LEUKEMIA, FLOWCY, RETICS, AUTOMATION, OTHERS Flashcards
Overproduction of various types of immature or mature cells in the bone marrow and or peripheral blood
Leukemia
Frequently involves WBCs of the myelogenous or lymphocytic cells types
Leukemia
Malignant cells easily trespass the blood brain barrier
Leukemia
Solid malignant tumors of the lymph nodes and related WBCs in the bone tissue
Lymphomas
The distinctive cell type is the lymphocyte
Lymphomas
Malignant cells are initially confined to the organs containing mononuclear phagocytic cells such as lymph nodes, spleen, liver, and bone marrow
Lymphomas
This can spill over into the circulating blood and demonstrate a leukemia-appearing picture on a peripheral blood film
Lymphoma
Leukemia has increased WBC count with shit to the ____
Left
Leukemia M:E ratio
10:1
Type of anemia usually present in cases of acute leukemia
Normocytic normochromic
Numerous immature cell forms in the bone marrow and or peripheral blood; increased total WBC count; symptoms of short duration
Acute leukemias
Mostly mature cell forms in the bone marrow and or peripheral blood; total WBC counts range from extremely elevated to lower than normal ; symptoms of long duration
Chronic leukemia
The FAB classification of leukemia is based on morphology of cells in what stained smear
Romanowsky-stained smear
Cytochemical stains used in FAB classification
Myeloperoxidase
Sudan Black B
Enzyme found in primary granules of neutrophils and eosinophils and monocytes
Myeloperoxidase
Used in differentiating blasts of AML from those of ALL
MYELOPEROXIDASE
Peroxidase stain reaction of ALL
POSITIVE
POSITIVE MPO
Neutrophilic granulocytes (Except normal blasts)
Auer rods
Leukemia blast in FAB M1, M2, M3
Eosinophils
POSITIVE MPO
Neutrophilic granulocytes (Except normal blasts)
Auer rods
Leukemia blast in FAB M1, M2, M3
Eosinophils
Reactions parallels those of the MPOs
Sudan black B
Stains sterols, neutral fats, phospholipids (found in the primary and secondary granules of neutrophils and lysosomal granules of monocytes)
Sudan black B
Most sensitive stain for granulocytic precursors
SBB
SBB POSITIVE
STRONGLY POSITIVE:
Promyelocyte, myelocyte, metamyelocytes, bands, and segmented neutrophils
Leukemic blasts
Auer rods
Eosinophils
Weakly positive or negative MPO
MONOCYTES
WEAKLY POSITIVE OR NEGATIVE SBB
Myeloblasts
Monocytic cells
NEGATIVE MPO
Myeloblasts
Basophils
Lymphocytic cell series
Erythrocytic cell series
NEGATIVE SBB
Lymphocytes and its precursors
NEGATIVE SBB
Lymphocytes and its precursors
Megakaryocytes and platelets
Erythrocytes
Peroxidase positive granules may produce what color
Red brown , dark brown, or black color
Rbcs develop diffusely __ color because of the pseudoperoxidase activity of hgb
Brown
A carcinogenic substrate used in one of the MPO methods
3,3-diaminobenzidine tetrahydrochloride
Peroxidase enzyme is _____ to light
Sensitive
May be used to detect eosinophilic leukemia ;
Cyanide-resistant peroxidase stain
Lymphocytic leukemias are generally MPO _ and SBB _
Both negative
Most common form of childhood leukemia
ALL
70% of childhood ALL
L1
70% of ADULT ALL
L2
ALL type that is rare in children and adults
L3
ALL L1 immunologic markers
CALLA (CD10)
TdT
CD19
CD20
ALL L2 immunologic markers
TdT
ALL L3 immunologic markers
slg
CD19
CD20
CD22
CD24
A homogeneous population of small blasts - ALL type
L1
Heterogenous population of large blasts - ALL type
L2
Homogenous population of large blasts (with nuclear and cytoplasmic vacuoles) - ALL type
L3
PAS, MGP, ORO reaction of ALL L1
+ - +
PAS, MGP, ORO reaction of ALL L2
+ - +
PAS, MGP, ORO reaction of ALL L3
- +
E rosettes positive
T-ALL
Surface Ig positive
B-ALL
Serum Anti-ALL positive
Common ALL
Most common type of leukemia in elderly characterized by persistent lymphocytosis
CLL
In CLL, these cells are present in the pbs
Smudge cells
Render cells
Examples of clinical variations of CLL
Hairy-cell leukemia
Lymphosarcoma cell leukemia
Prolymphocytic leukemia
Solid tumor counterpart of ALL
Lymphoma, poorly differentiated ; lymphocytic
Solid tumor counterpart of CLL
Lymphoma, well-differentiated ; lymphocytic
Solid tumor counterpart monocytic leukemia
Reticulum cell sarcoma
Solid tumor counterpart of acute myelogenous granulocytic leukemia
Chloroma
Solid tumor counterpart of plasma cell leukemia
Myeloma/ plasma cell myeloma
Stem cell solid tumor counterpart
Lymphoma, undifferentiated
Non-lymphocytic leukemias/myelogenous leukemia are generally MPO _ and SBB __
Both positive
AML, minimally differentiated ; MPO and SBB negative
M0
AML without maturation ; may demonstrate Auer rods
M1
AML with maturation ; most common subtype of AML ; may demonstrate Auer rods
M2
Acute promyelocytic leukemia ; DIC ; Faggot Cells
M3
Acute promyelocytic leukemia, microgranular variant that has a characteristic “butterfly”, “bow tie”, “coin-on-coin” or “apple core” nuclei
M3V
Acute myelomonocytic leukemia ; aka naegli monocytic leukemia ; 2nd most common ; may demonstrate Auer rods
M4
An Acute myelomonocytic leukemia with increased marrow eosinophils
M4E
Acute monocytic leukemia ; schilling leukemia
M5
Acute monocytic leukemia, poorly differentiated ; seen in children ; >80% monoblasts in bone marrow
M5a
Acute monocytic leukemia, well differentiated ; seen in children ; <80% monoblasts in bone marrow
M5b
Acute eryhtroleukemia ; Diguglielmo’s syndrome ; may demonstrate Auer rods ; normocytic normochromic ; PAS strongly positive
M6
Acute megakaryocytic leukemia ; required immunocytochem for accurate diagnosis ; factor VIII stain positive
M7
Acute basophilic leukemia
M8
Cytochem reactions of M1, M2, M3
+ + + - - -
Cytochem reactions M4
+ + + + + -
M7 reaction on a-naphthyl acetate esterase (NSE) and factor VIII stain
Localized positivity and positive
Chronic myelogenous leukemia is also known as
Chronic granulocyte leukemia
Characterized by the presence of Philadelphia chromosome
Chronic myelogenous leukemia
Three clinical phases of CML
Chronic phase, accelerated phase, blast crisis
___% of patients with CML have Philadelphia chromosome
90%
Presence of Philadelphia chromosome indicates ?
Good prognosis
CML must be differentiated from
Leukomoid reaction
Philadelphia chromosome is first described by ____ in the year ____
Peter C. Nowell in 1960
Philadelphia chromosome - due to reciprocal translocation involving the ___ arms of chromosome _ and __ (results in the formation of the _____ fusion gene)
Long ; 9 and 22 ; BCR-ABL1
Excessive leukocytic response in the peripheral blood ; confused with CML ; WBC ct greater than 50 x 10^9/L (with neutrophilia and a marked left shift presence of immature neutrophilic forms)
Leukomoid reaction
most frequently refers to neutrophils, but the increased count may be due to an increase in other types of WBCs
Leukomoid reaction
Generally used to distinguish Leukomoid reaction from CML
LAP/NAP TEST (LEUKOCYTE (NEUTROPHIL) ALKALINE PHOSPHATASE TEST
This can be observed in neutrophils that have undergone normal growth
Increased LAP activity
Hydrolysis of sodium alpha naphthyl phosphate by alkaline phosphatase produces a colored precipitate with a diazotised amine
Kaplow’s method
Kaplow’s fixative
Methanol and formalin
Kaplow’s buffer
Propanediole
Kaplow’s substrate
Sodium alpha napthyl phosphate
Kaplow’s initial stain
Brentamine-fast garnet salt
Kaplow’s counterstain
Aqueous Mayer’s hematoxylin
Kaplow’s positive color
Reddish brown to black precipitate of alkaline phosphate granules
LAP score 0
NO reddish brown to black precipitate precipitate
LAP score of 1+
SLIGHTLY DIFFUSED reddish brown to black precipitate
LAP SCORE 2+
MODERATELY DIFFUSED reddish brown to black precipitate
LAP SCORE 3+
HEAVILY DIFFUSED reddish brown to black precipitate
LAP SCORE 4+
VERY HEAVILY DIFFUSED reddish brown to black precipitate
Normal Kaplow’s score
15-100
Within normal or high LAP score (LR or CML?)
LR
Low LAP score (LR or CML?)
CML
Examples of high LAP score
Third trimester pregnancy
PV
Infections
Intoxication
Examples of low LAP score
CML
PNH
Sideroblastic anemia
Myelodysplastic syndrome
Most common clinical application of flow cytometry
Diagnopposis of leukemias and lymphomas
Most significant discovery that led to the improvement of flow cytometry suggests the measurement of a cell, this procedure is successfully applied to study in clinical practice
Development of monoclonal antibodies
LASER stands for
Light amplified by stimulated emission of radiation
The basic unit of all radiation is
Photon
Peripheral blood and bone marrow specimens for flow cytometry must be processed within
24-48 hours from time of collection
Preferred anticoagulant for flow cytometry
Heparin
This can be detected directly by flow cytometer
Fluorochromes
Tumor markers for immature lineage
CD34
CD 117
Terminal deoxynucleotidyl transferase
Tumor markers for granulocytic/monocytic lineage
CD 33
CD 13
CD 15
CD 14
Tumor markers for eryhthroid lineage
CD 71
Glycophorin A
Tumor markers for megakaryocytic lineage
CD 41
CD 42
CD 61
Tumor markers for B lymphocytes
CD 19
CD 20
CD 22
Kappa light chain
Gamma light chain
Tumor markers for T lymphocyte
CD 2
CD 3
CD 4
CD 5
CD 7
CD 8
Study of chemical constituents of cells ; useful for acute leukemias
Cytochemistry
Differentiate acute granulocytic leukemias from monocytic leukemias
Esterase
Nonspecific esterase
Alpha naphthyl acetate and butyrate
Specific esterase
Naphthyl AS-D chloroacetate
With strong positive reaction in a-naphthyl ACETATE esterase
Monocytes
Negative for both a-naphthyl acetate/butyrate esterase
Monocytes ((with NaF inhibition)
Immature non-nucleated RBC which contains 2 or more blue stained granulofilamentous materials after supravital staining
Reticulocyte
Permits effective assessment of RBC production by the bone marrow ; a measure of EFFECTIVE ERYTHROPOIESIS
Reticulocyte count
Retics reference range for Adult
0.5% to 1.5%
Retics reference range in newborns
1.8% to 5.8%
Considered as the first sign of accelerated erythropoiesis and observed in hemolytic anemias
Increased retics
Supravital stains for retics
New methylene blue
Brillian cresyl blue
Large square A of Miller disk is used for?
Counting retics
Small square B of Miller disk is used for?
Counting RBCs
Minimum number of RBCs that should be counted in small square B
112
For routine light microscopy, equal amounts of blood and supravital stain are allowed to incubate at ____ for ____
Room temp for 3 to 10 mins
Retics formula
of retics observed / 1000 rbcs x 100 = retics (%)
Calibrated Miller Disk method formula
Total retics in square A / (total RBCs in square B x 9) x 100
Most rapid accurate and precise method for retic count ; e.g. Sysmex R-3500
Flow cytometry
Sysmex R-3500 uses what kind of supravital fluorescent dye
Auramine O
Sum of the middle-fluorescence, or high-fluorescence ratios
Immature reticulocyte fraction (IRF)
Color of Howell-Jollly bodies in NMB
Deep purple
Color of papenheimer bodies in NMB
PURPLE
Color of hemoglobin in NMB
GREENISH BLUE
Nuclear fragments (DNA)
Howell-Jolly bodies
Usual present at the peripheral edge of the red cell ; denatured and precipitated hemoglobin
Heinz bodies
Color of Heinz bodies in NMB
LIGHT BLUE GREEN
Generally appear as several granules in a small cluster ; a wright stained or a Prussian blue stained smear may be examined to confirm their presence ; hemosiderin in the mitochondria
Papenheimer bodies
Multiple small dots ; seen in a form of alpha thalassemia
Hemoglobin H bodies
Multiple small dots ; seen in a form of alpha thalassemia
Hemoglobin H bodies
Actual number of Reticulocyte in 1 liter of whole blood
Absolute reticulocyte count
Absolute reticulocyte count formula
Retics (%) x RBC ct (x10^12/L) / 100 x 1000
Absolute reticulocyte count formula
Retics (%) x RBC ct (x10^12/L) / 100 x 1000
Reference range for absolute reticulocyte count
20 to 115x10^9/L
Sometimes referred to as reticulocyte index, hematocrit correction, poor man’s bone marrow aspirate
Corrected recticulocyte count
Corrects the observed reticulocyte count to a normal HCT of 0.45 L/L to allow correction for the degree of patients anemia
Corrected rectic count
CRC formula
Retics (%) x (HCT in L/L / 0.45 L/L)
Also known as shift correction which provides a further refinement of the CRC
Reticulocyte production index
A general indicator of the rate of erythrocyte production increase above normal in anemias
Reticulocyte production index
RPI formula
CRC / Maturation time in the peripheral blood
Maturation time of Hct of 40 to 45%
1.0
Maturation time of Hct of 35 to 39%
1.5
Maturation time of Hct of 25 to 34%
2.0
Maturation time of Hct of 15 to 24%
2.5
Maturation time of Hct of <15
3
RPI that generally indicates adequate bone marrow response
> 3
RPI that generally indicates inadequate bone marrow response
<2
Aka electronic resistance or low voltage direct current or “Coulter Principle”
Electrical impedance
_____ is proportional to the change in direct current
Total volume of cell
_____ is proportional to pulse size/change in the radiofrequency signal
Cell interior density
Plots conductivity (RF) and impedance (DC) of the cells
2D distribution cytogram or scatterplot
Displays clusters of cells ; Number of dots represents the concentration of a specific cell type
Scatterplot
Most common problem in cell counting ; produce POSITIVE ERROR
APERTURE PLUGS
Produces negative error in automated cell counting
Excessive lysing of RBC
In blood cell histogram, the number of cells is on what axis
Y-axis
In blood cell histogram, the cell size is on what axis
X-axis
If the RBCs are larger than normal, the curve will shit toward ___
The right
If the RBCs are smaller than normal, the curve will shift to the
Left
Distribution peak with small mononuclear population of cells (lymphocytes)
Fist peak (45-90 fL)
Distribution peak with small mononuclear population of cells (lymphocytes)
Fist peak (45-90 fL0
Distribution peak with minor population of large mononuclear cells ; an increase in the number of cells in this size range can also represent abnormal cell types
Second peak (90-160 fL)
Minor population of LARGE MONONUCLEAR cell ( monocytes ) ; an increase in the number of cells in this size range can also represent an abnormal cell types
Second peak (90-160 fL)
Distribution peak with normal mature types of granulocytes
Third peak (160-450 fL)
Signal irregularities in the WBC distribution and will appear next to the differential parameters that are in error
Region code flags (R)
Region code : warns of increased interference in the area left of the lymphocyte peak (approx 35 fL); typically caused by sickled RBCs, nucleated RBC, or clumped and giant platelets being counted in the WBC aperture bath
R1
Region code: warns of excessive overlap of cell populations at the lymphocyte/mononuclear cell boundary (approx 90 fL) caused by the presence of abnormal cell types, such as atypical lymphocytes, blast, or plasma cells
R2
Region code : warning is caused by excessive overlap of cell populations at mononuclear/granulocyte boundary (Approx 160 fL) which is due to the increased presence of immature granulocytes (bands, metamyelocytes)
R3
Region code ; warning is caused by the extension of the cell distribution past the upper end of the threshold (approx 450 fL); this most commonly occurs when the granulocyte population is very high
R4
Region code ; is the error code for MULTIPLE REGION OVERLAP
R5
The actual counting of platelet histogram takes place in the ____ aperture
RBC
Master of regulatory hormone of systemic iron metabolism
Hepcidin
Protein produced by hepatocyte membrane
hemochromatosis protein (HFE)
transferrin receptor 2 (TfR2)
Bone morphogenic protein receptor (BMPR)
Hemojuvelin (HJV)
Secreted product of macrophage
Bone morphogenic protein (BMP)
Produced by hepatocyte (and other cells) cytoplasm
Sons of mothers against decapentaplegic (SMAD)
Sites of maximal absorption of iron
Duodenum and upper jejunum
These enhance the reduction of ferric form to the ferrous form and release of iron from those that bind it
Gastric acid
Acidic food such as citrus
Duodenal cytochrome B
A form of iron that is more readily absorbed than ionic iron
Heme
A form of iron that is carried across the luminal side of the enterocyte by divalent metal transporter 1 (DMT1)
FERROUS IRON
Once ferrous iron is absorbed into enterocytes, it requires ______ to deliver it into the blood
Ferroportin
Reoxidizes ferrous into ferric form as it exits for transport into the blood
Hephaestin
Partially degraded ferritin ; appears in cases of iron overload ; may be seen microscopically using the Prussian blue iron stain
Hemosiderin
A plasma protein that is able to save free hemoglobin
Haptoglobin
A plasma protein that is able to save heme
Hemopexin
Gold standard stain for assessment of body iron
Prussian blue stain
Indicator of functional iron available in cells
Soluble transferrin receptor level (sTfR)
Log ferritin index
Aka bronze diabetes
Hereditary hemochromatosis
Characterized by increased iron accumulation in body tissues leading to tissue damage
Hemochromatosis
Most common form of iron overload disease ; autosomal recessive disorder that cause body to absorb and store too much iron ; arthritis, liver cirrhosis, congestive heart failure, impotence, bronze skin, diabetes, thyroid deficiency
Hereditary hemochromatosis
Hereditary hemochromatosis is caused by the mutations in the ___ gene
HFE gene
The gene that regulates the amount of iron absorbed from food
HFE gene
Hereditary hemochromatosis treatments
PHLEBOTOMY
Avoiding food that contains iron
DEFEROXAMINE (DESFERAL)
Increased tissue iron stores without accompanying tissue damage ; may progress to hemochromatosis
Hemosiderosis
