Chapter 15 Examination of the Peripheral Blood Film and Correlation with the Complete Blood Count A well stained

ed peripheral blood can provide information about: a) WBC and platelet estimates b) Relative proportions of the different types of WBC c) Evaluation for the abnormalities of the morphology of all three cell lines The peripheral blood film evaluation is the capstone of a panel of test called the complete blood count (CBC) or hemogram The CBC includes: a) Enumeration of cellular elements b) Quantification of hemoglobin c) Statistical analysis that provides a snapshot of cell appearances Peripheral Blood Films Specimen Collection EDTA Specimen for peripheral blood film is collected in lavender (purple) top tubes which contains Etyhylenediaminetetraacetic acid (EDTA) EDTA anticoagulates the blood by chelating the calcium that is essential for coagulation Specimen from EDTA tubes should be done within 2 to 3 hours of drawing the specimen Blood film from EDTA tubes that remains in room temperature for more than 5 hours have unacceptable blood artifacts Main advantages of making blood film for EDTA tubes: a) Multiple slides can be made if necessary b) Do not have to be prepared immediately after draw

1 c) Generally prevents platelets from clamping to the slide Platelet satellitosis is a phenomenon where in the platelets surround or adhere to neutrophils, which potentially causes pseudothrombocytopenia when counted is done by automation Low platelet counts and falsely increased WBC counts can result from EDTA induced platelet clumping Sodium citrate EDTA induced problems can be eliminated by recollecting specimens in light blue top tube which contains sodium citrate and ensuring the proper ratio of nine parts blood to one part anticoagulant is observed Platelet counts and WBC counts from sodium citrate specimens must be corrected for the dilution of blood with the anticoagulant Platelet counts and WBC counts from sodium citrate specimens, are multiplied to 1.1 to obtain accurate results Finger and heel puncture Platelet clumping must be expected if films are made directly from drop of finger-stick or hell stick or if blood is collected in heparinized microhematocrit tubes Only a few films can be made directly from blood from a skin puncture before site stops bleeding Peripheral Film Preparation Types of films Manual Wedge Technique Easiest to master Most convenient and commonly used method for making peripheral blood film

 Requires at least two 3-inch by 1-inch clean glass slides High quality beveled edge microscopic slides with chamfered corners for good lateral borders are recommended The size of the blood is important: a) Too large a drop creates a long or thick film b) Too small a drop creates a short or thin blood film It is important that the whole drop be picked up and spread Moving the pusher slide forward too slowly accentuates poor leukocyte distribution by pushing larger cells to the very end of the slide of the film Maintaining an even, gentle pressure on the side is essential It is crucial to keep the same angle all the way to the end of the film When the hematocrit is higher than normal the angle should be lowered so the film is not too short and thick For extremely low hematocrit, the angle may be raised Procedure A drop of blood about 2-3mm is placed at one end of the slide The drop may be delivered using a Diff-Safe dispenser The Diff-Safe dispenser is inserted into the rubber stopper of the EDTA tube which eliminates the need to remove the rubber stopper Push type wedge preparation where in spreader slide is pulled into the drop of blood then the film is made by pushing the blood along the slide Pull type wedge preparation where in spreader slide is pushed into the drop of blood then the film is made by pulling the blood along the slide Features of a well made wedge peripheral blood film 1. The film is two thirds to three fourths the length of the slide

2.

3. 4. 5. 6.

2 The film is finger shaped, very slightly rounded at the feathery edge, not bullet shape, this provides the widest area for examination The lateral edge is visible The film is smooth without irregularities (holes or streaks) Thin feathery edge has rainbow appearance when held against the light Whole blood is picked up and spread

Common causes of poor blood smear 1. Drop of blood too large or too small. 2. Spreader slide pushed across the slide in a jerky manner. 3. Failure to keep the entire edge of the spreader slide against the slide while making the smear. 4. Failure to keep the spreader slide at a 30 angle with the slide. 5. Failure to push the spreader slide completely across the slide. 6. Irregular spread with ridges and long tail: Edge of spreader dirty or chipped; dusty slide 7. Holes in film: Slide contaminated with fat or grease 8. Cellular degenerative changes: delay in fixing, inadequate fixing time or methanol contaminated with water. Biologic cause of poor blood smear 1. Cold agglutinin - RBCs will clump together. Warm the blood at 37 C for 5 minutes, and then remake the smear. 2. Lipemia - holes will appear in the smear. There is nothing you can do to correct this. 3. Rouleaux - RBCs will form into stacks resembling coins. There is nothing you can do to correct this Coverslip Technique Older technique compared to wedge technique Rarely used for peripheral blood films but is sometimes used for making bone marrow aspirate smears

 Advantage is that this method produces excellent leukocyte distribution, which leads to more accurate determination of differential counts Procedure The technique requires one drop of blood or bone marrow aspirate placed on a clean coverslip (22X22mm) and another coverslip placed on top, with the blood allowed to spread across the two slides Slight pressure is applied to the cover slip between the index finger and the thumb to help spread the bone marrow spicule before the two smear are pulled apart (crush preparation) Too much pressure on the cover slip can cause cell rupture Inadequate pressure prevents the spicule from spreading to a satisfactory monolayer Automated Slide Making and Staining Sysmex SP-1000i An automated slide making and staining machine Films can be produced approximately every 30 seconds Drying Films Before staining all blood films and bone marrow smears should be dried as quickly as possible to avoid drying artifacts Blowing breath on a slide is counter productive, because the moisture in breath causes RBCs to become echinocytic (crenated) or to develop water artifact (also called drying artifacts Staining of Peripheral Blood Films Staining method used for staining peripheral blood: Pure wright stain or Wright Giemsa stain (Romanowsky stain)

3 Considered polychrome stains because of eosin and methylene blue content May also contain methylene blue azure Purpose of staining is to makes cells more visible and allows their morphology to be evaluated Methanol in the stain fixes the cell to the slide Actual staining occurs when the buffer is added which is 0.05M sodium phosphate (pH 6.4) or aged distilled water Oxidized methylene blue and eosin form a thiazine-eosinate complex, which stains neutral components Staining reaction are pH dependent Free methylene blue is basic and stains acidic cellular components Free eosin is acidic and stains basic cellular components Water or drying artifact has been a long-lived nuisance to hematology laboratories Water or drying artifacts can appear as: a) Mouth eaten look to the RBC b) Heavily demarcated central pallor c) Refractile blotch on the RBCs d) Echinocytes (crenation) seen in the areas of the slides that dries most slowly Factor that contribute to water or drying artifact problem: Humidity in the air as the slide dries may add to the punched out, moth eaten or echinocytic appearance of the RBC Manufacturers have used 10% volume to volume methanol to minimize water or drying artifact

Wright Staining Method Manual technique Performed over a sink or a pan with a staining rack It is important to flood the slide completely

4 Stain should remain at least 1 to 3 minutes to fix the cells to the glass Approximate equal amount of buffer is added to the slide A metallic sheen (green scum) should appear on the slide if mixing is correct Bone marrow smears take longer to stain than peripheral blood film When staining is complete, slide is rinsed with a steady but gentle stream of neutral pH neutral then the slide is air dried in a vertical position Use of manual Wright staining technique is desirable for staining peripheral blood films containing very high WBC counts Under staining is common when a leukemia slide is placed on an automated slide stainer Disadvantage of manual technique are the increased risk of spilling stain and the longer time required to complete the procedure Automated slide stainers It takes 5 to 10 minutes to stain a batch of slides The slides may be automatically dipped in stain and then in buffer and a series of rinses or propelled along a platen surface by two conveyors In a Hema-Tek device, stain, buffer and rinses are pumped through holes in the platen surface flooding the slide at the appropriate time Slides can be added anytime to HemaTek stainer, and stains package are stable for only6 months Disadvantage of the dip-type batch stainers a) Stat slides can not be added to the batch once staining process had begun b) Working or aqueous solution of stain are stable for only 3 to 6 hours Quick stains Fast and easy which takes about 1 minutes Modified Wright or Wright Giemsa stain Required quantity can be filtered into a coplin jar or a staining dish, depending on the quantity of slides to be stained Quantity is often the concern of quick stain, however color quantity is acceptable Features of a well-stained peripheral blood film Should be pink to purple RBC should appear orange to salmon pink WBC nuclei should be purple to blue Cytoplasm of neutrophils should be pink to tan with violet or lilac granules Eosinophils should have bright orange refractile granules Best staining result are obtained on fresh slides because the blood itself acts as a buffer in the staining process Slides stained after 1 week or longer turn out blue Specimen that have increased levels of protein produce bluer staining blood film Peripheral Film Examination Evaluates the platelet and WBC count and differential, along with WBC, RBC, and platelet morphology Macroscopic Examination Film that is bluer overall than normal may indicate that the patient has increased blood proteins, as in multiple myeloma and that rouleaux may be seen in the film A grainy appearance to the film may indicate RBC agglutination Holes all over the film may indicate increased lipid levels Increased WBC counts and platelet counts may be detected from the blue specks out at the feathery edge

Microscopic Examination Proper adjustment of the microscope is done for proper blood film examination 10X Objective (LPO) Examination 10X or low power objective with a total magnification of 100X Not much time needs to be spent at this magnification Things observed under LPO: a) Overall film quality b) Color c) Distribution of cells Snow-plow effect - poor leukocyte distribution; large cells such as monocytes and neutrophils are pushed to the end of the slide; "snowplow effect" Check to see if there are good counting areas available free of ragged edges and cell clumps. Check the WBC distribution over the smear. Check that the slide is properly stained. Check for the presence of large platelets, platelet clumps, and fibrin strands. 40X Objectives (HPO) Examination 40X high dry objective lens with a total magnification of 400X Choose a portion of the peripheral smear where there is only slight overlapping of the RBCs. Count 10 fields take the total number of white cells and divide by 10. To do a WBC estimate by taking the average number of white cells and multiplying by 2000. 100X Oil Immersion Objectives (OIO) Examination 100X oil immersion objectives provides the highest magnification on most standard binocular microscope

5 which has a total magnification of 1000X WBC differential count is usually done under OIO RBC, WBC and platelet morphology evaluation and platelet estimate are done under OIO RBC inclusions such as Howell-Jolly bodies, and WBC inclusions such as Dhle bodies can be seen easily if present 50X Oil Immersion Objectives (OIO) Examination Larger view allows cells to be evaluated faster Optimal Assessment area Observation is done between the thick area (heel) and the thin area (feathery edge) Microscopically the RBCs are uniformly and singly distributed with few overlapping and have normal biconcave appearance Normal RBC count per 100X OIO is 200 to 250 RBC Performance of the White Blood Cell differential These counts are done in the same area as WBC and platelet estimates with the red cells barely touching This takes place under 100 (oil) using the back and forth serpentine or battlement tract pattern method of counting Count 100 WBCs including all cell lines from immature to mature. To increase accuracy it is advisable to count 200 cells when WBC is higher than 40 X109L Results are reported in percentage In addition to counting the cells, evaluators assesses their appearance Because differential alone provides only partial information, the absolute

cell counts are calculated for each cell type Red blood Cell Morphology Includes the assessment of cell size, variability in size (anisocytosis), cell color, cell shape (poikilocytosis) and cellular inclusions Degree of abnormal morphology is reported as: a) Slight, moderate or marked b) Scale form (1+ to 3+) Platelet Estimate Performed under 100X OIO 10 OIO field is counted in the same area as WBC and platelet estimates with the red cells barely touching Formula:

follow up testing recommended

can

6 be

Organization of Complete Blood Count Results The CBC panel is essentially divided into WBC, RBC, and platelet parameters Generally all the parameters interpreted together provide the best information, so a complete summary of the results should be generated Assessing Hematology Results Relative to Reference Ranges Strategies that can help determine the significance of the result: a) If the result are very far from the reference range, it is more likely that they are truly outside range and represent pathologic process b) If two or more diagnostically related parameters are slightly or moderately out of range in the same direction this suggests that the results are clinically significant and associated with some diagnostic process Some healthy individuals always have results slightly outside the range, the best comparison for their result is their results from prior time when they where healthy Summarizing White Blood Cell Parameter The WBC-related parameters of a routine CBC include the following: 1. Total WBC count (WBCs X 109/L) 2. WBC differential count values expressed as percentages (relative count) 3. WBC differential count values expressed as the actual number of each type of cell (absolute count) 4. WBC morphology

200 id the average number if RBCs per OIO field in the optimal assessment area Summarizing Complete Blood Count Result CBC (complete blood count) includes the assessment of WBCs, RBCs and platelets CBC provides information about hematopoietic system CBC plays a role in screening of those organs for disease One of the most frequently ordered laboratory test The process of interpreting the CBC test results has two phases a) PHASE 1: The numbers and description generated by the testing are summarized using appropriate terminology b) PHASE 2: Is to recognize consistent with various disease and to be able to narrow the diagnosis for the given patient or perhaps even to pinpoint it so that appropriate

STEP 1: Ensure that the WBC count is accurate. The presence of NRBC may require correction of WBC count STEP 2: Examine the WBC count for variation in the total number of WBCs; compare the patients count with the laboratorys established reference range STEP 3 and 4: Examine the differential information (relative and absolute) on variations in the distribution of WBCs STEP 5: Make note of immature cells in any cell line reported in the differential that should not appear in normal peripheral blood STEP 6: Make note of any morphologic abnormalities and correlate film findings with numerical values Summarizing Red Blood Cell Parameter The RBC-related parameters of a routine CBC include the following: 1. RBC count (RBCs X 1012/L) 2. Hb (g/dL) 3. Hct (% or L/L) 4. MCV (fL) 5. MCH (pg) 6. MCHC (% or g/dL) 7. RBC distribution width (RDW, %) 8. Morphology STEP 1: Examine the Hb concentration to assess anemia STEP 2: Examine the MCV to assess cell size STEP 3: Examine the MCHC to assess cell Hb concentration STEP 4: Examine the RDW to assess anisocytosis STEP 5: Examine the morphologic description and correlate with the numerical value. Look for evidence of a reticulocyte response

7 STEP 6: Review remaining information (RBC count and MCH) Summarizing Platelet Parameter The platelet-related parameters of a routine CBC include the following: 1. Platelet count (platelets X 109/L) 2. Mean platelet volume (MPV, fL) 3. Morphology STEP 1: Examine total platelet count STEP 2: Examine the MPV to assess platelet size STEP 3: Examine platelet morphology and correlate with the numerical values