Hematology Exam 4
What law describes the correlation between absorbance of cyanmethemoglobin at 540 nm and hemoglobin concentration?
Beer's law
What are the four RBC indices?
MCV (mean corpuscular volume) MCH (mean corpuscular hemoglobin) MCHC (mean corpuscular hemoglobin concentration) RDW (red cell distribution width)
What scientific notation do we use for platelets when doing hemocytometer counts?
no decimals x 10^3/mm3
What calculation do we use to correct the WBC count from a hemocytometer if NRBCs are present?
(100 x uncorrected WBCs) / (100 + number of NRBCs per 100 WBCs)
What are the two possible calculations we can use to estimate cell numbers with the hemocytometer?
(average # of cells counted / volume counted) x dilution factor (average # of cells counted x depth (10) x dilution factor) / # of primary squares counted
What dilution is used for counting RBCs on a hemocytometer?
1:200
What are some sources of error in the cyanmethemoglobin method? How might these affect your results? How could you fix some of these?
-Drabkin's reagent exposed to light -high WBC or platelet count (adds turbidity, decreases values, try diluting) -Lipemia (adds turbidity, decreases values, use ultracentrifuge or blank plasma) -intravascular hemolysis (increases values) -icterus (excess bilirubin, decreases values) -abnormal Hgbs (eg S and C) could resist lysis (decreases values) -abnormal Igs in MM/Waldenstrom's may precipitate (decreases values) -Carboxy-Hgb takes one hour to convert (seen in smokers, usually minimal effect)
How is a near native WBC analysis achieved on an automated differential?
-Red cells are removed via lysis -chemical is applied that prevents alteration of white cells
What are some sources of error in reticulocyte counts?
-Retics floating to top of blood-stain mixture during incubation -Moisture in air, poor slide drying (RNA remnants should not be refractile) -Confusing precipitate with other inclusions: HJ bodies, Heinz bodies, Pappenheimer bodies
What does the acronym VCS stand for? What method does each letter use, and what is measured for each?
-Volume (cell size, impedance) -Conductivity (internal cell structure, alternating current radiofrequency) -Scatter (cell shape and surface characteristics, flow cytometry)
What factors can alter hematocrit?
-age -sex -altitude -nutrition -smoking
What are some sources of error when using a hemocytometer?
-failure to mix blood well before pipetting -wiping tip of pipette -failure to let cells settle in hemocytometer -letting count chamber start to dry -flooding counting chamber -over or underdrawing blood into pipette to place in diluent (wrong dilution)
In what diseases do you see an increase in ESR?
-multiple myeloma -inflammatory disease -rheumatoid arthritis and collagen diseases -chronic infections -tissue damage or necrosis -autoimmune diseases
Define flow cytometry. Can it measure more than one parameter? What kind of 'flow' does it use, and what is the name of the instrument part through which the 'flow' occurs?
-technique for counting, examining and sorting microscopic particles suspended in stream of fluid (called sheath fluid) - lasers pick up details -uses LAMINAR flow -occurs in the hydrodynamically focused FLOW CELL
What is the reference range for reticulocyte percentage, corrected reticulocyte percentage, and RPI?
0.5% - 1.5%
What is the volume of an entire grid on a Neubauer hemocytometer?
0.9 mm3 (3mmx3mmx0.1mm)
How do we perform a manual hematocrit measurement?
1. Fill capillary tube 3/4-4/5 of the way with EDTA blood 2. Seal the capillary tube with Critoseal (preferably twice) 3. Repeat for a second sample 4. Place in hematocrit centrifuge (clay pointing towards edges!) for 5 minutes 5. Line up volume of tube on chart and read percentage at plasma-buffy coat line
How are reticulocyte counts performed with the Miller disc method?
1. Mix 2 parts methylene blue: 3 parts EDTA blood by inversion 2. Let tube stand 10 minutes 3. Make peripheral smear of the mixture and let dry 15 minutes 4. Count at 100x oil: total number of RBCs in small square, total number of retics in large square - want 50-70 RBCs in field 5. Move fields and continue counting until 111 RBCs are reached -follow same 'edge' rules as with hemocytometer
How long must we let blood sit in diluent before adding it to a hemocytometer? How long is it good for?
10 minutes 2 hours
How much can counts from either side of the hemocytometer deviate?
10%
How many tertiary squares are in a secondary square of a hemocytometer? Do we use these for our purposes?
16 No
What dilution is used for counting WBCs and platelets on a hemocytometer?
1:100
How many secondary squares are on a hemocytometer grid? What is their volume?
25 (contained in center primary square) Volume: 0.004mm3 (0.2mmx0.2mmx0.1mm)
What is the reference range for mean corpuscular hemoglobin?
29.0-32.0 pg
What is the reference range for mean corpuscular hemoglobin concentration?
33.4-35.5 g/dL (or %)
What is the WBC reference range for adults?
4.4 - 11.0 x 10^3/mm3
How long do we let a hemocytometer sit in order for all the cells to settle?
5-10 minutes
What does mean corpuscular hemoglobin tell us? How is it calculated? What is a problem with this index?
Average weight of hemoglobin per RBC (hemoglobin x 10) / RBC (millions) Can't tell us anything about 'chromia' because does not take into account cell size
What is the reference range for mean corpuscular volume?
80.0-96.0 fL
How many primary squares are on a hemocytometer grid? What is their volume?
9 Volume: 0.1mm3 (1mmx1mmx0.1mm)
What do we compare cyanmethemoglobin values to? How do we develop this?
A standard curve Use dilutions of cyanmethemoglobin standard
What does red cell distribution width tell us? How is it determined?
Amount of red cell size variation (quantitative anisocytosis) Determined from a histogram, reported as coefficient of variation or standard deviation
What does mean corpuscular hemoglobin concentration tell us? How is it calculated?
Average amount of red cell volume occupied by hemoglobin - indicates chromias (hemoglobin / hematocrit) x 100
What does mean corpuscular volume tell us? How is it calculated?
Average size of red blood cells (hematocrit x 10) / RBC (millions)
What is the principle of the hemocytometer method of counting?
Blood is diluted with 1% ammonium oxalate to lyse RBCs. White blood cells and platelets are then counted on a hemocytometer using a microscope.
How much time to reticulocytes spend in the bone marrow in healthy individuals? In the blood?
Bone marrow: 2 days Blood: 1 day
How does the Sysmex count RBCs and determine Hct?
Counts RBCs via impetance Calculates Hct via RBC number and MCV
What does Accugate software do?
Creates boundaries between cell populations on a scatterplot
What does coincidence correction do in reference to a Coulter counter?
Detect pulses that indicate that two cells may have passed through aperture at a time or cell may have reentered, adjust reports accordingly
What would an RBC histogram look like after transfusion?
Dimorphic (two peaks)
How are the number of reticulocytes calculated using the Miller disc method? What is the reasoning behind this?
Divide number of reticulocytes by 10, express as percentage -Large square is 9x larger than small square -retics per 111x9 RBCs have (theoretically) been counted (is ~1000 RBCs) -Divide retics by 10 to get number of retics per 100 RBCs
What type of anticoagulant do we use for the hemocytometer method? What else could we use?
EDTA Fresh blood from a capillary puncture
How do we calculate RPI?
First, calculate amount of time retics are spending to mature -(patient hct - 45)/2 -multiply this number by 0.1 -add resulting number to 1 Next, divide corrected reticulocyte percentage by the number you obtained above
What is the cyanmethemoglobin method used for?
Hemoglobin determination - hemoglobin concentration
What does the reticulocyte production index (RPI) tell us? What do the numbers '2' and '0.1' mean in the calculation?
How many reticulocytes are produced on a daily basis For every 2% difference in patient hematocrit and normal hematocrit, there is a 0.1 day increase in reticulocyte maturation time
What does an increased reticulocyte count mean? A decreased reticulocyte count?
Increased: Bone marrow is compensating for anemia Decreased: Bone marrow lacks RBCs to put into circulation
Is the raw reticulocyte percentage relative or absolute? Why?
It is relative - % could rise or fall due to number of mature RBCs present, even if number of retics remains constant
What would an MCHC <30% indicate? An MCHC >36%? What could be some erroneous causes of MCHC >36%?
Less than 30%: something's wrong with the assay (not a real result) Higher than 36%: if real, spherocytosis (there is no such thing as hyperchromia) Erroneous causes: cold agglutinins, lipemia, hemolysis, icterus
What level of light intensity should we use when performing hemocytometer counts? How can this be achieved?
Low intensity Lower the condenser Narrow the field diaphragm Lower the light control
What might a WBC histogram look like if a patient has a high level of variant lymphs?
Lymphocyte peak will have shifted to the right, reflecting larger cell size
In what order, from left to right, will you see white blood cell peaks on a WBC histogram? Is this reflective of their size in vivo?
Lymphocytes (50-90 fL) Basophils Monocytes (90-160 fL) Eosinophils Neutrophils (160-450 fL) -Not reflective of size in vivo: diluent affects tonicity
What are the reference ranges for ESR?
Male: 0-20 mm/hr Female: 0-30 mm/hr So they may not move at all!
What is the hemoglobin concentration reference range for adults?
Men: 13.5-17.5 g/dL Women: 12.0-16.0 g/dL
What is the RBC reference range for adults?
Men: 4.50-5.90 x 10^6/mm3 Women: 4.00-5.20 x 10^6/mm3
What is the reference range for hematocrit for adults? How close must the percentages be from both tubes used?
Men: 41.0-53.0% Women: 36.0-46.0% Must be within 1%
What is ESR useful for?
Monitoring course of existing inflammatory disease or differentiating between similar diseases
What does a negative flag mean on the Sysmex? A positive flag?
Negative flag: sample is all good (according to machine) Positive flag: the sample is abnormal and may need to be analyzed manually
What would an RBC histogram look like if agglutination was present?
Numerous peaks beyond the main RBC peak
What are the steps in the cyanmethemoglobin method?
Oxidization reagent: potassium ferricyanide Cyanization reagent: potassium cyanide Potassium ferricyanide and potassium cyanide are both contained in the diluent, Drabkin's reagent
What would an RBC histogram look like if there were microcytes, schistocytes, or giant platelets in the sample?
Peak to left of main RBC peak
What would an RBC histogram look like if there was a higher RDW?
Peak would be broader
How is cell surface granularity measured with flow cytometry?
Photocell picks up light that is scattered off the cell at many angles
What does sweep flow help us with when using a Coulter counter?
Prevent more than one cell from passing through aperture at a time Prevent cells from re-entering aperture
What prevents settling in an ESR? What promotes it?
Prevents: Increased albumin, zeta potential (negative charges from sialic acid on RBCs cause repulsion) Promotes: Increased fibrinogens, globulins (higher ESR with rouleaux)
What are some non-disease and RBC factors that affect ESR?
RBC factors: anemia, microcytes vs. macrocytes, poikocytosis Patient age and sex Technical factors: tilted tube, vibrations, temperature, size and shape of tube
What is the rule of 3? Are the values exact?
RBC x 3 = Hgb Hgb x 3 = Hct No, the values are not exact, but they are good for troubleshooting (don't use to calculate RBC indices!)
What is the formula for RDW-CV? How do we determine RDW-SD?
RDW-CV = (1SD/MCV) x 100 RDW-SD = width in fL at 20% height level on RDW curve
What is the reference range for RDW-CV? For RDW-SD?
RDW-CV: 11.5-15.0 g/L (%) RDW-SD: 39-47 fL
What is the erythrocyte sedimentation rate (ESR)? How is it reported? What is it directly and inversely proportional to?
Rate of RBCs settling (falling) over 1 hour, reported as mm/hr Directly proportional to RBC mass Inversely proportional to plasma viscosity
What kind(s) of cell(s) do we count in secondary squares? How many squares must we count?
Red blood cells: at least 5 squares Platelets: at least 10 squares
What is triplicate counting on a Coulter counter? What does it help us ensure?
Sample will flow through apertures three times and thus be measured via the impedance method three times. Helps us ensure precision, reducing repeats
What do thresholds help us do when using a Coulter counter?
Set a size limit to automatically select a pulse size In this example, all pulses are included with threshold 1, B and C are included in threshold 2, and none are included in threshold 3
What is a safer alternative for Drabkin's reagent? How does it work?
Sodium lauryl sulfate (SLS) Lyses RBCs and WBCs (can help with high WBC levels!) Alters hemoglobin and oxidizes the heme group (methemoglobin) Combines with methemoglobin to form colored complex that absorbs at 555nm
How does the Sysmex measure hemoglobin concentration?
Sodium lauryl sulfate method - like cyanmethemoglobin method but doesn't use cyanide
What do channels help us do when using a Coulter counter?
Sort cells into subpopulations by size In this example, pulses A and D are in channel 1 and pulses B and C are in channel 2
What does the term 'five part differential' mean?
The analyzer can distinguish between five different kinds of white blood cells: lymphocytes, basophils, monocytes, eosinophils, and neutrophils
How much of the grid do we count when performing eosinophil counts with a hemocytometer? Do we take the average of both grids for calculations?
The whole grid No
What kinds of RBC morphology can the Sysmex detect?
Three grades, increasing based on severity, for Aniso, micro, macro, hypo Dimorphic RBC population Schistocytes (reports as fragments) Agglutination
How many dots must be in a cell to count it as a reticulocyte?
Two
Describe the Coulter principle.
Two compartments filled with blood in saline diluent are separated save for a tiny aperture. Note that these compartments each have an electrode. A vacuum pulls blood cells from the external compartment to the internal compartment through the aperture, one at a time. As a cell passes through the aperture, it displaces some of the saline, causing an increase in resistance to the electrode in the internal compartment. The number of increases indicates the number of cells in the blood, and the size of the increases is directly proportional to the size of the cell.
How do we count cells that fall on the lines of the hemocytometer?
Upper and left lines: include in count Lower and right lines: do not include in count
What aspects of white blood cells does the Sysmex determine? What methods does it use?
Uses flow cytometry and fluorescent staining Cell size: forward scatter Internal cell structure: side scatter RNA/DNA: side fluorescence light
How do we calculate hemoglobin concentration?
Using Beer's law! (concentration unknown / concentration known) = (absorbance unknown / absorbance known) Multiply answer by dilution factor
What is the principle behind reticulocyte counts?
Using supravital stain on reticulocytes reveals ribosome precipitate that is visible via light microscopy. This allows for quantification of reticulocytes, which is directly proportional to an individual's level of RBC production
How is the platelet histogram created on a Coulter counter? Why is this process used?
Via curve fitting - helps us count more accurately when platelets larger than 20fL are present On this histogram, actual line is blue while fit curve is green - note how blue line stops at 20fL
What magnifications do we use to count cells with a hemocytometer?
WBCs: 10x (100x with eyepiece) RBCs and platelets: 40x (400x with eyepiece)
When would you perform a corrected reticulocyte count?
When the patient's hematocrit is less than (not more than!) 45%
What kind of cells do we count in primary squares? What is the minimum number of squares that must be counted?
White blood cells Must count at least 4 squares per side
What is on the X axis on a Coulter counter histogram? The Y axis?
X axis: femtoliters (fL, measure of size/amount of fluid displaced) Y axis: relative number (number of events/cells)
What is on each axis on a scatterplot turned out by a VCS system?
X axis: light scatter Y axis: volume z axis: conductivity
Do you wish you could use Cellavision for your diffs?
YES
Do we need to count the same number of squares on both sides of the hemocytometer?
Yes!
Do automated differential systems count more cells than we do with manual differentials?
Yes! They can count thousands of cells!
What units do we use for hemoglobin concentration?
g/dL (must convert if in mg/dL!)
What scientific notation do we use for white blood cells when doing hemocytometer counts?
one decimal place x 10^3/mm3
What does hematocrit measure?
percentage of red blood cells in the blood
How is the corrected reticulocyte percentage calculated?
raw retic % x (patient hct / 45)
What scientific notation do we use for red blood cells when doing hemocytometer counts?
two decimal places x 10^6/mm3
What scientific notation do we use for eosinophils when doing hemocytometer counts?
whole number / mm3