Hema 2 Lab M1 Flashcards
Main disease associated with Eosinopenia
Cushing’s Syndrome or Hyperadrenalism
In a Diluting Fluid
What is the function of Propylene Glycol
Lyses RBCs
In the Neubauer chamber, where do we count the eosinophils?
9 Large Squares
Give me the components of Randolph’s Diluting Fluid
Phloxine, Propylene Glycol, & Calcium Chloride
State the step-by-step procedure of Absolute Eosinophil Count
- Draw well-mixed anticoagulated whole blood to the 1 mark of the WBC pipette.
- Wipe the outside portion of the pipette and draw the diluting fluid to the 11 mark.
- Mix for 10 minutes using a pipette shaker
- Expel the first 4 drops and fill both sides of the counting chamber.
- Allow the cells to settle for at least 3 minutes before counting.
- Count the eosinophils in the entire ruled area of both sides of the
hemocytometer - Get the average count and compute for the absolute count.
Give me the components of Manner’s Diluting Fluid
Phloxine, Urea, Trisodium citrate, & Distilled Water
In a Diluting Fluid
What is the function of Heparin
Inhibits leukocyte clumping
The formula for Absolute Eosinophil Count
Eosinophils/cu.mm = (number of eosinophils counted / 9) x 100
Main disease associated with Eosinophilia
Addison’s Disease or Hypoadrenalism
What is the test for the normal adrenal function that usually involves eosinophil count?
THORN’S TEST (Eosinophil Depression Test)
In a Diluting Fluid
What is the function of sodium carbonate
Lyses all WBCs except eosinophil
Eosinophils are high in terms of (1) _______ (give examples) & (2) _________ (alongside basophils)
(1) Respiratory diseases (ex. Pulmonary infiltrates, Hay Fever)
(2) Allergic reactions
In a Diluting Fluid
What is the function of Phloxine
Stains eosinophils red
Give me the components of a Phloxine Diluting Fluid
Phloxine, Propylene Glycol, Sodium Carbonate, & Distilled Water
Give me the components of the Pilot’s Solution
Phloxine, Propylene Glycol, Sodium Carbonate, Distilled Water, & Heparin
State three reasons why eosinophilia happens
Parasitic Infections
Allergic Reactions
Certain Leukemias
Increase or Decrease
What happens to the number of eosinophils when there is high level of cortisol?
Decrease
Cortisol induces apoptosis, meaning if high ang cortisol mo (due to stress or etc.) mababa eosinophil count mo due to apoptosis (cushing’s syndrome)
State the possible situations where basophil count increases
Allergic/Inflammatory Reactions
Hypersensitivity Reactions
Other name for Toluidine Blue Method
Cooper and Cruickshank Method
State the function of Cetylpirimidium Chloride
Part of the Principle of Cooper and Cruickshank Method
Lyses the erythrocytes and render basophilic granules insoluble
State the function of Ammonium sulfate
Part of the Principle of Cooper and Cruickshank Method
Used as a mordant to improve the staining qualities of toluidine blue
What does EDTA do?
Part of the Principle of Cooper and Cruickshank Method
Prevent platelet agglutination
What is the color of basophils under the Toluidine Blue Method?
Purple red metachromatically-stained cells
Reference Range of Basophil Count
0–2 x 10^9/L
State two purpose of Nitroblue Tetrazolium Test
- Distinguish between systemic bacterial infections from non-bacterial infections
- Detect neutrophil function defect
When the Neutrophils are incubated with colorless soluble dye (Nitroblue Tetrazolium), reduction produces ________ precipitates, and the number of neutrophils that reduced the dye is quantified.
Color
Blue-black formazan precipitates
State the interpretation
> 10 NBT
Positive neutrophils
Systemic bacterial infection
State the interpretation
<10 NBT
Positive Neutrophils
Normal Individuals
State the interpretation
Negative NBT
Congenital Defect of Neutrophil Function
It is an older technique used to diagnose acute leukemia.
Cytochemical Staining
- It’s inexpensive
- Can be used by the whole world
3 mostly used stains in diagnosing Leukemia based on affected cell lineage
- Myeloperoxidase (MPO)
- Sudan Black B (SBB)
- Esterases
What is MPO and where is it found?
Myeloperoxidase
It is an enzyme found in primary granules of the WBCs
Myeloperoxidase is positive for what kind of cells? Also state the color.
Dark Brown
myeloblasts, promyelocytes, weak activity in
monocytes
What is SBB and where is it found?
Sudan Black B
Found in the lipid portion of primary and secondary granules
State the positive color for Sudan Black B and cells that are positive for this stain
BROWN to BLACK
myeloblasts, promyelocytes, weak activity in
monocytes
Cells that are negative for MPO
lymphoid cells, megakaryocytes, immature RBCs
Red blood cells may stain diffusely brown because of pseudoperoxidase activity of hemoglobin
Cells that are negative for SBB
lymphoid cells, megakaryocytes, immature RBCs
Same with MPO
State the two Specific Esterases
Chloroacetate Esterase and Napthol AS-D Acetate
Under the microscope you see a Bright Red Neutrophils and Mast Cells which is a positive result for a certain stain, what stain do you think the MedTech used?
Specific Esterase
Can be Chloroacetate Esterase and Napthol AS-D Acetate
These esterases are mainly found staining the monocytes. What are these?
Nonspecific Esterase
Give 2 Nonspecific Esterase
ANAE and ANBE
Alpha Naphthyl Acetate Esterase and Alpha Naphthyl Butyrate Esterase
What cells are negative for Specific Esterase?
eosinophils, monocytes, lymphoid cells
What cells are positive for Nonspecific Esterase? State the color.
Monoblast and Monocyte
Dark Red/ Reddish Brown color
Esterase found in Neutrophils
Specific Esterase
Esterase found in Monocytes
Nonspecific Esterase
What cells are negative for Nonspecific Esterase?
granulocytes, megakaryocytes, lymphoid cells
What do you need to add in the ANAE to differentiate monocytes from other cells that occasionally show positivity?
Sodium Fluoride
It inhibits the monocytic enzyme
What is CRP?
Cyanide-Resistant Peroxidase
Peroxidase in eosinophils
Sodium cyanide is added to inhibit peroxidase in WBCs other than those found in eosinophils
It is added to inhibit peroxidase in WBCs other than those found in eosinophils
Sodium Citrate
What is TRAP
Tartrate-Resistant Acid Phosphatase
Inhibits acid phosphatase in leukocytes, except acid phosphatase isoenzyme 5
Positive color for TRAP?
Purple to Dark Red
What cells does TRAP stains?
hairy cells in hairy cell leukemia
Mnemonics: Harry is TRAP
Negative lahat maliban dito
What cells does TRAP stains?
hairy cells in hairy cell leukemia
Negative lahat maliban dito
What is PAS?
Periodic Acid Schiff
Stains Carbohydrates like Glycogen and Mucoproteins
Positive cells and color for PAS
Magenta - Multiple Cell Types
These cells are negative for PAS
normal erythroblasts & lymphoid cells (L1 & L2)
What is LAP?
Leukocyte Alkaline Phosphatase
Enzyme in secondary granules of mature neutrophils
What color is the Neutrophils stained with LAP
Black Brown
Lap Score
No granules
0
Lap Score
4+
Intense, cytoplasm is packed with granules (100%)
Lap Score
Moderate (50-75%)
2+
Lap Score
3+
Strong numerous (75%)
Lap Score
1+
Faint, few granules (<50%)
LAP Score Reference Range
30-100
- Leukemoid reaction (LR)
- Chronic neutrophilic leukemia (CNL)
- Bacterial infections
- Polycythemia vera (PCV)
- 3rd term pregnancy
- CGL-blast crisis or with infection
Increase LAP Activity
Give Results or Diagnosis
Decreased LAP Activity
- Chronic granulocytic / myelogenous leukemia (CGL / CML) * Paroxysmal nocturnal hemoglobinuria (PNH)
- Sideroblastic anemia
- Eosinophilia
LR & CML/CGL are the same in morphology, the only way to differentiate them is through _____
LAP Activity
Primitive marker for lymphoblast but not for mature lymphoblast
Terminal Deoxynucleotidyl Transferase (TdT)
What is TdT?
Terminal Deoxynucleotidyl Transferase
DNA polymerase inside cell nuclei
TdT is positive in what cells?
T-Lymphoblasts (Acute Lymphoblastic Leukemia [ALL] L1 &
L2), pre-B cells
TdT is negative in what cells?
myeloblasts, monoblasts, & ALL-L3
What are the staining techniques to differentiate acute myeloid leukemia (AML) & acute lymphoblastic leukemia (ALL)
MPO, SBB, PAS, TdT
It is used to used to differentiate monocyte & granulocyte cell linkages.
Esterase
Reaction of AML in SBB
+
REVIEW ACUTE LEUKEMIA CYTOCHEMICAL REACTION CHART
Reaction of ALL in MPO
-
REVIEW ACUTE LEUKEMIA CYTOCHEMICAL REACTION CHART
Reaction of Megakaryocytic Leukemia in MPO, SBB, NASDA, ANBE, ANAE
- +(localized)
respectively
REVIEW ACUTE LEUKEMIA CYTOCHEMICAL REACTION CHART
MPO Positive or Negative
Auer rods found in leukemic blasts
Strongly MPO Positive
Auer Rods are found in primary or azurophilic granules
Between MPO and SBB what is more sensitive for the early myeloid cells?
SBB
What do you call the enzyme that accelerates the hydrolysis and synthesis of esters?
Esterase
How many isoenzymes are present in leukocytes?
9
Chloroacetate esterase is present in ____
Primary granules of the neutrophils
Except for MPO, Auer Rods also show positivity in what type of cytochemical stain?
CAE
Chloroacetate Esterase
A patient would be positive for Acute Monocytic Leukemia (AMoL) if ___% or more of the blast are nonspecific esterase positive and specific esterase negative
80%
A patient would be positive for Acute Myelomocytic Leukemia (AMML) if __% shows monocytic differentiation using the sodium fluoride
20
Differentiate ANAE and ANBE
Butyrate is more sensitive and more specific while Acetate can be mixed with sodium fluoride which inhibits the monocytic enzyme
