Hematology Laboratory: WBC and RBC Counts, Differentials, and Diagnostic Workup

Comprehensive practice flashcards covering laboratory procedures, calculation formulas, normal reference ranges, cell morphology, and diagnostic methodologies for white blood cell counts, red blood cell evaluations, hemoglobin assays, reticulocyte analysis, hemoglobin electrophoresis, osmotic fragility, and erythrocyte sedimentation rate.

What is the general principle of the manual white blood cell (WBC) count?

Whole blood is mixed with a weak acid solution (such as Acetic Acid) that lyses red blood cells. Following adequate mixing, the diluted specimen is loaded into a counting chamber where white blood cells are counted.

To what mark is blood initially drawn in the white blood cell diluting pipette, and to what mark is the diluting fluid drawn?

Blood is drawn to the $0.5$ mark, and diluting fluid is drawn to the $11$ mark (providing a $1:20$ dilution).

How long must the contents of the WBC diluting pipette be mixed, and how many drops are discarded before charging the hemocytometer?

The pipette is mixed for $3 - 5\,\text{minutes}$, and the first $4\,\text{drops}$ of unmixed, cell-free fluid are expelled.

What is this piece of equipment used to perform manual cell counts under a microscope?

An Improved Neubauer hemocytometer (counting chamber).

What is the formula used to calculate the manual WBC count per $\mu\text{L}$ ($\text{c\,mm}$) from a hemocytometer count of four corner squares?

$$\text{WBC Count} = \frac{\text{\# cells counted} \times 20 \times 10}{4} = \text{\# cells counted} \times 50$$

What is the reference range for white blood cell count in adults?

$4.4 - 11 \times 10^9/\text{L}$ ($4.4 - 11 \times 10^3/\mu\text{L}$, or $4,400 - 11,000\,\text{c\,mm}$).

What are the quality control criteria regarding cell variation between squares and chamber sides in a manual WBC count?

There should not be a difference greater than $10\,\text{cells}$ between each of the $4\,\text{squares}$ counted, nor greater than $20\,\text{cells}$ between the two sides of the hemocytometer.

When must a WBC count be corrected for nucleated red blood cells (nRBCs), and what formula is used?

It must be corrected if there are $5\,\text{or more nRBCs}$ per $100\,\text{WBCs}$, using the formula: $\text{Corrected WBC} = \text{Uncorrected WBC Count} \times \frac{100}{\text{nRBCs per 100 WBCs} + 100}$.

What device is shown here, used to classify leukocytes during a differential count?

A Schilling's counter.

What angle should be maintained between the spreader slide and the blood smear slide during peripheral blood smear preparation?

$30^\circ - 40^\circ$.

What are the primary characteristics of an ideal blood smear?

It occupies $\frac{3}{4}$ of the length of the slide, has an initial thick area and spreads to a thin feathery area, and contains no holes or streaks.

What are the morphological features of an ideal Wright's or Leishman stained blood smear?

Indigo in color with uniform staining; no vacuoles in blood cells; red blood cells are pink; nuclei of WBCs are purple; eosinophilic granules are red-orange; and minimal precipitates are present.

How is WBC count estimated from a stained peripheral blood smear under high dry power ($40\times$)?

Count the number of WBCs across $10\,\text{fields}$ in an area where RBCs do not overlap, calculate the average per high-power field (HPF), and multiply by $2,000$ to obtain WBC per $\text{mm}^3$.

What automated counting technology is illustrated in this diagram?

Impedance counter (Coulter principle), where cells pass between two electrodes through a narrow aperture, producing impedance changes proportional to cell volume.

In flow cytometry counters, what cellular characteristics are measured by forward scatter and side scatter?

Forward scatter measures cell size/diameter, while side (multi-angle) scatter measures internal complexity and granularity.

What diluting fluid, pipette marks, and dilution factor are used for a manual red blood cell count?

Hayem's diluting fluid is used with an RBC pipette (containing a red bead). Blood is drawn to $0.5$ and diluting fluid to $101$, resulting in a $1:200$ dilution.

What squares are counted for a manual RBC count on a hemocytometer, and what is the calculation formula?

Five medium squares in the center grid (the 4 corner medium squares and 1 center medium square). Calculation: $\text{RBC (cu.mm.)} = \text{\# cells counted in 5 squares} \times 5\,(\text{area factor}) \times 10\,(\text{depth factor}) \times 200\,(\text{dilution factor}) = \text{\# cells} \times 10,000$.

What are the adult reference ranges for red blood cell counts in males and females?

Male: $4.5 - 5.9 \times 10^6/\mu\text{L}$; Female: $4.5 - 5.1 \times 10^6/\mu\text{L}$.

When collecting blood into capillary tubes for hematocrit, when are blue-tip versus red-tip capillets used?

Blue-tip capillets are used if the blood sample already contains anticoagulant; red-tip capillets are used if the sample is without anticoagulant.

What are the centrifuge settings and reference ranges for a microhematocrit determination?

Centrifuged for $5\,\text{minutes}$ at $10,000\,\text{rpm}$. Reference ranges: Male $41.5\% - 50.4\%$, Female $35.9\% - 44.6\%$.

In the automated cyanmethemoglobin method, what chemical reactions convert hemoglobin to cyanmethemoglobin?

Drabkin's solution contains ferricyanide, which oxidizes hemoglobin iron to methemoglobin; methemoglobin then binds cyanide to form cyanmethemoglobin, which is measured spectrophotometrically.

What point-of-care hemoglobin testing device is shown here, and what reagent system does it use to avoid toxic cyanide?

HemoCue (Hb 201+), which utilizes a sodium nitrite and sodium azide reagent system.

What manual hemoglobin method is demonstrated by this comparator apparatus?

Sahli's Acid Hematin method, where blood mixed with dilute hydrochloric acid converts hemoglobin into a brown acid hematin complex, diluted with distilled water to match the permanent glass comparator.

What are the principle and criteria for the Copper Sulfate specific gravity method for hemoglobin screening?

A drop of blood is dropped from about $1\,\text{cm}$ height into Copper Sulfate solution with a specific gravity of $1.053$. Blood samples with hemoglobin $> 12.5\,\text{g/dL}$ sink within $15\,\text{seconds}$.

What are the adult reference ranges for hemoglobin concentration in males and females?

Male: $14 - 17.5\,\text{g/dL}$; Female: $12.3 - 15.3\,\text{g/dL}$.

What are the formulas and reference ranges for MCV, MCH, and MCHC?

$\text{MCV} = \frac{\text{Hematocrit (\%)}}{\text{RBC count (millions/cu\,mm)}} \times 10$ (Reference: $80 - 96\,\text{fl}$); $\text{MCH} = \frac{\text{Hemoglobin (g/dL)}}{\text{RBC count (millions/cu\,mm)}} \times 10$ (Reference: $27.5 - 33.2\,\text{pg}$); $\text{MCHC} = \frac{\text{Hemoglobin (g/dL)}}{\text{Hematocrit (\%)}} \times 100$ (Reference: $33.4 - 35.5\,\%$).

What type of red blood cell is indicated by the curved arrow, characterized by a lack of central pallor and smaller diameter than a lymphocyte nucleus?

Spherocyte.

What poikilocyte is shown, featuring irregular projections with bulbous ends and no central pallor, associated with liver disease and abetalipoproteinemia?

Acanthocyte (spur cell).

What poikilocyte is pointed out here, characterized by central pallor and regular, relatively even-length projections, commonly associated with renal disease?

Echinocyte (burr cell).

What crescent-shaped, pointed red blood cell lacking central pallor is shown in this field?

Sickle cell (drepanocyte).

What abnormally shaped, fragmented red blood cells created by mechanical shearing in hemolytic anemia are shown in this smear?

Schistocytes.

What poikilocyte ('mouth cell') displays a slit-like central pallor, associated with alcoholism, drug toxicity, and Rh null disease?

Stomatocyte.

What poikilocyte, also known as a 'target cell' or 'Mexican hat cell', is shown here?

Codocyte (target cell), seen in hemoglobinopathies, iron deficiency anemia (IDA), and thalassemia.

What figure-of-eight RBC inclusion representing mitotic spindle remnants in megaloblastic anemia is indicated by the arrow?

Cabot ring.

What red blood cell inclusion consisting of fine blue specks of ribosomal RNA, seen in lead poisoning, is indicated by the arrow?

Basophilic stippling.

What round, dark purple nuclear remnants indicated by the arrows are characteristically seen after splenectomy or in asplenia?

Howell-Jolly bodies.

What immature red blood cells displaying a reticular network of ribosomal RNA under new methylene blue stain are shown here?

Reticulocytes (polychromatophilic erythrocytes).

What is the normal reference range for circulating reticulocytes and the formula for calculating reticulocyte percentage?

Normal range: $0.5 - 2.5\%$ (typically around $1\%$). Formula: $\text{Reticulocyte \%} = \frac{\text{\# reticulocytes per 1000 RBC}}{10}$.

On cellulose acetate hemoglobin electrophoresis at alkaline pH, what is the order of hemoglobin migration from cathode ($-$) to anode ($+\text{)}$?

From cathode to anode: Hb C, Hb S, Hb F, Hb A (mnemonic: 'Crawl, Slow, Fast, Accelerate').

What are the normal salt concentration ranges for onset and completion of hemolysis in the Osmotic Fragility Test?

Onset of hemolysis: $0.45 - 0.50\%\,\text{NaCl}$; Complete hemolysis: $0.30 - 0.33\%\,\text{NaCl}$.

According to the osmotic fragility curves shown, which condition shows increased osmotic fragility (shifted to the right of normal)?

Hereditary Spherocytosis (and Auto-immune Hemolytic Anemia).

What two standard tubes are used to measure the Erythrocyte Sedimentation Rate (ESR) over a one-hour period?

Westergren tube and Wintrobe tube.

What plasma and cellular factors accelerate or retard the Erythrocyte Sedimentation Rate (ESR)?

Factors that accelerate ESR: fibrinogen and cholesterol (counteract zeta potential). Factors that retard ESR: albumin and lecithin (reinforce negative zeta potential).

What is the principle of the manual white blood cell (WBC) count?

A sample of whole blood is mixed with a weak acid solution (acetic acid) that lyses red blood cells, and the white blood cells in a diluted volume are counted in a counting chamber (hemocytometer).

What diluting fluid is used in the manual WBC count, and what is its specific action?

Acetic acid (HAc), which acts as a weak acid solution that selectively lyses mature red blood cells while leaving white blood cell nuclei intact for counting.

To what mark is blood drawn, and to what mark is diluent drawn in a WBC diluting pipette?

Blood is drawn exactly to the $0.5$ mark, and diluting fluid is drawn immediately to the $11$ mark, yielding a dilution of $1:20$.

How long must the contents of the WBC pipette be mixed, and how many drops should be expelled before charging the hemocytometer?

The pipette is mixed for $3 - 5\text{ minutes}$, and the first $4\text{ drops}$ of unmixed, relatively cell-free fluid from the stem are expelled.

At what angle should the WBC diluting pipette be held when charging a hemocytometer, and how long must cells settle before counting?

The pipette is held at a $45^\circ$ angle to the junction of the cover glass and counting chamber, and the cells are allowed to settle for about $3\text{ minutes}$.

Which areas of the hemocytometer are counted for a manual WBC count?

The white blood cells are counted in the four large corner squares (often marked as "W" or "pink" areas).

What is the formula for calculating the total manual WBC count per microliter?

\text{WBC Count} = \text{# cells counted} \times 20 \times \frac{10}{4} = \text{# cells counted} \times 50

What is the normal reference value range for total leukocyte count in adults?

$4.4 - 11 \times 10^9/\text{L}$ ($4.4 - 11 \times 10^3/\mu\text{L}$ or $4,400 - 11,000\text{ cmm}$).

What are the procedural error limits for manual WBC hemocytometer counting?

There should not be a difference greater than $10\text{ cells}$ between each of the $4\text{ squares}$ counted, or greater than $20\text{ cells}$ between opposite sides of the hemocytometer.

When must a leukocyte count be corrected for nucleated red blood cells (nRBCs), and what formula is used?

When there are $5\text{ or more nRBCs}$ per $100\text{ white blood cells}$, using the formula: $\text{Corrected WBC} = \text{Uncorrected WBC Count} \times \frac{100}{\text{nRBCs per 100 WBCs} + 100}$

At what angle should the spreader slide be held against the smear slide when preparing a peripheral blood smear?

At an angle of $30^\circ - 40^\circ$.

What are the three essential physical features of an ideal peripheral blood smear?

It occupies $\frac{3}{4}$ of the length of the slide, has a thick and a thin area, and features a smooth, feathery edge with no holes or streaks.

What are the key microscopic features of an ideally stained peripheral blood smear?

The slide is indigo and uniform in color, red blood cells appear pink, white blood cell nuclei are purple, eosinophilic granules are red-orange, blood cells lack vacuoles, and there is minimal background precipitate.

Where on a peripheral blood smear should a differential WBC count be performed, and what counting pattern is used?

In the monolayer area near the feathery edge where red blood cells do not touch or overlap, using a serpentine (battlement) track until $100\text{ leukocytes}$ are counted.

How is a rapid WBC estimation performed on a stained peripheral blood smear under high dry power ($40\times$)?

Count the number of WBCs in $10\text{ high-power fields (HPFs)}$ in an area where RBCs do not overlap, determine the average per HPF, and multiply by $2,000$ to obtain the estimated WBCs per $\text{mm}^3$.

What are the standard finger assignments on a Schilling counter during a differential WBC count?

Index finger on neutrophil, middle finger on lymphocyte, ring finger on monocyte, and little finger on eosinophil.

What benign hereditary anomaly is shown here, characterized by hyposegmented, bilobed "pince-nez" neutrophil nuclei?

Pelger-Huët anomaly.

What are Döhle bodies and toxic granules in neutrophils?

Döhle bodies are pale blue ribosomal cytoplasmic inclusions, while toxic granules are dark, coarse azurophilic granules; both are seen in severe infections, burns, or inflammatory states.

What is the operating principle of electrical impedance automated cell counters, and what type of differential do they produce?

Blood cells suspended in an electrolyte pass one at a time through a narrow aperture between two electrodes. The resulting change in electrical resistance (impedance) is directly proportional to cell volume, producing a three-part differential (PMNs, lymphocytes, and monocytes).

What is the principle of flow cytometry cell counters, and what type of differential do they generate?

A single-cell stream is passed through a laser beam; forward light scatter (cell size) and side scatter at multiple angles (granularity and internal complexity) are measured, producing a 5-part WBC differential (neutrophils, basophils, eosinophils, lymphocytes, and monocytes).

What diluting fluid is used for manual RBC counting, and what is its composition/role?

Hayem's diluting fluid, which preserves red blood cells, prevents clumping, and provides an isotonic medium for counting.

In a manual RBC count using an RBC pipette, to what marks are blood and Hayem's fluid drawn, and what is the final dilution factor?

Blood is drawn to the $0.5$ mark and Hayem's diluent to the $101$ mark, resulting in a dilution factor of $1:200$ within the bulb.

On this hemocytometer grid, which squares are used for counting white blood cells and which are used for manual red blood cell counts?

The 4 large corner squares (marked "W") are used for the WBC count, and the 5 medium squares in the center grid (the 4 corners and the center square, marked "R") are used for the RBC count.

What rule is used when counting cells touching boundary lines on a hemocytometer grid to avoid double counting?

Cells touching or lying across the upper and left boundary lines are counted (included); cells touching the lower and right boundary lines are excluded.

What is the formula used to calculate the manual RBC count in $\text{cells/mm}^3$ from 5 medium hemocytometer squares?

\text{RBC} = \text{# cells in 5 squares} \times \text{area factor (5)} \times \text{depth factor (10)} \times \text{dilution factor (200)} = \text{# cells} \times 10,000

What are the normal reference ranges for the RBC count in adult males and females?

Males: $4.5 - 5.9 \times 10^6/\mu\text{L}$ ($4.5 - 5.9 \times 10^{12}/\text{L}$). Females: $4.5 - 5.1 \times 10^6/\mu\text{L}$ ($4.5 - 5.1 \times 10^{12}/\text{L}$).

What is the definition of hematocrit (Hct), and what are the microhematocrit centrifugation settings?

Hematocrit is the ratio of the volume of packed red blood cells to the total volume of whole blood. The microhematocrit is centrifuged for $5\text{ minutes}$ at $10,000\text{ rpm}$.

What is the clinical difference between blue-tip and red-tip capillary tubes used for microhematocrit?

Blue-tip capillets are plain/non-heparinized (used for blood already containing an anticoagulant like EDTA), whereas red-tip capillets contain heparin (used for capillary fingerprick blood collected directly without prior anticoagulant).

What three distinct layers appear in a centrifuged hematocrit tube from top to bottom?

1. Plasma (top)
2. Buffy coat consisting of white blood cells and platelets (thin middle layer)
3. Packed red blood cells (bottom layer)

What are the adult reference ranges for hematocrit in males and females?

Males: $41.5\% - 50.4\%$. Females: $35.9\% - 44.6\%$.

What chemical reactions occur in the automated cyanmethemoglobin method?

Blood is mixed with Drabkin's solution; potassium ferricyanide oxidizes hemoglobin iron ($\text{Fe}^{2+}$ to $\text{Fe}^{3+}$) to form methemoglobin, which then combines with potassium cyanide to produce stable cyanmethemoglobin measured spectrophotometrically.

What reagent system does the point-of-care HemoCue device use to avoid toxic cyanide?

A sodium nitrite and sodium azide reagent system, providing a quantitative readout within $15 - 60\text{ seconds}$.

What manual hemoglobin measuring instrument is pictured here?

Sahli Haemometer (used in the Sahli Acid Hematin method).

How does the copper sulfate screening method evaluate hemoglobin in blood donors?

A drop of blood is dropped from a height of $1\text{ cm}$ into copper sulfate solution with a specific gravity of $1.053$. If the blood hemoglobin level is $> 12.5\text{ g/dL}$, the drop will sink within $15\text{ seconds}$.

What are the normal adult reference ranges for hemoglobin in males and females?

Males: $14 - 17.5\text{ g/dL}$. Females: $12.3 - 15.3\text{ g/dL}$.

What is the formula and reference range for Mean Corpuscular Volume (MCV)?

$\text{MCV} = \frac{\text{Hematocrit (\%)}}{\text{RBC count (millions/cumm)}} \times 10$ Reference value: $80 - 96\text{ fl}$ (normocytic; $< 80\text{ fl}$ microcytic, $> 96\text{ fl}$ macrocytic).

What is the formula and reference range for Mean Corpuscular Hemoglobin (MCH)?

$\text{MCH} = \frac{\text{Hemoglobin (g/dL)}}{\text{RBC count (millions/cumm)}} \times 10$ Reference value: $27.5 - 33.2\text{ pg}$ (normochromic; $< 27\text{ pg}$ hypochromic).

What is the formula and reference range for Mean Corpuscular Hemoglobin Concentration (MCHC)?

$\text{MCHC} = \frac{\text{Hemoglobin (g/dL)}}{\text{Hematocrit (\%)}} \times 100$ Reference value: $33.4 - 35.5\%$ (normochromic; $< 33.4\%$ hypochromic).

What internal ruler on a peripheral blood smear is used to gauge RBC diameter?

The nucleus of a small, resting lymphocyte (normally approximately $7 - 8\,\mu\text{m}$, identical to the diameter of a normal normocytic erythrocyte).

What abnormal red blood cell morphology is shown, characterized by irregular projections with bulbous ends and no central pallor?

Acanthocyte (spur cell), commonly seen in severe liver disease and abetalipoproteinemia.

What abnormal red blood cell morphology is indicated by the arrow, characterized by regular, even-length spicules and central pallor?

Echinocyte (burr cell), frequently associated with renal disease or uremia.

What abnormal RBC poikilocyte is shown here, characterized by a slit-like, mouth-shaped central pallor?

Stomatocyte ("mouth cell"), seen in alcoholism, drug toxicity, and Rh null disease.

What poikilocyte is depicted here, also known as a target cell or "Mexican hat cell"?

Codocyte (target cell), commonly observed in hemoglobinopathies, thalassemia, and iron deficiency anemia.

What sheared, fragmented red blood cell morphologies are shown in this smear?

Schistocytes, formed by mechanical shearing across fibrin strands in microangiopathic hemolytic anemias.

What crescent-shaped poikilocytes lacking central pallor and ending in sharp points are shown here?

Sickle cells (drepanocytes), pathognomonic for sickle cell anemia.

What is a blister cell and with what disease is it characteristically associated?

An erythrocyte with a peripherally located, vacuole-like area of pallor resembling a blister; characteristically seen in Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency.

What figure-eight-shaped RBC inclusion is indicated by the arrow, and what does it represent?

Cabot ring; it represents remnants of the mitotic spindle and is found in megaloblastic anemia.

What erythrocyte inclusion is indicated by the arrow, and what clinical condition is it classically associated with?

Basophilic stippling (fine blue specks of aggregated RNA and ribosomes), classically associated with lead poisoning and thalassemias.

What small, round, dense nuclear remnants indicated by the arrows are seen in RBCs following splenectomy or in asplenia?

Howell-Jolly bodies.

What immature red blood cell stage is visualized using supravital stains such as new methylene blue, displaying a ribosomal RNA reticulum?

Reticulocyte (or polychromatophilic erythrocyte).

What is the normal circulating reference percentage and lifespan of reticulocytes?

Reticulocytes circulate for approximately $2\text{ days}$ in the bloodstream before becoming mature RBCs, comprising $0.5 - 2.5\%$ (typically about $1\%$) of total circulating erythrocytes.

What is the formula used to calculate the reticulocyte percentage?

\text{Reticulocyte \%} = \frac{\text{# reticulocytes per 1,000 RBCs} \times 100}{1,000} = \frac{\text{# reticulocytes per 1,000 RBCs}}{10}

On cellulose acetate alkaline hemoglobin electrophoresis, what is the migration pattern from cathode (-) to anode (+)?

From cathode (-) to anode (+): $\text{Hb C} \rightarrow \text{Hb S} \rightarrow \text{Hb F} \rightarrow \text{Hb A}$.

Which hemoglobin variants migrate together with Hb C and Hb S on cellulose acetate electrophoresis at alkaline pH?

At the Hb C band: $\text{Hb } A_2$, $\text{Hb E}$, $\text{Hb O}_{\text{Arab}}$, and $\text{Hb C}_{\text{Harlem}}$. At the Hb S band: $\text{Hb D}$, $\text{Hb G}$, and $\text{Hb L}$.

How do hemoglobin electrophoresis findings differ between Sickle Cell Trait and Sickle Cell Anemia?

Sickle Cell Trait shows both $\text{Hb A}$ and $\text{Hb S}$ bands (with $\text{Hb A} > \text{Hb S}$); Sickle Cell Anemia lacks $\text{Hb A}$ entirely and exhibits a prominent $\text{Hb S}$ band along with variable $\text{Hb F}$.

What are the hallmark electrophoresis findings in Beta-Thalassemia minor (trait) versus Beta-Thalassemia major?

Beta-Thalassemia trait shows an elevation in $\text{Hb } A_2$ ($> 3.5\%$); Beta-Thalassemia major shows markedly elevated $\text{Hb F}$ ($up to 90\%$ or more), elevated/variable $\text{Hb } A_2$, and markedly decreased or absent $\text{Hb A}$.

What is the biological principle of the Osmotic Fragility Test (OFT)?

It evaluates the RBC surface area-to-volume ratio by exposing erythrocytes to progressively decreasing concentrations of hypotonic saline (NaCl) until osmotic water influx causes cell swelling and lysis.