our behind the scenes partner: the clinical laboratory carolyn fiutem, mt(ascp), cic apic chapter 26...
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Our Behind the Scenes Partner:The Clinical LaboratoryThe Clinical Laboratory
Carolyn Fiutem, MT(ASCP), CICCarolyn Fiutem, MT(ASCP), CICAPIC Chapter 26 – July 20, 2010APIC Chapter 26 – July 20, 2010
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Objectives:
• The attendee will be able to discuss at least 3 methodologies used in the microbiology lab for diagnosing infections/infectious diseases
• The attendee will be able to discuss the role of each of the 5 immunoglobulins on the patient’s response to an infection
• The attendee will be able to list at least 3 tests, not performed in the microbiology lab, used to aid in the diagnosing of infections/infectious diseases
• The attendee will be able to discuss the implications of various types of white blood cells (WBCs) on a differential
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Methodologies in Microbiology• Microscopy• Culture• Antigen Detection• Molecular Tests
Microbiology/Immunology Laboratory• Blood tests/Antibody Levels
Chemistry/Hematology• Cell Counts/Differentials/Chemistries
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Microscopy:
• Light• Fluorescent• Confocal• Electron
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Light Microscopy
• Bright-field – most common – uses incandescent light sources; stains
• Dark-field – examine fresh material – look for motile organisms not able to be stained, i.e. treponemes (syphilis); objects appear against a dark background for better resolution
• Phase-Contrast – examine fresh material – takes advantage of the different densities of cellular elements and makes them stand out; CSF examination for Naegleria
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Light Microscopy
Dark-Field
Phase-Contrast
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Fluorescent Microscopy• Utilizes a UV light
source• Coupled with
antibodies• Ease of interpretation• Quicker review of slide• Calcofluor White• Acridine Orange• Auramine-Rhodamine• DFA – Direct
Fluorescent Antibody
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Confocal Microscopy
• A type of fluorescent microscopy
• Uses a laser to provide the excitation light to get higher intensities
• Light eventually passes through a pinhole and is picked up by photomultiplier tubes
• Builder 3-D images
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Electron Microscopy
• Utilizes a beam of electrons produced by an electron-emitting tungsten filament
• Increased resolution• Special gold or
palladium stains used to prepare the specimens
• Images viewed on a computer monitor
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Cultures• Artificial Media – solid,
semi-solid, liquid• Tissue Culture
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Evaluate:• Colonial morphology• Cellular morphology• Growth rates• Growth conditions• Biochemical reactions• Susceptibility patterns
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Antimicrobial Susceptibility Testing
• Disk Diffusion (Kirby-Bauer)• Broth Dilution (MICs)• Disk Diffusion-Broth Dilution – E-test• Beta-Lactamase• Disk Approximation Test – D-Test for
inducible clindamycin resistance• Synergy Testing – Hodge Test for ESBL
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Epsilometer Test – E-Test
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Beta-Lactamase
D-Test
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Hodge Test
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Antigen Testing
• Molecular structures, proteins or polysaccharides
• stimulate the immune system to produce antibodies
• Utilize immunologic or serologic procedures• Early diagnosis before cultures positive or not
practical
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Antigen Testing Methodologies
• Agglutination Tests (i.e., Hemagglutination, Cold Agglutinin, MHA-TP)
• Precipitin Assays (i.e., Double Diffusion, Countercurrent Immunoelectrophoresis)
• Complement Fixation (Q-Fever)• Immunofluorescence (i.e., DFA, IFA)• ELISA (Enzyme-linked Immunosorbant Assay)• Radioimmunoassay (Hepatitis A)• Neutralization (C. difficile)• Immunoelectroblot (Western Blot)
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Molecular Techniques
• Gene Probes• PCR (Polymerase Chain Reactions)• RT-PCR (Reverse Transcriptase PCR)• PFGE (Pulsed Field Gel Electrophoresis)• Molecular Hybridization (Southern [DNA],
Northern [RNA], Western Blots [proteins])
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Immune System - Immunoglobulins• A complex of lymphoid
organs• Highly specialized cells• Circulatory system
separate from the blood circulatory system
• Lymph in Greek means a pure, clear stream
• Lymph – a fluid containing WBCs, primarily lymphocytes
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Four Primary Functions
• Recognition of self^self tolerance^immunologic privilege
• Immunosurveillence• Intracellular
Hormones• Defense against
infection
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Immune System Components
B-Cells• Mature in bone marrow• Produce antibodies• Part of antibody-
mediated (humoral) immunity
• Greeks called blood and lymph the “body’s humors”
T-Cells• Mature in the thymus• Attack and destroy• Responsible for cell-
mediated (cellular) immunity
• Regulate and coordinate the overall immune response
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Types of Antibodies
• Isotypes:IgMIgGIgAIgEIgD
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IgM – there to Mop up the infection
• Largest antibody• Most important for IPs• Indicates current disease process• First at the site of initial exposures
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IgG – I got it and its Gone
• Second at the site of the first exposure• First at the site of subsequent exposures
• Acute and Convalescent Sera1. Acute sera with elevated IgM2. Convalescent sera with 4-fold rise in titer
• Single specimen with elevated IgG just means there has been an infection
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IgA
• Secreted on the lining of the mucous membranes
• Protection against respiratory and gastrointestinal (GI) infections
• Infants have an immature IgA system, hence infections with agents like RSV and Rotavirus
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IgE
• Least abundant immunoglobulin• Works with basophils and mast cells• Involved in allergic reactions and immediate
hypersensitivity• Mast cells – reside in connective tissues,
release granules rich in histamine and heparin
• Benefit in wounds healing and defense against pathogens
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IgD
• Secreted in small amounts in serum by the B-lymphocytes’ cell surface, with IgM
• Responsible to signal the B-cells to be activated
• Also binds to basophils and mast cells to activate them
• They produce antimicrobial factors to participate in the respiratory immune defense
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Mechanical Action of Immune System• Skin: fatty acids (pH 1), competitive
colonization, enzymes (hydrolyze)• Flushing: tears, saliva, micturation,
peristalsis, coughing• Antimicrobial Agents: enzymes, bile salts,
gut pH• Mucous Membranes• Inflammatory Response• Fever
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Vascular Phase Inflammatory Response
• Pathogen invades• Tissue damage results• Causes degranulation of mast cells• Release of heparin & histamine into the area• Increased blood flow to the area• Results in dilated capillaries• We see “red skin”
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Cellular Phase Inflammatory Response
• Phagocytic cells squeeze through dilated capillaries
• Temperature at site increases• Bradykinin & serotonin release, causing
edema• Fibrin leaks into area; clot formation• Enzymes and dead WBCs form pus• Goal to wall off infection and prevent
spreading
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Staphylococcal organisms cause a rapid inflammatory response resulting in a localized infection
Streptococcus organisms have a slow response, allowing for disseminated infections
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Immune Suppression• Genetic deficiencies• Drug-induced• Cancer-induced – alters electrical charges of the
brain (bidirectional effect b/w brain and immune system) – psychoneuroimmunology
• Viral Infections – similar to cancer-induced• Bacterial Infections• Malnutrition• Stress• Iatrogenic Factors
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Immune System – Friend or Foe?
• Allergy – response to a false alarm• Auto-immune Diseases• Antigen-Antibody Complex – adverse effect occurs
when the infection is overwhelming• DIC (Disseminated Intravascular Coagulation) – over
activation of complement• Cytokine Storm – result of persistent fever; releases
tumor necrosis factor (TNF) and interleukins resulting in hypoglycemia and shock
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Non-Specific Immunity
• Leukocytes• Complement:Complex linear proteinCalled an “effector” moleculeIt helps to stimulate some immune activities
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Neutrophils (Polys)
•Most dominant WBC (40-70%)
•“First Responder”
•Acts against pyogenic (pus-forming) bacteria
•Life expectancy ~ 7 hours in circulatory system
•Large reserve in bone marrow
•Leukopenia – can be poor prognostic indicator
•Hypersegmentation – suggest B12 or folate deficiency
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Neutrophil RatiosDegenerative Left Shift – increase in bands with no leukocytosis; poor prognosis
Regenerative Left Shift – increase in bands with leukocytosis; good prognosis
Right Shift – few bands with increase in segmented neutrophil seen in liver disease, hemolysis, drugs, cancers, allergies, or megaloblastic anemia
Hypersegmentation – with no bands is seen in megaloblastic anemia and chronic morphine addiction
Myeloid Left Shift – Bands, Metamyelocytes (Metas), Myelocytes, Promyelocytes (Pros), Blasts
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Neutropenia
•Acute overwhelming bacterial infections – poor prognosis
•Viral infections
•Rickettsial and some parasitic diseases
•Drugs, chemicals, radiation, toxic chemicals
•Anaphylactic shock
•Severe renal disease
•Sepsis due to E. coli – reduced survival of polys
•Hormonal Disorders
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Neutropenia in Neonates
•Maternal neutropenia
•Maternal drug ingestion
•Maternal isoimmunization to fetal WBCs
•Inborn errors of metabolism (i.e., maple syrup urine disease)
•Immune deficits
•Myeloid disorders
•Defective intrinsic factor secretion
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WBC Abnormalities…
• Toxic Granulation – infections or inflammatory diseases – WBC is active
• Dohle Bodies – infections, inflammatory dieases, burns, Myeloproliferative disorders, cyclophosphamide therapy
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Lymphocytes
• 25-40% of WBCs• Fight viral infections• Pertussis• Chronic granulomatous
diseases, i.e., TB• Crohn’s disease• Ulcerative Colitis• Addison’s Disease• Brucellosis
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Lymphopenia
• Chemotherapy• After administration of cortisone• Obstruction of lymphatic drainage, Whipple’s
disease or tumors• Hodgkin’s disease• HIV/AIDS• Trauma
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Monocytes
• Fight severe infection via phagocytosis
• 3-7% of WBCs• Bacterial infections• TB• SBE• Syphilis• Parasitic, fungal,
rickettsial diseases
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Basophils• 0-1% of WBCs• Mast Cells are tissue basophils• Secrete histamine, seratonin,
& prostaglandins – increase blood flow to area
• Hodgkin’s Disease• Parasitic infections• Inflammation• Allergy• Sinusitis• After splenectomy• TB• Smallpox, Chickenpox• Influenza
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Eosinophils
• 1-4% of WBCs• Are cytotoxic• NAACP….• Neoplasm• Asthma/Allergy• Addison’s Disease• Collagen/Vascular
Disease• Parasitic Infections• Chronic Skin Conditions
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Absolute WBC Counts• Relative Number = percentage• Absolute Count = Percentage X Total WBC Ct.• Can have normal WBC count yet be neutropenic• Need to look at WBC count and differential• Normal WBC ranges:1. Adults ~ 3.5-10, 0002. Newborns ~ 9-30,0003. 2 weeks ~ 5-20,0004. 1 yr ~ 6-18,0005. 4 yr ~ 5500-17,0006. 10 yr ~ 4500 – 13,500
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Cell Count and Differential• CSF should be clear &
colorless• Glucose 40-70 mg/dl• Protein 15-45 mg/dl• CSF Glucose = ~2/3
serum glucose• Bacterial Meningitis:1. WBC = increased2. Diff – neutrophils3. Protein = marked
increase4. Glucose = markedly
decreased
• Viral (Aseptic) Meningitis:1. WBC = increased2. Diff – lymphs3. Protein = moderate increase4. Glucose = Normal• TB/Fungal Meningitis:1. WBC = increased2. Diff – Lymphs and Monos3. Protein = moderate to
marked increase4. Glucose = Normal to
decreased
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C-Reactive Protein (CRP)
• Protein virtually absent from serum of healthy person
• Seen in inflammatory process• Classical, most dramatic acute-phase reactant• Present within 18-24 hours of tissue damage• Can be used for monitoring wound healing,
especially burns and organ transplant• Normal = ~<0.8 mg/dl
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Liver Enzymes
• ALT/SGPT – Alanine Aminotransferase/Serum Glutamic-Pyruvic Transaminase
• AST/SGOT – Aspartate Transaminase/Serum Glutamic-Oxaloacetic Transaminase
• Total Bilirubin• Direct Bilirubin• GGT – Gamma-Glutamyl Transpeptidase• Alkaline Phosphatase
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ALT/SGPT AST/SGOT
Elevated in:• Viral/infectious
hepatitis• Infectious Mono• Severe Shock• BurnsDecreased in:• UTI• Malnutrition
Elevated in:• Acute/chronic hepatitis• Infectious Mono• Trama• Trichinosis• Cardiac Cath• Gangrene• Mushroom poisoningDecreased in:• Chronic Renal Disease
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Bilirubin
Total Bili – damage to parenchymal cells; viral hepatitis, Infectious Mono
Indirect Bili (unconjugated) – protein bound bilirubin; pulmonary infarcts, large hematomas
Direct Bili (conjugated) – circulates freely; pancreatic cancer
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Gamma GT Alk Phos
Elevated in:1. Hepatitis2. Alcoholism3. Infectious Mono
Normal in:1. Bone growth2. Strenuous exercise3. Renal Failure
Elevated in:1. Hepatitis2. Infectious Mono3. Diabetes mellitus4. Ulcerative colitis5. Bowel perforation6. Chronic renal failure
Decreased in:1. Malnutrition
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D-Dimer
• Are produced only by the action of plasmin on cross-linked fibrin (clots)
• Presumptive evidence of DIC• Normal = <250 ng/ml• Positive after surgery• Late in pregnancy, postpartum
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Fibrinopeptide A (FPA)
• To determine thrombin actionIncreased in:• DIC• Infections• Post-op patients
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Platelet Counts
Increased in:• Splenectomy• Acute infections, inflammatory diseases• TB• Renal FailureDecreased in:• Viral, bacterial, rickettsial infections• HIV• DIC• Hypersplenism
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Sepsis• An exaggeration of the body’s normal response to
infection• Activate coag cascade• Acute renal failure/Oliguria• ARDS• Oxygen demand can increase 12 fold• Decreased platelet count• Increased aPTT & INR• Decreased pH• Increased PaCO2 (hypoxemia)
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BUN Creatinine
• The amount of urea excreted varies directly with dietary protein intake, increased excretion in fever, diabetes and adrenal gland activity
• Increased with UTI• Decreased in hepatitis
• By-product in the breakdown or muscle creatine phosphate
• Increased in impaired renal function
• Decreased in severe liver disease, pregnancy
• Decreased BUN:Creatinine ratio in renal failure
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Lactate/Lactic Acid
Increased in:• Sepsis/Shock• Liver disease• Diabetes• Pulmonary Failure
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Cortisol
Increased in:• Pregnancy• Stress (trauma, surgery)• ObesityDecreased in:• Hepatitis• Cirrhosis• Taking steroids
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Ammonia
• An end-product in protein metabolism• Bacteria act on intestinal proteinsIncreased in:1. Liver Disease2. Sepsis/Shock3. Renal Disease4. GI tract infection with distention and stasis
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Diarrhea
Metabolic Acidosis:1. Decreased pH2. Decreased Bicarbonate3. Normal PaCO24. Hyponatremia5. Hypokalemia (if dehydrated, hyperkalemia)
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Vomiting
Metabolic alkalosis:1. Increased pH2. Increased Bicarbonate3. Normal PaCO24. Hyponatremia5. Hypokalemia
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