ASCP Clinical Microscopy 2 — Questions and Answers

Question 1: Which urinary crystal is pathognomonic for cystinuria?

Coffin-lid (triple phosphate) crystals
Flat hexagonal (benzene ring-shaped) crystals (Correct answer)
Calcium oxalate crystals
Uric acid crystals

Correct answer: Flat hexagonal (benzene ring-shaped) crystals

Cystine crystals in urine appear as flat, colorless hexagonal plates (benzene ring shape) and are pathognomonic for cystinuria — an autosomal recessive disorder causing defective renal tubular transport of cystine, ornithine, lysine, and arginine.

Cystinuria is caused by defective SLC3A1 and SLC7A9 cystine transporters in the proximal tubule, leading to accumulation of cystine, ornithine, lysine, and arginine (COLA) in urine. Cystine is poorly soluble and crystallizes, forming kidney stones including staghorn calculi. Crystals appear as flat, colorless hexagons that may stack, detected in acidic urine. The nitroprusside (cyanide-nitroprusside, Brand's) test is positive (magenta color) with above 75 mg cystine per gram creatinine. Management includes high fluid intake, urine alkalinization to pH above 7.5, and D-penicillamine or tiopronin.

Question 2: On a peripheral blood smear, target cells (codocytes) are most commonly associated with:

Iron deficiency anemia
Liver disease, thalassemia, hemoglobin C disease, and post-splenectomy (Correct answer)
Vitamin B12 deficiency
Autoimmune hemolytic anemia

Correct answer: Liver disease, thalassemia, hemoglobin C disease, and post-splenectomy

Target cells have excess membrane relative to hemoglobin content, creating a bull's-eye appearance. They are classic for liver disease (altered lipid composition), thalassemia, hemoglobin C or SC disease, and post-splenectomy states.

Target cell (codocyte) formation involves membrane excess relative to hemoglobin content, creating a central pale area with a hemoglobin bullseye. Mechanisms: (1) Increased membrane lipids in liver disease (excess cholesterol or lecithin expanding membrane) and obstructive jaundice; (2) Decreased hemoglobin or MCHC in thalassemia (reduced globin chains), severe iron deficiency, and hemoglobin C or SC (HbC crystallizes causing dehydrated cells); (3) Post-splenectomy (spleen normally remodels excess membrane). Liver disease targets are thin and lacy; thalassemia targets are more prominent. Association with HbC electrophoresis pattern is diagnostically important.

Question 3: Wright-Giemsa stain produces its polychromatic differential staining because it contains:

Hematoxylin for nuclei only
A mixture of acidic (eosin) and basic (methylene blue) dyes that react with acidic and basic cellular components (Correct answer)
Silver stain for reticulin fibers
Periodic acid-Schiff reagent

Correct answer: A mixture of acidic (eosin) and basic (methylene blue) dyes that react with acidic and basic cellular components

Wright-Giemsa is a Romanowsky stain containing eosin (acidic, orange-red, staining hemoglobin and eosinophilic granules) and methylene blue or azure B (basic, blue-purple, staining nuclei and basophilic granules). Together they produce the polychromatic differential staining of blood cells.

Romanowsky stain components: Eosin B or Y (acidic dye, anionic) binds basic cellular structures and stains hemoglobin orange-red, eosinophil granules red-orange, and RBCs pink. Methylene blue and oxidation products (azure A, azure B — basic dyes, cationic) bind acidic cellular structures and stain DNA, RNA, and acidic granules blue-purple. Resulting color matrix: neutrophil granules (lilac), basophil granules (dark blue-purple), lymphocyte cytoplasm (pale blue), monocyte cytoplasm (blue-gray), platelet granules (purple). Buffer pH of 6.4–6.8 is critical; too acidic causes too much red (eosin dominates); too alkaline causes too much blue (methylene blue dominates).

Question 4: Hypersegmented neutrophils (5 or more lobes in more than 5% of neutrophils, or any neutrophil with 6 or more lobes) are most characteristic of:

Iron deficiency anemia
Megaloblastic anemia (B12 or folate deficiency) (Correct answer)
Sickle cell disease
Polycythemia vera

Correct answer: Megaloblastic anemia (B12 or folate deficiency)

Hypersegmented neutrophils are a hallmark of megaloblastic anemia, reflecting impaired DNA synthesis (from B12 or folate deficiency) that delays nuclear division relative to cytoplasmic maturation, resulting in larger cells with more nuclear segments.

Megaloblastic hematopoiesis: impaired DNA synthesis through the thymidylate synthesis pathway requiring 5,10-methylene-THF dependent on folate, and methionine synthase requiring B12 for THF regeneration, leads to nuclear maturation lagging behind cytoplasmic maturation with abnormally large cells with immature-appearing nuclei. Peripheral smear shows oval macrocytes (distinct from round macrocytes of liver disease) and hypersegmented PMNs per Cooke's criterion of more than 5% PMNs with 5 or more lobes or any PMN with 6 or more lobes. Bone marrow shows megaloblastic changes and hypersegmented meta-myelocytes. Hypersegmentation may persist for weeks after B12 or folate treatment begins. Also seen in uremia, myelodysplasia, and hydroxyurea therapy.

Question 5: Schistocytes on a peripheral blood smear are most consistent with:

Liver disease
Microangiopathic hemolytic anemia (MAHA) such as TTP, HUS, or DIC (Correct answer)
Spherocytosis
Aplastic anemia

Correct answer: Microangiopathic hemolytic anemia (MAHA) such as TTP, HUS, or DIC

Schistocytes (red cell fragments, helmet cells) form when red cells are mechanically sheared by fibrin strands in microthrombi or damaged endothelium. They are pathognomonic for microangiopathic hemolytic anemia (MAHA): TTP, HUS, DIC, malignant hypertension, and HELLP syndrome.

MAHA mechanism: fibrin strands in DIC, TTP, or HUS microthrombi, or damaged endothelium in malignant hypertension, HELLP, or mechanical heart valves physically cut RBCs into helmet cells, triangular fragments, and bite cells (schistocytes). Criteria for MAHA: schistocytes on smear at 1% or more by ICSH guidelines, negative Coombs (non-immune hemolysis), elevated LDH and bilirubin, and low haptoglobin. TTP: severe ADAMTS13 deficiency below 10%, typically antibody-mediated. HUS: Shiga toxin from STEC O157:H7 causing endothelial injury with MAHA, thrombocytopenia, and AKI predominant. DIC: coagulation activation plus MAHA.

Question 6: In CSF analysis, lymphocytic pleocytosis with elevated protein and normal glucose is most consistent with:

Bacterial meningitis
Viral (aseptic) meningitis or early tuberculous meningitis (Correct answer)
Subarachnoid hemorrhage
Multiple sclerosis (uncomplicated)

Correct answer: Viral (aseptic) meningitis or early tuberculous meningitis

Viral meningitis classically shows CSF lymphocytic pleocytosis (10–1000 cells/µL, predominantly lymphocytes), mildly elevated protein (50–100 mg/dL), and normal glucose (at least 2/3 of serum glucose). Bacterial meningitis has predominantly PMNs, very high protein, and low glucose.

CSF interpretation framework: Normal CSF has 0–5 lymphocytes per microliter, protein 15–45 mg/dL, glucose 60–80% of serum (45–80 mg/dL), no PMNs. Bacterial meningitis: PMNs predominant (100 to 10,000 or more), protein above 100 mg/dL (often above 500), glucose below 45 mg/dL or CSF:serum ratio below 0.4, elevated opening pressure, turbid appearance, positive Gram stain (60–90%), positive culture. Viral or aseptic meningitis: lymphocytes predominant, mildly elevated protein (50–100 mg/dL), glucose normal, clear CSF, negative culture; enterovirus is most common. TB meningitis: lymphocytes, elevated protein (100–500 mg/dL), low glucose (CSF:serum below 0.5), AFB smear has low sensitivity (approximately 40%), culture is gold standard.

ASCP Clinical Microscopy 2

Which urinary crystal is pathognomonic for cystinuria?