1 Chapter 14 Routine and Point of Care Testing in Hematology: Manual and Semiautomatic Methods Manual Cell Counts Manual

al cell counts are performed using a hemacytometer, and manual dilutions made with calibrated, automated pipettes and diluents The principle for the performance of cell counts is essentially the same for leukocytes, erythrocytes and platelets; only the dilution, diluting fluid, and area counted varies Equipment Hemacytometer Also known as counting chamber Heart of manual cell count Most common is the Levy chamber with improved Neubauer ruling Composed of: a) Two raised surfaces, each in the shape of a 3mm X 3mm square (a total of 9 mm2) separated with a Hshaped moat b) Large square: nine 1mm X 1mm squares c) Corner or WBC squares: divided further into 16 squares d) Center square: Subdivided into 25 smaller squares e) Smallest square: 1/25 or 0.04 mm f) Distance of each counting surface and the cover slip is 0.1mm g) Total volume is 0.9mm3 Calculations
Cell type Leukocyte Erythrocyte Platelet Diluting fluid Weak acid Isotonic saline Ammonium oxalate Dilution 1:20 1:100 1:100 1:100 Objective 10X 40X 10X 40X or Area counted 4mm2 9 mm2 0.2 mm2 1 mm2

40 phase

White Blood Cell Count Represents the number of WBCs in 1 L of whole blood Diluting fluid: contains a weak acid (acetic acid or hydrochloric) or a buffered ammonium oxalate solution to lyse the non nucleated erythrocytes Typical dilution for the WBC count is 1:20 Procedure: 1. Clean the equipments with alcohol and dry with a lint free tissue 2. Make a 1:20 dilution in the test tube 3. Cover the tube and mix by inversion 4. Allow the dilution to sit for 10 minutes; leukocyte count should be done within 3 hours of dilution 5. Mix again and fill a plain microhematocrit tube 6. Charge both side of the hemacytometer by holding the microhematocrit tube at a 45 degree angle 7. After charging place the counting chamber in a moist chamber for 10 minutes before cell counting to allow cells to settle 8. Place the equipment under the microscope 9. Lower the condenser on the microscope and focus on LPO objective (10X) 10. Count cells in the 4 corner squares (top and right cells that touch the borders are counted) 11. Difference between both sides should be less than 10% 12. Average the counted cells on both sides of the hemacytometer then compute with the equation

2 Reference range: Male: 4.5 to 11.5 X 109 /L Female: 4.5 to 11.5 X 109 /L Source of error: 1. Dust and fingerprints may cause difficulty in distinguishing the cells 2. Diluting fluid must be free from contaminants 3. If the count is low, greater area may be counted to improve accuracy 4. Counting chamber must be charged properly 5. Allow the cells to settle for 10 minutes before counting 6. Accuracy of the manual WBC count can be assessed by performing a WBC estimate on the peripheral blood smear 7. Any nucleated erythrocytes (NRBCs) are counted as WBCs 8. If 5 or more NRBCs per 100 WBC are discovered on differential count it is corrected by following this formula: Procedure 1. Make 1:100 dilution 2. Mix the dilution then charge the chambers *Note: special thin flat-bottomed counting chamber is used in phase platelet counts 3. Place charged hemacytometer in a moist chamber for 15 minutes to allow platelets to settle 4. Platelets are counted at HPO objectives (40X) 5. Platelet count should be verified by performing an estimate on the Wright stained peripheral blood smear 6. Count the 25 small squares in the center grid; difference between both sides of the hemacytometer should be less than 10% Reference range: Male: 150 to 450 X 109 /L Female: 150 to 450 X 109 /L Sources of error: 1. Inadequate mixing and poor sample collection can cause platelets to clamp to the hemacytometer 2. If fewer than 50 cells are counted repeat the procedure by diluting 1:20 3. If more than 500 cells are counted repeat the procedure by diluting 1:200 4. Platelet satellitosis a) Occurs when EDTA anticoagulant is used b) Refers to the adherence of platelets around neutrophil, producing a ring or satellite effect c) Using sodium citrate should correct the problem (multiply platelet count by 1.1 for accuracy) Erythrocyte count Rarely performed because of the inaccuracy and questionable necessity More accurate manual RBC parameters, such as the microhematocrit and hemoglobin concentration is desirable

Platelet Count Phase contrast microscope is used as described by Brecher and Cronkite Platelets are adhesive to foreign objects and to each other Platelets are small and easily confused with dirt Diluting fluid: 1% ammonium oxalate which lyses the non nucleated erythrocytes Typical dilution for the platelet count is 1:100

Hemoglobin determination Cyan methemoglobin (hemoglobincyanide) method for hemoglobin determination is the reference method approved by the Clinical and Laboratory Standard Institute Principle Blood is diluted in an alkaline Drabkin solution of potassium Ferricyanide, potassium cyanide and a surfactant Hemoglobin is oxidized to methemoglobin (Fe3+) by the potassium Ferricyanide K3Fe(CN)6 Potassium cyanide (KCN) then converts the methemoglobin to cyanmethemoglobin Absorbance of the cyanmethemoglobin at 540 nm is directly proportional to the hemoglobin concentration Sulfhemoglobin is not converted thus it can not be measured Procedure 1. Create a standard curve a) When a standard containing 80mg/dl of hemoglobin is used, the following dilutions should be made:
Hb conc. (g/dL) Cyanmethemoglobin standard (ml) Cyanmethemoglobin reagent (ml) Blank 0 6 5 1.5 4.5 10 3 3 15 4.5 1.5 20 6 0

2. 3. 4. 5. 6. 7.

3 f) Standard curve should be set up with each new lot of reagents Control should be run with each batch of specimens Make a dilution of 1:251 Cover and mix well by inversion or using a vortex machine Stand for 10 min in room temperature to allow full conversion of hemoglobin to methemoglobin Read the patients sample and record the percent transmittance Determine the hemoglobin value from the standard curve

Reference range: Male: 14 to 18 g/L Female: 12 to 15 g/L Sources of error: 1. Cyanmethemoglobin reagent is sensitive to light 2. A high leukocyte and platelet count can cause turbidity and a false high result The solution can be centrifuged and the supernatant measured 3. Lipemia can also interfere and cause false result Adding 0.01ml of the patients plasma to 5ml of the cyanmethemoglobin reagent and using this as blank 4. Cells containing Hb S and Hb C may be resistant to hemolysis causing turbidity Making 1:2 dilution with distilled water and multiplying the result from the standard curve by 2 5. Abnormal globulins, such as those found in patients with multiple myeloma or Waldenstrm macroglobulinemia may precipitate Add 0.1gof potassium carbonate to the cyanmethemoglobin reagent 6. Carboxyhemoglobin takes 1 hour to convert to cyanmethemoglobin and theoretically could cause erroneous result in samples from heavy smoker

b) Transfer solution to cuvettes c) Set wavelength of the spectrophotometer to 540nm and use the blank to set to 100% transmittance d) Plot percentage transmittance on the y axis and the hemoglobin concentration on the x axis e) The hemoglobin concentrations of the control and patient samples can be read from the standard curve

7. Hemoglobin reagent contains lethal cyanide, minimum of 4L is lethal 8. Commercial absorbance kits are available to calibrate spectrophotometers Microhematocrit The volume of packed RBCs that occupies a given volume of whole blood Often referred to as packed cell volume (PCV) Reported either as a percentage or in liters per liter Procedure 1. Fill two plain capillary tubes approximately three quarters full with blood anticoagulated with EDTA or heparin (Mylar wrapped tubes are recommended) 2. Seal the end of the tube with the colored ring using nonabsorbent clay Plug should be at least 4mm long 3. Balance the tubes in the centrifuge 4. Tighten the head cover on the centrifuge and close the top 5. Centrifuge the tubes at between 10000g and 15000g 6. Determine the hematocrit by using a microhematocrit reading device 7. The value of the duplicated hematocrits should agree within 1% 90.01L/L Reference range: Male: 0.40 to 0.54 L/L Female: 0.35 to 1.49 L/L Sources of error: 1. Improper sealing of the capillary tube causes a decreased hematocrit reading as a result of loss of blood during centrifugation 2. An increased concentration of anticoagulant decreases the hematocrit reading as a result of erythrocyte shrinkage

3. 4. 5.

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4 A decreased or increased result may occur if the specimen was not mixed properly The time and speed of the centrifugation and the time when the results are read are important The buffy coat of the specimen should not be included in the hematocrit reading because this falsely elevates the result A decreased or increased in the reading may be seen if the microhematocrit reader is not used properly A temporary low hematocrit reading may result immediately after blood loss because plasma is replaced faster than erythrocytes Fluid loss associated with dehydration causes a decreased in plasma volume and falsely increased hematocrit reading Proper specimen collection is an important consideration

Rule of Three The rule applies only to specimens that have normocytic normochromic erythrocytes States that the value of the hematocrit should be three times the value of the hemoglobin plus or minus three ( ) If values do not agree, the blood smear should be examined for abnormal erythrocytes A control specimen should be run when such a discrepancy is found Red Blood Cell Indices Calculated to determine the size and hemoglobin content of the average RBC Serve as a quality control check May be used to differentiate anemias

5 Mean Cell Volume Average volume of the RBC Expressed in femtoliters (fL) or 10-15 L ( ) ( Reference range is 80 to 100 fL Microcytic: less than 80fL Macrocytic: more than 100fL Mean Cell Hemoglobin Average weight of hemoglobin on an RBC Generally not considered in the classification of anemias Expressed in pictograms (pg) or 10-12 g ( ) ( ) ) Reticulocyte Count Reticulocyte is the last immature erythrocyte stage Reticulocyte spends 2 to 3 days in the bone marrow and 1 day in the peripheral blood before developing into a mature erythrocyte Reticulocyte contains remnant cytoplasmic ribonucleic acid (RNA) and organelles such as mitochondria and ribosomes Reticulocyte count is used to asses the erythropoietic activity of the bone marrow Principle Whole blood with EDTA is stained with supravital stain, such as methylene blue Any nonnucleated erythrocyte that contains two or more particles of blue stain granulofilamentous material after new methylene blue staining is defined as a reticulocyte Procedure 1. Mix equal amounts of blood and new methylene blue stain (2to 3 drops) 2. Allow to incubate at room temperature for 3 to 10 minutes 3. Remix the preparation 4. Prepare two wedge films 5. Count 1000 RBCs under OIO objectives in the area where cells are close to each other but not overlapping 6. For accuracy count the other smear, value should agree within 20% 7. Calculate the reticulocyte count by:

Reference range for adults is 28 to 32pg Mean Cell hemoglobin Concentration Average concentration of hemoglobin in each individual erythrocyte Expressed in gamma per deciliters ( ) ( )

Reference range of normochromic cells is 32 to 36 g/dL Hypochromic a) Less than 32 g/dL b) Thalassemia and iron deficiency Hyperchromic a) Greater than 36 g/dL b) Cell appears full because of spherocytic shape c) Increased MCHC could be the presence of a cold agglutinin

Or the number of reticulocyte counted can be multiplied by 0.1 (100/1000) to obtain the result

6 Miller disk Designed to reduce labor intensive in reticulocyte count Composed of two squares with the area of the smaller square measuring 1/9 the area of the larger square The disk is inserted in the eyepiece of the microscope RBCs are counted in the smaller square Reticulocytes are counted in the larger square A minimum of 112 cells should be counted in the small square because this is equivalent to 1008 red cells in the large square Calculation formula: c) Pappenheimer bodies Hemosiderin in the mitochondria whose presence can be confirmed with an iron stain, such as Prussian blue Absolute Reticulocyte count Principle: The ARC is the actual number of reticulocyte in 1 L of whole blood Calculations: ( )

Reference range: Values between 25 X 109 /L and 75 X109 /L Reference range Male: 25 to 75 X 109 /L Female: 25 to 75 X 109 /L Sources of error: 1. If patient is very anemic or polycythemic the proportion of dye to blood should be adjusted 2. Error may occur if the blood and stain are not mixed properly before smear is made 3. Refractile appearance from air moisture or poor drying could be confused with reticulocytes 4. Other RBC inclusions that stains supravitally include: a) Heinz bodies Precipitated hemoglobin, usually appears round or oval, and tends to cling to the cell membrane b) Howell-Jolly bodies Round nuclear fragments and are usually singular Corrected Reticulocyte Count Principle: In specimens with a low hematocrit, the percentage of reticulocytes may be falsely elevated because whole blood contains fewer RBCs. A correction factor is used with the average normal value hematocrit considered to be 45% Calculations:

( ) Reference range: The corrected reticulocyte count depends on the degree of anemia Patients with a Hct of 35% are expected to have a corrected reticulocyte count of 2% to 3% In patients with less than 25% Hct, the count should increase to 3% to 5% to compensate for anemia

7 Reticulocyte Production Index Principle: Reticulocytes that are released from the marrow to the peripheral blood prematurely to compensate for anemia is called shift reticulocyte Normal reticulocyte take 1 day to lose their reticula shift reticulocytes take 2 to 4 days to lose their reticula Cells shifted to the peripheral blood prematurely stay longer as reticulocytes and contribute to the reticulocyte count for more than 1 day Reticulocyte count is falsely increased when polychromasia is present The patients hematocrit is used to determine the appropriate correction factor Hct (%) Correction factor (Maturation) (Days) 40-45 1 35-39 1.5 25-34 2 15-24 2.5 <15 5 Calculations: ( ) ( ) Flow cytometry Automated reticulocyte counts evaluate reticulocytes based on optical scanner or fluorescence after RBCs are treated with fluorescent dyes or nucleic acid stains to stain residual RNA in the reticulocytes These results are statistically more valid because of the large number of cells counted Other reticulocyte parameters: a) Maturation index b) Immature reticulocyte fraction c) Reticulocyte indices Useful in monitoring erythropoietic activity in patients undergoing: a) Chemotherapy b) Stem cell c) Bone marrow transplantation d) Patients with chronic renal failure Erythrocyte Sedimentation Rate ESR is commonly used as a general screening test ESR is useful for monitoring the course of an existing inflammatory disease or for differentiating between similar disease ESR can be used to asses the activity of pulmonary tuberculosis Principle ESR is the distance in milliliters that the RBCs fall in 1 hour ESR is affected by erythrocytes, plasma, and mechanical and technical factors Erythrocytes have a net negative surface charge and tend to repel one another The repulsive forces are partially or totally counteracted if there are increased quantities of positively charged plasma protein; the erythrocyte settle more rapidly as a result of the formation of RBC aggregate or rouleux

Reference range: Adequate bone marrow erythropoietic response: RPI >3 Inadequate bone marrow erythropoietic response: RPI <2

or or

Reticulocyte control Most of the controls are available at three levels Control samples are treated in the same manner as the clinical specimens Control can be used to verify the laboratorians accuracy and precision

 Example of macromolecules that can produce this reaction: a) Fibrinogen b) B-globulin c) Pathologic immunoglobulin Normal erythrocyte have a relatively small mass and settle slowly Alteration changes the erythrocyte surface, which leads to aggregation of the RBCs, increased RBC mass, and a more rapid ESR ESR is directly proportional to the RBC mass and inversely proportional to plasma viscosity Common methods of ESR Modified Westergren Erythrocyte Sedimentation Rate Most common method used Advantage: taller column height allows detection of highly elevated ESR Procedure 1. Use blood collected in EDTA and dilute at four parts blood to one part 3.8% sodium citrate or 0.85% sodium chloride 2. Place the diluted specimen in a 200 mm column with an internal diameter of 2.55 mm or more 3. Place the pipette into a rack and allow to stand undisturbed for 60 minutes 4. Record the number in mm the RBC has fallen. Buffy coat should not be included in the reading. 5. ESR is reported as 1 hour = __ mm Wintrobe Erythrocyte Sedimentation Rate Past: Sample used was oxalateanticoagulated whole blood which was placed in a 100 mm column Present Sample used was EDTA treated or citrated whole blood used with shorter column

8 Shorter column height allows a somewhat increased sensitivity in detecting mildly elevated ESRs Procedure 1. Use a minimum of 2ml whole blood collected in an EDTA anticoagulated tube 2. After mixing the blood thoroughly, fill a Pasteur pipette using a rubber pipette bulb 3. Place the pipette in a Wintrobe tube until tip reaches the bottom tube 4. Squeeze the bulb and expel the blood into the Wintrobe tube while pulling the Pasteur pipette up from the bottom of the tube Steady and even pressure should be implied in expulsion of the blood to avoid air bubbles into the column of blood 5. Fill the Wintrobe tube to the 0 mark 6. Place the tube in a Wintrobe rack and allow to stand undisturbed for 1 hour at room temperature 7. Record the number of millimeters the RBCs have fallen 8. Read the tube from the bottom of the plasma meniscus to the top of the sedimented red cells 9. The result is reported in milliliters per hour Reference range: Male: 0-50 yo 0-15mm >50yo 0-20mm Female: 0-50 yo 0-20mm >50yo 0-30mm Sources of error: Increase of the concentration of anticoagulant can cause falsely low ESR as a result of sphering of the RBCs, which inhibits rouleaux formation Anticoagulant sodium or potassium oxalate and heparin cause the RBCs to shrink and falsely elevate the ERS

 Change in temperature can increase the ESR Slight tilt in the pipette can cause the ESR to increase Prolonged standing of specimen (more than 2 hours) can cause sphering of the RBCs, which inhibits rouleaux formation Bubbles in the column of blood invalidate results The blood must be filled properly to the zero mark at the beginning of the test A clotted specimen cannot be used Hematologic disorders prevent formation of rouleaux thus decreasing ESR The ESR of patients with severe anemia is of little diagnostic value, because it will be falsely elevated Disposable kits Available for ESR testing Include safety caps for pipettes that allow the blood to fill precisely to the zero mark Safety cap makes the pipette a closed system and eliminates the error involved in manually setting the blood to the zero mark Automated Erythrocyte Sedimentation Rate Ves-Matic system Bench top analyzer Designed to determine ESR by use of an optoelectric sensor, which measures the change in opacity of a column of blood as sedimentation of blood progress Blood is collected in special Ves-Tec or Vacu-Tech tubes, which contains sodium citrate and are compatible with the vacutainer system Acceleration of sedimentation is achieved by positioning the tubes at an 18-degree angle in relation to the vertical axis

9 Results comparable with Westergren 1-hour values are obtained in 20 minutes Sedimat 15 Uses the principle of infrared measurement Capable of testing one to eight specimens randomly or simultaneously Provides result in 15 minutes ESR STAT PLUS system Based on centrifugation Advantage: a) Smaller sample volume b) Shorter testing time c) More suitable for a pediatric patient population Disadvantage: a) Number of exacting pre analytical steps that must be strictly followed to prevent erroneous results Point of Care Testing Defined as diagnostic testing at or near the site of patient care Offers the ability to produce rapid and accurate results that help facilitate faster treatment Most often carried out by nurses Testing site neutrality means regardless of where the diagnostic is being performed or who performs the test, all testing sites must follow the same regulatory requirements based on the complexity of the test POCT is classified as waived or moderately complex Test are classified as waived if they are determined to be simple test with an insignificant risk of erroneous result, most, but not all POCT is waived For POCT program to be successful, key elements such as clear administrative responsibility, well written procedures, quality control,

proficiency testing and equipment maintenance must be present Paramount to POCT is patient safety Point of Care Tests Hematocrit Most common methods for determining the hematocrit include: a) Microhematocrit centrifuge b) Conductometric methods c) Calculation by automated cell counts Examples of centrifuge based devices are the Hemastat II and STAT Crit iSTAT 1 and Epoc a) Use the conductivity method to determine the hematocrit Sources of error: 1. Conductivity of a whole blood sample is dependent upon the amount of electrolytes in the plasma portion 2. Conductivity does not distinguish RBCs from other nonconductive elements such as proteins lipids and WBCs 3. Low protein level will falsely decrease the hematocrit 4. Presence of lipids can interfere with the hematocrit 5. An increased WBC count will falsely increase the hematocrit 6. Presence of cold agglutinins can falsely decrease hematocrit Hemoglobin Level In POCT hemoglobin level is measured by modified hemoglobinometers or by oximeters integrated with a blood gas analyzer HemeCue hemoglobinometers uses a small cuvette that contains a lysing agent and reagent to form a hemoglobin azide which is measured by a photometer AVOX 1000E measures total hemoglobin by a spectrophotometric method

10 STAT-Site MHbg Meter and Hgb Pro use reflectance photometer to measure reflected light in the rest area Cell and Platelet Count Traditional cell counting can be employed at the point of care for the analysis of WBCs, RBCs, and platelets Ichor Hematology Analyzer performs a complete blood count along with platelet aggregation Quantitative buffy coat analysis involves centrifugation in specialized capillary tubes designed to expand the buffy coat layer Platelets, mononuclear cells and granulocytes can be measured with the assistance of fluorescent dyes and a measuring device