Chapter 1:  Table of Contents
Preface
For most of us, the perfect day might well be one spent
hugging a microscope in quiet contemplation. Inevitably,
however, this does not describe a typical day. Instead,
such moments must be stolen between the myriad clinical
laboratory difficulties that crop up every day. Even to those of
us with keen interest in it, the clinical laboratory is a mystery.
The truth is that many of us spend our days in the realm of
anatomic pathology, where through immersion we are able to
keep most important facts at the forefront of our brain. But
when heaved into clinical pathology, we often must relearn
things, comb through patient charts, ask questions, and
synthesize it all into a plan of action. When the problem is first
posed, we don’t always have a good answer; in this instant, we
sympathize, promise to investigate, and promise to follow up.
This state of affairs comes as a surprise to most young
pathologists. It is in fact normal and is no reflection upon
one’s training or dedication. A willingness to engage with and
assist clinicians in such moments is in large measure the value
of the local pathologist. The help we offer in these situations
is a large part of what defines us as a specialty. Furthermore,
Acknowledgements
The existence of Practical Clinical Pathology is credited to
Joshua Weikersheimer, who saw the value of expanding from
the Quick Compendium idea to create a book better suited to the
everyday needs of general pathologists. My mentor, Dr James
Kelley, exemplified the real value one could bring as a general
pathologist: by being always available to assist or consult,
always forthright in explanations and corrections, and fearless
ISBN 978-089189-5985
–3 SD –2 SD –1 SD mean +1 SD +2 SD +3 SD
small improvements in the clinical pathology laboratory are
quickly leveraged to impact a large number of patients in ways
that anatomic pathology can rarely achieve. Last, it is our
duty; in that humming and whirring place toil the too-often
nameless face shield-wearing people who are the long arm of
our own medical licenses.
The original Quick Compendium began as a publication of my
personal notes, begun when I was a resident, because I thought
that somebody might like to use them. Unexpectedly, many
came to depend upon them not only for board preparation but
also, alarmingly, for the practice of clinical pathology. That
certainly was outside the intended scope of use!
Practical Clinical Pathology hopes to provide a comprehensive
and up-to-date working review of clinical pathology; the
making of it required help from experts in a variety of
disciplines. So please pay attention to the Contributors’ page
that faces this Preface. With their help, we mean to bring a
text that encompasses what, to date, is of greatest practical
necessity for general pathologists and others wanting more
than can ever be included in a Quick Compendium.
Daniel Mais
in taking on new problems and in correcting entrenched old
ones. I hope that this is the sort of book that he might have
made good use of.
Last, I acknowledge those close to me who put up with me
(and without me) for so long while I was absorbed in doing
this book.
v
 Table of Contents
Chapter 1:  Table of Contents

Dedication . . . . . . . . . . . . . . iv Acute myocardial infarction (MI). . . . . . . . . . . . 9

–3 SD –2 SD –1 SD mean +1 SD +2 SD +3 SD

Contributors . . . . . . . . . . . . . . iv Cardiac reperfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Myocarditis. . . . . . . . . . . . . . . . . . . . . . . .9
Preface . . . . . . . . . . . . . . . . v Chemotherapy induced cardiac toxicity . . . . . . . . 10
Acknowledgements . . . . . . . . . . . v Proteins. . . . . . . . . . . . . . . 10
Chapter 1:  Chemistry Prealbumin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Anand Dighe, Dina Greene, Daniel Holmes & Daniel Mais
The liver. . . . . . . . . . . . . . . . 1
Liver function tests. . . . . . . . . . . . . . . . . . . .1
Aspartate aminotransferase (AST) & alanine aminotransferase (ALT) . . . . . . . . . 1
Lactate dehydrogenase (LD) . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Alkaline phosphatase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
γ-glutamyl transferase (GGT) . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
5′ nucleotidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Ammonia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Bilirubin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Prothrombin time (PT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
γ globulins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Albumin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Neonatal jaundice . . . . . . . . . . . . . . . . . . . .4
Acute hepatic injury . . . . . . . . . . . . . . . . . . . 5
The pancreas . . . . . . . . . . . . . . 6
Pancreatic enzymes. . . . . . . . . . . . . . . . . . . .6
Amylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Lipase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Laboratory evaluation of acute pancreatitis. . . . . . .6
Confirming acute pancreatitis . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Prognosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Etiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Tests of pancreatic exocrine function. . . . . . . . . . 7
Secretin-CCK test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Noninvasive tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
The heart . . . . . . . . . . . . . . . 7
Myocardial markers . . . . . . . . . . . . . . . . . . . 7
Creatine kinase (CK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Troponin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Myoglobin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
B-type natriuretic peptide (BNP) . . . . . . . . . . . . . . . . . . . . . . . . . 8
Acute coronary syndrome (ACS). . . . . . . . . . . . .9
Major serum proteins . . . . . . . . . . . . . . . . . 10
Albumin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
α1-antitrypsin (AAT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
α1-acid glycoprotein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
α2-macroglobulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Ceruloplasmin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Haptoglobin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Transferrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Fibrinogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
C-reactive protein (CRP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Patterns in serum protein electrophoresis. . . . . . . 12
Normal serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Bisalbuminemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
α1-antitrypsin (AAT) deficiency . . . . . . . . . . . . . . . . . . . . . . . . 12
Nephrotic syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Acute inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
β-γ bridging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Monoclonal gammopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
CSF protein electrophoresis . . . . . . . . . . . . . . 14
Urine protein electrophoresis . . . . . . . . . . . . . 15
Glomerular proteinuria pattern . . . . . . . . . . . . . . . . . . . . . . . . . 15
Tubular proteinuria pattern . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Overflow proteinuria pattern . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Cryoglobulinemia. . . . . . . . . . . . . . . . . . . . 15
Cryoglobulins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Mixed cryoglobulinemia (types II & III) . . . . . . . . . . . . . . . . . . . . . 15
Laboratory methods . . . . . . . . . . . . . . . . . . 15
Protein electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Immunofixation & immunotyping . . . . . . . . . . . . . . . . . . . . . . . 16
Acid-base & electrolytes . . . . . . . . . 16
Sodium . . . . . . . . . . . . . . . . . . . . . . . . . 16
Hyponatremia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Hypernatremia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Potassium. . . . . . . . . . . . . . . . . . . . . . . . 18
Potassium measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Hypokalemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Hyperkalemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18

vi Practical Clinical Pathology


Calcium. . . . . . . . . . . . . . . . . . . . . . . . . 18
Calcium measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Hypercalcemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Hypocalcemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Acid-base disorders. . . . . . . . . . . . . . . . . . . 20
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Henderson-Hasselbalch equation . . . . . . . . . . . . . . . . . . . . . . . . 21
Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Classifying an acid-base disorder . . . . . . . . . . . . . . . . . . . . . . . . 21
Renal function . . . . . . . . . . . . . . . . . . . . . 22
Renal function tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Laboratory screening for chronic kidney disease . . . 23
Laboratory evaluation in acute renal failure . . . . . 23
Hepatorenal syndrome. . . . . . . . . . . . . . . . . 24
Nephrogenic systemic fibrosis . . . . . . . . . . . . . 24
Laboratory tests in pregnancy. . . . . . 24
Amniotic fluid bilirubin (ΔOD 450). . . . . . . . . . 24
Human chorionic gonadotropin. . . . . . . . . . . . 25
hCG in normal gestation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
hCG in ectopic pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
hCG in spontaneous abortion . . . . . . . . . . . . . . . . . . . . . . . . . . 26
hCG in gestational trophoblastic disease (GTD) . . . . . . . . . . . . . . . . . . 26
Prenatal screening for trisomy & neural tube defects. 26
Available approaches to screening . . . . . . . . . . . . . . . . . . . . . . . 26
Analytes & risk calculation . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Clinical considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Assessing risk of preterm birth. . . . . . . . . . . . . 27
Determination of fetal lung maturity. . . . . . . . . 27
Lecithin/sphingomyelin (L/S) ratio . . . . . . . . . . . . . . . . . . . . . . . 28
Phosphatidylglycerol (PG) concentration . . . . . . . . . . . . . . . . . . . . 28
Foam stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Lamellar body count (LBC) . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Fluorescence polarization assay (S/A ratio)* . . . . . . . . . . . . . . . . . . . 28
Laboratory evaluation of diseases in pregnancy. . . . 28
Physiologic changes & altered reference ranges in pregnancy . . . . . . . . . . . 28
Medical conditions of particular importance in pregnancy . . . . . . . . . . . . . 28
Toxicology . . . . . . . . . . . . . . 30
Pharmacokinetics. . . . . . . . . . . . . . . . . . . . 30
Half life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Free vs bound . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Volume of distribution (Vd) . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Drugs of abuse screening (forensic toxicology) . . . . 30
Cocaine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Opiates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Barbiturates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Amphetamines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Phencyclidine (PCP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Ethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
ISBN 978-089189-5985
Table of Contents
Overdose . . . . . . . . . . . . . . . . . . . . . . . . 32
General aspects of laboratory evaluation . . . . . . . . . . . . . . . . . . . . 32
Toxic alcohol (ethylene glycol, methanol & isopropyl alcohol) poisoning . . . . . . 34
Lead poisoning (plumbism) . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Carbon monoxide (CO) poisoning . . . . . . . . . . . . . . . . . . . . . . . . 35
Acetaminophen (Tylenol) poisoning . . . . . . . . . . . . . . . . . . . . . . 35
Cyanide poisoning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Salicylate (aspirin) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Arsenic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Tricyclic antidepressants (TCAs) . . . . . . . . . . . . . . . . . . . . . . . . 37
Organophosphates & carbamates . . . . . . . . . . . . . . . . . . . . . . . . 37
Mercury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Therapeutic drug monitoring (TDM) . . . . . . . . . 37
Digoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Procainamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Aminoglycosides (eg, gentamicin) . . . . . . . . . . . . . . . . . . . . . . . 38
Vancomycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Quinidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Phenytoin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Lithium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Amiodarone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Lipids & carbohydrates. . . . . . . . . 38
Lipids . . . . . . . . . . . . . . . . . . . . . . . . . 38
Brief review of lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Lipid disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Carbohydrates . . . . . . . . . . . . . . . . . . . . . 41
Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Hypoglycemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Types of diabetes mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Diagnosis & monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Diabetic ketoacidosis (DKA) . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Hyperglycemic hyperosmolar nonketotic coma (HHNC) . . . . . . . . . . . . . . 44
The metabolic syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . .44
Tumor markers. . . . . . . . . . . . 44
Prostate-specific antigen (PSA) . . . . . . . . . . . . 44
Prostate cancer screening . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Adjunctive PSA indices . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Prostate cancer prognosis & recurrence detection . . . . . . . . . . . . . . . . 45
Carcinoembryonic antigen (CEA)
& colorectal carcinoma screening. . . . . . . . . . . 45
Thyroglobulin. . . . . . . . . . . . . . . . . . . . . . 46
Cancer antigen (CA) 125. . . . . . . . . . . . . . . . 46
CA 27.29 & 15 ‑ 3 . . . . . . . . . . . . . . . . . . . . 46
CA 19 ‑ 9. . . . . . . . . . . . . . . . . . . . . . . . . 46
α fetoprotein (AFP) . . . . . . . . . . . . . . . . . . 47
Human chorionic gonadotropin (hCG). . . . . . . . . 47
β2 microglobulin (β2M) . . . . . . . . . . . . . . . . 47
Alkaline phosphatase. . . . . . . . . . . . . . . . . . 47
Markers of neuroendocrine tumors . . . . . . . . . . 47
Carcinoid tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Markers of medullary thyroid carcinoma . . . . . . . . . . . . . . . . . . . . 47
Paraganglioma & pheochromocytoma . . . . . . . . . . . . . . . . . . . . . 48
Neuroblastoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Urine markers for urothelial carcinoma. . . . . . . . 48
vii
Table of Contents
Endocrine. . . . . . . . . . . . . . . 48
Thyroid chemistry . . . . . . . . . . . . . . . . . . . 48
Thyroid function tests (TFTs) . . . . . . . . . . . . . . . . . . . . . .
Hyperthyroidism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Hypothyroidism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Neonatal hypothyroidism . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Nonthyroidal illness syndrome (euthyroid sick syndrome) . . . . . . . . . . . . . 50
Exogenous estrogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Medications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Adrenal cortex . . . . . . . . . . . . . . . . . . . . . 50
Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Cushing syndrome (hypercortisolism) . . . . . . . . . . . . . . . . . . . . . . 51
Addison disease (primary adrenal insufficiency) . . . . . . . . . . . . . . . . . 51
Secondary adrenal insufficiency . . . . . . . . . . . . . . . . . . . . . . . . 52
Conn syndrome (hyperaldosteronism) . . . . . . . . . . . . . . . . . . . . . 52
Congenital adrenal hyperplasia . . . . . . . . . . . . . . . . . . . . . . . . 52
Renal artery stenosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Pituitary . . . . . . . . . . . . . . . . . . . . . . . . 53
Growth hormone (GH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
FSH & LH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Prolactin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
ACTH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Antidiuretic hormone (ADH) . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Postmortem chemistries . . . . . . . . . 54
Samples. . . . . . . . . . . . . . . . . . . . . . . . . 54
Analytes. . . . . . . . . . . . . . . . . . . . . . . . . 54
Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
BUN & creatinine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Sodium & chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Potassium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Digoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Tryptase & the postmortem diagnosis of anaphylaxis . . . . . . . . . . . . . . . 55
Body fluids. . . . . . . . . . . . . . 55
Urine . . . . . . . . . . . . . . . . . . . . . . . . . 55
Macroscopic examination . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Urine chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Nephrolithiasis (kidney stones) . . . . . . . . . . . . . . . . . . . . . . . . . 56
Urine microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Urine microscopy in the patient with acute renal failure . . . . . . . . . . . . . 58
Cerebrospinal fluid (CSF) . . . . . . . . . . . . . . . 59
CSF chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .59
CSF microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Pleural fluid . . . . . . . . . . . . . . . . . . . . . . 59
Pleural fluid chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Pleural fluid microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Peritoneal fluid (ascites fluid) . . . . . . . . . . . . . 60
Peritoneal fluid chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Peritoneal fluid microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Synovial fluid. . . . . . . . . . . . . . . . . . . . . . 61
Synovial fluid chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Synovial fluid microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Methods in enzymology . . . . . . . . . . . . . . . . 61
Enzyme activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Enzyme antigen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
References . . . . . . . . . . . . . . 62
viii
Chapter 2:  Blood Banking/
. . . . . . . . . . 48 Transfusion Medicine
Kimberly Sanford & Daniel Mais
Blood donation . . . . . . . . . . . . 69
Donor history & physical examination . . . . . . . . 69
Registration & donor identification . . . . . . . . . . . . . . . . . . . . . . . 69
Timing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Physical examination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Autologous donors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Apheresis donors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Blood obtained from therapeutic phlebotomy . . . . . . . . . . . . . . . . . . 70
Collecting blood from donor . . . . . . . . . . . . . . 70
Information given to donor . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Blood collection system . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Volume drawn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
After collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Donor adverse reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Laboratory testing of donor blood. . . . . . . . . . . 71
ABO & Rh testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Antibody screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Infectious disease screening . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Pretransfusion procedures . . . . . . . . 72
Routine pretransfusion procedures . . . . . . . . . . 72
Blood collection from recipient . . . . . . . . . . . . . . . . . . . . . . . . . 72
ABO & Rh testing of recipient blood . . . . . . . . . . . . . . . . . . . . . . . 72
The antibody screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Comparison with prior transfusion records . . . . . . . . . . . . . . . . . . . . 73
ABO & Rh testing of donor blood . . . . . . . . . . . . . . . . . . . . . . . . 73
The crossmatch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Compatible products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
Visual inspection of blood prior to issue . . . . . . . . . . . . . . . . . . . . . 75
Transfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Nonroutine pretransfusion procedures:
investigation of a positive antibody screen
or unexpected crossmatch incompatibility . . . . . . 76
The reagent RBC panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Types of reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Phases of testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Direct antiglobulin test (DAT) & indirect antiglobulin test (IAT) . . . . . . . . . . 77
Clinical significance of detected antibodies . . . . . . . . . . . . . . . . . . . 77
How to interpret the routine panel . . . . . . . . . . . . . . . . . . . . . . . 77
The nonroutine panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Recipient phenotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Recipient genotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Likelihood of finding compatible blood . . . . . . . . . . . . . . . . . . . . . 80
The positive crossmatch . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Illustrative nonroutine cases . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Other unusual findings . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Autoantibodies, a common cause of nonroutine findings. . 82
When to consider autoantibody . . . . . . . . . . . . . . . . . . . . . . . . 82
Warm reacting autoantibodies . . . . . . . . . . . . . . . . . . . . . . . . . 82
Cold reacting autoantibodies . . . . . . . . . . . . . . . . . . . . . . . . . . 82
Mixed type autoantibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Paroxysmal cold hemoglobinuria (PCH) . . . . . . . . . . . . . . . . . . . . . 83
Blood banking considerations with a cold autoantibody . . . . . . . . . . . . . 83
Drugs may induce a positive DAT by several mechanisms . . . . . . . . . . . . . 83
Practical Clinical Pathology

Transfusion in special clinical circumstances.84
Transfusion in SCD . . . . . . . . . . . . . . . . . . 84
Shock: fluid resuscitation, emergency release
& massive transfusion . . . . . . . . . . . . . . . . 84
Shock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
Fluid resuscitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
Emergency release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Massive transfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Therapeutic apheresis . . . . . . . . . . . . . . . . . 86
Definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Indications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Replacement fluid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Vascular access . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Medication interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Neonatal & intrauterine transfusion. . . . . . . . . . 86
Special blood requirements neonatal & intrauterine transfusion . . . . . . . . . . 86
Maternal immune thrombocytopenic purpura (ITP) . . . . . . . . . . . . . . . 87
Neonatal alloimmune thrombocytopenic (NAIT) . . . . . . . . . . . . . . . . . 87
Hemolytic disease of the fetus & newborn (HDFN) . . . . . . . . . . . . . . . . 87
Blood components . . . . . . . . . . . . . . . . . . . . . . . . . 89
RBC components . . . . . . . . . . . . . . . . . . . . 89
How prepared & stored . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Storage lesion. . . . . . . . . . . . . . . . . . . . . . 90
Transport & reissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Whole Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
RBCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Apheresis RBCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
RBC manipulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Indications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Contraindications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Platelets. . . . . . . . . . . . . . . . . . . . . . . . . 91
How prepared & stored . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
What it contains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Indications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Contraindications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Dosing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Prevention of platelet-transmitted infection . . . . . . . . . . . . . . . . . . . 91
Compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
Granulocyte concentrates . . . . . . . . . . . . . . . 92
How prepared & stored . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
What it contains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
Indications & contraindications . . . . . . . . . . . . . . . . . . . . . . . . . 92
Dosing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
Plasma products . . . . . . . . . . . . . . . . . . . . 92
How prepared & stored . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
What it contains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
Indications & contraindications . . . . . . . . . . . . . . . . . . . . . . . . . 92
Dosing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Cryoprecipitated anti‑hemophilic factor
(cryoprecipitate or cryo) . . . . . . . . . . . . . . . 93
How prepared & stored . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
What it contains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Indications & contraindications . . . . . . . . . . . . . . . . . . . . . . . . . 93
Dosing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Plasma derivatives . . . . . . . . . . . . . . . . . . . 93
Estimating the dose of factor VIII . . . . . . . . . . . . . . . . . . . . . . . . 94
Calculating the dose of factor VIII . . . . . . . . . . . . . . . . . . . . . . . . 94
ISBN 978-089189-5985
Table of Contents
Irradiated products. . . . . . . . . . . . . . . . . . . 94
How prepared & stored . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Indications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Leukoreduced products. . . . . . . . . . . . . . . . . 94
How prepared & stored . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Indications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Washed RBCs. . . . . . . . . . . . . . . . . . . . . . 94
How prepared & stored . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Indications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Blood substitutes . . . . . . . . . . . . . . . . . . . . 94
Synthetic oxygen carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Blood avoidance strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Blood group antigens. . . . . . . . . . 95
ABO & the carbohydrate antigens. . . . . . . . . . . 95
ABO, Lewis, H, I & i . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
ABO phenotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
The relationship of Le, Se, H, I, i & ABO . . . . . . . . . . . . . . . . . . . . . 96
Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
P/GLOB blood group. . . . . . . . . . . . . . . . . . 97
Antigens & phenotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Rh . . . . . . . . . . . . . . . . . . . . . . . . . 97
Rh antigens & phenotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Rh antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Kidd blood group system. . . . . . . . . . . . . . . . 99
Kidd antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Kidd antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Duffy . . . . . . . . . . . . . . . . . . . . . . . . . 99
Duffy antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Duffy antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
MNS system . . . . . . . . . . . . . . . . . . . . . . 99
MNS antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .99
MNS antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Kell blood group . . . . . . . . . . . . . . . . . . . . 100
Kell antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Kell antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Lutheran . . . . . . . . . . . . . . . . . . . . . . . . 100
Lutheran antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Lutheran antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Human leukocyte antigens (HLA). . . . . . . . . . . 100
Some hard to remember antigen/antibody facts. . . 101
Transfusion complications. . . . . . . . 101
Suspected transfusion reaction. . . . . . . . . . . . 101
Clinical signs & symptoms . . . . . . . . . . . . . . . . . . . . . . . . . . 101
What to do . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Febrile, nonhemolytic transfusion reactions (FNHTR) . 102
Allergic transfusion reactions . . . . . . . . . . . . . 102
Acute HTRs. . . . . . . . . . . . . . . . . . . . . . 102
DHTR & delayed serologic transfusion reaction (DSTR).103
Bacterial contamination. . . . . . . . . . . . . . . . 103
TA-GVHD. . . . . . . . . . . . . . . . . . . . . . . 103
Transfusion related acute lung injury (TRALI) . . . 104
Posttransfusion purpura (PTP) . . . . . . . . . . . . 104
Platelet refractoriness . . . . . . . . . . . . . . . . . 105
ix
Table of Contents
Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Cytomegalovirus (CMV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
West Nile Virus (WNV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Syphilis (Treponema pallidum) . . . . . . . . . . . . . . . . . . . . . . . . 106
Chagas disease (Trypanosoma cruzi) . . . . . . . . . . . . . . . . . . . . . . 106
Malaria (Plasmodium species) . . . . . . . . . . . . . . . . . . . . . . . . . 106
Babesiosis (Babesia microti) . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Creutzfeldt-Jakob disease (CJD) . . . . . . . . . . . . . . . . . . . . . . . . 106
Transfusion associated metabolic derangements . . . 106
Transfusion-associated circulatory overload (TACO) . . . . . . . . . . . . . . . 106
Hypocalcemia (citrate toxicity) . . . . . . . . . . . . . . . . . . . . . . . . 106
Hypothermia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Hyperkalemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Hypokalemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Iron overload . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Immunomodulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Medical directorship considerations unique
to blood banking: Hospital Transfusion Virology . . . . . . . . . . . . . . . 126
Committee, Process Control & Blood Bank
Equipment. . . . . . . . . . . . . . 107 Laboratory methods . . . . . . . . . . . . . . . . . . 126
Hospital Transfusion Committee. . . . . . . . . . . 107
Regulatory requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Transfusion Committee purpose . . . . . . . . . . . . . . . . . . . . . . . . 107
Conduct of the transfusion committee . . . . . . . . . . . . . . . . . . . . . 107
Documentation of transfusion committee proceedings . . . . . . . . . . . . . 107
Other regulatory requirements. . . . . . . . . . . . 107
Component identification & traceability . . . . . . . . . . . . . . . . . . . . 107
Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Records . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
Blood bank regulatory authority . . . . . . . . . . . . . . . . . . . . . . . . 108
Common calls. . . . . . . . . . . . . 108
Transfusion reactions . . . . . . . . . . . . . . . . .
Platelet shortage . . . . . . . . . . . . . . . . . . .
Compatible blood cannot be found
for an anemic patient. . . . . . . . . . . . . . . .
Mislabeled specimen . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . 109
Chapter 3:  Microbiology
John Branda, Bobbi Pritt, Patricia Simner & Daniel Mais
Clinical syndromes & causative agents . . 111
Urinary tract infection (UTI) . . . . . . . . . . . . . 111
Differential diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Laboratory approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Specific agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
Infectious diarrhea. . . . . . . . . . . . . . . . . . 112
Differential diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
Specific agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
Infectious diarrhea in AIDS . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Laboratory evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Pneumonia . . . . . . . . . . . . . . . . . . . . . . 115
Differential diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Specific agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
x Practical Clinical Pathology
Infective endocarditis (IE) . . . . . . . . . . . . . . 117
Differential diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Specific agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
Laboratory approach to diagnosis . . . . . . . . . . . . . . . . . . . . . . . 118
Meningitis . . . . . . . . . . . . . . . . . . . . . . . 119
Differential diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Specific agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
Laboratory evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Prosthetic joint infections . . . . . . . . . . . . . . . 121
Definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Causative agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Laboratory evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Sepsis . . . . . . . . . . . . . . . . . . . . . . . . . 122
Does this patient have sepsis? . . . . . . . . . . . . . . . . . . . . . . . . . 122
Catheter related sepsis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Neonatal sepsis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Causative agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
Other methods . . . . . . . . . . . . . . . . . . . . . 127
Serology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Direct antigen detection . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Histology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Molecular techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Human herpesviruses (HHV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Herpes simplex virus (HSV) . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Varicella-zoster virus (VZV) . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Cytomegalovirus (CMV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Epstein-Barr virus (EBV) . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
108
HHV6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
108 Adenovirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
Parvovirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
108 Polyomaviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
109 Papillomavirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
Poxviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Hepatitis viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
Orthomyxovirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
Paramyxoviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Picornaviruses (small [pico-] RNA viruses) . . . . . . . . . . . . . . . . . . . 138
Arboviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
Arenavirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
Retroviridae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
Parasitology. . . . . . . . . . . . . 142
Laboratory methods . . . . . . . . . . . . . . . . . . 142
Direct examination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
Serology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Molecular methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Parasites . . . . . . . . . . . . . . . . . . . . . . . . 144
Protozoa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
Apicomplexa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
Metazoa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Cestodes (tapeworms) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Additional pearls of parasitology - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

Mycology . . . . . . . . . . . . . . 158
Laboratory methods . . . . . . . . . . . . . . . . . . 158
Direct examination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
Fungal culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
Initial examination of a fungal isolate . . . . . . . . . . . . . . . . . . . . . 159
Fungal isolate growing as a mold in 25 ‑ 30°C culture:
thermally dimorphic fungi
& thermally monomorphic molds. . . . . . . . . . 160
Thermally dimorphic fungi (also called endemic fungi) . . . . . . . . . . . . . 160
Thermally monomorphic molds . . . . . . . . . . . . . . . . . . . . . . . . 164
Dematiaceous molds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Zygomycetes (pauciseptate molds) . . . . . . . . . . . . . . . . . . . . . . 172
Fungal isolate growing in culture at 25 ‑ 30°C
as yeast: yeast & yeastlike fungi . . . . . . . . . . 174
Approach to identification . . . . . . . . . . . . . . . . . . . . . . . . . . 174
Notes on specific yeasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Bacteriology. . . . . . . . . . . . . 177
Laboratory methods . . . . . . . . . . . . . . . . . . 177
Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Direct examination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Culture media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
Culture temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
Key biochemical tests. . . . . . . . . . . . . . . . . 180
Catalase test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
Coagulase test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
Novobiocin susceptibility . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
Optochin susceptibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
CAMP test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
PYR test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Bile-esculin test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Oxidase test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Indole test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Rapid urease test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
β-glucuronidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
Hippurate hydrolysis test . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
Lysozyme test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
Specific bacteria: key characteristics. . . . . . . . . 182
Gram+ cocci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
Gram– cocci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
Gram+ bacilli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
Gram– bacilli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
Spirochetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
Chlamydiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
Rickettsiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
Mycoplasma . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mycobacteria . . . . . . . . . . . . . 206
Laboratory methods . . . . . . . . . . . . . . . . . . 206
Direct examination of clinical specimens . . . . . . . . . . . . . . . . . . . . 206
Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
Nucleic acid amplification tests (NAATs) . . . . . . . . . . . . . . . . . . . . 207
Important species . . . . . . . . . . . . . . . . . . . 207
Mycobacterium tuberculosis (MTB) . . . . . . . . . . . . . . . . . . . . . . . 207
Nontuberculous mycobacteria . . . . . . . . . . . . . . . . . . . . . . . . . 209
References . . . . . . . . . . . . . . 211
Table of Contents
Chapter 4:  Hematopathology
Daniel Mais
Diseases of red blood cells . . . . . . . 215
Cytoskeletal disorders . . . . . . . . . . . . . . . . . 215
Hereditary spherocytosis (HS) . . . . . . . . . . . . . . . . . . . . . . . . . 215
Hereditary elliptocytosis (HE)/hereditary ovalocytosis . . . . . . . . . . . . . . 216
Hereditary stomatocytosis (HSt) . . . . . . . . . . . . . . . . . . . . . . . . 216
Enzyme disorders. . . . . . . . . . . . . . . . . . . 217
Glucose-6-phosphate dehydrogenase (G6PD) deficiency . . . . . . . . . . . . . 217
Pyruvate kinase (PK) deficiency . . . . . . . . . . . . . . . . . . . . . . . . 217
Pyrimidine 5′ nucleotidase deficiency . . . . . . . . . . . . . . . . . . . . . 218
Structurally abnormal hemoglobin variants
(hemoglobinopathies). . . . . . . . . . . . . . . . 218
Hemoglobin S (β6 glu→val) . . . . . . . . . . . . . . . . . . . . . . . . . 218
Hemoglobin C (β6 glu→lys) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .221
Hemoglobin E (β26 glu→lys) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
Hemoglobins D & G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
Hemoglobin Lepore . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
Hemoglobin constant spring (CS) . . . . . . . . . . . . . . . . . . . . . . . 221
Altered oxygen affinity hemoglobins . . . . . . . . . . . . . . . . . . . . . . 221
Unstable hemoglobins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
Methemoglobin (Hi, hemiglobin) . . . . . . . . . . . . . . . . . . . . . . . 222
Sulfhemoglobin (SHb) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
Carboxyhemoglobin (HbCO) . . . . . . . . . . . . . . . . . . . . . . . . . . 222
Thalassemia . . . . . . . . . . . . . . . . . . . . . . 223
General features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
α thalassemia syndromes . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
β thalassemia syndromes . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
Hemoglobin A2 prime (HbA2′ ) . . . . . . . . . . . . . . . . . . . 224
Hereditary persistence of fetal hemoglobin (HPFH) . . . . . . . . . . . . . . . 224
Immune hemolytic disorders. . . . . . . . . . . . . 224
Warm autoimmune hemolytic anemia (WAIHA) . . . . . . . . . . . . . . . . 224
Cold autoagglutinins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
Cryoglobulinemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
Paroxysmal nocturnal hemoglobinuria (PNH) . . . . . . . . . . . . . . . . . . 227
Disorders of marrow production. . . . . 228
Iron deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
Folate & vitamin B12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
Anemia of chronic disease (ACD) . . . . . . . . . . . . . . . . . . . . . . . 230
Sideroblastic anemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
Cong enital dyserythropoietic anemia (CDA) . . . . . . . . . . . . . . . . . . 231
Fanconi anemia (FA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
Dyskeratosis congenita (Zinsser-Engman-Cole syndrome) . . . . . . . . . . . . 231
. . . . . . . 205 Pure red cell aplasia (PRCA) . . . . . . . . . . . . . . . . . . . . . . . . . . 232
Congenital amegakaryocytic thrombocytopenia (CAMT) . . . . . . . . . . . . . 232
Thrombocytopenia with absent radii (TAR) syndrome . . . . . . . . . . . . . . 232
Congenital neutropenia (Kostmann syndrome) & cyclic neutropenia . . . . . . . 232
Shwachman-Diamond syndrome . . . . . . . . . . . . . . . . . . . . . . . 233
Myelokathexis (& WHIM syndrome) . . . . . . . . . . . . . . . . . . . . . . 233
Barth syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
Autoimmune neutropenia . . . . . . . . . . . . . . . . . . . . . . . . . . 233
Aplastic anemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
ISBN 978-089189-5985 xi
Table of Contents
Approach to the diagnosis
of quantitative abnormalities. . . . . . . . . . . . 233
Anemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
Erythrocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
Neutrophilia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
Lymphocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
Monocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
Eosinophilia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
Neutropenia (agranulocytosis) . . . . . . . . . . . . . . . . . . . . . . . . 238
Lymphopenia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
Monocytopenia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
Thrombocytopenia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
Thrombocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
Neoplastic hematopathology. . . . . . . 241
B cell neoplasms . . . . . . . . . . . . . . . . . . . . 241
General clinical considerations . . . . . . . . . . . . . . . . . . . . . . . . 241
Small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) . . . . . . 241
Mantle cell lymphoma (MCL) . . . . . . . . . . . . . . . . . . . . . . . . . 244
Follicular lymphoma (FL) . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
Marginal zone lymphoma (MZL) . . . . . . . . . . . . . . . . . . . . . . . . 247
Hairy cell leukemia (HCL) . . . . . . . . . . . . . . . . . . . . . . . . . . . 248
Prolymphocytic leukemia (PLL) . . . . . . . . . . . . . . . . . . . . . . . . 250
Lymphoplasmacytic lymphoma (LPL)/Waldenström macroglobulinemia . . . . . 250
Heavy chain disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
Diffuse large B cell lymphoma (DLBCL) . . . . . . . . . . . . . . . . . . . . . 250
Distinct clinicopathologic types of DLBCL . . . . . . . . . . . . . . . . . . . . 251
Immunodeficiency associated lymphoproliferative disorders . . . . . . . . . . . 254
Burkitt lymphoma/leukemia (BL) . . . . . . . . . . . . . . . . . . . . . . . 254
Lymphoblastic leukemia & lymphoma (ALL/LBL). . 255
General features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
Precursor B-ALL/LBL, NOS . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
B-ALL/LBL with recurrent genetic abnormalities . . . . . . . . . . . . . . . . 256
T-ALL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
Acute leukemia with mixed lineage . . . . . . . . . . . . . . . . . . . . . . 257
Plasma cell neoplasms . . . . . . . . . . . . . . . . 257
Plasma cell myeloma/multiple myeloma (MM) . . . . . . . . . . . . . . . . . 257
Plasma cell leukemia (PCL) . . . . . . . . . . . . . . . . . . . . . . . . . . 259
Solitary plasmacytoma (osseous & extraosseous) . . . . . . . . . . . . . . . . 259
Monoclonal gammopathy of unknown significance (MGUS) . . . . . . . . . . . 259
Osteosclerotic myeloma (POEMS syndrome) . . . . . . . . . . . . . . . . . . 259
T cell neoplasms . . . . . . . . . . . . . . . . . . . . 259
General features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
Peripheral T cell lymphoma (PTCL) . . . . . . . . . . . . . . . . . . . . . . . 260
Adult T cell leukemia/lymphoma (ATCL) . . . . . . . . . . . . . . . . . . . . 260
Angioimmunoblastic T cell lymphoma (AITL) . . . . . . . . . . . . . . . . . . 260
Anaplastic large cell lymphoma (ALCL) . . . . . . . . . . . . . . . . . . . . . 261
Large granular lymphocytic leukemia (LGL leukemia) . . . . . . . . . . . . . . 261
Aggressive NK cell leukemia (ANKL) . . . . . . . . . . . . . . . . . . . . . . 262
Nasal type NK/T cell (NTNKT) lymphomas . . . . . . . . . . . . . . . . . . . 262
Blastic NK cell lymphoma . . . . . . . . . . . . . . . . . . . . . . . . . . . 262
Enteropathy associated T cell lymphoma (EATCL) . . . . . . . . . . . . . . . . 262
Hepatosplenic T cell lymphoma (HSTCL) . . . . . . . . . . . . . . . . . . . . 262
Subcutaneous panniculitic T cell lymphoma . . . . . . . . . . . . . . . . . . 262
Cutaneous T cell lymphoma (CTCL), mycosis fungoides (MF) & Sézary syndrome . . 263
Hodgkin lymphoma (HL). . . . . . . . . . . . . . . 263
Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) . . . . . . . . . 263
Classic Hodgkin lymphoma (CHL) . . . . . . . . . . . . . . . . . . . . . . . 264
xii Practical Clinical Pathology
Myeloid neoplasms. . . . . . . . . . . . . . . . . . 266
Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266
Myelodysplastic syndromes (MDS) . . . . . . . . . . . . . . . . . . . . . . . 266
Myelodysplastic/myeloproliferative neoplasms (MD/MPN) . . . . . . . . . . . . 268
Myeloproliferative neoplasms (MPN) . . . . . . . . . . . . . . . . . . . . . 268
Myeloid & lymphoid neoplasms with eosinophilia
with abnormalities of PDGFRα, PDGFRβ, or FGFR1 . . . . . . . . . . . . . . . . 272
Acute myelogenous leukemia (AML) . . . . . . . . . . . . . . . . . . . . . . 272
Congenital acute leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . 277
Mast cell neoplasms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
Methods . . . . . . . . . . . . . . . 278
RBC indices. . . . . . . . . . . . . . . . . . . . . . 278
Manual techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
Automated techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
Counting reticulocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
Leukocyte indices. . . . . . . . . . . . . . . . . . . 280
Total leukocyte count . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
Leukocyte differential . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
Platelet indices . . . . . . . . . . . . . . . . . . . . 280
Detection of normal & variant hemoglobins. . . . . 281
Rapid detection of hemoglobin S . . . . . . . . . . . . . . . . . . . . . . . 281
Detection of hemoglobin F . . . . . . . . . . . . . . . . . . . . . . . . . . 281
Hemoglobin electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . 281
High pressure liquid chromatography (HPLC) . . . . . . . . . . . . . . . . . . 282
Molecular methods for hemoglobin identification . . . . . . . . . . . . . . . . 282
Hemoglobin oxygen saturation . . . . . . . . . . . . . . . . . . . . . . . . 282
Histochemistry & cytochemistry. . . . . . . . . . . 283
Wright stain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
Cytochemical stains for typing blasts . . . . . . . . . . . . . . . . . . . . . . 283
Leukocyte alkaline phosphatase (LAP) score . . . . . . . . . . . . . . . . . . 283
Immunophenotyping . . . . . . . . . . . . . . . . . 283
Flow cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
Immunohistochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
Immunophenotypic evolution in hematolymphoid cells . . . . . . . . . . . . . 286
Conventional cytogenetics & molecular techniques . 287
Cytogenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
Molecular examination of lymphocytes . . . . . . . . . . . . . . . . . . . . 287
Bone marrow biopsy . . . . . . . . . . . . . . . . . 288
Sites of hematopoiesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
Indications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
Peripheral blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
Bone marrow aspirate & touch imprints . . . . . . . . . . . . . . . . . . . . 289
Bone marrow biopsy & clot section . . . . . . . . . . . . . . . . . . . . . . 289
References . . . . . . . . . . . . . . 289
Chapter 5:  Coagulation
Daniel Mais
Hemostasis. . . . . . . . . . . . . . 295
Normal hemostasis occurs in 3 steps. . . . . . . . . 295
Vasoconstriction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
Platelet aggregation (primary hemostasis) . . . . . . . . . . . . . . . . . . . 295
Fibrin formation (coagulation) . . . . . . . . . . . . . . . . . . . . . . . . 295

Laboratory evaluation of hemostasis . . . 296
Laboratory evaluation of platelets. . . . . . . . . . 296
The bleeding time (BT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296
PFA-100 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
Platelet aggregometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
Platelet flow cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
Laboratory evaluation of coagulation . . . . . . . . 298
Activated clotting time (ACT) . . . . . . . . . . . . . . . . . . . . . . . . . 298
Activated partial thromboplastin time (aPTT) . . . . . . . . . . . . . . . . . . 298
Activated protein C resistance (APCR) screening assay . . . . . . . . . . . . . . 300
Anticardiolipin antibody (ACA) . . . . . . . . . . . . . . . . . . . . . . . . 300
Anti‑Xa assay (heparin antifactor Xa assay) . . . . . . . . . . . . . . . . . . . 300
Antithrombin (AT, previously antithrombin III) . . . . . . . . . . . . . . . . . 300
Bethesda assay (factor VIII inhibitor assay, anti‑factor VIII antibody assay) . . . . . 300
Clot retraction test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
Clot stability test (urea solubility test, factor XIII deficiency screen) . . . . . . . . 300
D-dimer & fibrin-degradation products (FDPs) . . . . . . . . . . . . . . . . . 301
Factor assays (II, V, VII, VIII, IX, X, XI, XII) . . . . . . . . . . . . . . . . . . . . 301
Fibrinogen activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
Lupus anticoagulant (LAC) assays . . . . . . . . . . . . . . . . . . . . . . . 301
Protein C activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
Protein S activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
Prothrombin variant (prothrombin 20210) test . . . . . . . . . . . . . . . . . 302
Prothrombin time (PT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
Thrombin time (TT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
von Willebrand factor assays . . . . . . . . . . . . . . . . . . . . . . . . . 302
Causes of excessive bleeding (hemophilia). 302
Laboratory evaluation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
Clinical clues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
Morphologic examination of platelets . . . . . . . . . . . . . . . . . . . . . 303
Platelet disorders. . . . . . . . . . . . . . . . . . . 304
Bernard-Soulier syndrome (BSS) . . . . . . . . . . . . . . . . . . . . . . . . 304
Platelet storage pool disorders . . . . . . . . . . . . . . . . . . . . . . . . 304
Glanzmann thrombasthenia . . . . . . . . . . . . . . . . . . . . . . . . . 305
von Willebrand disease (vWD) . . . . . . . . . . . . . . . . . . . . . . . . 305
Aspirin & NSAIDs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
Platelet defects in myeloproliferative neoplasms . . . . . . . . . . . . . . . . 308
Uremia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
Cardiopulmonary bypass . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
Paraproteinemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
Thrombocytopenia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
Coagulation defects. . . . . . . . . . . . . . . . . . 308
Hemophilia A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
Hemophilia B (Christmas disease) . . . . . . . . . . . . . . . . . . . . . . . 310
Inherited deficiency of factors II, V, VII, X, or XI . . . . . . . . . . . . . . . . . 310
Factor XIII deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
Contact factor deficiency (Factor XII, HMWK, prekallikrein) . . . . . . . . . . . . 311
Inherited combined factor deficiency . . . . . . . . . . . . . . . . . . . . . 312
Fibrinogen defects: afibrinogenemia, hypofibrinogenemia & dysfibrinogenemia . . 312
Acquired factor deficiencies . . . . . . . . . . . . . . . . . . . . . . . . . . 312
Disseminated intravascular coagulation (DIC) . . . . . . . . . . . . . . . . . . 312
Nonhemostatic causes of excessive bleeding. . . . . 313
Vascular & connective tissue disorders . . . . . . . . . . . . . . . . . . . . . 313
Causes of excessive thrombosis
(thrombophilia). . . . . . . . . . . 314
Clinical considerations. . . . . . . . . . . . . . . . 314
When to suspect thrombophilia . . . . . . . . . . . . . . . . . . . . . . . . 314
Clinical clues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314
Laboratory evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314
Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314
ISBN 978-089189-5985
Table of Contents
Specific causes of thrombophilia. . . . . . . . . . . 314
Activated protein C resistance (factor V Leiden) . . . . . . . . . . . . . . . . . 314
Prothrombin variant (prothrombin G20210A mutation) . . . . . . . . . . . . . 315
Antithrombin deficiency (antithrombin III deficiency) . . . . . . . . . . . . . . 315
Protein C & protein S deficiency . . . . . . . . . . . . . . . . . . . . . . . . 316
MTHFR gene mutation & hyperhomocysteinemia . . . . . . . . . . . . . . . . 316
Paroxysmal nocturnal hemoglobinuria (PNH) . . . . . . . . . . . . . . . . . . 317
Heparin cofactor II (HC-II) deficiency . . . . . . . . . . . . . . . . . . . . . . 317
Factor XII deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
Elevated plasminogen activator inhibitor type 1 (PAI-1 gene polymorphisms) . . . 317
Antiphospholipid (APL) syndrome: anticardiolipin antibody (ACA)
& lupus anticoagulant (LAC) . . . . . . . . . . . . . . . . . . . . . . . . 317
Platelet glycoprotein polymorphisms . . . . . . . . . 318
Wien-Penzing defect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
Sticky platelet syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
Heparin induced thrombocytopenia (HIT) . . . . . . . . . . . . . . . . . . . 319
Thrombotic thrombocytopenia purpura (TTP) . . . . . . . . . . . . . . . . . . 320
Hemolytic uremic syndrome (HUS) . . . . . . . . . . . . . . . . . . . . . . 320
Thrombocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
Therapeutic agents & monitoring. . . . . 321
Anticoagulants . . . . . . . . . . . . . . . . . . . . 321
Warfarin (Coumadin) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
Unfractionated heparin (UH) . . . . . . . . . . . . . . . . . . . . . . . . . 322
Low molecular weight heparin (LMWH) . . . . . . . . . . . . . . . . . . . . 323
Fondaparinux (Arixtra) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
Direct thrombin inhibitors (DTI) . . . . . . . . . . . . . . . . . . . . . . . . 323
New oral anticoagulants . . . . . . . . . . . . . . . . . . . . . . . . . . . 324
Antiplatelet agents . . . . . . . . . . . . . . . . . . 324
Aspirin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324
Thienopyridines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324
Dipyridamole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324
GPIIb/IIIa receptor antagonists . . . . . . . . . . . . . . . . . . . . . . . . 324
References . . . . . . . . . . . . . . 325
Chapter 6:  Immunology
Kimberly Sanford & Daniel Mais
Immune system . . . . . . . . . . . . 329
Cellular constituents . . . . . . . . . . . . . . . . . 329
Stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
B cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330
NK cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
Antigen presenting cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
Granulocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
Complement. . . . . . . . . . . . . . . . . . . . . . 331
Effect of formed complement . . . . . . . . . . . . . . . . . . . . . . . . . 331
Complement pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
MHC complex & HLA antigens. . . . . . . . . . . . 332
Evaluation of immune function. . . . . . 332
Screening tests . . . . . . . . . . . . . . . . . . . . . 332
History & physical examination . . . . . . . . . . . . . . . . . . . . . . . . 332
Global tests of immune system . . . . . . . . . . . . . . . . . . . . . . . . 333
Specific tests . . . . . . . . . . . . . . . . . . . . . . 333
Testing B cell function . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
Testing T cell function . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
Testing NK cell function . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
Testing neutrophil function . . . . . . . . . . . . . . . . . . . . . . . . . . 334
Testing complement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
HLA testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
xiii
Table of Contents
Transplantation specific considerations. . . . . . . 335
Transplantation testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
Transplant rejection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336
Graft vs host disease (GVHD) . . . . . . . . . . . . . . . . . . . . . . . . . 337
Primary immunodeficiency disorders . . . 337
B cell defects . . . . . . . . . . . . . . . . . . . . . . 337
General features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337
Bruton (X-linked) agammaglobulinemia . . . . . . . . . . . . . . . . . . . . 337
Common variable immunodeficiency (CVID) . . . . . . . . . . . . . . . . . . 338
Selective IgA deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338
Hyper-IgE syndrome (Job syndrome) . . . . . . . . . . . . . . . . . . . . . . 339
T cell defects . . . . . . . . . . . . . . . . . . . . . . 339
General features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339
DiGeorge syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339
Severe combined immunodeficiency (SCID) . . . . . . . . . . . . . . . . . . . 339
Hyper-IgM syndrome (X-linked immunodeficiency with hyper-IgM) . . . . . . . 339
Wiskott-Aldrich syndrome (WAS) . . . . . . . . . . . . . . . . . . . . . . . 340
Ataxia-telangiectasia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340
Chronic mucocutaneous candidiasis . . . . . . . . . . . . . . . . . . . . . . 340
Duncan disease (X-linked lymphoproliferative disease) . . . . . . . . . . . . . 340
Neutrophil/phagocytic defects. . . . . . . . . . . . . 340
General features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340
Chronic granulomatous disease (CGD) . . . . . . . . . . . . . . . . . . . . . 340
Chediak-Higashi syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . 340
Other neutrophil/phagocyte defects . . . . . . . . . . . . . . . . . . . . . . 341
Complement deficiencies . . . . . . . . . . . . . . . 341
C1 esterase inhibitor (C1 Inh) deficiency & hereditary angioedema (HAE) . . . . . 341
Autoimmunity & rheumatologic disease . . 342
Pathophysiology . . . . . . . . . . . . . . . . . . . . 342
Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342
Autoimmunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342
MHC disease associations . . . . . . . . . . . . . . . . . . . . . . . . . . . 342
Predisposing factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342
Notes on laboratory testing . . . . . . . . . . . . . . 342
General laboratory abnormalities in autoimmune disease . . . . . . . . . . . . 342
Synovial fluid & synovial tissue analysis . . . . . . . . . . . . . . . . . . . . 342
Autoantibody detection by immunofluorescence . . . . . . . . . . . . . . . . 343
Antinuclear antibodies (ANA) . . . . . . . . . . . . . . . . . . . . . . . . . 343
Antibodies to cytoplasmic constituents . . . . . . . . . . . . . . . . . . . . . 344
Automated methods for autoantibody detection . . . . . . . . . . . . . . . . 345
Other tests for autoimmune diseases . . . . . . . . . . . . . . . . . . . . . 346
Autoimmune diseases. . . . . . . . . . . . . . . . . 346
Systemic lupus erythematosus (SLE) . . . . . . . . . . . . . . . . . . . . . . 346
Rheumatoid arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
Seronegative spondyloarthropathies . . . . . . . . . . . . . . . . . . . . . . 348
Celiac disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348
Inflammatory bowel disease . . . . . . . . . . . . . . . . . . . . . . . . . 349
Progressive systemic sclerosis (scleroderma) . . . . . . . . . . . . . . . . . . 349
IgG4-related sclerosing disease . . . . . . . . . . . . . . . . . . . . . . . . 349
Autoimmune hepatitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350
Primary biliary cirrhosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350
Autoimmune cholangitis . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
Primary sclerosing cholangitis (PSC) . . . . . . . . . . . . . . . . . . . . . . 351
Sjögren syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
Vasculitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
Inflammatory myopathies . . . . . . . . . . . . . . . . . . . . . . . . . . 355
Myasthenia gravis (MG) . . . . . . . . . . . . . . . . . . . . . . . . . . . 356
Familial Mediterranean fever (FMF) . . . . . . . . . . . . . . . . . . . . . . 356
Hypersensitivity reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
References . . . . . . . . . . . . . . 358
xiv Practical Clinical Pathology
Chapter 7:  Molecular Pathology
George Leonard & Daniel Mais
Molecular biology . . . . . . . . . . . 361
The structure of nucleic acids (DNA & RNA) . . . . 361
Nucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
The phosphodiester bond . . . . . . . . . . . . . . . . . . . . . . . . . . . 362
Double stranded DNA (hydrogen bonding) . . . . . . . . . . . . . . . . . . . 362
Types of nucleic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362
Nucleic acid-modifying enzymes. . . . . . . . . . . 363
Polymerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
Ligase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
Nuclease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
Mitochondrial DNA . . . . . . . . . . . . . . . . . . 363
Mitochondrial genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
Heteroplasmy/homoplasmy . . . . . . . . . . . . . . . . . . . . . . . . . . 363
Maternal inheritance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
Fundamentals of expression . . . . . . . . . . . . . 364
Cell signaling basics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
Transcription & its regulation . . . . . . . . . . . . . . . . . . . . . . . . . 364
Translation & its regulation . . . . . . . . . . . . . . . . . . . . . . . . . . 365
Basics of protein structure. . . . . . . . . . . . . . 366
Primary structure (peptide bonds & amino acids) . . . . . . . . . . . . . . . . 366
Secondary structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
Higher-order structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
DNA replication & cell division . . . . . . . . . . . . 366
DNA replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
Cell cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
Meiosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
Classification of genetic anomalies . . . . . . . . . . 367
Classification schemes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
Effect on organism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
Concepts in human genetics. . . . . . . . . . . . . . 368
Chromosome structure & nomenclature . . . . . . . . . . . . . . . . . . . . 368
Molecular techniques. . . . . . . . . . 368
Karyotyping/G-banding . . . . . . . . . . . . . . . . 368
Basic techniques in molecular pathology . . . . . . . 368
Specimen requirements & isolation of nucleic acid . . . . . . . . . . . . . . . 368
DNA amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
Signal amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
Restriction enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372
Blotting (Southern, northern, western) . . . . . . . . . . . . . . . . . . . . 372
DNA sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372
Hybridization techniques (FISH, CGH, array CGH) . . . . . . . . . . . . . . . . 373
Applications . . . . . . . . . . . . . . . . . . . . . . 374
Parentage, forensic identity & chimerism using short tandem repeats . . . . . . . 374
Haplotyping using single-nucleotide polymorphisms . . . . . . . . . . . . . . 374
Whole-genome applications . . . . . . . . . . . . . . . . . . . . . . . . . 374
Pharmacogenomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375
Practical aspects of molecular pathology . . . . . . . . . . . . . . . . . . . . 375
Patterns of inheritance. . . . . . . . . . . . . . . . 376
Inherited vs acquired genetic disorders . . . . . . . . . . . . . . . . . . . . . 376
Inheritance patterns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376

Genetics of nonneoplastic disease . . . . 376
Renal . . . . . . . . . . . . . . . . . . . . . . . . . 376
Inherited nephritic syndrome . . . . . . . . . . . . . . . . . . . . . . . . . 376
Inherited nephrotic syndrome . . . . . . . . . . . . . . . . . . . . . . . . . 377
Inherited tubular disorders . . . . . . . . . . . . . . . . . . . . . . . . . . 378
Polycystic kidney disease . . . . . . . . . . . . . . . . . . . . . . . . . . . 378
Sporadic & multifactorial congenital renal disorders . . . . . . . . . . . . . . . 379
Cardiovascular . . . . . . . . . . . . . . . . . . . . 380
Channel conduction disorders . . . . . . . . . . . . . . . . . . . . . . . . . 380
Myocardial disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381
Structural cardiac disorders as a part of a larger genetic syndrome . . . . . . . . 381
Endocrine. . . . . . . . . . . . . . . . . . . . . . . 382
Adrenal cortex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382
Pituitary gland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
Parathyroid gland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
Thyroid gland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
Diabetes mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
Gastrointestinal, hepatobiliary & pancreatic . . . . . 385
Hirschsprung disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
Osler-Weber-Rendu syndrome (hereditary hemorrhagic telangiectasia) . . . . . . 385
Microvillus inclusion disease (MID) . . . . . . . . . . . . . . . . . . . . . . 386
Multifactorial disorders & syndromic disorders affecting the GI tract . . . . . . . 386
Biliary fibrocystic diseases (Caroli disease & congenital hepatic fibrosis) . . . . . . 387
Syndromic paucity of bile ducts (Alagille syndrome, arteriohepatic dysplasia) . . . 388
Hereditary hemochromatosis (HH) . . . . . . . . . . . . . . . . . . . . . . . 388
Wilson disease (hepatolenticular degeneration) . . . . . . . . . . . . . . . . 389
α1 antitrypsin deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
Bilirubin excretion disorders . . . . . . . . . . . . . . . . . . . . . . . . . 390
Inherited pancreatitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390
Neuromuscular . . . . . . . . . . . . . . . . . . . . 392
Central neurodegenerative disease . . . . . . . . . . . . . . . . . . . . . . 392
Peripheral neuropathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394
Skeletal muscle diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . 394
Congenital hearing loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396
Disorders of mitochondria. . . . . . . . . . . . . . 396
Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396
Structural mitochondrial DNA defects . . . . . . . . . . . . . . . . . . . . . 397
Microdeletion disorders . . . . . . . . . . . . . . . 397
Solid tumors & inherited tumor syndromes . . . . . 397
Gastrointestinal tract tumors . . . . . . . . . . . . . . . . . . . . . . . . . 397
Gastrointestinal stromal tumor (GIST) . . . . . . . . . . . . . . . . . . . . . 402
Pancreatic tumors . . . . . . . . . . . . . . . . . . . 403
Ductal adenocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403
Neuroendocrine tumors (Islet cell tumors) . . . . . . . . . . . . . . . . . . . 403
Hepatobiliary tumors. . . . . . . . . . . . . . . . . 403
Hepatocellular carcinoma (HCC) . . . . . . . . . . . . . . . . . . . . . . . . 403
Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404
Breast cancer . . . . . . . . . . . . . . . . . . . . . 404
HER2 (Neu, ERB-B2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404
TP53 tumor suppressor gene . . . . . . . . . . . . . . . . . . . . . . . . . 405
Steroid receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405
BRCA-associated tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . 405
Other inherited influences upon breast cancer . . . . . . . . . . . . . . . . . 406
Molecular classification of breast carcinoma (gene expression profiling) . . . . . . 406
ISBN 978-089189-5985
Table of Contents
Genitourinary tumors . . . . . . . . . . . . . . . . . 407
Renal cell carcinoma syndromes . . . . . . . . . . . . . . . . . . . . . . . . 407
Sporadic renal cell carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . 407
Wilms tumor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 408
Prostate cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 408
Urothelial (transitional cell) carcinoma . . . . . . . . . . . . . . . . . . . . . 409
Testicular tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409
Soft tissue & bone. . . . . . . . . . . . . . . . . . . 409
Ewing sarcoma family of tumors . . . . . . . . . . . . . . . . . . . . . . . 410
Neuroblastoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410
Rhabdomyosarcoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411
Synovial sarcoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411
Low grade fibromyxoid sarcoma (LGFMS)/hyalinizing spindle tumor
with giant rosettes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412
Tumors of adipocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412
Osteochondromatosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412
Head & neck tumors . . . . . . . . . . . . . . . . . . 412
Squamous cell carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . 412
Salivary gland tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412
Thyroid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413
Skin tumors. . . . . . . . . . . . . . . . . . . . . . 414
Basal cell carcinoma (BCC) . . . . . . . . . . . . . . . . . . . . . . . . . . 414
Melanoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414
Dermatofibrosarcoma protuberans (DFSP) . . . . . . . . . . . . . . . . . . . 414
Central nervous system tumors . . . . . . . . . . . . 415
Gliomas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415
Retinoblastoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416
Meningioma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416
Embryonal tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416
Pulmonary tumors . . . . . . . . . . . . . . . . . . 416
Gynecologic tumors. . . . . . . . . . . . . . . . . . 417
Inherited gynecologic tumor syndromes . . . . . . . . . . . . . . . . . . . . 417
Cervix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418
Endometrium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418
Gestational trophoblastic disease . . . . . . . . . . . . . . . . . . . . . . . 418
Other tumor syndromes . . . . . . . . . . . . . . . . 419
Tuberous sclerosis complex (Bourneville syndrome) . . . . . . . . . . . . . . . 419
Nevoid basal cell carcinoma syndrome (Gorlin Goltz syndrome) . . . . . . . . . 419
Neurofibromatosis type 1 (NF1; von Recklinghausen disease) . . . . . . . . . . 419
Neurofibromatosis type 2 (NF2; bilateral acoustic neuroma syndrome) . . . . . . 419
Li-Fraumeni syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . 420
Aniridia/WAGR syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . 420
Beckwith-Wiedemann syndrome . . . . . . . . . . . . . . . . . . . . . . . 420
Chromosomal breakage syndromes . . . . . . . . . . . . . . . . . . . . . . 420
PTEN-related disorders: Cowden syndrome,
Bannayan-Riley-Ruvalcaba syndrome & Proteus syndrome . . . . . . . . . . . 421
Carney complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421
References . . . . . . . . . . . . . . 422
xv
Table of Contents
Chapter 8:  Medical Directorship
Mary Mayo & Daniel Mais
Legislation & regulation,
agencies & oversight . . . . . . . . . 433
Legislation & regulations relating to laboratories. . 433
Clinical Laboratory Improvement Amendment of 1988 (CLIA ’88) . . . . . . . . . 433
Medical devices & biologics . . . . . . . . . . . . . . . . . . . . . . . . . . 434
Agencies that set nonbinding standards . . . . . . . . . . . . . . . . . . . . 435
Medicare, Medicaid & the Prospective Payment System . . . . . . . . . . . . . 435
Reimbursement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 436
Direct billing law, physician self-referral law (Stark law) & antikickback law . . . . 437
The Privacy Act & the Privacy Rule (HIPAA) . . . . . . . . . . . . . . . . . . . 437
The Occupational Safety & Health Administration (OSHA) . . . . . . . . . . . . 437
Financial considerations in the laboratory. 438
Types of costs & calculation of the breakeven point. 438
Budgeting. . . . . . . . . . . . . . . . . . . . . . . 439
Statistical considerations in the laboratory.439
Definitions. . . . . . . . . . . . . . . . . . . . . . . 439
Gaussian distribution, estimates of central tendency & estimates of variation . . . 439
Analytical accuracy & precision . . . . . . . . . . . . . . . . . . . . . . . . 440
Clinical sensitivity & specificity . . . . . . . . . . . . . . . . . . . . . . . . 440
Predictive value . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 440
Relative risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 440
Types of variables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 440
Diagnostic accuracy: receiver operating
characteristic (ROC) curves. . . . . . . . . . . . . 440
Diagnostic accuracy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 440
ROC curves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441
Reference intervals. . . . . . . . . . . . . . . . . . 441
Purpose of reference intervals . . . . . . . . . . . . . . . . . . . . . . . . . 441
Establishing reference intervals . . . . . . . . . . . . . . . . . . . . . . . . 441
Factors that confound reference intervals . . . . . . . . . . . . . . . . . . . 442
Implementation of new methods. . . . . 442
Overview . . . . . . . . . . . . . . . . . . . . . . . 442
Elements of method verification/validation. . . . . . 443
Calibration & calibration verification . . . . . . . . . . . . . . . . . . . . . . 443
Precision & establishment of quality control (QC) ranges . . . . . . . . . . . . . 443
Accuracy & inaccuracy (bias), method comparison,
analytic specificity & interference . . . . . . . . . . . . . . . . . . . . . . 443
Analytical sensitivity & functional sensitivity . . . . . . . . . . . . . . . . . . 444
Carryover tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444
Clinical reportable range (CRR) & analytical measuring range (AMR) . . . . . . . 444
Reference intervals & critical values . . . . . . . . . . . . . . . . . . . . . . 444
Specimen stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444
Information systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445
Written procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445
Education of laboratory staff & clinical staff . . . . . . . . . . . . . . . . . . . 446
Quality management. . . . . . . . . . 446
Definitions. . . . . . . . . . . . . . . . . . . . . . . 446
Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446
Quality management, quality improvement, quality control . . . . . . . . . . . 446
xvi Practical Clinical Pathology
Quality & safety initiatives. . . . . . . . . . . . . . 447
Statistical quality control . . . . . . . . . . . . . . . 447
Control specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447
Testing & evaluating controls . . . . . . . . . . . . . . . . . . . . . . . . . 448
Addressing “out of control” tests . . . . . . . . . . . . . . . . . . . . . . . 448
Proficiency testing (external quality assessment) . . 449
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449
Nonanalytic variables in laboratory
medicine: preanalytic & postanalytic . . . 450
Nonanalytic variables . . . . . . . . . . . . . . . . . 450
Preanalytic variables. . . . . . . . . . . . . . . . . 450
The preanalytic phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450
Patient identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450
Age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450
Gender . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450
Food intake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450
Exercise . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450
Smoking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450
Posture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450
Time of day . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450
Tourniquets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
Order of draw . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
Storage & transport conditions . . . . . . . . . . . . . . . . . . . . . . . . 451
Serum vs plasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
Underlying hematologic malignancy . . . . . . . . . . . . . . . . . . . . . . 451
Postanalytic variables . . . . . . . . . . . . . . . . . 451
The postanalytic phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
Result reporting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
Critical values reporting . . . . . . . . . . . . . . . . . . . . . . . . . . . 452
Laboratory calculations . . . . . . . . . . . . . . . . 452
Reticulocyte proliferation index (RPI) . . . . . . . . . . . . . . . . . . . . . 452
International normalized ratio (INR) . . . . . . . . . . . . . . . . . . . . . . 452
Corrected (platelet) count increment (CCI) . . . . . . . . . . . . . . . . . . . 452
Anion gap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452
Osmolal gap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452
Friedewald equation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452
Creatinine clearance (ClCr) . . . . . . . . . . . . . . . . . . . . . . . . . . 452
Fractional excretion of sodium (FENa) . . . . . . . . . . . . . . . . . . . . . 452
Henderson-Hasselbach equation . . . . . . . . . . . . . . . . . . . . . . . 452
Standard deviation (SD) . . . . . . . . . . . . . . . . . . . . . . . . . . . 452
Coefficient of variation (CV) . . . . . . . . . . . . . . . . . . . . . . . . . . 452
Corrected serum sodium in hyperglycemia: . . . . . . . . . . . . . . . . . . . 452
Corrected serum sodium in hyperlipidemia: . . . . . . . . . . . . . . . . . . 452
Corrected serum sodium in hyperproteinemia: . . . . . . . . . . . . . . . . . 452
Body surface area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452
References . . . . . . . . . . . . . . 453
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455
3:  Microbiology

laminated material. Clinical and radiologic features, micro-
scopic and macroscopic morphology and serology are com-
monly combined for definitive diagnosis.
Echinococcosis is acquired when ingesting food contami-
nated with eggs from the stool of an infected dog, the defini-
tive host. Sheep, cattle, and other herbivores are intermediate
hosts; thus, the infection is commonest in sheep and cattle-
raising areas (pastoral infections). There is also a wild (sylvatic)
life cycle that occurs between foxes and wolves and their her-
bivore prey. Echinococcus infection results in the formation of
cysts in various organs, particularly the liver. A single hydatid
cyst, sometimes containing daughter cysts, is seen in E granu-
losis infection, while multilocular cysts are seen with E multi-
locularis. Polycystic hydatid cyst disease is seen with E vogeli.
Hepatic cysts may rupture through the diaphragm, giving rise
to pulmonary cysts.
Additional pearls of parasitology t3.23-t3.26
t3.23  Dual infections involving parasites
Ascaris lumbricoides & Trichuris trichiura
Pinworm & Dientamoeba fragilis
Babesia, Lyme disease & Anaplasma phagocytophilum
Lepromatous leprosy or HTLV-1 & Strongyloides stercoralis hyperinfection
t3.24  Parasitic oculocutaneous infections
Loa loa—disease is caused by the adult worm
Onchocerca volvulus—disease is caused by the larvae
The microscopic morphology of fungi in direct wet prepara-
tions can help with initial classification. Yeasts are unicellular
organisms, most of which display budding, and many of
which form pseudohyphae. Molds are multicellular organisms
which form true hyphae. Hyphal structures may be further
classified as septate, aseptate or pauciseptate.
India ink is used for the presumptive identification of
Cryptococcus neoformans, particularly in CSF. A small amount
of India ink is added to a drop of CSF on a slide. The slide
is examined for the presence of encapsulated yeasts, which
appear round, with wide variation in size, and narrow-based
budding. A clear halo around the yeast cells indicates the pres-
ence of a capsule, which strongly suggests Cryptococcus as
few other yeast species are encapsulated.
In tissue sections, fungi are difficult to detect by examining
routine H&E-stained sections. The exception are the intrinsi-
cally pigmented (dematiaceous) molds. For sensitive detection
of most fungi, special stains are needed. Commonly, either
a Gormori methenamine silver (GMS) or periodic acid schiff
(PAS) stain is used.
Fungal culture
For general purpose cultures, several media are typically
inoculated and incubated simultaneously, including brain
heart infusion (BHI) agar, Sabouraud dextrose agar, and inhibi-
tory mold agar t3.27. Cultures are incubated at 25 ‑ 30°C for 4 - 6
weeks. Special culture techniques may be applied either for
initial isolation or for differentiation, in some circumstances t3.28

t3.27  Fungal media

t3.25  Parasitic infections capable of person to person spread
Enterobius vermicularis
Fungal medium Principle Purpose
Sabouraud dextrose Acid pH & high dextrose General-purpose medium for cultivation/
Hymenolepis nana
agar (SDA) concentration inhibits bacterial isolation of many fungi
growth but permits growth of
fungi; some formulations also
t3.26  Parasitic infections in immunodeficient patients contain antibiotics
Type of immunodeficiency Susceptibility Inhibitory mold agar Nutrient-rich medium Selective isolation of fungi from
T cell (cellular) immunodeficiency Many, eg, Toxoplasmosis, Cryptosporidium, (IMA) containing chloramphenicol specimens that may contain commensal
Cystoisospora, Cyclospora, microsporidia, are & sometimes gentamicin or bacterial flora
more common ciprofloxacin; chloramphenicol
Others, eg, Strongyloides, are more severe suppresses the growth of many
B cell (humoral) immunodeficiency Giardia more common bacteria
Splenectomy Babesiosis more severe Brain heart infusion Nutrient-rich medium The nonselective formulation is a
agar (BHI) containing brain/meat general purpose medium used in the
infusion, peptone & dextrose cultivation/isolation of bacteria, yeasts
Chloramphenicol & gentamicin & molds
can be added for selectivity When supplemented with
chloramphenicol & gentamicin, it is
Mycology used for selective isolation of fungi from
Laboratory methods specimens that may contain commensal
bacterial flora
Direct examination Cycloheximide- Cycloheximide inhibits the Selective isolation of slow growing
For examination of primary specimens, wet preparations containing media growth of many saprophytic pathogenic fungi that may be
are typically made using a stain for fungi, such as the cal- fungi, while permitting overgrown by rapidly growing
cofluor white stain. Calcofluor white is a fluorochrome that growth of most (but not all) saprophytic fungi
pathogenic fungi Notably, cycloheximide also inhibits the
selectively binds to the cellulose and chitin in fungal cell walls. Chloramphenicol & gentamicin growth of certain pathogenic fungi,
Human cells, which lack these targets, remain unstained. The can be added to inhibit including C neoformans, many Candida
slide is then examined using fluorescence microscopy. Fungal bacteria species, Aspergillus species & the
elements, whether yeasts or hyphae, appear bright white, blue zygomycetes, among others
or apple green, depending on the microscope’s UV filter f3.35. It is frequently used to target
dermatophytes or thermally dimorphic
The calcoflour white stain will highlight all fungi, including fungi
Pneumocystis, plus a few nonfungal organisms, including
Prototheca and the cysts of Acanthamoeba.

158 Practical Clinical Pathology

 3:  Microbiology

Initial examination of a fungal isolate t3.29

The first clue is the colony morphology: yeast form creamy or
mucoid colonies; molds make fuzzy colonies. Thermally dimor-
phic fungi grow as yeast when incubated at 37°C, but grow as
molds when incubated at 25°C or 30°C.
A fuzzy colony usually indicates the presence of a mold. The
major considerations in the differential diagnosis are the hyaline
septate molds (also called hyaline hyphomycetes), the dema-
tiaceous molds, the zygomycetes, and the thermally dimorphic
fungi. In order to differentiate among these, 3 major character-
istics are considered—rate of growth, hyphal septations, and
pigmentation.
ƒƒ The hyaline septate molds tend to grow moderately to
rapidly, and have hyphae with frequent septations. The
surface of the colonies may be white or colored, due to
various (nonmelanin) pigments in their reproductive
structures, but the reverse side of the plate is usually light,
as the hyphae do not contain dark melanin pigment.
f3.35  Fungal hyphae stained with calcofluor white, viewed under UV light ƒƒ The dematiaceous molds grow moderately to slowly, and
have hyphae with frequent septations. Typically, these
molds have melanin pigment in both their hyphae and their
reproductive structures, and so the surface and reverse side
of the plate are both dark.
t3.28  Specialized fungal culture media
ƒƒ The zygomycetes are extremely rapidly growing molds that
Medium Purpose may cover the surface of the dish after overnight incubation
Cornmeal or potato Promotion of characteristic reproductive structures & pigmentation
dextrose agar useful for morphologic identification of mold isolates, when general- (lid lifters). They are aseptate or, more commonly,
purpose media prove inadequate for a particular isolate pauciseptate. Like the hyaline molds, they do not contain
Cornmeal or rice agar Promotion of characteristic structures (eg, chlamydospores, melanin pigment and the reverse side of the plate is light.
with Tween 80 arthroconidia) useful for morphologic identification of yeast isolates,
when more routine identification methods prove inadequate
ƒƒ Thermally dimorphic fungi are slow growing, sometimes
Sabouraud dextrose Isolation/cultivation of Malassezia species, all of which (except requiring several weeks for colonies to develop. When
agar, Dixon medium, M pachydermatis) require lipid supplementation for growth incubated at 30°C, the usual incubation temperature for
or Leeming-Notman fungal culture, young colonies tend to be white, and
medium overlaid with hyphae are septated. In order to confirm that the fungus
sterile olive oil
is thermally dimorphic, it can be converted from the mold
Trichophyton agars Differentiation between species of Trichophyton, which can be difficult
to speciate based on morphology alone form to the yeast form by reincubating the colonies at 37°C.
Bird seed (niger seed) Demonstration of phenol oxidase activity in Cryptococcus neoformans DNA-based testing is commercially available for some
agar isolates of the more common dimorphic fungi (eg, H capsulatum,
B dermatitidis and C immitis). These tests employ labeled
oligonucleotide probes complementary to segments of
species-specific ribosomal RNA (rRNA).

t3.29  Classification of fungi based on morphology

Yeast Mold Thermally dimorphic
Morphology Blastoconidia Blastoconidia with Arthroconidia Septate hyphae Pauciseptate hyphae
only pseudohyphae Hyaline molds (hyphae & other Dematiaceous molds
structures are non-melanized) (hyphae and/or conidia
are darkly pigmented
with melanin)
Important Cryptococcus Candida species (except Trichosporon Aspergillus Alternaria Zygomycetes (eg, Rhizopus, Histoplasma capsulatum
examples Candida C glabrata) Geotrichum Penicillium P boydii/S boydii Mucor, Cunninghamella, Blastomyces dermatitidis
glabrata Saccharomyces cerevisiae Fusarium Scedosporium prolificans Rhizomucor) Coccidioides immitis/posadasii*
Rhodotorula Dermatophytes Curvularia Paracoccidioides brasiliensis
Malassezia (Epidermophyton, Microsporum, Sporothrix schenkii
Trichophyton) Penicillium marnefii
*In routine culture, Coccidioides produces the same structures (septate hyphae & arthroconidia) whether grown at 25°C or 37°C; it can technically be considered thermally dimorphic, though, because the structures it produces in
tissue (spherules) can be induced in culture at 37°C using special media

ISBN 978-089189-5985 159

3:  Microbiology

ƒƒ Once a fungus has been placed into one of these broad

categories, examination of its reproductive structures a b
(conidia or spores) often provides the basis for a genus- or
species-level identification.
A smooth, creamy or pasty colony usually indicates the
presence of a yeast. Cryptococcus species may produce mucoid
colonies, because they are encapsulated. Candida albicans may
form “feet” or star-like projections, because of its ability to form
true hyphae in addition to pseudohyphae and blastoconidia.
The presence of yeast colonies can be confirmed by making c d
a simple wet preparation, which will demonstrate budding
yeast (blastoconidia) and may also demonstrate pseudohyphae
and occasionally, true hyphae as well. A few yeast also pro-
duce arthroconidia. Once a yeast has been detected, it can be
fully identified using several methods including biochemical
testing, MALDI-TOF mass spectrometry or morphologic clas-
sification after growth on special yeast morphology medium.

f3.36  H capsulatum
Fungal isolate growing as a mold in a GMS stained, b PAS stained tissue sections demonstrate small yeasts with narrow based budding;
25 ‑ 30°C culture: thermally dimorphic c H&E stained, d Wright stained smears demonstrate intracellular yeast forms
fungi & thermally monomorphic molds
Thermally dimorphic fungi (also called endemic fungi) t3.30

t3.30  Summary of dimorphic fungi

Common sites
Yeast form Mold form Route of of disseminated
Species characteristics characteristics infection infection
H capsulatum 2 ‑ 4 µm, narrow- Septate hyphae with tear Inhalation Reticuloendothelial
var based budding shaped microconidia & system,
capsulatum thick-walled, tuberculate mediastinum
macroconidia
B dermatitidis 8 ‑ 15 µm thick- Septate hyphae with Inhalation Skin, mucous
walled, broad- lollipop-like conidia membranes
based budding atop unbranched
conidiophores
C immitis 10 ‑ 100 µm Barrel shaped Inhalation Skin, bone, joints
spherules arthroconidia alternating
containing 2 ‑ 5 µm with empty cells
(nonbudding)
endospores
P brasiliensis 10 ‑ 50 µm Septate hyphae with Inhalation or Skin, mucous
mariner’s wheel intercalary & terminal traumatic membranes, bone
(circumferential chlamydospores inoculation marrow, lymphatics
f3.37  H capsulatum cultured at 25-30°C colony morphology—powdery or cottony white mold
budding)
S schenckii 4 ‑ 6 µm elongated Rosettes of microconidia Traumatic Regional lymphatics
(cigars) with at the apex of swollen, inoculation
narrow-based delicate conidiophores
budding smooth, microconidia and large (7 ‑  15 μm), thick  walled,
P marneffei 3 ‑ 5 µm ovoid, Colonies producing Inhalation Bone marrow, skin spiny macroconidia f3.38. The latter are extremely helpful for
divide by fission diffusible red pigment morphology-based identification, but may be absent in imma-
ture cultures. This makes yeast conversion, exoantigen tests, or
Histoplasma capsulatum molecular confirmatory tests very important.
In tissue sections f3.36 or primary wet preparations H  cap- Histoplasmosis caused by H  capsulatum var capsulatum is
sulatum var capsulatum yeast cells typically are found within endemic in the eastern regions of the United States, and espe-
histiocytes or reticuloendothelial cells, where they are seen as cially the Ohio and Mississippi River valleys. It is also found
2 - 4 µm ovoid yeast with narrow-based budding. A retraction throughout Latin America. In the environment, the fungus
artifact in tissue sections may mimic encapsulation. H capsu- favors soil contaminated by droppings from chickens, other
latum var duboisii is contrasted with H  capsulatum var capsu- birds, and bats, because of its high nitrogen content. H capsu-
latum in t3.31. latum var duboisii is found in Central and western Africa.
In cultures incubated at 25 ‑ 30°C, H  capsulatum is a very
slow growing, powdery or cottony white mold f3.37 that forms
septated, hyaline hyphae with intermittent small (2 ‑ 5 μm),

160 Practical Clinical Pathology

 3:  Microbiology

a b a b

f3.38  H capsulatum; in mold form, demonstrates a septated, hyaline hyphae with intermittent small f3.39  B dermatitidis
(2 μm), smooth, microconidia & b large (7-15 μm), thick walled, spiny macroconidia a & b Smears demonstrate uniform, large (8-15 μm) yeasts with broad based budding; the yeast cell
walls are thick & double contoured

t3.31  Histoplasma capsulatum

var capsulatum var duboisii
Geography Worldwide, but most common in North and Central and western Africa, especially
Central (Latin) America; eastern US repre- Nigeria, Senegal, the Congo and
sents the area of highest endemicity Angola
Disease Pulmonary, with or without dissemination to May be localized or disseminated;
the reticuloendothelial system most frequently involves skin,
subcutaneous tissue & bone
Culture Slowly growing, white cottony colonies; micro- Colony and microscopic morphol-
scopic examination reveals hyaline, septate ogy is indistinguishable from var
mold with smooth microconidia and thick- capsulatum
walled, tuberculate (spiked) macroconidia
Tissue Often intracellular within histiocytes or re- Often intracellular within giant cells
ticuloendothelial cells; oval, small (2 ‑ 4 µm) or macrophages
yeast bud on a narrow base Round to oval, thick-walled yeast
measuring 7 ‑ 15 µm
Bud on a narrow base, unlike
B dermatitidis, the yeast of which
are similar in size

Histoplasmosis caused by H capsulatum var capsulatum may
be asymptomatic, or may cause acute or chronic pulmonary
infection. Disseminated histoplasmosis may follow pulmo-
nary infection, particularly in HIV/AIDS patients and other
immunocompromised hosts. Disseminated infection may
lead to oropharyngeal ulcers, hepatic and/or splenic involve-
ment, or infection of the bone marrow, central nervous system,
major arteries or cardiac valves. Histoplasmosis caused by
H capsulatum var duboisii may be acquired by inhalation or by
direct inoculation of the skin. It most often presents with skin
(chronic ulcers), subcutaneous tissue involvement (nodules), or
bone involvement (osteolytic lesions), and pulmonary infec-
tion is not a typical feature.
In addition to culture, the diagnosis of histoplasmosis can
be made with an antigen test performed on urine or serum.
Blastomyces dermatitidis
In tissue sections, touch imprints f3.39, primary wet prepara-
tions, or culture at 37°C, B dermatitidis appears as fairly uni-
form, large (8 - 15 µm) yeasts with broad-based budding. The
yeast cell walls are thick and double-contoured.
When cultured at 25 ‑ 30°C, B dermatitidis is a slow growing
mold, with a cottony, white surface that darkens to tan with
age f3.40. Microscopic examination of the mold colonies reveals
septate, hyaline hyphae with short, unbranched conidiophores
producing single, pyriform to round, smooth conidia that mea-
sure 2 ‑ 10 μm. The conidiophores together with their solitary
conidia are sometimes referred to as “lollipops.” Because this
morphology is similar to that of certain nondimorphic molds
ISBN 978-089189-5985
f3.40  B dermatitidis; the mold form has a cottony white surface that darkens to tan with age
(eg, Chrysosporium), definitive identification of B  dermatitidis
rests either on conversion to its yeast form at 37°C, or exoan-
tigen tests or molecular confirmatory tests.
Blastomycosis has a geographic distribution that partly
overlaps with that of Histoplasmosis, being endemic in the
Mississippi and Ohio River valleys. It is also endemic in the
Southeastern US and in areas bordering the Great Lakes
(several states and Canadian provinces), and the St Lawrence
Seaway (parts of New York and Canada). It is thought to thrive
in the soil of wooded areas.
B dermatitidis initially infects the lung, where it can produce
acute and/or chronic infection, although many infections are
asymptomatic. Severe pulmonary disease and disseminated
infection occur more frequently in immunocompromised
hosts. Disseminated infection most often involves the skin or
mucous membranes of the nasopharynx and mouth. Other
common sites of disseminated infection include the bones,
prostate, and central nervous system. Rare primary cutaneous
blastomycosis may arise as a result of direct inoculation fol-
lowing trauma or dog bite. In addition to culture, the diagnosis
can also be made with an antigen test performed on urine or
serum.
161
3:  Microbiology

a b

c d

f3.41  C immitis f3.42  C immitis; in culture, mature arthroconidia are barrel shaped & alternate with empty cells
a, c-d In tissue sections stained with H&E, it appears as large (10-100 μm) spherules with thick, hyaline
walls that enclose numerous tiny (2-5 μm) endospores, which do not bud
b The spherules are highlighted by GMS stain

Coccidioides immitis/posadasii
In tissue sections f3.41 or primary wet preparations, Coccidioides
is seen as large (10 - 100 µm) spherules with thick, hyaline walls
that enclose numerous tiny (2 - 5 µm) endospores. The spher-
ules may be confused with the sporangia of Rhinosporidium
seeberi or Prototheca species. Sometimes the endospores are
seen spilling from the spherules, and individual endospores
may be confused with small yeasts such as those produced by
H capsulatum. However, in contrast to yeast, the endospores of
Coccidioides do not bud. Septated hyphae may also be seen in
tissue, particularly in association with foreign material.
In routine culture, whether incubated at 25 ‑ 30°C or 37°C,
C immitis grows moderately to rapidly in its mold form, initially
forming glabrous, moist, gray colonies that become white and
cottony when mature. Microscopic examination reveals thin,
hyaline, septate hyphae and arthroconidia. Mature arthroco-
nidia are barrel shaped, and alternate with empty cells f3.42.
However, immature arthroconidia are not barrel shaped and
may resemble the arthroconidia of Malbranchea species. If spe-
f3.43  P brasiliensis yeast form with circumferential budding
cial media are used, incubation at 37°C can produce the same
morphology seen in tissue sections, including spherules.
Coccidioides has recently been divided into 2 separate species:
C  immitis, which is found mostly in the San Joaquin Valley of
California, and C posadasii, which is found outside of California factor for severe disease. In addition to culture, the diagnosis
in the Southwestern United States (Arizona and New Mexico) can also be made with an antigen test performed on urine or
as well as parts of Mexico and Central America. The 2 species serum, or with antibody testing.
are morphologically indistinguishable and produce the same Coccidioides is a risk to laboratory personnel and can be con-
clinical syndrome. The infectious arthroconidia are present in tracted from laboratory cultures.
soil, and soil disruption or soil exposure are risk factors.
Coccidioides is acquired through inhalation of environmental Paracoccidioides brasiliensis
arthroconidia, causing pulmonary infection. It may also dis- In tissue sections or primary wet preparations, the diagnostic
seminate, most commonly to skin but also to bone, joints, the form is a round, large (10 ‑ 50 µm) yeast cell with circumferential
meninges or other organs. Compromise of the immune system budding, giving the appearance of a mariner’s wheel f3.43. The
predisposes infected persons to dissemination (eg, HIV/AIDS, daughter cells bud on a narrow base from the mother cell.
corticosteroid use), and for some reason those in certain ethnic When cultured at 25 ‑ 30°C, P  brasiliensis is a slow  growing
groups (Filipino and African American) are at greater risk for mold with a white to tan surface and a variable texture that may
dissemination. Pregnancy in the third trimester is another risk be leathery, velvety or glabrous. Microscopic examination of the

162 Practical Clinical Pathology


f3.44  S schenckii mold colonies cultured at 25-30°C
f3.45  S schenckii mold form; conidiophores topped by clusters of microconidia (“rosettes”)
cultured mold reveals septate, hyaline hyphae with terminal and
intercalary chlamydospores, and infrequent, pear shaped micro-
conidia arranged along the hyphae. Definitive identification
requires conversion of the mold form to the yeast form.
Paracoccidioidomycosis is encountered in the rainforests
of Central and South America. It can be acquired either by
inhalation or by direct inoculation during penetrating injury.
In adults, pulmonary infection is chronic and indolent, and
untreated infection often leads to dissemination involving
the skin, oral mucosa, lymphatics, viscera or central nervous
system. In children and immunocompromised hosts, the infec-
tion progresses more rapidly and frequently disseminates to
involve bone marrow, lymphatics and the abdominal organs.
ISBN 978-089189-5985
3:  Microbiology
f3.46  S schenckii; yeast form cultured at 37°C
f3.47  S schenckii yeast form; “cigar bodies”
Sporothrix schenckii
In tissue sections or primary wet preparations, S schenckii
appears as 4  - 
6 µm elongated (“cigar shaped”) yeasts with
narrow based budding, usually seen in a background of puru-
lent inflammation.
When cultured at 25 ‑ 30°C, the mold form grows moder-
ately to rapidly. Colonies are moist and white to pale orange
initially, turning brown with age f3.44. Microscopic examina-
tion of the cultured mold reveals very delicate, hyaline, sep-
tate hyphae producing conidiophores topped by clusters of
microconidia (“rosettes”) f3.45. Definitive identification requires
conversion of the mold form to the yeast form, which exists as
tan to brown, creamy colonies f3.46. Yeast cells are oval or long
and thin (“cigar bodies”), and bud on a narrow base f3.47.
163
3:  Microbiology

Sporotrichosis is seen worldwide, although the majority of

cases occur in North and South America. Infection is usually
acquired by penetrating injury from contaminated plant mate-
rial (“rose gardener’s disease”) or soil, although infection via the
respiratory route occurs rarely. The most common clinical mani-
festation is lymphocutaneous infection, with nodular, ulcerative
skin lesions following lymphatics.

Penicillium marneffei
In tissue sections or primary wet preparations, P  marneffei
appears as elongated, ovoid, small (3 - 5 µm) yeast that divide by
fission rather than by budding. They are frequently found within
histiocytes.
When cultured at 25 ‑ 30°C, P marneffei forms rapidly growing,
tan mold colonies that are initially powdery or velvety on the
surface, and become colored with maturity (typically blue/green
centrally, but other colors are also seen). A red pigment diffuses
into the agar around and underneath the mold colonies; this is an
important feature for correct identification. Microscopic exami- f3.48  Aspergillus species in tissue—narrow septate hyphae with acute-angle dichotomous
nation of the mold colonies reveals features indistinguishable evenly spaced branching is characteristic but not genus-specific
from other Penicillium species, including hyaline, septate hyphae
with conidiophores and metulae producing brushlike clusters
of phialides. Chains of small, oval conidia form at the terminal
ends of the phialides. For concrete identification, thermal dimor-
a b
phism should be demonstrated by converting the mold form to
the yeast form at 37°C. Yeast colonies are off-white to pink, and
consist of small (3 ‑ 5 μm), oval yeastlike cells that reproduce by
fission rather than by budding. Although they are yeastlike in
appearance, they are actually single celled arthroconidia.
Penicilliosis marneffei is endemic in Southeast Asia, and is not
found elsewhere. At particular risk are HIV/AIDS patients, among
whom the infection is most prevalent. The usual portal of entry is the
respiratory route, but pulmonary infection is not always a prominent f3.49  Aspergillus species, when growing in an air-filled space (in this case a paranasal sinus) may
clinical feature. Most cases lead to involving the bone marrow, skin, demonstrate the formation of fruiting heads; in this case, A niger
lymphatics, liver and spleen.

Thermally monomorphic molds

On routine fungal media, Aspergillus is rapidly growing.
Hyaline septate molds (hyaline hyphomycetes)
Microscopically, all cultured Aspergillus species have in
Aspergillus species
common that they produce a swollen vesicle (“aspergillum”)
Aspergillus species are ubiquitous in soil and on decaying
at the end of each conidiophore. This feature is distinct to the
vegetable matter. Hundreds of species have been described,
genus. Identification to the species level is based on character-
but only a few are pathogenic for humans.
istic colony and microscopic morphology.
In tissue sections, Aspergillus species produce narrow
(3 ‑ 6  μm), uniform, hyaline, septate hyphae with character- ƒƒ A fumigatus colonies are blue-green with a distinct white
istic acute-angle (45°) dichotomous, evenly-spaced (arboreal) apron f3.50a and a light reverse f3.50b. Microscopically,
branching f3.48. However, this appearance is not specific for the species is notable for conidiophores that terminate
aspergillosis and can represent other fungal species. The his- in a swollen vesicle having a single row of phialides
tologic differential diagnosis should also include Fusarium, (uniseriate) that cover only the top 2/3 of the vesicle. Each
Scedosporium apiospermum (P boydii), and many others. The phialide gives rise to a chain of small (2 ‑ 4 μm), round
exception is the rare circumstance in which the diagnostic conidia f3.51.
fruiting heads of Aspergillus are seen in tissue sections. ƒƒ A flavus produces yellow-green to olive colonies f3.52a
Normally, fruiting heads of Aspergillus are produced only in with a light reverse f3.52b. Microscopic examination
culture, and are not seen in vivo. However, if Aspergillus grows demonstrates circumferential phialides f3.53. Some strains
in an air-filled tissue pocket (as is sometimes the case in asper- are uniseriate, while others are biseriate. Importantly,
gilloma), its reproductive structures are produced and a defini- when A flavus contaminates grains and other foodstuffs,
tive identification can be made by histology f3.49. it may produce aflatoxins, which are carcinogenic.
Chronic exposure to aflatoxins by ingestion can lead to
hepatocellular carcinoma.

164 Practical Clinical Pathology

 3:  Microbiology

a a

b b

f3.50  A fumigatus colonies a are blue-green with a distinct white apron & b a light reverse f3.52  A flavus colonies a are yellow-green to olive with b a light reverse

f3.51  A fumigatus: swollen vesicles with single row of phialides (uniseriate) that cover only the top f3.53  A flavus: circumferential phialides
2/3 of the vesicle

ISBN 978-089189-5985 165

3:  Microbiology
f3.54  A niger colonies are dark brown to black; the reverse side is light
f3.55  A niger: circumferential biseriate pigmented phialides
ƒƒ A niger cultures have a dark brown to black surface
f3.54, due to the dark (non-melanin) pigmentation of
the conidia, but the reverse side is light. This contrasts
with cultures of dematiaceous molds, which tend to be
darkly pigmented front and back, and contain melanin.
Microscopic examination reveals vesicles with 2 rows
of phialides (biseriate) covering the entire surface of the
vesicle f3.55. Phialides produce chains of rough, round,
dark conidia. Of note, A niger pulmonary infection
is associated with oxalosis (calcium oxalate tissue
deposition).
166
b
f3.56  A terreus colonies a are cinnamon brown on the surface with b a yellow or orange reverse
ƒƒ A terreus colonies are cinnamon brown on the surface
f3.56a, with a yellow or orange reverse f3.56b. Microscopically,
A terreus looks superficially like A fumigatus, in that its
phialides cover only the top 2/3 of the vesicle. In contrast
to A fumigatus, though, A terreus is biseriate and typically
has longer chains of conidia. A terreus is intrinsically
resistant to amphotericin B, unlike A fumigatus, A flavus
or A niger.
Aspergillosis most commonly affects the respiratory tract.
The type of respiratory disease caused by Aspergillus depends
on host factors. An immunocompetent individual with cavi-
tary lung disease, such as from bronchiectasis or old tubercu-
losis, is prone to develop the saprophytic form of aspergillosis:
an aspergilloma (fungus ball), which is a noninvasive coloni-
zation. Allergic bronchopulmonary aspergillosis (ABPA) is a
condition in which Aspergillus colonizes the airway, without
invasion, and elicits a marked allergic response characterized
by mucus impaction, peripheral eosinophilia, and reactive
airways. The inflammatory response can be sufficiently severe
Practical Clinical Pathology

a b
f3.57  Allergic fungal sinusitis
a A clue is eosinophil-permeated mucin containing b fungal hyphae on GMS stained sections
to cause hemoptysis and eventual bronchiectasis. This form of
aspergillosis is often seen in patients with an atopic history;
there is also an increased incidence of the cystic fibrosis trans-
membrane regulator (CFTR) gene mutations in patients with
this condition. The diagnosis can be supported with tests for
serum anti‑Aspergillus IgE, elevated total serum IgE, Aspergillus
skin tests, and peripheral eosinophilia. Allergic sinonasal
aspergillosis (allergic fungal sinusitis) is a related condition
which can be diagnosed on the basis of sinus curetting his-
tology: inflamed sinonasal mucosa and eosinophil-permeated
mucin containing fungal hyphae f3.57. An individual with
emphysema or other structural lung disease, who is receiving
steroid therapy, is prone to develop semi-invasive pulmo-
nary aspergillosis, also called chronic necrotizing pulmonary
aspergillosis. Here, the mold colonizes cavitary regions of
the emphysematous lung parenchyma, and invades superfi-
cially into the surrounding lung tissue. Finally, profoundly
immunosuppressed hosts are susceptible to invasive broncho-
pulmonary aspergillosis (IBPA) or invasive fungal sinusitis,
very serious conditions in which the mold invades tissue and
blood vessels, and may disseminate. Aspergillus species and
the zygomycetes have a propensity to invade vessel walls
(angioinvasion) and give rise to tissue infarction. Confirming
the diagnosis of IBPA is difficult and may require lung biopsy.
Therefore, there has been growing use of an ELISA assay
for the serum marker galactomannan, a cell wall constituent
released during tissue invasion by Aspergillus. The assay is best
used as a screening tool for early detection in asymptomatic,
immunosuppressed patients. Another fungal antigen assay
can detect serum 1 ‑ 3-β-D-glucan, a marker for invasive fungal
infection by a wide range of agents, including Aspergillus spe-
cies. A positive serum 1 ‑ 3-β-D-glucan test can complement
a positive serum galactomannan test, increasing the positive
predictive value.
Other hyaline hyphomycetes
This group of organisms is known for causing such infec-
tions as mycotic keratitis, onychomycosis, and eumycotic
mycetoma, and some genera are associated with more serious
invasive infections. Fusarium species cause a spectrum of
infections overlapping with those caused by Aspergillus spe-
cies, including invasive pulmonary and sinus infections, and
disseminated infection, in immunocompromised hosts, and
fungus ball or allergic bronchopulmonary disease in immuno-
competent hosts. Fusarium species also cause fungal keratitis,
particularly in contact lens wearers, as well as onychomycosis.
ISBN 978-089189-5985
3:  Microbiology
f3.58  Fusarium species
In addition, Fusarium has recently emerged as an impor-
tant opportunistic pathogen in burn wounds. Pseudallescheria
boydii/Scedosporium boydii causes eumycotic mycetoma after
penetrating trauma, fungal keratitis, and pneumonia after
near-drowning accidents. In immunocompromised hosts,
P  boydii/S  boydii causes invasive pulmonary or sinus infec-
tions, and disseminated infection. It is intrinsically resistant to
amphotericin B.
The hyaline hyphomycetes can be distinguished based on
their colony and microscopic morphology. One key aspect
of their microscopic morphology is the arrangement of
their conidia. Useful information can be acquired simply by
observing whether the conidia occur singly, in clusters, or in
branching chains.
Conidia arranged in clusters: Fusarium, Acremonium,
Gliocladium. Fusarium species form canoe shaped, multicellular
macroconidia with 3 ‑  6 cells each, often clumping together
f3.58. Some strains may also produce smaller, 1 ‑ 2 celled micro-
conidia in clusters at the apex of unbranched conidiophores.
Acremonium species are characterized by long, threadlike,
unbranched phialides bearing clusters of single  celled micro­
conidia. Gliocladium species have branching conidiophores
bearing flask shaped phialides, resembling Penicillium.
However, microconidia do not chain but rather cluster in a ball,
resembling a golf ball held at the end of outstretched fingertips.
Conidia arranged in chains: Penicillium, Paecilomyces,
Scopulariopsis. Penicillium species are characterized by a brush-
like arrangement of flask shaped phialides that give rise
to unbranching chains of microconidia. The phialides are
present on metulae (secondary branches of conidiophores).
Paecilomyces species have branching conidiophores with elon-
gated, flask shaped phialides arranged in pairs or brush-like
groups. Long chains of oval or spindle shaped microconidia
emanate from the phialides. Scopulariopsis species have single
or branched conidiophores that give rise to annellides (similar
to phialides). Annellides are often clustered in a brush-like
167
3:  Microbiology
f3.59  Trichophyton rubrum; red reverse
arrangement. Each annellide bears a chain of rough-walled,
round or truncated microconidia.
Conidia arranged singly: Chrysosporium species, Sepedonium
species, and Beuveria species. Chrysosporium species form
single, cutoff microconidia directly on hyphae or on the tips of
simple conidiophores. They sometimes also produce interca-
lary, cylindrical conidia resembling arthroconidia. Sepedonium
species have single-borne, hyaline conidia at the ends of
branched or unbranched conidiophores. The conidia are large,
thick walled and echinulate, resembling the macroconidia of
the mold form of H capsulatum. Beuveria species are character-
ized by single, small, round or oval microconidia emerging
from each inflection point in a zigzagging (geniculate), flask
shaped conidiophore. Although the conidia appear singly, the
conidiophores themselves typically cluster together.
Dermatophytes
Dermatophytes are considered separately from other hya-
line septate molds because they share certain unique features.
They are keratinophilic, meaning that they are able to digest
keratin as nutrient source using keratinases. This special
ability informs their pathogenicity, which is mostly limited
to infection of superficial, keratinized structures such as hair,
nails, and the stratum corneum of skin. In addition, they are
uniformly resistant to cycloheximide, a feature that is lever-
aged in culture strategies by including a cycloheximide-con-
taining medium when culturing for dermatophytes.
Rapid diagnosis of dermatophytosis can be made with a
bedside KOH prep or calcofluor white prep of skin scrap-
ings. This does not permit species identification, which is not
always necessary to guide therapy.
Dermatophytes are speciated based upon characteristic
colony and microscopic morphology after culture. Trichophyton
rubrum has a particularly distinctive colony morphology,
because it produces a red pigment that causes the reverse
side of the plate to appear red f3.59. Microscopically, the der-
matophytes appear as hyaline, septate hyphae that produce
168 Practical Clinical Pathology
f3.60  Trichophyton rubrum; birds on a wire
microconidia, macroconidia, or both. These reproductive
structures are often the key to a species-level identification.
ƒƒ Trichophyton rubrum: tear shaped microconidia are
arranged along the hyphae, producing a “birds on a
telephone wire” appearance f3.60.
ƒƒ Trichophyton mentagrophytes: microconidia are arranged
in clusters, and occasional spiral hyphae may be seen.
Club shaped macroconidia are sometimes present,
containing 1 ‑ 6 cells each. T mentagrophytes has the
ability to penetrate hair shafts, unlike T rubrum, which
can appear morphologically similar to some strains of
T mentagrophytes.
ƒƒ Trichophyton tonsurans is distinctive for the marked size
and shape variability of its microconidia. Microconidia
may be round, tear shaped, cigar shaped, or swollen
and enlarged. Intercalary chlamydospores are common,
particularly in mature cultures.
ƒƒ Microsporum canis: macroconidia are spindle shaped and
rough (echinulate), and taper to a knob-like end. Each
macroconidium contains >6 cells, separated by transverse
septae.
ƒƒ Microsporum gypseum: macroconidia are oval shaped
structures with rounded ends and transverse septae. Each
macroconidium contains 3-6 cells.
ƒƒ Epidermophyton floccosum: macroconidia are smooth,
club shaped structures with rounded ends, which
may be found singly or in characteristic clusters.
Each macroconidium contains 2 ‑ 6 cells, separated by
transverse septae. Microconidia are never produced.
Dermatophyte infection may take many forms, including
tinea capitis (scalp ringworm), tinea corporis (ringworm),
tinea cruris (jock itch), tinea pedis (athlete’s foot), and tinea
unguium (onychomycosis). Multiple dermatophyte species can
cause the same clinical manifestation.

b f3.62  Alternaria species form chains of conidia with transverse & longitudinal (muriform) septations;
f3.61  Dematiaceous molds are typically pigmented a on the surface & b reverse side of the plate
Dermatophytes may be classified according to their
natural habitat, which informs the mode of transmission.
Anthropophilic dermatophytes principally infect humans,
and are transmitted between individuals either directly or
via fomites. Major examples include T  rubrum, T  tonsurans
and Epidermophyton floccosum. Zoophilic dermatophytes infect
animals, and can be transmitted to humans by animal con-
tact. Important examples include T  mentagrophytes (rodents),
M  canis (cats and dogs), T  verrucosum (cattle), and T  equinum
(horses). Geophilic dermatophytes are soil inhabitants that
rarely infect humans, with the exception of M gypseum.
Dematiaceous molds
The unifying feature among dematiaceous molds is pro-
duction of melanin pigment, conferring dark pigmentation.
Dematiaceous molds typically are darkly pigmented on the
surface and reverse side of the plate, because both their hyphae
and conidia are melanized f3.61. However, some species are
melanized only in their conidia, resulting in a dark surface
ISBN 978-089189-5985
3:  Microbiology
one end of each conidium is blunt & the other pointed
with a lighter reverse side, and other species do the opposite.
The first 2 groups below (conidia with internal septae) grow
relatively rapidly, while the last group is a short list of dispa-
rate slow growers. In tissue, there are no features that permit
distinction to the genus level, but intrinsic dark pigmentation
is a clue to the presence of a dematiaceous mold.
Conidia with transverse septae: Bipolaris, Drechslera,
Exserohilum, Helminthosporum, Curvularia.
ƒƒ Bipolaris is characterized by oval, transversely septated
conidia that arise from bent (geniculate) conidiophores.
Bipolaris gets its name from the production of germ tubes
from both ends of the conidia in saline mounts incubated
for 12 - 24 hours. Each conidium contains 3 ‑ 5 septations.
ƒƒ The genus Drechslera produces similar conidia but is
distinguished from Bipolaris by its lack of production
of bipolar germ tubes in saline incubation; instead, it
produces germ tubes along the sides of the conidia.
ƒƒ Exserohilum also resembles Bipolaris, except that its conidia
are longer and thinner, and have more septations (5 ‑ 12).
ƒƒ Helminthosporium has a highly characteristic bottle
brush-like microscopic appearance, with side by side
conidia arranged in whorls along the conidiophores.
ƒƒ Curvularia is easy to recognize, since its transversely
septated conidia curve distinctly when mature.
Conidia with transverse & longitudinal septae: Alternaria,
Ulocladium, Stemphilium.
ƒƒ Alternaria species produce chains of transverse and
longitudinally septated (muriform—resembling a brick
wall) conidia that have alternating blunt and pointed
ends f3.62.
ƒƒ Ulocladium is characterized by oval muriform conidia
borne singly on bent (geniculate) conidiophores. The
conidiophores bend in a zigzag fashion.
169
3:  Microbiology
b f3.64  Pseudallescheria boydii/Scedosporium boydii complex forms a mold colony with a light brown
f3.63  A slow growing dematiaceous mold whose a early colony morphology is smooth & moist
(yeastlike) & whose b late colony morphology is fuzzy (moldlike); this would be typical of Exophiala,
Wangiella & Hortaea
ƒƒ Stemphilium conidia resemble those of ulocladium but are
borne upon straight conidiophores, and frequently the
conidiophores will each produce only a single, terminal
conidium.
Slow growing dematiaceous molds may be grouped into 2
categories: those whose early growth is yeastlike (becoming
moldlike with age), and those whose early and late growth is
moldlike. Those with yeastlike early growth produce smooth,
moist colonies in early culture f3.63a, composed of budding
yeastlike cells. With age, the colonies become fuzzy f3.63b and
the microscopic morphology converts to septate hyphae with
conidia formation. Examples include Exophiala, Wangiella, and
Hortaea. Those with moldlike early growth produce fuzzy
colonies from the beginning. Examples include Pseudallescheria
boydii/Scedosporium boydii complex and Scedosporium prolificans.
The name “P boydii” refers to the sexual state of the mold,
whereas S  boydii refers to its asexual state. In either state,
the mold has a light gray or brown surface f3.64, owing to
170 Practical Clinical Pathology
melanized surface
f3.65  Pseudallescheria boydii/Scedosporium boydii complex: oval, truncated, melanized microconidia
with non-melanized hyphae
melanization of its oval, truncated microconidia f3.65. Its sep-
tate hyphae are hyaline (non-melanized), resulting in a light
reverse, which can darken with age. In its sexual state, the mold
produces large (50-200 μm), dark cleistothecia f3.66, whereas the
asexual form lacks this feature but is otherwise indistinguish-
able. An alternative asexual form of the same mold, called
Graphium, is also sometimes seen. Its colony morphology is
indistinguishable from P boydii or S boydii, but microscopically
it is characterized by thick mats of long conidiophores stuck
together side by side, resembling the bristles of a broom f3.67.
P  boydii/S  boydii complex molds are intrinsically resistant to
amphotericin B, but are usually susceptible to triazoles such
as voriconazole and posaconazole. A closely related species,
Scedosporium apiospermum was thought until recently to be an
 3:  Microbiology

f3.66  Pseudallescheria boydii/Scedosporium boydii complex: in the sexual state, the mold produces b
large dark cleistothecia

f3.68  Scedosporium prolificans has a gray or black a surface & b reverse

f3.67  Pseudallescheria boydii/Scedosporium boydii complex: the alternative asexual form, Graphium,
characterized by thick mats of long conidiophores stuck together side by side, resembling the bristles
of a broom

asexual form of P boydii, but is newly recognized as a separate

species. It is morphologically identical to S boydii, and is best
distinguished using molecular methods.
Scedosporium prolificans has a gray or black surface and
reverse f3.68. Unlike P boydii/S boydii, its growth is inhibited by
cycloheximide, and it has no sexual state. Microconidia are
oval and truncated, forming clusters at the end of annellides
(conidiogenous cells) f3.69. The annellides have swollen bases
and thin necks. S  prolificans is intrinsically resistant not only
to amphotericin B, but also to the azoles and echinocandins,
making antifungal therapy ineffective.

f3.69  Scedosporium prolificans microconidia are oval & truncated, forming clusters at the end of
annellides, which have swollen bases & thin necks

ISBN 978-089189-5985 171

3:  Microbiology

f3.70  Chromoblastomycosis, demonstrating muriform bodies (sclerotic bodies having internal b

septations in more than one plane)

Infections caused by dematiaceous molds can be classified

as chromoblastomycosis, mycetoma, or phaeohyphomycosis,
based on the appearance of the fungus in tissue and the associ-
ated clinical manifestations.
Chromoblastomycosis is a subcutaneous mycosis associ-
ated with prominent pseudoepitheliomatous hyperplasia of
the overlying skin (cauliflower-like or warty lesions). In tissue,
one sees pigmented hyphae and sclerotic bodies. The latter are
small (5 ‑ 12 μm) round, dark structures with internal septa-
tions in more than 1 plane (muriform) f3.70. Fonsecaea pedrosoi,
Phialophora verrucosa, and Cladophialophora carrionii are the
principal agents of chromoblastomycosis, which is found in
tropical and subtropical areas, particularly where shoes are
not regularly worn. The organisms gain entrance through f3.71  Rhizopus species in culture: colonies are rapidly growing (lid lifters) & quickly cover the entire
puncture wounds, so infections tend to be found on the lower agar surface; they are initially cottony & white, turning light brown with sporulation
extremities. Lesions are usually solitary and may take the form
of verrucous lesions, nodules, or massive tumors.
Mycetoma (also known as Madura foot or Maduromycosis)
is another form of subcutaneous infection, in which a slowly- Zygomycetes (pauciseptate molds)
developing subcutaneous nodule is formed, often with The zygomycetes grow extremely rapidly both in vivo and
draining sinus tracts to the skin. Mycetoma can be caused by in culture. The hyphae are broad and have few septations,
filamentous bacteria (actinomycotic mycetoma) or by molds making them floppy and ribbonlike. Branching is infrequent,
(eumycotic mycetoma). Eumycotic mycetoma is frequently haphazard, and nondichotomous. Care must be taken not to
caused by dematiaceous molds, although it can also be caused grind specimens prior to culture if zygomycosis is a clinical
by certain hyaline molds. Within the subcutaneous nodule are consideration, as the grinding process can easily disrupt all
granules composed of clusters of the infectious agent within the viable hyphae. Tissue specimens should be minced instead.
a proteinaceous matrix. Like chromoblastomycosis, myce- The zygomycetes can be distinguished from one another
toma is usually the result of a puncture wound. The causes of based on certain microscopic features. It is important to note
eumycotic mycetoma include Madurella, Exophiala, Wangiella, the presence or absence of rootlike structures called rhizoids,
and P boydii/S boydii. Actinomycotic mycetomas are caused by and their location, and to determine whether the sporangio-
aerobic actinomycetes, such as Nocardia species phores are branched or unbranched (simple). The presence or
Phaeohyphomycosis is a catch-all term for infections caused absence of an apophysis at the sporangiophore’s apex should
by dematiaceous molds that do not fit into the chromoblasto- also be noted. Distinguishing features of some commonly iso-
mycosis or eumycotic mycetoma categories. Infections may lated zygomycetes are described below.
involve any organ system. In tissue, agents of phaeohyphomy- ƒƒ Rhizopus species f3.71, f3.72 produce rhizoids and
cosis form dark, septate hyphae, as well as yeastlike forms and unbranched sporangiophores that arise directly over
forms that resemble pseudohyphae. the rhizoids. Their sporangia (sack-like structures that

172 Practical Clinical Pathology

 3:  Microbiology

a b a b

f3.72  Rhizopus species produce rhizoids & unbranched sporangiophores that arise directly over the c d
rhizoids; their sporangia are prominent spherical structures that tend to collapse when mature,
resembling a collapsed umbrella; their sporangiophores lack an apophysis

f3.75  Zygomycetes
a A necrotic vessel containing angioinvasive ribbonlike hyphae seen better b at high magnification in
H&E stained sections
c The trichrome stained section highlights the residual vessel wall
d A residual thrombosed vessel in cross section, with numerous mural hyphae
contain spores) are prominent spherical structures full
of tiny spores which tend to collapse when mature,
resembling a collapsed umbrella. Sporangiophores lack
an apophysis.
ƒƒ Mucor species f3.73, f3.74 do not produce rhizoids.
Sporangiophores can be branched or unbranched, but
lack an apophysis. Their sporangia are large spherical
structures that tend to fall apart, releasing their numerous
spores.
f3.73  Mucor species: colonies are rapidly growing (lid lifters), fluffy & become pigmented with the
development of sporangia ƒƒ Lichtheimia (formerly Absidia) produces rhizoids but its
sporangiophores arise at points between rhizoids, rather
than over the rhizoids. Sporangiophores are branched
and form a conical apophysis at the top.
ƒƒ Cunninghamella species differ from most zygomycetes in
that their branched sporangiophores are topped by large
vesicles. The vesicles are covered with spines (denticles),
each of which supports a single spore contained within a
round sporangiolum.
Zygomycetes may produce several forms of invasive
infection: rhinocerebral, pulmonary, gastrointestinal, and
cutaneous. Hosts are typically immunocompromised, and
important risk factors include uncontrolled diabetes (espe-
cially ketoacidosis), stem cell or solid organ transplantation,
neutropenia, corticosteroid therapy or severe burns. Like
Aspergillus species, the zygomycetes characteristically invade
vessel walls and thereby produce parenchymal infarction and
sometimes disseminated infection. They can be seen in rou-
tine H&E-stained sections f3.75.

f3.74  Mucor species: note the absence of rhizoids

Sporangiophores can be branched or unbranched, but lack an apophysis
Their sporangia are large spherical structures that tend to fall apart, releasing their numerous spores

ISBN 978-089189-5985 173

3:  Microbiology
Fungal isolate growing in culture at
25 ‑ 30°C as yeast: yeast & yeastlike fungi
Approach to identification
Presumptive identification methods
Chromogenic agar plates (Chromagar) are available that
are selective for yeast, while inhibiting the growth of bac-
teria, and allowing for differentiation between some common
Candida species on the basis of colony color after incubation.
Chromogenic compounds within the medium are used to
demonstrate enzyme activity that is species specific. If pri-
mary specimens are directly cultured on this medium, a pre-
sumptive identification of C albicans, C tropicalis, or C krusei can
be made after 24 ‑ 48 hour incubation at 35 ‑ 37°C. This method
is particularly useful in fungal culture of specimens from the
vagina, oropharynx, stool or urine, where Candida species are
frequently encountered along with resident bacterial flora.
Germ tube test
This assay can be performed on yeast isolates (after initial
culture) to rapidly identify C albicans presumptively. The test
relies on 2 principles
1. Yeast cells of C albicans begin to produce hyphal elements
(pseudohyphae and true hyphae) more rapidly than most
other species
2. C albicans yeast cells are able to produce true hyphae
without a constriction between the mother cell and the
hyphal element, whereas most yeast species can produce
only pseudohyphae, which do have a constriction
Briefly, yeast are suspended in serum and incubated at 37°C
for up to 3 hours (no more). A wet mount is prepared
and examined for the formation of germ tubes—yeast cells
producing hyphae with no constriction at the juncture with the
yeast cell. Sensitivity is good but not 100%, and the absence of
germ tubes does not rule out C albicans. Specificity is also high,
but C dubliniensis also produces germ tubes within 3 hours.
Rapid trehalose assimilation
This test can presumptively identify C  glabrata, using yeast
isolates after initial culture. Unlike most other yeast, C glabrata
has the ability to ferment trehalose rapidly at 42°C. To perform
the test, a yeast isolate is inoculated into broth containing treha-
lose and a pH indicator. After a short (3 hour) incubation at 42°C,
fermentation of the substrate causes a color change, indicating a
positive result. The test is very sensitive, and specificity is high,
particularly if one only tests isolates with appropriate micro-
scopic morphology (small budding yeast producing no pseudo-
hyphae, and germ tube negative).
Urease test
Detection of urease enzyme activity can distinguish asco-
mycetous yeasts, which do not have urease activity, from
basidiomycetous yeasts, which do. It is most commonly used
to presumptively identify Cryptococcus species isolates after
initial culture. Urea disks are available for rapid testing and
can detect urease activity by a color change within a few hours.
An alternative method involves the use of urea agar slants, but
this requires a longer incubation. A positive urease test can aid
in the presumptive identification of Cryptococcus species, when
174 Practical Clinical Pathology
accompanied by appropriate colony morphology and micro-
scopic morphology. Importantly, other commonly isolated basid-
iomycetous yeasts are also urease+, including Rhodotorula species
and Trichosporon species, so a positive urease test alone cannot be
relied upon to presumptively identify Cryptococcus species
Phenol oxidase test
Demonstration of phenol oxidase enzyme activity can dif-
ferentiate Cryptococcus neoformans from nonneoformans
Cryptococcus species and from other yeast genera. C neoformans
is uniquely able to oxidize diphenolic compounds such as caffeic
acid, dopamine, and dopa to produce darkly pigmented melanin
or melanin precursors. The classic phenol oxidase test involves
culturing a yeast isolate on bird seed agar, a natural source of
caffeic acid. Growth of brown-pigmented yeast colonies after
overnight incubation supports the presumptive identification of
C neoformans, whereas white or nonpigmented colonies indicate
a negative result. A more rapid method involves the use of caf-
feic acid disks, which are inoculated with a yeast isolate and can
demonstrate brown pigment production within a few hours.
Definitive identification methods
Assimilation tests
These tests assess the ability of an isolate to use a particular
carbohydrate as its sole carbon source, or its ability to use nitrate
as its sole source of nitrogen. Several commercially-available,
biochemical identification systems are available that combine
a battery of assimilation tests for yeast identification. Because
these systems rely on yeast metabolism, a prolonged incubation
period (18 - 48 hours) is required to obtain results. However, they
are accurate and provide a species-level identification along
with a confidence score, to help interpret the results.
MALDI-TOF mass spectrometry
This method allows for the analysis of yeast isolates without
the need for growth and metabolism, and therefore can provide
a full identification in a matter of minutes. In this method, an
isolate taken from an agar plate is transferred to a target slide.
The sample on the target slide is then overlaid with a matrix
solution, and loaded into the ionization chamber of a matrix-
assisted laser desorption/ionization time of flight (MALDI-TOF)
mass spectrometer. The sample is irradiated by a laser, causing
the sample/matrix mixture to vaporize. During this process,
proteins in the sample acquire an electrical charge. Electric
fields in the instrument then push the charged proteins into
a vacuum tube. The time of flight through the vacuum tube
is measured, and used to determine the mass of each protein
within a certain size range. From these data, a mass spectrum is
compiled, which serves as a type of fingerprint that can be used
to identify the organism. The mass spectrum generated from
the sample is compared to a database of spectra generated from
known yeast species, to provide a species-level identification
and an associated confidence score. The same methodology can
be used to identify bacteria, mycobacteria, and molds.
Demonstration of specific morphology using specialized
media: Certain media, especially rice or cornmeal agar supple-
mented with Tween 80, will reliably induce formation of char-
acteristic yeast structures such as blastoconidia, pseudohyphae,
true hyphae, arthroconidia or chlamydospores (chlamydoco-
nidia). Microscopic examination can be performed by dropping

f3.76  Pseudohyphae: note the constriction between the yeast & the hyphal structure
f3.77  C albicans colonies frequently form filamentous extensions (“feet”) around the edges
a coverslip directly on the agar and examining the yeast struc-
tures through the coverslip. This method can be used alone to
identify yeast, but is more often used as a supplemental test
when more convenient identification methods prove inad-
equate for definitive identification of a particular isolate.
Notes on specific yeasts
Candida species
C  albicans is by far the most common Candida species iso-
lated from humans, regardless of specimen type or host. On
direct examination, C  albicans appears as 3 ‑ 5 µm budding
yeast with accompanying pseudohyphae f3.76, and occasionally
true hyphae as well. Colonies grown on solid medium fre-
quently form filamentous extensions (“feet”) around the edges
ISBN 978-089189-5985
3:  Microbiology
f3.78  C albicans on yeast morphology medium: pseudohyphae with clusters of blastoconidia at
septations & single terminal chlamydospores
f3.79  C glabrata: slow growing smooth creamy colonies lacking “feet”
f3.77—a characteristic that helps to differentiate C albicans from
nonalbicans species. When cultured on yeast morphology
medium (cornmeal or rice agar with Tween), C albicans forms
pseudohyphae with clusters of blastoconidia present at the
septations. In addition, single, terminal chlamydospores are
seen f3.78. C albicans is positive by the germ tube test and forms
green colonies on chromogenic agar. Most clinical isolates are
susceptible to azole agents, echinocandins, and amphotericin
B.
C glabrata is frequently isolated from blood and urine. Direct
examination reveals small (2 ‑ 4 µm) budding yeast (blastoco-
nidia), without pseudohyphae or other structures. In culture,
isolates are relatively slow growing in comparison to other
Candida species, and may take an extra day of incubation for
175
3:  Microbiology

a b

f3.81  C neoformans: a Wright stain & b calcofluor white show spherical, narrow-budding yeasts that
vary greatly in size

a b

f3.80  C glabrata on yeast morphology medium: budding yeast with no additional structures formed

mature colony formation f3.79. Yeast morphology medium does

f3.82  C neoformans: India ink preparation at a low & b high magnification show spherical, narrow-
not induce structures other than budding yeast f3.80. C glabrata budding yeasts with thick capsule
is positive by the rapid trehalose assimilation test. A signifi-
cant proportion of clinical isolates has reduced susceptibility
or frank resistance to azole agents (both imidazoles and tri-
azoles), but most isolates are susceptible to echinocandins and
amphotericin B.
Certain other Candida species are predictably resistant to
antifungal agents. C  krusei is intrinsically resistant to fluco-
nazole, but isolates are generally susceptible to the triazoles,
echinocandins and amphotericin B. C lusitaniae is considered
resistant to amphotericin B, despite low MICs in many cases
by in vitro testing. C parapsilosis, C krusei, C guilliermondii and
C lusitaniae sometimes exhibit relatively high MICs for echino-
candins, but frank resistance is unusual.

Cryptococcus neoformans
Although there are numerous species within the genus,
the main human pathogens are C  neoformans and C  gattii.
Previously, C  gattii was considered a variety of C  neoformans,
but these have now been classified as separate species. C neo-
formans is the cause of most infections in the US. C gattii, pre-
viously confined to tropical zones, has now been reported as
an emerging infection in the Pacific northwest region of the f3.83  C neoformans: mucoid colonies on solid agar
United States.
In histologic sections or direct wet mounts, cryptococci
present as spherical, narrow-budding yeasts that vary in
The cryptococcal capsular polysaccharide antigen can be
size from 3 - 15 µm f3.81. The yeast are encapsulated (although
detected in either serum or body fluids (usually CSF). Latex
rare capsule-deficient strains exist), a feature that can be
particles coated with antibodies against cryptococcal capsular
highlighted in tissue using a mucicarmine stain, or in wet
antigen form the basis for this simple agglutination test.
mounts using india ink f3.82. The yeast cell walls contain
C neoformans is acquired by inhalation during exposure to
melanin, which makes them positive by the Fontana-Masson
contaminated soil, particularly soil containing bird excreta (eg,
stain in tissue sections. Colonies grown on solid agar are fre-
pigeon, chicken). C gattii appears to be associated with trees,
quently mucoid f3.83, owing to the capsular material. Whether
especially eucalyptus trees. C gattii has become increasingly
visualized directly in primary specimens or after culture,
relevant in the US for 2 reasons: it is an emerging infection in
Cryptococcus forms only budding yeast, and never forms other
the Pacific northwest, and seems capable of causing infection
structures like pseudohyphae.
in immunocompetent hosts.

176 Practical Clinical Pathology


a toms, especially tachypnea, bilateral interstitial infiltrates, and
b c
d e
f3.84  Pneumocystis: characteristic exudative material is seen within alveolar airspaces on a an H&E
stained lung section & b on Pap stained & c Wright stained sputum smears; note the central dot visible
within the empty spaces on the Wright stained preparation
In GMS stained preparations at d low & e high magnification, the organisms are approximately the
size of yeast but do not bud; they tend to cluster & have a central dark staining dot when stained with
silver stains; cyst forms are round or cup shaped & have been likened to crushed ping pong balls
Pneumocystis jiroveci
Pneumocystis jiroveci (formerly known as P carinii) is an atyp-
ical fungus that cannot be cultured in vitro. Laboratory diag-
nosis depends upon direct examination of clinical specimens.
The organism was first recognized as a cause of human dis-
ease a century ago, at which time it mainly affected the severely
malnourished. In this population it was generally associated
with interstitial plasma cell pneumonitis or granulomatous
pneumonitis. In the late 20th century, P  jiroveci emerged as
a major pathogen in HIV-infected patients. In HIV infected
patients, the strongest risk factor is a CD4 count <200 cells/mL.
In addition to the HIV infected, populations at risk include those
with primary immunodeficiency, particularly severe combined
immunodeficiency (SCID), patients undergoing chemotherapy,
especially for lymphoma, and patients receiving immunosup-
pression for transplant or collagen vascular disease.
ISBN 978-089189-5985
3:  Microbiology
Infected patients typically present with respiratory symp-
hypoxemia. Serum LD is usually very high. Serologic testing
is not helpful. Respiratory samples such as bronchoalveolar
lavage (BAL) are ideal, but induced sputum may provide suf-
ficient sensitivity in severely immunocompromised hosts.
The organisms, within typical exudate f3.84, are easily seen on
Pap-stained, Giemsa-stained, and GMS-stained preparations.
Immunofluorescent staining with anti‑Pneumocystis mono-
clonal antibodies can provide greater specificity.
Bacteriology
Laboratory methods
Specimens
Routine aerobic and anaerobic cultures can be performed
on a wide variety of specimens. Specimens for anaerobic
culture should be transported to the lab immediately, or in
anaerobic transport containers. Specimens transported anaer-
obically can be used for aerobic cultures as well. The following
sites normally harbor indigenous anaerobic flora and should
not be cultured for anaerobes: stool, skin, oropharynx, vagina,
and urethra. Anaerobic culture is best reserved for specimens
obtained from normally-sterile sites.
Blood culture bottles are ideally inoculated with 10 mL of
blood per bottle (in adults), and typically a 2- bottle set (con-
sisting of 1 aerobic and 1 anaerobic bottle) is collected from a
given anatomic site (20 mL per set). Inoculation of <10 mL per
bottle in adults reduces yield; likewise, collecting only a single
set of bottles per episode is discouraged, because the total
volume cultured is inadequate and interpretation of results is
more difficult. The optimal number of blood cultures per epi-
sode is 3 sets, but the minimum should be 2 sets (40 mL total)
drawn from separate sites. Bacteremic children tend to have a
higher concentration of organisms in their blood than adults,
and so collection of smaller quantities of blood for culture in
children is acceptable. In children, the volume collected should
not exceed 1% of total blood volume.
Direct examination
Gram staining is routinely performed on certain specimen
types, especially those collected from normally-sterile sites.
Blood and urine specimens are not examined directly, because
Gram  staining is too insensitive to be practical. For CSF, the
specimen must be concentrated by cytocentrifugation prior to
staining and examination. t3.32, t3.33
Gram stain procedure
ƒƒ Heat-fix a smeared slide
ƒƒ Apply crystal violet for 20 seconds, then wash with water
ƒƒ Add iodine for 20 seconds, then wash with water
ƒƒ Decolorize with acetone-alcohol, then wash with water
ƒƒ Apply safranin for 20 seconds, then wash with water
ƒƒ Blot dry and examine at 1,000× magnification (oil
immersion light microscopy)
177
3:  Microbiology

hydrophobic barrier and stain the mycolic acids in the cell

t3.32  Gram stain morphology of selected organisms
wall. Once stained, the organisms resist decolorization, even
Organism Gram stain morphology with harsh decolorizers like hydrochloric acid. This is why
Gram+ cocci they are termed “acid-fast.”
Staphylococci Gram+ cocci in grapelike clusters
In the classic Ziehl-Neelsen technique, a red carbol fuchsin
Pyogenic streptococci Gram+ cocci in pairs & short chains
Nonpyogenic streptococci Gram+ cocci in long chains dye containing phenol is heated during application, to aid pen-
Streptococcus pneumoniae Gram+ lancet shaped diplococci etration and staining of the bacterial cell wall. A strong acid,
Enterococcus species Gram+ diplococci 3% HCl, is used for decolorization. A methylene blue counter-
Gram– cocci stain is then added to provide contrast with the background.
Neisseria species Gram– coffee-bean shaped diplococci The slide must be examined at 1,000× magnification using oil-
Gram+ rods See t3.33 immersion light microscopy.
Gram– rods The Kinyoun (cold) acid-fast staining technique is a modifi-
Vibrio species Gram– curved, comma shaped short rods cation of the Ziehl-Neelsen method. Use of a higher concentra-
Campylobacter species Gram– curved, thin, rods (S-shaped, seagull shaped or
tion of phenol in the carbol fuchsin dye obviates the need for
corkscrew shaped)
Yersinia pestis Gram– rods with bipolar staining (“closed safety pin”) heating the stain, making the technique more convenient. The
Legionella species Gram-invisible in primary smear decolorization and counterstaining steps are identical to the
Ziehl-Neelsen method.
An excellent alternative to fuchsin-based acid-fast stains
t3.33  Gram stain morphology: Gram+ rods for mycobacteria is the fluorescent auramine-rhodamine stain.
Short, irregularly Regularly shaped, monomorphic Auramine and rhodamine stain mycolic acids, and fluoresce
shaped, Small to Branching, when exposed to ultraviolet light. The staining technique
pleomorphic medium Large filamentous involves application of an auramine-rhodamine stain con-
Aerobic or Corynebacterium Listeria species Bacillus species Nocardia taining phenol, followed by an HCl decolorization step and
facultatively species Erysipelothrix species sometimes a potassium permanganate counterstain. The slide
anaerobic Other coryneforms* species is examined using fluorescence microscopy, with mycobacteria
Non-Nocardia aerobic appearing bright yellow-green on a black background. The
actinomycetes† contrast is so great that the slide may be examined at relatively
Aerotolerant Actinomyces species‡ Aerotolerant
Bifidobacterium Lactobacillus
low magnification (200×), allowing more rapid slide review
species strains than traditional fuchsin based staining methods.
Anaerobic, Propionibacterium Eubacterium Clostridium Actinomyces Modified acid-fast staining techniques are useful for direct
nonspore- species species perfringens israelii visualization of weakly acid-fast bacteria, particularly Nocardia
forming Lactobacillus species and related aerobic actinomycetes. The cell wall of such
species organisms contains fewer mycolic acids than Mycobacteria, and
Anaerobic, spore- Clostridium Other Clostridium
forming ramosum species§ strong decolorization with HCl washes away the fuchsin stain,
*Examples include Arcanobacterium species, Rothia species & Arthrobacter species making them invisible by traditional acid-fast staining tech-
†Examples include Rhodococcus species, Gordonia species & Tsukamurella species niques. Modified acid-fast staining methods employ a weaker
‡Examples include Actinomyces neuii, A odontolyticus, A viscosus
decolorizing agent, such as 1% H2SO4, allowing these bacteria
§Examples include C septicum, C difficile, C sordellii, C sporogenes, & C bifermentans
to retain the fuchsin stain. Modified acid-fast staining is also
used to visualize the intestinal coccidia (Cryptosporidium,
Gram+ organisms have a thick peptidoglycan cell wall
Isospora, Cyclospora and Sarcocystis) in smears prepared from
without a surrounding outer membrane. Bacteria with this
concentrated stool specimens. Legionella micdadei is also modi-
structure will retain the purple crystal violet/iodine stain
fied acid-fast, unlike other Legionella species and other Gram–
during the decolorization step. Gram– organisms have a much
organisms in general.
thinner peptidoglycan cell wall layer, surrounded by an outer
membrane. Bacterial with this structure lose the crystal violet/
iodine stain during decolorization, and are visualized using Culture media t3.34, t3.35, t3.36
the red safranin counterstain. Media may be liquid or solid. Liquid media are espe-
The nuclei of human cells, such as neutrophils or squamous cially helpful for isolating organisms present in rare amount,
cells, stain red (Gram–). If present, these cells can be used to whereas solid media (in general) are less sensitive in this
detect under-decolorization of the smear, in which case their regard. However, solid media allow for separation of mixed
nuclei will appear purple (Gram+). organisms into pure-cultured isolates.
Nonselective media are designed to allow the growth of
Acid-fast stains many organism types, limited only by the nutritional growth
Mycobacteria have the cell-wall structure of Gram+ bac- factors present in the medium’s formulation. Selective media
teria, but are usually Gram-invisible. Their cell walls contain are intended to inhibit the growth of nontarget organisms,
mycolic acids, which create a hydrophobic barrier that pre- while allowing the growth of target organisms. Many nonse-
vents the crystal violet/iodine stain from reaching the pep- lective media are also available with antimicrobial additives,
tidoglycan cell wall. Such organisms can be visualized using making them selective. Differential media are designed to help
various acid-fast staining techniques. Acid-fast staining relies differentiate among organisms whose growth is supported by
on heat and/or phenol to allow a fuchsin dye to penetrate the the medium, by eliciting a phenotypic feature present in some
but not others.

178 Practical Clinical Pathology

 3:  Microbiology

t3.34  Commonly used nonselective media t3.36  Commonly used differential media
Medium Type Purpose Medium Type Basis for differentiation Purpose
Sheep blood Solid General bacteriology; supports the growth of most bacteria, with MacConkey Solid Lactose fermentation results in Differentiating between lactose
agar important exceptions (eg, N gonorrhea, H influenzae & Legionella). (MAC) agar pink or red coloration of colonies fermenting & nonlactose
Chocolate agar Solid Cultivation/isolation of fastidious bacteria like Neisseria species & Lactose nonfermenters form fermenting enterics
Haemophilus species, but not Legionella species translucent colonies
Buffered Solid Recovery of Legionella species; contains cysteine & iron Eosin methylene Solid Lactose fermentation results in Differentiating between lactose
charcoal-yeast supplementation, necessary to support growth of Legionella blue (EMB) purple-black colonies or colonies fermenting & nonlactose
extract (BCYE) Activated charcoal helps bind & sequester growth inhibitors that may be with a green metallic sheen fermenting enterics
agar present in the specimen Lactose nonfermenters form
Mueller-Hinton Solid Antimicrobial susceptibility testing of many common bacteria translucent colonies
agar Hektoen enteric Solid Lactose and/or sucrose Differentiating between lactose
Thioglycolate Liquid Cultivation of bacteria, including microaerophilic & obligately anaerobic (HE) agar fermentation results in yellow or and/or sucrose fermenting
broth bacteria; oxygen tension decreases toward bottom of tube, permitting orange coloration of colonies enterics & nonlactose or
growth of obligate anaerobes & microaerophilic bacteria without Lactose & sucrose nonfermenters nonsucrose fermenting
incubation in an anaerobic atmosphere form translucent colonies enterics
. H2S production results in black Differentiating between
coloration of colonies H2S-producing & non-H2S-
producing enterics
t3.35  Commonly used selective media Salmonella- Solid Lactose fermentation results in Differentiating between
Shigella (SS) pink or red colonies lactose fermenting enterics
Medium Type Basis for selectivity Purpose agar Lactose nonfermenters form & nonlactose fermenting
MacConkey agar (MAC) Solid Bile salts & crystal violet inhibit Cultivation/isolation of hardy translucent colonies enterics
growth of Gram+ bacteria & enteric Gram– rods H2S production results in black Differentiating between
delicate Gram– bacteria coloration of colonies H2S-producing & non-H2S-
Eosin methylene blue Solid Aniline dyes inhibit growth of Cultivation/isolation of hardy producing enterics
(EMB) Gram+ bacteria enteric Gram– rods Thiosulfate- Solid Sucrose fermentation results in Differentiating between
Campylobacter blood Solid Antimicrobials to which Recovery of Campylobacter citrate-bile yellow colonies sucrose fermenting Vibrios
agar (Campy-BAP) Campylobacter species are species salts-sucrose Sucrose nonfermenters form (eg, Vibrio cholerae) & sucrose
resistant (cephalothin, (TCBS) agar translucent colonies nonfermenting Vibrios
vancomycin, trimethoprim, Cefsulodin- Solid Mannitol fermentation results in Differentiating between
amphotericin B, polymyxin B) Irgasan- characteristic “bull’s eye” colonies mannitol fermenting
Hektoen enteric (HE) Solid Bile salts & the dyes bromthymol Enhanced recovery of Novobiocin (colorless with red center) Yersinia species (eg, Yersinia
agar blue & acid fuchsin inhibit the Salmonella & Shigella, (CIN) agar Mannitol nonfermenters form enterocolitica) & mannitol
growth of Gram+ organisms & compared with MAC or EMB translucent colonies nonfermenting Yersinia
some Gram– strains species
Salmonella-Shigella (SS) Solid Bile salts, sodium citrate & Recovery of Salmonella & Motility test Semi-solid Motile organisms show growth Differentiating between motile
agar brilliant green dye inhibit Shigella, although Shigella agar spreading away from the line & nonmotile bacteria
Gram+ bacteria & many enterics strains may be inhibited or inoculation (stab), clouding
other than Salmonella Not recommended for the agar
primary isolation of Shigella Nonmotile organisms stay within
Selenite broth Liquid Sodium selenite inhibits growth Recovery & enrichment of the stab, leaving the remaining
of Gram+ bacteria & many Salmonella agar clear
enterics other than Salmonella
Thiosulfate-citrate-bile Solid Bile salts inhibit Gram+ bacteria Recovery of Vibrio species
salts-sucrose (TCBS) Alkaline pH inhibits most enterics Culture temperature
agar & enhances growth of Vibrio The optimal temperature for initial incubation is usually
species
Cefsulodin-Irgasan- Solid Antimicrobials (cefsulodin, Recovery of Yersinia species
37°C. Yersinia enterocolitica and certain Pseudomonas species (eg,
novobiocin (CIN) agar Irgasan, novobiocin) & crystal Pseudomonas fluorescens, Pseudomonas putida) grow optimally at
violet inhibit most Gram– & 25°C to 30°C, while the Campylobacter species most commonly
Gram+ bacteria other than associated with diarrheal illness (C  jejuni and C  coli) grow
Yersinia species optimally at 42°C. Listeria monocytogenes grows optimally at
Anaerobic colistin- Solid Antimicrobials (colistin, nalidixic Recovery of anaerobic 37°C, but displays its characteristic motility only at 25°C, and is
nalidixic acid (CNA) agar acid) inhibit Gram– bacteria streptococci; blood in the
agar allows differentiation notoriously able to multiply at refrigeration temperature (4°C).
based on hemolytic reactions
Lim broth Liquid Antimicrobials (colistin, nalidixic Recovery of Group B
acid) inhibit Gram– bacteria streptococci (S agalactiae)
Regan-Lowe medium Solid Antimicrobials to which Recovery of Bordetella
Bordetella species are resistant pertussis & Bordetella
(cephalexin) parapertussis
Thayer-Martin medium Solid Antimicrobials to which Recovery of Neisseria species
Neisseria species are resistant from nonsterile sites
(vancomycin, colistin, nystatin,
& SXT)

ISBN 978-089189-5985 179

7:  Molecular Pathology

particularly within the background of oxidative stress that is

common to many of the underlying disorders. The acquisi-
tion of mutations within regenerative nodules gives rise to
dysplastic nodules, the precursor of hepatocellular carcinoma.
The molecular events underlying HCC are incredibly com-
plex and differ significantly from one case to the next. For
example, HBV induced HCC is strongly associated with
TP53 alterations, but these are present in only a minority of
non-HBV tumors. Furthermore, HBV-driven tumors quite
often have portions of the HBV genome incorporated into the
host cell genome, but this is not seen in HCV-driven tumors.
Expression of the HBV gene HBX leads to suppression of the
p53 protein. In all, dozens of genetic alterations have been
described in HCC, and subsets of these are found together in
individual cases. Nascent efforts to sub-classify HCC on the
basis of the molecular substrate are under way.
The approach to treatment of HCC has traditionally relied
upon surgical resection, when feasible, and locoregional abla-
tion. Sorafenib, a tyrosine kinase inhibitor (TKI), was the first f7.38  Her2 by immunohistochemistry, showing circumferential membranous staining
molecular targeted therapy to be approved for the systemic
treatment of HCC. Unlike most TKI therapy, sorafenib is not
targeted at the neoplastic hepatocytes; instead, its target is the
endothelial cells involved in angiogenesis.
Furthermore, Her2 overexpression is seen almost exclusively in
invasive ductal carcinoma of the usual type. Her2 is rarely over-
Cholangiocarcinoma
expressed in ductal carcinoma variants such as mucinous (col-
Risk factors for both intrahepatic and extrahepatic chol-
loid) and tubular carcinoma, and within the spectrum of lobular
angiocarcinoma include infection with liver fluke (Clonorchis
carcinoma is seen mainly in pleomorphic variants, if at all.
sinensis, Opisthorchis viverrinii), primary sclerosing cholangitis,
Much hinges upon the result of Her2 testing. Therapeutic
Caroli disease, choledochal cysts, thorotrast exposure, and
benefits can be great; however, a course of therapy is extremely
a variety of genetic polymorphisms. Risk factors mainly for
expensive, and there is a risk of serious cardiotoxicity. As with
intrahepatic cholangiocarcinoma include hepatolithiasis, HCV
all tests, the pathologist must ensure the validity of the result
and HBV infection, and hepatic schistosomiasis. Risk factors
through meticulous control of the conditions under which the
for extrahepatic cholangiocarcinoma include duct obstruction
test is performed and interpreted. At the very least, one should
(choledocholithiasis), and obesity.
participate in interlaboratory proficiency testing and conform
Virtually 100% of cholangiocarcinomas harbor telomerase
to guidelines published jointly by the American Society of
abnormalities. Mutations in many of the same genes involved
Clinical Oncologists (ASCO) and the College of American
with the development of HCC can also be seen in cholangio-
Pathologists (CAP).
carcinoma including WNT pathway factors, p53, and p16, with
In assessing Her2 expression by IHC, only circumferen-
similar patterns of epigenetic inhibition. Mutations in EGFR
tial membranous staining f7.38 is considered. Expression is
have been found in a minority (~10%) of cases of cholangiocar-
usually reported as 0 ‑ 3+, depending upon the proportion of
cinoma and <5% of hepatocellular carcinoma cases. Targeted
cells displaying strong circumferential membranous staining.
small molecule inhibitor therapy, similar to lung adenocarci-
Scores of 0 and 1+ are considered negative, 3+ is positive, and
noma harboring amenable EGFR mutations of EGFR-mutant
2+ is equivocal. A 3+ result is defined as strong circumferen-
cholangiocarcinoma has shown some early success.
tial staining in >30% of tumor cells. An equivocal result (2+)
is defined as complete circumferential membrane staining,
Breast cancer that is either nonuniform or weak, in >10% of tumor cells.
HER2 (Neu, ERB-B2) Occasionally, a 2+ result can be due to strong membranous
The HER2 receptor is a member of the epidermal growth staining in >10% but <30% of tumor cells. 1+ is defined as weak
factor receptor (EGFR) family. About 20% of breast cancers incomplete (noncircumferential) staining in any proportion
exhibit overexpression of the HER2 receptor, usually through of tumor cells. Strong (3+) IHC staining is closely associated
a mechanism of gene amplification. Amplification of HER2 is with amplification of HER2. Equivocal staining (2+) is asso-
associated with high nuclear grade, increased mortality rate, ciated with HER2 amplification ~25% of the time, and such
and increased rate of recurrence; however, the addition of results are an indication for FISH testing. Negative (0+) or very
anti‑Her2 (trastuzumab) to adriamycin based chemotherapy weak (1+) staining is very infrequently associated with HER2
mitigates these aggressive features. amplification.
With regard to the correlation of histology with Her2 status, HER2 amplification by FISH is determined by calculating
a few generalizations can be made. As noted, Her2+ tumors the ratio of signals from HER2 (located on chromosome 17) to
tend to have a higher nuclear grade; grade 1 tumors are rarely a chromosome enumeration probe (CEP) for chromosome 17
Her2+, and the vast majority of Her2+ tumors have a grade of 3. f7.39. Amplification is defined as a ratio >2.2; a negative result

404 Practical Clinical Pathology


a b
f7.39  Her2 by fluorescence in situ hybridization (FISH)
a The ratio of Her2 signals (red) & chromosome 17 signals (green) is 1.0; no amplification is present
b Large clusters of Her2 signals (red) are present in this case with Her2 amplification
is a ratio <1.8. Equivocal FISH results (ratio between 1.8 and
2.2) should be repeated or more cells counted. If repeatedly
equivocal FISH results are obtained, IHC should be considered
(if not already done).
TP53 tumor suppressor gene
The TP53 gene (17p) encodes the transcription factor p53. It
is the normal function of p53 to bind to several specific DNA
sequences and enhance their transcription. The genes it nor-
mally enhances—such as those which promote apoptosis and
others which inhibit entry into the cell cycle—work in concert
to control cell division. Clinically significant TP53 muta-
tions usually affect the DNA binding domain and result in
loss of specificity of p53 protein; thus, this effective promoter
of transcription becomes less inclined to promote its usual
targets and becomes free to activate other genes.
Mutated TP53 encodes a p53 protein that is resistant to deg-
radation (so-called TP53 stabilized mutant protein); thus TP53
mutations, though associated with decreased p53 functional
activity, are associated with increased p53 immunohistochem-
ical (IHC) staining.
In breast cancer, TP53 mutations correlate with tumor
aggressiveness, higher tumor grade, and lower rates of ER/
PR expression. There is some evidence that TP53 mutation
may reduce the efficacy of chemotherapeutic agents whose
mechanism of action involves the induction of apoptosis; eg,
adriamycin, 5-fluorouracil (5-FU).
Steroid receptors
The steroid receptors ER and PR, like all steroid recep-
tors, reside within the nucleus and, when bound to steroid
hormone, activate transcription of specific genes. There is an
α form and a β form for both ER and PR. In the case of ER,
these are encoded by separate largely homologous genes. In
contrast, PRα and PRβ are encoded by the same gene, one with
more 3′ (amino-terminal) material than the other. In routine
immunohistochemical assays for ER, only ERα is measured;
whereas the PR assay measures both PRα and PRβ.
A clinical response to hormonal therapy (tamoxifen, aroma-
tase inhibitors, or LHRH agonists) is largely, but not entirely,
dependent upon the expression of steroid hormone recep-
tors. In fact, ~70% of tumors expressing both ER and PR will
respond to hormonal therapy, and only ~5% of tumors nega-
tive for ER and PR will respond. ER+, PR– tumors respond at
ISBN 978-089189-5985
7:  Molecular Pathology
a b c
f7.40  Basal-like invasive breast carcinoma
a Low magnification view shows a somewhat circumscribed tumor with central collagenous scarlike zone
b Intermediate magnification shows that the tumor is composed of high grade epithelial cells
arranged in somewhat syncytial trabecular groups
c Strong & diffuse expression of CK5/6
roughly the same rate as those positive for both; however, ER–,
PR+ tumors respond in only ~15% of cases.
BRCA-associated tumors
~1/2 of familial breast cancer is caused by inherited defects
in one of the BRCA genes. In most of the remaining familial
cases, no defined genetic defect has been identified.
There are 2 BRCA genes, BRCA1 located on chromosome
17q21, and BRCA2 on chromosome 13q12 ‑ 13. Each encodes
a tumor suppressor protein. Each of the genes is large, with
thousands of described mutations, making mutation detec-
tion difficult. BRCA mutations are especially prevalent in
the Ashkenazi Jewish population. A quarter of Ashkenazi
women with breast cancer harbor BRCA mutations. Within
this population, mutation detection is facilitated by a small
number of founder mutations that account for 90% of all cases:
185delAG mutation in BRCA1, 5385insC mutation in BRCA1,
and 6174delT mutation of BRCA2.
A mutated BRCA gene (BRCA1 or BRCA2) is expressed in an
autosomal dominant fashion, with high penetrance. In fami-
lies harboring germline BRCA gene mutations, there are often
2 or more generations of women with premenopausal breast
cancer, sometimes arising bilaterally. In addition, there is an
increased risk of neoplasms of the ovary, fallopian tube, colon,
uterus, and pancreas. In particular, the BRCA2 6174delT muta-
tion is strongly associated with familial pancreatic cancer. In
families with BRCA2 mutations, there is a high rate of prostate
cancer. BRCA mutations increase the lifetime risk of breast
cancer to 80%, compared to the 10% risk seen in the general
population. Such mutations increase the risk of ovarian cancer
to 50%, especially BRCA1 mutations. The lifetime risk of breast
cancer in a male who carries a BRCA mutation is ~6%; BRCA2
carries a higher male breast cancer risk than does BRCA1.
About 5% of all women with breast cancer and 25% of
Ashkenazi Jewish women with breast cancer have a germ-
line BRCA mutation. Additionally, in a woman with breast
cancer who is found to harbor a BRCA mutation, the risk of
cancer developing in the contralateral breast is 25%. BRCA-
associated breast cancers often have distinctive though not
entirely specific histologic features. The tumors tend to have a
high nuclear grade, a central acellular scar-like zone, pushing
rather than infiltrating margins, and tumor-infiltrating lym-
phocytes (medullary carcinoma-like histology) f7.40. They are
most negative for ER, PR, and HER2 (triple-negative), nega-
tive for luminal cytokeratins (CK8/18), and positive for basal
405
7:  Molecular Pathology

cell markers (CK5/6). This constellation of findings has been 2. basal-like—expressing high molecular-weight
described as the basal-like phenotype. While typical of BRCA- cytokeratins (CK5/17 or 5/6), having a poor prognosis,
associated tumors, basal-like carcinoma is frequently seen often ER– and high nuclear grade, but sensitive to
sporadically. chemotherapy
3. HER2+—also associated with a poor prognosis and
Other inherited influences upon breast cancer sensitivity to chemotherapy, almost always associated
Germline mutations in P53 (Li-Fraumeni syndrome), PTEN with amplified HER2
(Cowden syndrome), CDH1 (hereditary diffuse gastric cancer It is clear depending on the nature of the clustering that some
syndrome), and STK11 (Peutz-Jeghers syndrome) are associ- of these groups may have subgroups (luminal-A, luminal-B,
ated with an increased risk of breast cancer. luminal-C) but the significance of these subgroups is unclear
in relationship to the parent group. Note also that “basal-like”
Molecular classification of breast carcinoma (gene and “triple-negative” are not synonymous terms. While most
expression profiling) basal-like tumors are in fact triple-negative, a wide variety of
It has long been acknowledged that certain histologic sub- triple-negative tumors do not have basal-like features.
types of breast cancer demonstrate particular clinical courses, Predictive panels to determine response to chemotherapy
while some seemingly histologically disparate tumors behave also promise to improve response to particular agents. By
similarly. In addition it is known that the benefit of adjuvant testing tumors for genes that are associated with response to
chemotherapy is variable between patients. Classification of individual agents, such as tamoxifen or one of the taxols it can
breast carcinomas by gene expression patterns and the utiliza- be predicted whether a patient may respond and a tailored
tion of gene expression panels to make clinical decisions have chemotherapeutic regimen can be designed. A further chal-
gained support as a means to better characterize tumors and lenge is to combine the results of prognostic and predictive
better predict their behavior. Some of the first work was done panels to determine whether a patient with a certain molecular
with predictive quantitative multigene panels. In an effort to expression group responds to a particular chemotherapeutic
stratify patients who would derive the most benefit from adju- reagent.
vant hormone therapy, panels of genes including proliferation Much is still unknown, particularly about how robust
markers, hormone receptors and some downstream targets molecular classification will be, but the promise exists for
of hormone receptors, and genes associated with metastatic tailoring therapeutic options and prognosis based on gene
disease among others were constructed. Given the quantitative expression patterns. Of note, it has recently been demonstrated
basis the results are given as a quantitative “score.” Patients that a combination of morphology and immunohistochemical
with certain test values are predicted to derive the most benefit analysis can be used to derive results comparable to those
from hormone therapy while others less so. Since the initial obtained by gene expression profiling t7.14.
description of this test many others with similar testing strate-
gies have evolved with likely more in the future. The design t7.14  Breast cancer subtyping by morphology & IHC
of markers to determine whether any individual tumor will
Subtype Morphology ER, PR, Her2 Other IHC Notes
respond to a particular chemotherapeutic regimen, such as
Luminal A Low grade ER+, PR±, Low Ki67 Sensitive to endocrine
the quantitative multigene assays previously described, is a ductal, NOS Her2– CK8/18+ therapy, variable response to
form of supervised (directed) classification. If a large panel of chemotherapy, overall good
expressed genes is grouped according to their expression pat- prognosis
terns (eg, upregulated, downregulated), this is referred to as Luminal B High grade ER+, PR±, Mod to high Ki67 Tend to be sensitive to
unsupervised classification. ductal, NOS Her2+ CK8/18+ endocrine therapy, variable
response to chemotherapy,
Unsupervised classification of gene expression by math- more aggressive than
ematical tools such as hierarchical clustering allows patterns luminal A
to evolve and groups to form based on shared molecular Her2+ ER–, PR–, High Ki67 Sensitive to trastuzumab
expression features. Surprisingly, once tumors are grouped Her2+
into clusters the gene expression patterns within these groups Basal-like Triple negative High Ki67 Resistant to everything,
CK5/6+, P63+ require aggressive
is fairly consistent. In addition the expression pattern of most
chemotherapy
genes is consistent between groups. There are, however, genes BRCA1-related
that can be used to distinguish certain groups with high Triple-negative Triple negative
reproducibility. The most basic groups based on hierarchical
clustering are
1. luminal—expressing low molecular-weight cytokeratins
(CK8/18), harboring a favorable outcome, and most often
associated with ER positivity

406 Practical Clinical Pathology

 7:  Molecular Pathology

Genitourinary tumors
Renal cell carcinoma syndromes
Von Hippel-Lindau syndrome (VHL syndrome) is an auto-
somal dominant condition causing predisposition to a variety
of tumors, including clear cell renal cell carcinoma, hemangio-
blastoma of the central nervous system (cerebellum, retina, or
spinal cord), pheochromocytoma, pancreatic islet cell tumors
and cysts, cystadenomas of the epididymis or broad liga-
ment, and the papillary tumor of endolymphatic sac origin.
Germline mutations in the gene VHL located on chromosome
3p25 ‑ 26 account for the majority of cases. Alteration of 3p is
extremely common in sporadic clear cell renal cell carcinoma.
A form of the syndrome having all the usual features except
pheochromocytoma is known as type 1 VHL. It is associated
with nonproduction of the VHL gene product, resulting from
gene deletion or missense mutation. Forms of the syndrome
with a high risk of pheochromocytoma are known as type 2
VHL, and these are associated with missense mutations. The
VHL gene product is a part of a ubiquitin-dependent complex f7.41  Birt-Hogg-Dubé syndrome-associated renal tumor
that is responsible for the degradation of the hypoxia inducible
factor (HIF-1). Increased HIF-1 leads to increased angiogenesis
and promotion of tumor growth.
Birt-Hogg-Dubé syndrome is an autosomal dominant con- upon prognosis, while loss of 14q, 9p, and 8p each correlates
dition predisposing to renal cell carcinoma. Skin lesions with higher stage and a worse prognosis.
and spontaneous pneumothorax complete the diagnostic Papillary renal cell carcinoma accounts for ~15% of renal
triad. Skin manifestations include multiple fibrofolliculomas, cell carcinomas. Like many of the nonclear variants, the gross
trichodiscomas, and acrochordons, especially affecting the appearance is that of a uniform brown tumor. Papillary renal
head, neck, and upper trunk. The lung contains numerous cell carcinoma is often multifocal. There are 2 histologic
cystic parenchymal spaces in addition to blebs and bullae, subtypes—type 1, characterized by bland cuboidal cells on
causing recurrent spontaneous pneumothorax. The spon- tubular or papillary formations, and type 2, which has larger
taneous pneumothoraces tend to involve the lower lobes of pseudostratified cells lining papillary cores. The vascular
the lung, unlike most other conditions that predispose to cores in each tend to have foamy interstitial macrophages.
pneumothorax (α1 antitrypsin deficiency, lymphangiomyoma- Type 2 papillary RCC is more often associated with multiple
tosis, Langerhans cell histiocytosis, Ehlers-Danlos syndrome, chromosomal anomalies, but both can be associated with loss
Marfan syndrome). The renal tumors tend to be bilateral and of the Y chromosome and gains of chromosomes 7 and 17
multifocal and have features of a combined chromophobe (similar to urothelial carcinoma). These chromosomal abnor-
renal cell carcinoma and oncocytoma f7.41. Mutations of the malities help differentiate papillary RCC from the histologi-
BHD (FLCN) gene on chromosome 17p11.2 encoding the pro- cally similar mucinous tubular and spindle cell carcinoma of
tein folliculin are responsible for the syndrome. the kidney.
Familial clear cell renal cell carcinoma is associated with
mutations of 3p and lacks the other features of the VHL syn- t7.15  2004 WHO classification of renal tumors (modified)
drome. A hereditary form of papillary renal cell carcinoma
is associated with bilateral papillary renal cell carcinomas Tumor Immunophenotype Genetic anomalies Behavior
Clear cell renal cell CD10+, vimentin+, RCC Ag+ del(3p) Aggressive
and gain of function mutations of the protooncogene C-MET carcinoma
on chromosome 7q31. C-MET functions in a manner similar Papillary renal cell CD10+, vimentin+, Loss of Y, gains of 7 & 17 Indolent
to C-KIT, with mutation resulting in a constitutively acti- carcinoma AMACR+, CK7+
vated tyrosine kinase. Tuberous sclerosis is associated with Chromophobe renal cell CD10–/+, vimentin–, Loss of Y, 1, 2, 6, 10, 13, Indolent
increased incidence of renal cell carcinoma, but is much more carcinoma CD117+, CK7+ 17 & 21
commonly associated with the renal angiomyolipoma. Collecting (Bellini) duct CD10–, vimentin–, high MW del(1q) Aggressive
carcinoma keratin+
Renal medullary carcinoma CD10–, vimentin–, CEA+ Unknown Aggressive
Sporadic renal cell carcinoma Xp11.2 translocation TFE3+ (nuclear) Xp11.2 translocation Indolent
The most common type of renal cell carcinoma t7.15 is con- carcinoma CD10+
ventional clear cell carcinoma. Portions of the short arm of Oncocytoma CD10–/+, vimentin±, CK7 Highly variable: loss of Benign
chromosome 3 are deleted in most cases. 3 particular loci are rare focal +, CD117+ 1, 14. t(5;11)
disproportionately affected: 3p14, 3p25.3 (the location of the Mucinous, tubular & CD10–, vimentin+ Loss of 1, 4, 6, 8, 13, 14 Indolent
spindle cell carcinoma
VHL gene), and 3p21.3. Only a small percentage of cases have Angiomyolipoma HMB45+ LOH at TSC2 on 16p Benign
3p  anomalies as the sole abnormality; most tumors start
with 3p  mutations and collect additional ones, commonly in
14q, 9p, 8p, and 6q. Furthermore, del(3p) by itself has no impact

ISBN 978-089189-5985 407

7:  Molecular Pathology
a b
f7.42  Renal carcinoma with Xp11.2 translocation
Chromophobe RCC has clear to microvacuolated cytoplasm
and distinct “plant cell” borders. Hale colloidal iron and CD7
preferentially stain chromophobe RCC and help to distinguish
it from oncocytoma. CD117 helps to distinguish it from clear
cell RCC. Most chromophobe RCCs are hypodiploid with
common losses of multiple chromosomes including Y, 1, 2, 6,
10, 13, 17, and 21.
Renal carcinoma with Xp11.2 translocation most commonly
affects children and young adults. The tumor has a distinc-
tive appearance with alveolar (nested) and papillary architec-
ture, clear cells, and psammoma bodies f7.42. Based upon the
foregoing, therefore, TFE3-associated carcinoma should be
thought of whenever
1. a diagnosis of a RCC with a mixed histologic pattern is
entertained
2. any RCC is seen in a child
The tumor is characteristically negative for EMA (a marker
uniformly positive in conventional clear cell carcinoma) but
positive for CD10. Translocations of the transferrin receptor
TFE3 gene is responsible for the disease. Several Xp11.2
abnormalities have been described in connection with these
tumors, including t(X;1)(p11.2;q21), producing the TFE3-PRCC
fusion gene, t(X;17)(p11.2;q25), producing TFE3-ASPL, and
t(X;1)(p11.2;p34), producing TFE3-PSF. The TFE3-ASPL gene
fusion of t(X;17) is also seen in alveolar soft part sarcoma
(ASPS); however, a key difference is that the translocation in
ASPS is an unbalanced one, while that in renal carcinoma
is balanced. The TFE-ASPL renal tumors characteristically
present at advanced stage but, like ASPS, follow an indolent
course marked by very late relapses/metastases. Renal cell car-
cinomas with t(6:11)(p11.2;q12) involves the TFEB gene which
is closely related in function to the TFE3 gene. The resulting
tumor is essentially identical to Xp11.2 tumors.
408 Practical Clinical Pathology
Wilms tumor
Nephroblastoma or Wilms tumor is most often caused by
a mutation in the WT1 gene on chromosome 11p13. Wilms
tumor is a feature of WAGR syndrome, which is caused by a
microdeletion that involves the WT1 gene and adjacent PAX6
gene. Loss of PAX6 is associated with the aniridia of WAGR
syndrome.
Prostate cancer
When studied by conventional cytogenetics, most pros-
tatic adenocarcinomas have a normal karyotype. However,
numerous small abnormalities are demonstrable by FISH or
comparative genomic hybridization (CGH), the most common
of which is loss of 8p. More recently, a recurrent translocation
t(4;6)(q22;q15) has been demonstrated. Found in ~10% of cases,
this translocation creates the fusion transcript TMPRSS2:ERG,
and is associated with high stage at presentation, high tumor
volume, and high baseline PSA levels. Several oncogenes are
overexpressed in prostatic adenocarcinoma, MYC and BCL2
especially, and many cases demonstrate mutations in PTEN,
GSTP1, and P53.
Some genes overexpressed in carcinoma have been
exploited for diagnostic purposes. The prostate cancer 3
(PCA3) gene encodes a nontranslated transcript called DDT3
that is excreted in urine. The DDT3 transcript is elevated in
>95% of prostate cancers (by 66-fold on average). The gene
encoding PSA, KLK3, is not overexpressed (increased PSA
in cancer is probably caused by increased release of the pro-
tein into the extracellular matrix). Using quantitative reverse
transcriptase PCR, the quantity of the PCA3 transcript can be
measured and compared to the urine PSA (to correct for the
number of prostatic cells obtained).
About 5% of cases are associated with a strong family his-
tory. The proportion may be as high as 40% in cases diagnosed
before age 55. Presently, most cases are thought to be linked
to the RNASEL gene (encoding ribonuclease L). Additionally,
families with germline mutations of the BRCA1 or BRCA2
genes have a higher rate of prostatic adenocarcinoma.
From a practical standpoint, specimen identity testing is
occasionally required in the practice of prostate pathology
and other types of biopsy pathology. Specimen identity
testing refers to the use of molecular techniques to ensure
that a specimen belongs to a particular patient. Most patholo-
gists are familiar with the uniquely alarming circumstance
in which cancer cannot be found in a prostatectomy after a
positive biopsy. Often the issue can be resolved with thorough
sectioning, but there are rare cases in which even after exhaus-
tive efforts one is left with the predicament of an unequivo-
cally positive biopsy and a negative prostate gland. In 1995,
Goldstein referred to this situation as the “vanishing cancer”
phenomenon. While there are a number of possible explana-
tions, one that must occasionally be entertained is a specimen
mix-up. One approach to this problem is the comparison of
DNA microsatellites, either between biopsy and patient or
between biopsy and prostatectomy. Microsatellites are regions
 7:  Molecular Pathology

of the genome composed of a short repetitive sequence; eg, a Testicular tumors

run of adenine bases, a run of alternating cytosine-adenine Several chromosomal regions, including the Y chromosome
(CA) couplets. They are usually found in noncoding segments gr/gr deletion have been shown to be associated with an inher-
of DNA, and each person inherits, for every microsatellite ited risk of developing testicular germ cell tumors. No clear
locus, one maternal and one paternal allele. The parental candidate genes involved with an inherited predisposition for
alleles are usually of differing length (heterozygous), but they testicular have been yet identified.
are occasionally the same (homozygous); within a particular The majority of germ cell tumors are associated with gains
person, every cell in the body has microsatellites of equal of chromosome 12p, most often as an isochromosome, i(12p).
length. From person  to  person, microsatellites are usually of Gain of 12p can be seen in all types of germ cell tumors and
different lengths, and, particularly if several microsatellite loci may represent an early step in tumor development. CKIT
are looked at simultaneously, they can be used to very reliably mutations are frequent in seminoma, as a result of which many
distinguish one person from another. This analysis can be seminomas exhibit strong immunohistochemical staining for
performed on formalin-fixed, paraffin-embedded tissue from c-kit (CD117). The membranous staining pattern is strongly
which DNA is extracted. The recovered DNA is subjected to associated with seminoma, other staining patterns can be
multiplex (multiple primers) PCR designed to amplify several found in nonseminomatous germ cell tumors.
microsatellite markers. The amplified products can be sub-
jected to electrophoresis on a gel and analyzed visually. The
pattern derived from a prostate biopsy should match exactly Soft tissue & bone
that obtained from the prostatectomy. Tumors of ectodermal or epithelial origin often demon-
strate one or several gene mutations, but with rare exception
they lack specific chromosome structural rearrangements. In
Urothelial (transitional cell) carcinoma
contrast, tumors of mesodermal origin, including the hema-
Biologically speaking there appear to be ≥2 distinct path-
tolymphoid system, soft tissue, and bone, often demonstrate
ways to arrival at urothelial carcinoma. The first pathway
specific and reproducible structural rearrangements, such as
leads to the development of low  grade papillary transitional
translocations t7.16.
cell carcinoma, with almost no metastatic potential. Low grade
lesions may occasionally be a precursor to high grade tumors,
but mainly serve as a marker of risk. The other pathway t7.16  Molecular features of soft tissue tumors
involves high  grade papillary carcinoma and flat urothelial Most common
carcinoma in situ, each of which may give rise to a high grade Tumor rearrangement(s) Genes
invasive tumor. The low grade tumors often have loss of P16 Chondroid lipoma t(11;16)(q13;p12 ‑ 13) C11orf95/MKL2
on chromosome 9p21 and mutations in the FGFR3 gene. While Schwannoma del(22q12) NF2
high  grade tumors frequently have loss of P16, they usually Ewings/PNET t(11;22)(q24;q12) EWS/FLI1
t(21;22)(q22;q12) EWS/ERG
lack FGFR3 mutations. Instead they demonstrate inactiva-
t(7;22)(p22;q12) EWS/ETV1
tion of P53, chromosomal instability, and an often complex t(17;22)(q21;q12) EWS/ETV4
karyotype with gains or losses of 6p, 7, 9, and 17. Diagnosis has t(2;22)(q33;q12) EWS/FEV
been facilitated with a recent application of FISH technology Neuroblastoma del(1p), +17 n-MYC
to urine specimens in patients with microhematuria or recur- Alveolar rhabdomyosarcoma t(2;13)(q35;q14) PAX3/FOX01
rent tumors. Enumeration probes for chromosomes 3, 7, and 17 t(1;13)(p36;q14) PAX7/FOX01
along with a locus-specific indicator for the P16 gene on 9p21 Alveolar soft part sarcoma der(17)t(X;17)(p11;q25) ASPSCR1/TFE3
form the basis of the UroVysion assay. Examination of cytolog- Desmoplastic small round cell tumor t(11;22)(p13;q12) EWS/WT1
Myxoid & round cell liposarcoma t(12;16)(q13;p11) FUS/DDIT3
ically abnormal cells for quantitation of FISH signals provides
Low grade fibromyxoid sarcoma, t(7;16)(q33;p11) FUS/CREB3L2
high specificity and sensitivity. In initial reports, specificity hyalinizing spindle cell tumor with
approached 100%, with positive FISH results in some patients giant rosettes
preceding the development of clinically apparent tumor Dermatofibrosarcoma protuberans, t(17;22)(q22;q13) COL1A1/PDGFB
(“anticipatory positives”). Subsequent studies have not con- giant cell fibroblastoma
firmed this degree of specificity. Infantile fibrosarcoma, congenital t(12;15)(p12;q25) ETV6/NTRK3
mesoblastic nephroma
Myxoid chondrosarcoma, t(9;22)(q22;q12) EWS/NR4A3
extraskeletal
Liposarcoma t(12;16)(q13;p11) FUS/CHOP
Angiomatoid fibrous histiocytoma t(12;22)(q13;q12) EWS/ATF1
t(2;22)(q33;q12) EWS/CREB1
Clear cell sarcoma t(12;22)(q13;q12) EWS/ATF1
t(2;22)(q33;q12) EWS/CREB1
Synovial sarcoma t(X;18)(p11.2;q11.2) SSX1/SYT
t(X;18)(p11.2;q11.2) SSX2/SYT
Inflammatory myofibroblastic tumor t(1;2)(q25;p23) TPM3/ALK
t(2;19)(p23;p13) TPM4/ALK

ISBN 978-089189-5985 409

7:  Molecular Pathology
a b
c d
e f
f7.43  Ewing family tumors
a-c Ewing sarcoma/PNET
d Extraskeletal myxoid chondrosarcoma
e & f Intraabdominal desmoplastic small round cell tumor
Ewing sarcoma family of tumors
Ewing sarcoma f7.43 is a member of a large family of tumors,
all of which have fusion genes involving the EWS gene. Ewing
sarcoma is one of the most common soft tissue tumors of chil-
dren, accounting for 20% of soft tissue tumors in this popula-
tion. Primitive neuroectodermal tumor (PNET) is essentially
identical to Ewing sarcoma with the addition of neuroecto-
dermal features as demonstrated by histologic, immunohisto-
chemical, or ultrastructural means.
The EWS gene often undergoes translocation to a member
of the ETS transcription family f7.44. The ETS sites function
as DNA binding domains; thus, such translocations localize
the strong transcriptional activation domain of the EWS to
ETS inducible genes. Almost 95% of cases of Ewing sarcoma
express a particular translocation of the EWS gene on chromo-
some 22q12 to the FLI1 gene on 11q24. There is some variation
in the exact breakpoints but most commonly the breaks occur
between exon 7 of EWS and exon 6 of FLI1. Less common (5%
of cases) is the t(21;22)(q22;q12) translocation of EWS with ERG.
Rare cases have been associated with the translocations t(7;22),
t(17;22), t(2;22), inversion of 22q, and even a non-EWS translo-
cation of t(16;21)(p11;q22) resulting in a TLS-ERG fusion. The
TLS gene shares extensive homology with EWS. Additional
cytogenetic abnormalities (most often gains of 1q, 8, 12) can be
found in 1/2 of the cases of Ewing sarcoma and portend a poor
prognosis. Genetic diagnosis can be made with RT-PCR, which
provides high sensitivity and specificity when primers and
410 Practical Clinical Pathology
a b
f7.44  Ewing sarcoma FISH utilizing dual color break apart probes; the red & green probes flank the
region of known breakpoints
a A normal nucleus contains 2 red-green fusion signals (2F)
b An abnormal nucleus demonstrates 1 red-green fusion signal & separate red & green signals
(1R,1G,1F)
probes to cover all the different breakpoints are used. Other
common modalities used for diagnosis include karyotyping,
FISH with EWS break apart probes, or Southern blotting.
Intraabdominal desmoplastic small round cell tumor is a
recently described tumor, most commonly presenting as an
abdominal mass in a young male. It is consistently associated
with the translocation t(11;22)(p13;q12) fusing the genes EWS
with WT1. The tumor appears histologically as a small round
blue cell tumor; its only distinctive feature is growth within
strikingly desmoplastic stroma. A unique immunohistochem-
ical profile usually aids in identifying this tumor, which is
found to be NSE+, EMA+, keratin+ and desmin+ in the vast
majority of cases.
Clear cell sarcoma of tendons and aponeuroses (malig-
nant melanoma of soft parts) is another member of the EWS
tumor family. Aside from presenting in the deep soft tissue of
young adults, it shares many histomorphologic, immunohisto-
chemical, and ultrastructural features with melanoma. Unlike
malignant melanoma, nearly 90% of cases of clear cell sarcoma
are associated with a specific translocation, t(12,22)(q13;q12)
fusing EWS to the transcription factor ATF1. There are 3 major
fusion protein subtypes involved, the major one involving
exon 8 of EWS to exon 4 of ATF1.
Extraskeletal myxoid chondrosarcoma is another member of
the EWS family of tumors. It is a rare tumor whose chondroid
differentiation is subtle, consisting largely of a cording pattern
of growth. Extraskeletal myxoid chondroscarcoma is com-
monly associated with the translocation t(9;22)(q22;q12) that
fuses the EWS gene with the CHN (TEC) gene. The remaining
cases are found to harbor 1 of 2 additional translocations,
t(9;12)(q22;q11) that fuses CHN to TAF2N, or t(9;15)(q22;q21) that
fuses CHN to the gene TCF12 These translocations are specific
for extraskeletal myxoid chondrosarcoma and have not been
seen with any other chondroid related tumors.
Neuroblastoma
Neuroblastoma f7.45 is the most common malignancy in
patients under the age of 5 years. It presents as an abdominal
mass or a posterior mediastinal mass, as it usually arises from
the adrenal medulla or sympathetic chain. Amplification of
the MYCN (n-Myc) protooncogene is found in 30% of neu-
roblastomas and is a marker of aggressive behavior. The
mechanism of amplification is primarily as extrachromosomal
 7:  Molecular Pathology

a b a b

c d
f7.45  Adrenal neuroblastoma

double minutes, though in some cases there is in  situ gene

duplication and in others gene duplication and insertion into
random chromosomes, leading to segmental chromosome e
gain that may be visible as homogeneously staining regions
(HSRs). Greater than 10-fold amplification is correlated with
poor prognosis. The overall proportion of tumors with this
degree of MYCN amplification is 30%, but 40% in high stage
tumors and only 10% in low stage tumors. Several modali-
ties are available for detecting MYCN duplication, including
FISH, CISH, Southern blotting, and quantitative RT-PCR. In
addition to MYCN amplification, neuroblastomas may display
aneuploidy and/or a complex set of structural chromosomal f7.46  Synovial sarcoma
a & b Monophasic synovial sarcomas
anomalies. Abnormalities of 17q23 are present in >50% of c & d Biphasic synovial sarcomas
cases, deletion of 1p36 in 30 - 40%, and deletion of 11q23 in e Immunohistochemical expression of TLE1
40 - 50%. The 1p deletion is associated with poor prognosis. The
DNA content (ploidy) of the tumor has been shown to correlate
with outcome, particularly in infants. Those with hyperdip-
contains a single primer to both PAX3 and PAX7 conserved
loid tumors (DNA index >1.0) have a more favorable outcome
sequence, it is important to identify the PAX fusion gene.
(similar to what is seen in acute lymphoblastic lymphoma).
Tumors associated with the PAX3 translocation are associated
with a very poor prognosis (<10% survival at 4 years), while
Rhabdomyosarcoma tumors harboring PAX7 have a relatively favorable prognosis.
While there are several subtypes of rhabdomyosarcoma,
the only one that is consistently associated with a chromo-
Synovial sarcoma
somal rearrangement is alveolar rhabdomyosarcoma (ARMS),
Synovial sarcoma f7.46 is a tumor with a predilection for
in which is found the PAX-FOXO1 (formerly FKHR) fusion.
young adults and adolescents. Typically arising within the
Approximately 60% of cases are associated with t(2;13)(q35;q14)
para-articular deep soft tissue in an extremity, these tumors
which fuses PAX3 with FOXO1, while ~20% of cases are associ-
can be either monophasic or biphasic. Monophasic tumors are
ated with t(1;13)(p36;q14), fusing PAX7 with FOXO1. Similar to
highly cellular spindle cell lesions without the characteristic
EWS-FLI, the fusion protein in ARMS joins the DNA binding
herringbone features of fibrosarcoma. Biphasic tumors are
region of the PAX genes with the transcriptional activa-
less common and in addition to a spindle cell component also
tion region of FOXO1. The breakpoints in the translocations
have an epithelial component composed of glands or papil-
associated with alveolar rhabdomyosarcoma are consistent,
lary structures. In 90% of synovial sarcomas is associated the
involving intron 7 of PAX3 or PAX7 with intron 1 of FOXO1.
characteristic t(X;18)(p11;q11) translocation. Most commonly
Amplification of the PAX7-FOXO1 fusion gene is seen in a
the translocation is not the sole abnormality and aneuploidies
majority of cases with the t(1;13) translocation and to a much
of 3–, 7+, 8+, and 12+ have been described. The genes involved
lesser extent with the PAX3-FOXO1 fusion from t(2;13).
are the SSX genes (predominantly SSX1, SSX2, and SSX4) on
Detection of the fusion gene for diagnosis is limited by
the X chromosome and SS18 (also known as SYT or SSXT)
the sensitivity of RT-PCR for nucleic acid isolated from fixed
on chromosome 18. The most common fusion, SS18-SSX1
tissue (~50%) and the relative high incidence (up to 20%) of
accounts for 2/3 of cases, while SS18-SSX2 accounts for most
tumors that fail to demonstrate the presence of a translocation.
of the remaining cases. Rare case reports exist describing the
Detection of the translocation, however, is very specific as it
SS18-SSX4 fusion. TLE1 immunohistochemistry, which shows
has not been demonstrated in any other tumor type. Despite
strong nuclear reactivity in synovial sarcoma, can act as a sur-
the ability to detect both fusion genes with a primer set that
rogate marker, though TLE1 expression is not entirely specific.

ISBN 978-089189-5985 411

7:  Molecular Pathology
Low grade fibromyxoid sarcoma (LGFMS)/hyalinizing
spindle tumor with giant rosettes
These tumors f7.47 are now considered extremes of the mor-
phologic spectrum of tumors with the translocation t(7;16)
(q34;p11). The translocation produces a fusion gene of the 5′
transcriptional activation domain of TLS with the 3′ DNA
binding and leucine zipper region of the CREB3L2 gene.
Another related translocation t(11;16)(p11;p11) of TLS with
CREB3L1 has to be found in a small minority of cases.
Tumors of adipocytes
Most lipomas are associated with complex cytogenetic
anomalies, often involving high mobility group protein genes,
such as HMGA2 on chromosome 12q13-q15. The most common
translocation is t(3;12)(q29;q15) fusing the genes HMGA2 and
LPP. Rearrangements of the chromosome 8q12 region with the
PLAG2 gene underlie a significant portion of lipoblastomas.
An additional mechanism for PLAG2 overexpression occurs in
~20% of lipoblastomas with chromosome 8 polysomy.
Well differentiated liposarcoma and atypical lipomatous
tumor involve alterations of the region 12q14-q15 and take the
form of supernumerary ring chromosomes, translocations, or
giant marker chromosomes with amplifications.
The most common rearrangement observed in myxoid
and round cell liposarcoma is t(12;16)(q13;p11) which makes
a fusion gene of TLS (previously called FUS) on 16p11 with
DDIT3 (previously known as CHOP or GADD153) on 12q13.
Less common is the translocation t(12;22)(q13;q12) which fuses
DDIT3 with the promiscuous EWS.
Osteochondromatosis
Osteochondromatosis is an autosomal dominant condition
with a hereditary predisposition to the formation of multiple
bony exostoses (osteochondromas), differing from solitary spo-
radic osteochondromas in their location and malignant poten-
tial. In patients with osteochondromatosis, osteochondromas
develop on the femur in 70% of cases. While this site is typical
for sporadic osteochondromas, syndromic lesions tend to be
bilateral and symmetrical. Furthermore, osteochondromas are
found in uncommon sites such as the forearm, scapula, pelvis,
vertebrae, phalanges, wrist, and ankle. Sarcomatous change
occurs in ~1% of cases. Other frequent bony lesions include
short stature, bone deformities, and premature osteoarthritis.
The most common mutation associated with osteochondroma-
tosis affects the gene EXT1 on chromosome 8q (70%), with the
remaining cases attributed to the EXT2 gene on 11p.
Head & neck tumors
Squamous cell carcinoma
More than 95% of head and neck malignancies are squa-
mous cell carcinomas. The risk factors for head and neck
squamous cell carcinoma have traditionally been tobacco and
alcohol. Increasingly, human papillomavirus (HPV) is being
recognized as causing a subset of head and neck squamous
cell carcinomas. HPV-driven tumors often arise in younger
patients who are nonsmokers, and their behavior is more indo-
lent. HPV status and smoking history may in fact be stronger
predictors of long term outcome than traditional TNM staging.
As rates of smoking have been decreasing, HPV-associated
412 Practical Clinical Pathology
a b c
f7.47  Low grade fibromyxoid sarcoma (LGFMS)—hyalinizing spindle tumor with giant rosettes
(HSTGR) spectrum tumors
a Tumor with predominantly LGFMS features
b Tumor showing intermediate features
c Tumor with predominantly HSTGR features
carcinoma has become relatively more prevalent. High risk
serotypes, especially HPV 16, are typically involved.
In smoking-associated squamous cell carcinoma, the most
common molecular anomalies are found in the tumor sup-
pressor genes, including loss of genetic material in 3p, 9p, and
17p (TP53 locus). Overexpression of p53 by immunohistochem-
istry is associated with poor prognosis. EGFR is overexpressed
in the majority of tobacco related head and neck squamous cell
carcinoma; however, EGFR genetic anomalies are uncommon.
Neither molecular nor immunohistochemical assessment of
EGFR status correlates with response to anti‑EGFR therapy.
HPV-associated squamous cell carcinoma (HPV-SCC) is
usually found in the oropharynx, including base of tongue
and tonsils. Up to 70% of squamous cell carcinomas in these
sites are positive for HPV. Tumors of the oral tongue have a
low incidence of HPV. HPV-SCC is significantly more likely
to have nonkeratinizing and basaloid histology. As in cervical
HPV-driven SCC, integration of portions of the HPV genome,
including HPVE6 and HPVE7, leads to suppression of p53 and
Rb and overexpression of p16. TP53 is un-mutated and not over-
expressed by immunohistochemistry. Immunohistochemistry
for p16 is commonly used as a surrogate for HPV infection.
Up to 15% of p16+ tumors show no evidence of HPV infection
by in situ hybridization. Some studies have shown that these
apparently HPV–/p16+ tumors behave similarly to HPV+/
p16+ tumors. P16 overexpression appears to be a more useful
marker than HPV in situ hybridization.
Salivary gland tumors
The most common malignant tumor of the salivary glands
is mucoepidermoid carcinoma (MEC). The translocation
t(11;19)(q21;p13) fusing the transcriptional activators MECT2
on chromosome 19 to MAML2 on chromosome 11. This trans-
location can be found by cytogenetic analysis in ~1/2 of cases,
especially the low and intermediate-grade tumors. RT-PCR
detection is more sensitive and can detect the translocation
in almost 90% of cases. The presence of the translocation in
Warthin tumors is controversial.
The most common benign salivary gland tumor is pleomor-
phic adenoma (PA). The majority of cases have demonstrable
karyotypic abnormalities, most commonly rearrangements
of 8q12 involving the gene PLAG1. Translocation t(3;8) of
PLAG1 to the β-catenin gene, CTNNB1, is the most common,
followed by the translocation t(5;8) of PLAG1 to LIFR, the
leukemia inhibitory factor receptor. The end result of these
 7:  Molecular Pathology

translocations is the overexpression of PLAG1. Some tumors

demonstrate PLAG1 overexpression in the absence of demon- a b
strable chromosomal abnormalities. The second most common
gene involved with PA is HMGA2 on chromosome 12q15. It is
most commonly rearranged as t(3;12), resulting in fusion of
HMGA2 with FHIT (fragile histidine triad).
NUT midline carcinoma is a tumor uniquely defined by its
molecular features. Rearrangements in the NUT (nuclear pro-
tein in testis) gene on chromosome 15q14 are definitive for the
tumor, as demonstrated by either RT-PCR or FISH. Most cases f7.48  Medullary carcinoma of thyroid
exhibit the translocation t(15;19)(q14;p13.1), fusing the NUT
gene to BRD4. Because of the reliance on the molecular fea-
tures for accurate diagnosis the tumor is often misdiagnosed.
Most often they are poorly differentiated tumors with abrupt a b
keratinization that are easily confused with other small blue
cell tumors. Currently, promising results are being seen with
immunohistochemistry using a NUT-specific antibody, which
may facilitate accurate diagnosis.

Thyroid
Familial thyroid carcinoma
The cardinal manifestations of multiple endocrine neoplasia
type 1 (MEN 1; Wermer syndrome) are parathyroid adenomas, c d
pituitary adenomas, and pancreatic islet cell tumors. The most
common and earliest manifestation is usually primary hyper-
parathyroidism, caused by a PTH-secreting pituitary adenoma.
Most of the pituitary tumors are prolactinomas. Pancreatic tumors
often produce gastrin (gastrinomas) or insulin (insulinomas).
Nonendocrine lesions in MEN1 include facial angiofibromas,
collagenomas, lipomas, and meningiomas. A predisposition
exists to endocrine tumors in the lung, adrenal cortex, thymus,
and gastrointestinal tract. The MEN1 gene on chromosome e f
11q13 encodes the protein menin and 90% of the time a germline
MEN1 mutation can be found in patients with MEN 1 syndrome.
Somatic MEN1 mutations are found in 15 - 20% of sporadic para-
thyroid adenomas, islet cell tumors, and gastrinomas.
Mutations in the RET protooncogene on chromosome
10q are a feature common to multiple endocrine neoplasia
type 2A (MEN2A; Sipple syndrome), MEN2B, and familial
medullary thyroid carcinoma (FMTC). The 3 syndromes—
f7.49  Papillary carcinoma
MEN2A, MEN2B, and FMTC—have some things in common, a Classic papillary carcinoma, papillary variant
all imparting a high risk for medullary thyroid carcinoma, b Tall cell variant
all autosomal dominant, and all due to a RET gene muta- c Columnar variant
d-f Follicular variant
tion. These features alone comprise FMTC. These features
plus pheochromocytoma and parathyroid adenoma comprise
the MEN2A syndrome; and these plus pheochromocytoma,
mucosal neuromas, ganglioneuromatous intestinal polyps, 40% of sporadic papillary thyroid carcinomas have somatic
and Marfanoid body habitus comprise MEN2B. While the mutations in RET.
histology of medullary thyroid carcinomas f7.48 is not par-
ticularly distinctive in these syndromes, the appearance of Papillary thyroid carcinoma
the background thyroid is: C-cell hyperplasia and numerous Papillary thyroid carcinoma (PTC) is the most common spo-
microscopic foci of medullary carcinoma. MEN2A is caused radic cancer of the thyroid, accounting for >75% of all thyroid
by mutations affecting exons 10 ‑ 11 of the RET gene, most often malignancies. Mutations in BRAF, RET, and RAS, all of which
affecting a particular cysteine residue (634 Cys). MEN2B is are capable of causing unregulated MAPK stimulation, have
most often due to a RET mutation affecting exon 16, encoding been found in PTC in mutually exclusive distribution.
the tyrosine kinase domain. FMTC is caused by RET muta- The most common mutation found in PTC is the BRAF
tions affecting other cysteine residues, particularly 609, 611, V600E mutation, which is seen in almost 1/2 of cases. BRAF
618, and 620 Cys. Mutations of the RET gene are associated mutation is found with highest frequency in conventional
with Hirschsprung disease (often gain of function), and (60%) and tall cell (80%) variants of PTC f7.49, and it is found
almost 1/2 of sporadic papillary thyroid carcinoma. About
ISBN 978-089189-5985 413
7:  Molecular Pathology
in only 10% of follicular variant. Several studies have dem-
onstrated that the accuracy of thyroid FNA diagnosis can be
significantly improved by adding BRAF testing. In this setting,
the BRAF mutation appears to have a specificity of >99% for
PTC. Lastly, BRAF mutation serves as a prognostic marker,
with BRAF positive PTCs demonstrating more aggressive
behavior.
There are several structural chromosomal rearrangement
involving the RET gene that result in marked RET overex-
pression. Collectively, these rearrangements are denoted RET/
PTC, but individual rearrangement partners are termed PTC1
through PTC11. The vast majority of PTCs harboring such a
rearrangement have either RET/PTC1 or RET/PTC3, with PTC1
being the CCDC6 gene and PTC3 the NCOA4 gene. Nonclonal
RET/PTC rearrangements can be found in benign thyroid
tissue, and clonal rearrangements are found in 20  - 
30% of
PTCs, being more common in radiation-associated PTC and in
PTC arising in children. Tumors with RET/PTC have classic
architecture and a high rate of lymph node metastases.
Nearly all PTCs with RAS mutations have follicular archi-
tecture (follicular variant of PTC). Interestingly, nearly 1/2 of
follicular carcinomas also have RAS mutations, as do 1/3 of
follicular adenomas. RAS mutations appear to correlate with
metastatic potential, especially to bone.
Skin tumors
Basal cell carcinoma (BCC)
BCC, the most common cutaneous malignancy, often
appears on sun-damaged skin in adults. Several inherited
disorders are associated with an increased risk of basal
cell carcinoma. Gorlin syndrome (nevoid basal cell carci-
noma syndrome) is due to germline mutations in the PTCH
gene. The protein product of the PTCH gene is involved in
sonic-hedgehog (SHH) signaling, functioning as an inhibitor
of signal transduction. Mutations in PTCH lead to SHH-
independent signaling. PTCH mutations are found in a high
proportion of sporadic BCC.
Xeroderma pigmentosa-affected individuals are prone to
several different types of cutaneous malignancies, including
BCC and squamous cell carcinoma.
Melanoma
Less common than either squamous cell carcinoma or basal
cell carcinoma but accounting for far more morbidity and
mortality than the 2 combined, melanoma is one of the worst
human cancers. Emerging molecular data suggests that the
entity we had been referring to as melanoma may actually rep-
resent a class of tumors sharing similar morphologic features.
It is difficult to predict behavior of the tumors as metastatic
disease can occur with even thin melanomas. Even molecular
characterization of melanoma is fraught with difficulty as
many of the changes seen in melanoma, such as BRAF muta-
tions, are present in benign melanocytic nevi.
Increased risk for the development of cutaneous mela-
noma can be due to several factors—fair skin and a tendency
to freckle, having multiple nevi, environmental exposure
(deep sunburns), and familial predisposition. The latter is a
feature of several familial melanoma syndromes, including
familial atypical mole and melanoma (FAMM) syndrome. It is
414 Practical Clinical Pathology
estimated that ~10% of melanoma is due to a familial predispo-
sition. Dysregulation of p16, the product of the gene CDKN2A
(cyclin-dependent kinase inhibitor 2A) underlies FAMM.
Another form of familial melanoma is based upon defects in
the CDK4 gene on chromosome 12q14, which functions as an
oncogene in the retinoblastoma pathway.
In sporadic melanoma, comparative genomic hybridization
(CGH) studies have elucidated certain reproducible chromo-
somal gains and losses that may help to differentiate Spitz nevi
and other ambiguous melanocytic tumors from melanomas,
while also demonstrating that different types of melanomas
have different genetic anomalies with which they are often
associated. Based on the array CGH findings a refined set of 4
FISH probes has shown initially promising results in differen-
tiation of melanocytic.
Approximately 1/3 of mucosal and acral melanomas are
associated with mutations in C-KIT. This presents an attractive
therapeutic target since C-KIT-mutation tumors may respond
to the tyrosine kinase inhibitor imatinib. Additionally, strong
immunohistochemical staining for c-kit has been shown to
have a close association with mutation status, opening a poten-
tial screening test for C-KIT mutation analysis.
Mutations in BRAF are more closely associated with cuta-
neous melanoma. Specifically, BRAF mutations (pV600E pre-
dominantly) are common in cutaneous melanomas that are
not associated with sun damage. The BRAF pV600E mutation
results in a constitutively active protein that sends proliferative
signals to the nucleus in the absence of mitogen activation.
Successes in the treatment of metastatic melanoma with a
specific small molecule inhibitor of BRAF kinase herald a new
era in therapy.
An additional issue with molecular testing in melanoma
is lymph node evaluation. As lymph node status is a major
determinant in prognosis, accurate assessment for the pres-
ence of melanoma is critical. With current histomorphologic
and immunohistochemistry techniques it is clear that some
potentially microscopically-positive lymph nodes are going
undetected because a significant number of node-negative
cases develop recurrent disease. Specific mRNA markers for
the detection of melanoma have been developed for paraffin-
embedded tissue and show some promise in detecting occult
metastatic melanoma in these lymph nodes.
Dermatofibrosarcoma protuberans (DFSP)
Like many other soft tissue tumors, DFSP is associated
with a characteristic chromosomal translocation. Fusion of the
type I collagen α-1 chain gene, COL1A1, to the platelet derived
growth factor β-chain gene, PDGFB, either via linear translo-
cation (most common) or supernumerary ring chromosome
formation is the hallmark of DFSP. While there are several
specific breakpoints leading to translocation heterogeneity,
the fusion gene is present in almost all cases of DFSP. In addi-
tion, the same translocation is seen in the pediatric giant cell
fibroblastoma tumor, providing evidence that in fact that DFSP
and giant cell fibroblastoma are the same tumor with different
ages of presentation.
 7:  Molecular Pathology

Central nervous system tumors a b

Gliomas
Diffuse gliomas (astrocytoma & oligodendroglioma)
The most common primary neoplasms of the central ner-
vous system (CNS) are the diffuse gliomas (astrocytoma and
oligodendroglioma). Diffuse gliomas are categorized by the
WHO into grades II through IV, with grade I lesions being
benign well-circumscribed tumors. Grade IV glioma, also
called glioblastoma multiforme (GBM), may arise de novo (pri-
mary GBM) or from preexisting grade II/III glioma (secondary c d
GBM). Primary GBM is a rapidly progressive tumor that gen-
erally arises in older patients, while secondary GBM arises in
younger patients and has a somewhat more protracted clinical
course. The accumulated evidence suggests that a small
number of genetic events gives rise to grade II glioma, and a
series of additional genetic events accrues in the progression
from grade II to grade IV glioma.
Early events in tumorigenesis include mutations in the
f7.50  Oligodendroglioma FISH; in a & b there is a red marker for 1p & a green marker for 1q
IDH1 and/or IDH2 genes. IDH mutations are rare in tumors a There is a normal 2 red, 2 green (2R2G) signal, indicative of retention of 1p
outside the CNS and, importantly, not found in benign CNS b There is only 1 red signal (1R2G) indicating 1p deletion
conditions such as reactive gliosis. Direct (Sanger) sequence Likewise, in c & d red (19q) & green (19p) markers are used to assess the status of 19q, with
c a normal result & d 19q deletion
analysis is generally required to detect IDH mutations, but
recently a promising new immunohistochemical marker has
become available. A divergence in paths occurs somewhat 1p and 19q are very specific for oligodendroglioma f7.50. The
later, with tumors acquiring 17p13 (TP53) anomalies differen- frequency 1p/19q co-deletion is up to 90% in grade II oligoden-
tiating into grade II astrocytomas, and those developing 1p drogliomas, 60% in grade III (anaplastic) oligodendrogliomas,
and/or 19q deletions differentiating into grade II oligodendro- and up to 50% in mixed oligoastrocytomas. The presence of
glioma. Note that both types of glioma retain their original the combined loss of 1p/19q is important, as these tumors tend
IDH mutations. Additional anomalies are found in grade III to respond better to chemotherapy (including temazolamide)
lesions, including deletion of 9p and CDKN2. Lastly, progres- and have an overall better prognosis than oligodendrogliomas
sion to grade IV glioma (secondary GBM) is usually marked by (and astrocytomas) lacking these genetic anomalies. PCR-
the loss of material from 10q. As might be expected, primary based assays for microsatellite regions within 1p and 19q or
GBM often lacks many of these early abnormalities, demon- locus-specific FISH probes can be used to assess for loss of
strating instead amplification of EGFR, mutation of PTEN, and heterozygosity.
loss of 10q.
About 1/3 of astrocytomas harbor mutations in TP53.
t7.17  Oligodendroglioma vs astrocytoma
TP53 mutations are rare in oligodendrogliomas. As expected,
Li-Fraumeni syndrome (germline TP53 mutation) is associated Oligodendroglioma Astrocytoma
with the development of astrocytoma. Immunohistochemical Imaging Peripheral (cortical), well demarcated, Central (subcortical), infiltrative
calcified
evidence of p53 nuclear accumulation correlates well with TP53 Histology Round, regular nuclei Elongated, irregular nuclei
mutation status. Furthermore, TP53 mutations are operant in Paucity of glial processes Abundant glial processes
the development of secondary GBM but are not found com- Perineural satellitosis
monly in secondary GBM in which EGFR amplification is Microcysts filled with mucin
found. EGFR amplification and TP53 mutations rarely co-exist WHO types Grade II (oligodendroglioma) Grade II (low grade astrocytoma)
Grade III (anaplastic Grade III (anaplastic astrocytoma)
in these tumors. EGFR immunohistochemistry is an effective
oligodendroglioma) Grade IV (GBM)
means of assessing for EGFR mutation status. EGFR mutated IHC Not generally useful, although astrocytomas are more likely to express strong
tumors display aggressive behavior, are associated with a GFAP & p53, with considerable overlap
small cell phenotype, but respond to small molecule EGFR Genetics Loss of 1p in 80% Loss of 1p in 30 - 40%
kinase inhibitors. (by LOH or FISH) Loss of 19q in 80% Loss of 19q in 10 - 15%
Because of their infiltrative nature, surgical cure is often dif- Loss of 1p & 19q in 60 - 80% Loss of 1p & 19q in 5%
Losses in 9p & 10q increase with grade Losses in 9p & 10q increase with grade
ficult to achieve. Adjuvant chemotherapy and radiation therapy No EGFR amplification EGFR amplification in high grade
have an important role, and in general oligodendrogliomas astrocytomas (esp GBM)
respond better to adjuvant therapy. Therefore it is important to Prognosis Better, with combined loss of 1p & 19q Worse, with unclear significance of
distinguish between astrocytoma and oligodendroglioma t7.17. associated with chemosensitivity combined loss of 1p & 19q cases
While many oligodendrogliomas are quite straightforward,
presenting as a proliferation of cells with rounded nuclear The MGMT (O6-methylguanine-DNA methyltransferase)
contours and perinuclear halos within a background of arbo- gene, which is involved in DNA repair, plays an interesting
rizing “chicken-wire” capillaries, a significant number of cases role in glioma management. In some but not all gliomas, the
have borderline or mixed features. Fortunately, the loss of the MGMT gene undergoes epigenetic silencing through promoter
hypermethylation. The status of the MGMT gene profoundly
ISBN 978-089189-5985 415
7:  Molecular Pathology
impacts clinical response to treatment with temozolomide and
radiotherapy; that is, an unsuppressed MGMT gene can miti-
gate the DNA-damaging effect of combined therapy, thus res-
cuing the tumor cells from harm. The mechanism of action of
temozolomide, an alkylating agent, is the addition of a methyl
group to the O6-position of nucleotide guanine residues,
which results in DNA damage; the MGMT protein is capable
of repairing this damage. In 2006, Hegi et al reported that,
in GBM patients treated with temozolomide and XRT, 49%
of those with methylated MGMT were alive at 2 years, com-
pared to 15% of those with unmethylated MGMT. The status
of the MGMT gene can be assessed by a variety of methods,
including methylation-specific PCR.
Pilocytic astrocytoma
The BRAF gene, encoding the MAPK pathway protein RAF,
is involved in tumorigenesis throughout the body. In many
tumors, BRAF activation arises as a result of a point muta-
tion, most commonly the BRAF V600E mutation. In pilocytic
astrocytoma, BRAF mutation appears to be the sole inciting
genetic event. Both BRAF point mutations and BRAF gene
duplication have been found. BRAF anomalies are found in up
to 80% of pilocytic astrocytomas, but they are rare in diffuse
astrocytomas.
Retinoblastoma
Retinoblastoma is a rare tumor closely associated with
mutations of the RB1 gene on chromosome 13q14. Over 90% of
cases of retinoblastoma are sporadic, the remainder are associ-
ated with an inherited defect in one copy of the RB1 gene. The
presence of bilateral retinoblastomas in a young patient should
raise suspicion of an inherited cause. Patients with inherited
mutations in RB1 are at high risk for later development of
osteosarcoma, pineal gland tumors, and primitive neuroecto-
dermal tumors (PNETs).
Meningioma
Monosomy of chromosome 22 is the most common abnor-
mality found in meningiomas, and this was one of the first
chromosomal rearrangements described in solid tumors.
The key region appears to be 22q12.2 where the NF2 gene is
located. Recall that meningioma is one of the features of the
NF2 syndrome (caused by germline NF2 mutations). Merlin is
the protein encoded by the NF2 gene. Merlin is found in the
cell membrane where its function is to mediate cell-cell contact
and cell contact inhibition.
The frequency of NF2 anomalies varies from one histologic
variant to the next. NF2 abnormalities are found in 80% of
transitional and fibroblastic meningiomas but in only 25% of
meningothelial meningiomas and in virtually no secretory
meningiomas. The overall rate is ~50%.
NF2 anomalies are an early event in tumorigenesis and
that additional molecular defects underlie tumor progres-
sion. Higher grade (atypical and anaplastic) meningiomas are
found to have NF2 abnormalities at roughly the same rate as
low grade meningiomas, but have additional complex anoma-
lies, including numerous genetic gains and losses. The losses
involve 1p, 9p, 14, and others, and the gains include 1q, 9q,
and others. In fact, del 1p is the second most common chro-
mosomal abnormality found in meningiomas. Deletion of 14
416 Practical Clinical Pathology
(monosomy 14) and/or deletion of 9p21 have been associated
with shortened survival.
Particular histologic subtypes are thought to have a higher
rate of progression, including clear cell, chordoid, rhabdoid,
and papillary. With regard to the more aggressive subtypes,
only chordoid meningioma so far appears to have a molec-
ular signature. An unbalanced translocation—der(1)t(1;3)
(p12 - 13;q11)—has been found in this variant and preliminarily
promises to be a specific marker.
Embryonal tumors
Embryonal tumors belong to a group defined by the pres-
ence of undifferentiated-appearing small blue cells, which
account for the majority of primary pediatric brain tumors. 3
of the embryonal tumors—medulloblastoma, primitive neu-
roectodermal tumor, and atypical teratoid/rhabdoid tumor—
have characteristic molecular alterations.
Medulloblastoma is a tumor of the posterior fossa affecting
the cerebellum with a tendency to metastasize within the CNS.
There are several subtypes with varying histologic appear-
ances and prognoses. Certain inherited tumor predisposition
conditions are associated with a higher risk of developing
medulloblastoma, including Turcot syndrome secondary to
mutations in APC, Gorlin syndrome, and Li-Fraumeni syn-
drome. In sporadic medulloblastoma, the most common aber-
ration, seen in 1/2 of tumors, is isochromosome 17q. The
alteration most strongly associated with prognosis is loss
of chromosome 17p, present in 1/3 of tumors. Loss of 17p is
associated with more aggressive tumor behavior, shortened
survival, and a relatively poor response to chemotherapy.
Atypical teratoid/rhabdoid tumor (AT/RT) is a particularly
aggressive pediatric CNS tumor that usually affects the very
young (<2 years old). AT/RT is prone to extensive metastatic
disease and lack of response to chemotherapy. Loss of the
SMARCB1 gene on chromosome 22q11.2, which encodes INI1,
is found in majority of AT/RTs. The INI1 protein is a compo-
nent of a SWI/SNF chromatin remodeling complex involved
in DNA replication and transcription. Loss of INI1 enables
confident distinction from other related and similar-appearing
tumors, such as medulloblastoma or PNET, as this mutation
is not frequently seen in either. Immunohistochemistry for
INI1 is available, as are FISH probes for the affected region on
chromosome 22.
Pulmonary tumors
Lung carcinoma can be divided histologically into 2 major
categories: small cell lung cancer (SCLC) and for lack of a
better name, non-small cell lung cancer (NSCLC). The latter
group encompasses the majority of lung tumors and includes
adenocarcinoma and squamous cell carcinoma. For many
years there was little or no clinical benefit obtained from
efforts to distinguish among non-small cell carcinoma types.
Studies published in 2004 and 2006 changed this, as it became
clear that patients with squamous cell carcinoma might suffer
fatal hemorrhagic complications if treated with antiangiogen-
esis agents such as bevacizumab (Avastin). As a result of this
finding, it became critical to distinguish squamous cell carci-
noma from other varieties of non-small cell lung cancer. For
poorly differentiated tumors, an immunohistochemical panel

can be helpful in this regard; eg, a panel consisting of CK5/6
and P63 (both positive in squamous cell carcinoma) vs TTF1
and BerEP4 (both positive in adenocarcinoma).
A demand for even sharper distinction among non-small
cell carcinomas emerged as a result of success with EGFR-
targeted therapy in some patients. In particular, it seems that
adenocarcinomas show the best response to these agents, and
further histologic subtyping of adenocarcinoma can roughly
predict response. Tumors with a lepidic component are likely
to have EGFR-susceptible mutations, as are tumors with pre-
dominantly micropapillary or solid growth patterns. There
appears to be no correlation, positive or negative, with acinar
growth, and tumors with mucinous differentiation and enteric
differentiation are very unlikely to respond.
Mutation analysis is the best predictor of response. 3 genes
in particular have been shown to be frequently mutated in
lung adenocarcinoma—EGFR, KRAS, and ALK—all mutually
exclusive of one another.
EGFR mutations in this setting most commonly affect exons
18 through 21, which encodes a portion of the tyrosine kinase
domain. In particular, one common mutation in exon 21 con-
sists of a leucine to arginine substitution at amino acid 858
(L858R). Also frequent is a deletion in exon 19. These anoma-
lies have the result of constitutive activation and signaling.
Adenocarcinomas with these mutations are most commonly
seen in patients with specific demographic features—young
Asian females and/or never-smokers. Anti‑EGFR agents come
in 2 varieties, including small molecular tyrosine kinase
inhibitors (EGFR-TKIs) such as gefitinib (Iressa) and erlotinib
(Tarceva), and anti‑EGFR monoclonal antibodies such as cetux-
imab (Erbitux). Tumors with mutations in exons 18 through
21 have a high likelihood of response to EGFR-TKIs but are
unlikely to respond to anti‑EGFR monoclonal antibodies.
EGFR status by immunohistochemistry and FISH has not reli-
ably predicted response, and at this time mutation analysis
by PCR is recommended, targeting exons 18 - 21. As discussed
below, mutations in the genes encoding downstream pro-
teins in the EGFR signaling cascade (KRAS, BRAF) are found
exclusively in tumors that lack EGFR mutations. Lastly, some
patients develop secondary resistance to anti‑EGFR therapy.
Secondary resistance is associated with certain additional
mutations in EGFR (T790M) as well as mutations in other
genes such as MET.
KRAS mutations can lead to signaling downstream and
independent of EGFR. There is a very high KRAS mutation
rate in mucinous adenocarcinoma (75%) as compared to non-
mucinous adenocarcinoma (45%). Since they are mutually
exclusive, the presence of KRAS mutations would theoretically
be associated with nonresponsiveness to anti-EGFR therapy
(as is the case in colon cancer); on the contrary, this has not
consistently proven to be the case. While KRAS mutations do
appear to predict lack of response to EGFR-TKIs (gefitinib,
erlotinib), there is no correlation with response to anti‑EGFR
monoclonal antibodies (cetuximab).
The small percentage of lung adenocarcinomas not associ-
ated with EGFR or KRAS mutations may have mutations in
the anaplastic lymphoma kinase gene, ALK. While uncommon
in lung cancer overall (1 - 5%), ALK rearrangements have been
described in up to 20% of high-stage tumors. ALK, another
receptor tyrosine kinase, was originally identified as part of the
ISBN 978-089189-5985
7:  Molecular Pathology
t(2;5)(p23;35) translocation in anaplastic large cell lymphomas
and subsequently shown to be rearranged in inflammatory
myofibroblastic tumor. Oncogenic ALK fusion genes are ones
that result in overexpression and constitutive activation ALK.
In non-small cell lung cancer, the EML4-ALK translocation
(and less commonly, TFG-ALK and KIF5-ALK) causes the for-
mation of a fusion gene of the transcription activation domain
of ALK with the dimerization domain of EML4, leading to
constitutive activation. The translocation is the result of an
interstitial inversion in the short arm of chromosome 2. So far,
it appears that IHC correlates poorly with response to small-
molecule ALK-TKIs, and FISH is recommended.
Reduced expression of the miRNA, let-7 is a stage-indepen-
dent poor prognostic finding. It is hypothesized that one of
the targets of let-7-mediated control of gene expression, RAS
may be the effector molecule for this poor prognosis. Other
miRNAs, including the miR-17 ‑  92 cluster and miR-31 act
as oncogenes and have been demonstrated to be aberrantly
expressed in a number of non-small cell lung cancer cases.
Gynecologic tumors
Inherited gynecologic tumor syndromes
Most of the cases of inherited predisposition to gynecologic
tumors are due to germline mutations in mismatch repair
proteins (Lynch syndrome) or mutations in BRCA1/BRCA2
(hereditary breast and ovarian cancer). Peutz-Jeghers can
present with particular gynecologic manifestations, such as
adenoma malignum of the cervix and ovarian sex cord tumor
with annular tubules (SCTAT).
Lynch syndrome is caused by an inherited germline muta-
tion in the gene for one of the proteins involved in mismatch
repair, most commonly MLH1, MSH2, or MSH6. Women with
Lynch syndrome have a nearly 50% lifetime risk of endome-
trial cancer (often of the lower uterine segment) and a 10% risk
of an epithelial ovarian cancer. The risk for endometrial cancer
in Lynch syndrome is equivalent to the risk for colorectal car-
cinoma. Ovarian cancer in Lynch syndrome tends to present
at an early age, usually before 50, and is often of the clear cell
type. While most screening guidelines (Bethesda, Amsterdam)
are focused more on colorectal carcinoma, there are also
guidelines proposed by Society of Gynecologic Oncologists
that address screening for an increased risk of endometrial
and ovarian carcinoma. Similar to colorectal carcinoma it has
been proposed that universal screening of all endometrial car-
cinomas for defects in mismatch repair be performed.
Hereditary Breast/Ovarian Cancer (HBOC) is caused by
mutations in the tumor suppressor genes BRCA1 or BRCA2. It
is estimated that a patient with BRCA mutations has a 10-fold
increased risk of developing an epithelial ovarian carcinoma,
and ~10% of ovarian cancers are caused by underlying BRCA
mutations. The majority of high  grade serous ovarian carci-
nomas due to mutations in BRCA also harbor mutations in
P53. These high  grade or type  II serous carcinomas are dif-
ferent from low grade or type I serous carcinoma in behavior,
appearance, and underlying mutations. Type I tumors are less
likely to be associated with mutations in BRCA, instead har-
boring mutations in KRAS or BRAF. Recently it has become
clear that these high  grade tumors in BRCA patients arise
from a precursor lesion in the fallopian tube, serous tubal
417
7:  Molecular Pathology

intraepithelial carcinoma (STIC). STIC lesions often predate
high grade serous tumors and have similar mutations in P53.
Prophylactic bilateral salpingo-oophorectomy is reported to
reduce the risk of developing pelvic cancer by as much as 90%.
Cervix
In the United States, the incidence of invasive cervical
cancer is relatively low due to the success of Pap screening.
Still, in some countries of southern Asia, central America,
and sub-Saharan Africa cervical cancer is still for women the
most common cause of death from cancer. The most common
cause of cervical carcinoma is human papillomavirus (HPV)
infection.
HPV is a double stranded DNA virus with many different
serologic subtypes. These subtypes are grouped according
to their risk for developing squamous carcinoma. The HPV
types that are considered carcinogenic are referred to as high
risk HPV (HRHPV) and include HPV 16, 18, 31, 33, 35, 39, 45,
51, 52, 56, 58, 59, 68, 73, and 82. Others, such as 6 and 11 are
extremely common causes of human infection but do not pose
a great cancer risk. The likelihood of malignant transforma-
tion appears to relate to 2 variables:
1. the ease with which the viral genome becomes integrated
into the host genome
2. the particular gene sequence (alleles) of the viral E6 and
E7 genes
That is, HPV infections that result in benign processes
are usually associated with an episomal viral DNA, whereas
HPV infections that result in malignancy are usually associ-
ated with integration of the viral DNA into the host genome.
Such integration results in unchecked transcription of 2 viral
genes in particular—E6 and E7—that trigger malignant trans-
formation. The E6 and E7 gene products act through several
mechanisms, important among them being the inhibition of
the retinoblastoma (Rb) and p53 tumor suppressor proteins.
The E6 protein is known to bind to the p53 protein, leading
to its subsequent degradation through the ubiquitin pathway.
The E7 protein interacts with the Rb-E2F complex, blocking Rb
inhibition. Uninhibited, E2F can function as a transcriptional
activator of the genes involved in cell cycle progression. This
biphasic attack on the regulatory apparatus of the cell leads
to unregulated cell division. Various serotypes differ in the
effectiveness of their respective E6 and E7 proteins to abrogate
cellular regulatory machinery. A surrogate marker of unregu-
lated cell cycle progression, p16, can be used to detect cells that
may be infected with high risk HPV subtypes.
The approach most widely advocated for HPV screening is
cervical cytology. When a cytologic diagnosis of either low- or
high  grade squamous intraepithelial lesion (LSIL or HSIL)
is rendered, colposcopy is indicated. When a cytologic diag-
nosis of atypical squamous cells (ASC) is rendered, molecular
testing for the presence of high  risk HPV (HRHPV) is indi-
cated, followed by colposcopy and biopsy if this is positive.
In all 3 instances, the purpose of colposcopy is to exclude a
high grade lesion. Several assays are currently available for the
molecular detection of HPV in cervical samples, most of which
are performed on the same liquid-based collection system
used to prepare the slide for cytologic screening. The most
common assay is a solution-phase hybridization using labeled
RNA probes. Successful hybridization of the probes (with any
viral DNA present in the sample) is detected by enzymatic
reaction. This assay may be used to detect HRHPV specifically,
or it may be used to detect low-risk HPV. Such assays have
much higher sensitivity than cytology, but they are positive
in a large number of women with no lesion (low specificity);
hence the recommendation for testing only in the presence of
an atypical cytology.
Endometrium
Type I endometrial adenocarcinomas, including those with
endometrioid, mucinous, and secretory morphology, are often
estrogen-dependent and low  grade. Type  I tumors typically
demonstrate mutations in mismatch repair genes, with associ-
ated microsatellite instability, and mutations in PTEN, KRAS,
and CTNNB1 (β-catenin). Type II tumors, serous papillary and
clear cell carcinoma, are high  grade estrogen unresponsive
tumors. These tumors are more likely to have mutations of
HER2 and P53.
Endometrial stromal tumors arise from the mesenchymal
stroma of the uterus. The translocation t(7;17)(p15;q21) has
been found in a significant number of cases of endometrial
stromal sarcoma (ESS). The translocation creates a chimeric
protein between the JAZF1 gene on chromosome 7 and the
JJAZ1 on chromosome 17. The translocation is found in the
majority of cases of ESS, but has also been found in a number
of endometrial stromal nodules and neoplastic undifferenti-
ated endometrial sarcomas, thereby limiting its diagnostic
specificity for ESS.
Gestational trophoblastic disease
Partial moles are most commonly derived from the fertiliza-
tion of an egg by 2 sperm. Partial mole is triploid (usually 69,
XXY) and may be found in association with a triploid fetus.
Complete moles are derived from the fertilization of a blighted
ovum (one without genetic material), either by a single sperm
followed by duplication of genetic material or by 2 sperm.
Complete moles are diploid, but composed entirely of paternal
genetic information. Historically, morphology and ploidy
analysis has been used to distinguish between the complete
and partial mole. Chromosomal enumeration can distinguish
partial moles from hydropic abortuses. However, hydropic
products of conception can resemble complete molar pregnan-
cies, and both are diploid.
Immunohistochemical stains for a paternally-imprinted
gene, P57 have shown promise as a way of distinguishing com-
plete molar pregnancies. Complete moles show no staining for
P57, while the nuclei of intermediate trophoblastic cells will
stain in a partial mole or hydropic pregnancy. In addition,
STR analysis of potential molar pregnancies has been used
for classification purposes. By comparing the STR haplotype
of the presumed molar pregnancy to maternal tissue or blood
one can determine the origin of the genetic material in the
products of conception. If a diploid conceptus has evidence of
maternal STRs, then one can exclude a complete mole.

418 Practical Clinical Pathology


Other tumor syndromes
Tuberous sclerosis complex (Bourneville syndrome)
Named for 2 cardinal features of the disorder, the tuber-like
lesions of the cerebral cortex and the periventricular calcifica-
tion, tuberous sclerosis presents with a myriad of findings. The
Tuberous Sclerosis Alliance has proposed criteria for the clin-
ical diagnosis of tuberous sclerosis. The major criteria include
facial angiofibroma (“adenoma sebaceum”), subungual or peri-
ungual fibroma (“Koenen tumor”), 3 or more hypomelanotic
macules (“ash leaf spots”), connective tissue nevus (“Shagreen
patch”), retinal hamartomas, cerebral cortical tuber, subepen-
dymal nodule, subependymal giant cell astrocytoma, cardiac
rhabdomyomas, lymphangioleiomyomatosis (LAM) f7.51, and
renal angiomyolipoma. Minor criteria include dental enamel
pits, hamartomatous rectal polyps, bone cysts, cerebral white
matter radial migration lines, gingival fibromas, nonrenal
hamartomas, retinal achromic patch, cutaneous symmetric
hypopigmented macules, and multiple renal cysts.
As is evident from the listed criteria, tuberous sclerosis is
a multisystem disorder with a predilection to development
of mainly benign lesions characterized by a variety of mostly
benign tumors. There is, however, an increased risk of malig-
nant renal neoplasms, including clear cell renal cell carcinoma.
TSC is caused by autosomal dominant inheritance or spo-
radic mutation in 1 of 2 genes. Most (80%) cases are due to
mutations in the TSC1 gene, located on chromosome 9q34,
which encodes the protein hamartin. The remaining cases
are due to mutations in the TSC2 gene, located on chromo-
some 16p13, which encodes the protein tuberin. ~60% of TSC
cases arise sporadically, reflecting the high rate of de  novo
mutations.
Nevoid basal cell carcinoma syndrome
(Gorlin Goltz syndrome)
Nevoid basal cell carcinoma syndrome, as first described by
Gorlin and Goltz, is characterized primarily by odontogenic
keratocysts, multiple basal cell carcinomas, and bifid ribs.
Additional anatomic findings may include calcification of the
falx cerebri, palmoplantar pits, and frontal bossing with coarse
facies. The tumor predisposition also includes a high rate of
medulloblastoma, rhabdomyosarcoma, and meningioma. In a
large study of affected individuals, features present in >1/2 of
patients included palmar pits, odontogenic keratocysts, basal
cell carcinomas, and calcification of the falx cerebri. Anomalies
in the PTCH1 gene on chromosome 9q22.3 underlie nearly all
cases of Gorlin-Goltz syndrome. Mutations in the PTCH1 gene
are inherited in an autosomal dominant manner; penetrance
approaches 100%, but expression is variable.
Neurofibromatosis type 1
(NF1; von Recklinghausen disease)
NF1 is caused by mutations in the NF1 gene encoding
neurofibromin on chromosome 17q11.2. Neurofibromatosis
is inherited in an autosomal dominant fashion, although 1/2
of cases are sporadic. It is most often diagnosed by a constel-
lation of clinical features. These features, codified in an NIH
consensus statement in 1988, include the presence of 6 or more
café au lait spots, 2 or more neurofibromas or any single plexi-
form neurofibroma, groin/axilla freckling (Crowe sign), 2 or
ISBN 978-089189-5985
7:  Molecular Pathology
a b
f7.51  Lymphangiomyomatosis of lung
more ocular Lisch nodules, one of the distinctive bony lesions
(eg, dysplasia of the sphenoid wing), and a first degree affected
relative.
In childhood, NF1 initially presents with multiple café
au lait spots and intertriginous (groin, axilla) freckling.
Neurofibromas typically begin to emerge in adolescence or
early adulthood (diagnosis is often delayed into the second
decade as a result). Many women with NF1 experience a rapid
increase in the number and size of neurofibromas during
pregnancy. Ocular Lisch nodules are fairly common but
innocuous. Inconsistent features include vertebral dysplasia,
sphenoid wing dysplasia, scoliosis, bone cortical thinning,
pseudarthrosis, mental retardation, pulmonic stenosis, and
NF1 vasculopathy. The latter most often manifests as renal
artery stenosis (arterial dysplasia), with associated hyperten-
sion. An appearance resembling Noonan syndrome is seen in
~12% of individuals with NF1.
NF1 results in a predisposition toward certain tumors,
including malignant peripheral nerve sheath tumor (MPNST),
optic glioma, leukemia, medulloblastoma, pheochromocy-
toma, ampullary adenocarcinoma of the small intestine, and
breast cancer. The lifetime risk for MPNST is 10%. Optic
gliomas nearly always take the form of pilocytic astrocytomas
and may cause visual loss.
Most mutations of the NF1 gene result in protein truncation
and loss of function of the NF1 tumor suppressor gene. Most
clinically significant NF1 mutations are nonsense mutations,
missense mutations, or microdeletions that, while distributed
throughout the gene, cause protein truncation. Discrete point
mutations are responsible for only ~10% of cases. Thus, while
there are an enormous variety of mutations, the laboratory can
screen for ~80% of them with protein truncation tests. FISH
can detect the 5 - 10% of cases that are caused by microdele-
tions (conventional cytogenetic testing is not sensitive enough).
Genetic testing is primarily indicated for prenatal testing and
rarely for confirmation in individuals who do not fulfill the
diagnostic criteria.
Neurofibromatosis type 2
(NF2; bilateral acoustic neuroma syndrome)
Mutations in the NF2 gene on chromosome 22q, which
encodes the protein merlin, is the cause of NF2. Up to 1/3
of NF2 cases are sporadic (simplex) cases. Much about the
nomenclature of this syndrome is confusing. NF2 is character-
ized by bilateral vestibular nerve (not acoustic nerve) schwan-
nomas (neither neurofibromas nor neuromas occur), and it
419
7:  Molecular Pathology
has almost no relationship to NF1. The disease first manifests
around the age of 20, and nearly all affected individuals have
bilateral vestibular schwannomas by the age of 30. Early detec-
tion is important, to prevent deafness. Schwannomas may
arise in association with other nerves as well, and there is an
increased tendency to develop meningiomas, ependymomas,
and pilocytic astrocytomas.
Li-Fraumeni syndrome
Mutations in the tumor suppressor gene TP53 (p53) are
found in ~50% of malignancies, regardless of type, and are
the most common genetic anomaly found in malignant
tumors. An inherited (germline) mutation in TP53 causes
Li-Fraumeni syndrome; as might be predicted, Li-Fraumeni is
associated with a tendency to develop numerous widespread
malignancies,
Early reports focused on the association of Li-Fraumeni
syndrome with a number of tumors, including osteosarcoma,
soft tissue sarcoma, breast cancer, adrenal cortical carcinoma,
and acute leukemia. It seems now that there is no limit to the
variety of tumors, however, with well-documented docu-
mented increased risk for gastric adenocarcinoma, colorectal
carcinoma, pancreatic carcinoma, esophageal carcinoma, germ
cell tumors, melanoma, Wilms tumor, and malignancy of the
central nervous system. Perhaps the only characteristic fea-
tures of Li-Fraumeni are (1) that any given tumor type arises
at a younger age than would be expected for a sporadic tumor
of the same type, and (2) that multiple disparate tumors may
arise in the same person. About 40% of those with Li-Fraumeni
have developed a malignancy by the age of 20 years, 60% by
age 40, and 90% by age 60.
Especially concerning for Li-Fraumeni is the appearance
of adrenocortical carcinoma in a young adult; >1/2 such cases
are associated with Li-Fraumeni syndrome. Adrenocortical
carcinoma is rare overall, and they often occur in children
with Li-Fraumeni syndrome. Thus, the occurrence of an adre-
nocortical carcinoma in a child or young adult is sufficient
indication for Li-Fraumeni testing.
About 85% of cases are due to TP53 mutations (chromosome
17p); mutations in the CHEK2 gene (whose product is one of the
intracellular targets of p53) has been identified in a minority of
cases. While there are a variety of TP53 mutations, most arise
between exons 4 through 9 (or amino acids 91 through 309).
This permits good (95%) sensitivity for sequencing analyses
confined to this portion of the gene.
Immunohistochemistry can be performed for p53 expres-
sion. Most mutated forms of p53, though nonfunctional as
tumor suppressor proteins, have a prolonged half life within
the cell (the mutated forms are able to avoid protein degrada-
tion). Thus, tumors with TP53 mutations (and decreased p53
activity) have overexpression of p53.
Aniridia/WAGR syndrome
Aniridia may be a sporadic or inherited trait, usually
occurring as an isolated finding; however, in some it occurs
as part of a Mendelian syndrome that includes Wilms tumor,
aniridia, genitourinary anomalies, and mental retardation
(WAGR syndrome). The WAGR syndrome is a contiguous gene
syndrome in which a microdeletion spanning 11p13 affects
several genes. In ~20 - 30% of cases, the deletion is detectable by
420 Practical Clinical Pathology
high-resolution chromosome studies. FISH studies, utilizing
several probes that span the affected band, can also be diag-
nostically useful.
Beckwith-Wiedemann syndrome
Beckwith-Wiedemann syndrome can be caused by any
one of several defects, the common thread being abnormal
transcription of genes within the 11p15.5 band. 11p normally
is an imprinted domain (expression depends upon whether
inherited from the mother or from the father), one in which
maternally derived alleles are preferentially expressed. Several
genes are located within 11p, including:
1. KCNQ1, encoding a potassium channel, and the
same gene implicated in Romano-Ward and Jervell
Lange-Nielsen syndromes
2. IGF2, encoding an insulin-like growth factor (IGF)
3. H19, encoding a nontranslated mRNA
4. CDKN1C, encoding a cyclin-dependent kinase inhibitor
Embryonal tumors (Wilms tumor and hepatoblastoma, in
particular) occur with a high rate in Beckwith-Wiedemann
syndrome. The syndrome may become apparent in utero, with
a fetus that is large for gestational age (LGA) and has polyhy-
dramnios. At birth, the placenta is large, and the umbilical cord
is abnormally long. Additional features that may be identified
at birth include macrosomia, macroglossia, hemihypertrophy
(asymmetric growth), omphalocele (exomphalos), and anterior
ear creases or pits. A peculiar adrenocortical cytomegaly has
been described in patients with Beckwith-Wiedemann, and
renal anomalies (renal medullary dysplasia, nephrocalci-
nosis, medullary sponge kidney, and nephromegaly) are very
common.
About 80% of cases are inherited, and the remaining are
sporadic (simplex) cases. Beckwith-Wiedemann is primarily
a clinical diagnosis. The molecular diagnosis is problematic,
as no single finding defines it. Conventional cytogenetics
can identify an anomaly at 11p15 in only ~1% of cases. FISH
can identify another 1 - 2%. Methylation assays are capable
of finding abnormalities in >1/2 of patients (either gain or
loss of methylation). Uniparental disomy studies can identify
abnormalities in ~15% of cases. Lastly, a single gene defect in
CDKN1C appears to underlie around 10% of simplex cases and
up to 40% of familial cases.
Chromosomal breakage syndromes
Among this group of disorders, which are usually trans-
mitted in an autosomal recessive fashion, the unifying feature
is a tendency in cell culture to exhibit elevated rates of chro-
mosomal breakage or instability. Underlying this tendency are
defects in DNA repair mechanisms, and the clinical effect is
a predisposition to cancer. Chromosomal breakage disorders
are numerous and include Bloom syndrome, ataxia telangiec-
tasia, Nijmegen syndrome, Fanconi syndrome, and xeroderma
pigmentosa. This group of disorders should be conceptually
distinguished from the trinucleotide repeat disorders associ-
ated with the presence of “fragile sites” which do not cause a
cancer predisposition.
 7:  Molecular Pathology

Xeroderma pigmentosa (XP) is characterized by a 1,000-

fold increased risk of cutaneous malignancy, including basal a b
cell carcinoma, squamous cell carcinoma, and melanoma,
resulting from extreme sensitivity to ultraviolet (UV) light.
Freckling begins by the age of 2 years, and a cutaneous tumor
typically arises by the age of 20 years. XP is caused by auto-
somal recessive inheritance of a mutation in one of the genes
of the nucleotide excision repair complementation groups,
including XPA through XPG, whose function is to repair UV
induced DNA damage. Some forms of XP are associated with
neurologic deficits and others with ocular manifestations.
Affected patients must assiduously avoid UV light exposure.
c d
PTEN-related disorders: Cowden syndrome, Bannayan-
Riley-Ruvalcaba syndrome & Proteus syndrome
This family of disorders is associated with mutations in the
gene PTEN on 10q23. PTEN, which stands for phosphatase and
tensin homolog, encodes a phosphatidylinositol 3,4,5-trispho-
sphate 3-phosphatase negatively regulating a key compound
in the AKT signaling pathway. All the PTEN-related disorders
demonstrate a tendency to form hamartomatous tumors.
In Cowden syndrome these are most commonly hamar- f7.52  Lhermitte-Duclos lesion
tomatous intestinal polyps. In addition there can be multiple a MRI shows cerebellar thickening with enlarged folia & cystic-appearing areas
b-d Histology demonstrates a thickened molecular & internal granular area in which there are nodular
lipomas and fibromas, malformations of the genitourinary collections of dysplastic ganglion cells
tract, and mucocutaneous lesions such as facial trichilem-
momas, papillomas, palmoplantar keratoses, and palmoplantar
hyperkeratotic pits. Microcephaly and mental retardation are
common, and these are strongly associated with cerebellar
dysplastic gangliocytoma (Lhermitte-Duclos lesion) f7.52, which
is considered pathognomonic. Patients with Cowden syn- Carney complex
drome are at an increased risk of carcinoma, including fol- Carney complex is an autosomal dominant condition also
licular carcinoma of the thyroid gland and carcinomas of the known as LAMB syndrome (an acronym denoting lentigines,
breast, colon, and endometrium. atrial myxoma, and blue nevi) or NAME syndrome (nevi, atrial
Bannayan-Riley-Ruvalcaba syndrome has a different pre- myxoma, myxoid neurofibroma, and ephelides). Cutaneous
sentation with macrocephaly and mental retardation, high lentigines (simple lentigos) are the most common presenting
birth weight, myopathy, joint hypermobility, pectus exca- finding, located on the face (particularly the oral and con-
vatum, and scoliosis. Associated tumors include hamartoma- junctival mucosa), vagina, and penis. Blue nevi, are common,
tous intestinal polyps and pigmented macules of the glans particularly the cellular blue nevus. Cardiac myxomas occur
penis. frequently, at a young age, and may affect any chamber. In
As the name suggests, Proteus syndrome has a highly vari- addition to the previously mentioned features, there may also
able presentation and demonstrates a pattern of affected indi- be myxomas of the breast, female genital tract, and skin, as
viduals consistent with mosaicism; ie, some organs are heavily well as endocrine tumors such as follicular adenomas of the
affected whereas others are entirely spared. Some examples thyroid gland, pituitary adenomas, and primary pigmented
of the syndrome manifest connective tissue nevi (considered nodular adrenocortical disease (PPNAD), which presents
pathognomonic), asymmetric limb growth, skull hyperostosis, as Cushing disease. Large  cell calcifying Sertoli cell tumors
megaspondylodysplasia of the vertebrae, or visceral over- arise in most affected males. Note that this unusual tumor
growth (especially of spleen and thymus). is also seen in Peutz-Jeghers syndrome; however, the SCTAT
of Peutz-Jeghers is not seen in females with Carney complex.
Psammomatous melanotic schwannoma, rare as a sporadic
tumor, is common in the Carney complex. Nearly 1/2 the cases
of Carney complex are due to mutations in the PRKAR1A
gene on 17q24. No other genes have yet to be implicated in the
remaining cases. Note that the similarly named Carney triad
is an entirely different syndrome (triad of gastric GIST, pulmo-
nary chondroma, and extraadrenal paraganglioma).

ISBN 978-089189-5985 421