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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
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 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)
What is the formula for calculating the WBC count?
(#cells counted x dilution factor x depth factor)/
Why do Nucleated RBCs appear?
Severe stress causes bone marrow to "dump" nucleated RBCs before they mature.
Abnormally increased number of RBCs
(can look like false decrease in plasma volume)
Happens with high altitude or heart disease
What is the diluting solution for a manual RBC count?
Isotonic solution of 1.99mL saline with .01L of blood
Dilution is 1:200
Components of CBC
-Differential White Cell Count
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 % 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?
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 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
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:
-AGE AND SEX DEPENDANT!
-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
-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%
What gives Low Hematocrit Results? And why?
-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.
- 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
x = 20
.-. 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
Calculating the WBC Count Formula
(#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 (4.0 to 5.5 million/mm3)
adult male (4.5 to 6.0 million/mm3 )
(dehydration, diarrhea, vomiting)
.-. no increase in actual number of red cells, decrease in plasma volume
(erythrocytosis, increase in RBC production)
.-. low oxygen tension (high altitude or pulmonary disease)
.-. slowing of circulation due to heart disease
.-. defects in hemoglobin molecule
.-. Erythemia (leukemia of red blood cells)
.-. 7-10 million cells/mm3
.-. Unknown disorder of the bone marrow
.-. bone marrow disorders - aplastic anemia
.-. therapeutic drugs
.-. acute hemorrhage
.-. increased cell distruction (hemolytic anemia)
.-. 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 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
.-. 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
.-. 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
.-. (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
.-. 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
Red Cell Maturation
.-. oxygen content of blood controls rate of RBC formation
.-. under hormone control (erythropoietin)
.-. produced in the kidney
Stages Red Cell Maturation
-> basophilic normoblast ->polychromatophilic normoblasts (last stage of mitosis)
-> 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
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
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)
= 60,000 retics/mm3
Reticulocytes Clinical Application
Determines if bone marrow is functioning
.-. .-. 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
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
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
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.
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?
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
microscope to its clinical use:
_____ Used with fluorochrome dyes to identify lymphocyte subsets
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
Twenty cells are counted in one of the nine large squares of a hemocytometer. The sample is diluted 1:10. How many cells are present per mL?
What error was probably made if red cells are seen when attempting to manually count white blood cell (WBCs) on a hemocytometer?
The Unopette was not allowed to sit for 10 minutes for complete red blood cell (RBC) lysis
Which of the following would cause a manual cell count to be falsely high?
Not wiping excess blood from outside of pipette when filling
Ten nucleated red blood cells (NRBCs) are found in a blood film when differentiating 100 white cells. What is the corrected white blood count if the automated cell counter printed a count of 10.0 ´ 109/L?
9.1 ´ 109/L
Why is it important to have a scrupulously clean hemocytometer when manually counting platelets?
Their small size makes them easily confused with dirt.
In the center square on one side of a hemocytometer, 100 platelets were found; 200 were found in the center square on the other side. Select the best course of action.
Repeat the procedure—the two counts are not close enough.
Which of the following could cause the microhematocrit to be falsely decreased?
A) Specimen contaminated with intravenous fluid
B) Trapped plasma from abnormally shaped red cells
D) Insufficient centrifugation
A) Specimen contaminated with intravenous fluid
The MCV on a patient is calculated to be 115 fL and the MCHC is 35 g/dL. What, in general terms, must be true of the MCH if it is consistent with the previous two indices?
It should be above reference range.
On a standard reticulocyte preparation with new methylene blue, 100 cells are counted that contain blue-stained granulofilamentous material. The red blood count is 3.22 ´ 1012/L, and the hematocrit is 30%. Calculate the reticulocyte count as a percentage.
What cellular component of the erythrocyte is its control center?
The nucleus is the control center of the cell, is responsible for all its chemical reactions, and is the site of deoxyribonucleic acid (DNA) replication and transcription.
How does the genetically active portion of the nuclear chromatin look when stained with Wright stain?
Diffuse, uncondensed and pale blue
The genetically active portion of the nuclear chromatin, composed of DNA plus supporting proteins, appears diffuse and uncondensed and stains pale blue with Wright stain. It is also called euchromatin. Chromatin, which is condensed and stains darkly, is also called heterochromatin; it is the genetically inactive portion of the nucleus.
If the cytoplasm of a cell is very basophilic or blue when stained with Wright stain it contains:
Large number of ribosomes
Ribosomes, which are made up of ribonucleic acid (RNA), stain blue or basophilic with Wright stain. If a cell's cytoplasm contains many ribosomes, then it will stain very blue.
How do cells get from the bone marrow into peripheral circulation?
Mature cells squeeze through spaces in the vessel endothelial lining.
As mature blood cells come into contact with endothelial cells in the bone marrow, they bind to the surface via a receptor-mediated process. The binding creates a space between the luminal adventitial and endothelial cells. This allows the bone marrow cells to escape into circulation.
When hematopoiesis occurs in the liver in an adult, it is called:
Whenever hematopoiesis occurs outside the bone marrow in an adult, other than lymphopoiesis in lymphatic tissue, it is called extramedullary. This is abnormal.
Where are developing red cells located in the bone marrow?
Adjacent to the outer surfaces of the vascular sinuses
Maturing red cells are found in two places in the bone marrow: (1) adjacent to the outer surfaces of the vascular sinuses and (2) surrounding iron-laden macrophages
Why are erythrocyte cellular inclusions such as Howell-Jolly bodies found after surgical removal of the spleen?
The absent spleen cannot "pit" or remove the normally formed inclusions.
Macrophages in the spleen normally remove abnormal inclusions from red cells, which includes Howell-Jolly bodies. This process is called pitting.
What condition is most associated with autosplenectomy?
Sickle cell anemia
In sickle cell anemia, repeated sickling events in the spleen, where the red cells are stressed, result in multiple splenic infarcts. Eventually, the spleen is no longer functional. This results in functional loss of the spleen and is called an autosplenectomy.
Programmed cell death
is a normal physiologic process eliminating unwanted, abnormal, or harmful cells.
What directs the mitosis and maturation action of progenitor neutrophil cells in the bone marrow?
Colony-stimulating cytokines bind to receptors on progenitor cells to direct them to undergo mitosis and to mature to identifiable neutrophil precursor cells. Erythropoietin is the primary cytokine for red cell development. Neither the number of stem cells nor the number of mature cells in circulation function in this role.
What is the earliest stage of maturation where a neutrophil can be distinguished from an eosinophil?
Eosinophil granules differentiate the eosinophils from the neutrophil at the early myelocyte stage. Before this, the myeloblast and promyelocyte stages are indistinguishable by morphology.
Which of the following neutrophils is capable of chemotaxis?
A) Progenitor cells
B) Stem cells
The band neutrophil is capable of chemotaxis (i.e., it can move along a chemical gradient). It is an almost mature polymorphonuclear neutrophil, and has full motility, active adhesion properties, and some phagocytic ability.
Which of the following has the most controversial morphologic identification system?
A) Progenitor cells
B) Stem cells
At least three systems exist for morphologic identification of band neutrophils. They vary with the shape of the nucleus. Hematology analyzers that perform automated differentials classify them (bands) with polymorphonuclear (segmented) neutrophils; therefore many laboratories now use the same criteria when identifying cells using the microscope.
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