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Hemocytometer: Manual Cell Counting

By Edited Mar 25, 2016 0 0
Hemocytometer
Credit: Jeffrey M. Vinocur through Wikimedia Commons

Counting cells in blood

Counting the different types of cells present in blood is one of the oldest practices in hematology. They also permit the detection of quantitative alterations (in relative and absolute terms) of the different blood cells.

Platelet count. In certain pathological conditions (changes in the size of platelets) automatic counting is ineffective, since the increasing size of such cells makes it impossible for the apparatus to differentiate from other blood cells of a larger size.

Cell Study in other biological samples. There are other biological samples (cerebrospinal fluid, peritoneal and many others), for which it is often necessary to carry out the counting of erythrocytes or leukocytes (that are present due to the disease) manually.

Materials

Neubauer chamber

Also called counting chamber or hemocytometer (hemacytometer, haemocytometer). It consists of a thick plate-shaped glass holder, whose central portion is divided in 3 axes perpendicular to the longitudinal axis of the camera. Of these, the two sides are uplifted 0.1 mm from the center, and in the latter there is a square lattice engraved (single chamber).

The central part may be, in turn, divided into two identical half bands separated by a dent parallel to the longitudinal axis of the chamber (double chambers). There is an identical grid engraved in each of these two half bands to facilitate the cell counting.

Not all counting chambers are the same; the most commonly used in hematology is the Neubauer chamber. It is a double chamber, whose grid (grids if it is a double chamber) are called Neubauer grids.

Each grid is divided into nine x1mm large squares. The corner squares are the ones used for WBC (because there are fewer WBC than erythrocytes, the grids used can have fewer reference lines), which are in turn subdivided into 16 smaller squares that measure 0.25 mm in width/height.

The central square is used to count red blood cells and platelets. It is divided into 25 squares (0.2mm), and these in turn are subdivided into 16 small squares. Thus the total number of small squares in the central box is 400.

Coverslips

Preferably, it should be slightly thicker than those normally used for the microscope. It has to rest on the two sidebands of the central portion of the chamber. Thus, a space is delimited between the central strip and the cover in which the sample is deposited and whose thickness is 0.1 mm.

Some chambers also have two special clips to ensure that the cover won’t move.


Diluting pipettes of Thoma

These special pipettes consist of a capillary tube and an ampullary dilation or bulb.

The capillary tube is sharpened at one of its ends, and is continuous until it reaches the bulb at the other end. Furthermore, it is divided into 10 equal parts, with an highlighted 5th division (with a 0.5) 10th division (a 1).

The bulb contains a glass bead to facilitate the mixing of the blood with the dilution liquid, and ends in a short capillary tube. The glass bead is red in the pipettes used for erythrocyte count, and white on those used for the leukocyte count. In addition, the capacity of the bulb in erythrocyte pipettes is 100 times bigger than the one of the longer capillary tub. In leukocyte pipettes, the bulb capacity is 10 times bigger than the capillary tube length, so there is a mark at 11 in the shorter tube.


Rubber suction and nozzle

It is a rubber tube that allows the aspiration of the sample and the diluting liquid.

At one end is fitted a nozzle, and the other is attached to the any of the Thoma pipettes’short capillary. The tube is applied to a nozzle through which the technician will aspirate.

Reagents

There are several types of dilution liquids. Its composition varies depending on the type of cells that are intended.

Those used for erythrocyte count contain sodium chloride to make them isotonic to plasma and to avoid hemolysis. But some also incorporate anticoagulants such as sodium citrate, and even antiseptics such as formalin.

Those used for counting leukocytes contain a substance such as glacial acetic acid that breaks the erythrocytes and a dye, such as crystal violet, which stains the nuclei of leukocytes.

Those used for the platelet count can incorporate a hemolytic substance and an anti-clotting agent for platelets.

The most commonly used dilution fluids are Hayem to count erythrocytes, and Turck, to count leukocytes.

Sample

Capillary or venous blood anticoagulated with EDTA. This blood will be used at an appropriate dilution.

Cleaning of equipment

With regards to the cleaning of the Neubauer chamber: after its use, it should be rinsed with warm water and then wiped with a soft cloth.

Following the use of pipettes Thoma, they should be washed as follows:

    First, by passing water through them, once.

    Then, by passing distilled water through them 3 times.

    Finally, by passing acetone or alcohol (95º) through them.

Thereafter, they should be dried inside, preferably with an electric dryer.

Manual counting

Depending on the type of blood cell to count, we will have to choose the appropriate dilution liquid but also a dilution ratio that will take into account the realtive amounts of each blood cell type in blood.

In all cases, once we perform the corresponding cell counts in the appropriate regions of the camera (depending on the cell type under study), the results should be referred to a volume (related to the area of the squares counted but also to the height of the chamber) and of course corrected by the dilution factor. We express the results as cell density: number of cells (leukocytes, erythrocytes or platelets) / mm3.

On the other hand, it is necessary to emphasize the wide margin of error introduced by this method. The manual counting technique is characterized by having a high margin of error motivated by:

    Errors in the dilution.

    Dilution fluid contamination.

    Errors in the assembly and / or filling of the chamber.

    Errors in calculations to perform.

    Errors in the count itself.

The margin of error can be reduced with a good practice and skill of the operator, but there is a protocol to follow to avoid or reduce the error in the count itself, since otherwise a cell could be counted twice or not counted at all.

The rule to follow is:

You must conform to certain rules so that cells that are on or near the boundary lines are not counted twice or excluded in the counting. All cells that are within a defined measurement zone should be counted. Cells that are touching the left or top lines should be counted as well, but not the ones that are touching the bottom or right lines.

You should also establish a direction of counting, for example a zigzag in which you go left on one line and right on the other (so that you don’t count squares twice).

 

For more information on hemocytometer calculation visit www.hemocytometer.wordpress.com.

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