms of Anovulation
Tests Used to Determine the Cause of Anovulation
Hormonal Tests for Diagnosing the Cause of Anovulation
CONDITIONS THAT CAN INTERFERE WITH OVULATION AND MENSTRUATION
Practical Evaluation of Hormonal Status
ALGORITHM FOR EVALUATING AMENORRHEA (Figs 24.12 to 24.18)
IMMUNOASSAYS FOR LH, FSH AND PRL
SENSITIVITY
Detection Limits of Various Immunoassays
Conditions in which LH/FSH levels can go below 2.0 mIU/ml
STREPTAVIDIN-BIOTIN ASSAY SYSTEM
Calibrator Matrix
WHO Std. Reference
CROSS REACTION
MONOCLONAL CAPTURE
Normal Ranges
Sample Collection and Storage
LH and FSH
Prolactin
Hyperprolactinemia
Causes for Hyperprolactinemia
Symptoms of Hyperprolactinemia
Diagnosis
Treatment
ADRENAL CORTEX
ALDOSTERONE
Aldosterone, Serum and Urine
Normal values
Method
Test Significance
Clinical Relevance
Interfering Factors
Clinical Disorders of Mineralocorticoids
Primary Hyperaldosteronism
Secondary Hyperaldosteronism
Isolated Hypoaldosteronism
ADRENAL MEDULLA
TESTES
STEROIDS
ESTRIOL
17-β-ESTRADIOL
DHEAS (DEHYDROEPIANDROSTERONE SULFATE)
Δ4-ANDROSTENEDIONE
PROGESTERONE
17αOH PROGESTERONE
TOTAL TRIIODOTHYRONINE (T3)
EXPECTED VALUES FOR THE T3 EIA TEST SYSTEM
Interpretation of Total T3 in ng/ml
FREE TRIIODOTHYRONINE (FT3)
Interpretation
Expected Ranges of Values
T-UPTAKE
Interpretation
Expected Ranges of Values
TOTAL THYROXINE (T4)
Interpretation
Expected Range of Values
Normal Values (μg/dl)
FREE THYROXINE (FT4)
Interpretation
Expected Range of Values
THYROTROPIN (TSH)
Interpretation
Expected Ranges of Values
Normal Values (μIU/ml)
EXAMPLE OF CHEMILUMINESCENCE IMMUNOASSAY METHOD—TSH ESTIMATION
THYROGLOBULIN ANTIBODIES
Interpretation
Expected Range of Values
THYROID PEROXIDASE ANTIBODIES
Interpretation
Expected Range of Values
LUTEINIZING HORMONE (LH)
Interpretation
Expected Range of Values
Usage
Values are Increased in
Values are Decreased in
FOLLICLE STIMULATING HORMONE (FSH)
Interpretation
Normal Values in mIU/ml
Values are Increased in
Values are Decreased in
PROLACTIN HORMONE (PRL)
Interpretation
Expected Range of Values
HPRL Levels are Increased in
HPRL Levels are Decreased in
CHORIONIC GONADOTROPHIN (HCG)
Interpretation
Normal Values in mIU/ml
Values are Increased in
Values are Decreased in Abortion and Ectopic Pregnancy
Factors that Affect Results
Other Data
CIA™ INSULIN
C-PEPTIDE
Expected Values
ANTI-INSULIN
Clinical Relevance
Indications
Normal Values
25:
Histopathology
PREPARATION OF TISSUES
FIXATION
DECALCIFICATION
Nitric Acid Method
Formic Acid Sodium Citrate Method
Electrolytic Method
PROCESSING OF TISSUES
Method I
Preparation of Sections
Cutting Sections
ATTACHING SECTIONS TO SLIDES
Mayer's Egg Albumin
Egg Adhesive from Dried Albumin
TECHNIQUE FOR FROZEN SECTIONS
REMOVAL OF PIGMENTS AND PRECIPITATES
Mercury Precipitate
Formalin Produced Precipitate
Method I
Method II
Melanin Pigment
Malarial Pigment
Method I
Method II
Method III
ROUTINE STAINING PROCEDURES
COMMONLY EMPLOYED HEMATOXYLINS
Harris's Alum Hematoxylin
Mayer's Hematoxylin
Counterstains for Hematoxylin Stains
Stock 1% Aqueous Eosin Solution
Stock 1% Alcoholic Eosin Solution
Working Eosin Solution
Routine Hematoxylin and Eosin Stain
Solution
Staining Procedure
SPECIAL STAINS
Stains for Connective Tissue
a. Mallory's Phosphotungstic Acid Hematoxylin Stain
b. Mallory's Phosphomolybdic Acid Hematoxylin Stain
c. Heidenhain's Iron Hematoxylin Stain
d. Mallory's Aniline Blue Collagen Stain
e. Lee-Brown's Modification of Mallory's Aniline Blue Stain
f. Van Gieson's Stain for Collagen Fibers
g. Barbeito-Lopez Trichrome Stain
h. Gomori's One-Step Trichrome Stain
i. Heidenhain's Aniline Blue Stain
j. Gallego's Iron Fuchsin Stain
k. Weigert's Resorcin Fuchsin Elastic Stain
l. Gomori's Aldehyde Fuchsin Stain
m. Verhoeff's Elastic Stain
n. Wilder's Reticulum Stain
o. Koneff's Stain for Bone and Cartilage
Stains for Cytoplasmic Granules
a. Gomori's Chromaffin Stain
b. Acid Fuchsin Aniline Blue Method for Pituitary Granules
c. Fontana-Masson Stain for Argentaffin Granules
d. Luna's Mast Cell Stain
Stains for Fats and Lipoids
a. Osmium Tetraoxide Stain for Fat
b. Oil Red O Fat Stain
c. Sudan Black B Stain for Fat
Stains for Carbohydrates and Mucoproteins
a. Best's Carmine Stain for Glycogen
b. Periodic Acid-Schiff (PAS) Reaction
c. Mayer's Mucicarmine Stain
d. Alcian Blue
e. Alcian Blue-PAS Stain
f. Crystal Violet Amyloid Stain
g. Bennhold's Congo Red Amyloid Stain
h. Toluidine Blue Metachromasia Stain
Stains for Pigments and Minerals
a. Modification of Mallory's Reaction for Iron
b. Gomori's Iron Reaction
c. von Kossa's Method for Demonstrating Calcium
d. Svain's Bile Pigment Stain
e. De Galantha's Method for Demonstration of Urate Crystals
Stains for Bacteria, Fungi and Inclusion Bodies
a. Kinyoun's Acid-Fast Stain
b. Ziehl-Neelsen Stain for Acid-Fast Bacteria
c. Fite-Faraco Stain for Acid-Fast Bacilli
d. Brown and Brenn Stain for Bacteria in Tissue
e. Levaditi's Method for Staining Spirochetes in Blocks
f. Warthin-Starry Method for Staining Spirochetes
g. Silver Method for Spirochetes and Donovan Bodies
h. Gridley's Stain for Fungi
i. Gomori's Methanamine-Silver Nitrate Technique (Grocott's application to Fungi)
j. Phloxine Toluidine Blue Stain for Malarial Parasites
k. Parson's Stain for Negri Bodies
l. Hematoxylin-Shorr S3 Stain for Inclusion bodies
m. Giemsa's Stain for Rickettsiae
SOME STAINING TECHNIQUES IN DETAIL
MALLORY'S PHOSPHOTUNGSTIC ACID HEMATOXYLIN STAIN: PTAH
Solutions
Phosphotungstic Acid Hematoxylin
Staining Procedure
Results
VAN GIESON'S STAIN FOR COLLAGEN FIBERS
Solution
Weigert's Iron Hematoxylin
Working Solution
van Gieson's Solution
Staining Procedure
Results
MASSON'S TRICHROME STAIN
Solutions
Bouin's Solution
Weigert's Iron Hematoxylin
Biebrich Scarlet-Acid Fuchsin Solution
Phosphomolybdic-Phosphotungstic Acid Solution
Aniline Blue Solution
Light Green Solution
1% Acetic Water Solution
Staining Procedure
Results
VERHOFF'S ELASTIC STAIN
Solutions
Elastic Tissue Stain
Ferric Chloride Solution
Van Gieson's Stain
Sodium Thiosulphate (Hypo) Solution
Staining Procedure
Results
WILDER'S RETICULIN STAIN
Solutions
Phosphomolybdic Acid Solution
Uranium Nitrate Solution
Ammoniacal Silver Solution
Reducing Solution
Gold Chloride Solution
Sodium Thiosulphate (Hypo) Solution
Nuclear Fast Red (Kernechtrot) Stain
Staining Procedure
Results
FONTANA-MASSON STAIN FOR ARGENTAFFIN GRANULES
OIL RED O FAT STAIN
Solutions
Oil Red O solution
Glycerin Jelly
Harris's Hematoxylin for counterstain
Staining Procedure
Results
BEST'S CARMINE STAIN FOR GLYCOGEN
Solutions
Carmine Stock Solution
Working Solution of Carmine
Differentiating Solution
Staining Procedure
Results
PERIODIC ACID-SCHIFF (PAS) REACTION
Solutions
Coleman's Feulgen Reagent
Schiff's Leuco-Fuchsin Solution
Test for Schiff's Leuco-Fuchsin Solution
Normal Hydrochloric Acid Solution
0.2% Light Green counterstain (Stock)
Working Light Green Solution
Diastase Solution
Staining Procedure
Results
MAYER'S MUCICARMINE STAIN
Solutions
Weigert's Iron Hematoxylin
Working Solution
Metanil Yellow Solution
Mucicarmine Stain
Staining Procedure
Results
CRYSTAL VIOLET AMYLOID STAIN
Solutions
Stock Crystal Violet Solution
Working Crystal Violet Solution
Abopon Mounting Medium
Staining Procedure
Results
VON KOSSA'S METHOD FOR DEMONSTRATING CALCIUM
Solutions
5% Silver Nitrate Solution
5% Sodium Thiosulphate Solution
Nuclear Fast Red (Kernechtrot) Stain
Staining Procedure
Results
ZIEHL-NEELSEN STAIN FOR ACID-FAST BACTERIA
Solutions
Carbol Fuchsin solution
1% Acid Alcohol
1% Sulphuric Acid Water
Working Methylene Blue Solution
Staining Procedure
Results
AUTOMATION IN HISTOPATHOLOGY
26:
Cytology
27:
Microbiology and Bacteriology
CLASSIFICATION
PROTOPHYTA
Schizomycetes (Bacteria and related forms)
Actinomycetales
Eubacteriales
Spirochetales
Microtatobiotes (The smallest living things)
Virales
BACTERIAL CELL CONSTITUENTS
Capsule
Flagella
Spores
Inclusion Granules
Shape of Bacteria
1. Cocci
2. Bacilli
Bacterial Reproduction
Bacterial Physiology
1. Food
2. Moisture
3. Hydrogen-ion-concentration or pH
4. Oxygen Needs
5. Carbon dioxide
6. Temperature
7. Light
8. Symboisis or mutual beneficial coexistence
Products of Bacterial Growth
1. Bacterial toxins
2. Bacterial enzymes
3. Bacterial pigments
KOCH'S POSTULATES
MORPHOLOGY AND STAINING REACTIONS
1. Loeffler's Methylene Blue
Method
2. Dilute Carbolfuchsin
Gram's Stain
Method
Results
Reagents
Ziehl-Neelsen Stain
Method
Results
TROUBLESHOOTING (AFB-STAINING)
Problem: False Positive Results
Problem: False Negative Results
Modified Ziehl-Neelsens Stain
SPECIAL STAINS
Stains for Diphtheria Bacillus
Ponder's Stain
Albert's Stain
Modified Neisser's Method
Staining of Capsules
Hiss's Method
India Ink Method
Staining of Spores
Modified Ziehl-Neelsen Method
Staining of Spirochetes
Fontana's Method
Staining of Fungi
Lactophenol Cotton Blue
Staining of Flagella
Loeffler's Method
Negative Staining
India Ink method
MOTILITY OF BACTERIA
Hanging Drop Method
CULTURE
Media
1. Basic Media
2. Enrichment Media
3. Differential and Selective Media
4. Indicator Media
Temperature
Atmosphere
Cultural Characteristics
1. Shape
2. Surface
3. Size
4. Contiguity
5. Consistency
6. Pigmentation
7. Opacity
8. Elevation
9. Media Changes
BIOCHEMICAL REACTIONS
1. Sugar Fermentation
2. Other Biochemical Tests
Serology
PREPARATION OF CULTURE MEDIA
Peptone Water
Nutrient Broth
Nutrient Agar
Blood Agar
Chocolate Agar
Sugar Media
Preparation of Andrade's Indicator
Hiss's Serum Water Sugars
Loeffler's Serum Slopes
Media for Identification of Fungi
Sabouraud's Glucose Agar
READY TO POUR, STERILIZED POUCHED MEDIA FOR MICROBIOLOGICAL APPLICATIONS
TROUBLESHOOTING
Problem: Smooth Gelled Media Not Formed
MEDIA FOR GROWTH OF ANAEROBES
Robertson's Cooked Meat Medium
MacCONKEY'S MEDIUM
LOWENSTEIN JENSEN MEDIUM
Mineral Salt Solution
METHOD OF INOCULATION
Streak Plate Method
Method I
Method II
Method III
PRECAUTIONARY MEASURES TO BE UNDERTAKEN IN A MICROBIOLOGY LABORATORY
SPECIMEN COLLECTION
Urine
EASYBACT
TROUBLESHOOTING
Problem: Twisting and Swirling the Media
Problem: Discoloration
Problem: Discrepancy in Results
Feces
Sputum
Throat and Nasal Smears
Pus Swabs
Blood Cultures
CSF, Pleural Fluid and Other Body Fluids
Ear Discharge Cultures
Eye Cultures
GENERAL INSTRUCTIONS FOR MICROBIOLOGY
STORAGE OF ORGANISMS
PRECAUTIONS BEFORE AND DURING TESTING
SPECIMEN COLLECTION AND PREPARATION
INOCULATION OF SAMPLES
GRAM-POSITIVE COCCI
STAPHYLOCOCCI
Morphology
Culture
Coagulase Test
1. Slide Test
2. Tube Test
Pathogenicity
STREPTOCOCCI
Morphology
Alpha-hemolysis
Beta-hemolysis
Gamma-hemolysis
Culture
Pathogenicity
PNEUMOCOCCI
Morphology
Culture
Other Differences between Pneumococci and Strep. viridans
Pathogenicity
GRAM-NEGATIVE COCCI
NEISSERIA
Morphology
Culture
Morphology
Culture
Morphology
Culture
Comparison of meningococci and gonococci
VEILONELLA
ANAEROBIC SPORE BEARING BACILLI
CLOSTRIDIA
Pathogenic clostridia
Morphology
Culture
Laboratory Diagnosis
Toxin Produced
GAS GANGRENE CAUSING ORGANISMS
Morphology
Culture
Toxins produced
Morphology
Culture
Morphology
Culture
Diagnosis of Gangrene
Microscopy
Culture
Morphology
Culture
Laboratory Diagnosis
AEROBIC SPORE FORMING BACILLI
GRAM-POSITIVE BACILLI
CORYNEBACTERIA
Morphology
Culture
Diseases Caused
Laboratory Diagnosis
DIPHTHERIOID BACILLI
MYCOBACTERIA
STRAINS OF MYCOBACTERIA
MORPHOLOGY
CULTURES
Cultural Charactristics
LABORATORY DIAGNOSIS
CONCENTRATION METHODS
Petroff's Method
Oxalic Acid Method
Trisodium Phosphate Method
OTHER PATHOGENIC MYCOBACTERIA
NON-PATHOGENIC SPECIES
ANIMAL INOCULATION
OVERVIEW OF M. TUBERCULOSIS DIAGNOSTIC APPROACH AFB STAINING, CULTURE AND SENSITIVITY
INTRODUCTION
BRIEF MICROBIOLOGY
DIAGNOSIS OF MYCOBACTERIUM TUBERCULOSIS INFECTION
SPECIMEN SELECTION
SAMPLE CONCENTRATION AND DECONTAMINATION
MUCOLYTIC, DISINFECTANT, SPECIMEN PRETREATMENT AND BUFFERING SYSTEM FOR AFB STAINING AND CULTURE
TROUBLESHOOTING
Problem: False Negative Results in LJ Media
Problem: False Positive Results in LJ Media
Problem: Turbidity in Phosphate Buffer
Problem: No Liquefaction of Mucoid Sputum
The AFB Smear
RAPID TWO STEP COLD AFB STAIN
AFB CULTURE AND ISOLATION
READY TO USE LJ SOLID MEDIUM FOR MYCOBACTERIUM TUBERCULOSIS ISOLATION
TROUBLESHOOTING
Problem: Growth on the Slant within First Week of Incubation
Problem: Collapse of Slants
COMBIPACK OF SOLID AND LIQUID MEDIUM FOR MYCOBACTERIUM TUBERCULOSIS ISOLATION
TROUBLESHOOTING
Problem: Growth on the Slant within First Week of Incubation
Problem: Collapse of Slants
SUSCEPTIBILITY TESTING OF MYCOBACTERIUM TUBERCULOSIS
Pattern of Drug Resistance for Mycobacterium tuberculosis
Susceptibility Testing Methodology
Susceptibility Testing of Mycobacteria
Drug Concentrations for Proportion Method Susceptibility Testing using various culture media*
Drug Concentration (µg/ml)
PRIMARY/SECONDARY DRUG CONTAINING LOWENSTEIN-JENSEN MEDIA PANEL MTB SENSITIVITY TESTS
OTHER MARKERS
TROUBLESHOOTING LB
Problem: Growth not obtained on LJ control slant after diluting the culture suspense to 1:10000 of the Standard 0.5 McFarland turbidity
Problem: Collapse of Slants
IN DETERMINATION OF ADENOSINE DEAMINASE ACTIVITY IN SERUM, PLASMA AND BIOLOGICAL FLUIDS
TROUBLESHOOTINGS
Problem: False Positive Results
Problem: False Positive Results
Problem: Reagent not working
GRAM-NEGATIVE BACILLI
ACHROMOBACTERIACEA
ENTEROBACTERIACEAE
Methyl Red
Voges-Prokaeur Reaction
Citrate Test
Morphology
ENTERIC FEVER BACILLI-TYPHOID AND PARATYPHOID
Morphology
Cultures
Pathogenicity
Diagnosis of Enteric Infections
Blood Culture
Feces Culture
Urine Culture
Widal Reaction
ORGANISMS OF BACTERIAL ENTERITIDIS OR FOOD POISONING
Diagnosis of Salmonella Food Poisoning
GROUP OF DYSENTERY BACILLI
Morphology
Culture
BRUCELLACEAE
Morphology
Culture
Morphology
Culture
Morphology
Culture
Laboratory Diagnosis
HEMOPHILUS
PSEUDOMONADACEAE
Morphology
Culture
BACTERIACEAE
Morphology
Culture
Laboratory Diagnosis
Methods of Examination
Cultural Methods
SPIROCHETES
Primary Stage
Secondary Stage
Tertiary Stage
Morphology
Laboratory Diagnosis of Syphilis
Material Collection
Leptospira icterohemorrhagiae
Laboratory Diagnosis
Cultivation
Laboratory Diagnosis of Relapsing Fevers
GRAM STAINER
COLOR ATLAS—MEDIA AND COLONIES
QUALITY ASSURANCE IN BACTERIOLOGY
QUALITY CONTROL OF MEDIA AND STAINS
QUALITY CONTROL OF MEDIA
Sources of Media
Dehydrated Media
Dehydration with Additive
Commercially Prepared Media
Sources of Error
Inappropriate Medium
Water
Weighing
Dispensing
Proper Sterilization
Glassware
Quality Control
Physical Appearance
Sterility
Growth
Biochemical Response
Quality Control of Media
Selective Media
Spectrum of Quality Control
Performance of Plated Media
Quality Control of Stains
Quality Control of Bacteriological Techniques
Quality Control of commonly used media suggested control organisms and expected reactions
Quality Control procedures for commonly used tests
28:
Mycology
SUPERFICIAL MYCOSES AND DERMATOPHYTES
INTERMEDIATE SUPERFICIAL DEEP MYCOSES
DEEP OR SYSTEMIC MYCOSES
FUNGI USUALLY PRESENT AS CONTAMINANTS BUT WHICH RARELY CAUSE DISEASE—USUALLY IN PATIENTS CHRONICALLY ILL FROM OTHER DISEASES
MYCOLOGICAL METHODS
29:
Diagnostic Skin Test
TECHNIQUE OF SKIN TESTS
INTRACUTANEOUS INJECTION
TRANSCUTANEOUS ADMINISTRATION
PATCH TESTS
IMMUNOLOGIC BASIC FOR SKIN TESTS
TOXIN—ANTITOXIN NEUTRALIZATION
ANAPHYLATIC TYPE OF HYPERSENSITIVITY (IMMEDIATE REACTIONS)
TUBERCULIN TYPE OF HYPERSENSITIVITY (DELAYED REACTIONS)
COMMON SKIN TESTS
TOXIN—ANTITOXIN NEUTRALIZATION TESTS
Schick Test
1. Material
2. Technique
3. Interpretation
4. Test for Toxoid Sensitivity
Dick Test
Schültz-Charlton Reaction
IMMEDIATE REACTION TYPE OF SKIN TESTS
Test for Sensitivity to Horse Serum
1. Technique
2. Unfavorable Reactions following Serum Administration
3. If the Skin Test is Positive
4. Desensitization is Done as follows
5. Treatment of Severe Reactions
Direct Skin Tests for Allergens (Respiratory, contact, drug or food)
1. Technique
2. Interpretation
Prausnitz-Kustner Reactions
1. Technique
2. Interpretation
Penicillin Hypersensitivity
Foshay's Test
Extracts of Parasitic Worms (e.g. Trichinella)
DELAYED REACTION TYPE OF SKIN TESTS
Tuberculin Test (Mantoux Test, Pirquet Reaction)
1. Test Materials
Approximate Tuberculin Equivalents
2. Technique
3. Reading and Interpretation
Candida
Filariasis
Ducrey Test (Ito-Reenstierna test, chancroid skin test, dmelcos test; for the diagnosis of chancroid)
Brucellergen Test (For the diagnosis of brucellosis)
Tularemia Skin Test
Frei Test (For the diagnosis of lymphogranuloma venereum)
Mumps and Herpes Simplex Tests
Echinococcus Skin Test (Casoni reaction; for the diagnosis of hydatid disease)
Trichinella Skin Test (For the diagnosis for trichinosis)
Coccidioidin Test (For diagnosis of past infection with Coccidioides immitus)
Histoplasmin Test (For the diagnosis of past infection with Histoplasma capsulatum)
Blastomycin Test (For the diagnosis of past infection with Blastomyces)
Kveim-Siltzbach Test (For the diagnosis of sarcoidosis)
Toxoplasma Skin Test
Nonbacterial Regional Lymphadenitis (“Cat Scratch Fever”) Test
30:
Cytogenetics
INTRODUCTION
CELL DIVISION
BLOOD LYMPHOCYTE CULTURE
CHROMOSOME PREPARATION FROM BONE MARROW
CHROMOSOME PREPARATION FROM WHOLE BLOOD CULTURE
Harvesting of Cultures
Preparation of Chromosome Slide
Cleaning and Preparation of Slides
Procedure of Giemsa Staining
Preparation of Phosphate Buffer Solution
KARYOTYPING
G AND Q BANDINGS
G BANDING
Q BANDING (QUINACRINE BANDING TECHNIQUE)
C BANDING TECHNIQUE
IMPORTANCE OF CHROMOSOMAL STUDIES
VARIATIONS IN THE CHROMOSOME NUMBER
Polyploidy
Aneuploidy
BARR BODY ANALYSIS BUCCAL SMEAR FOR STAINING SEX CHROMATIN MASS
Appendix
INDEX
TOC
Index
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