Normal Lab Values
Normal Lab Values
Normal Lab Values
All procedures have institution-established reference intervals....a range of values expected for an analyte or parameter
in healthy individuals that are based on the population served. Normal reference ranges vary with age, sex, ethnicity,
geographical location and/or test methodology. Reference ranges must be determined and verified by each laboratory.
3
EQUIVALENT UNITS cmm (cubic millimeters) = mm = uL (microliters) = mcL
3
thousands/uL = x10 /cmm = x10E3/uL = K/cmm
6
millions/uL = x10 /cmm = x10E6/uL = M/cmm
DIFF Adults
Segs 43-74% }Neutrophils 45-75%
Bands 0-10% Absolute neutrophils 1800-7500/cmm or 1.8-7.5 K/uL
Lymphs 15-45% Absolute lymphocytes 1000-3400/cmm
Monos 1-12% Absolute monocytes 100-800/cmm
Eos 0-6% Absolute eosinophils 50-400/cmm
Basos 0-2% Absolute basophils 0-200/cmm
Children
Segs 25-70% (5-16y)
Bands 5-11% (7d-14y)
Lymphs 45-75% (1-4y) 25-55% (5-14y)
Monos 1-12%
Eos 0-8% (0-14y)
Basos 0-2%
FORMULAS
MANUAL CELL COUNTS # cells/cmm = total # cells (both sides) x dilution factor x 10^ mm (depth)
2
total area counted mm (both sides) ^10 = inversion of 0.1 mm
RBC INDICES MCV (fL) = HCT x 10 MCH (pg) = HGB x 10 MCHC (%) = HGB x 100
RBC RBC HCT
RELATIVE RETICS # retics x 100 OR # retics in 1000 RBCs = Relative Percent reticulocytes (%)
1000 RBCs 10
e.g. 4.0% retic (40 retics in 1000 RBCs) & RBC of 3.01 million/uL gives an absolute retic count of:
e.g. 31% lymphs and WBC count of 8,000/cmm would give an absolute lymphocyte count of:
3
31 x 8000 = 2,480/cmm = 2,500/cmm OR 0.31 x 8.0 = 2.5 x 10 /uL
100
WBC ESTIMATE (from smear) Find average # WBCs per high power field (HPF); Avg # WBC/HPF x 2,000 = WBC estimate/uL
PLT ESTIMATE (from smear) Find average # of platelets per oil immersion field (OIF):
•Take the 1st 10 PLTs (from Avg # PLT/OIF) x 20,000 each (= 200,000) PLUS each additional
PLT above 10 x 10,000 each = PLT estimate/cmm
WBC CORRECTION FOR 100 = Corrected WBC/ul OR Corrected WBC/ul = uncorrected WBC/ul x 100
NUCLEATED RBCS (if needed) 100 + #NRBCs/diff Uncorrected WBC/ul 100 + # NRBCs/diff
CRITICAL VALUES VERIFIED, CALLED & DOCUMENTED - VALUES VARY PER INSTITUTION POLICY
WBC < 1,000/cmm OR > 40,000/cmm
HGB < 7.0 g/dl OR > 18.5 g/dl
PLT < 40,000/cmm OR > 1,000,000/cmm
Differential Absolute neutrophil count (ANC) < 500/cmm; Blasts, lymphoma cells, highly unusual findings
PT INR > 4.0
PTT > 100 seconds
TEST INCLUDES:
CBC without DIFF/Hemagram WBC, RBC, HGB, HCT, RBC INDICES (MCV, MCH, MCHC, RDW), PLT
CBC with DIFF WBC, RBC, HGB, HCT, RBC INDICES, PLT, WBC DIFFERENTIAL, CELL MORPHOLOGY
CLS416 Clinical Hematology 2
Quality Control (QC)
Quality control systems have been developed to guarantee the reliability of measurements and define the
significance of patient changes. QC is based on utilizing control samples with known values to verify the
proper operation of a testing system. The control material has allowable limits of variation established.
When values deviate outside the predictable limits, there is strong reason to doubt the validity of results.
Besides confirming the accuracy of results, quality control is a process that evaluates all parts of the
actual testing process including reagents, equipment/instrument function and individual testing technique.
Terms:
Accuracy – closeness of the measured value to the true value.
Precision – reproducibility of the result, i.e., the spread between replicate determinations.
Reliability – ability to maintain both accuracy and precision.
Mean – average of all observations (X)….determined by assay.
Confidence limits/intervals – range of values from acceptable low to acceptable high limits…usually
established at two standard deviations (SD).
Gaussian curve – used to derive standard deviation, which is the dispersion of values around the
mean……+ 2 standard deviations should include about 95% of results.
Levey-Jennings chart – gives rapid, visual display of quality control data and aids in the detection of
“out of control” situations. The chart displays the assay value of controls versus time and shows the
control mean value with the range of acceptable control limits.
Levey-Jennings Chart
Standards:
Pure substance of known composition that is used to standardize/calibrate an instrument or testing
procedure.
Controls:
Material whose composition closely resembles unknown patient samples and is tested in conjunction
with unknown samples. Purchased from manufacturer. The control material is assayed to determine
a known mean or target value.
Testing often utilizes a control level with normal analyte concentrations and abnormal low or high
control levels. In certain tests, known positive and negative control samples are run in conjunction
with unknown patient samples to assure valid results. Note: If only 1 control level is run, the control
value must fall within 2 SD of the mean to be acceptable.
►A control that ‘reads’ is “in control” and falls within the acceptable value range of + 2 standard deviations
of the mean or shows the correct positive & negative results…the results of unknown patient samples are
assumed to be correct and can be reported.
►If the control value is not within 2 standard deviations of the mean (i.e., does not ‘read’) or the control
fails to show the correct positive & negative results, the results of unknown patient samples are doubtful
and cannot be reported.
Summary: Laboratory quality control employs control materials to confirm accuracy, monitor precision,
detect analytical errors, and check reliability of results. Controls do not detect pre-analytical errors.
CLS416 Clinical Hematology 3
PROCEDURE LIST
Refer to the NORMALS sheet for procedures in which the reference ranges are not listed.
Important NOTE:
For ALL procedures performed, important steps are:
1. Assuring that the sample to be analyzed is properly labeled AND meets the specimen
requirements for the test being performed….detect pre-analytical errors.
2. Verifying the lot# and/or expiration date of all reagents AND control levels….prevent
analytical errors.
3. Checking that your unknown test results are realistic and reported in the proper units….prevent
post-analytical errors. A number without units is meaningless.
B. FOLLOW STEPS as listed on the capillary blood collection COMPETENCY CHECKLIST and refer to the
information provided regarding capillary draws in the Phlebotomy CLS424 packet.
C. TECHNIQUE:
1. On children and adults use 3rd or 4th finger...area just to side of ball of fingertip, not close
to the nail/knuckle or in the center of the fingertip. Do not use cyanotic or edematous
tissue as a puncture site. Cold extremities may be warmed ~ 3 minutes in warm water.
2. Cleanse area with 70% alcohol and let completely dry. A moist surface will not allow
formation of a rounded drop. Twist & remove cap (Microgard closure) of the microtainer.
3. Gently massage prior to puncture; hold finger firmly. Place the puncture device at an
angle and just to the right (or left) side of the fingertip as shown in the picture below.
Press down on the top of the device to make the puncture (you will hear a click).
Immediately discard the puncture device in a sharps container.
4. The *first drop of blood should be wiped away with dry cotton, as it is diluted with tissue
fluid. After a drop of blood has formed at puncture site, hold microtainer at an angle
below site and touch collector end to the drop of blood. The blood will flow down wall to
bottom of tube by capillary action. Avoid ‘scooping’ the blood off the finger as this can
result in blood flow that “resists” going into the microtainer.
5. Slight pressure may be applied a short distance from the wound. Gentle "milking" is
acceptable but excessive squeezing is not. Squeezing dilutes the blood with tissue juice
and invalidates the tests to be performed.
6. To stimulate a better blood flow, you can wipe the blood away and start with a fresh drop.
When collection is complete, replace cap. Apply pressure to the site.
8. ●Minimum draw is 500 ul - 2nd line on microtainer. Over or underfilling of tube may
result in erroneous results due to alterations of the blood to anticoagulant ratio.
D. SOURCES OF ERROR:
1. Failure to wipe away the first drop of blood and/or excessive squeezing.
2. Clotting of the sample (e.g., micro-clots) due to inadequate mixing of the microtainer,
difficulty obtaining the blood or overfilling the microtainer (making it hard to properly mix).
CLS416 Clinical Hematology 5
2
MICROSCOPE USE and CARE
1. Eyepiece (ocular): produces the secondary image magnification (x10) of the specimen
2. Objectives: produces the primary image of the specimen; directs image to the eyepiece
3. Stage: adjustable component that holds the specimen slide (front to back, side to side)
4. Iris diaphragm: controls the amount of light passing through the condenser to specimen
5. Condenser: gathers and focuses the illumination light onto the specimen
6. Coarse and fine adjustment knobs: focuses the image for optimal viewing
7. Light source: provides illumination light
B. MAGNIFICATION
Total magnification of the lens system is the magnification of the eyepiece (10x) times the
magnification of the objective lens; each lens system is used for a specific task:
C. FIELD of VIEW
Adjust the binocular eyepiece for maximal viewing by adjusting the interpupillary distance so that
you see only one circle (field of view) through the eyepiece when viewing with both eyes open.
If you can only see the field of view with one eye open, check that eyepieces are the same height
and adjust if necessary.
1. Place microscope on a level, vibration-free surface, plug it in and turn on light source (adjust
light intensity to at least 2/3 maximum with rheostat). To avoid scratching or damaging the
objective lens, move the stage to its lowest position using the coarse adjustment knob before
placing a specimen onto the stage or rotating objectives into position. Rotate the low power
(10x) objective into the light path until it ‘clicks’.
Adjust light by raising or lowering condenser AND opening or closing the iris diaphragm.
2. The height of the condenser is controlled by a knob located below the stage and towards the
rear of the scope. Move the condenser to the position stated below for optimal viewing with:
a. Low power (10x) objective = the condenser should be low
b. High dry power (40x) objective = the condenser should be high
c. Oil immersion (100x oil) = the condenser should be at its highest point
3. Adjust the iris/aperture diaphragm of the condenser from one-third open (10x) to fully
open (100x), depending on the objective lens to be used.
a. Generally, more light is needed as a higher objective lens is used.
b. The iris diaphragm is adjusted using a small lever or rotating ring on the condenser.
Focusing
4. Place a stained blood slide (or hemocytometer) on the stage using holder clips to secure.
a. Using the stage adjustment knobs, move the slide directly under the low power (10x)
objective and over the central opening for the light source (light path).
b. Check that you have moved the slide to the correct area for viewing cells on a blood
smear. You should be in the rainbow area of the slide, NOT in a thick area by the label.
5. With the low power (10x) objective in place, slowly raise the stage while looking through the
eyepiece with both eyes (the objective should NEVER touch the slide).
a. Slowly turn the coarse adjustment knob towards you until you see the object(s) on the
slide (e.g., WBCs and RBCs). Then use the fine adjustment knob to focus the object(s).
b. Less light is needed so the condenser should be low and iris diaphragm ~one-third open.
6. Next, move the high dry (40x) objective into place if needed (e.g., WBC estimate). Do NOT
use the coarse adjustment knob. You should only use the fine adjustment knob to bring the
object(s) into sharper focus. The condenser should be high and iris diaphragm ~half open.
7. Oil must be added to the slide to use the oil immersion (100x) objective. Move the 40x
objective aside and place a drop of oil on the slide directly over the circle of the light path.
Be careful not to get oil on the high dry (40x) objective lens.
a. Next, move the oil immersion (100x) objective into place and use ONLY the fine
adjustment knob to focus. Adjustment should be minimal.
b. High light is needed so the condenser should be fully raised and diaphragm fully open.
E. MAINTENANCE
1. After each use, CLEAN and thoroughly dry the eyepieces, all objectives, the stage and
condenser lens using ONLY lens cleaner and lens paper.
a. Lower the stage, check that stage is empty, and store with low power objective in place.
b. Carefully clean coverslipped slides with lens cleaner after use. For slides without a
coverslip, do NOT use lens cleaner but gently clean off oil with a tissue.
Note: If you are not going to use the microscope for a period of time, NEVER leave the scope
with a slide under the oil objective and always turn off the light source.
2. If you can’t focus and/or ‘everything’ looks hazy, you may need to clean eyepieces and/or
objectives (most likely an oil problem). If the eyepiece is dirty, dirt will rotate when the
eyepiece is turned.
a. If you focus on a hemocytometer and don’t see any cells, you likely have too much light.
b. If you can’t focus on a slide, check that the blood (and coverslip) on the slide is facing up.
PRINCIPLE:
The well made peripheral blood smear is essential to the morphologic evaluation of hematologic
disorders. Poorly made smears are misleading and may cause erroneous findings. EDTA whole blood
or capillary blood may be used. Heparinized blood is unacceptable as it causes a blue background.
The ‘IDEAL SMEAR’ has a straight "feather" edge and becomes gradually thicker toward the point of origin
(blood drop). The feather edge should include a large rainbow area with no ridges, tails, streaks or holes.
EQUIPMENT:
Clean 3 x 1" quality slides, untreated microhematocrit tubes, plastic pipets or wooden sticks, pencil
PROCEDURE:
1. Place a small drop of blood on a clean slide at one end. The drop of blood should be small
enough so that the entire drop can be spread, but large enough so the smear goes 1/2 to 1 inch
from the opposite end. The size of the blood drop will affect smear thickness and length.
2. Take a pusher slide and place it just in front of the drop of blood at a 30o angle from the smear
slide. The angle of the pusher slide will affect smear thickness and length.
3. Draw the pusher slide back into the drop of blood and allow the blood to spread evenly. Gently
push it forward with very little pressure and a moderate amount of speed across the surface of
the smear slide. ●Use a new pusher slide edge for each smear.
2. Slides must be properly labelled before staining with Wright=s stain. Use pencil to label (first
and last name) on frosted end. ■NEVER PRE-LABEL.
SPECIAL CONSIDERATIONS:
1. EDTA tube must be mixed before obtaining an aliquot of blood. Tilt to mix, do not shake.
2. The blood should be spread immediately. The drop of blood should not rest on the slide for
longer than 5 seconds before spreading or the bigger cells will be carried to slide edges which
may distort the WBC differential result.
3. For normal bloods, a 30o angle is best along with moderate speed.
a. The smaller the angle of the pusher to the smear slide and the slower the "push" of the
pusher slide, the thinner the smear. The larger the angle of the pusher to the smear
slide, the thicker the smear.
b. Decrease the angle of the pusher slide to make a longer, thinner smear and increase
the angle of the pusher slide to make a shorter, thicker smear.
4. ●Do not apply pressure to the pusher slide as this will break cells and/or distort the distribution
and morphology of the cells. The pusher slide should literally be glided gently across the smear
slide with only enough pressure to guide and hold the pusher slide over the smear slide.
5. Use clean slides only. Chipped slides cause tails and any blood on the pusher slide causes tails.
CLS416 Clinical Hematology 8
3
BLOOD SMEAR PREPARATION/WRIGHT’S STAIN PROCEDURE
PRINCIPLE:
Wright’s stain is widely used for staining peripheral blood smears. Wright’s stain is a Romanowsky stain
that contains Eosin and Methylene blue. Eosin (acid dye) stains basic cell structures such as hemoglobin,
eosinophil granules, and primary granules a red-orange color. Methylene blue (basic dye) stains acid cell
structures such as RNA in nucleolus and cytoplasm, nuclear chromatin, and basophil granules a blue color.
A combination of both dyes stains neutral cell structures such as neutrophil granules a pinkish-tan color.
Prepare and LABEL smears that meet the criteria of a properly made blood smear.
4. Wipe off stain on backside of smears. Let smears air dry in rack.
^^More than one slide can be stained at a time AND stain times are not exact.
1. Dip blood smears for five, one second dips into the Fixative Solution. Allow excess fixative to
drain back into the cuvette, or blot the backside of the smear.^^
2. Dip blood smears for five, one second dips into Solution I. Allow excess stain to drain back into
the cuvette, or blot the backside of the smear.
3. Dip blood smears for five, one second dips into Solution II. Allow excess stain to drain back into
the cuvette, or blot the backside of the smear.
4. Rinse blood smears with deaminized water. Let smears air dry in rack.
■If jar appears very blue, dump and refill with fresh deaminized water.
^^More than one slide can be stained at a time if placed back to back. Dip times are not exact.
Staining notes: ●A properly stained smear looks pinkish-purple macroscopically; the red cells appear
red-orange microscopically. For the Quick Wright’s stain, methanol in the stain fixes cells to the slide;
deaminized water is the buffer.
1. The RBCs may appear blue and the WBC nuclei deeply stained if:
a. The staining time is too long.
b. Washing is inadequate.
c. The stain or buffer is excessively alkaline.
d. The smear is too thick.
●Correct by decreasing stain time, decreasing pH of buffer, or making a thinner slide.
2. The RBCs may appear excessively red and the WBC nuclei poorly stained if:
a. The staining time is inadequate.
b. Washing of slide is excessive.
c. The stain or buffer is too acidic.
●Correct by increasing stain time or increasing pH of the buffer.
A. PRINCIPLE:
The hematocrit measures the volume of packed red cells in a given volume of whole blood. This
method uses EDTA anticoagulated whole blood or capillary blood obtained by fingerstick.
B. PROCEDURE:
1. MIX EDTA blood specimen and whole blood control vial well before taking blood sample.
TILT to mix – do NOT shake.
2. Fill TWO microhematocrit tubes 2/3 full for each EDTA sample and/or control. Bubbles
are OK. Each test is done in duplicate.
3. Clay the dry end of each tube and put tubes in HCT sheet holes. Label the sheet with
your name and carefully transport to centrifuge area.
4. Balance tubes in centrifuge, PUT ON LID, and centrifuge 5 mins. Record the centrifuge #
and groove #'s used for each tube on sheet.
5. After centrifugation, use the HCT reader (e.g. card) to set 0 and 100, then read the
HCT% at the top of the packed red cells; read to the nearest 0.5%. Record the HCT%
obtained for each tube on the labsheet.
Refer to the diagram below of layers present in a spun hematocrit tube.
C. QUALITY CONTROL:
1. Duplicate HCT tubes must agree + 1% (1% = 1 HCT percentage point) to accept results
or another HCT tube must be centrifuged.
2. The control must be within + 2 standard deviations of the assayed control mean to accept
patient HCT results. Check that the HCT control value is within the acceptable limits
given on the HCT QC chart. If the control does not "read", testing must be repeated^.
^If the HCT control is not within the acceptable range, first check that you are using the HCT
reader properly.
D. REPORTING:
1. Average the final patient HCT results IF the control is acceptable and duplicate HCT
tubes agree.
2. Report manual hematocrit results to the nearest 0.5%. Do not average results if, for
example, the patient HCT results are 32.5% and 33.0%, report either.
E. LIMITATIONS:
●Pre-analytical/blood collection errors, e.g. clotted blood, hemolysis, EDTA tube filled less than
half full with blood.
●Analytical/technical errors, e.g. inadequate mixing of blood sample, insufficient centrifugation,
poor duplication of results, improper use of HCT reader or including buffy coat in HCT reading.
A. PRINCIPLE:
The hemoglobin concentration in a fresh capillary or anticoagulated blood sample (EDTA
preferred) is determined photometrically using a dry reagent system. The red cells are lysed
and hemoglobin is converted to azidemethemoglobin by sodium nitrite and sodium azide. This
method of HGB measurement is a widely used point-of-care test.
B. HEMOCUE OPERATION:
1. Turn the Hemocue on using the switch in the back. The display screen should read
"Hb". Pull the black cuvette holder out to the insertion position. After about 6 seconds,
the screen should read "READY".
2. Place a cuvette into the holder and insert to the "measuring" position. The HGB results in
g/dl will be displayed in 45-60 seconds (results remain on screen about 5 minutes).
C. QUALITY CONTROL:
1. Perform an electronic calibration check of the instrument prior to each use and verify that
the value falls within the assigned range. The RED control cuvette must read within +
0.3 gm/dl of the assigned value for the specific instrument to assure proper function or
the instrument cannot be used. Record the Hemocue instrument you are using AND the
value obtained for the RED electronic calibration cuvette on your labsheet.
2. A hemoglobin whole blood control sample (normal level) must be run once per day on
each Hemocue instrument. The control HGB value must read within the assayed range
to accept patient hemoglobin results.
a. Run the HGB whole blood control ONLY IF the control has not yet been run
on the Hemocue you are using. Follow the PROCEDURE below and check
that the HGB control value obtained reads within the limits on the HGB QC
chart. Repeat the control if it is not within established limits. Document the
control result on the Hemocue Daily QC Sheet for the specific Hemocue
instrument you are using AND record the HGB control value on your labsheet.
b. If the daily HGB whole blood control has already been run on the Hemocue
you are using, do NOT run. Check that the HGB control result documented on
the Daily QC Sheet for the Hemocue instrument you are using reads within limits
on the HGB QC chart AND record the HGB control value on your labsheet.
D. PROCEDURE:
1. Fill the cuvette with blood (obtained from a mixed EDTA tube or control vial) in a
CONTINUOUS process WITHOUT BUBBLES. ●The cuvette needs to be filled all
at once because the chemical reaction starts immediately and any delay in filling the
cuvette results in incomplete red cell lysis.
2. ●Air bubbles in the center of the cuvette require repeating.
3. Wipe any excess blood from the outside of the cuvette, being careful not to touch the
curved edge...important. Cuvettes must be read within ten minutes of filling with blood
to prevent alteration of the result by drying.
4. Place a cuvette (control or unknown) into the holder and insert to the "measuring"
position. The results in g/dl will be displayed in 45-60 seconds. Record results to the
nearest tenth on your labsheet.
E. LIMITATIONS:
●Pre-analytical/specimen collection errors, e.g. clotted blood or wrong patient identification.
●Analytic/technical errors, e.g. filled cuvette is not read within 10 minutes or reagent deterioration.
PRINCIPLE: Manual cell counts may be performed when a parameter is below the automated
instrument’s linearity, to verify a doubtful result “flagged” by the instrument or when smear findings don’t
agree with the automated result.
Manual cell counts are performed with the use of a hemocytometer and blood dilutions (or dilutions of body
fluids such as CSF). The principle is the same for leukocytes, erythrocytes and platelets, however,
the dilution, diluting fluid and area counted can VARY. Dilutions are often made with Unopettes.
2. PROCEDURE:
Diluting and plating
a. Puncture a PLT/WBC unopette reservoir and remove shield from pipet.
b. MIX EDTA whole blood specimen before obtaining aliquot of blood (tilt to mix).
The pipet will fill with blood by capillary action. It is IMPORTANT that there are
NO BUBBLES in pipet and that any excess blood is wiped from outside of pipet.
c. Completely rinse blood from pipet into unopette reservoir and mix blood with
diluent. LABEL; let unopette stand 10 minutes to allow for complete RBC lysis.
d. CLEAN hemocytometer with alcohol and dry well; place coverslip on top of the
hemocytometer.
e. After 10 minutes, invert unopette several times to mix blood with diluent before
converting to dropper assembly and plating dilution on the hemocytometer.
f. Discard a few drops and plate/load the unopette dilution by placing the pipet in
groove and slowly squeezing. Smoothly fill the entire area under the coverslip on
each side of the hemocytometer without bubbles; be careful not to overfill.
g. Allow cells to settle about 3 minutes in a petri dish with a damp cotton ball before
placing hemocytometer on the microscope stage for counting.
Counting
h. ●Using 10x (low power) objective and LOW light, focus on hemocytometer
groove, then move to the counting area. Check for even cell distribution in
the counting chamber squares before counting.
i. For consistency in counting – count dark cells which touch the top and left
boundary lines; exclude light cells touching bottom and right lines…see diagram
@ bottom right which also has arrows showing a systematic way to count cells.
j. ●Count all 9 squares on each side of chamber…total area counted = 18 mm2.
Use 2 counter/tabulator keys…one key for counting the cells on each side. You
can use 40x (high power) if there is difficulty distinguishing cells from debris.
k. Record the # of cells counted in each counting chamber side (and the total #) on
the chambercount worksheets on the labsheet.
l. The # of cells counted between sides must agree +20% to accept (or replate).
m. Calculations:
WBC/cmm = # cells (both sides) x 100 or # cells x 100 x 10 mm
0.1 mm x 18 sqmm 18 sqmm
n. ■Report WBC counts to nearest hundred if no decimal or nearest tenth if a
decimal. See Normals & Calculation sheets.
Counting chamber (one side)
2. PROCEDURE:
Diluting and plating
a. Puncture a PLT/WBC unopette reservoir and remove shield from pipet.
b. MIX EDTA whole blood specimen before obtaining aliquot of blood (tilt to mix).
The pipet will fill with blood by capillary action. It is IMPORTANT that there are
NO BUBBLES in pipet and that any excess blood is wiped from outside of pipet.
c. Completely rinse blood from pipet into unopette reservoir and mix blood with
diluent. LABEL; let unopette stand 10 minutes to allow for complete RBC lysis.
d. CLEAN hemocytometer WELL with alcohol (to avoid confusing junk with platelets
when counting) and dry well; place coverslip on top of hemocytometer.
e. After 10 minutes, invert unopette several times to mix blood with diluent before
converting to dropper assembly and plating dilution on the hemocytometer.
f. Discard a few drops and plate/load the unopette dilution by placing the pipet in
groove and slowly squeezing. Smoothly fill the entire area under the coverslip on
each side of the hemocytometer without bubbles; be careful not to overfill.
g. Allow cells to settle 10 minutes in a petri dish with a damp cotton ball before
placing hemocytometer on the microscope stage for counting.
Counting
h. Focus on hemocytometer groove using 10x (low power), then move to counting
area. ●Using 40x (high dry power) objective and LOW light (adjust light for best
contrast), check for even cell distribution in the counting chamber squares.
i. For consistency - count dark cells which touch the top and left boundary lines;
exclude light cells touching bottom and right lines...see diagrams on page 13.
j. ●Count the center 1 sq mm on each side of hemocytometer (divided into 25
squares)...total area counted = 2 mm2. Use 2 counter keys…one key for each
side. Focus up & down while counting; platelets are greenish, NOT shiny.
k. Record the # of cells counted in each counting chamber side on the chambercount
worksheets.
l. The # of cells counted between sides must agree +20% to accept (or replate).
m. Calculations:
PLT/cmm = # cells (both sides) x 100 or # cells x 100 x 10 mm
0.1 mm x 2 sqmm 2 sqmm
n. ■Report PLT counts to nearest thousand. See Normals & Calculations sheets.
o. The manual platelet count must agree with the platelet estimate from the blood
smear + 20% if the PLT count is > 50,000/cmm and + 10,000/cmm if the PLT
count is < 50,000/cmm to accept. If agreement is acceptable, the platelet count
is reported, NOT the estimate from the blood smear.
●REMEMBER to adjust light (raise condenser & open diaphragm) when changing from manual cell
counting (low light) to looking at stained blood smears (oil immersion and high light). ■Scan the smear on
low power (10x) to check cell distribution and smear quality (or “BAD” cells) before going to oil immersion.
A. Manual DIFFERENTIALS:
Performed on a properly prepared Wright's stained blood smear using OIL immersion (100x) and
HIGH light in the "rainbow" area where the red cells just touch.
The leukocyte differential is done to classify the type of leukocytes present in the blood. The slide
is scanned from side to side counting & identifying 100 consecutive WBC's using counter keys =
relative (%).
NOTE: IF SEEN, Nucleated RBC’s are reported # per diff & immature WBC=s are included in 100 cell diff.
B. Cell MORPHOLOGY:
The white cells, red cells and platelets are observed for normal size, shape, inclusions and/or
granulation. Any variations from normal are quantitated or noted per institution policy. SEE
GUIDELINES for GRADING ABNORMAL MORPHOLOGY (pg 16).
C. PLATELET ESTIMATE:
The number of platelets are estimated from the Wright's stained smear where the red cells just
touch by the following method:
1. Count the # of platelets in each of 10 fields using OIL immersion (OIF's)...use counter
keys when counting platelets. Find the average (don=t round) = Avg # PLTS per OIF.
●Take 1st 10 PLTS (from Avg # PLTs/OIF) x 20,000 each (or 200,000/cmm) PLUS each
additional PLT (above 10) x 10,000/cmm each = PLT estimate/cmm.
2. The platelet estimate should agree with the PLT count and corresponds to a platelet range.
a. Should agree + 20% if the PLT count is > 50,000/cmm.
b. Should agree + 10,000 if the PLT count is < 50,000/cmm.
c. The platelet estimate corresponds to a platelet comment range, as listed on the
bottom of page 16.
D. WBC ESTIMATE:
The number of WBC's are estimated from the stained smear using the high power lens and in an
area where the red cells are slightly overlapping.
1. Count the # of WBC's in 10 HIGH (40x) power fields (HPFs); find average.
2. The average # WBC'S per HPF x 2,000 = WBC estimate/cmm.
3. The estimate should agree + 20% with the WBC count.
You will be using the following criteria for grading/quantitating abnormal cell morphology seen on
the blood smear. Memorization is not necessary. Always follow your institution established policy.
Specific Poik - Schistocytes, Spherocytes, Acanthocytes, Echinocytes (Crenated/Burr cells), Target cells,
Teardrops, Ovalocytes, Pencil cells (ONLY reported if micro/hypo red cells), Stomatocytes:
Occ: 1-5% -- Use for schistocytes and spherocytes ONLY
Few: 5-10%
Several: 10-25%
Many: greater than 25%
Hypochromia: By definition, this means the central pallor area of the RBC is greater than 1/3 the cell
diameter. If the central pallor area is greater than 3/4 the red cell diameter, increase the hypochromia
category one degree from that used to quantitate the number of hypochromic red cells seen.
Slight: 5-15%
Moderate: 15-40%
Marked: greater than 40%
Polychromasia: Try to exempt polychromatophilic red cells (which are larger than mature red cells) from
your sizing judgement of macrocytosis.
Slight: 2-3%
Moderate: 3-5%
Marked: greater than 5%
Hypersegmented Neutrophils:
Occasional: On scan to 1%
Few: 1-2%
Several: 3-5%
Many: greater than 5%
A. PRINCIPLE: The reticulocyte is a non-nucleated immature red cell containing residual RNA. A
supravital stain, new methylene blue, is used to precipitate the RNA into dark-blue filaments
or granules to identify retics.
B. SPECIMEN and REAGENTS: EDTA whole blood is the preferred anticoagulant; New
Methylene Blue Staining solution, 12x75mm tubes, pipets, glass slides.
C. PROCEDURE:
1. Put 2 drops of new methylene blue in the bottom of a 12x75mm tube. Using a pipette,
add 2 drops of well-mixed EDTA blood to the tube.
2. Mix blood/stain mixture. The mixture color should be smoky-gray. Adjust if needed,
i.e., add more blood if mixture is too blue.
3. Incubate mixture at least 5 minutes but no longer than 10 minutes.
4. MIX solution again….important! Prepare 2-4 good smears, LABEL and let dry.
5. Counting: Using oil/100x power, count 500 total red cells separating mature RBC's from
retics (use two counter keys). Retics are greenish with blue precipitates of RNA. Two
“dots” or more is a retic. Go from feather edge to body of smear, making sure you are
not counting too thick.
6. Two techs count 500 RBC's on different retic smears for a total of 1000 RBC's counted.
7. Quality control: The number of retics/500 RBC's must agree + 2 retics between techs
to accept results or another slide is counted. Controls must read within the assayed
range to accept results.
D. CALCULATIONS:
1. The relative reticulocyte count uses the sum of the two techs answers and the percent is
reported to the nearest tenth (one decimal):
2. The absolute reticulocyte count is reported to the nearest thousand/cmm using the
following calculation:
E. SOURCES OF ERROR:
1. Inadequate mixing before making smears
2. Counting artifact or other inclusions as retics……black/shiny inclusions are “junk”.
3. Improper ratio of blood to stain.
4. Not counting all of the retics…..two blue “dots” or more is a retic.
5. Wrong calculation
A. PRINCIPLE: The HGB S prep is a screening test based on the insolubility of HGB S when
oxygen is removed as compared to other hemoglobins. Blood is added to the reagent which
causes red cell lysis, hemoglobin release, and deoxygenation. Blood containing HGB S will
form a cloudy, turbid suspension while other hemoglobins (A,F,C) are soluble in the
reagent.
C. CONTROLS: Positive (AS) and Negative (AA) controls are done with each patient run to check
the reagent.
E. PROCEDURE:
1. Make a 2ml mark on 12x75mm tubes for each control or patient to be run and LABEL.
Dispense 2mL of reagent solution into marked tubes. The solution should be at room
temperature before adding the blood.
2. Add 20 ul (0.02 mL) of mixed control or patient blood to the tube using a pipet. Mix tubes
by inversion using parafilm or by capping.
3. Allow each tube to stand in test tube holder for 10 minutes. Test interpretation can be
done between 10 and 20 minutes. The reaction is read macroscopically by looking
through the tube at ruled lines.
F. INTERPRETATION:
■Solution cloudy (lines not visible) - POSITIVE for HGB S.
■Solution transparent (lines visible) - NEGATIVE for HGB S.
■Report patient as POSITIVE or NEGATIVE for HGB S ONLY IF controls read.
G. SIGNIFICANCE:
1. Positive S preps occur for individuals with Hgb SS, SA, SC.
2. Negative S preps occur for individuals with Hgb AA, AC, CC or other non-sickling
hemoglobins.
H. LIMITATIONS:
1. False positives may occur if the test sample is lipemic or contains high levels of globulins,
presence of Hgb C-Harlem or Polycythemia (too much blood/reagent).
2. False negatives may occur if the Hgb S level is below 10% (e.g., infants <3 months old or
recent transfusion), reagent deterioration or severe anemia (too little blood/reagent).
NOTE: The HGB S prep is sensitive to HGB S levels as low as 10%. All positive screens should be
followed by a hemoglobin electrophoresis to determine disease versus trait condition (i.e.,
quantitate the amount of Hgb S).
A. PRINCIPLE: The heterophile antibodies of infectious mononucleosis may appear by the 4th
day but almost always by the 21st day of illness persisting for several months. The SERADYN
method uses horse erythrocytes (Reagent B) to detect the heterophile antibodies (IgM class)
associated with IM and guinea pig kidney antigen (Reagent A) to absorb out interfering antibodies
(Forssman or serum sickness antibodies). The etiologic agent of infectious mononucleosis (IM)
is usually the Epstein-Barr virus (EBV) via infectious secretions.
B. SPECIMEN: Serum is the preferred patient sample but plasma can be used.
D. QUALITY CONTROL: Run the Positive and Negative Control sera whenever a patient is tested.
Controls are run exactly like a patient. BOTH controls must read correctly. If controls do not
read, the kit may be defective or there was an error in technique.
E. PROCEDURE: Bring reagents to room temperature. One test circle is required for each control
or patient sample to be tested...you will need one test card. Pipets and reagent droppers should
always be held VERTICALLY when delivering drops.
1. Gently mix Reagent A; add one drop to left side of each test circle.
2. Invert Reagent B several times to mix; add one drop to the right side of each test circle.
Note that reagents are added to all testing circles at each step.
3. Using pipets provided, add one drop of a control serum or patient's serum next to
Reagent A on left side of each test circle.
4. Invert pipet and use the "paddle" portion to thoroughly mix Reagent A and serums
(controls or patient). Then gradually mix this solution into Reagent B (reddish-brown)
while covering entire test circle.
5. Rock card slowly and gently for exactly one minute.
6. Read results immediately.
F. INTERPRETATION:
■A POSITIVE IM reaction will have dark agglutinins (clumps).
■A NEGATIVE reaction will have no agglutination or may have fine granules.
■Report the patient as POSITIVE or NEGATIVE for infectious mononucleosis ONLY IF the
Positive and Negative controls read correctly.
G. LIMITATIONS:
1. If testing too early, a low titer results in a negative test. Repeat later. About 10% of
young adults and 50% of children under age 4 do not produce detectable IM heterophile
antibodies.
2. ●The test result should be correlated with the patient's blood count findings and
clinical symptoms to make a diagnosis of infectious mononucleosis…false positive and
negative results can occur. Cytomegalovirus, toxoplasmosis, viral hepatitis or HIV may
show a similar blood picture. Positive heterophile tests have been rarely reported in
certain pathologic conditions such as hepatitis, rubella, leukemia, rheumatoid arthritis and
Burkitt’s lymphoma.
A. PRINCIPLE: The ‘sed rate’ is a non-specific indicator of disease and is commonly performed.
ESR refers to the rate red cells settle in a vertical tube and is expressed as the distance the red
cells fall in mm/time. This test is primarily used to monitor patients with inflammatory disease
particularly rheumatoid arthritis.
B. SPECIMEN: EDTA anticoagulated whole blood that is less than 4 hours old; the EDTA tube must
be at least half full.
C. EQUIPMENT: Sediplast system including sedivials (with 0.2 ml sodium citrate), autozero
Westergren tubes and Sediplast rack; plastic pipets.
D. CONTROLS: Both normal and abnormal ESR control samples are run daily and must fall within
established limits.
E. PROCEDURE:
1. Mix EDTA whole blood sample or control vial. LABEL sedivial and remove sedivial cap.
Using a transfer pipet, obtain an aliquot of blood and fill the sedivial with blood (0.8 ml) to
the fill line. Recap and mix thoroughly.
2. Place sedivial in rack on a level surface. Insert the autozero tube into the sedivial (either
with cap removed or directly through cap). Continue inserting until the tube rests at the
bottom of sedivial.
3. Verify that the blood is at the zero mark and that there are no bubbles in the tube.
4. Allow the sample to stand undisturbed for exactly one hour and then read the results of
the sed rate in millimeters (distance the red cells have fallen in one hour).
F. SIGNIFICANCE:
■In normal individuals, sedimentation or falling of the red cells is slow.
NORMAL Reference Ranges: Males 0-10 mm/hr >50yo 0-20 mm/hr
Females 0-20 mm/hr >50yo 0-30 mm/hr
■In conditions with increased concentrations of certain plasma proteins that promote
rouleaux (such as FIBRINOGEN, an acute phase reactant), red cell falling is accelerated
causing an abnormal (increased) ESR.
■Abnormal/increased ESR results are seen in acute and chronic infections, chronic
inflammatory disorders (RA), malignancies especially multiple myeloma, tissue necrosis and
pregnancy.
G. SOURCES OF ERROR: Erroneous results occur if the blood is clotted or over four hours old; if
the blood tube is not at least 1/2 full; if the tube is slanted or there are bubbles in the tube.
12
WHOLE BLOOD CLOTTING TIME (WBCT) and CLOT RETRACTION PROCEDURES
Obtain blood by venipuncture using a syringe. Draw 5 ml of blood and slowly place 1 ml of blood into each
of two 12x75mm glass tubes; parafilm each tube. Put the other 3 ml of blood into an EDTA tube, mix and
label [for use later].
A. Whole blood clotting time (WBCT) - Tilt one glass tube every 30 secs until the blood clots =
WBCT. The glass contact activates coagulation; it will take longer for the other tube to clot. After
the blood in both tubes has clotted, place the tubes in a 37oC waterbath for the clot retraction test.
B. Clot Retraction - Evaluate tubes at 1 hour for retraction of the clot from the sides of the tubes.
Although rarely performed, the clot retraction test measures the ability of platelet contractile
proteins (actomyosin, thrombasthenin) to reduce clot size.
A. PRINCIPLE: The bleeding time is an in vivo measure of platelet plug formation following
capillary injury. It is a screening test for disorders of platelet function and may be affected by
vessel defects or low platelet numbers (e.g., PLT count below 100,000/cmm).
B. SPECIMEN: This procedure is performed at the bedside. No special patient prep is required; the
patient’s arm must not have IVs.
D. PROCEDURE:
1. Place the patient’s arm on a supportive surface with palm up. Cleanse the area selected
with alcohol being careful to avoid surface veins.
2. Place blood pressure cuff on patient’s forearm and inflate cuff to 40 mm Hg. This
pressure should be frequently monitored during the procedure.
3. Remove Surgicutt device from blister pack and remove safety clip. Rest the device lightly
on the forearm so that the cut is made parallel to the fold in the elbow.
4. Push the trigger and simultaneously start the stopwatch. Remove the device and
dispose of in a biohazard container.
5. Wick the flow of blood with filter paper every 30 seconds. DO NOT TOUCH the paper
directly to the incision to avoid disturbing the formation of the platelet plug.
6. Continue blotting until blood no longer stains the filter paper. Stop the stopwatch and
record time to nearest 30 seconds. Discontinue procedure if bleeding does not stop
within 15 minutes.
7. Remove cuff, clean arm and apply butterfly bandage across the incision.
F. SOURCES OF ERROR:
1. Failure to maintain pressure cuff at 40 mm Hg.
2. Touching the wound site when Awicking@ away the blood.
G. PRECAUTIONS:
1. A bleeding time ordered on a patient with a platelet count below 40,000/cmm must be
approved before doing the bleeding time.
2. Recent aspirin ingestion may prolong the BT result.
A. PRINCIPLE: The PT and APTT tests measure certain plasma proteins which participate
in clot formation. The time for clot formation is detected by completion of an electrical
circuit by a fibrin strand (endpoint). Neither test measures platelets, calcium, or factor XIII.
C. PROCEDURE:
1. Prothrombin time/PT - Measures factors in the extrinsic and common pathways.
a. Reagent: Tissue thromboplastin (a tissue phospholipid source, i.e., TF)
with calcium.
b.. Add 0.1 ml patient citrated plasma (prewarmed at 37o) to 0.2 ml reagent
(prewarmed at 37o) and time for clot formation.
c. Normal range: 11.0-13.4 seconds.
D. CONTROLS: Both normal and abnormal control levels are run and must "read" to
report patient results.
E. SOURCES OF ERROR: ‘Short draw’ (i.e., citrate tube not full), clotted sample, heparin
contamination, traumatic draw, hemolyzed sample.