Sect 2 - Manual Differential

Wright's stain

stain used for performing manual differentials

aka Romanowsky stain

Methylene Blue

Eosin

Glycerin

components of the Wright's stain

Phosphate Buffer

used to determine the pH of the stain

6.4

pH of the stain

Deionized water

What type of water is used for rinsing during staining?

Methylene Blue

absorbs onto the acidic portion of the cell

Eosin

absorbs onto the basic portion of the cell

RBCs

reddish/pink cells in a good stain

neutrophils (segs) & lymps

nuclei will stain purple-pink in a good stain

eosiniphils

granules will stain orange-pink in a good stain

basophils

granules will stain dark blue-black in a good stain

pH <6.0 / too acidic

RBCs will be orange

excess amount of buffer to stain

insufficient staining time

over-washing

What causes pH to be too acidic (pH

pH >6.0 / too alkaline

RBCs will stain purple/blue/gray

excess stain to buffer

not adequately rinsed

What causes pH to be too alkaline (pH >6.0)?

insufficient staining

prolonged washing

too high acidity of the stain or buffer

Stain will be too pink or light if there is:

Granulocytic

Erythrocytic

Monocytic

Megakaryocytic

What cell lines are denoted:

CFU-G

CFU-E

CFU-M

CFU-M

100 cell differential

RBC morphology

WBC morphology

Platelet morphology

WBC estimate

Platelet estimate

What are the 6 components of manual differentials?

10x

Objective magnification that is used to look for even distribution in the differential procedure

40x

Objective magnification used to perform the WBC estimate in the differential procedure

100x

Objective magnification used to perform a 100-cell differential and perform the platelet estimate in the differential procedure

RBCs, WBCs, platelets

What 3 morphologies are included in the differential?

Cell size

Nucleus

Cytoplasm

Granules

Nuclear:Cytoplasm ratio

What are the 5 characteristics to be considered for WBC differentiation?

chromatin/non-chromatin pattern

presence of a nucleolus

shape of the nuclear membrane

3 characteristics to be considered of the nucleus to differentiate the cell

color

inclusions

vacuoles

3 characteristics to be considered of the cytoplasm to differentiate the cell

nuclear:cytoplasm ratio

Amount of space occupied by the nucleus in relationship to the space occupied by the cytoplasm

Differs from cell size

Polymorphonuclear

What does PMN stand for?

**hint: pertaining to a white blood cell with a multi-lobed; Segmented Neutrophils

phagocytosis

function of neutrophil

a type of endocytosis in which a cell engulfs large particles or whole cells

true

T or F:

If vacuoles are seen in the neutrophils, we need to report them

Neutrophilia

increase in neutrophils

Conditions associated:

Leukemoid reactions, Bacterial infections, Chronic myelogenous leukemia, Myelofibrosis, Pregnancy

Neutropenia

decrease in neutrophils

Conditions associated:

Viral illness, Malaria, Thyroid disorders, Aplastic anemia, Typhoid fever

horseshoe

shape of the nucleus in the band neutrophil

Band Neutrophil

immature neutrophil

seen in peripheral smear when cells are being pushed out in increased numbers as a response to specific conditions

natural killer cells

lymphocytes that contain azurophilic (red) granules

T-cells & B-cells

two types of lymphocytes

T-cells

cellular immunity

B-cells

humoral immunity

Lymphocytosis

increase in lymphocytes

Conditions associated:

Viral infection, Pertussis, Toxoplasmosis, TB, Syphilis

Lymphocytopenia

decrease in lymphocytes

Conditions associated:

AIDS, Hodgkin's Lymphoma, Lupus, Renal failure, Administration of certain drugs

RBC

What cell is generally used to compare to the size of a normal lymphocyte?

Monocyte

an agranulocytic leukocyte that performs phagocytosis to fight infection

vacuoles can be observed during phagocytosis

ground glass appearance, light blue, or lacy

color of cytoplasm of a monocyte

purple & irregular shaped

nuclear color and chromatin pattern of a monocyte

phagocytosis

function of monocyte

vacuoles can be observed during this process (don't need to report out)

Monocytosis

increase in monocytes

Conditions associated:

Recovery phase of an acute infection, Ulcerative Colitis, SLE (lupus), Rheumatoid arthritis, TB syphilis, and many hematological neoplasms (Myelodysplastic syndrome)

true

T or F:

Eosinophilic granules are secondary granules

enhance or suppress the immunoinflammatory reaction

mildly phagocytic

function of eosinophils

Eosinophilia

increase in eosinophils

Conditions associated:

NAACP

CML, Polycythemia Vera, Myelofibrosis, Hodgkin's Lymphoma, Essential Thrombocythemia, Allergic disorders, Asthma, Parasitic infections

Eosinopenia

decrease in eosinophils

Conditions associated:

Shock, Trauma, Surgery, Administration of corticosteroids or epinephrine

purple-black

color of the secondary granules of the basophil

heparin and histamine

two chemicals contained in the granules of basophils

enhance the immunoinflammatory response

function of basophils

Basophilia

increase in basophils

Conditions associated:

Allergic reaction, Chicken pox, Ulcerative colitis, Various myeloproliferative disorders (CML, Polycythemia Vera, Myelofibrosis)

relative ranges

ranges express the specific type of WBC as a percentage of the total WBC count

50-70%

relative range of neutrophils

20-40%

relative range of lymphocytes

1-10%

relative range of monocytes

0-5%

relative range of eosinophils

0-2%

relative range of basophils

absolute ranges

express the quantification (number) of a specific type of WBC

5,000-10,000ul

normal range for WBCs

lymphocyte

name the cell:

basophil

name the cell:

neutrophil

name the cell:

monocyte

name the cell:

eosinophil

name the cell:

myelocytic stage

Stage of granulopoiesis where secondary granules appear