ALL (PAS stain) If you stain lymphoblasts, will be PAS+ and peroxidase negative.

Myeloid Precursors:

Above (right): Immature granulocytes (left shift). If you have lots of granulocytes (in this case, dont see any blasts), do MPO stain will be positive but wont be helpful because you can tell theyre granulocytes by looking at them. Need to do leukocyte alkaline phosphatase (LAP) test. If normal granulocytes, test will be positive. If CML, LAP will be negative.

CML see more blasts w/nucleoli.

LAP is + in leukemoid rxn, neg. in CML.

AML: many blasts.

Auer rods = AML. Nothing else makes Auer rods.

Megakaryocytic, erythroid, monocytic series do not make Auer rods. If you see Auer rods, will be AML M2-3-4.

Above (left): More undifferentiated AMLs. M1 wont make Auer rods, M2 might. Above (middle): M3 promyelocytic; see granules; has 15;17 translocation. Above (right): If you stain w/MPO, granules will be positive (generally M2, M3, M4).

Above: Sudan Black stain. Will be + in some AMLs.

Above: Nonspecific esterase. If you go on to the monocytic series, nonspecific esterase is the best marker. These are the ones that involve the gums and oral cavity.

Above: M6 erythroid (RBC). M7 megakaryocytic version.

Case 7. 6 y.o. male with URIs, headache, bone pain and easy bruising; axillary and cervical lymphadenopathy; hepatosplenomegaly. Young kid w/lots of blasts = ALL. Any time there are 40% blasts, is NOT a benign condition is leukemia. CBC: Hgb 9 MCV 82 WBC 92,000 (86% abnormal) Plt 18,000 7a. Diagnosis? A. ALL B. AML C. CLL D. CML

7b. What test results are expected on a bone marrow aspirate? A. MPO (+), PAS (+) B. MPO (-), PAS (-) C. TdT (+), Precursor B cell (+) D. TdT (-), Precursor T cell (+) E. Sudan black B (+); CD 10 (-) C. TdT positive and precursor B cell positive.

Case 8. 15 y.o. male with flulike symptoms; axillary and cervical lymphadenopathy; gums are thick and bleeding. CBC: Hgb 9 MCV 90 WBC 42,000 (88% abnormal) Plt 22,000 BM Bx results: MPO (-) PAS (-) Sudan Black B (-) Non-specific esterase (++) marker for monocytic series. Serum lysozyme 162 (4-15 normal) Lymphomas: Hodgkin RS Cells Often curable Stage important Few malignant cells < cell-mediated Localized radiation Non-Hodgkin Low grade- slowly progressive, rare cure High grade- rapidly progressive; a minority are curable Grade important Mostly lymphoma < humoral Systemic chemotherapy 8. A. B. C. D. E. Diagnosis: AML (FAB M1) AML (FAB M3) AML (FAB M5) ALL (L1) Infectious mononucleosis

One of the lymphomas will be on the glass exam need to recognize Hodgkins, follicular lymphoma, follicular hyperplasia, SLL, and Burkitts. Abdominal lesions will also be on glass slide exam Burkitts, Wilms, or neuroblastoma.

Above (both): Follicular hyperplasia equal in size, rim of blue cells, look in middle see tingible body macrophages. Benign condition due to bacterial infection. Also called adenitis. Bacterial adenitis/lymphoid adenitis = bacterial infection w/hyperplasia of the follicles.

Follicular hyperplasia, lymph node. See mantle zone, dark zone, and light zone. See tingible body macrophages.

Follicular hyperplasia, lymph node. See tingible body macrophages, mantle zone, and lymphoycytes.

Above (both): Follicular lymphoma. Less defined follicles, malignant. No rim of blue cells, no tingible body macrophages in the middle. Lymphocytes are monotonous. No variation, no monocytes.

Follicular lymphoma, lymph node. See lymphocytes.

SLL/CLL, lymph node. See lymphocyte infiltration.

Above (right): SLL/CLL lots of little round blue cells everywhere. In SLL and CLL, these cells are CD5/CD23+. B cell lymphoma that marks w/T cell marker.

SLL/CLL, spleen see lymphocyte infiltration.

SLL/CLL, lymph node. See lymphocytes.

Above (both): Burkitts lymphoma. See tingible body macrophages (stars) and lymphocytes (sky). Have many large malignant lymphoblasts. Tingible body macrophages are present as in follicular hyperplasia, but in this case, they accompany malignant cells. Make sure you can recognize.

Nodular Sclerosis Hodgkin Lymphoma, Lymph Node. See fibrous bands, nodules.

Hodgkin Lymphoma. See eosinophils, lymphocytes, and Hodgkin cells.

Need to be able to recognize Hodgkins disease microscopically and descriptively.

Hodgkin Lymphoma, Spleen. See lymphocytes and Reed-Sternberg cells.

Radiograph of Plasma Cell (Multiple) Myeloma. See punched out lesions.

Above (left): In Hodgkins, mainly see benign inflammatory cells eosinophils, plasma cells, lymphocytes, and an occasional large cell. May see RS classic form. Any time you see a description of mixed inflammatory cells and several large atypical cells w/prominent nucleoli = Hodgkins disease. Above (right): Multiple myeloma. Proliferation of plasma cells punched out lesions in the skull. Will also have light chains in the urine and in the serum.

Plasma cells and bone spicule.

Plasma Cell (Multiple) Myeloma, Bone. See plasma cells.

Microbiology/Infectious Disease Review: Need to know the difference between G(+) and G(-). G(+) have lipoteichoic acid. G(-) have lipid A and lipopolysaccharides that give rise to toxic shock, etc.

Most common bacteria that cause infections:

Peptidoglycan: Sugar + cross-linked peptides Osmotic protection Penicillin and cephalosporin inhibit CW synthesis Lysozyme in tears cleaves peptidoglycan PG is in all bacteria. Is the basis of the mechanism of action of all beta-lactam antibiotics (cephalosporins, penicillin, etc.) When the components of the cell wall mutate, the antibiotics no longer work. Ex: MRSA has MecA mutation that changes bacterial cell wall structure. Beta lactam antibiotics no longer work. Outer membrane of Gram(-): Lipopolysaccharide (LPS) Lipid A induces IL-1 and TNF; also the ENDOTOXIN component Polysaccharide (O-antigens of Gram bacteria) Pili for attachment LPS and Lipid A cause sepsis and shock. In G(+), teochoic acid is most important for attachment to human cells. Capsule: Polysaccharide except B. anthracis which is D-glutamate Inhibits phagocytosis Used to make vaccines (S. pneumoniae pneumovax; H. influenzae Hib vaccine) Highly antigenic Some organisms have a capsule, and all the capsules are polysaccharides except for anthrax (capsule is protein). Encapsulated organisms tend to be more resistant but also more antigenic. Almost all the organisms that we have vaccines for have a capsule.

Glycocalyx: Slimy capsule of Staphylococcus epidermidis coagulase (-) staphylococci) allows adherence to catheters, for example (also artificial joints, heart valves, etc.) Polysaccharide extra layer Spore: Bacillus and Clostridium Resist heat and chemicals Dipicolinic acid Special Stains: Gram Stain- stains many bacteria but NOT Treponema (Dk Field or silver), the Rickettsia, Mycoplasma, Chlamydia, Legionella (silver), Mycobacterium (acid-fast), fungi (GMS silver) and viral inclusions

If organisms are cocci first do catalase test to differentiate between Staph and Strep. If catalase +, then do coagulase test. If coagulase + = Staph aureus. Coagulase is the enzyme that splits fibrinogen fibrin and causes a clot.

For G(-) organisms, one of the important tests is lactose fermentation. If fermenters, they turn pink on the plates. If not, remain colorless. If lactose (-), possibly Salmonella, Shigella, Proteus, Pseudomonas

Obligate aerobes: Oxygen-dependent ATP M. tuberculosis (lung apex in 2ndary TB), Nocardia, Pseudomonas Obligate anaerobes: Oxygen is toxic Clostridium, Bacteroides, Actinomyces One of the key differences between Nocardia and Actinomyces is aerobic vs. anaerobic (both are filamentous). Intracellular microorganisms: Rickettsia, Chlamydia- energy (ATP) parasites Neisseria Mycobacterium tuberculosis Salmonella Listeria, Brucella, Francisella, Yersenia Histoplasma capsulatum Microorganisms with capsules: Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis (*Vaccines) Klebsiella IgG2 required for immune response B. anthracis- protein capsule Cryptococcus neoformans- India Ink or latex agglutination

Above (left): Classic picture of S. pneumoniae. If you get a sputum sample, will see neutrophils and gram+ diplococci have halo around them (capsule). Pneumococcal pneumonia. Above (right): Capsule of Cryptococcus (encapsulated yeast). Can demonstrate w/India Ink, but Latex agglutination test has better sensitivity and specificity. Hemolytic bacteria: Alpha- incomplete hemolysis (turns a little bit green); Streptococcus pneumoniae (catalase optochin-S); Viridans Group (alpha strep) are optochin-R. Beta- totally break down RBCs, see cleared areas; Staphylococcus aureus, Streptococcus pyogenes (Group A), Streptococcus agalactiae (Group B) and Listeria (tumbling motility) Gamma- no hemolysis; Enterococcus Catalase and Coagulase: Streptococci are catalase Staphylococci are catalase + S. aureus is also coagulase + Hemolysis patterns:

Catalase + (bubbles):

Case 1: 20 y.o. female has 103 C fever 2 days after menses. She is hypotensive and has a rash on her chest. Desquamating rash may be on trunk, palms, anywhere. Toxic shock Usually due to S. aureus but also can be S. pyogenes. Methicillin-resistant S. aureus or MRSA: Nosocomial (MecA 1-3) MRSA are resistant to many antibiotics; treat w/Vancomycin Community acquired (MecA4,5) MRSA can be treated with TMP-SMX (Septra trimethoprim-sulfamethoxazole) Staphylococcus epidermidis (CNS): Coagulase-negative (so aka coagulase-negative Staph) Infections in neonates or on indwelling medical devices; adhesion polysaccharide (slime or glycocalyx) Streptococcus pyogenes: M-protein is antigenic and associated with virulence; ASO titer and other lab tests; Bacitracin-S Pyogenic- Pharyngitis, impetigo, cellulitis Toxigenic- TSS, scarlet fever Immunologic- RF and GN Penicillin G Associated w/rheumatic heart disease, post-streptococcal glomerulonephritis Case 2: 50-y.o. female had a bad strep infection as a child and now has aortic and mitral stenosis Rheumatic heart disease - mitral valve is most nd rd commonly involved, aortic 2 , both 3 . Beta hemolysis, S. pyogenes (left) Aschoff body in the heart (right) Remember that in rheumatic heart disease, there is no bacterial endocarditis in the acute phase. The valves are damaged, but there arent any viable organisms there is an immunologic reaction. It is only later on that they get infected w/other organisms. Case 2A: Young adult with hx. of rheumatic fever develops mitral valve endocarditis after dental extraction. The most likely etiology is: A. Streptococcus pyogenes B. Staphylococcus aureus C. Streptococcus bovis D. Streptococcus mutans E. Streptococcus viridans There is no such species as E. The alpha strep are all called viridans strep, but streptococcus viridans does not exist. The answer here is D because it is one of the alpha streptococci. Streptococcus agalactiae: Group B Neonatal sepsis Neonatal meningitis Bacitracin-R; CAMP+ Newborn with seizures- now rare do to treatment of colonized moms Used to be most common cause of neonatal meningitis. Now we test pregnant women for GBS, so the cases of neonatal meningitis from GBS have gone way down.

Streptococcus pneumoniae Encapsulated, Gram+ diplococci, alpha-hemolytic Optochin-S More virulent if pt. is Sickle cell (SS) Disease and/or post splenectomy spleen is important in defending against encapsulated microorganisms Rusty-colored sputum, lobar consolidation, alcoholic Not in strep viridans group in its own group. Viridans streptococci are also alpha hemolytic. Viridans Group (alpha streptococci): Oral flora Optochin-R Endocarditis on damaged valves Splinter hemorrhages (underneath fingernails), Oslers nodes (fingers and toes), Janeway lesions (palms and soles), Roths spots o All indicate septic emboli from endocarditis An example is Streptococcus intermedius This group would cause endocarditis on someone w/a hx of rheumatic heart disease. Two organisms involved original damage caused by S. pyogenes and current infection usually caused by alpha streptococci.

Case 5: 20-y.o. man had recurrent pharyngitis as a child now has fever. Oslers nodes and Janeway lesions are present. Echo shows mitral valve vegetations. Blood cultures grow GPCs Hx of rheumatic heart disease this patient has endocarditis. Alpha hemolytic strep will cause it (usually viridans group). Figure: A-C. Oslers Nodes; D. Janeway lesion Oslers nodes big and painful Janeway lesions small, painless Both on fingers/toes, palms, and balls of feet.

Clostridium: C. tetani (painful muscle spasms) and C. botulinum (upper body paralysis) C. perfringens- alpha toxin (lecithinase) with myonecrosis/gas gangrene C. difficile- makes cytotoxin that kills enterocytes; pseudomembranous enterocolitis; looks like mushroom on H&E slide; secondary to ampicillin or clindamycin; **#1 cause of nosocomial diarrhea Case 6: 30-y.o. construction worker presents with stiff neck and shoulders. He relates earlier difficulty chewing his food, opening and closing his mouth. LP is performed and is normal. Hands and feet are normal except for an old puncture wound on the bottom of his right foot. = C. tetani (tetanus)

Listeria monocytogenes Gram+ rods; tumbling/umbrella motility Newborn with meningitis shows G+ rod in CSF, cultures show tumbling motility (under microscope) Unusual because it is a G+ bacillus. Dairy food, refrigerated items, ice cream (on occasion). Falls head over heels tumbles. In semi-solid media, grows like an umbrella. Figure: Umbrella motility

Diphtheria: Corynebacterium diphtheriae Exotoxin inhibits protein synthesis and causes myocarditis Pharyngeal pseudomembrane; Gram+ rods with metachromatic granules that look like Chinese letters Immigrant girl with sore throat and difficulty breathing; mucous film on oropharynx Bacillus anthracis: Malignant pustule Woolsorters disease, gastrointestinal disease, inhalation anthrax Gram+ sporeformer; protein capsule; non-hemolytic Toxin with edema factor and lethal factor both on A subunit Example: Young woman with skin ulcers who works with wool and/or animal hides Not much lab risk for inhalation in the lab. Safe to work with.

Spores

Cutaneous ulcers

Causes pneumonia, but cant see spores here.

Bacillus cereus: Spores; aerobic; Gram+ rods Heat-stable toxin (nausea, vomiting) Heat-labile toxin (diarrhea) Example: Young man gets Nausea & frequent Vomiting soon after eating fried rice (6-10 hours) B. cereus and S. aureus cause food-poisioning in short time preformed toxins. Actinomyces and Nocardia: Both are Gram+ FILAMENTOUS bacteria which are often beaded israelii- G+ ANAEROBE causes chronic pneumonia and draining ulcers and fistulas in the neck (lumpy jaw); also lungs: poor dental hygiene; yellow-colored sulfur granules in tissue; NOT acid-fast Nocardia asteroides; aerobe; weakly acid-fast; pneumonia and brain abscess; lung and/or brain abscess in transplant pt. o Nocardia not tradititonal acid-fast stain. Must be modified acid-fast stain If you see this, you can tell that it is filamentous G+ bacteria. Need modified acid-fast stain to tell Actinomyces apart from Nocardia

Red beaded organisms = Nocardia. If we didnt see these, would be Actinomyces.

Case 7: 45-y.o. male has draining sinuses with purulent discharge on the left side of his face. He had dental surgery last week. Gram stain shows G+ filamentous bacteria. Modified Acid-fast stain is negative. Lumpy jaw from Actinomyces filamentous bacteria, non-acid fast. Figure (left): Microscopically can see sulfur granules To naked eye, are yellow. Figer (right): Gram-positive, filamentous Actinomyces. If you look REALLY close (2000x), can see filaments.

Neisseria: N. gonorrhoeae (GC) No capsule No maltose fermentation No vaccine Gonorrhea, septic arthritis and neonatal conjunctivitis Ceftriaxone If CT also suspected, add doxy. or azithromycin

N. meningitidis Polysaccharide Capsule Maltose+ Vaccine Meningococcemia, meningitis; WaterhouseFriderichsen Syndrome Penicillin

Haemophilus influenzae: Bacteria with Type B capsule most invasive Chocolate agar; requires X (hematin) AND V (NAD) factors (see bottom of plate) o Requires heme for growth Life-threat ceftriaxone Other AmCl (Augmentin), Septra Vaccine between 2 and 18 mos Case 10: 2-y.o. child living in a commune has meningitis. LP shows GNR and growth only on chocolate agar; requires hemin AND NAD; treatment is ceftriaxone.

Case 10-A: 27-year-old AIDS patient has bilateral pneumonia. CD4 count is 250. Gram stain shows gram-negative diplococci. G(-) diplococci could be Neisseria meningitis or Moraxella catarrhalis (especially in someone w/AIDS). If G(+) diplococci, would be S. pneumo.

Enterobacteriaceae: Lactose (-): Salmonella- motile; more invasive; 100,000 for infection Shigella- non-motile; 1-10 for infection (virulent) Proteus- swarmer; UTIs; UREASE+ Pseudomonas Lactose(+): Klebsiella- capsule; UTI; Lobar Pn.; antibiotic-R coli- UTI; neonatal meningitis; HURS Enterobacter

Lactose negative will be clear colonies on the selective media. Lactose positive will be pink. Klebsiella G(-) organism, heavily encapsulated. Patients often have reddish/brownish sputum.

Above (left): Selective media. Left lactose positive (E. coli, Klebsiella). Right lactose negative (Shigella, Salmonella). Above (middle): Swarms Proteus. Above (right): Pink slimy half of plate (currant jelly) due to capsule Klebsiella.

Above: If lactose negative (clear on selective media) and produces hydrogen sulfide on this medium, its Salmonella. Figure: Causes of diarrhea E. coli 0157 associated w/HUS. Blood and WBCs in stool = invasive!

C. Difficile:

C. difficile mushroom:

In C. difficile, do not see flask see mushroom on top of mucosa. Amoebic dysentery E. histolytica: Eryhrophagocytosis by amoebae:

H&E Stain of the flask-shaped ulcer. Other Enterobacteriaceae: Bacteroides fragilis- anaerobe; abdominal infec.; metronidazole Vibrio cholera- ADP-ribosylates Gs protein with adenylate cyclase; Lactose-(or slow); non-bloody rice water stool; doxy. Cholera is non-invasive. Produces toxin. Treat by replacing fluid (more important to tx w/fluid than give antibiotics) Campylobacter jejuni- growth at 42 C; maybe #1 cause of diarrhea; erythromycin Helicobacter pylori- gastritis, ulcer, MALT lymphoma; urease+; Triple therapy = metro.+ tetr.+ Bismuth Hyperplasia of lymphoid tissue MALT lymphomas (just like in Hashimotos)

RBCs in macrophages

Above: From AIDS patient w/diarrhea could be Cryptosporidium (dots coating small intestine malabsorption). Could also be Giardia, but it would be much bigger and looks like old man w/two big eyes. Do modified acid-fast stain on stool to demonstrate oocysts (Crypto).

TUBERCULOSIS: Caused by M. tuberculosis and less often by M. bovis; (granulomas) M. bovis acquired from milk or cattle with infection via the GI tract; also bladder CA patients Primary TB: mid-lungs areas Secondary TB/Reactivation TB: apical/upper lobes; more necrosis