1. Specimen dilution 2. Hemocytometer charging 3. Microscopy (Viewing the ruled areas)

Hemocytometer Flashcards by Kyra Catedral

Manual Cell Counting is used in the following circumstances:

Validity check of electronic methods for calibration purposes

Validity check of electronic counts in patients with profound leukopenia or thrombocytopenia

For blood specimens with platelet counting interferences

Back-up method

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GENERAL PROCEDURES

Specimen dilution
Hemocytometer charging
Microscopy (Viewing the ruled areas)

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BLOOD DILUTION
Purpose

To avoid crowding of cells during manual counting

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Name of pipette

Thoma pipette

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Purpose of bead

For mixing

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RBC Bulb units

100

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WBC bulb unit

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why the last calibration ends in 101 (for RBC) and 11 (for WBC)

One unit =_______

from the tip to the 1.0 mark

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Size of the bulb

RBC - Larger
WBC -Smaller

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Color of the bead

RBC - Red
WBC - White

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Volume of the bulb

RBC - 100 units
WBC -10 units

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Size of the bore

RBC - Smaller
WBC - Larger

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Calibration

RBC - 0.5; 1; 101
WBC - 0.5; 1; 11

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Dilution

RBC - 1:200
WBC - 1:20

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Dilution factor

RBC - 200
WBC - 20

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Diluting Fluids for WBC

2-3% Glacial Acetic Acid

1% Hydrochloric Acid

Turk’s Solution

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The____ is the counting chamber used for manual counting of (3)

hemacytometer

white blood cells, red blood cells, and platelets.

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The principle for counting all three cells are basically the same, only the (3) have differed.

dilution
diluting fluid
area for counting

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Nowadays, the new improved_______ is the most commonly used hemacytometer for enumerating blood cells

Neubauer counting chamber

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There are depressions, called “_____”,
“, that surround the platform.

A______ is positioned so that it covers both ruled areas of the hemocytometer.
This also allows the fluid in the chamber.

moats

coverglass

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The two ruled areas of the hemocytometer have specific dimensions.

Each ruled area is consist of a large square (______). The total_____ area is divided into____ equal squares, each with____

3mm x 3 mm

9mm^2

nine

1mm^2

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According to Rodak’s, all____ squares in the center are for platelet count).

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-Depth of chamber =

0.1 mm

Before using the hemocytometer,____ of the blood sample are first made.

dilutions

T or F Dilution for red cells, white cell, and platelet counts are done separately as each procedure uses different diluting fluids and equipment.

True

T or F The diluting fluids and pipettes used for each type of cell have different characteristics that allow the counting of one cell type of interest only

True

RBC Diluting Fluids

Hayem's Gower's Toisson’s Bethel's Normal Saline Solution, 3.8% Sodium Citrate Formol Citrate

Characteristics of the Diluting Fluid

RBC - Isotonic solution WBC - Will hemolyze red blood cells except the nucleated RBCs; Hypotonic solution

Area counted

RBC - 0.2 mm2 WBC (1:20) 4 mm2 (1:100) 9 mm2

Objective

RBC - 40x HPO WBC - 10x LPO

Charging the hemocytometer Clean the hemocytometer and coverslip with______. Place the coverglass on the hemocytometer such that it covers______. Charge the hemocytometer by touching the tip of the pipette to the point where the______ and ______ on one side meet. Allow the fluid from the pipette to flow into one side of the hemocytometer by____ action. The other side of the chamber is charged the same manner. Before counting, the charged chamber should be allowed to stand for_____ in a moist chamber to allow settling of cells.

alcohol both ruled areas coverglass and the raised platform capillary 10-15 minutes

Viewing the ruled areas Mount the charged hemocytometer on the microscope with________ in place. Bring the stage closer to the objective using course adjustment until the etched lines come into view. Use the fine adjustment knob for sharp focus of the etched lines.

low power objective (10x)

The rule in counting When counting, follow the _____pattern, where the______ and _____ are considered significant.

inverted L upper and left boundaries

Rule in counting • For three-line boundary…

do not count the cells that touch only the outermost boundary

Rule in counting • If the cell touches both the mid and outermost boundary…

include it in the counting

Rule in counting • For two-line boundary, count the cells that…

touch up to the second boundary line

Rule in counting • For one-line boundary, count the cells that touch…

that single boundary line

Rule in counting • Whether the box has three-, two, or one-boundary line, do not count any cells that…

touch the right and lower boundary lines

Rule in counting • For the______ direction, start from the upper left corner box going to the right

serpentine

Why must RBC solution be ISOTONIC?

Prevent lysis of RBC

Why must WBC solution be ACIDIC or Hypotonic?

Will lyse ALL red blood cells except nucleated RBCs Enhances WBC nucleus

Dilution =

volume of solute total volume of solution

RBC DILUTION Computation for RBC Dilution a. solute (pertains to the amount of blood sipped) -____ or _____ b. total volume of the solution (pertains to the volume capacity of the BULB) -_____ c. SOLVENT - just subtract_____ units from 100 units

0.5 units or 1.0 units 100 units 0.5

cells/uL =

number of cells counted x dilution factor ————— area counted x depth * (.1mm)

Shortcut for solving

RBC = x1000 WBC = x50

DETERMINING THE ALLOWABLE VARIATION IN THE COUNTS Whether RBC, WBC, or platelets, both sides of the counting chamber should be counted. The counts of the two sides need NOT be exactly the same. There is an allowable variation. The counts of the two sides of the hemocytometer should agree by______

10% or less less.

To determine the allowable variation:

1. Get the clifference of the two total counts 2. Get the average of the two counts 3. Divide the difference by the average and multiply by 100

ALLOWABLE VARIATION IN THE COUNTS the example, the answer is 5.97%. Therefore, the variation between the total counts of the two sides of the counting chamber is______.

acceptable

When the outcomes in allowable variation is not acceptable,_________. In the case that the variation is still not acceptable after repeat charging, this warrants repeat______ or ______

repeat charging the hemocytometer blood dilution or repeat blood collection.

NOTE: If the average count obtained is in decimal (e.g. 112.5), always…

round up to whole number

= 3.34 x 10^6uL = 3.34 x 10^12/L

Conventional unit S.I unit

Why discard first 3 drops?

Conc. cells

express with___ decimal points for RBC Count express with___ decimal points for WBC Count No decimal points for Platelet Count

2 1

For a Standard leukocyte count within the reference range, there should be NO MORE THAN____-CELL DIFFERENCE between the highest and lowest total number of cells found among the eight 1-mm-corner squares (2 chambers) counted.

For Standard erythrocyte count, there should be NO MORE THAN___-CELL DIFFERENCE between the highest and lowest total number of cells counted in the ten-0.04-mm^2 ( 2 chambers). Counts that do not meet these standards should not be reported. Rather, the procedure should be repeated.