Hematology Final Exam Review

Cell Counts Using the Hemacytometer

• Formula for total count is: Total Count = \frac{\text{#cells counted} \times \text{dilution factor} \times \text{depth factor}}{\text{Area counted based on 1 large square (mm}^3)}

• Volume of area: 9 \times 0.1 = 0.9 \text{mm}^3

Hemoglobin Electrophoresis

• Cellulose Acetate (pH 8.4)

  • Patient sample is loaded at the cathode and migrates toward the anode (positive).

  • Patterns observed in normal, sickle trait, Hemoglobin D trait, SC disease, SE disease, and normal cord blood.

• Hemoglobin Composition

  • Hemoglobin A: (α2β2) Adult: >95%. Newborn-2 Years: 10-40%

  • Hemoglobin A2: (α2δ2)Adult: <3.5%. Newborn: <1%

  • Hemoglobin F: (α2γ2) Adult: 1-2%. Newborn to 2 years: 60-90%

  • Bart's Hemoglobin: 4 gamma chains

  • Hemoglobin H: 4 Beta Chains

• Citrate Agar Electrophoresis (pH 6.0-6.2):

  • Used to differentiate hemoglobins.

  • Patterns for normal, sickle trait, Hemoglobin D trait, SC disease, SE disease, normal cord blood and C-Harlem trait are assessed.

WBC Differentiation

• Myelocyte: The last granulocyte to undergo mitosis.

• Primary Granules (Non-Specific): First seen in Promyelocyte; contains Myeloperoxidase.

• Secondary Granules (Specific): First observed in the Myelocyte; contains lysozymes.

Cell Differentiation

• Band

• Monocyte

• Myeloblast vs. Lymphocyte

• Lymphocyte vs. Monocyte

Module 1 Questions

• Question 1: Patient with many large cells, high N/C ratio, multiple nucleoli, no granules, and fine nuclear chromatin likely has Leukemia.

• Question 2: Patient with 3% cells with huge red-orange granules and segmented nucleus likely has Parasitic Infection/Allergy.

• Question 3: Which cells can be found in the blood of a normal healthy adult? Possible correct answer is: Plasma Cell.

Module 4 Questions

• Question 1: A 5-year-old with WBC 9,000/µL and 75% lymphocytes, with one reactive lymphocyte per 100 WBCs, likely has a normal condition or Probable bacterial infection, or Probable infectious mono.

Module 2 Questions

• Question 1: Indicate if cells are usually found in both peripheral blood and bone marrow or only in bone marrow:

  • Plasma Cells: Bone Marrow

  • Lymphocytes: Peripheral Blood and Bone Marrow

  • Macrophage with iron: Peripheral Blood and Bone Marrow

  • Bands: Peripheral Blood and Bone Marrow

  • Rubricytes: Bone Marrow

  • Heinz Bodies: Peripheral Blood and Bone Marrow

  • Segmented neutrophils: Peripheral Blood and Bone Marrow

  • Metarubricytes on a newborn: Peripheral Blood

• Question 2: Hematocrit of 37% is normal for an adult female. Reference ranges: Female: 35-49%, Male: 40-54%, Child: 36-46% (4-7 years), 35-49% (8-13 years).

• Question 3: NOT Typically Part of a Differential: Estimation of red cell number.

• Question 4: When red cell morphology shows agglutination, the following would be affected on the automated cell counter: Red Count.

Corrected WBC Calculation

• Normal Newborn N-RBC range: 2-24/100 WBC

• It is not normal to see N-RBC in adult peripheral blood.

• If more than 5 N-RBC are seen on peripheral smear, must correct the count.

• Corrected WBC Formula: Corrected WBC = \frac{WBC \times 100}{(\text{#NRBC} + 100)}

• Reported in #cells/µL.

Module 1 Question

• Question: A patient has 5 orthochromic normoblasts (metarubricytes) in the peripheral blood per 100 white blood cells. Which of the following can be said regarding the normalcy of this finding? Normal for a newborn; abnormal for an adult

Module 4 Question

• Question: If a patient has a white count of 1,000/µL with 60% neutrophils, what term would be correct? Absolute neutropenia. Calculation: 1,000 \times 0.60 = 600

• Note: Absolute neutropenia.

• Absolute reference range:

  • Neutrophil: 1700-7500

  • Lymphocyte: 1000-3200

  • Monocyte: 100-1300

  • Eos:0-300

  • Baso: 0-200

Absolute Cell Count Example

• Example: Patient has a white count of 150,000/µL with 40% neutrophils. Term: Absolute neutrophilia. Calculation: 150,000 \times 0.40 = 60,000

• Absolute reference range:

  • Neutrophil: 1700-7500

  • Lymphocyte: 1000-3200

  • Monocyte: 100-1300

  • Eos:0-300

  • Baso: 0-200

M:E Ratio (Bone Marrow)

• Myeloid to Erythroid Ratio.

• Includes Myeloid cells and Erythroid cells but not lymphoid.

• Normal: 3:1.

• Decreased in Erythroid Hyperplasia: 1:1.

• Increased in Myeloid Leukemia: 20:1.

Tests for Diagnosis: Myeloproliferative/Chronic Myeloproliferative Disorders

• Clinical presentation for all: fatigue, malaise, bone pain, splenomegaly, and symptoms related to cell counts.

Module 4 Question

• Question: A low Leukocyte Alkaline Phosphatase (LAP) may help distinguish: CML from a leukemoid reaction.

AML (Acute Myeloid Leukemia)

• All acute leukemias have variable WBC with decreased platelet.

• Patient presents with bruising.

• Prognosis is still poor; remission is between 8-18 months.

• FAB classification >30% Blasts in Bone Marrow.

• Hypercellular BM.

• Most common type of leukemia in adults.

• Incidence increases with age.

Tests for Diagnosis: Cytochemical Stains

• Cytochemical Stains for Acute Leukemia include: Sudan Black; Peroxidase, Alpha-Naphthyl Acetate Butyrate, Non-Specific Esterase, Naphthol-AS-D Chloroacetate, Periodic Acid Schiff (PAS), Specific Esterase

Module 4 Question

• Question: Acute leukemia almost always has which of the following blood counts? Low platelet count.

Tests for Diagnosis: Other Leukemias

• ALL (L1-L3):

  • TdT, PAS, and CALLA positive.

  • Mostly blasts with one nucleolus and scanty cytoplasm.

  • Best chance for survival for acute leukemias.

  • L1-seen in children; present with bruising.

• CLL:

  • Best prognosis of all leukemias among adults.

  • Majority of cells are lymphocytes found in peripheral blood.

  • Small, mature lymphocytes with smudge cells.

  • High WBC with low/normal PLT.

  • Found in older adults.

• Hairy Cell Leukemia:

  • Pancytopenia with hairy cells.

  • Dry bone marrow type.

  • TRAP positive (Tartrate Resistant Phosphatase).

Multiple Myeloma

• Malignant plasma cells with high serum immunoglobulins.

• Found in older adults.

• Observe punched-out bone lesions.

• Increased calcium and protein.

• Increased IgA and IgG.

• Peripheral pancytopenia with rouleaux.

• Abnormal plasma cells in bone marrow (Flame, Mott, Grape Cells).

Lymphoma

• Hodgkin’s Lymphoma:

  • Found in younger patients.

  • Reed-Sternberg cell found in Bone marrow.

  • Present with fever of unknown origin, enlarged nodes.

  • Blood counts often normal.

  • Good prognosis.

• Non-Hodgkin’s Lymphoma:

  • Found in Older patients.

  • Many different varieties.

  • Blood is abnormal only if bone marrow is abnormal.

  • May see Butt Cells.

  • Immature Bizarre lymphoid cells.

Module 4 Question

• Question: What is the typical blood picture in lymphoma without bone marrow involvement? Normal.

Qualitative WBC disorders

• Infections and Burns:

  • Toxic granulation.

  • Dohle Bodies.

  • Vacuoles.

Reactive Lymphocytes

• WBC will be high to normal.

• Relative and absolute lymphocytes increase.

• Know the description for reactive (atypical) lymphocyte:

  • Nucleus is often stretched out.

  • Ballerina skirt.

  • May have granules.

  • Monospot and heterophile positive.

  • Cold agglutinin may be present.

  • RBC agglutination.

Module 3 Question

• Question: A patient has a hemoglobin value of 7.8 g/dL. The patient's MCH will be? Could be low, normals, or high

Macrocytic Anemia

• Megaloblastic Anemia

  • Defective DNA synthesis from B12 or Folate poor diet.

  • Bone marrow will show asynchrony, karryorhexis, giant metamyelocytes and bands.

  • Hypersegmented Neutrophils.

  • Pancytopenia.

  • Treat with replacement vitamin therapy.

  • \downarrow Retic

  • \uparrow Direct

  • \uparrow Indirect bilirubin and LD

• Pernicious Anemia:

  • Lack of intrinsic factor which is involved in the absorption of B12.

  • Schilling Test.

Microcytic Anemia

• Impaired Hemoglobin Synthesis

  • Iron deficiency (Hypo, Micro)

    • Chronic blood loss the most common reason in adults

    • Poor diet

    • \downarrow Serum Fe

    • \uparrow TIBC

    • \downarrow Ferritin

    • Retic increases only after therapy

  • Anemia of Chronic disease

    • \downarrow Serum Fe

    • \downarrow TIBC

    • \uparrow Ferritin

    • Treat underlying condition first GI Bleed

  • Beta Thalassemia

    • Hypo,micro

    • Beta Chains reduced or absent

    • Increase in alpha chains

    • Common in Mediterranean, South East Asians, and Black

    • Peripheral blood: Target cells Basophilic stippling

    • Electrophoresis \uparrow A2 and F

  • Alpha Thalassemia

    • Hypo,micro

    • Hydrops fetalis (alpha- thalassemia major) : 4 gene deletion (Incompatible with life)

    • Hgb H (4 Beta chains): 3 gene deletion

    • Alpha Thalassemia Minor: 2 gene deletion

    • Silent Carrier: single gene deletion

    • Hg A, A2, and F are absent due to lack of alpha chains

    • Hgb Barts present ( 4 Gamma Chains)

    • Peripheral blood: Target cells Basophilic stippling

Module 3 Question

• Match the following:

  • Microangiopathic Anemia: Serious life threatening disease.

  • Sickle Cell Trait (Hemoglobin AS): No or minimal clinical disease.

• Sports Anemia (march Hemoglobinuria):

  • Sports Anemia (march Hemoglobinuria): Loss of 10% of total blood from an acute bleed

  • Alpha Thalassemia 4 gene deletion

Module 3 Question

• Which anemia would produce the most polychromasia on the blood smear? Pernicious Anemia.

Module 3 Questions

• Beta Thalassemia Major: Reduced synthesis of structurally normal globin chains.

• Hb SC Disease: Structural defect ( amino acid substitution) in globin chains.

• Paroxysmal Nocturnal Hemoglobinuria: Cells sensitive to lysis by complement.

• Microangiopathic Anemia (DIC): Cells fragmented by fibrin in vessels.

Module 3 Question

• Which of the following is a product involved in heme synthesis? Coproporphyrin.

Hemoglobin Breakdown

• Macrophage

• Iron (Fe)

• Unconjugated bilirubin

  • Circulation

  • Conjugated bilirubin

    • Liver

      • Some reabsorbed into circulation

    • Kidney

      • Urine urobilinogen: normal

      • Urine bilirubin: negative

    • Intestine

      • Urobilinogen

      • Fecal urobilinogen: Normal

• Red blood cell

  • Heme or metheme

    • Unconjugated bilirubin

Other Anemias

• Hemolysis

  • \downarrow Haptoglobin

  • \uparrow Bilirubin

  • \uparrow LD

  • \uparrow Retics

• Intravascular Hemolysis

  • Hemosiderin in urine

  • Urine Free Hemoglobin

• Hereditary Spherocytosis

  • Inherited defect in spectrin

  • Decreased cell surface

  • Peripheral smear: Spherocytes (MCHC >36%) Polychromasia

  • \uparrowOsmotic fragility with hemolysis beginning at a NaCl concentration >0.5\%.

Module 3 Question

• Question: Select the disorder that best fits this case:

  • F.M. is a 50-year-old white female experiencing shortness of breath, numbness, yellow tinge. Lab results: HGB= 4.7, HCT = 13.6%, RBC = 1.1 x 10 6/µL, WBC = 2.0 x 10 3/µL, PLT = 87.0 x 10 3/µL. Pernicious Anemia

Module 3 Question

• Question: At 0.55% NaCl there was a tinge of color in the supernatant and at 0.35% NaCl there was a dark red color with no red cell button. This osmotic fragility test is indicative of: Hereditary Spherocytosis.

Other Anemias

• Paroxysmal Nocturnal Hemoglobinuria

  • Clonal stem cell disorder with cells sensitive to complement lysis

  • Red urine

  • Urine hemosiderin +

  • Sucrose + (Sugar Water)

  • Ham’s test +

• Auto Immune Hemolytic Anemia

  • Warm

    • Secondary to autoimmune

    • Idiopathic

    • Peripheral Smear: +DAT, Spherocytes, Polychromasia

• G-6-PD

  • Sex-linked, decrease in enzyme

  • Oxidant drugs cause hemoglobin to denature and precipitate

  • Most often occurs in black males

  • Heinz bodies seen with Methylene Blue

• Pyruvate Kinase

  • Reduced ATP production results in alterations of RBC membrane, failure of cation pumps causing potassium loss as well as sodium loss, which leads to cell dehydration

  • Peripheral Smear: Echinocytes

• Lead Poisoning

  • Usually Normochromic, Normocytic

  • Chronic exposure can lead to Hypochromic, Microcytic

  • Basophilic Stippling

  • Lead will interfere with incorporation of iron into protoporphyrin IX, which will lead to excess iron and protoporphyrins

• Sickle Cell

  • Sickle Cell Beta Chain substitution ( β7[A3]Glu→Val)

  • Cells form rods at low O_2

  • Sickle Dex +

  • Confirm with Hb electrophoresis

  • Peripheral Smear: Sickle Cells Target Cells

Significance of Reticulocytes

• Increased in our hemolytic anemias

• Decreased in untreated anemias due to nutritional deficiencies Iron deficiency Megaloblastic

• Absolute Retic Count: \frac{\%Retic \times RBC Count}{ 100} = # cells/μL

• Corrected Retic Count: \frac{\%Retic \times (Patient HCT\%/45)}{}

Module 3 Question

• Low or normal reticulocyte count (untreated): Iron Deficiency Anemia.

• Decreased hemoglobin and hematocrit: Both.

• Low MCV and MCHC: Iron Deficiency Anemia.

• Hypersegmented neutrophils: Pernicious Anemia.

• Schilling test to diagnose: Pernicious Anemia.

RBC Inclusions

• Howell Jolly Body: DNA; seen in post-splenectomy, megaloblastic anemias, some hemolytic anemias

• Reticulocyte: RNA seen with New Methylene Blue associated with decreased RBC survival/hemorrhage, erythroid hyperplastic marrow, Referred to as Polychromasia when seen on Wright Giemsa Stain

• Pappenheimer body: Iron; seen in sideroblastic anemia, thalassemia

• Heinz Body: Precipitated Hemoglobin; seen in G6PD deficiency, Can only be seen with supravital stain

• C Crystal: Crystalized hemoglobin C

• Ringed Sideroblast: Iron

• Basophilic stippling: RNA aggregates; seen in thalassemia and lead poisoning

• NRBC: DNA

Module 2 Question

• Indicate the stain most often used to observe the following:

  • Howell Jolly Bodies

  • Reticulocytes

  • Polychromasia

  • Heinz Bodies

  • Pappenheimer Bodies

  • Siderocytes

  • Basophilic stippling

ESR (Erythrocyte Sedimentation Rate)

• A mechanism for cells falling (settling)

• A screening test, not diagnostic at all

• Factors affecting:

  • Serum Fibrinogen/protein \uparrow

  • Sickle Cell \downarrow

  • Polycythemia and PV \downarrow

  • Rouleaux \uparrow

  • Serum: Inflammation, infections, malignancies \uparrow

  • Cold temperature \downarrow

Module 2 Question

• All of the following cause an elevated ESR EXCEPT: Polycythemia

Directly Measured Automated Parameters

• WBC: Impedence, All cells >35 fL are counted, Cells are categorized by pulse height, Cytoplasm is collapsed around nucleus to differentiate lymph, mono, granulocyte

• RBC: Impedence, Cells >36 fL are counted, WBC are included in this count!!

• Platelet: Impedence, Counted in same bath as RBC, Anything between 2-20fL, MPV.

• Hemoglobin: Cyanmethemoglobin Method, WBC bath containing lyse drains into hemoglobin cuvette, Light passes through cuvette, Read at 525nm

Calculated Parameters

• HCT (RBC x MCV)/10 Lower than manual due to no trapped plasma

  • 37-47% Females

  • 42-52% Males

• MCV Measured from the pulse height, Calculated from area under RBC curve (HCT/RBC) \times 10 80-100 fL

• MCH (HGB/RBC) \times 10 27-31pg

• MCHC (Hgb/Hct) \times 100 31-35%

• RDW red cell distribution curve width 12-15%

Module 2 Question

• Which of the following would have a normal MCH? Macrocyte. MCHC correlates the hemoglobin content with the volume of the cell. Macrocytes will have increased MCH, but normal MCHC.

Module 3 Question

• Which of the following would have a low MCH? Thalassemia trait.

Module 2 Question

• Red count = 3.80 \times 10^6/µL, Hemoglobin = 15.0 g/dL, Hematocrit = 37 %, MCV = 97 fL. Are these results reportable?

Module 2 Question

• Which of the following is most responsible for maintaining the biconcave shape of RBCs? Peripheral Proteins (spectrin).

Module 2 Question

• How is the hematocrit determined on most automated cell counters? Calculated using red count and MCV; HCT= (RBC x MCV)/ 10

Module 2 Question

• Which of the following measurements must have the red cells lysed? Hemoglobin.

Module 2 Question

• Polychromasia would most likely be associated with: Acute blood loss, a couple of days later.

Automated Histograms

• WBC Histogram Cytoplasm has been collapsed

  • 35-90fL: Lymphs

  • 90-160fL: Mononuclear Cells (Monocytes, Blasts, Immature Granulocytes, reactive lymphs)

  • 160-450fL: Granulocytes

• WBC Histogram Review Criteria

  • R1 Lymphocyte population does not begin at baseline (35fL) Clumped or Giant Platelets, NRBC, Intracellular parasites

  • R2 No valley between lymphocyte and mononuclear population (90fL) Variant Lymphs or Blasts

  • R3 No valley between mononuclear and granulocyte population (160fL) Immature granulocytes, eosinophilia, Basophilia

  • R4 Granulocyte population does not return to baseline (450fL) Granulocytosis

  • RM Interference detected in multiple positions

Interfering Substances for Cell Counters

• The slide has a detailed table outlining how various interfering substances affect WBC, RBC, HGB, MCV, RDW, and PLT counts. Substances include High Glucose, Clumped or large Platletes, Hemolyzed Specimen, Lipemic Specimen, NRBCs, Platelet Satellites, RBC agglutination, RBC fragments, and Tiny RBCs

Module 3 Question

• Which of the following will NOT affect the automated hematocrit? Lipemia. HCT= (RBC x MCV)/ 10

Module 5 Question

• What should be done before reporting these results? Warm the sample to 37 degrees and rerun.

Module 5 Question

• A patient with CML has a WBC of 300 \times 10 ^3/µL on the cell counter. Which of the following results would be correct and would require no further testing. PLT

Module 4 Question

• A 78-year-old female with severe vomiting and diarrhea. Admission hematocrit was 35%, next morning 30%. Cell counter controls were within 2SD. The doctor called concerned. The best course of action: Report the results-it makes sense in view of the therapy.

Plasma to cell Volume

• Normal, Anemia, Polycythemia, and dehydration visual examples of haematocrit levels.

Miscellaneous Items to Review

• Anisocytosis: Refers to variation in cell size.

• Poikilocytosis: Refers to a variation of RBC shape.

• Know how to calculate the indices and normal ranges for each MCV, MCH, MCHC.

• Be able to classify the anemia based on these results Prussian Blue stains Iron Be able to calculate absolute values

Review Questions

• What is the most likely cause of polychromasia on a blood smear? Reticulocytes

• What will interfere with hemoglobin determination on the Coulter analyzer? Lipemia, High WBC, decreased RBC lysing reagent

• Will spherocytes interfere with the hemoglobin concentration? No!

• Where will NRBC be indicated on the Coulter WBC histogram? Before the lymphocyte region

• What cells are increased in Myeloid Hyperplasia? Blasts, Pros, Myelocytes, Metas, Bands and Segs

• What would the M:E ratio be in myeloid hyperplasia? Increased

• An 80 year old male with 98% lymphs, 2% segs and smudge cells most likely has? CLL

• Which leukemia is associated with the Philadelphia Chromosome? CML

• Which leukemia is found in children? ALL

• What must RBCs be able to do to escape the macrophages in the spleen? Change their Shape

• What characteristic distinguishes a myeloblast from a lymphocyte? Nuclear clumping

• Are sickle cells a normal finding in adults? No!

• Is an occasional echinocyte a normal finding in adults? Yes!

• Could a differential be performed on a dried, stained slide without gloves? Yes.

• What is the defect in Beta Thalassemia Major? Rate of globin chain synthesis

• What is the defect in Hemoglobin SC disease? Structural defect in globin chain

• What is the problem in microangiopathic anemia? Cells fragmented by fibrin in the vessels

• What is the problem in PNH? Cells are sensitive to lysis by complement

• What is the best test to distinguish IDA from anemia of chronic disease? Ferritin

• What is the best test to distinguish PV from secondary polycythemia? Oxygen saturation

• Which AML is more likely to develop DIC? M3

• If a patient has peripheral smear containing blasts that react with the non- specific esterase stain, a positive murimadase, and bleeding gums, what is the most likely leukemia? M5 (monocytic)

• Which cell has the highest nuclear to cytoplasm ratio? Myeloblast

• Which cell has a round nucleus and specific granules? Myelocyte

• Which cell has the most condensed chromatin clumping? Segmented neutrophil

• Which WBC has nucleoli with no granules? Myeloblast

• Will the presence of urine hemosiderin indicate extravascular hemolysis occurring in the spleen? No.

• If 100 particles are counted in the center large square of a hemacytometer and the dilution is 1:10, how many particles are present in 1 µL? 100 X 10 X 10/1= 10,000/µL

• If a patient is found to have dimorphic RBCs and ringed sideroblasts in the bone marrow, what is the probable diagnosis? Sideroblastic Anemia

• Administering Chloramphenicol often causes what? Aplastic Anemia

• A TB patient that has both \downarrow Iron and \downarrow TIBC has what? Anemia of chronic disease

• A patient that has TTP, thrombocytopenia, and shistocytes has what? Microangiopathic Anemia

• A patient that has an increased osmotic fragility result, increased retic and \uparrow MCH and MCHC has? Hereditary Spherocytosis

• Why should Pelguer-Huet anomaly be identified? Distinguish from a left shift

• An older person with a massive spleen, decreased WBC with left shift, a rare blast, occasional NRBC, and tear drop shaped RBC most likely has what? Myelofibrosis

Iron Studies

• A table provides differential diagnosis of hypochromic, microcytic anemias based on RDW, Serum Fe, TIBC, Ferrtin, FEP, Electrophoresis, Marrow Sideroblasts, and PBS findings for Iron deficiency, Thalassemia, Chronic disease, Sideroblastic, and Lead poisoning.