Lecture 2

Manual Hemocytometry Errors

Manual Hemocytometry Errors

• Random / Human

• Standard / Systematic (equipment)

• Inherent / Field

  • random settling of cells

  • Poisson’s Law

RBC diluting fluids

• Hayem’s + HgCl

• Gower’s + Glycerine

• Toisson’s + Methyl violet

• Bethel’s + Sodium thiosulfate

• Formol citrate / Dacie’s best

• Normal saline solution

Formol citrate / Dacie’s best

prevents the growth of molds

Normal Saline Solution

• cheapest diluting fluid

• 0.85% NaCl conc

• lyse WBC

WBC diluting fluids

• 2.3% HAc / Ch3COOH with methyl violet

• 1% HCl with methyl violet

2.3% HAc / Ch3COOH with methyl violet

1% HCl with methyl violet

to differentiate from RBC diluting fluid

acids don

Red bead

thoma pipet for RBC

White bead

thoma pipet for WBC

Nucleated Red Blood Cells

these are seen in newborns or patients with leukemia

>10 NRBCs (immature) / 100 WBCs in the differential count

corrected WBC

CWBC = uncorrected count x no. NRBCs / 100 + no. of NRBCs

long method CWBC

CWBC = 100 / (100 + NRBCs) x uncorrected count

short method CWBC

Differential count

counting various types of WBC in % in a blood smear

True

human is better than any machine to recognize cells. true or false?

• ½ or 2/3 of the slide

• feathery edge

• head, body, tail (zone of morphology)

• no holes

• no overlapping cells

• thick to thin (gradual transition)

a blood smear must:

False. venous blood causes holes

holes may be caused by using capillary blood instead of venous blood. true or false?

• Wrigth’s

• Giemsa

  • MB

  • Eosin

stain used in blood smear

Hema Quick

what is the stain used in our laboratory?

• 2-slide method

• Slide and coverslip method

• 2-coverslip method

methods of blood smear for differential count

Affects the quality of the smear

• size of the drop of blood

• angle

• speed of spreading

• pressure on the slide

• surface of the slide and spreader

these factors affects _________

30 degrees

ideal angle for smearing

• 2-field meander

• 4-field meander

• Exaggerated battlement

• Crenelation / Strip differential

methods of counting

cells/liter

SI unit

mm³

conventional unit

Male: 4.5 - 5.9 × 10¹² cells/liter

Female: 4.0 - 5.2 × 10¹² cells/liter

RBC reference value (SI unit)

Male: 4,500,000 - 5,900,000 cells/mm³

Female: 4,000,000 - 5,200,000 cells/mm³

RBC reference values (conventional)

Male: 13.5 - 17.5 grams/deciliter

Female: 12 - 16 grams/deciliter

Hb reference values (not the SI unit)

Male: 135 -175 grams/liter

Female: 120 - 160 grams/liter

Hb reference values (SI unit)

Male: 0.41 - 0.53 liter/liter

Female: 0.36 - 0.46 liter/liter

Hct reference values (SI unit)

Male: 41-53%

Female: 36-46%

Hct reference values (percentage)

B¹² deficiency

more than 100 femtoliters indicates

Iron deficiency

less than 80 femtoliters indicates

1 × 10⁹ - 3 × 10⁹ cells/liter

platelet count reference values (SI unit)

100,00 - 300,000 cells/mm³

platelet count reference values (conventional)

Reticulocyte count

this count helps differentiate if the anemia is hemolytic or non-hemolytic

0.5-1.5%

reticulocyte/retics count reference values for adult

2-6%

reticulocyte/retics count reference values for infants

4.5 - 11 × 10⁹ cells/liter

WBC reference values - male & female (SI unit)

4,500 - 11,000 cells/mm³

WBC reference values - male & female (conventional)

Estrogen doesn’t affect WBCs

why are WBC reference values the same for male and females?

1. Neutrophils (segmenters)

2. Lymphocytes

3. Monocytes

4. Eosinophils

5. Basophils

6. Band/Stab/Staff

WBC differential count (pre 200 or 100 cells)

• Neutrophils - 47-79.5%

• Lymphocytes - 12.5-40%

• Monocytes - 2-11%

• Eosinophils - 0-7%

• Basophils - 0-2%

• Band/Stab/Staff - 0-5%

WBCC reference values

Bacteria

neutrophils higher than 80% indicates

Neutrophils

• lobulated nuclei

• 3-6 lobules

• lilac granules

Viral infection

high lymphocyte count indicates

Monocytes

• TB, syphilis

• has vacuoles

• largest WBC

Eosinophils

• parasitic infections

• orange granules

Basophils

• allergies with eosinophils

• elevated in cancers

• least occurring cell

• blue-black granules

Band/Stab/Staff

immature neutrophil

Polycythemia

an increase number of red blood cells

• oil immersion field

• 8-10 count

platelets must be estimated

Estimated WBC and Differential Count

WBC/HPF WBC/LITER

2-3 = 4×10³ - 7×10³

4-6 = 7×10³ - 10×10³

7-10 = 10×10³ - 13×10³

11-20 = 13×10³ to 18×10³