Section 14 Flashcards
Cyanmethemoglobin Method
Standard curve developed
-Using dilutions of a cyanmethemoglobin standard
Measure absorbance of controls and patients:
-Determine Hgb values of control samples and patient samples from the standard curve.
Method recommended by ICSH
-(International Committee for Standardization in Hematology)
Using Beer’s Law calculate the Hgb Concentration
concentration of unknown /
concentration of known
=
absorbance of unknown/
absorbance of known
Multiply answer by dilution
Hemoglobin Reference Range
Male: 13.5 – 17.5 g/dl
Female: 12.0 – 16.0 g/dl
Sources of Error in Hgb Determination
-Drabkins Rgt is sensitive to light
-High WBC or PLT
-Lipemia
-Intravascular hemolysis
-Icterus
-Abnormal Hgbs may resist lysis (S and C)
-Abnormal Igs in MM
- Waldenstroms may precipitate
-Carboxy-Hgb takes one hour to convert
(smokers – usually minimal effect)
Sodium Lauryl Sulphate (SLS) Method
-Reagent lyses RBCs and WBCs
-Alters hemoglobin and oxidizes the heme group
(Fe +2 to Fe +3)
-Combines with sodium lauryl sulfate to become sodium lauryl sulfate-hemoglobin hemachrome molecule – a colored complex which absorbs at 555 nm
Hematocrit
% of whole blood which is occupied by rbcs
Varies with age, sex, altitude, nutrition, and smoking
Hematocrit
OHSU Reference Range
Male: 41.0 – 53.0%
Female: 36.0 – 46.0%
Rule of Three
RBC x 3 ~ Hgb
Hgb x 3 ~ Hct
Note: Do not use the rule of three when calculating RBC indices.
Mean Cell Volume (MCV)
Average size of red blood cells
Indicator of microcytosis or macrocytosis
MCV = Hematocrit x 10
/ RBC in Millions
Mean Cell Hemoglobin (MCH)
Average weight of hemoglobin per rbc
not an indicator of “chromia” since there is no correlation with cell size
MCH = Hemoglobin x 10
/RBC in millions
Mean Cell Volume (MCV) Reference Range
OHSU reference range = 80.0 -96.0 fl
Mean Cell Hemoglobin (MCH)
Reference Range
OHSU reference range = 29.0 – 32.0 pg
Mean Cell Hemoglobin Concentration (MCHC)
Average amount of red cell volume occupied by hemoglobin
indicates normochromia or hypochromia
MCHC = Hemoglobin x 100
/Hematocrit
Mean Cell Hemoglobin Concentration (MCHC) Reference Range
OHSU reference range = 33.4 – 35.5 g/dl ( or %)
Mean Cell Hemoglobin Concentration (MCHC) outside range
MCHC < 30 % should not occur
MCHC > 36 % should not occur except in spherocytosis. No such thing as hyperchromia.
Causes of erroneous MCHC >36 %
-Cold Agglutinins
-Lipemia
-Hemolysis
-Icterus
Red Cell Distribution Width (RDW)
Amount of red cell size variation
-Quantitate anisocytosis
Red Cell Distribution Width (RDW)
Reference Range
OHSU reference range = 11.5 - 15.0 g/L (%)
Red Cell Distribution Width (RDW) - CV
Directly calculated from the RBC histogram. Mathematically, it is the coefficient of variation
RDW (CV)% = 1SD/MCV x 100%
Red Cell Distribution Width (RDW) - SD
The RDW-SD is measured by calculating the width in fL at the 20% height level on the RBC curve
Reference range = 39-47 fL in adults
Reticulocytes
Last Immature RBC Stage
-Stage immediately after rbc extrudes its nucleus
-Normally spends 2 days in bone marrow and 1 day in peripheral blood before becoming a mature rbc
-Contains remnant cytoplasmic RNA
-On Wright’s stained smear appears slightly more blue or lavender than mature rbc (polychromasia)
Reticulocyte Count
Supravital stain
-New Methylene Blue
Measures erythropoietic activity of the bone marrow
-increased - bone marrow compensating
-decreased - bone marrow lacks rbcs to put into circulation
Reticulocyte Counts
(B) Count total red cells (mature and retics)- 6
(A) Count only retics (includes small square) - 7
-Continue until 111 rbcs counted in B (small)
-Divide # of retics counted by 10 for retic %
Reticulocyte Count
Sources of Error
Mixing of blood and stain
-Reticulocytes float to top of mixture during incubation
Moisture in air and poor slide drying
-RNA remnants in retics are NOT refractile
Other rbc inclusions
-Heinz bodies
-Howell-Jolly Bodies
-Pappenheimer bodies
Corrected Retic Count
(Corrects for abnormal Hct)
Corrected Retic %
= raw retic % x patient Hct/
normal Hct (45%)
Reticulocyte Production Index (RPI)
RPI = corrected retic count/
# of days to mature
A Hct of 45% is considered normal. For every two percentage point drop in the Hct, the reticulocyte maturation time rises by 0.1 days
Example
Patient Hct = 35%
45% - 35% = 10% difference
10/2 = 5 (2 % pts difference)
5 x 0.1 = 0.5 day increase in maturation
1 day normal maturation \+ 0.5 day increase = 1.5 days to mature in blood
Reference Range for Corrected Retic, Raw Retic, and RPI
0.5% - 1.5%
Erythrocyte Sedimentation Rate (ESR)
Rate of RBCs settling (falling) within 1 hour
-Reported in millimeters- mm/ hr
Directly proportional to RBC mass
Inversely proportional to plasma viscosity
Useful for monitoring the course of existing inflammatory disease or differentiating between similar diseases.
Erythrocyte Sedimentation Rate (ESR)
Forces that prevent settling
Plasma viscosity
-increased albumin decreases ESR
Zeta potential - sialic acid groups within rbc membrane create overall negative charge - rbcs repel each other
Erythrocyte Sedimentation Rate (ESR)
Forces that promote settling
(increasing the ESR)
Plasma Proteins (generally effect zeta potenital)
-Fibrinogen
-Globulins
Erythrocyte Sedimentation Rate (ESR)
Diseases which increase plasma proteins
(increasing the ESR)
-Multiple Myeloma
-Inflammatory Disease
-Rhematoid Arthritis and Collagen diseases
-Chronic Infections
-Tissue damage or necrosis
-Autoimmune diseases
Erythrocyte Sedimentation Rate (ESR)
Other Factors affecting the ESR
RBC factors
-Anemia
-Microcytes vs. Macrocytes
-Poik
Patient age and sex
Technical factors
-Tilted tube
-Vibrating or bumping tube
-Temperature
-Size and shape of tube
Erythrocyte Sedimentation Rate (ESR)
OHSU Reference Ranges
Male: 0-20 mm/hr
Female: 0-30 mm/hr
