Section 14

Question 1

Cyanmethemoglobin Method

Answer 1

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)

Question 2

Using Beer’s Law calculate the Hgb Concentration

Answer 2

concentration of unknown /
concentration of known
=
absorbance of unknown/
absorbance of known

Multiply answer by dilution

Question 3

Hemoglobin Reference Range

Answer 3

Male: 13.5 – 17.5 g/dl
Female: 12.0 – 16.0 g/dl

Question 4

Sources of Error in Hgb Determination

Answer 4

-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)

Question 5

Sodium Lauryl Sulphate (SLS) Method

Answer 5

-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

Question 6

Hematocrit

Answer 6

% of whole blood which is occupied by rbcs

Varies with age, sex, altitude, nutrition, and smoking

Question 7

Hematocrit
OHSU Reference Range

Answer 7

Male: 41.0 – 53.0%
Female: 36.0 – 46.0%

Question 8

Rule of Three

Answer 8

RBC x 3 ~ Hgb

Hgb x 3 ~ Hct

Note: Do not use the rule of three when calculating RBC indices.

Question 9

Mean Cell Volume (MCV)

Answer 9

Average size of red blood cells
Indicator of microcytosis or macrocytosis

MCV = Hematocrit x 10
/ RBC in Millions

Question 10

Mean Cell Hemoglobin (MCH)

Answer 10

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

Question 11

Mean Cell Volume (MCV) Reference Range

Answer 11

OHSU reference range = 80.0 -96.0 fl

Question 12

Mean Cell Hemoglobin (MCH)
Reference Range

Answer 12

OHSU reference range = 29.0 – 32.0 pg

Question 13

Mean Cell Hemoglobin Concentration (MCHC)

Answer 13

Average amount of red cell volume occupied by hemoglobin
indicates normochromia or hypochromia

MCHC = Hemoglobin x 100
/Hematocrit

Question 14

Mean Cell Hemoglobin Concentration (MCHC) Reference Range

Answer 14

OHSU reference range = 33.4 – 35.5 g/dl ( or %)

Question 15

Mean Cell Hemoglobin Concentration (MCHC) outside range

Answer 15

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

Question 16

Red Cell Distribution Width (RDW)

Answer 16

Amount of red cell size variation
-Quantitate anisocytosis

Question 17

Red Cell Distribution Width (RDW)
Reference Range

Answer 17

OHSU reference range = 11.5 - 15.0 g/L (%)

Question 18

Red Cell Distribution Width (RDW) - CV

Answer 18

Directly calculated from the RBC histogram. Mathematically, it is the coefficient of variation

RDW (CV)% = 1SD/MCV x 100%

Question 19

Red Cell Distribution Width (RDW) - SD

Answer 19

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

Question 20

Reticulocytes
Last Immature RBC Stage

Answer 20

-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)

Question 21

Reticulocyte Count

Answer 21

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

Question 22

Reticulocyte Counts

Answer 22

(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 %

Question 23

Reticulocyte Count
Sources of Error

Answer 23

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

Question 24

Corrected Retic Count
(Corrects for abnormal Hct)

Answer 24

Corrected Retic %
= raw retic % x patient Hct/
normal Hct (45%)

Question 25

Reticulocyte Production Index (RPI)

Answer 25

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

Question 26

Reference Range for Corrected Retic, Raw Retic, and RPI

Answer 26

0.5% - 1.5%

Question 27

Erythrocyte Sedimentation Rate (ESR)

Answer 27

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.

Question 28

Erythrocyte Sedimentation Rate (ESR)
Forces that prevent settling

Answer 28

Plasma viscosity
-increased albumin decreases ESR

Zeta potential - sialic acid groups within rbc membrane create overall negative charge - rbcs repel each other

Question 29

Erythrocyte Sedimentation Rate (ESR)
Forces that promote settling
(increasing the ESR)

Answer 29

Plasma Proteins (generally effect zeta potenital)
-Fibrinogen
-Globulins

Question 30

Erythrocyte Sedimentation Rate (ESR)
Diseases which increase plasma proteins
(increasing the ESR)

Answer 30

-Multiple Myeloma
-Inflammatory Disease
-Rhematoid Arthritis and Collagen diseases
-Chronic Infections
-Tissue damage or necrosis
-Autoimmune diseases

Question 31

Erythrocyte Sedimentation Rate (ESR)
Other Factors affecting the ESR

Answer 31

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

Question 32

Erythrocyte Sedimentation Rate (ESR)
OHSU Reference Ranges

Answer 32

Male: 0-20 mm/hr
Female: 0-30 mm/hr