Hematology Unit 1 Exam

Hematopoiesis

The dynamic process of blood cell production and development of the various cells of the blood

Bone Marrow Function

• main site of cell production in fetus (from 2-7 months) 

• Primary site of hematopoiesis

Core functions:

• Generating rbcs for O2 transport

• Producing platelets for clotting

• Creating all leukocytes, including lymphocyte precursors

• Serving as a primary lymphoid organ where B-cells mature and become immunocompetent

• Provide stem cell reservoir (hematopoietic stem cells) from which all blood lineages derive

Spleen Function

• Second largest lymphoid organ and serves as a blood filter and immune surveillance hub

• Filters the blood: removes old, damaged, or abnormal rbcs

• Immune surveillance: traps blood-born Ag and presents them to lymphocytes

• Mounts immune responses to blood-borne pathogens (bacteria, viruses, parasites)

• Stores a reserve of monocytes and platelets

• In fetal life, major site of hematopoiesis

Liver Function

• Primary site of fetal hematopoiesis before the bone marrow takes over

• Produces majority of complement proteins

• Synthesizes acute-phase proteins during inflammation

• Houses Kupffer cells: resident macrophages that phagocytose pathogens and debris from portal blood

• Metabolizes and clears immune complexes and endotoxins

• Produces clotting factors essential for hemostasis

Thymus Function

• Primary lymphoid organ critical for T-cell development and self-tolerance

• Site where T-cell precursors mature into functional, immunocompetent T-cells

• Oversees positive selection — ensuring T cells can recognize self-MHC molecules

• Oversees negative selection — eliminating T cells that react too strongly to self-antigens (preventing autoimmunity)

• Exports mature naïve T cells to the peripheral circulation

• Most active during childhood; undergoes involution (shrinkage) with age, though it retains some function throughout life

Lymph Nodes

• Lymph nodes are small, bean-shaped secondary lymphoid organs distributed throughout the body along lymphatic vessels:

• Filter lymph fluid, trapping pathogens, cancer cells, and foreign antigens

• Serve as sites of antigen presentation — where dendritic cells present antigens to T and B cells

• Facilitate adaptive immune responses — clonal expansion of antigen-specific lymphocytes occurs here

• Produce antibody-secreting plasma cells (from activated B cells) and effector/memory T cells

• The characteristic swelling of lymph nodes during infection ("swollen glands") reflects active immune responses

Reticuloendothelial system (RES)

• The reticuloendothelial system (RES) — also called the mononuclear phagocyte system (MPS) in modern terminology — is a diffuse network of phagocytic cells and supporting stromal (reticular) cells distributed throughout the body.

• Rather than a discrete anatomical organ, it is a functional system spanning multiple organs and tissues, unified by its roles in hematopoiesis, waste clearance, and immune defense

RES and Cell Production

• Hematopoiesis is the generation of all cellular blood components from hematopoietic stem cells (HSCs). The RES both produces and supports this process through reticular stromal scaffolding and cytokine signaling.

• Red bone marrow = site of adult hematopoiesis

• Role of Reticular Stroma

• The reticular cells and fibers of the bone marrow, lymph nodes, and spleen create the microenvironmental niche (hematopoietic niche) that:

• Physically anchors developing blood cells

• Provides growth factors (SCF, IL-3, GM-CSF, EPO, TPO)

• Regulates the balance between self-renewal and differentiation

RES and Cell Destruction

• The RES is the body's primary system for removing aging cells, debris, pathogens, and abnormal cells through two overlapping mechanisms.

• Phagocytosis: the active engulfment and degradation of particles by professional phagocytes — chiefly macrophages, monocytes, neutrophils, and dendritic cells.

• Mechanism: Recognition, opsonization, engulfment, killing, processing (apc activity)

• Apoptosis (programmed cell death): the intrinsic death program of cells — but the RES plays a critical role in recognizing and clearing apoptotic cells (efferocytosis) before they become inflammatory

• Key RES sites and phagocytic roles in photo

How the RES handles apoptosis

• Apoptotic cells display "eat-me" signals 

• RES macrophages recognize these via receptors (integrins) and phagocytose the cell corpse silently, without triggering inflammation

• This is critical in the thymus, where ~95% of developing T cells undergo apoptosis during negative/positive selection — thymic macrophages and dendritic cells rapidly clear these corpses

• In germinal centers of lymph nodes, B cells that fail to bind antigen with sufficient affinity undergo apoptosis and are cleared by tingible body macrophages (macrophages visible with engulfed nuclear debris)

• Failure to clear apoptotic debris efficiently (as in lupus) can lead to autoimmune disease

RES and immunologic defense

• RES sits at the intersection of innate and adaptive immunity, both executing immediate defense and orchestrating long-term immune responses

Bridge to Adaptive Immunity - Ag presentation

• A defining immunologic role of the RES is antigen processing and presentation, linking innate sensing to adaptive (T and B cell) responses:

1. Dendritic cells (the most potent APCs) in skin, mucosa, and tissues capture antigens

2. They migrate to lymph nodes and spleen — both RES organs

3. Antigenic peptides are loaded onto MHC class I (for CD8⁺ T cells) or MHC class II (for CD4⁺ T helper cells)

4. Macrophages in lymph nodes and spleen present antigen and provide co-stimulatory signals

5. This initiates clonal expansion of antigen-specific T and B cells → effector cells and immunological memory

Describe the function of nuclear and cytoplasmic cell structures (e.g., chromatin, nucleolus, mitochondria, Golgi, lysosomes, ribosomes).

• Generally, they regulate gene expression and produce proteins necessary for cellular survival. 

• Chromatin: packaged DNA and proteins that condense into chromosomes during cell division

• Nucleolus: sub-structure

• Mitochondria: generates most of the cell’s ATP for energy

• Golgi: packages proteins and lipids for delivery to designated locations

• Lysosomes: break down worn-out cell parts, foreign invaders like bacteria, and biomolecules (proteins, lipids, and carbohydrates) into simpler building blocks for the cell to reuse or expel

Ribosomes: make proteins based on instructions delivered from nucleus

erythropoiesis

The production of red blood cells in the bone marrow

Describe factors involved in the regulation of erythrocyte production, including substances needed for erythropoiesis and hemoglobin synthesis

• Production of rbcs is controlled by rate of oxygen delivery

• Regulated by EPO, EPO response takes ~5 days

• Androgens influence RBC production

Leukopoiesis

Production of leukocytes (WBCs)

Neutrophils

• the primary first responders of the innate immune system. Produced daily in the bone marrow, they circulate in the blood and quickly migrate to sites of infection or injury to destroy invading pathogens and clear cellular debris

• Circulation is less than 24 hours, but lifespan can be 3-5 days in tissues.

• Differential percentage range: 45-75%

Eosinophils

• fighting parasitic infections, regulating allergic responses, and maintaining tissue homeostasis

• Circulate 8-18 hours, but can live in tissues for 2-5 days

• Differential percentage: 0-6%

Basophils/Mast Cells

• release active chemical granules—most notably histamine and heparin—to defend the body against parasites, manage allergic reactions, and regulate inflammation. One of the first responder cells for immune system. 

• Circulate 24-48 hours, so 1-2 days

• Differential percentage: 0.2%

Monocytes/Macrophages

• engulfing and destroying invading pathogens (phagocytosis), regulating inflammatory responses, cleaning up dead or damaged cells, and presenting antigens to activate the adaptive immune system

• Circulate 1-3 days, then live in tissues for months to years

• Differential percentage: 1-12%

Lymphocytes

• identify, target, and destroy pathogens (bacteria, viruses), abnormal cells (cancer), and toxins. They also retain "memory" of past infections to prevent you from getting sick from the same threat twice. B-cells and T-cells

• Lifespan: weeks to months, but memory cells can last years

• Differential percentage: 15-45%

Plasma Cells

• secrete massive quantities of immunoglobulins (antibodies) to neutralize pathogens, playing a central role in humoral immunity

• A few days to months, but long-lived plasma cells in bone marrow can live from decades to an entire lifetime

• Differential percentage 1-3%

Thrombopoiesis

the physiological process of blood platelet (thrombocyte) formation in the bone marrow, primarily driven by the hormone thrombopoietin (TPO).

Platelet lifespan and pools

• Platelets live 7-10 days

• Circulating pool: Makes up about 70% of the total platelet mass. These are the platelets actively flowing in the bloodstream

• Splenic pool: Makes up the remaining 30%. The spleen acts as a dynamic reserve that freely exchanges with the circulating pool

Platelet function

clot

List the common specimen types analyzed in Clinical Hematology

Blood, bone marrow, synovial fluid, pleural fluid, serous fluid

Explain the action of the anticoagulants most commonly used to obtain whole blood or plasma specimens for Clinical Hematology testing

• EDTA: prevents blood coagulation by chelating calcium (removing it from blood)

• Sodium Citrate: works by chelating (binding to) calcium ions in the blood, which inhibits the clotting cascade and prevents the blood from coagulating outside the body

Routine Tests for Clinical Hematology

HGB: 

• Male: 13.0 - 17.0 g/dL

• Female: 11.5 - 15.5 g/dL

• Report to nearest 10th

HCT: 

• Male: 37.0 - 51.0%

• Female: 35.0 - 46.0%

• Report to nearest 10th

RBC: 

• Male: 4.40 - 5.80 x 10^6/uL or M/uL or M/cmm

• Female: 3.80 - 5.20 x 10^6/uL

• Report to nearest 100th

WBC: 

• Adult: 4.0 - 11.0 x 10^3/uL or K/uL or K/cmm

• Report to nearest 10th if decimal (e.g. 7.7 x 10^3/uL)

• Report to nearest hundred if no decimal (e.g. 7,700/uL)

PLT: 

• 150 - 45 x 10^3/uL or K/uL or K/cmm

• Report to nearest thousand (e.g. 150,000/uL or 150 x 10^3/uL)

RBC Indices:

• MCV: 82.0 - 98.0 fL → report to nearest 10th 

• MCH: 27.0 - 33.0 pg → pg = picograms

• MCHC: 32.0 - 36.0%

Evaluate specimen requirements for each routine test performed in Clinical Hematology (e.g., preferred anticoagulant for cell counts), including stability times and minimum blood draw requirements

Stability Time

• Analyze within 24 hours, otherwise, store at 2-8°C if testing is delayed

Blood Draw Requirements: 

• EDTA, purple top tube

• Venipuncture is preferred

Specimen Type: whole blood, no clots or hemolysis

Molecular structure, synthesis (e.g., protoporphyrin) and breakdown/catabolism of hemoglobin

Molecular structure:

• B-chains, a-chains, Heme, iron, polypeptide chains

• Major 3 are protoporphyrin, iron, and globin

Synthesis: 

• 65% is synthesized during nucleated RBC maturation

• 35% during reticulocyte stage

Hemoglobin requires the following for production:

• Adequate iron delivery and supply

• Adequate synthesis of protoporphyrin (heme precursor)

• Adequate globin synthesis

Catabolism/Breakdown

• Protoporphyrin ring of Heme is cleaved and is broken down to bilverdin, then is reduced to unconjugated bilirubin

• Albumin transports unconjugated bilirubin to liver and converts it to conjugated bilirubin

• Conjugated bilirubin is excreted into the bile and then reduced by bacteria in the colon into urobilinogens. 

• Iron is released, bound to transferrin, and recycled for hgb production or goes into storage

• Globin polypeptides are hydrolyzed to amino acids and returned to amino acid pool for protein synthesis

Iron metabolism and iron pools/compartments, including storage forms (e.g., ferritin) and the role of the transport protein transferrin

Iron Metabolism:

• Iron is released, bound to transferrin, and recycled for hgb production or goes into storage

Iron Compartments

• ⅔ hemoglobin (largest storage)

• Ferritin = storage iron

• Myoglobin

• Tissue Iron (cytochrome enzymes)

• Transport proteins (smallest storage) → bound to transferrin

Storage forms

• ferritin

Role of transport protein Transferrin

• Dietary Iron (ferric) → reduced to ferrous (Fe2+) by HCl → ferrous absorbed through intestinal mucosa oxidized back to ferric → ferric is transported by transferrin to body tissues and storage compartments

Hemoglobin A

• 97% in adults

• 2 alpha chains

• 2 beta chains

Hemoglobin A2

• 2% of adult hgb

• 2 alpha chains

• 2 delta chains

Hemoglobin F

• fetal hemoglobin

• 1% of adult hgb

• 2 alpha chains

• 2 gamma chains

percentage of fetal hemoglobin at birth

80%

Normal hemoglobin forms with ferrous iron and oxygen carrying function (oxyhemoglobin, deoxyhemoglobin)

Oxyhemoglobin (Fe2+ with O2)

• Expelled 2,3-DPG

Deoxyhemoglobin (Fe2+ w/out O2)

• Binding 2,3-DPG

Iron must be in Ferrous state (Fe2+) to transport O2

• 2,3-diphosphoglycerate (2,3-DPG): important for loading and unloading oxygen

• Binding 2,3-DPG unloads O2, and expelling loads O2

Hemoglobin forms unable to transport oxygen (methemoglobin, carboxyhemoglobin)

• These hgb are unable to transport or deliver O2

• At toxic levels, tissue O2 decreases → cyanosis → hypoxia → coma/death

Methemoglobin:

• Iron is oxidized to Fe3+ state (ferric)

• Ferric iron can’t carry O2

• Brown color of blood, reversible

Carboxyhemoglobin:

• O2 replaced with carbon monoxide

• Cherry red color of blood, reversible

• CO is binding 200x greater than O2

Hemoglobin types with abnormal structure (S, C), including altered properties (e.g., solubility, electrophoretic mobility)

Hemoglobin S

• Point mutation replaces hydrophilic glutamic acid with hydrophobic valine at the 6th position of the B-globin chain

• Under low O2 conditions, the hydrophobic valine residues fit into complementary hydrophobic pockets on adjacent HbS molecules

• This causes abnormal polymerisation, where the hemoglobin molecules bond together to form long, rigid, helical fibers.

• This distorts RBCs, forcing them into a crescent or sickle cell shape. This makes the cells fragile, leading to chronic hemolytic anemia.

Hemoglobin C

• Point mutation at same 6th position of B-globin chain replaces glutamic acid with basic lysine

• Substitution of lysine decreases solubility of the Hgb molecule

• Bc it lacks the polymerization tendency of HbS, it doesn’t cause sickling. The molecules tend to aggregate, and intracellularly crystallize (often forming hexagonal or rod-shaped crystals), particularly when the cells are dehydrated. 

• Causes mild chronic hemolytic anemia and splenomegaly (enlarged spleen) due to the spleen removing these rigid, crystal-containing cells. 

• Protects against severe malaria

Describe the principle and accuracy of the cyanmethemoglobin method (or modifications) for determination of hemoglobin concentration, including hemoglobin forms measured

• Principle: it’s the reference method for measuring hemoglobin concentration in the blood. Uses Drabkin’s reagent (potassium ferricyanide). It converts hemoglobin to cyanmethemoglobin. Photometric absorbance of cyanmethemoglobin is measured at 540 nm. Measures all clinically significant forms of Hgb.

• Considered the international reference method for hemoglobin determination.

• Highly accurate because nearly all hemoglobin forms are converted to a single stable pigment.

• Follows Beer-Lambert principles over a wide analytical range.

• Provides excellent reproducibility and standardization between laboratories.

Evaluate possible sources of error when measuring hemoglobin photometrically (e.g., lipemia, bilirubinemia, WBC count over linearity)

Lipids & Bilirubin: affect photometric measurements that check for hemoglobin → can make Hgb results inaccurate because it makes the solution cloudier than it should be

Evaluate a manual spun hematocrit determination, taking into account the principle, acceptable specimen, and sources of error caused by pre-analytical, mechanical and technical variables

• Percentage of red cells in a given volume of whole blood.

• Manual spun HCT centrifuges blood into layers

• Volume of packed red cells reported to nearest 0.5%

Sources of Error

• Pre-analytic/blood collection errors

• Not wiping off first blood drop if collected by fingerstick

• EDTA tube must be at least half full for manual HCTs

Analytic/technical errors

• Improper use of HCT card reader

•  Control sample “out”

• Centrifuging at improper speed or time

• RBC count, Hgb and HCT measurements parallel each

Evaluating the control for a manual spun hematocrit

• Control samples are run to verify the manual spun hematocrit procedure is producing accurate and reliable results. When one level is analyzed, the result is compared with the manufacturer’s est. acceptable range. 

• When the control is within acceptable limits: the system is functioning properly, and technique is acceptable, patient results may be reported. 

• Control outside acceptable limits indicates a possible problem in the analytical phase and patient results should not be reported until the issue is resolved.

Causes of erroneous results when running the hematocrit controls

• centrifuge malfunction: verify RPM and timer settings and repeat centrifugation

• improper centrifugation time: centrifuge using correct procedure

• faulty reader: check calibration and reread specimen

• improper tube filling: recollect or prepare a properly filled tube

• poor mixing of specimen: remix specimen thoroughly and repeat test

• Excess plasma trapped between cells: ensure proper centrifugation conditions

• Deteriorated or expired control material: use fresh control material

• Clotted specimen: reject specimen and obtain a new sample

 Evaluate the significance of RBC, hemoglobin and hematocrit results in terms of age and sex (e.g., anemia, polycythemia).

• ↓ RBC, HGB and/or HCT values….Anemia

• Decreased production, increased loss/destruction

• ↑ RBC, HGB and/or HCT values….Polycythemia

• Increase production

• Critical values: Hgb <7.0 or ≥21.0 g/dL (varies)

Evaluate laboratory data using correlations between RBC, hemoglobin and hematocrit values.

• Rule of Three

• HGB x 3 = HCT +/- 3%

• RBC x 3 = HGB

• RBC x 9 = HCT

• Used to estimate values or check data correlation

• Rules only apply if red cells are normal in size and hgb content

Calculate the RBC indices including MCV, MCH and MCHC using proper reporting format

• MCV (mean cell volume)

• MCH (mean cell hemoglobin)

• MCHC (mean cell hemoglobin concentration)

• Calculated using RBC, HGB, and HCT values

• Invalid if erroneous values used for calculation

• MCV and MCHC are used to describe the red cells and classify types of anemia

• Correlate with appearance of red cells seen on blood smear

Correlate normal and abnormal RBC size and hemoglobin content with MCV

MCV: RBC Size

• Normal MCV (82.0-98.0 fL): normocytic red cells, about the size as the nucleus of a small lymph

• Small MCV (<82.0 fL): microcytic red cells

• Large MCV (98.0 fL): macrocytic red cells

Correlate normal and abnormal RBC size and hemoglobin content with MCH

MCH: average weight of hemoglobin per RBC in picograms

• Varies with both RBC size and hgb content → not used dto describe the red cells

• Normal MCH 27.0-33.0 pg = red cells are usually normocytic and normochromic

• MCH <27 pg = microcytic and/or hypochromic red cells

• If red cells are microcytic, MCH is low due to decreased size

• If microcytic red cells are also hypochromic, MCH is lower

• MCH >33 pg = macrocytic and normochromic red cells

• MCV and MCH values generally parallel each other, e.g., if MCV is increased, MCH is increased

Correlate normal and abnormal RBC size and hemoglobin content with MCHC values

• Normal Hgb concentration (32.0 – 36.0%): normochromic red cells

• Low Hgb concentration (MCHC <32.0%): hypochromic red cells with increased pallor area

• High Hgb concentration (>36.0%): red cells are spherocytes with no pallor area → NOT HYPERCHROMIC

MCV, MCH, MCHC Equations

RBC Indices Equations

Evaluate the significance of WBC counts and PLT counts (e.g., leukopenia, thrombocytosis).

Preparation and staining of peripheral blood smears using Wright’s stain

Principle of Romanowsky-type stains

• Well-made smears are essential to the evaluation of hematologic disorders

• These are pH-dependent reactions

Action of the reagents/dyes

Methylene blue

• Stains acid cell structures a blue color

• RNA in cytoplasm, basophil granules

Eosin

• Stains basic cell structures a red-orange color

• Hgb in RBCs, eosinophil granules

Combo of Both Dyes

• Stains neutral cell structures a pinkish-tan color

• Neutrophil granules

Characteristics of a properly made smear

• Proper length

• Proper width

• Proper thickness

• Presence of squared feathered edge with rainbow area

• Angle of pusher and/or size of blood drop is critical and determines length and thickness

Determine actions to correct for poor smear quality (e.g., too thick) and improperly stained Wright’s stained smears

Adjust the angle of the pusher for length of smear. If it’s too short, decrease the angle of the pusher

Explain the procedure(s) for performing a WBC and platelet estimate from a Wright’s stained blood smear, including proper area, objectives used, appropriate light adjustment and sources of error.

WBC estimates are done to verify the validity of automated WBC counts

• 40x HPF

• Count WBCs in 10 HPFs where red cells slightly overlap (2s and 3s)

• Include fields with no cells in estimate; count broken cells

• Take average #/HPF x 2000

• Should agree with WBC count +/- 20%, the WBC estimate is never reported

Platelet Estimates: done to verify the validity of automated PLT counts

• Oil immersion and high light

• Count platelets in 10 fields where red cells just touch

• Include fields with no cells in estimate

• Find average #/OIF

• The platelet estimate is never reported

Calculate WBC and/or PLT estimates to verify the accuracy of WBC and/or PLT counts.

WBC estimate

• WBC estimate average x 2000 → must be within +/- 20% of count

PLT estimate

• PLT estimate average

• Take first 10 of average x 20,000

• Take the rest of the average x 10,000

• add those values together → must be within +/- 20% if > 50,000/uL and +/- 10,000 if < 50,000/uL

Explain the procedure for performing a manual leukocyte differential, including proper area, objectives used, appropriate light adjustment and sources of error.

• Manual differentials are done if significant patient abnormalities exist and/or automated differential results are flagged

• Requires well-made stain/smear:

• Oil immersion and high light

• Rainbow are where red cells just touch, correct FOV ~150-200 RBCs

Differentials include: 

• WBC differential to classify WBC types

• Slide is scanned from side-to-side, counting and identifying 100 consecutive WBCs = %

Usual Differential Sequence: Seg neutrophils → Band neutrophils → Lymphocytes → Monocytes → Eosinophils → Basophils

• Cell Morphology/appearance

• Evaluation of WBCs, RBCs, and platelets → 10 fields

Platelet estimate: done to verify the accuracy of automated platelet counts

Recognize the normal differential percentage reference ranges (including age variations) of each type of leukocyte

• Deviations from normal reference ranges may indicate disease → provide clues to diagnosis

• No immature cells should be present

• May see occasional smudge, broke, or dying cells

• Critical Value: Blast Cells

Discuss manual reticulocyte counts including each of the following:

• Procedure and principle of supravital stains

• Sources of error

• Normal reference values expressed in both relative and absolute numbers

Procedure and principle of supravital stains

• Principle: supravital stains are used to count retics. 

• Supravital “living cell” stain → NEW methylene blue

• Precipitates RNA into filaments or granules

• 2 blue “dots” or more is a retic

• Don’t count red cells with black/shiny inclusions

• Using Oil Immersion, count 500 red cells in area of slide where red cells just touch

• Separate mature RBCs vs retics using 2 keys

• retics/500 RBCs must agree +/- 2 retics on 2 different slides or another slide is counted

Sources of error

• Absolute # is more reliable than relative %

• Improper blood to stain ratio or inadequate mixing

• Improper counting → counting artifact or not counting red cells with 2 “dots” as retics

• Wrong calculation → especially absolute

Normal reference values expressed in both relative and absolute numbers

• Adult: 

• 0.5 - 2.0%

• 25-100,000/cmm Absolute #

• Newborn

• 2.0 - 6.0%

Calculate relative and absolute retic counts, including proper reporting format.

Evaluate the significance of increased or decreased reticulocytes in the blood, including causes and/or examples of conditions (e.g., hemolytic anemia, aplastic anemia, renal disease).

↑ absolute Retic count….Reticulocytosis

• Increased RBC production… ↑ erythropoietin stimulation following acute blood loss or corrective therapy

• Retic response is seen in about 3-5 days

• Will observe increased amount of polychromasia on the Wright’s-stain blood smear

• Causes: hemolytic anemias, acute or chronic blood loss, response to anemia treatment, erythroblastosis fetalis, kidney tumors/cysts

↓ absolute Retic count….Reticulocytopenia

• Decreased RBC production…↓ EPO stimulation, bone marrow injury, deficiency of essential supply

• Causes: Nutritional deficiencies, CKD, Bone Marrow Disorders, Anime of chronic disease, liver cirrhosis or dysfunction, medications and treatments

• Causes of increased retic % can be either an increased number of retics OR, a decreased number of total red cells

Correlate retic findings with the presence or absence of polychromasia on a Wright’s stained blood smear.

• On a Wright’s-stained blood smear, retics appear slightly larger than mature red cells with a grayish-blue tinge that is reported as polychromasia

• The amount of polychromasia seen correlates with the # of retics in the blood

• A retic count must be done to obtain a definitive number

Explain the dimensions of the Neubauer hemocytometer.

Chamber dimensions: 3mm x 3mm x 0.1mm

Identify the appropriate diluent used for performing manual cell counts.

• Blood dilution is prepared using a Thrombo-tic reagent vial and capillary pipet

• Vial contains 1% ammonium oxalate (diluent)

• 1:100 dilution (x100)

• RBCs will lyse leaving WBC and platelets

• Fill entire counting area under coverslip on each side of hemocytometer.

Correct magnification and light adjustment for manual cell counts

• 40x objective and low light, count cells in all 9 squares on each side of hemocytometer = 18 mm^2 

• Cells counted on each side must agree +/- 20%

Manual cell count appearance of cells

platelets appear greenish, not shiny

Manual cell count sources of error

Pre-analytic/blood collection errors, e.g., clotted blood

Analytic/technical errors

• Poor diluting or plating technique

• Calculation error…is the result believable??

• Counting wrong area, using wrong light, or counting junk as cells

• High WBC count may make counting difficult-secondary dilution?

• Important that chamber is well cleaned (if using glass)

Focusing problems for manual cell count

• Clean lens

• Adjust condenser → open condenser

• Adjust light intensity → decrease light intensity

• Clean eyepiece

• Start over

Manual cell count formula

• Area (squares) to count and/or blood dilution to make is determined by cell number

• Formula to obtain #cell/cm or #cells/uL

Describe instances when manual methods of cell counting might be used

• Automated cell count is flagged as doubtful

• Estimate from blood smear doesn’t agree w/ result

Describe the theory of the Coulter electronic impedance principle of cell counting and sizing.

• The Coulter Principle (electrical impedance method) is the most widely used method for automated counting and sizing of blood cells in hematology analyzers.

• The principle is based on:

• Blood cells are poor conductors of electricity.

• The diluent surrounding the cells is a good electrical conductor.

• When a cell passes through a small aperture (opening) between two electrodes carrying an electric current, it temporarily displaces an equivalent volume of conductive diluent.

• This causes a brief increase in electrical resistance (impedance) and a corresponding decrease in current flow.

• Cell Counting

• Each pulse generated = one cell counted.

• The number of pulses produced is proportional to the number of cells passing through the aperture.

• Therefore, the analyzer determines the cell count by counting the pulses.

• Cell Sizing

• The height (amplitude) of the pulse is proportional to the volume of the cell.

• Larger cells produce larger pulses.

• Smaller cells produce smaller pulses