LEUKEMIA, FLOWCY, RETICS, AUTOMATION, OTHERS Flashcards by Eliseo John Posion

Overproduction of various types of immature or mature cells in the bone marrow and or peripheral blood

Leukemia

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Frequently involves WBCs of the myelogenous or lymphocytic cells types

Leukemia

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Malignant cells easily trespass the blood brain barrier

Leukemia

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Solid malignant tumors of the lymph nodes and related WBCs in the bone tissue

Lymphomas

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The distinctive cell type is the lymphocyte

Lymphomas

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Malignant cells are initially confined to the organs containing mononuclear phagocytic cells such as lymph nodes, spleen, liver, and bone marrow

Lymphomas

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This can spill over into the circulating blood and demonstrate a leukemia-appearing picture on a peripheral blood film

Lymphoma

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Leukemia has increased WBC count with shit to the ____

Left

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Leukemia M:E ratio

10:1

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Type of anemia usually present in cases of acute leukemia

Normocytic normochromic

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Numerous immature cell forms in the bone marrow and or peripheral blood; increased total WBC count; symptoms of short duration

Acute leukemias

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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

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The FAB classification of leukemia is based on morphology of cells in what stained smear

Romanowsky-stained smear

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Cytochemical stains used in FAB classification

Myeloperoxidase
Sudan Black B

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Enzyme found in primary granules of neutrophils and eosinophils and monocytes

Myeloperoxidase

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Used in differentiating blasts of AML from those of ALL

MYELOPEROXIDASE

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Peroxidase stain reaction of ALL

POSITIVE

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POSITIVE MPO

Neutrophilic granulocytes (Except normal blasts)
Auer rods
Leukemia blast in FAB M1, M2, M3
Eosinophils

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POSITIVE MPO

Neutrophilic granulocytes (Except normal blasts)
Auer rods
Leukemia blast in FAB M1, M2, M3
Eosinophils

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Reactions parallels those of the MPOs

Sudan black B

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Stains sterols, neutral fats, phospholipids (found in the primary and secondary granules of neutrophils and lysosomal granules of monocytes)

Sudan black B

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Most sensitive stain for granulocytic precursors

SBB

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SBB POSITIVE

STRONGLY POSITIVE:
Promyelocyte, myelocyte, metamyelocytes, bands, and segmented neutrophils

Leukemic blasts
Auer rods
Eosinophils

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Weakly positive or negative MPO

MONOCYTES

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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

70% of ADULT ALL

ALL type that is rare in children and adults

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

Heterogenous population of large blasts - ALL type

Homogenous population of large blasts (with nuclear and cytoplasmic vacuoles) - ALL type

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

AML without maturation ; may demonstrate Auer rods

AML with maturation ; most common subtype of AML ; may demonstrate Auer rods

Acute promyelocytic leukemia ; DIC ; Faggot Cells

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

An Acute myelomonocytic leukemia with increased marrow eosinophils

M4E

Acute monocytic leukemia ; schilling leukemia

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

Acute megakaryocytic leukemia ; required immunocytochem for accurate diagnosis ; factor VIII stain positive

Acute basophilic leukemia

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?)

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

RPI that generally indicates adequate bone marrow response

RPI that generally indicates inadequate bone marrow response

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

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

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)

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

Region code ; is the error code for MULTIPLE REGION OVERLAP

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