Monday, 19 October 2015

WBCs and RBCs count in sterile body fluids (such as CSF)

The quantity of white blood cells and red blood cells in sterile body fluids (such as CSF) can be obtained by performing a cell count on the uncentrifuged specimen, preferably the last specimen taken, using a modified mirrored Fuchs Rosenthal counting chamber. The cell count of sterile body fluids specimen such as cerebrospinal fluids (CSF) usually gives important clues of presence of infection or not and if present, the infecting organism. Sequential specimens 1 to 4 are usually obtained from one lumbar puncture and specimen 1 is sent to Clinical Biochemistry laboratory while 2 – 4 are sent to Microbiology laboratory. Red cell count is done on the entire 3 Specimen sent to Microbiology laboratory and both RBC and WBC count is performed on specimen 4. Uniform blood staining of all samples suggests previous haemorrhage into the subarachnoid space, whereas reducing counts in sequentially obtained samples suggest bleeding induced by the tap procedure.  CSF obtained more than 12 hours post intra-cranial haemorrhage may show raised WBC counts of up to 500 x 106/l as a result of an inflammatory response.
The procedure for performing a cell count starts with drawing a line with a chinagraph pencil on the external supports of the clean and dry counting chamber and then gently pushes the cover glass onto the counting chamber from the front. The formation of interference lines (Newton rings) between the external support and the cover glass shows that the cover glass is correctly positioned. Fill the counting chamber with the specimen using a fine tip sterile pipette and allows to settle for 5 minutes. Observe under x10 objective lens microscope to focus and the x10 or x40 to count cells.
The modified Fuchs Rosenthal counting chamber has nine (9) large triple lined squares; each divided in 16 small squares and has a depth of 0.2 mm. as shown in Figure below.  Each large square is 1 mm2; therefore 5 large triple lined squares are counted to get the count /mm3. The four corner squares and the middle are also counted.  If cells are lying on the triple lines between squares count only the cells lying on the inner two lines of the top and the left side lines.
The ratio of WBC and RBC in a normal blood in WBC1-2: 1000 RBC and under normal conditions the number of white cells in a CSF is <5/mm3 in children and <20/mm3 in adults. In some situations where the sample is turbid or blood stained, with a high expectation of RBC count of >200 mm3, dilution is often performed in sterile saline before loading on the counting chamber. The cell count result must then be multiplied by the dilution factor.

Normal CSF values

Normal CSF values              Neonates                   Adults

Leucocytes                         Neonates              0-30  cells / cu mm   ( x 106/l)
                                           1-4yr old                0-20  cells / cu mm   ( x 106/l)
                                           5yr-puberty            0-10  cells / cu mm   ( x 106/l)
                                           Adults                     0-5   cells / cu mm   ( x 106/l)
Erythrocytes                Newborn                     0-675 cells / cu mm   ( x 106/l)
                                      Adults                        0-10  cells / cu mm   ( x 106/l)

The counting chamber grid

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