Myeloid Precursors:
Above (right): Immature granulocytes (left shift). If you have lots of granulocytes (in this case, don’t see any blasts), do MPO stain – will be positive but won’t be helpful because you can tell they’re 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.
ALL (PAS stain)
If you stain lymphoblasts, will be PAS+ and peroxidase negative.
Above (left): More undifferentiated AMLs. M1 won’t 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: 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
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.
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)
8. Diagnosis: A. AML (FAB M1) B. AML (FAB M3) C. AML (FAB M5) D. ALL (L1) E. Infectious mononucleosis Lymphomas: Hodgkin Non-Hodgkin
RS Cells Low grade- slowly progressive, rare cure
Often curable High grade- rapidly progressive; a minority are curable Stage important Grade important
Few malignant cells Mostly lymphoma < cell-mediated < humoral
Localized – radiation Systemic – chemotherapy
One of the lymphomas will be on the glass exam – need to recognize Hodgkin’s, follicular lymphoma, follicular
hyperplasia, SLL, and Burkitt’s. Abdominal lesions will also be on glass slide exam – Burkitt’s, Wilm’s, 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 Follicular hyperplasia, lymph node. See zone, dark zone, and light zone. See tingible tingible body macrophages, mantle zone, and
body macrophages. 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): Burkitt’s 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. Hodgkin Lymphoma. See eosinophils, lymphocytes, See fibrous bands, “nodules”. and Hodgkin cells.
Hodgkin Lymphoma, Spleen. See lymphocytes and Radiograph of Plasma Cell (Multiple) Myeloma.
Reed-Sternberg cells. See “punched out” lesions.
Above (left): In Hodgkin’s, 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 = Hodgkin’s 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
commonly involved, aortic 2nd, both 3rd. 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 aren’t 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), Osler’s nodes (fingers and toes), Janeway lesions (palms and soles), Roth’s 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. Osler’s 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. Osler’s Nodes; D. Janeway lesion Osler’s 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.
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
– Woolsorter’s 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 can’t 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 didn’t 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; Waterhouse-Friderichsen 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
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, it’s 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. RBCs in macrophages 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 Hashimoto’s)
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
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).
