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Hematology Lecture 2 and 1 other from honeybee0710. Power Points with notes - me Quiz questions Study Guide questions whatever else I feel like putting in here.

What is a normal WBC count?

4,500-12,000 for adults.
This count varies throughout the day, can rise with meals, exercise, stress, temperature extremes and pregnancy


Fluxes twice daily
If the count is more active, there are more RBCs

What is Leukocytosis?

Abnormally high WBC count
12,00-20,000 indicates bacterial infections
>50,000 is usually leukemia


Abnormally low WBC count
<4,500 indicates viral infections
<1,000 is dangerously low, must do manual count to confirm

What is the most common manual count?

Body Fluids are 2nd most common.

What is the diluting solution for a manual WBC count?

Whole blood added to 3% acetic acid diluting solution. Reservoir contains 475uL and 25uL Blood.
Dilution is 1:20 (part:whole)

When counting Body Fluids, how many squares in the Hemacytometer should be counted?

9 squares

What is the formula for calculating the WBC count?

(#cells counted x dilution factor x depth factor)/
(area counted)

Why do Nucleated RBCs appear?

Severe stress causes bone marrow to "dump" nucleated RBCs before they mature.

What is the corrected count for NRBCs?


What is a normal RBC count?

Adult Female= 4-5.5 million/mm3
Adult Male= 4.5-6 million/mm3


Abnormally increased number of RBCs
(can look like false decrease in plasma volume)
Happens with high altitude or heart disease


Leukemia of RBCs
Because of unknown disorder of Bone Marrow


Abnormally low number RBCs
Can have 3 different causes.

Aplastic Anemia

Chronic, complete bone marrow shutdown

What is the diluting solution for a manual RBC count?

Isotonic solution of 1.99mL saline with .01L of blood
Dilution is 1:200

Most required hematology test

Complete Blood Count (CBC)

Components of CBC

-Hemoglobin Concentration
-RBC Count
-WBC Count
-Differential White Cell Count
-Platelet Count

What is the majority weight of the RBC?


What is Hemoglobin made of?

- 4 polypeptide chains (each has 2 pairs of globin chains)
- 4 Heme molecules (1 attached to each chain)
- 4 Oxygen molecules (1 on each Heme molecule)
- 4 Iron molecules (1 on the center of each Heme molecules)

What molecule must be present in order to carry oxygen on Hemoglobin?

Iron in the Ferrous state (Fe+2)

Where/When does Hemoglobin synthesis take place?

Where: In the Endoplasmic Reticulum of RBCs
When: During the early stages of Erythropoiesis

What hormone controls Erythropoiesis?

Erythropoietin, which is produced by the Kidney

What % of the body's required oxygen is carried by hemoglobin?
What else carries oxygen?

5% is dissolved in plasma

What types of chain make up normal adult hemoglobin?
What is it known as?

2 alpha and 2 beta chains
AKA: Hemoglobin A1

What is the secondary, "normal" functioning hemoglobin?
What is it made up of?

Hemoglobin A2
2 alpha and 2 delta chains

What types of chains make up normal fetal hemoglobin?
What is it known as?

2 alpha and 2 gamma chains
AKA: Hemoglobin F

In the first 3 months of gestation, what two forms are hemoglobin known as?
What are they made of?

Gower 1: 2 zeta and 2 epsilon chains
Gower 2: 2 alpha and 2 epsilon chains

Which of the transitions in hemoglobin is the most important within gestation?

The alpha gene is Gower 2.
If alpha does not switch from zeta, life is not compatible.

What is anemia?

Deficiency in circulating hemoglobin

What are the 3 types of anemia?

1. Low amount of RBCs (due to trauma or bleeding, hemoglobin is normal)
2. Decrease in amount of hemoglobin per RBC, AKA: Iron Deficiency Anemia (you have the building blocks, but genetic code which doesn't support)
3. Defect in hemoglobin molecule, AKA: Sickle Cell Anemia (enough RBCs, but not enough building blocks of Heme)

What is Heredity Persistence?

When Hemoglobin F will still occur in large amounts after birth. Individual will still be functional and "normal", will only matter if participating in electrophoresis.

What are the symptoms of Anemia?
Why do they occur?

-Weakness=not enough oxygen
-Fatigue=not enough oxygen
-Low blood pressure=low amount of RBCs
-Fainting=not enough oxygen to brain

Why is oxygen important?

Needed for ATP in Krebs cycle

What are the types of abnormal Hemoglobins?

Hemoglobin S
Hemoglobin C
Hemoglobin E
S-C Disease

What is Hemoglobin S?
What does it cause?

-An abnormal Hemoglobin where valine is substituted for glutamic acid at the 6th position of the globin chain.
-It causes sickle cell disease or sickle cell trait

What is sickle cell trait?

There is 1 S gene present and 1 normal gene present, only matters if body is stressed

What is Hemoglobin C?
What does it cause and what population is it popular in?

-An abnormal Hemoglobin in which RBCs break down earlier than normal
-Causes extreme splenomegaly, intense arthalgia (due to RBCs blocking blood vessels in joints), and chronic hemolytic anemia
-Common in African-Americans

What is Hemoglobin E?
What does it cause and what population is it popular in?

-A single point mutation on the Beta chain that can result in smaller RBCs associated with a resistance to malaria
-Causes a very mild form of hemolytic anemia
-Common in India and SE Asia

What are the 2 normal hemoglobin derivatives?

-Oxyhemoglobin=Oxygen is bound to hemoglobin molecule (Lung->Tissue)
-Reduced Hemoglobin=Carbon Dioxide is bound to hemoglobin (Tissue->Lung)

What are the harmful hemoglobin derivatives?


What is Carboxyhemoglobin?

When carbon monoxide attaches to hemoglobin rather than oxygen. CO has higher affinity and will always win. Causes CO poisoning.

What is Methemoglobin?

When Iron is in the Ferric State (Fe+3) therefore the hemoglobin molecule is unable to carry oxygen. It is constantly formed, but is reduced by enzymes in the RBC. Won't necessarily cause death, but will cause chronic mild anemia.

What is Sulfhemoglobin?

When Sulfur binds to hemoglobin. It is irreversible, and hemoglobin cannot carry O for life of cell. Can happen with sulfonamides, usually with no more than 5% of hemoglobin is affected.

What is Cyanmethemoglobin?

When Cyanide converts all forms of hemoglobin derivatives (except sulfhemoglobin) to this stable irreversible form of cyanomethemoglobin. Causes Cyanide poisoning, like gas chambers.

What is the Specific Gravity Test?

A cheap screeing test for blood donation. It applies the weight of blood compared to the weight of water, using a copper sulfate solution.

What is the Cyanmethemoglobin Test?

A test using Drabkin's reagent (cyaninde) to read the amount of hemoglobin in lysed RBCs using a spectrophotometer. More intense red color, more hemoglobin.

What are normal Hemoglobin values?
(for adult males, adult females, newborns, 3MO, and 10YO)

Adult males= 14-18 g/dL
Adult females= 12-16 g/dL

Newborns= 17-23 g/dL
3-month-olds= 9-14 g/dL
10-year-olds= 12-14.5 g/dL





What is Hyperbilirubinemia?

Yellow plasma from icteric patients, causes falsely elevated values because of altered color read by spectrophotometer.

What is Lipemia?

Milky plasma from high cholesterol patients, only some machines will correct for error.

What is Leukemia?

WBC cancer in bone marrow, producing high WBC counts. Interferes with production of RBCs gives lower hemoglobin value.

Conditions that affect Hemoglobin values:

-Can change based upon where location of patient. More RBCs give more hemoglobin, which make a higher normal range
-Newborns have larger RBCs, so they have higher hemoglobin values

What is Hematocrit?

Percent volume of red blood cells per volume of whole blood expressed as a decimal. Also referred to as packed cell volume. It is a fast macroscopic determination as a quality control check on hemoglobin. Manual method is done in duplicate, must agree within 2% of hemoglobin determinations.

Hemoglobin= 9g/dL, then Hematorcrit should be 27%.

What two types of tubes are used to manually measure hematocrit? (Microhematocrit)

-Heparanized= at bedside POC, directly from patient
-Plain Tube= from anti-coagulated specimen in purple top. (EDTA is anticoagulant)

What are the cell layers of microhematocrit?

-Red Cells= heaviest because of hemoglobin, has small amount of plasma between packed cells
-Buffy Coat= WBCs & Platelets

How is automated Hematocrit different than Microhematocrit?

Automated tells you the average cell size, and doesn't include the empty space between RBCs. These values will be slightly less than manual values (1.1-3% less)

What gives False Low Hematocrit Results?

-Blood sample is QNS, excess EDTA causes cells to shrink
-Hemolyzed Samples
-Inadequate centrifugation sealing prevents proper packing of RBCs

What gives False High Hematocrit Results?

-Clotted blood, gives false packing of RBCs
-Improper centrifuge calibration of speed/time prevents proper packing of RBCs

What are normal Hematocrit values?
(for adult males, adult females, newborns, and 1YO)

Adult males= 42-52%
Adult females= 36-46%

Newborns= 50-62%
1-year-olds= 31-39%

What gives Low Hematocrit Results? And why?

-Anema=low RBCs
-Retaining water or IV fluids=increased plasma volume
-Low Blood Pressure=not enough RBCs

What will give High Hematocrit Results? And Why?

-Trauma/Surgery=loss of blood
-Burns=loss of fluids -> Low plasma volume increases hematocrit. Must be corrected or will be falsely high.

Microhematocrit (image)

Microhematocrit Method (image)

Spectrophotometer Principle (image)


- abnormally low count
-viral infections, <4,500
- exposure to radiation
- 1,000 WBC/mm3 or lower is considered dangerously low
.-.-.seen in AIDS
.-.-.very low counts are not reliable on automated machines


whole blood added to diluting solution (3% acetic acid)
reservoir contains 0.475 mL (475 ml)
25 mL of blood
dilution is 1:20
475 + 25 = 500mL
25 = 1
500 x
x = 20

Unopette Diagram (image)

Hemacytometer Method

.-. Counting chamber must be free of dust and grease
.-. Expel first 2 drops from Unopette, then charge both sides of chamber
.-. Let filled chambers stand 2 minutes to let cells settle and stabilize

WBC Counting Method

.-. Count cells in each of four corner squares
.-. Each corner square is 1mm2
.-. Count cells on border of two sides only
.-. Cell counts on each side of hemacytometer should be within 10 cells

WBC Counting Method (image)

Calculating the WBC Count Formula

WBC/mm3 =

(#cells counted x dilution factor x depth factor ) / (area counted)

Correction for Nucleated Red Blood Cells

.-. NRBCs are mistaken for WBCs and will increase WBC count
.-. Corrected= WBC count - nuRBC count

.-. count number of NRBC/100 WBC counted (differential)
NRBC/mm3 =(NRBC x WBC/ mm3) / (100 + NRBC)

Normal Value for Red Cell Count
adult female:
adult male:

adult female (4.0 to 5.5 million/mm3)

adult male (4.5 to 6.0 million/mm3 )


.-. increase in normal number of RBCs
.-. Abnormally High RBC Count

Relative polycythemia

(dehydration, diarrhea, vomiting)
.-. no increase in actual number of red cells, decrease in plasma volume

Absolute polycythemia

(erythrocytosis, increase in RBC production)
.-. low oxygen tension (high altitude or pulmonary disease)
.-. slowing of circulation due to heart disease
.-. defects in hemoglobin molecule

Polycythemia vera

.-. Erythemia (leukemia of red blood cells)
.-. 7-10 million cells/mm3
.-. Unknown disorder of the bone marrow


.-. due to fewer red cells
.-. Abnormally Low RBC Count

Anemia Causes

.-. bone marrow disorders - aplastic anemia
.-. therapeutic drugs
.-. acute hemorrhage
.-. increased cell distruction (hemolytic anemia)

Isotonic solution

prevents hemolysis in manual RBC Count

Center square

Area counted
.-. 5 small squares in center square
.-. 1/5 mm2

Counting Area Formula

RBC/mm3 = (#cells counted) x (dilution factor) x (depth) x (area counted)

Tracking Errors

.-. Contaminated diluted fluid
.-. Dirty hemacytometer
.-. Too much time elapsed between drawing the sample and performing the procedure
.-. Too much time elapsed between diluting the sample and charging the hemacytometer
.-. Misinterpretation of the counting area on the hemacytometer

Red Cell Indices

.-. Useful in classifying anemias
.-. Quantitative measurements of average cell size
.-. Hemoglobin content
.-. Hemoglobin concentration of RBCs

Mean Cell Volume (MCV)

.-. Average volume of a red cell
.-. Units expressed in femtoliters, fL

Mean Cell Volume Formula

MCV = (Hct (%) x 10) / (RBC count (millions))

Mean Cell Clinical Significance

Indicates RBC size
.-. Microcytic
.-. Macrocytic
.-. Normocytic

Microcytic MCV

.-. MCV < 80 fL are small
.-. iron deficiency anemia

Macrocytic MCV

.-. MCV > 102 are big
.-. Vitamin B12 deficiency or pernicious anemia

Normocytic MCV

.-. MCV >80<102 are normal size

Mean Cell Hemoglobin (MCH)

.-. Average weight of hemoglobin inside the cell
.-. Normal range is 27.0 to 33.0 pg
.-. Elevated in pernicious anemia
.-. Decreased in iron-deficiency anemia

Mean Cell Hemoglobin Formula

.-. MCH = (Hgb(g/dL) x 10) / (RBC count (millions))
.-. Units expressed in picograms (pg)

Mean Cell Hemoglobin Concentration (MCHC)

.-. Average concentration of hemoglobin in a given volume of packed red cells
.-. Units expressed in g/dL or %
.-. Concentration of hemoglobin in cell is indicated by color intensity

Mean Cell Hemoglobin Concentration Formula

MCHC= (Hgb x 100) / (Hct)

Normocytic MCHC

MCHC >33.0<38.0 is normal

Hypochromic MCHC

MCHC <33.0 is pale

Hyperchromic MCHC

.-. MCHC > 38.0 means there is too much hemoglobin in the cell
.-. can't happen, there is no hyperchromic cell
.-. spherocyte cells look hyperchromic, but it is because of a shape change in the cell

Direct method - Unopette

Manual Platelet Counts
.-. dilution is 1:20, 1:100 or 1:200
.-. diluent will lyse RBCs
.-. hemacytometer(count all smaller squares of central square, 1mm3)

Direct Method Formula

.-. Platelet count = #platelets counted x dilution x depth factor x area counted

Sources of Error for Manual Platelet Counts

.-. Improper dilution of the sample
.-. Contaminated reagent
.-. Blood clots
.-. Improper filling of hemacytometer
.-. Counting cell fragments or dust particles
.-. Incorrect calculation

Indirect method - Procedure

.-. make and stain blood smear
.-. using 100X objective, count # .-. platelets/1,000 RBCs
.-. use EDTA sample

Indirect Method Formula

platelet count = (# platelets x RBC count) / (1000)

60 platelets/1,000 RBCs counted
RBC count = 5,000,000

platelet count = (60 x 5,000,000)/(1,000)
. . . . . . = 300,000 platelets/mm3

Erythrocyte Sedimentation Rate (ESR)

.-.Rate at which RBCs settle out of plasma when placed in a vertical tube

Clinical Significance
.-. Used as a screening procedure
.-. Is nonspecific
.-. Used as an index of the presence of active diseases
.-. .-. inflammatory disorders that Lupus, rheumatoid arthritis
.-. Infectious diseases increase globulins and fibrinogen
.-. .-. increase ESR

Stages of erythrocyte sedimentation

.-. initial period of aggregation
.-. period of rapid settling
.-. packing

Initial period of aggregation (ESR)

1st ten minutes, rouleaux formation)

Period of rapid settling (ESR)

50 minutes, sedimentation occurs at a fairly constant rate)

Packing (ESR)

10 minutes, sedimented RBCs are packed at the bottom of the column

Measuring ESR



.-. (100mm tube)
.-. fill Wintrobe tube with 1ml of blood
.-. use long-stemmed Pasteur pipette
.-. tube set vertical for 1 hour


.-. (200mm tube)
.-. 4 volumes of blood to 1 volume of anticoagulant (sodium citrate)
.-. tube set vertical for 1 hour

Sources of Error in ESR

.-. Reading time
.-. Temperature
.-. Increased ESR
.-. .-. tilting
.-. .-. vibrations
.-. .-. heparin alters the charge on RBC
.-. Decreased ESR
.-. .-. clots or bubbles
.-. .-. if test is not set up within two hours, rouleaux will not form

RBC Factors Influencing ESR

.-. Rate of settling directly proportional to density
.-. .-. hypochromic cells will not settle as fast
.-. Countered by buoyancy, tendency to float is directly proportional to their surface area to volume ratio
.-. .-. Large cells have smaller surface area in relation to volume than small cells
.-. .-. abnormal shapes fall slower, rouleaux formation slower
.-. Anemia increases ESR, polycythemia lowers ESR

Plasma Factors Influencing ESR

.-. Fibrinogen and globulin lower negative charge on red cell
.-. .-. favor rouleaux, increase ESR
.-. Albumin slows rouleaux formation, decreases ESR

Technical Errors in ESR


Red Cell Maturation
.-. oxygen content of blood controls rate of RBC formation
.-. under hormone control (erythropoietin)
.-. produced in the kidney

Stages Red Cell Maturation

stem cell
-> basophilic normoblast ->polychromatophilic normoblasts (last stage of mitosis)
->nucleated RBC
-> mature RBC


.-. Remain in bone marrow for 1 day then go to peripheral circulation
.-. .-. low hemoglobin takes longer maturation
.-. 2 days to form mature RBC
.-. Retic contains ribosomal RNA
.-. .-. ribosomal RNA stained with supravital stains
.-. .-. these stains will only stain living cells

Reticulocyte Count (RC) Procedures

.-. Preparing the smear
.-. Counting reticulocytes

Preparing the smear (RC)

.-. mix equal parts of brilliant cresyl blue or new methylene blue with blood
.-. incubate the mixture
.-. prepare a blood smear in the usual measure and allow it to air dry

Counting Reticulocytes

.-. use oil immersion
.-. count 1000 cells (both RBCs and retics)

Reticulocytes (image)

Percent of Reticulocyte Formula

(% retics) = (#retics counted x 100) / (1000 (RBCs counted)]
(% retics) = (# retics counted)/ (10)

Normal Percent RANGE
males 0.5 to 1.5
females 0.5 to 2.5

Absolute # of Reticulocytes Formula

Absolute # of reticulocytes =
(% retics) x (RBC count)

Normal Absolute
= 60,000 retics/mm3

Reticulocytes Clinical Application

Determines if bone marrow is functioning
.-. reticulocytopenia
.-. .-. pernicious anemia
.-. .-. aplastic anemia
.-. .-. bone marrow failure
.-. increase in reticulocytes
.-. .-. positive response to anemia
.-. .-. response to therapy

The morphology of blood cells is important to evaluate:

When flag limits established by clinical laboratory scientists in the laboratory are exceeded

The primary function of platelets is to

Facilitate blood clotting

Which of the following can be evaluated only through the microscopic examination of a stained blood film?
A) White blood cell (WBC) count
B) Reticulocyte count
C) Hemoglobin concentration
D) Presence or absence of blood cell inclusions

Presence or absence of blood cell inclusions

What is the best substance to use for cleaning oil from microscope objectives

Alcohol-based products

Where must fluid-resistant laboratory coats be worn?

Walking to a patient room to collect a sample

Universal standard precautions written by OSHA are designed to:

Protect employees from possible infectious contamination.

Which of the following is acceptable safe practice?

Disposing of used glass slides in sharps container

Fire drills should be conducted _____


Personal protective clothing, which must be provided by the employer, includes:

The employer must provide gloves for all employees. These should be latex, and alternatives should be available for those employees with latex sensitivity or allergy

A 10% solution of household bleach is used to clean contaminated surfaces in the clinical laboratory. This 10% solution should be prepared:


What can increase the resolution of an object under the microscope?

Enlarging the numeric aperture of the objective lens


is the ability to distinguish two objects closely situated to one another as distinct entities. Increasing the numeric aperture increases resolution.

Where should the condenser be located when using the ´100 oil objective?

Highest position

What is the most likely reason for cells on a hemocytometer appearing "fuzzy" when switching from low power to the ´40 objective?

Oil on the 40´ lens

What immersion oil is used most commonly in hematology?

Type B

The different types of oil differ with respect to their viscosity (i.e., thickness). The one most commonly used in hematology is type B, which has high viscosity. Type A has a low viscosity, and type C has a very high viscosity

What solution is best for cleaning lenses?

Commercial glass cleaning solution

microscope to its clinical use:
_____ Routine differentials


microscope to its clinical use:
_____ Used with fluorochrome dyes to identify lymphocyte subsets


microscope to its clinical use:
_____ Identifying crystals in synovial fluid

Polarized light

microscope to its clinical use:
_____ Counting platelets manually

Phase contrast

microscope to its clinical use:
_____ Viewing spirochetes in microbiology


Which of the following procedures is safe from the production of an aerosol mist?

Manual differentials on blood films

Manual cell counting methods in automated hematology laboratories are used for all of the following except:
A) Routine testing
B) Very low counts
C) Malfunctioning automated instruments
D) Counting spinal fluids

A) Routine testing

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