Hematology 425 PB Smear Examination

1. Hematology 425 PB Smear Examination Russ Morrison October 20, 2006

2. PB Smear • The well-made, well-stained blood smear can provide valuable information • More can be learned from this procedure than any other single hematologic test • WBC and platelet counts can be estimated • Proportions of different types of WBCs can be observed • Morphology of the three circulating cell lines (RBC, WBC, platelet) can be evaluated for abnormalities

3. PB Smear Preparation • EDTA-anticoagulated blood is collected in a purple top tube • EDTA anticoagulates the blood by chelating calcium required for coagulation • Smears should be prepared within 2 to 3 hours of collection the EDTA anticoagulated sample • Blood more than 5 hours old shows RBC artifact (echinocyte, spherocyte, necrobiotic leukocytes, vacuolated neutrophils and monocytes)

4. PB Smear Preparation • Advantage of making smears from a tube of blood is that multiple slides can be made and the timing of slide making can be postponed • Platelet estimates are more accurated when anticoagulated blood is used to prepare the smear • Some still believe that direct preparation of smears is best to evaluate blood cells and their morphology

5. PB Smear Preparation • EDTA can be a source of error • Platelet satellitosis can cause a pseudothrombocytopenia • Platelet clumping can cause low platelet and increased WBC dounts • These problems may be eliminated using sodium citrate as an anticoagulant

6. Platelet satellitosis, (platelets encircling a neutrophil) occurs when a patient has a serum factor that reacts to the anticoagulant EDTA

7. PB Smear Preparation • PB smears may also be made directly from finger or heel punctures at the patient’s side. • Limitations of direct prep • Platelet clumping can be expected • Limited number of smears can be made

8. PB Smear Preparation • Techniques for making PB Smears • Manual wedge technique • Coverslip technique • Automated slide maker • Regardless of method, smears should be dired quickly to avoid drying artifact (moth-eaten looking red cells)

9. Staining of PB Smears • Ronamowsky Stains such as Wright’s or Wright-Giemsa stain are most often used • They are polychrome stains because both eosin and methylene blue are used • Methanol in the slide fixes the cells to the slide • A buffer facilitates the staining action • Staining reactions are pH-dependent

10. Staining of PB Smears • Methylene blue is basic and stains acidic cellular components such as RNA • Eosin is acidic and stains basic components such as Hgb or eosiniphilic granules • Slides may be stained manually or with automated stainers

11. PB Smear Examination • 10X or Low Power • Assess the overall quality of the smear, including abnormal distribution of RBCs, agglutination or a disproportionate number of large nucleated cells at the edge of the smear. If any of the above conditions exist, a new smear should be prepared. While scanning with this lens, check to see if abnormal cells such as blasts, reactive lymphs or parasites are present. Also check for clumps of platelets which may indicate inadequate anticoagulation of the sample (partial clotting).

12. PB Smear Examination • 40X or High Dry • Using the 40X (high dry) objective, find an area of the smear in which the RBCs are evenly distributed and barely touch one another. Scan 8 to 10 fields in this area of the smear and determine the average number of white blood cells (WBCs) per field. The average number of WBCs per high power field multiplied by 2000 gives an approximation of the total WBC count/mm3. Discrepancies between the WBC estimate and the machine count should be resolved.

13. PB Smear Examination • 100X or Oil Immersion • In the same area of the smear as used for 40X review, perform the WBC differential. Count 100 consecutive WBCs in a systematic manner using the “battlement” track (Figure 1-6) of the Hematology atlas. Results are reported as a % for each type of cell seen during the differential. NRBCs are also counted during the differential, if seen, and reported as # NRBCs/100 WBCs. Evaluate the RBC morphology in relation to size, shape and color of the RBCs on the smear. Results of the RBC evaluation should be consistent with the indices reported by the hematology instrumentation.

14. PB Smear Examination • 100X or Oil Immersion • The final step with the 100x objective is to estimate the platelet count. In the same area where the differential was performed, count the number of platelets in 10 oil immersion fields. The average number of platelets in those 10 fields multiplied by 20,000 will provide an estimate of the platelet count which should match the automated count within reason. The platelet estimate is reported as decreased, adequate or increased (using 150 to 450 x 103/ul as the reference range).

15. PB Smear Examination • Platelet number is reported as adequate, increased or decreased • RBC morphology is evaluated in regard to presence of anisocytosis and poikilocytosis • Anisocytosis is quantitated when greater than normal, poikilocytosis is quantitated and the poikilocytes present are identified