Using the 40X objective -
finding an area where 2-3 RBC overlap but most are lying out by themselves
scan 8 -10 fields and count the number of WBC seen in each
calculate the average WBC seen per field and multipy by 2000(this number will approximate the WBC value obtained by the automated instrument)
Using the 100X objective - scanning the monolayer for WBCs in a systematic manner. When a WBC is found press the appropriate button on the manual differential counter , 100 WBC are counted and the differential is reported in percentages; for example 55% segs, 4% bands, 26% lymphs, 9% monocytes, 5% eosinophils and 1% basophils. As a quality check the technician should always analyze the results to ensure that they equal 100%.
observe the monolayer for RBC morphology, Scan at least 8 - 10 fields identifying any RBC abnormalities and / or inclusions. Simultaneously, review the RBC indices and verify that the microscopic analysis corelates with the indice values reported from the automated instrument (ie. MCV >100fl = macrocytes, MCHC < 31.0 g/dl = hypochromia, RDW > 14.5% = presence of anisocytosis, etc).
100X objective find an area of the smear where the RBC barely touch (somewhere between the "swiss cheese" and the monolayer) and count the number of platelets seen in at least 10 OIF (Oil Immersion Fields). Find the average number of platelets seen per field and multiply this number by 20,000 (this value will approximate the platelet value obtained from the instrument).
reference range for platelets
150 - 450 x 109/L
Types of Unopette Systems-WBC
* 20 ul pipet -1:100 dilution AMMONIUM OXALATE
* 25ul pipet -1:20 dilution ACETIC ACID
Types of Unopette Systems- RBC count
10ul pipet - 1:200 dilution with SALINE
Types of Unopette Systems-Platelet count
same dilution that is utilized for manual WBC counts.
20ul pipet / 1:100dilution / AMMONIUM OXALATE
final WBC value Calculations
The total number of cells counted from one side of the hemacytometer is averaged with the total number of cells counted from the second side of the hemacytometer
# cells counted x dilution x 10 /Number of "quadrants" counted
Hgb F Hgb A2
2 alpha & 2 beta chains
2 alpha & 2 gamma chains
2 alpha & 2 delta chains
Hgb Gower I
Hgb Gower II
2 zeta & 2 gamma chains
2 zeta & 2 epsilon chains
2 alpha & 2 epsilon chains
RBC maturation series
"Perry Browns Pet Ox Ran Everywhere"
Reference ranges for RBCs
4.00 - 6.00 x 1012 / L.
Howell Jolly Bodies
very round, purple-staining, single inclusion
Composed of DNA
Seen in: Splenectomy, hemolytic anemia, megaloblastic anemia and other nuclear maturation defects
Many, coarse or fine, purple-staining granules in the RBC that are fairly evenly distributed throughout the cell
Composed of RNA
Seen in: Lead poisoning, thalassemia
Pappenheimer Bodies (Siderotic Granules)
Generally, there are multiple granules or they can be found in a small cluster (granules are more than one but less than what is found in basophilic stippling)
Granules tend to be near the periphery (outside) of the cell
Composed of Iron
Seen in: Splenectomy, hemolytic anemia, megaloblastic anemia, hemoglobinopathies and sideroblastic anemia