Clinical Pathology Ain Shams

Table of contents :
Cover
......Page 1
Preface
......Page 3
Intended Learning Objectives 1......Page 8
Red Blood Cells 2......Page 9
-- Red Blood Cell Abnormalities 3......Page 10
-- Microcytic Hypochromic Anaemia 4......Page 11
-- Macrocytic Anaemia 6......Page 13
-- Anaemia of Chronic Disorders 7......Page 14
-- Haemolytic Anaemia 8......Page 15
Polycythaemia 14......Page 21
Erythrocyte Sedimentation Rate 15......Page 22
Leucocytes 16......Page 23
-- Reactive Disorders of Leucocytes 18......Page 25
Leukemias 20......Page 27
-- Acute Leukemia 21......Page 28
-- Chronic Leukemia 22......Page 29
Multiple Myeloma 25......Page 32
Myelodysplastic Syndrome 26......Page 33
Hypersplenism 27......Page 34
Bone Marrow Examination 28......Page 35
-- Physiology of Haemostasis 29......Page 36
-- Laboratory Evaluation of Homeostatic Function 34......Page 41
-- Vascular Disorders 37......Page 44
-- Thrombocytopenia 38......Page 45
-- Qualitative Platelet Disorders 39......Page 46
-- Coagulation Disorders 40......Page 47
Blood Transfusion 43......Page 50
-- Bilirubin 50......Page 57
-- Serum Enzyme Assays 53......Page 60
-- Serum Proteins , Albumin & Prothrombin time 54......Page 61
Diabetes Mellitus 56......Page 63
-- Plasma Glucose 57......Page 64
-- Diabetes mellitus 58......Page 65
-- Laboratory Diagnosis 59......Page 66
-- Monitoring of Glycaemic Control 61......Page 68
-- Oral Glucose Tolerance Test 63......Page 70
Disorders of Plasma Lipids and Lipoproteins 66......Page 73
-- Lipoprotein Disorders 67......Page 74
Renal Diseases 69......Page 76
-- Urine Analysis 70......Page 77
-- Kidney Function Tests 74......Page 81
-- Biochemical Findings in Some Renal Disorders 78......Page 85
-- Respiratory Disturbances 79......Page 86
-- Metabolic Disturbances 80......Page 87
Sodium 81......Page 88
Potassium 82......Page 89
-- Cardiac Enzymes in Diagnosis of Myocardial Infarction 84......Page 91
-- Acid Phosphatase 87......Page 94
Clinical Disorders of Calcium and Phosphate 88......Page 95
-- Serum Phosphorus 89......Page 96
-- Hyperparathyroidism 90......Page 97
-- Hypoparathyroidism 91......Page 98
-- Metabolic Bone Diseases 92......Page 99
Thyroid gland 93......Page 100
-- Thyroid Function Tests 94......Page 101
-- Hyperthyroidism 96......Page 103
-- Non-Thyroidal Illness and the Sick Euthyroid Syndrome 97......Page 104
Autoimmune Diseases 99......Page 106
-- Systemic Lupus Erythematosus 101......Page 108
-- Thyroid Autoimmune Diseases 103......Page 110
-- Disorders of the Pancreas 104......Page 111
Immunological Diagnosis of Liver Diseases 105......Page 112
-- Acute Viral Hepatitis 106......Page 113
-- Chronic Hepatitis 108......Page 115
Hypersensitivity 110......Page 117
Immunologically Mediated Renal Diseases 112......Page 119
Immunodeficiency Diseases 113......Page 120
Miscellaneous Immunological Diseases 116......Page 123
Tumor Markers 117......Page 124
Normal Flora 119......Page 126
Microbilogy Laboratory and Pre-analytical Variables 122......Page 129
Diagnosis Of Infectious Diseaeses 126......Page 133
-- Central Nervous System (CNS) Infections 127......Page 134
-- Body Fluid Effusion 129......Page 136
-- Blood Stream Invasion 130......Page 137
-- Upper Respiratory Tract Infections 136......Page 142
-- Lower Respiratory Tract Infections 137......Page 143
-- Tuberculosis (T.B) 138......Page 144
-- Gastrointestinal Tract Infections 139......Page 145
-- Pyrexia of Unknown Origin 139......Page 146
-- Brucellosis 140......Page 147
-- Lower Genital Tract Infections and Sexually Transmitted Disease 141......Page 148
-- Transplacental Infections 144......Page 151
-- Abscesses and Wound Infections 145......Page 152
-- Anerobic Infections 146......Page 153
Antibiotics 148......Page 155
Infection Control and Safety Measures 151......Page 158
-- Jaundice (1) 157......Page 164
-- Jaundice (2) 158......Page 165
-- Diabetes Mellitus 159......Page 166
-- Hypothyroidism 160......Page 167
-- Hyperthyroidism 161......Page 168
-- Rheumatic Diseases 162......Page 169
-- Acute Hepatitis 163......Page 170
-- Chronic Hepatitis 164......Page 171
-- Immunodeficiency 165......Page 172
-- classification of bacteria 166......Page 173
Question Sheet
......Page 179
Curriculum
......Page 180
Q Arranged
......Page 181

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TABLE O F C O N TE N TS ... .• ... · ••· ·· · .. · •• •· · ·•. ... ... ... : es tiv ec bj O ng ni ar Le Intended Ch ap te r I. Haematology:

1

2

Red Blood Cells: ... .. 3 ... ... · · · · · · · · · · · · · · · · · · · · · · · · · · · ·.. ... 3 ... . ... .. ... ... ... ... is ies po ro ... .... Eryth s .. . . . .. . .. . . . . . .. .. .. . .. .. .. .. .... 4 Red Blood Cell Abnormalitie s er et m ra Pa ll Red Blood Ce Anaemias: 4 of Anaemia Morphological Classification 4 mia Microcytic Hypochromic Anae 6 Macrocytic Anaemia 7 aemia Normochromic Normocytic An 7 Anaemia of Chronic Disorders 8 Haemolytic Anaemia 14 Polycythaemia 15 Ra te Er yt hr oc yt e Se di me nt ati on

~ -----16

------·----- Leucocytes: ytes Reactive Disorders of Leucoc Infectious Mononucleosis Lc uk ac mi as : Acute Leukemia Chronic Leukemia M ul tip le Myeloma e Myelodysplastic Sy nd ro m Hy pe rsp len ism Bone M ar ro w Ex am in ati on Ha cm os tas is: Physiology of Haemostasis meostatic Function Laboratory Evaluation of Ho Bt ec dm g D1sordcrs: Vascular Disorders Thrombocytopenia Qualitative Platelet Disorders Coagulation Disorders Bl oo d Tr an sf us io n

18 20 20 21 22

25 26 27 28 29 29 34

""31 37 38 39 40 43

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Ch apt er 2. Clinical Chemistry: Hep atob iliar y Dis orde rs: . Bilirubin . Serum Enzyme Assays . Serum Proteins , Allumin & Prothuombim time Dia bete s Mel litus : Plasma Glucose Diabetes mellitus Laboratory Diagnosis Monitoring of Glycaemic Control Oral Glucose Tolerance Test

50 50 53

54 56 57 58

59 61

63

Dis orde rs of Plas ma Lipi ds and Lip opro tein s: Lipoprotein Disorders coal Diseases: Urine Analysis Kidney Function Tests Biochemical Findings in Some Renal Disorders Acid Bas e Bal anc e: Respiratory Disturbances Metabolic Disturbances

66 67

69 70

74 78 79 79

80 81

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Pota ssiu m Usc of Enz yme s in Clin ical Diagnosis: rction Cardiac Enzymes in Diagnosis of Myocardial Infa / Acid Phosphatase Serum Amylase Clinical Dis orde rs of Cal cium and Pho sph ate: Serum Calcium Serum Phosphorus Hyperparathyroidism Hypoparathyroidism Metabolic Bone Diseases

~gland :)_

82 84 87 88

88 89 89 90 91 92

93

yroid Function Tests: Hypothyroidism Hyperthyroidism drome Non -Thyroidal Illness and the Sick Euthyroid Syn I

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96 96 97

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/ Chapter 3. Clinical Immunology: 99 101 103 103 104 lOS lOS lOS 106 108 110 110 112 113 116 117

Autoimmune Diseases: Systemic Lupus Erythematosus Rheumatoid Arthritis Thyroid Autoimmune Diseases Disorders of the Pancreas Immunological Blood Diseases Myasthenia Gravis

Immunological Diagnosis of Liver Diseases: Acute Viral Hepatitis Chronic Hepatitis Hepatocellular Carcinoma

Hypersensitivity Immunologically Mediated Renal Diseases Immunodeficiency Diseases Miscellaneous Immunological Diseases -Tumor Markers

Chapter 4. Clinical Microbiology: ormal Flora icrobilogy Laboratory and Pre-analytical Variables iagnosis Of Infectious Diseaeses Central Nervous System (CNS) Infections Body Fluid Effusion Blood Stream Invasion Urinary Tract Infections Upper Respiratory Tract Infections Lower Respiratory Tract Infections Tuberculosis (T.B) Gastrointestinal Tract Infections Pyrexia of Unknown Origin Brucellosis Lower Genital Tract Infections and Sexually Transmitted Disease Transplacental Infections Abscesses and Wound Infections Anerobic Infections

Antibiotics Infection Control and Safety Measures

119 119 122 126 127 129 130 135 136 137 138 139 139 140 141 144 145 146 148 151

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Chapter 5. Flow charts: Flow chart for Investigating Jaundice (I) Flow chart for Investigating Jaundice (2) Flow chart tor Investigating Diabetes Mellitus Flow chart tor Investigating llypothyroidism Flow chart for Investigating Hyperthyroidism Flow chart for Investigating Rheumatic Diseases Flow chart for Investigating Acute Hepatitis Flow chart for Investigating Chronic Hepatitis flow chart tor Investigating Immunodeficiency Flow chart for classification ofbacteria.

157 158 159 160 161 162 163 164 165 166

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Hacm atolog y

---------------------------------------• M Q Extre me rise in ESR may be

seen in: Yelom a . . ---.--1. • M~araprotemaemia, and macroglobu maemt·a align a.nr- y---. • C~_Ilagen diseas es • ............... Activ e TB

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Clinical Chemistry

immunoglobulins by the reticuloendothelial system in addition to their decreased clearance. • serum albumin is decreased in the following conditions: a. Inadequate intake e.g., malnutrition. b. Decreased absorption e.g., malabsorption syndrome. c. Increased need e.g., pregnancy. d. Impaired synthesis e.g., liver diseases, chronic infections. e. Increased breakdown e.g., neoplasms, infections, trauma. f. Increased loss e.g., oedema, ascites, bums, nephrotic syndrome, protein- losing enteropathy. g. Congenital deficiency.

J.Scrum Immunoglobulins • In chronic liver diseases there is usually an overall increase in serum lgs. • In most types of cirrhosis, serum IgA is moderately increased and there are usually smaller increases in lgG and lgM. • In primary biliary cirrhosis, serum IgM increases greatly. • In chronic active hepatitis, serum lgG tends to be the most increased. NB: In liver cirrhosis serum protein electrophoresis shows hypoalbuminaemia and polyclonal hypergammaglobulinaemia with betagamma bridging (polyclonal gammopathy) (fig 2.2).

Serum Protein Electrophoresis l__ - - ·-- - - -

-- .

-

- - · -··- - - - - · -·· -- - - -

Liver Cirrhosis Hypoalbuminemia Polyclonal hypergammaglobulin emia Albumin

with

13- y bridging

Fig 2.2: serum protein electrophoresis in liver cirrhosis

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Clinical Chemistry 4·Pro thro

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•The liver is the majo r site of synthesis of co~gulation facto rs. Because ~ost of these factors are normally present ~~ exc~s s concentrations unpa ired coag ulatio n is usually seen in severe h:er dtsease o~ly. ' • Abno nnali ties of these factors can be most effictently determme d by th P~othrombin time (PT), which requires the integrity of most of th: VItamin K-dependent coagulation factors (II, VII, I_)C and X). PT is thus depe nden t on normal hepatic synthesis of coagulation factors as Well as sufficient intestinal uptake of vitamin K. • Prot hrom bin time may be prolonged in liver disea se due to: . a- Impa ired synthesis of one or more of the coagulatiOn factor s in sever e acute or chronic hepatocellular damage. Beca use these proteins ha~e a shorter half-life than that of albumin, PT. may be an earlier indic ator of severe liver injury than serum albumin and can be used to assess progress and prognosis. Prolongation in PT in such cases is a bad progn ostic finding. b- Deficiency offat soluble vitam in-K due to failure of absorption of lipids caused by cholestasis. This condition can be distinguish ed from hepa tic synthesis failure by demonstrating normalization of PT 24 - 48 · hrs after parenteral injection of vitamin K

DIABETES ME LLI TUS INTENDED LEARNING OBJECTIVES: 1. Define diabetes mellitus. 2. Identify the reference intervals of plasm a glucose in healthy subjects. 3. Enum erate the blood sample specifications for glucose assay . 4. List the various types of diabetes mellitus and other categ ories of glucose intolerance . 5. Enumerate the diagnostic criteria of diabetes mellitus and other categories of glucose intolerance . 6. Reco gnize the value of urine analysis in diabetes mellitus and its limitations. 7. Selec t the appropriate test to monitor glycaemic control; namely glyca ted haemoglobin and fruetosamine . . 8. Reco gnize the importance of glucose meters i-:-, self-monitoring of glyca emic control, and the difference betvreer whole. blood and plasm a gluco se levels.

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Clinical Chemistry

9. Enumerate the metabolic complications of diabetes mellitus. 1o.List the various metabolic derangements occurring in diabetic ketoacidosis and their underlying pathophysiologic mechanisms. lt.Enumerate the indications, normal criteria, and abnormalities of the oral glucose tolerance test (OGTT), the diagnostic criteria and causes of these abnormalities. 12.List the necessary precautions when performing an OGTI, and how to do the test. 13. Recognize who should be screened for gestational diabetes mellitus, when and how to do the screening, and how to perform the definitive test and interpret its results.

I- PLASMA GLUCOSE: A- Reference Intervals of Plasma Glucose in Healthy Subjects: 1- Fasting Plasma Glucose: - < 100 mg/dL

-Fasting means no caloric intake for at least 8 hours. 2- 2-hr Postload or 2-hr Postprandial Plasma Glucose: - < 140 mg/dL

- Glucose load is 75 g anhydrous glucose for a non-pregnant adult, and 1.75 g/Kg body weight for a child (Maximum 75 g). 3- Random Plasma Glucose: - < 200 mg/dL

- Random means at any time of the day, irrespective of the last meal.

8- Blood Sample Specifications: • Serum or plasma free of haemolysis. • Common anticoagulants as heparin, EDTA, citrate, and oxalate cause no interference. • Glycolysis decreases serum glucose by 5-7 % in 1 hour (5-10 mg/dL) in normal uncentrifuged coagulated blood at room temperature. This rate increases in the presence of leukocytosis or bacterial contamination. Hence, prompt separation of senim or plasma from RBCs is mandatory.

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Clinica l Chemi stry • In separated, non-haemolysed serum samples, glucose is stable for 8 hours at 25°C and 72 hours at 4°C.

II-DIABETES MELLITUS: A- Definition Diabetes mellitus (DM) is a state of diminished insulin act.ion which occurs due to decreased availability or diminished effectiveness of the produced insulin.

B- Classification of DM and Other Categories of Glucose Intole rance 1. Type 1 DM: Previously referred to as insulin-dependent DM (IDDM), juvenile type, or type I DM. 2. Type 2 DM: Previously referred to as non-insulin-dependent DM (NIDDM), maturity-onset type, or type II OM. 3. Other Specific Types of DM: Previously referred to as secondary DM. Hyperglycaemia is due to a specific underlying disorder, e.g., a. Diseases of the pancreas, e.g., trauma, pancreatectomy, pancreatitis, pancreatic tumours, and haemochromatosis. b. Hyperfunction of the endocrine glands producing the insulin antagonists, e.g., Cushing, acromegaly, phaeochromocytoma, glucagonoma, thyrotoxicosis, and prolactinoma. c. Drugs or hormones known to cause hyperglycaemia, e.g., oral contraceptives, thiazide diuretics, and corticosteroids. d. Stress. c. Cerebrovascular accidents. f. Certain genetic syndromes, e.g., Down' s syndrome, Turner's syndrome, porphyria, and Klinefelter' s syndrome. g. Insulin receptor abnormalities. 4. Impair ed Glucose Tolera nce: - This term applies to individuals who have a fasting plasma glucose level less than that required to diagnose diabetes mellitus, i.e. Clinical Chemistry

•Renal causes: Loss of functioning ncphrons, e.g., acute or chronic glomerulonephritis, and chronic renal failure. •Post- renal causes: Increased pressure in the tubular side of the nephron, e.g., prostatic enlargement.

•!• SERUM UREA Serum is the end product of protein metabolism. Reference Range: 15-40 mg/dL BUN = Blood urea nitrogen= Urea divided by 2.14 Causes of Increa sed Serum Urea: J- Pre-renal causes (increased format ion) • High protein diet. • Dehydration. •Congestive heart failure. •Catabolic states, e.g., fever, trauma and bums. •Gastrointestinal tract haemorrhage. •These conditions are accompanied by normal serum creatinine level 2- Renal causes (decreased excretion): same as creatinine Both urea & creatinine are increased proportionally. 3- Post-renal causes Obstruction of the urinary tract by: • Stone in the ureter. •Enlarged prostate . Both urea & creatinine are increased but the increase in urea is greater than the increase in creatinine.

•!• SERM URIC ACID It is the end product of purine and pyrimidine metabolism. Reference Range: 2 - 6 mg/dl in females. 3 - 7 mg/dl in males. Causes of Increas ed Serum Uric Acid: 1- Overproduction of uric acid • Essential hypeniricaemia: due to increased activity of pathways, which lead to the formation of urate. • Increased rate of nucleic acid breakdown due to increased cell hrrn-over or dcslruction (as in leukaemia). 2- Defective elimination of uric acid: •In chronic renal failure, with decreased GFR.

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\ Clinical Chemistry

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•ln ketoacidosis, and lactic acidosis.

b. Glomerular permeability_: filter of blood that restricts the p The glomerulus acts as a selecuve . of molecular weights great assage of macromolecules. In general protetnl~merulus. The nonnal urina er than 66 K.Das are retained by the healthy TdL & is made up of mostly a~ total protein excretion is less than 150 mg lltnj11 and some smaller proteins. Causes of Glomerular Proteinuria: .d s diabetes mellitus •Systemic diseases which affect the kt ney.da . . 'systCtnic d 'd · amylot osts; an rna1tgnancy lupus erythematosus, sarcot osts, , e.g., Hodgkin's lymphoma, multiple myeloma. hr. . h . • Renal disease: acute or chronic glomerulonep ths, nep rohc syndrotne ' graft rejection. d' . h .. . S b ute bacterial endocar ttts, epatttts B . • I n fi·echons: rna1ana, u ac · •Drugs/ toxins: Lithium. • Pre-eclampsia. Type of proteins:

. . Usually albumin (moderate molecular wetght). Wtt? pr~gress of the condition, high molecular weight proteins also appear m unne. N.B: In all the above cases, proteinuria may be present alone or in addition to casts which indicate kidney disease. '

2. Assessment of Tubular Function Sometimes the tubules are affected as part of a systemic di~ease, or as a result of congenital abnormalities. a. Loss of reabsorptive capacity of the proximal convoluted tubules • Aminoaciduria, e.g. cystinuria, Fanconi syndrome. • Glucosuria: renal glucosuria (benign condition). • Phosphaturia: e.g., vit D resistant rickets due to loss of P04 in unne. • Proteinuria: this is usually low molecular weight proteins. e.g., (a1• micr~globulin, B2-microglobulin, lysozyme, retinol-binding protem). In some cases, there are small amounts of albumin. All the above proteins need special tests for their detection in urine b. Concentrating power ofthe tubules: This can be tested by: • Measuring the specific gravity of a morning sample (most concentrated sample of the day). This is usually ~1.020.

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Clinical Chemistry

• fulecific g~vi~y of morni~g .sample after overnight fluid restriction. • fluid dcpnvahon test: Th1s Is done on an in-patient. • ~opressin test: This is done if there is no response in the water deprivation test.

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. pOLYURIA: a unne volume > 2000 mL /24hours • as defined . Jt IS . . . causes: • Diabetes mellitus: Polyuna +Increased specific gravity of urine. .chronic renal failure: Polyuria + fixed specific gravity of urine around

1 :I

1.010.

• Hysterical p~l~dypsi~: ~olyuria + dec.reased specific gravity. • Diabetes insipidus (Pituitary): Polyuna +decreased specific gravity. • Diabetes insipidus (nephrogenic): Polyuria+ decreased specific gravity.

Hysterical polydypsia: responds to water deprivation and to vasopressin test.

I

Pituitary diabetes insipidus does not respond to water deprivation, but responds to vasopressin test. Nephrogenic diabetes .inspidus does not respond to either water deprivation or vasopressm test. PROTEINURIA: It is defined as an increase in the amounts of protein in urine. Types: J- Glomerular proteinuria. 2- Tubular proteinuria. 3- Overflow proteinuria. 4- Asymptomatic proteinuria: Protetn appears transient in urine in normal persons in the following conditions:• Excessive exercise • Exposure to cold •Orthostatic proteinuria (prolonged standing). 5- Post.,Renal Proteinuria: It refers to protein arising from the urinary tract below the kidney. Urine contains proteins + cells (white cells, red cells, malignant cells) It is caused by:

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Clinical Chemistr y

• Infection. • Inflammation. • Haemorrh age. • Degenera tive lesions.

Biochemical Findings in Some Renal Disorders CHRON IC RENAL FAILUR E (CRF): Serum finding: • Increased serum. levels of urea (BUN), creatinine, and uric acid. • Increased organic acids~ metabolic acidosis ~ J.. pH of blood. • Increased serum magnesium (Mg). • Decrease d serum sodium. ./• • Increased serum potassium. ~ • Increased serum phosphorus due to decreased G .F .R. ' ..~r ~ " • Decrease d serum calcium due to reduced activity of the renal enzyrn responsib le for 1a-hydroxylation of 25(0H) cholecalciferol (vit D~ Calcium may be corrected to normal due to . t~e oc~u~ence of 2ry hyperpara thyroidism . If calcium is increased, this IS an mdtcation of the occurrence of bone disease or 3ry hyperpara thyroidism or overtreatment by active vitamin D and Ca.

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Urine finding: • Polyuria + fixed specific gravity ( 1.01 0). • Proteinur ia. •Haematu ria, pyuria and granular and cellular casts.

ACUTE GLOME RULONE PHRITIS (NEPHR ITIC SYNDROME): Causes: 1. . Following infections, the most common is streptococ cal infection. It represents hypersensitivity reaction to streptococcal protein immune complexes, which deposit on the basement membrane of Bowman' s capsule. 2. As a part of multi-system disease, e.g. SLE, polyarteritis nodosa. 3. As a complicat ion ofHenoch -Schonlei n' s purpura.

Serum jinlling: • Increased serum levels of urea~ BUN, and creatinine . • Diminish ed creatinine clearance.

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Clinical Chemi stry

Urine finding: .oliguria. • High urine specific gravity. • proteinuria ( 3g/day) proteinuria heavy include: criteria Diagnostic bypoalbuminaemia, hypercholesterolaemia and oedema. Serum finding: • Decreased serum albumin and total protein . • serum urea and creatinine are usually normal at presentation. • Increased alpha2_macroglobulin and beta globulins. •Hyperlipidaemia: increased serum cholesterol and triglycerides due to increased synthesis of VLDL in the liver, hence leading to increased circulating levels ofVLDL, IDL, and LDL. Urine finding: •Heavy proteinuria due to increased glomerular permeability (>3g/day). •Lipoid casts (fatty casts). •Mild microscopic haematuria may be present.

ACID BASE AND ELEC TRO LYTE DISTURBANCES Reference Range (arterial): pH= 7.35- 7.45 Serum bicarbonate (HC03): 21 - 28 mmol/L : 34 - 46 mmHg Blood PC02 I. Respiratory Disturb ances:

•Respiratory disturbances will produce initial changes in PC0 2, and compensatory changes of bicarbonate concentration will occur in the same direction, thus limiting the degree of pH change.

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Clinical Che mis try •In re_spira tory failure, the PC0 rises thus tendin~ to I_o wer the 2 (res ptra tory acidosis) but the bicarbonate concent ration Will also . Pli com pen sate . nse to • In_ove r-ve ntila tion , on the other hand, the exce ssive washout of C Will cau se PC02 to fall and the reverse chan ges occur: respirat 02 alka losi s with compensatory fall in bicarbonate. ory

A. Cau ses of Respiratory Acidosis: Thi s incl ude s all conditions leading to respirato ry failure: 1. Lun g dise ases : e.g., chronic bronchitis ~nd emphysema, ede~a, pul~onary fibrosis, pneumoma, advanced pulrnona tuberculos~ resp irato ry dtstress syndrome. ' 2. Airw ay obstruction and suffocation. 3. Diso rder s of thoracic cage: e.g., trauma or kyphoscoliosis. 4. Neu~omuscular disorders affecting respirato ry movements, e.g. Polio or h1gh spinal injuries . 5. Res pira tory center disturbances, e.g., drug (overdose) and cere brov ascu lar accidents. B. Cau ses of Respiratory Alkalosis: Thi s incl ude s conditions leading to over-ventilat ion: 1. Cen tral: e.g., stimulation of respiratory cent er by hypoxia, anxiety salicylates. ' 2. Peripheral: Improperly controlled mechani cal ventilation.

II. Metabolic Disturbances: Met abo lic disturbances involve primary chan ges in the level of bica rbon ate with a compensatory respiratory response. In metabolic acid osis , bica rbon ate level decreases thus tend ing to lower the pH with a com pen sato ry redu ctio n in PC0 . 2

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A. Cau ses of Metabolic Acidosis: Thi s may be due to retention of hydrogen ions or loss of bicarbonate. 1. Met abo lic Acidosis due to Retention of Hyd rogen Ions: Ret enti on of hyd roge n ions occurs in associat ion with som e anions not usu ally pres ent in high concentrations. Such condition may arise from "end oge nou s" disease processes or from loading by "exogenous" acids. a. End oge nou s anions :as in, renal failure, diab etic ketoacidosis and lact ic acidosis . b. Exo gen ous anions: as in aspirin overdose, met han ol poisoning and ethy lene glyc ol poisoning.

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Clinical ChemistrY 2. Metabolic Acidosis due to Bicarbonate Loss Bicarbonate loss may take place: a. From the kidney: as in, renal tubular acidosis, therapy with carbonic . anhydrase inhibitors as acetazolamide . · . . . b. From the intestine:. Where losses from below the pylorus contain bicarbonate. This occurs in severe diarrhea, high intestinal fistula and in case of implantation of the ureter into the colon.

B. Causes ofMetabolicAlkalosis: 1_ Ingestion of absorbable alkali (such as sodium bicarbonate for indigestion). 2- Loss of hydrogen ton due to severe vomiting (pyloric stenosis, prolonged aspiration) or prolonged diuretic therapy . or Cushing's syndrome.

SODIUM • Sodium ions (Nal are the major cations of extracellular fluid (ECF). •Reference Range: 136-145 mmol/L. HYPONATREMIA (Plasma Na+ concentration 10 mmol/L ~ A-V bloc VL h ld b · NB: Critical values (K+7.5 mmo ) s ou e unrnediatety reported.

USE OF ENZYMES IN CLINICAL DIAGNOSis I-CARDIAC MARKERS IN DIAGNOSIS OF ACUTE CORONARYSYNDROMES. .

Serum cardiac markers:

.

When myocytes become necrotic, .they l~ose thetr ~em?rane .i~tegnty, and intracellular macromolecules dtffuse mto the cardtac ~ntersttttum and ultimately into the cardiac microvasculature and .Iymphatt~s. Eventuan these macromolecules are detectable in t~e penpheral cuculation. ~~ term . currently used to collectively . descn~e these macromolecules q· serum cardiac markers. The diagnosis of acute myocardial infarction (AMI), as ~ormany established by the World Health Organization (WHO), requtres the presence of at least two of the following criteria: 1 A history of chest pain. 2 Evolutionary changes on the ECG. 3 Serial elevation of serial cardiac markers. ECG is usually the first test performed. If the ECG pattern is equivocal the physician must depend on serum markers of myocardial damag; Serum cardiac markers are useful to: 1 Confirm the diagnosis of AMI when the ECG changes are inconclusive. 2 Provide valuable prognostic information as the magnitude of rise in markers level correlates to the size of the infarction. Many cardiac markers have been used to assess cardiac injury. The most commonly available tests include creatine kinase isoenzymes, lactate dehydrogenase (LDH) and aspartate aminotransferase (AST). New cardiac markers (myofibrillar proteins) include myoglobin and cardiac troponins. I. Enzyme Markers a- Creatine kinase 1 This enzyme is present in high concentrations in cardiac and skeletal muscles and brain.

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Clinical Chemistry and MM. CK~MB is present 2 There .are three isoe~zyrnes: BB, MB, brain . solely m the myocardmm, and BB in the table in 3 In health, only MM (from cardiac and skeletal muscle) is detec serum. with 4 An elevated serum CK-MB activity (>6% of total CK activity) ased total CK > 2 X URL, points towards the heart as a cause of incre CK-MB in serum ( myocardial necrosis) for the 5 CK-MB rise and fall on serial sampling can provide evidence cardiac diagnosis of MI in the absence of availability of assay of troponins. Other Causes of Increased Total CK Activity: ;= Muscular dystrophy • Severe exercise • Muscle trauma, I.M. injection • Hypothyroidism b- Lactate dehydrogenase (LD) or (LDH).· d in the 1. This is widely distributed in tissues, the highest activity is foun kidney, skeletal muscle, liver, cardiac muscle, and erythrocytes. 2.Not recommended as it is nonspecific normally 3.There are five principal isoenzymes, LD-1 to LD-5, all detectable in serum. LD-5 4.1n the myocardium, LD-1 predominates, while in the liver . predominates. together S.ln myocardial infarction, there is an increase in LD-1 mainly with the increase in total LD. er for 6.Due to its prolonged half-life, LD-1 is a clinically sensitive mark late MI when to be used 24 hours after onset of infarction (for diagnosis) Other Causes of Increased LD Activity: • Megaloblastic anaemia • Hepatocellular disorders • Pulmonary infarction • Malignancy (all types)

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Clinical Chemistry

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c- Aspartate aminotransferase (AST):

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10

cells of the cardi l.This enzyme is present in high concentratiOns and skeletal muscles, the liver, kidney and erythrocytes. Damage to a~c Y of these tissues may increase serum AST levels. . is infarction myocardtal after 2.The pattern of release of AST Very similar to that of CK. 3.Not recommended as it is nonspecific

Other Causes of Increased AST Activity: • • • • •

Acute and chronic liver damage Muscular dystrophy Haemolysis Renal infarction Acute pancreatitis

In the investigation of AMI, blood s~ou~d be drawn promptly after the onset of symptoms. Serial determmatl~ns should be performed at appropriate intervals with the proper chotce of enzyme tests dependin on the time gap since the onset of the infarct. A guide to the sequence 0 cardiac enzyme changes is shown in Table 2.2.

1

2- Protein Markers (Table 2.3) a- Myoglobin

1. Myoglobin is a low- molecular weight haem protein found in cardiac and skeletal muscles. 2. It is rapidly released in serum after trauma to either skeletal or cardiac muscles as in crush injuries or AMI. 3. In AMI, myoglobin is a sensitive marker, it appears in the circulation earlier than CK-MB and may be detected as early as 2 hours after MI. However, because of its poor specificity, myoglobin is best used as a negative predictor of myocardial injury in ICU. 4. Can be usesd to monitor success of thrombolytic therapy (reperfusion)

b- Troponins The contractile proteins of all myofibrils include the regulatory protein troponin. Troponin is a complex of three protein subunits; troponin C (the calcium-binding component), troponin I (the inhibitory component) (Tnl),and troponin T (the tropomyocin-binding component)(TnT).

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Clinical Chemistry

~cific ~roponins(Tni&TnT) are characterized by: .. c spe d' I d ord13

. . to minor myocar ta amage. ostuve . MB , se . e simtlar to CK. , earlY ~s elevated for 5-10 days after AMI {cTnT), so can replace LD 310 , Rei11 me assay in late presenting MI patients.

jsoeozY

r•blc z.z=

--rotaJCK

~

~DH

~T

~bin

-

.

cardiac markers Pattern in AMI Peak Initial rise

cTnl cTnT

4-8 3-8 10-12 6-12 1-3 hr 3-8 hr 3-8 hr

12-24 12-24 48-72 18-36 6-9 hr 24-48 hr 24-48 hr ( 151peak) 72-100 hr (2" peak)

U- ACID PHOS~~ATASE

Back to normal 3-5 2-3 5-14 4-6 I day 3-5 days 5-10 days

[

This enzyme mainly ongmates from the prostate but may also arise from bone and red cells. Clinical Significance: t. Diagnosis of C~ncer Prost~te. In cancer prostate w1th metastasis the level of serum acid ~hospha.tase ay be increased 40 - 50 folds the normal range whereas m localized :ancer prostate the serum level is normal or slightly increased. 2. Follow Up of Treatment Of Cancer Prostate; Following successful surgery or oestrogen therapy the level of acid phosphatase returns to normal. In case of recurrence, a subsequent rise occurs. 3. Bone Disease. An increase in the non-prostatic acid phosphatase occurs in many bone diseases as: •Paget's disease. •Osteoclastoma. • Metastatic tumors to bone e.g., breast cancer. 4. Haematological Conditions: (increase in the non-prostatic acid phosphatase) as in: •Gaucher's disease. •Hairy cell leukemia.

II

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Ill- SERUM AMYL ASE

Serum am~lase originates front two so~rces, the panc~eas (P-amylase and the sahvary glands (S-amylase) and ts excreted m unne. )

Clinical Significance:

1- Acute Pancrea titis • Serum amylase starts to increase within 2 - 12 hours to reach a · peak at 12 - 72 hours. Values return to normal by the third or founh day. • Peak values greater than I 0 folds the nonnal values are diagnostic of acute pancreatitis. Peak values 5 folds the normal may be found but are less diagnostic, and nonnal amylase values may be found in 20o/o of cases. · • Urine amylase is increased in parallel to serum amylase. 11-Cases of Acute Abdom en In cases of acute abdomen the rise of serum amylase is 2- 5 folds the normal range e.g. • Perforated peptic ulcer. • Intestinal obstruction. • Acute appendicitis. • Peritonitis. • Mesenteric infarction. • Rupture d ectopic pregnancy. III- Drugs that cause spasm of the sphincter ofOddi e.g., morphine. IV- Salivar y gland causes •Mump s. • Salivary gland irradiation. •Obstru ction of salivary ducts.

CLIN ICAL DISO RDER S OF CALC IUM AND PHOS PHAT E INTEN DED LEARNING OBJECTIVES: 1. Recogn ize the reference range for calcium and its percentage distribu tion. · 2. List the causes of hypocalcaemia and hypercalcaemia in order of importa nce. 3. List the causes of hypophosphataemia and hyperphosphataemia. 4. Recogn ize the laboratory findings in primary, secondary and tertiary hyperpa rathyroi dism.

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Clinical Chemistry

tertiary and secondary pnmary, between 5. Differentiate hyperparathyroidism. laboratory the and 6. Identify findings primary m pseudohypoparathyroidism. 7. Identify the role of the laboratory in diagnosis of the following metabolic bone diseases: osteoporosis, osteomalacia, rickets, renal osteodystrophy and Paget's disease of bone.

SERUM CALCIU M Reference Range: 8.5 - I 0.5 mg/dL (50% is ionized, 40% is proteinbound mainly to albumin, and I 0% complexed with small anions). 1. HYPOCALCAEMIA Causes: a. Hypoalbuminaemia: the commonest cause of decreased total serum calcium. b. Rickets and osteomalacia. c. Chronic renal failure. d. Hypoparathyroidism e. Peudohypoparathyroidism f. Acute pancreatitis: due to Ca deposition in necrotic pancreatic tissue. g. Iatrogenic. 2. HYPERCALCAEMIA: Causes: a- Excessive intake of vit. D or Ca or both, b- Milk alkali syndrome. c- Hyperparathyroidism (lry and 3ry), which leads to, increased bone · resorption. d- Malignancy (direct tumour erosion of the bone or release of PTHrelated protein). e- Multiple myeloma f- Sarcoidosis.

SERUM PHOSPH ORUS Reference Range: Adults: Children:

2.7 - 4.5 mg/dL 4.5 - 5:5 mg/dL

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1. IIYPOPHOSPIIATAE MIA Causes· I· bsorption syndrome and vit . . a. D · · ecreased mtestinal absorption e.g., ma a D deliciency. . . b. Gil loss: e.g., vomiting and diarrhea. c. Renal tubular loss: e.g., 1ry and 2ry hyperparathyrOidism, Fanconi

(d . · h , . syndrome. d. Intracellular shift e.g., with glucose intake, msulm _t crapy unng treatment of diabetic ketoacidosis), respiratory alkalosis. 2. IIYPERPHOSPHATAE MIA Causes: a. Renal failure (decreased phosphate excretion). b. Hypoparathyroidism. c. Pscudohypoparathyroidism. d. Extracellular shift (acidosis). c. Cell lysis (haemolysis, cytotoxic therapy).

HYPERPARATHYROIDISM A) PRIMARY HYPERPARATHYRO IDISM: Excessive inappropriate PTH secretion. Causes: I . One or more parathyroid adenomas. 2. Hyperplasia of parathyroid glands. 3. Carcinoma of one gland. 4. Multiple endocrine neoplasia (MEN) syndromes: It occurs in 85 %of patients with MEN type I and 20 % of MEN type Ila. Biochemical Findings:

• Hypercalcaemia (may be intermittent). • Decreased plasma phosphate (PTH causes decreased renal tubular reabsorption of phosphate). • Increased urinary Ca and phosphate. •Increased plasma alkaline phosphatase (late in the disease due to increased osteoblastic activity). •I f yperchloraemic metabolic acidosis (PTH decreases renal tubular reabsorption of I-IC0 3 } •Increased PTH in blood.

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B) SECONDARY HYPERPARATHYROIDISM:

This is caused by the secondary increase in plasma PTH that is appropriate for the correction of hypocalcaemia e.g., as in cases of chronic renal failure and long-standing intestinal malabsorption disease, rickets and osteomalacia. Biochemical Findings: .ca level is never high, but tends to be low or normal. • Phosphorus is high (e.g., in chronic renal failure). • The response of the parathyroid gland can be suppressed by successful treatment of the primary condition. C) TERTIARY HYPERP ARATIIYROIDISM:

These patients develop functioning adenomas of the parathyroid gland as a complication of previously existing 2ry hyperparathyroidism. Therefore these glands are subjected to long-standing and sustained positive feedback by the low plasma free-ionized Ca concentration of secondary hyperparathyroidism. Biochemical Findings: •Serum Ca level is increased. •Serum PTH is always high. • Serum phosphate is increased.

HYPOPARATHYROIDISM A-PRIMARY IIYPOPARATHYROIDISM: •The diagnosis is supported by a serum calcium and increased serum phosphate. •Serum PTH is undetectable or reduced. • Alkaline phosphatase is usually normal. 8 - SECONDARY IIYI)OPARATHYROIDISM It occurs in response to high calcium e.g., due to vit. D excess, sarcoidosis and multiple myeloma. Biochemical Findings • Serum Ca level is increased. • Serum PTH is decreased.

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Clinical Chemistry C- PSEU DOHY POPARATHYROIDISM This is a rare inborn error of metabolism in which there is end ra~lure · to respon org d to parathyroid hormone. an Daagnosis is suggested by: I. Famil y history. 2 · Increased serum PTH. 3 · Reduc ed serum calcium. 4. Increa sed serum phosphate.

MET ABO LIC BON E DISE ASES _ These are diseases, which affect bone because of a disturbance calcm m metabolism. 0 f I. Osteoporosis (an osteoclastic reaction) Serum C:a, phosp hate .and alkaline phosph~tase. activi ty ~re ~11 nollbal e.g., semle osteop orosis (slow rate of osteoporosis) and agmg Is a rna· risk factor. If rapid osteoporosis occurs as in acute immobilization a~or an accident, this leads to increased Ca, increased phosphate, while p'l'TT ~r 1 11 IS suppressed.

2. Osteomalacia and Rickets In serum , there is reduced Ca leading to secondary hyperparathyroidisrn reduce d phosp hate and increased alkaline phosphatase activity (rnaj~r and earliest bioche mical finding and is due to increased bone turnover).

3. Renal Osteodystrophy This bone disease of chronic renal failure is an example of the compl exitie s of calcium and phosphate regulation. The commonly found bone abnormalities arc: a- Osteitis fibros a : due to 2ry hyperparathyroidism. b- Osteomalacia due to lack of active vit. D, and thus impaired calciu m absorption.

4. Paget 's Disease The diseas e is diagnosed by X-ray or by noting an elevated serum alkali ne phosp hatase level (up to 10 folds) , and increased bone turnover denot ed by high urinary hydroxyproline. Serum levels of calcium and phosp hate are usuall y normal.

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THYROID GLAND INTENDED LEARNING OBJECTIVES:

1. To describe the synthesis and metabolism of the thyroid hormones 2. To list the different circulatory forms of the thyroid hormones 3. To recognize the regulatory feedback system controlling the thyroid hormone production. 4. To enumerate the different causes of thyroid dysfunction 5. To list the different thyroid function tests and their clinical signilicancc 6. To be able to interpret the different thyroid function tests and correlate them with the different thyroid dysfunctions 7. To recognize non-thyroidal illness states and their influence on the thyroid hormones. 8. To list the other laboratory tests that may be affected with the thyroid dysfunction.

Synthesis of the Thyroid Hormones: •Trapping of serum iodide by the thyroid gland is the first step in the synthesis. • Tyrosine amino acid and iodide binding is the second step: . •Tyrosine + I iodide__. mono-iodo tyrosine(MIT) + 1 iodide__. di-iodo tyrosine (DIT) •MIT + DIT __. tri .. iodo tyrosine (T3) •DIT + DIT -> tetra-iodo tyrosine (T4) •The thyroid hormone is stored coupled with thyro-globulin (TBG) in the thyroid gland. • Proteolytic cleavage from TBG occurs so that the hormone gets released from the thyroid gland. •1 O% of T4 and T3 produced each day are excreted in bile. •Small amounts ofun-metabolized T4/T3 are excreted in urine. Circulating Forms of Thyroid Hormone:

•Thyroid gland secretes T3, T4, small amounts of inactive reversed T3 (rT3) and minute quantities of MIT and DIT. •T3: Bound to TBG (80%) +albumin •T4: Bound to TBG (70%) +to pre-albumin and albumin •Free T4 (:::::0.05%) •FT4 is the primary secretory product of the normal thyroid.

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Clinical Che mist ry •T3 is tour-folds more biologically potent than T4: . . •Cir cula ting T3 and rT3 are formed by T4 d_etodmatlOn .(rather than dire ct secretion from the thyroid gland) and thts occu rs penp hera lly Cie. Inside the different body tissues) .. • Peripheral de-iodination: if it occurs in 5' posttlon of oute r ring~1' 3 posi tion of inner ring -+ rT3

Reg ulat ory Feed back of Thy roid Hor mon e Prod ucti on:

Hypo thala mus

Ante rior Pitui tary

Thyr oid glan d

targ et cells thro ugh out bod y

TH YR OID FUN CTI ON TES TS 1- Tota l T 4 and Tota l T 3

Mea sure men t of total T 4 and total T is misleadi ng sinc e if thyroxine 3 bind ing glob ulin (TBG) is increased bound T will 4 incr ease with false elevations of total T . 4

(

I

I

i

~I

Causes of incr ease d TBG =Causes offalse high tota l T4 or total T3:

l. Preg nanc y. 2. Oes trog en ther apy. 3. Oes trog en prod ucin g tumours. 4 . Viral hepa titis . 5. I lero in addi ctio n. 6 . Here dita ry TBG exce ss or variants of albu min and prealbumin.

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Clinical Chemi stry

Causes of decreased TBG =Causes offalse low total T4 or total T3

1. Androgen therapy. 2. Testosterone producing tumours. 3. Liver cirrhosis. 4. Nephrotic syndrome. 5. Malnutrition.

2- Free T3 and Free T 4: It can better discriminate between hypo, hyper and Euthyroid states and isn't affected by changes in TBG. 3- TSII: • It is secreted fi·om the anterior pituitary gland. • It is the regulatory hormone of T3 and T4. 4-TRII Stimul ation test: It is used to differentiate between primary and secondary hypothyroidism. It is contra-indicated with preganacy. •Baseline TSH is measured then TRH is given I.V . • TSH measured at 20, 30, 40 min . • jTSH in euthyroid status :2 - 20 miU!L •jTSH >20 miU/L = (exaggerated response)= Primary hypo-thyroidism • jTSH 6 months is indicative of chronicity. ~:..=J (b) HBcAg: • Particle from the core of the vims. • Indicates viral replication. • Persistence for > 6 months is indicative of progression to chronicity. N.B.: The most sensitive test of viral replication is viral DNA by PCR

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Clinical Immunology and DNA polymerase. (c) HBcAb: • Anti-HBc precedes the appearance of anti-HBs by weeks to months. • It is a sole marker of H~V infection during the window period (i.e., the interval bet ween the disappearance of llBs/\g and the appearance of anti llBs) • Anti-llBc helps in the differentiation of recent and remote HBV infections.

• IgM anti-HBc predominates the first 6 months after acute infection. Therefore, lgM anti-HBc is detectable in patients with current or recent acute HBV infection (including patients who arc in the anti-IIBc window). • IgG anti-IIBc is predominant beyond 6 months of acute HBV infectionIn chronic hepatitis B and those who have recovered from HBV infection in the remote past, lgC anti-HBc is predominant. ' (d) HBcAb: • Scroconversion from HBcAg to HBcAb is a good prognostic sign. • Failure of seroeonversion indicates progression to chronicity. (c) IIBsAb: • Denotes past infection and natural immunity (+ve HbcAb lgG and i·vc HbsAb). • If alone denotes post vaccination (-ve HbcAb IgG and +ve HbsAb).

HEPATITIS C Laboratory Diagnosis: 1. Biochemical Tests: usually anicteric with mild elevation of the liver enzymes.But can take the picture of acute hepatitis with elevated liver enzyme and juandice. ~

Immunological Tests: : Antibody Detection: Anti-HCV by ELISA : It is screening II. However tests are usually negative antibody appears late. 1. False positive ELISA resul_ts a~e 11 .,... hepatitis, hypergammoglobuhne~ta rh~atoid factor. (false posttlve confirmed by RIBA). 1.

test in acute cases smcc the seen. with autoimmune and . m the presence of antibody test can be

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Clinical Immunol ogy

• Viru~ Detection: viral RNA b_y PCR. It is importan t for confirming the dtagnosis of h~s C

HEPAT ITIS D

=-

Virus : Small incomple te RNA virus (Delta agent) that exists only in the presence of HBV either simultane ously (i.e. coinfectio n) or superimposed (i.e. superinfe ction).

Laborato ry Diagnosi s: (A) Superinf ection: 1. Biochem ical Tests: Laborator y finding of acute hepatitis on top of chronic.

2. lmmuo/o gical Tests: • • • • •

Delta antigen (ELISA) within the first few days. HDV Ab: IgM +ve. HBsAg: +ve. HBcAb IgG:+ve. HDV-RNA : early & sensitive marker

(B) Coinfect ion: 1. Biochem ical Tests: Laborator y finding of acute hepatitis 2. lmmuolo gica/ Tests: • • • • •

Delta Ag: early. HDV Ab: + ve lgM then +ve IgG. IIBcAb: 1-ve lgM. HBsi\g: positive. IIDV-RN J\ : early & sensitive marker

NJl:Othe r causes of acute hepatitis arc: • HEY • Epstein- Barr virus • Cytomeg alovirus • Herpes simplex • Autoimm une hepatitis

2- CI IRONI C I-IEPATITIS

(Fig.5.12) :

It occurs as : • Post viral hepatitis B, C j D. • Autoimm une . • Others: a 1 antitrypsi n deficiency , Wilson's disease.

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Clinical Immunolog y

POST VIRAL CHRONIC HEPATITI S: Laboratory Diagnosis: J. Biochemical Tests_: Liver enzyme are usually only mild elevated 2 .Jmmuological Tests a) HCV • IICV Ab ·I vc by ELISA and by PCR for 1-ICV-RNA • IICV RNA is used for genotyping and quantitation of viral load for prognosis and follow up.

•

• • • •

b)HBV + ve HBsAg: 1-IBcAb lgG: 1-vc llbcAg: +vc I -vc HBcAb: +vc /-ve HBV DNA (PCR) and DNA polymerase: +ve /-ve

N.B. Inactive carriar state is charactarized by: +ve • IffisAg: • IIBcAb IgG: 1-ve • IlbeAg: -ve • Persistently normal ALT& AST levels • IIBV DNA (PCR) and DNA polymerase: -ve

AUTOIMUNE CHRONIC HEPATITI S (LUPOID HEPATITI S) Laboratory Diagnosis: /. Biochemical Tests : Laboratory findings of acute or chronic hepatitis. 1. Immunological Tests: • Negative markers for viral hepatitis. • Presence of one or more autoantibodies: - ANA - ASMA. - LKM (liver kidney microsomal). - Soluable Liver Antigen

PRIMARY BILIARY CIRRHOSI S (PBC):

.

Laboratory Diagnosis: I. Biochemical Tests: Laboratory finding of abstractive jaundice with

t

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Clinical Immunology

ALP and GGT.

2. lmmuo/ogica/ Tests:

• i i total

lgM. • vc J\MI\ (anti-mitochondrial antibodies) (high titers) by Indirect lmmutlO lluorcscncc. N.B:This differentiates it from primary sclerosing cholangitis with -ve AMI\. 1

IIEPATOCELLULAR CARCINOMA: l.For· Primary llcpatoma: Alpha fctoprotcin. 2. For· Li\'cr l\1ctastasis:Tumor markers of primary malignancy ( e.g C t-:A in colorcctal cancer). N.B.:

• Large elevation of AFP- > hepatoma. • Mild elevation of AFP +elevation ofCEA - >secondary liver cancer. • I :Jc,·ation of CEA without increased levels of AFP-> colorcctal cancer without hepatic malignancy.

1-IYPERSENSrfiVI'l'Y INTENDED LEARNING OBJJ.i:CTIVJ.:S: I. To know the different typee of hypersensitivity reactions , 2. To understand the pathogenesis of hyprsensitivity diseases and their c I i 11 ical presentation 3. To select different immunological tests to diagnose and follow-up hypersensitivity diseases.

There arc 4 types of hypersensitivity reactions. Types I, II, and III depend on the interaction of antigen with humoral antibody and tend to be called 'immediate' type reactions although some are more immediate than others. Type IV involves receptors bound to the T -lymphocyte surface and because of the longer time course this has been referred to as ·delayed-type' hypersensitivity.

Type 1: Anaphylactic Hypersensitivity: • This depends upon the reaction of antigen with specific IgE antibody bound through its Fe to the mast cell. • 1Jay fever and extrinsic asthma represent the most common atopic aiJcrg ic disorders.

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Clinical Immunology • There is a st~ong familial predisposition and the tendency of an individual to produc e htgh levels of lgE is an important contributing factor. • An elevate d total lgE confirm s diagno sis (in the absenc e of parasit ic infestation). Howev er a normal lgE docs not exclud e the diagno sis. • The offend ing antigen is identified by intradermal prick tests giving immedia~e wheal and crythae ma reactions, allergen specifi c IgE, or by provocatiOn tests.

Type II: Antib ody Depen dent Cytotoxic Hyper sensit ivity:

• This involve s the death of cells bearing antibod y attache d to a surf~tcc antigen. • The cells may be taken up by phagocytic cells or they may be lysed by the full comple ment system . Cells bearing IgG may also be killed by polymo rphs and macrop hages or by K-cells through an extrace llular mechan ism (antibo dy-dep endent cell-mediated cytotox icity "ADCC "). • Examples are : transf1tsion reactions, haemolytic disease of the newbor n through rhesus incompatibility, antibod y-medi ated graft rejection. • Immun ologica l diagnosis: Coomb s' test direct and indirect.

Type III: Immu ne Complex mediated Hypersensitivity:

• This results from the effects of deposit ion of antigen -antibo dy complexes. • Where circula ting antibod y levels arc high, the antigen is precipi tated ncar the site of entry into the body. Examples arc Farmer's lung, and pulmon ary aspergi llosis. • In relative antigen excess, soluble comple xes are formed which are deposited under circumstances of increased vascula r permea bility at certain preferr ed sites, the kidney glomerulus, the joints, the skin and the choroid plexus. Examp les are: glomerulonephritis associa ted with systemic lupus or infections with streptococci, malaria and other parasites, and polyarteritis nodosa linked to hepatitis B virus.

Type IV: Cell media ted (Delayed-type Hypersensitivity):

• This is based upon the interaction of antigen with primed T -cells and represents tissue damage resulting from inappro priate cell-me diated immune reactions. • The typical exampl e is the delayed hypersensitivity respon se such as the Mantou x reaction to tuberculin. This is a delayed appear ance of an indurated and erythae matous reaction which reaches a maxim um at 24-

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Clin ical Imm unol ogy 4H hour s . inclu de macrophage • In_ vitr? t~sts for cell- med iated hype rsens itivit y form ation . migr ation mhib ition and asse ssme nt of blast cell trans rial (tuberculosis • Exam ples are : tissu e dam age occu rring in bacte sis' l~prosy), viral (Sma ll pox, measles, herpes), fungal (candidia ) iasis ~Isto~Iasmosis) and parasitic (leishmaniasis, schistosom mfec t10n s, and cont act denn atitis . y by a persisting • Co~tinuing prov ocat ion of delayed hype rsens itivit antig en lead s to the form ation of chro nic gran ulom ata.

IM MU NO LO GIC AL LY ME DIAl'ED RE NA L DIS EA SES INT END ED LEA RNI NG OBJ ECT IVE S: respo nsibl e for I . To unde rtand the diffe rent hypersensitivity react ions imm une med iated kidn ey disea ses. ses: 2. To diag nose case s of imm une mediated kidney disea a. Anti -glom erula r Base men t Mem bran e (GB M) Glom erulo neph ritis b. Imm une Com plex Glomerulonephritis c. Tubu loint ersti tial Neph ritis tubu lo-in terst itial Imm unol ogic ally indu ced glom erulo neph ritis and s of end stage renal neph ritis are resp onsi ble for almo st 50% of all case failu re .

Mem bran e Bas eme nt Ant i-gl ome rula r 1Glo mer ulon eph ritis (Ty pe II hyp erse nsit ivity ):

(GBM)

lobu lins and often C3 • It is char acte rized by linea r depo sits of imm unog foun d in serum. al ong the GBM and anti- GBM antib odie s arc usua lly ritis, Goo dpas ture' s • Anti -GB M antib odie s can caus e glom erulo neph hem orrh age) and synd rom e (glom erulo neph ritis and pulm onar y occa sion ally idiop athic pulm onar y hem oside rosis .

11- Imm une Com plex Hyp erse nsit ivit y):

Glo mer ulon eph ritis

(Ty pe

III

unog lobu lins and • It is charahagocytic I>cficiency: Immun ologica l Diagno sis

• Leucocyte count with differential : measure total neutrophils • Tests for phagocytosis using either candida albicans killing test or nitrobluc- tetrazolium test. CHRO NIC GRAN ULOM ATOU S DISEA SE It is an immunodeficiency disease that results from a defect in neutrophil

killing. ACQU IRED IMMU NODE FICIE NCY

These deficiencies occur in previously healthy individuals. Almost any severe illness may lead to impairment in immune function. ACQU IRED IMMU NODE FICIE NCY SYNDR OME (AIDS): Diagno sis: 1- Labora to•·y Findin gs:

• Lymphopenia with decreased T helper cells. • Decreased T-cell function. hypergammglobulinaemia early show • B-cells hypogammaglobulinaemia.

and

late

2- Immun ologic al Diagno sis:

• HIV Antibody screening by ELISA and confirmation by Western blot. • HIV-RNA by PCR o Establish the diagnosis. o For treatment & follow up . o To demonestrate neonatal transmission .

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Clinical Immunol ogy

MISCE LLANE OUS IMMU NOLO GICAL DISEASES INTEND ED LEARNI NG OBJECT IVES: 1. To request and interpret immunological tests required for the diagnosis and follow up of different. immunlog ically mediated diseases. 2. To identify monoclon al gammopathy and their significan ce.

1- Infectio us Mononucleosis: Immuno logical diagnosis • • • •

Absolute lymphocy tosis. Up to 50-70% of the lymphocytes are atypical. Positive monospo t test Positive EBV- IgM antibodies by ELISA.

2- Multipl e Myelom a: • Diagnosi s is based on finding myeloma cells in bone marrow, character istic lytic lesions in bones~ and an associated serum or urine monoclon al protein. • Serum protein electroph oresis usually shows a monoclon al (M) ·band in the gamma or beta region . .. Less commonl y there is hypogam maglobul incmia (when only light chains arc produced by the neoplasti c plasma cells (light chain disease). • Immunoe lectropho resis or immuno!ixalion confirms the diagnosis to identify the para protein heavy chian (G,A,E &D) & light chain (kappa & lambda). · • 132 microglo bulin ( for follow up).

3- Waldcn strom's Macrog lobuline mia: The identification of the paraprotein is achieved by immunoe lectropho resis or immunofixation . The heavy chains of IgM and only one type of light chain is found either kappa or lambda.

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p Clinical Immunology

TUM OR MARKERS INTENDED LEARNING OBJECTIVES: follow 1. To und~rstand the ~ole of tumour markers in the diagnosis and

up of dtfferent malignancies. 2. To know relevant tumo r markers for different mal ignancies and their clinical relev ance . and These are subst ances which are produced or induced by tumo r cells es. are released into blood , or body fluids or are expressed on cell surfac They may be: 1- Tumo r deriv ed: secreted by the tumor cells. r. 2- Tumo r associated: secreted as a result of the presence of the tumo

Applications: • Detection and screening in high-risk population. • Diagnosis, togeth er with histopathologic studies. with • Prognosis and stagin g. Level of tumor mark er must correlate tumo r load. • Follow up (less than 10% of original level means good response). N.B : • Normal level (negative result) does not exclude malignancy. • Single determ inatio n does not allow de·finite conclusion. • Comb ining different mark ers can improve the diagnostic precision. early • The most valua ble function of tumor markers is to provi de an indication of recurrence. It depends on change in tumo r mark er concentration from recen t base line. h • None of the tumo r markers available are sensitive or specific enoug to be used as scree ning test in an asymptomatic popul ation.

Classification: 1- 1/ormones: tumo r A- Excessive production of normally secreted hormone by cells e.g., HCG in hydatiform mole, choriocarcinoma and testicular teratoma .

8- Ectopic Hormones, e.g.

lung, • Calcitonin: in medullary ·carcinoma of thyroid gland, cance r pancreas and prostate. • Antidiuretic horm one (ADH) in carcinoma of lung.

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• Erythr opoieti n in carcino ma of kidney. 2- Enzym es: e .g.Acid phosph atase (prosta tic isoenzy me) m cancer prostat e. 3- Protein s: e.g. Immun oglobu lins in multipl e myelom a.

4- Tumor Antige ns • • • •

Carcin oembry onic antigen (CEA) in carcinoma of gastroi ntestina l tract Alpha fetopro tein (AFP) in hepatom a, carcino ma of lung and GIT. Prostat ic specifi c antigen (PSi\) in cancer prostat e. Carboh ydrate antigen (CA): C A 125 in ovarian carcino ma. - CA 19-9 in GIT malign ancy and pancre atic carcino ma. - Ci\ 15-3 in cancer breast. - C i\50 in colorec tal carcino ma.

5- Cell Recept ors: • Oestro gen recepto rs in cancer breast and prostat e. • Proges terone recepto rs in cancer breast 6- Cell Surfac e Marke rs: used in classifi cation of lcukae mias and lympho mas . 7- Metabo lites: • Vanilly lmandc lic acid (VMA) in pheoch romocy toma. • 5 hydrox y indole acetic acid (5-I-IIA) in carcino id tumors . 8- Geneti c Marke rs as Philade lphia chromo some.

----

--

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[

J

MI CR OB IOL 0G Y NORMAL FLO RA

INTE NDE D LEA RNIN G OBJE CTIV ES: 1- Identify function of normal flora 2- Classify bacte ria accor ding to pathogenicity. 3- Deline prima ry patho gen. 4- Define oppor tunist ic patho gen.

Definition & Func tion of Norm al Flora :

on Normal Flora is defin ed as those organisms norm ally found nt skin/mucosal surfac es. They are non harmful bacteria or yeast that preve effect colonization by other potential pathogens; thus exert a beneficial e (Table 4.1). Howe ver they can cause disease when individuals becom usual inunu nocom prom ised or debilitated or when they chang e their true anatomic location. Virus es and parasites are always consi dered pathogens. Table (4.1): Predo minan t and potentially pathogenic Flora of Various body Sites.

i

Low VucuLENCE (REslom rr)

Boov SITE

NODe"'

Rluod

None

nuues

PropitNtibac~eri-.

Sua

Nuopha rynx

Co~cm-(~).

Streptoc occus p-lllftDII UK,

Nruseri a ntellinliJidis. HaLmophilus infLwaqJe, gruup A

coqulas c-aepliw : .._.,lac oa:i NaneriD sw~ ~ lllriiP'CJCOCCi. Moraull 4. Pep1D611 YpfDCOCC U NeuuriD spp~ viridMa ~i. Morwf'll a. PrptostiY piDcoccu

stre pcococci. Sti:Jplryl«occws

'

""""us (uulcrior narcs)

I

~

S~omach

None:

'

Snail intestine

None

. tf ~~~ iftfant Adult

I

t

'•

i V-a~ina j Prc:pubena.l and .1 Postmrno pwasal 1 Cbildbea rin«

Stn:ptoco a::i, Prptostrc piOCOCe ta. ochcnl from mouda Scanly, Yllriable

None

llncteroides frugilu. uclvric:I Ua coli. Pseu.lomONU, CUIIdida. Clostridi~m~ (C.

petfri•g'- IU,

C. di.Jiici/e)

Bi{ttJ.obocuTi- . I.AceohaciUu. BacteroUks, F~ri-. Enco:ru badcri- -. UuetOC «aU, ClostTidiwrt Diphther oids, Uphyloc oa:i.

C. afbicOJU

F.ntct' llbKtc "-

38.3°C on several occasions, lasting more than 3 weeks with no diagnosis despite 1 week of inpatient investigations or 3 weeks as an outpatient.

Causes: (Table 4.6) Table (4.6): Causes o f pyrexi a o f un k nown or!gin . Infectiv e causes Non specific Specific

A- Bacterial •Typhoid fever. • Brucellosis. el'.B.

B- Viral • Hepatitis . •Infectious mononuclcosis(EBV •Cytomegalovirus (CMV). C- Protozoal: Malaria and amoebic abscess and Toxoplasmosis. D- F11nga/ infections. Candida , Aspergillus and

• Hidden abscess (Pelvic, perinephric). •Infective endocarditis. • UTI (rare). • Ear sinus or dental infections.

Non infectiv e

• Neoplasms • Collagen diseases (Systemic lupus erythematosus) • Sarcoidosis • Thyrotoxicosis • Drug reactions.

!,

O J 'lJIVCOCC US .

Labor atory Diagn osis: ~ Microb iology I . Blood, urine, stool and sputum samples or specimens from any

other probable site of infection for culture and sensitivity. 2. Serological tests for any suspected causative agent. 139

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Clinical Microbiology ~ Hematology: CBC, ESR, and blood film for malaria and detection of atypical lymphocytes. ~ Chemistry: Liver Function Tests. 00 Immunology: Hepatitis markers (e.g. HBsAg) for diagnosis of viral hepatitis and autoantibodies for diagnosis of collagen disorders.

BRUCELLOSIS Brucellosis is a common disease in many developing countries and it is one of the differential diagnoses of PUO. It has been known by various names, including MediteiTanean fever, Malta fever, and undulant fever. Causative organisms: B. abortus (cattle), B. melitensis (sheep or goats), B. suis (pigs) and B. canis (dogs).

Mode of transmission: Humans acquire brucellosis by occupational exposure among farmers, slaughterhouse workers, and veterinarians through: I. Ingestion of undercooked meat or consumption of unpasteurized dairy products. 2. Bacteria can also enter wound in skin or mucous membranes, through contact with infected animals. 3. Inhalation of bacteria during handling of infected animal.

Clinical presentation: Brucellosis is a systemic infection that can involve any organ of the body and clinically presented as asymptomatic infection or as acute, subacute or chronic infection. Relapse is considered an important feature of brucellosis.

Laboratory diagnosis: 00 Direct Methods: a. Specimens for culture and sensitivity : Blood, bone marrow and other specimens as CSF, pleural and synovial fluids , and urine . It is essential that the clinical microbiology laboratory be notified whenever brucellosis is suspected to ensure that specimens arc cultivated in an appropriate manner for optimum recovery of the organism. b. Direct stains of clinical specimens are not particularly useful for the diagnosis of brucellosis.

c. Culture: ./ Blood culture: It should be collected in the first three weeks and it is recommended to use bottles of the automated blood culture system.

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Clinical Microbiology n should extend /con ven tion al .cultu~e of other specimens but incubatio sterile site). to 7 days . Flu~d enn~hment increase yield( if collected from ns of detecting d. Molecular d.aag~o~as are reliable and specific mea routine use. Brucella spp m chntcal specimens but not available for

00 Indirect Methods Serolo ical dia nosis :

detects antibodies a. Standard agglutination test [SAT). This technique s antibodies to B. abortus, B. melitensis, and B. suis, but not B. cani s to B. canis). (there is no serological test available to detect antibodie ive excess of SAT may give false negative results due to the relat due to nonantibodies against antigens (prozone phenomenon) and test (indirect agglutinated lgG in chronic cases. So, anti-human globulin to detect nonCoombs' test) should be performed after the SAT ucellosis . agglutinating antibodies in subacute and chronic cases ofbr for lgG and IgM b. Enzyme-linked immunosorbent assays (EL ISA ) antibodies.

l!J Complete blood count: Neutropenia with leukocytosis. brucellosis Tre atm ent: To prevent relapse of infection, patients with iotics that can should undergo prolonged treatment (6 weeks) with antib penetrate macrophages.

LO WE R GE NIT AL TR AC T INF EC TIO NS AND SEXUALLY TRANSMITTED DISEASES gh sexual Lower genital tract infections may be acquired either throu ns. contact with an infected partner or through non sexual mea Causative org anis ms (Table 4.7) and sexually Table (4.7): Common Causes of genital tract infections transmitted diseases Disease

Agent

Organism group

Genital and anal warts (condyloma accuminata); cervical dysplasia; cancer Vaginitis

Human papilloma virus

Viruses

-Gardnerella I Mobil uncus (Bacterial vaginosis), -Trichomonas vagina/is, -Candida albicans -Neisseria gonorrhea, -Chlamydia trachoma/is (D K), -Ureaplasma urealyticum & Mycoplasma genitatium .

Bacteria, parasites, fungi

Urethritis, cervicitis

Bacteria

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Clinical Microbiology Pt"tlstatitis \

- --

I Icrpcs gcnitalis (genital and skin ulcers) Syphilis . Chanc roid Lymph ogranu loma venere um Granu loma inguinale

-

Acute : Gram negative bacilli and enterococci. Also Neisseria gonorrhoeae and Chlamydia trachomatis infection in any male younger than 35 years. Chronic: Gram negative bacilli and enterococci, C. trachoma/is, Ureaplasma, Trichomonas vagina/is . Uncommon causes as M tuberculosis, I listoplasma, and Candida , Cytomegalovirus I !etprs simplex type II ( less comm only type I) Treponema pallidu m Haemophilus ducreyi Chlamydia trachoma/is (L 1- L3) Klebsiella granuloma/is (Donovania)

Mainl y bacteria

Viruses Bacter ia Bacter ia Bacter ia Bacteria

Notes: •HIV , human T-celllymphotropic virus type I [HUV -1], hepatitis B virus, Cytomegalovirus (CMV) and flerpes simples virus (HSV) are known sexually transmitted diseases. • Frequently, in sexually transmitted infections, patients are infected with more than one pathogen.

Diagnosis: A- Ureth ritis, Prost atitis , Cervicitis, and Vaginitis.

Spec imen s: • For female: Swab from uretheral discharge, vaginal discharge (high vaginal swab or aspirate) or endocervical swab. • For male: Uretheral discharge or swab or prostatic fluid and urine before and after prostatic massage. • All genital specimens should be sent immediately to the lab. and should never be refrigerated. Neisseria gonorrhea is fastidious and the use of transport medium is necessary. Micr oscop ic exam inatio n 1- Wet film: • Vagi nal disch arge: Trichomonas, Candida, pus cells and clue cells (epithelial cells covered by many gram negative bacilli). • Prost atic disch arge, pre-massage and post-massage urine for detection of pus cells. N.B: For the detection of Trichomonas vagina/is, s~mples must be delivered immediately after collection. After I 0 minutes of collection, 20% of samples became negative with gradual decline by more delay . 142

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Clinic al Microb i.ology

stained smear : Gram stained smear is diagno stic for: 2· Gra;0 norrhea in urethra l dischar ge, prostat ic discha rge or ' d cervical swab: pus cells with gram negativ e diploco cci intra and en o .. xtraccllular. hs (NGU) is . urethri tis caused by urethn a~ nococc Non-go ~.B: /amydia trachoma~zs, Ureaplasma urealytzcum or Mycoplasma 1 c~nitalium (in the stamed film, pus cells with no organis m detecte d). ~!so, T vagina/is, HSV can cause NGU. Freque ntly, patient s are with both N. gonorr hea and chlamydia trachomatis. ·nfectcd 1 candid a infecti on: presen ce of pus cells, buddin g yeasts and . . . . pseudohyphae. . swab: Interpr eted by vagmal H1gh m (BV) sJs vagmo al Bacteri Nugent's score which depend s on the abunda nce of small gramnc•Jativc or variabl e rod (G. vagina/is and Bacteroides spp ), curved gr:m-ncgative or ~~m variabl e rods (Mo~i/uncus) & clue cells, ~.ith the absence or mtmma l presen ce of medmm to large gram-p osthve rods (Lactobacilli). Score of 7 or more is diagno stic ofBV.

3- Culture: on variety of ordinar y and selectiv e media accord ing to the site of infection arid the suspec ted pathog ens. 4- Molecular diagno sis and serolog ical diagno sis for non-cu ltivable organisms on artifici al culture media as chlamydia and viral causes of infection .

B- For Syphilis:· On Primary and Secon dary lesion exudat e • Dark ground micros copy reveals movem ent of Treponema pallidu m. ' Direct Immun ofluore scent of exudat es for antigen detecti on.

In Secondary and tertiar y stages: serum antibod y detecti on: • Semi-quantitative non-tre ponem a} tests that detect antibod ies against cardiolipin as VDRL (Vener eal Diseas e Resear ch Labora tory) and RPR (rapid plasma reagin) tests. While not specifi c for syphili s, but they are useful for screeni ng purpos es and to follow up treatme nt respon se. • 7_'reponema pallidu m specifi c tests which measur e antibo dy specifi c for T. pallidum as Treponema pallidum . hemagg lutinati on tests and the fluorescent trepone ma! antibod y absorp tion test.

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Clinical Microbiology

'fRAN SPLA CENT AL INFEC TION S Causati ve organis ms A-Para sitic • Toxopla sma ro • Plasmo dium spp B= Viral • Ruhella R • Cy tomegal ovirus C • He1pes virus. II • Varicell a-zoster virus (VZV), • Enterov iruses. • Lympho cy tic choriomeningitis virus. • HIV • Measles • Parvovi rus B/9 • Hepatitis B C-Bactc rial causes • Treponema pa/Jidum causing Syphilis • Listeria

TORCH and Syphilis can produce congenital fetal abnorma lities if they

infect pregnan t women especial ly in the first trimester as the infection passes through the transplacental route to the fetus . Other viruses now known to cause congeni tal anomalies as varice/Ja-zoster virus (VZV), enteroviruses and Lympho cytic choriomeningitis virus.

Diagnosis (Table 4.8). . [iect10ns wit T a blc(4 8) . o·1ag_nosJs · o f congem.tal m . h TORCH an d Siypih ITIS. Pathoge n Isolation or Molecular Other Tests to be done detection CMV from urine, throat swab The persistence of lgG antibody or CSF, but diagnosi s in infant sera over six to 12 can only be made if the months (titer that does not drop virus is detected within at the expected rate of a two2-3 weeks of life. fold decline per month). from urine, throat, CSF HSV and any detected vesiculated lesion. Toxopla smosis in CSF.

. .

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Clin ical Micr obio logy

aen p~rho.,

Isolation or Molecular detection from nasal, blood, throat, urine and cerebrospinal fluid specimens

Other Tests to be done

• Detection of IgM antibodies in infant serum or CSF. • The persistence of IgG antibody in infant sera over six to 12 months. • VORL or RPR should be . :1 v.-;: IS syphl performed on infant serum (not umbilical cord blood) and, if reactive, the infant examined be should thoroughly for evidence of congenital syphilis. • Also, any suspicious lesions, body fluids, or tissues (e.g. umbilical cord, placenta) should be examined by dark field microscopy or PCR testing. ific IgM Single negative mate rnal seru m at time of delivery for Spec out the and IgG or othe r tests diag nost ic for the causative agen t rule diagnosis.

ABSCESSES AND WOUND INF EC TIO NS a result of I!JWound infections occur primarily from breaks in the skin as complications associated with surgery, trauma, and bites, or from diseases that interrupt the mucosal or skin surface. l!l Abscesses are collections of pus in confined tissue spaces. Causative Orga nism s: I. Mrobic orga nism s -negative •Gram-positive cocci as Staphylococcus aureus, Coagulase gram-positive staphylococci, Enterococci, other streptococci and other aerobes. ginosa, •Gram-negative bacilli as E.coli, Pseudomonas aeru mon iae. Enterobacter species, Prot eus mirabilis and Klebsiella pneu 2· .inacrobcs e.g. Clostridia, Bacteroides, and anaerobic cocci.

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Clinical Microbiology

Notes: • Human bite wounds are particularly subject to anaerobic infections, in contrast, infected bites of domestic animals (dogs, cats) are almost always due to Pasteurella multocida. • In abscesses, mixture of aerobic and anaerobic organisms could be the cause. The most commonly isolated aerobic organism is E. coli and staph. aureus, and the most commonly observed anaerobic organism is Bacteroides fragilis. 3. Fungal: Candida species infections in patients who receive prolonged antibiotic therapy. 4. Protozoal: Entamoeba histolytica in amoebic liv'e r abscess.

Lab. Diagnosis: Specimens: Pus may be collected in sterile syringe or by deep wound swab and specimen should be transported rapidly to the microbiology laboratory in a suitable transport medium. Specimens will be subjected to the followings: 1- Direct Film • Wet film for motile ameba in liver abscess. • Gram stain to detect causative organism. Gram stain can improve the accuracy of evaluating wound culture. The presence of PML is an indication of an inflammatory or infectious process, while the presence of epithelial cells indicates surface contamination of the specimen. 2- Culture both aerobic and anaerobic culture if specimen is suitable (refer to diagnosis of anaerobic infections).

ANAEROBIC INFECTION S Anaerobic organisms cannot grow except in the absence of 0 2 as it has a lethal effect on these organisms. Facultative anaerobes will grow in the presence or absence of 0 2. Microaerophili c . bacteria (Campylobacter ) can grow only m an atmosphere with reduced 02 and increased C02 • Strictly aerobic organisms will grow only in the presence of 0 2 •

Anaerobic microorganisms arc classified as: 1-Non-Spore Forming e.g. Bacteroides species, anaerobic cocci, Actinomyces and P. acnes. Bacteroides species are the most common cause of bronchiectasis and closed abscesses, anaerobic intraabdominal infections commonly following colon surgery. 11-Sporc-•'orm ing Organisms (Clostridia spp) (Table 4.9) 146

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Clinical Microbi ology

Ta blc (4.9)·. Diseases causedb>y d'fI ferent Clostridia spp. Organism C. perfringens

C. tetani Organism C. botulinum

C. diffici/e

-

Disease Ga~ gangren e or clostridial myonecrosis is an acute and raptdly ~rogressive invasive process producing marked changes m muscles. Anac~obic ceUulitis i~ a gradual necrotizing process of the soft ttssues. Gas is produced; but it does not involve muscles. N.B: Distinguishing between the two conditions is critical to avoid performing unnecessarily aggressive surgery in the (ormer condition. Food poisoning. Tetanus . Disease neurotoxin produce organism The : Botulism which results into dry food poisoning (without vomiting or diarrhea) . Antibiotic associated diarrhea is a non-bloody diarrhea occurring after broad spectrum antibiotics e.g. clindamycin. Suppression of normal flora by antibiotics aiJows proliferation of c. difficile with subsequent production of toxins and occurrence of pseudomembranous colitis. It can be treated by stopping the antibiotic intake with administration of oral vancomycin or metronidazole.

Lab. Diagnosis: 1- Specimens: Tissue, fluid or pus in sterile anaerob ic containe r.

N.B: Throat, nasopha ryngeal, sputum, gastric contents , stool, food remnant s, material adjacen t to skin or mucous membra nes, voided urine, and vaginal or cervical specime ns are not. suitable for anaerob ic culture. 2- Direct film stained with Gram stain: • The presenc e of numerou s, large, "boxcar "-shaped , gram-po sthve bacilli provide s presump tive con-firmation of the clinical diagnos is of Clostridium perfringes infection . • May be diagnos tic of anaerob ic streptoc occal myositis . • The presenc e of gram positive bacilli that have terminal bulging spore with drumsti ck appeara nce, support the clinical diagnos is of tetanus. 3- Culture on convent ional culture media under anaerob ic conditio ns. 4- Toxin Detectio n for Clostridium difficile toxins (A& B) and Clostridium perfringens toxin in stool and Clostridium botulinum toxin in food remnant s.

Antibiotic used for treatme nt of anaerob ic infections arc penicilli ns, clindam ycin, cefoxiti n, ceftriax one, cefotaxi me and tmtpcnc m.

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ANTIBIOTICS INTENDE D LEARNIN G OBJECTIV ES: l. List the mechanism of action of antibiotics. 2. Mention the differences between bacteriostatic and bactericidal antibiotics. . 3. Define the antibiotics according to spectrum of activity. 4. Define the clinical use of antibiotics whether prophylacti c or therapeutic. 5. Mention the suggested regimen for surgical prophylaxis . 6. Identify the importance of empirical therapy. 7. Define what is meant by synergism, antagonism and indifference in antibiotics combination. 8. Enumerate causes of antibiotic failure. 9. Define common multidrug resistant organisms.

Definition: Antibiotics are . . microorganisms.

Chemicals

that

kOI

or

inhibit

the

growth

of

Mechanis m of action: antibiotics exert their effect on bacteria by disturbing one of the following vital processes: protein synthesis, cell wall synthesis, genetic replication, cytoplasmic membrane integrity or folic acid synthesis.

Classification of antibiotics: Antibiotics arc classified according to 1-Whether it is bactaeriost atic or bactericida l :• Bacteriosta tic antibiotics that inhibit the multiplication of the organisms but the actual eradication of bacteria is done by the host immune system. • Bactericida l antibiotics that kill and eradicate bacteria completely. 2-The spectrum of activity: • Narrow spectrum: Active mainly against either gram positive or gram negative bacteria e.g. Cloxacillin & fucidic acid for staphlylococci. • Broad spectrum: Active against many gram positive and gram ncgati vc bacteria e.g. ampicillin & ccphalosporincs . • Extended spectrum: llavc wider spectrum of activity against many gram positive or gram negative bacteria, either aerobic or anaerobic e.g. imipcncm. 148

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clinical Use of Antib iotics : . refers to the use of antibi otics to preve nt ylaxis proph Antibiotic infections. Suggested regim en for surgic al proph ylaxis : • The use of new wide-s pectru m antibio tic should be avoide d to preve nt emergence of resista nt strains. • The choice of antibio tic depen ds on the site of operat ion and cost. • The short preope rative stay decreases the risk of acquis ition of resista nt strains hence suppo rt the prophylaxis.

1. Proph ylacti c:

• Dose and Time:

.; Dose should be calcul ated accord ing to patien t weigh t and given once immediately before the procedure at time of incisio n (e.g. with induction of anesthesia) excep t for vanco mycin should be administered one hour before the operat ion by slow infusi on . ./ Introduction of bacter ia into the surgical wound occurs not only at the time of incisio n but continuously throug hout the surgic al proced ure. So, to ensure that adequ ate antibiotic levels are mainta ined above the minimum inhibi tory concentration (MIC) of the pathog ens throug hout the surgical proced ure a supplementary dose of antibio tic should be given at double the half-life of that antibiotic. 2. Thera peutic : a- Empir ical: • Initial treatment is empirical in patients with severe life-th reaten ing infections in hospital, where antimicrobial treatment is indica ted as soon as the specimens have been collected for culture. • Direct smears can help to guide empirical therapy. Gram stain can identify gram positive and gram negative organisms as antibio tic therapy differs. Narro w spectrum bactericidal antibiotic is the best. It is also helpful in the rapid diagnosis of certain infections as gonor rhea in males, meningococcal meningitis, Candida, and Vince nt's angina. b- definitive therap y:• The antibiotic is given according to the results of culture and sensitivity, better to choose narrow spectrum antibiotic. Clinic al impro vemen t usually occurs after 48 hours of administrating antibiotic but it must be continued for 7-10 days according to the type and severi ty of infection.

Antibiotics comb inatio n: Antibiotics combination may be required in certain situati ons to delay the emergence of antibiotics resistance as for treatment of TB or for the treatment of serious infections as cntcrococcal endoc arditis and empir ical

149

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Clinical Microbiology

treatment in high risk patients. Drug combinations may exhibit synergism, antagonism or indifference. • Antagonism: The final effect is less than the effective agent alone. This is known upon most of the combination of bactericidal and bacteriostatic antibiotics e.g. penicillin with erythromycin. • Synergism: The final effect is much greater than the sum of the effects of both antibiotics with mo~e efficient clearance of infections (penicillin and gentamycin, amikacin with imipenem). • Indifference: The combined action of both equals the action of the effective one only.

Causes of •"'ailure of Antibiotic Therapy: I. Improper choice, dose, route, time and duration of therapy. 2. Presence of natural barrier as prostatic barrier in chronic prostatitis. 3. Antagonism upon use of certain combination of bactericidal and bacteriostatic antibiotics. 4. Antibiotic therapy alone without surgical drainage of abscess. 5. Development of antimicrobial resistance.

Examples of Emerging Antimicrobial Resistance: In recent years, the Infectious Diseases Society of America has highlighted a group of antibiotic-resistant bacteria "ESKAPE" pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudo.monas aeruginosa, and Enterobacter species). They currently cause the majority of hospital acquired infections and effectively "escape" the effects of antibacterial drugs. a. Gram positive cocci: • Enterococci: Vancomycin is the drug usually reserved as a last resort for treating lifethreatening infections caused by gram positive organisms that are resistant to all P-lactam drugs. One of the most dramatic examples of antimicrobial resistance that is mainly detected among Enterococcus spp, is the vancomycin-resistant enterococci (VRE).

• Staphylococcus aureus: Staphylococcus au reus, another common cause of nosocomial infections, is becoming increasingly resistant to antimicrobials. Methicillin-resistant Staphylococcus aureus (MRSA) are resistant to methicillin as well as all other P-lactam drugs. Infections caused by these strains are generally treated with vancomycin. Few hospitals have reported isolates that are no longer susceptible to normal levels of vancomycin; vancomycin-intermediate S. aureus (VISA) and vancomycin-resistantS. aureus (VRSA).

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Clinical Microbiology mechanisms of res ista nce GraJil negative bacilli: Th e ~ain

b. ;odu"ction of P-l act ma ses maml

are thr ou gh

y:

, Extended sp ec tru m P-lactmascs

(ESBLs)

ne, istance to cefotaxime, ceftriaxo res fer con t tha es ym enz are They , as well as old er ~-Iactam nam reo azt and me idi taz cef , me ccfpodoxi is com mo n ma inl y am on g nce ista res of m nis cha me is drugs. Th Enterobacteriaceae.

, Ca rba pc na ma scs

of the most important me cha nis m Resistance to car bap cne ms is ella, ative bacilli (E.coli, Klebsi resistance am on g gra m neg er). onas aeruginosa and Acinetobact Enterobacter as well as Pseudom cs, nt to mo st types of antibioti car bap ena ma ses con fer resista lifeare the last resort in treating including car bap ene ms wh ich threatening infections.

SA FE TY IN FE CT IO N CO NT RO L AND MEASURES CT IV ES : INTENDED LE AR NI NG OB JE althcare acquired infections). 1. Define nosocomial infections (he ocomial infections. 2. Enumerate different types of nos ocomial infections. 3. Enumerate possible sources of nos care acquired infections. 4. List adverse outcome of health l flora of the hand. 5. Identify both types of microbia d hygiene. 6. Highlight the importance of han e. 7. Enumerate types of han d hygien types. 8. Define isolation and mention its es of isolation. 9. Mention differences bet we en typ e. ste and give examples of each typ 1O.Enumerate types of hospital wa regation ll.Ciassify hospital waste for seg

IN FE CT IO NS (HAis) HEALTH CA RE ASSOCIATED I HO SP IT AL AC QU IR ED (NOSOCOMIAL INFECTIONS INFECTIONS) during : An infection which is acquired Health car e ass oci ate d infection e of present or incubating at the tim hospitalization and which was not becomes acquired in the hospital and admission. Infection which is sidered HAL evident after discharge is also con

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TABLE O F C O N TE N TS ... .• ... · ••· ·· · .. · •• •· · ·•. ... ... ... : es tiv ec bj O ng ni ar Le Intended Ch ap te r I. Haematology:

1

2

Red Blood Cells: ... .. 3 ... ... · · · · · · · · · · · · · · · · · · · · · · · · · · · ·.. ... 3 ... . ... .. ... ... ... ... is ies po ro ... .... Eryth s .. . . . .. . .. . . . . . .. .. .. . .. .. .. .. .... 4 Red Blood Cell Abnormalitie s er et m ra Pa ll Red Blood Ce Anaemias: 4 of Anaemia Morphological Classification 4 mia Microcytic Hypochromic Anae 6 Macrocytic Anaemia 7 aemia Normochromic Normocytic An 7 Anaemia of Chronic Disorders 8 Haemolytic Anaemia 14 Polycythaemia 15 Ra te Er yt hr oc yt e Se di me nt ati on

~ -----16

------·----- Leucocytes: ytes Reactive Disorders of Leucoc Infectious Mononucleosis Lc uk ac mi as : Acute Leukemia Chronic Leukemia M ul tip le Myeloma e Myelodysplastic Sy nd ro m Hy pe rsp len ism Bone M ar ro w Ex am in ati on Ha cm os tas is: Physiology of Haemostasis meostatic Function Laboratory Evaluation of Ho Bt ec dm g D1sordcrs: Vascular Disorders Thrombocytopenia Qualitative Platelet Disorders Coagulation Disorders Bl oo d Tr an sf us io n

18 20 20 21 22

25 26 27 28 29 29 34

""31 37 38 39 40 43

ii

\ /

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Ch apt er 2. Clinical Chemistry: Hep atob iliar y Dis orde rs: . Bilirubin . Serum Enzyme Assays . Serum Proteins , Allumin & Prothuombim time Dia bete s Mel litus : Plasma Glucose Diabetes mellitus Laboratory Diagnosis Monitoring of Glycaemic Control Oral Glucose Tolerance Test

50 50 53

54 56 57 58

59 61

63

Dis orde rs of Plas ma Lipi ds and Lip opro tein s: Lipoprotein Disorders coal Diseases: Urine Analysis Kidney Function Tests Biochemical Findings in Some Renal Disorders Acid Bas e Bal anc e: Respiratory Disturbances Metabolic Disturbances

66 67

69 70

74 78 79 79

80 81

/ /

Pota ssiu m Usc of Enz yme s in Clin ical Diagnosis: rction Cardiac Enzymes in Diagnosis of Myocardial Infa / Acid Phosphatase Serum Amylase Clinical Dis orde rs of Cal cium and Pho sph ate: Serum Calcium Serum Phosphorus Hyperparathyroidism Hypoparathyroidism Metabolic Bone Diseases

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yroid Function Tests: Hypothyroidism Hyperthyroidism drome Non -Thyroidal Illness and the Sick Euthyroid Syn I

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/ Chapter 3. Clinical Immunology: 99 101 103 103 104 lOS lOS lOS 106 108 110 110 112 113 116 117

Autoimmune Diseases: Systemic Lupus Erythematosus Rheumatoid Arthritis Thyroid Autoimmune Diseases Disorders of the Pancreas Immunological Blood Diseases Myasthenia Gravis

Immunological Diagnosis of Liver Diseases: Acute Viral Hepatitis Chronic Hepatitis Hepatocellular Carcinoma

Hypersensitivity Immunologically Mediated Renal Diseases Immunodeficiency Diseases Miscellaneous Immunological Diseases -Tumor Markers

Chapter 4. Clinical Microbiology: ormal Flora icrobilogy Laboratory and Pre-analytical Variables iagnosis Of Infectious Diseaeses Central Nervous System (CNS) Infections Body Fluid Effusion Blood Stream Invasion Urinary Tract Infections Upper Respiratory Tract Infections Lower Respiratory Tract Infections Tuberculosis (T.B) Gastrointestinal Tract Infections Pyrexia of Unknown Origin Brucellosis Lower Genital Tract Infections and Sexually Transmitted Disease Transplacental Infections Abscesses and Wound Infections Anerobic Infections

Antibiotics Infection Control and Safety Measures

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Chapter 5. Flow charts: Flow chart for Investigating Jaundice (I) Flow chart for Investigating Jaundice (2) Flow chart tor Investigating Diabetes Mellitus Flow chart tor Investigating llypothyroidism Flow chart for Investigating Hyperthyroidism Flow chart for Investigating Rheumatic Diseases Flow chart for Investigating Acute Hepatitis Flow chart for Investigating Chronic Hepatitis flow chart tor Investigating Immunodeficiency Flow chart for classification ofbacteria.

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2. How to investigate a case of microcytic hypochromic anaemia 36. Lab tests to evaluate anaemia of chronic disease 21. Lab. diagnosis of a case' of congenital hemolytic anaemia . 25. Investigation of a case of megaloblastic anaemia 26. Lab. diagnosis of pernicious anaemia 5. Aplastic anaemia (causes, Lab. diagnosis) 20. Pancytopenia (Causes, DD & Lab. diagnosis) e. Benign Neutrophil Disorders e. Benign Lymphocytosis, Causes, Lab.diag., Differential 27. Lab. diagnosis of CML 28. Lab. diagnosis of multiple myeloma 44. Classify thrombocytopenia 47. Lab. diagnosis of hyper- and hyposplenism 11 . Haemartharosis, how to investigate, enumerate possible causes,discuss Lab. diagnosis of one 18. Lab. diagnosis of bleeding tendency 29. Thrombocytopenic purpura (causes, Lab. diagnosis) e. Neonatal Hemorrhage , Aetiology, inv, Management 9. Lab. diagnosis of thrombocytosis 12. Causes, Lab. diagnosis of a case of repeated venous thrombosis 40. Lab Investigation in hyperviscosity syndrome 41. Mechanism of action & monitoring of oral anticoagulant 14. Discuss DIC (causes, Lab. diagnosis) 7. Lab. diagnosis of Polyuria 19. Kidney function tests 23. Lab. diagnosis of a case of Pyuria e. Lab.diag. of Diabetic Nephropathy 5. Enzymatic liver function tests 8. Lab. diagnosis hepatitis B & C 17. Lab. diagnosis of viral hepatitis e. how to Investigate a case of acute viral hepatitis e. Causes & Inv of a case of Elevated serum transaminases (Alt & Ast) 6. Jaundice (def., types, Lab. tests used for diagnosis) 24. Investigation of a case of hyperbilirobinaemia e. Neonatal Jaundice, Types, Causes, Lab Findings 1. Thyroid function testes 15. Lab. profile of post thyroidectomy 22. Lab. diagnosis of a case of thyroid gland enlargement 43 . Lab. diagnosis of thyrotoxicosis 30. Monitoring of D.M 46 . Lab. diagnosis of hyperglycemia

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34. Lab. diagnosis of acute pancreatitis 13. Lab. diagnosis of a case presented by chest pain 35. List cardiac markers & their order of appearance after MI 16. Values of analysis of CSF

31. Bacteremia (causes, clinical features of food poisoning) 37 Hematological & serological tests used for diagnosis of glandular fever 42. Discuss atypical pneumonia (causes, complication, and Lab. diagnosis) 45 . Lab. diagnosis of AIDS 48 . Lab. diagnosis of TB. 3. Widal test (def., limitation,interpretation) 4. Hyperlipidemia (risk factors, how to investigate) 32. Causes of hyperphsphataemia 33. Pathogenesis of R.A & its Lab. diagnosis 10. Lab. diagnosis of SLE & its interpretation?

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