Hematology, Leukocyte Disorders, Instrumentation, and Anemias

Comprehensive practice flashcards reviewing hematopoiesis, Romanowsky staining, WBC morphology, maturation stages, quantitative and inherited granulocyte disorders, monocytes, lipid storage diseases, lymphocyte subsets, hematology instrumentation, quality control, RBC morphology, and microcytic/macrocytic anemias.

Into which two major progenitor lineages do pluripotent hematopoietic stem cells differentiate?

The lymphoid and myeloid progenitor lineages.

Which mature cell types are produced by the myeloid progenitor lineage?

Erythrocytes, thrombocytes, granulocytes (neutrophils, eosinophils, basophils), and monocytes.

Which mature cell types originate from the lymphoid progenitor lineage?

T cells, B cells, and Natural Killer (NK) cells.

What are four named examples of Romanowsky staining systems?

Wright-Giemsa, Leishman, May-Grunwald, and RAL Systems.

What are the chemical charge characteristics and cellular targets of Eosin Y in Romanowsky stains?

Eosin Y is acidic/anionic ($-ve$ charge) and binds basic ($+ve$) cellular components, staining eosinophil granules and cytoplasm pink-red, and RBCs salmon pink.

What are the chemical charge characteristics and cellular targets of Methylene Blue / Azure B?

Methylene Blue / Azure B is basic/cationic ($+ve$ charge) and binds acidic ($-ve$) components like DNA/RNA, basophil granules, and nuclear chromatin to yield blue/purple tones.

What staining appearance do neutral cellular components, such as neutrophilic granules, produce?

They interact with both acidic and basic dyes to produce purple tones.

What is the adult leukocyte reference interval for total white blood cells (WBC)?

$$4.8 - 10.8 \times 10^9/\text{L}$$

What are the size, reference interval, relative percentage, and circulatory lifespan of adult neutrophils?

Size: $9–15 stage\mu\text{m}$; Reference interval: $2.0 - 7.5 \times 10^9/\text{L}$; Relative percentage: $50–70\%$; Lifespan: $6\text{ hours} – \text{few days}$.

What are the key morphological features of a mature neutrophil?

A multi-lobed nucleus ($2–5$ lobes connected by thin threads) with pale lilac/neutral granules masking clear-blue cytoplasm.

What are the adult reference interval, relative percentage, and key morphological features of eosinophils?

Reference interval: $0.0 - 0.6 \times 10^9/\text{L}$; Relative percentage: $0–4\%$; Morphology: bi-lobed ("Pince-nez" style) nucleus packed with bright orange-red specific granules.

What is the circulatory and tissue lifespan of eosinophils?

They circulate for $4–5\text{ hours}$ in blood and persist in tissues for $8–12\text{ days}$.

What are the adult reference interval, relative percentage, and key morphological features of basophils?

Reference interval: $0.0 - 0.2 \times 10^9/\text{L}$; Relative percentage: $0–2\%$; Morphology: bi/tri-lobed nucleus heavily obscured by large, dark blue/purple water-soluble granules.

What are the adult reference interval, relative percentage, and morphological features of monocytes?

Reference interval: $0.1 - 0.9 \times 10^9/\text{L}$; Relative percentage: $2–9\%$; Morphology: pleomorphic/kidney-shaped nucleus with blue-gray cytoplasm exhibiting a "frosted/ground-glass" appearance.

What are the adult reference interval, relative percentage, and morphology of normal small lymphocytes?

Reference interval: $1.2 - 3.4 \times 10^9/\text{L}$; Relative percentage: $20–44\%$; Morphology: high N:C ratio, dense clumped chromatin, and a clear medium-blue rim of agranular cytoplasm.

What are the six sequential morphological stages of granulocytic maturation?

1. Myeloblast, 2. Promyelocyte, 3. Myelocyte, 4. Metamyelocyte, 5. Band Cell, and 6. Segmented Neutrophil.

What are the size and nuclear-cytoplasmic characteristics of a myeloblast?

Size: $14–20\,\mu\text{m}$; high N:C ratio of $4:1$, lacy chromatin, nucleoli present, no granules (sometimes contains Auer rods in blasts).

What primary characteristic distinguishes the promyelocyte stage during granulopoiesis?

Size: $15–22\,\mu\text{m}$; contains abundant primary azurophilic (non-specific) granules of a burgundy/merlot color.

What morphological transition defines the myelocyte stage?

Size: $10–18\,\mu\text{m}$; beginning of secondary (specific) granules while primary granules decrease, and the nucleus starts to flatten and condense.

What are the key morphological features of a metamyelocyte?

Size: $10–15\,\mu\text{m}$; kidney- or bean-shaped nucleus, and cytoplasm filled entirely with specific (neutrophilic, eosinophilic, or basophilic) granules.

How is a band cell defined morphologically, and how are bands counted?

Size: $9–15\,\mu\text{m}$; C- or U-shaped curved nucleus with parallel sides throughout the indentation; all bands are added to mature cell counts.

What are the four sequential steps of phagocytosis?

1. Chemotaxis (chemical signals draw cells), 2. Opsonization (coating with complement/antibodies), 3. Ingestion (formation of phagosome), and 4. Killing/Digestion (fusion into phagolysosome and lytic enzyme release).

What is NETosis and what are neutrophil extracellular traps composed of?

A host defense mechanism where neutrophils release extracellular traps composed of decondensed DNA, histones, and antimicrobial proteins (myeloperoxidase, elastase) to capture extracellular bacteria.

What is toxic granulation in neutrophils and what is its primary composition?

Larger, darker, more prominent purple-black primary (azurophilic) granules persisting into mature stages, composed largely of myeloperoxidase.

What does the presence of toxic vacuolation indicate in neutrophils?

Active phagocytosis and degranulation resulting in round clear spaces in the cytoplasm (check for artifacts if sample is old).

What are Döhle bodies and what causes them to form?

Light blue-gray, oval cytoplasmic inclusions near the periphery composed of remnants of rough endoplasmic reticulum / ribosomal RNA due to accelerated maturation.

What is the hematological definition of a "left shift"?

The presence of immature granulocytes in peripheral blood, defined by bands $> 0.7 \times 10^9/\text{L}$, or the presence of metamyelocytes/myelocytes.

What is the formula used to calculate the Absolute Neutrophil Count (ANC)?

$$\text{ANC} = \frac{(\%\text{Neutrophils} + \%\text{Bands}) \times \text{Total WBC}\,(10^9/\text{L})}{100}$$

What numerical threshold defines neutrophilia and what are its common causes?

$\text{ANC} > 7.5 \times 10^9/\text{L}$; caused by bacterial infections, acute inflammation, stress, surgery, or CML.

What numerical threshold defines neutropenia and what are its common causes?

$\text{ANC} < 2.0 \times 10^9/\text{L}$; caused by chemotherapy, aplastic anemia, overwhelming sepsis, or viral infections.

What are the alert value thresholds for ANC and what action do they require?

$\text{ANC} < 1.0 \times 10^9/\text{L}$ or $> 50.0 \times 10^9/\text{L}$; requires same-day prompt verbal communication.

What are the critical value thresholds for ANC and what action is required?

$\text{ANC} < 0.5 \times 10^9/\text{L}$ or $> 100.0 \times 10^9/\text{L}$; life-threatening finding requiring immediate $24/7$ medical notification.

What laboratory protocol must be followed if a 100-cell differential yields zero neutrophils?

Count an additional $100$ cells ($200$ total); if still zero, scan the entire slide before confirming "rare neutrophil seen" or reporting $0\%$.

What are the inheritance pattern and nuclear features of Pelger-Huët Anomaly (PHA)?

Autosomal dominant; hyposegmentation where the nucleus pinches into a symmetrical bilobed ("Pince-nez" / eyeglasses) shape with highly condensed chromatin.

What percentage of bilobed cells distinguishes true Pelger-Huët Anomaly from pseudo-PHA?

True PHA has $> 70 - 80\%$ bilobed cells, whereas pseudo-PHA (seen in MDS) has fewer.

What are the inheritance pattern and hematological features of May-Hegglin Anomaly?

Autosomal dominant (MYH9 disorder); features large, hypogranular platelets (causing thrombocytopenia/bleeding) and prominent Döhle bodies in granulocytes.

What are the inheritance pattern and cellular inclusions found in Alder-Reilly Anomaly?

Autosomal recessive; dense, dark purple-staining cytoplasmic inclusions (Reilly bodies) resembling toxic granulation across neutrophils, monocytes, and lymphocytes; cells function normally.

What are the clinical and morphological features of Chediak-Higashi Anomaly?

Rare autosomal recessive disorder; features partial oculocutaneous albinism, photophobia, neutropenia with defective killing, giant grayish-red lysosomal granules in WBCs, bleeding, and neurological abnormalities.

What is the clinical diagnostic purpose of the Leukocyte Alkaline Phosphatase (LAP) score?

Differentiates a benign leukemoid reaction (high score) from Chronic Myelogenous Leukemia (CML - low/absent score).

What specimen types are acceptable for the LAP score, and why is EDTA unacceptable?

Fresh peripheral blood or bone marrow collected in Heparin or via capillary collection; EDTA is unacceptable because it inhibits the alkaline phosphatase enzyme.

How is the LAP score graded and calculated?

$100$ neutrophils are graded from $0$ to $4$ based on azurophilic dye precipitate intensity to calculate a total score.

What are the size and morphological features of monocytes compared to other blood cells?

Size is $12–22\,\mu\text{m}$ (largest cells on a normal film); features abundant light blue-gray "frosted/ground-glass" cytoplasm, a folded/indented/"three-pointed hat" nucleus, and occasional vacuoles.

What are the thresholds for monocytosis and monopenia?

Monocytosis: $> 0.9 \times 10^9/\text{L}$ (chronic infections, RA, IBD, SLE, CMML); Monopenia: $< 0.1 \times 10^9/\text{L}$ (aplastic anemia, chronic corticosteroids, hairy cell leukemia).

What general biochemical defect causes lipid storage diseases?

Autosomal recessive disorders where missing metabolic enzymes cause undigested lipids to accumulate in monocytes and macrophages within the reticuloendothelial system (RES).

What is the deficient enzyme, accumulating substrate, and presentation of Gaucher Disease?

Deficient enzyme: $\beta$-Glucocerebrosidase; accumulates glucosylceramide; features Gaucher cells in bone marrow, hepatosplenomegaly, bone pain/infarcts, and anemia with thrombocytopenia.

What enzyme is deficient in Niemann-Pick Disease, and what are its hallmark clinical features?

Deficient enzyme: Sphingomyelinase (accumulates sphingomyelin); features Niemann-Pick cells in bone marrow, hepatosplenomegaly, cherry-red spot in the eye, and severe neurodegeneration.

What enzyme is deficient in Tay-Sachs Disease, and what substrate accumulates?

Deficient enzyme: Hexosaminidase A; accumulating substrate: $\text{GM}_2$-ganglioside (fatal in early childhood with no distinct blood or bone marrow features).

What proportion of circulating lymphocytes do T cells and B cells constitute, and what are their roles?

T cells: $60–80\%$ (cell-mediated immunity); B cells: $10–20\%$ (humoral immunity; differentiate into antibody-secreting plasma cells).

What proportion do Natural Killer (NK) cells represent, and how are they handled on CellaVision?

They represent $< 20\%$ of lymphocytes; provide innate immune surveillance and are counted separately from regular lymphocytes on CellaVision.

What morphological features characterize reactive (variant/atypical) lymphocytes?

Enlarged size up to $15–18\,\mu\text{m}$, intense dark blue peripheral basophilia, and cytoplasm that molds or indents around neighboring red blood cells.

What is the etiology and classical hematological differential finding in Infectious Mononucleosis?

Caused by Epstein-Barr Virus (EBV / HHV-4); features absolute lymphocytosis with abundant reactive lymphocytes causing an inverted/reverse differential (lymphocytes outnumber neutrophils).

What principle does the Monospot Latex Agglutination Test use, and what can cause false positives?

Detects heterophile antibodies that agglutinate Paul-Bunnell antigen-coated latex; false positives occur with rheumatoid factor, autoimmune diseases, or lymphoid malignancies.

Which organisms cause mononucleosis-like syndromes in patients who test negative on the Monospot test?

Cytomegalovirus (CMV) or Toxoplasma gondii (a parasite from cat feces dangerous in pregnancy).

What are the morphological features and diagnostic significance of peripheral blood plasma cells?

Eccentric round nucleus with "cartwheel"/clock-face chromatin, deeply basophilic cytoplasm, and perinuclear hof; indicates Multiple Myeloma or plasma cell leukemia.

What are Mott cells and Russell bodies?

Mott cells are plasma cells containing multiple clear globular cytoplasmic inclusions called Russell bodies (accumulated immunoglobulins), associated with chronic inflammation or multiple myeloma.

What are smudge cells, with which malignancy are they associated, and how are they counted if $\ge 10$ are present?

Ruptured fragile lymphocytes characteristic of CLL; if $\ge 10$ per 100 WBCs, add them to the total lymphocyte count, write a comment, and use $22\%$ bovine albumin to remake slides.

What is the evaluation guideline for a $40\times$ field on CellaVision?

Each field contains roughly $500$ RBCs; an abnormal cell type must appear $5$ times to equal $1\%$, with slides slightly thinner (about $1/3$ of RBCs touching).

How does the Coulter Principle (Impedance) quantify cell count and cell size?

Passes cells through an aperture to create electrical impedance pulses; pulse count equals cell number and pulse height corresponds directly to cell volume.

Which CBC parameter is measured strictly using photometry, and what reagent is used?

Hemoglobin (HGB), where a lysing agent produces a clear hemolysate (the only parameter that uses a lamp).

In flow cytometry, what do Forward Scatter (FSC), Side Scatter (SSC), and Side Fluorescence Light (SFL) correlate with?

FSC correlates with cell volume; SSC correlates with internal complexity and granularity; SFL correlates with DNA/RNA content.

What three levels of Quality Control (QC) must be run on automated CBC analyzers?

Level 1: Low abnormal; Level 2: Normal; Level 3: High abnormal (must be run in both open and closed modes if independent).

How do red blood cell and platelet parameters change as commercial QC blood ages?

Parameters like HCT, MCV, RDW-SD, PLT-O, and PLT-I tend to increase because RBCs swell.

How do systematic and random errors differ in automated hematology QC?

Systematic error is a consistent, repeatable deviation in one direction (shifts/trends); random error is unpredictable fluctuation above and below the mean reflecting precision issues.

What are the golden rules of QC troubleshooting regarding recalibration and calculated parameters?

Recalibration is rarely the fix; always troubleshoot the underlying directly measured parameter (e.g., HGB or HCT) rather than calculated indices (e.g., MCHC).

What is the purpose of Patient Moving Averages (XM / Bull's Algorithm)?

It runs in the background using rolling patient data to continuously monitor systematic analyzer control without extra reagent cost.

What is a delta check and what is the primary cause of delta check flags?

An automated alert triggered when a current test result shifts beyond a predefined threshold compared to a previous result; pre-analytical human error is the primary cause.

Why is MCV the primary parameter used for delta checks?

MCV is the most stable parameter because it rarely changes drastically in an individual patient without significant cause.

What is the formula for calculating the PAD score in proficiency testing?

$$\text{PAD} = \frac{\text{Lab Result} - \text{Assigned Value}}{\text{Allowable Performance Limit}} \times 100$$

What actions do PAD scores of $\le 100\%$, $> 100\%$ to $150\%$, and $> 150\%$ require?

$\le 100\%$ requires no action; $> 100\%$ to $150\%$ triggers a warning (W1); $> 150\%$ requires mandatory investigative actions and proficiency provider reporting.

What do the Volume, Conductivity, and Scatter channels assess in Beckman Coulter VCS technology?

Volume (V) measures size via DC impedance; Conductivity (C) measures internal nuclear structure/composition via RF current; Scatter (S) measures surface structure, shape, and granularity via laser light.

What cell features are measured by the four scatter angles in Abbott Cell-Dyn MAPSS technology?

$0^\circ$: cell size/volume; $10^\circ$: nuclear-to-cytoplasmic complexity; $90^\circ$: cellular granularity; $90^\circ$ depolarized: eosinophilic granules.

How do leukocyte lineages react in the Siemens Advia Peroxidase Channel?

Neutrophils show the strongest myeloperoxidase reaction, eosinophils moderate, monocytes weak, and lymphocytes and basophils show no reaction.

What do the terms anisocytosis and poikilocytosis define?

Anisocytosis is variation in RBC size (measured by RDW); Poikilocytosis is variation in RBC shape.

What are Dacrocytes (teardrop cells) and Codocytes (target cells), and what causes them?

Dacrocytes are teardrop cells caused by cells squeezing through fibrotic marrow (myelofibrosis, thalassemia); Codocytes are target cells caused by excess cell membrane (liver disease, hemoglobinopathies, thalassemias, post-splenectomy).

What defect causes Stomatocytes?

Failure of the $\text{Na}^+/\text{K}^+$ pump, resulting in a slit-like central pallor (hereditary, alcoholism, liver disease, or artifact).

How do Acanthocytes and Echinocytes differ morphologically and clinically?

Acanthocytes (spur cells) have irregular, spiked projections (liver disease, abetalipoproteinemia); Echinocytes (burr cells) have blunt, evenly spaced projections (uremia, renal disease, or sample aging artifact).

What are the morphological features, MCHC threshold, and clinical associations of Spherocytes?

Microcytic, hyperdense cells lacking central pallor with $\text{MCHC} > 360\,\text{g/L}$; strongly indicative of autoimmune hemolytic anemia (AIHA) or hereditary spherocytosis.

What are Howell-Jolly bodies, Pappenheimer bodies, and Basophilic stippling composed of?

Howell-Jolly bodies: DNA remnants; Pappenheimer bodies: iron granules (confirmed by Prussian blue); Basophilic stippling: ribosomal RNA precipitates (lead poisoning, thalassemia).

What are Heinz bodies, how are they visualized, and what conditions cause them?

Denatured hemoglobin precipitates visible only with supravital stains (not Wright-Giemsa); associated with G6PD deficiency and oxidative hemolysis.

What peripheral blood changes occur following a splenectomy (Post-Splenectomy Changes)?

Persistent Howell-Jolly bodies, Pappenheimer bodies, target cells, leukocytosis, thrombocytosis, increased poikilocytosis, and aging echinocytes.

What are the normal reference interval, alert value, and critical value for platelets?

Reference interval: $150 - 400 \times 10^9/\text{L}$; Alert value: $< 50 \times 10^9/\text{L}$; Critical value: $< 20 \times 10^9/\text{L}$.

What causes EDTA-induced pseudothrombocytopenia and platelet satellitism?

In vitro autoantibodies reacting with platelet surface antigens in the presence of EDTA cause clumping or adherence to neutrophil membranes; redrawn in a citrate tube.

How is anemia defined and how is it classified morphologically by MCV and MCHC?

Reduction in oxygen-carrying capacity due to decreased RBCs or HGB; Normocytic Normochromic (MCV $80 - 100\,\text{fL}$, MCHC $320 - 360\,\text{g/L}$), Microcytic Hypochromic (MCV $< 80\,\text{fL}$), and Macrocytic (MCV $> 100\,\text{fL}$).

What is the normal reference interval for reticulocytes, and what does polychromasia indicate?

Reference interval: $0.5 - 2.0\%$ ($24 - 84 \times 10^9/\text{L}$); polychromatic macrocytes contain residual RNA and indicate the bone marrow red cell production rate.

What are the four main categories of microcytic hypochromic anemias?

1. Disorders of Iron Metabolism (IDA, ACI), 2. Disorders of Heme Synthesis (Sideroblastic Anemia), 3. Disorders of Globin Synthesis (Thalassemias), and 4. Disorders of Porphyrin Synthesis (Lead poisoning).

What do Ferritin, Transferrin, TIBC, and % Saturation represent in iron metabolism?

Ferritin stores iron intracellularly; Transferrin transports iron; TIBC measures available transferrin binding sites; % Saturation is the percentage of binding sites carrying iron ($20 - 50\%$ normal; $< 16\%$ indicates ineffective erythropoiesis).

Where does iron absorption occur, and what are its dietary enhancers, inhibitors, and hormonal regulator?

Absorbed in duodenum and proximal jejunum; enhanced by Vitamin C (reduces $\text{Fe}^{3+}$ to $\text{Fe}^{2+}$); inhibited by tannins and phytates; regulated by Hepcidin (inhibits absorption when iron is high).

What are the four sequential stages in the progression of iron deficiency?

1. Normal, 2. Iron Depletion (ferritin drops, TIBC rises), 3. Iron Deficient Erythropoiesis (serum iron drops, % saturation falls), and 4. Iron Deficiency Anemia (low HGB, MCV, MCHC; microcytosis and hypochromia).

What are the peripheral blood and bone marrow findings in Iron Deficiency Anemia (IDA)?

Microcytic, hypochromic RBCs with high RDW, thrombocytosis, absent stainable iron on Prussian blue, and an expanded erythroid precursor pool with a decreased M:E ratio (e.g., $1:1$ vs normal $2:1$ to $5:1$).

What is the pathophysiology and bone marrow finding in Sideroblastic Anemia (SBA)?

Defective mitochondrial heme synthesis traps iron in mitochondria; features ringed sideroblasts in bone marrow on Prussian blue stain, increased serum iron, and ferritin overload.

What is the genetic and clinical distinction between Beta-Thalassemia Minor and Beta-Thalassemia Major?

Controlled on Chromosome 11; Minor is heterozygous with mild microcytic anemia, increased $\text{Hb A}_2$ ($4 - 7\%$); Major (Cooley's Anemia) is homozygous with severe anemia, excess unpaired $\alpha$-chains, hepatosplenomegaly, and transfusion dependency.

What clinical syndromes result from missing 3 versus 4 alpha-globin genes on Chromosome 16?

3 genes missing ($--/-\alpha$): Hemoglobin H ($\text{Hb H}$) disease with $\beta_4$ tetramers ("golf ball" inclusions); 4 genes missing ($--/--$): Hydrops Fetalis producing $\text{Hb Barts}$ ($\gamma_4$), fatal at $35 - 40\text{ weeks}$, incompatible with life.

What is the Mentzer Index formula and how is it interpreted?

$\text{Mentzer Index} = \frac{\text{MCV}\,(\text{fL})}{\text{RBC count}\,(10^{12}/\text{L})}$; an index $< 13$ indicates Thalassemia Minor, while an index $> 13$ indicates Iron Deficiency Anemia.

How does the RDW differentiate Iron Deficiency Anemia from Thalassemia Minor?

In IDA, RDW is significantly increased due to progressive anisocytosis; in Thalassemia Minor, RDW is normal or only slightly increased due to a remarkably homogeneous microcytic population.

What defines Megaloblastic Macrocytic Anemia and its cellular asynchrony?

Impaired DNA synthesis (Vitamin $\text{B}_{12}$ or folate deficiency) causes delayed nuclear maturation while cytoplasmic RNA/protein synthesis continues normally, causing intramedullary destruction and pancytopenia.

What are the hallmark peripheral blood findings and biochemical cause of Megaloblastic Anemia?

Oval macrocytes, hypersegmented neutrophils ($> 5$ lobes), and large platelets; caused by failure to synthesize thymidine triphosphate (TTP) when folate is trapped as methyltetrahydrofolate.

What clinical symptom distinguishes Vitamin $\text{B}_{12}$ deficiency from pure Folate deficiency, and what is Pernicious Anemia?

$\text{B}_{12}$ deficiency causes neurological defects (neuropathy, dementia); Pernicious Anemia is an autoimmune lack of Intrinsic Factor treated with lifelong $\text{B}_{12}$ injections.

What are the characteristics and causes of Non-Megaloblastic Macrocytic Anemia?

MCV typically $100 - 110\,\text{fL}$ with round macrocytes and no hypersegmented neutrophils; caused by liver disease, alcoholism, hypothyroidism, hyperglycemia, or drugs (Methotrexate, Hydroxyurea).

What findings constitute a spurious count/pseudo-macrocytosis critical flag, and how does Cold Agglutinin Disease differ from Warm AIHA?

Flag: $\text{MCHC} > 360\,\text{g/L}$, decreased RBC, $\text{MCV} > 125\,\text{fL}$; Cold Agglutinin Disease is IgM-mediated (reacts $< 37^\circ\text{C}$, optimally $4^\circ\text{C}$), while Warm AIHA is IgG-mediated with spherocytosis and extravascular hemolysis.

What laboratory protocol is used to correct Cold Agglutination in an EDTA sample?

1. Incubate EDTA tube at $37^\circ\text{C}$ for $20 - 30\text{ minutes}$, re-smear, and re-assay warm; 2. If unresolved, perform Warm Plasma Replacement (centrifuge at $1500\,\text{rpm}$ for $10\text{ minutes}$, replace plasma with warm saline, incubate $15 - 20\text{ minutes}$ at $37^\circ\text{C}$, and re-assay; if still failing, report only HGB).