LABORATORY EVALUATION Flashcards
is the volume of packed RBCs that occupies
a given volume of whole blood.
hematocrit
Macromethods
- Wintrobe Method
- Haden’s Modification Method
- Van Allen’s Method
- Sanford – Magath Method
- Bray’s Method
1.1% Na citrate
Haden’s modification method
Heparin
Bray’s Method
1.3% NA citrate
Sanford - Magath Method
Double oxalate
Wintrobe Method
Anticoagulated whole blood is centrifuged, and
the total volume of the red cell mass is
expressed as a percentage or a decimal
fraction
hematocrit
normal value of hematocrit in women
35 % - 49 %
normal value of hematocrit in men
42 % - 54 %
Clinical Implication
- Decreased values are indicator of anemia
- Hct may or may not be reliable immediately after
even a moderate loss of blood or immediate
transfusion
Increased Hct values occur in
⚫Erythrocytosis
⚫Polycythemia vera
⚫Shock , when hemocentration rise
considererably
Interfering Factors
⚫High altitude
⚫NV vary with age and gender
⚫Lower value in men and women older than 60
y/o
⚫Severe dehydration from any cause falsely raises
the hct
Sources of Error in Hematocrit Determination
- Speed and duration of centrifugation
Decrease in centrifugal force will result in more trapped plasma
in between red cells. - Type and amount of anticoagulant
Excess anticoagulant causes shrinkage of cells - Integrity in the length and diameter of the tube
- Errors in the sample, improper techniques in the collection of venous and
capillary blood. - Failure to mix the blood properly before sampling
- Leakage of blood in the case of micro hematocrit.
- Errors in taking the reading and calculating the result
the process of enumerating blood cells
Hemocytometry
composed of two raised surfaces each in the shape of a
3-mm x 3-mm square separated by an H-shaped moat
Hemocytometry
represents the number of WBCs in 1 liter of whole blood
WBC count
unit for WBC ct
10^9/L
WBC Diluting Fluid
- 2-3% glacial acetic acid
- 1% HCl added with 1 drop of methyl violet or crystal violet
Criteria of Good WBC Diluting Fluid
- should be hypotonic
- should color/stain the nuclei of white blood cells
Reference Range for WBC ct:
4.5-11.5 x 10⁹/L
WBC Count must be corrected if 5 or more NRBCs are counted
on differential count since NRBCs present in the sample are not
lysed by the diluting fluid and counted as WBC
Corrected WBC Count
RBC ct formula
RBC Count = # of cells counted x area c.f. x depth c.f. x dilution factor
unit for RBC ct
10¹²/L
Reference Range for RBC ct:
Male: 4.6-6.0 x 10ˈ²/L
Female: 4.0-5.4 x 10ˈ²/L
RBC Diluting Fluids
- Hayem’s Fluid
- Gower’s Solution
- Toisson’s Fluid
- Dacie’s or Formol Citrate Solution
- Bethel’s Fluid
- NSS or Physiologic Salt Solution
- 3.8% Sodium Citrate
this is considered the best diluent.
It keeps for a long time and does not alter
the shape of the cells.
Dacies Fluid ( Formol Citrate)
component of dacies fluid
40 % formaldehyde - 10 ml
3% w/v disodium citrate - 990 ml
component of Hayem’s Diluting Fluid
Mercuric chloride - 1.0 gram
Sodium Sulfate Anhydrous - 4.4 grams
component of Gower’s Solution-
- Sodium sulfate anhydrous - 12.5 grams
- Glacial acetic acid - 33.3 ml
- Distilled water - 200 m
prevents rouleaux formation
Gower’s Solution-
high specific gravity and stains the
WBC
Toisson’s Fluid
used in emergency cases,
used in the presence of rouleaux formation and
autoagglutination of cells.
Normal Saline Solution
Criteria of Good RBC Diluting Fluid
- Must be an isotonic solution
- Has a good preservative
- Does not initiate the growth of molds
and yeast - With a high specific gravity
- With buffer action
- Cheap and easy to prepare
the value of the hematocrit should be three times the value of
hemoglobin
rule of three
Indices define the size and Hb content of the RBC
and consist of the mean corpuscular volume
Red Blood Cell
Indices
Individual cell size is the best index for classifying
anemias.
- Index expresses the volume occupied by a single
erythrocyte and measures in cubic micrometers(
femtoliters) of the mean volume - Indicates whether the rbc size appears normal
,smaller than normal or larger than normal.
Mean Corpuscular Volume
Normal Value of MCV
80-100 fl
Formula of MCV
MCV = Hct (%) x 10 / RBC in million
Measures the average concentration of Hb.
* most valuable in monitoring therapy for anemia
Mean Corpuscular Hgb Concentration
Normal Value of MCHC
32-36 g/dl
Formula of MCHC
Hb (g/dl) x 100 / Hct (%)
Automated method of
measurement is helpful in investigation of some
hematologic disorders and in monitoring response to
therapy.
Red cell size Distribution Width
normal value of RDW
11.5 – 14.5 CV of red cell size
Clinical Implication of RDW
Helpful in distinguishing uncomplicated
heterozygous thalassemia ( low MCV
use to asses erythropoietic activity of the bone marrow
- whole blood, anticoagulated with EDTA is stained with a supravital stain such as new methylene blue or brilliant cresyl blue
reticulocyte count
reticulocyte formula
% Reticulocyte = # of reticulocytes/1000 RBCs observed x 100
is the actual number of reticulocyte in 1 liter of whole
blood
Absolute Reticulocyte Count (ARC)
reference range for ARC
25-75 x 109 /L
in specimen with a low Hct, the percentage of
reticulocytes maybe falsely elevated because whole
blood contains fewer RBCs.
Corrected Reticulocyte Count
Increased Reticulocyte Count
- Hemolytic anemia
- Lead poisoning
- Malaria
- Parasitic infections
- Blood intoxication
- Kala-azar
- Erythroblastic anemia
- Sickle cell anemia
- Relapsing fever
- Leukemia
- Splenic tumor
Decreased Reticulocyte
Count
- Aplastic anemia
- Acute benzol poisoning
- Chronic infections
- Anaplastic crisis of hemolytic anemia
Physiologic Increase of Reticulocytes - Pregnancy
- At birth
- Menstruation
refers to the speed of fall of the erythrocyte to settle
down from their plasma.
- useful in monitoring the course of an existing
inflammatory disease or differentiating between
similar diseases.
Erythrocyte Sedimentation Rate
2 ways of measurement
- Measuring the length of fall from the top of the
column of RBC in a specified period of time - Determining the time required for the red cells to
reach a specified point
- initial period of aggregation
- few cells sink under gravity but the majority form
agglomerates (rouleaux ) of various sizes. - takes place during the first 10 minutes
Agglomeration Phase
- the agglomerates sink rapidly
- the rate of fall depends on size
- takes place for about 40 minutes
Phase of Fast Settling
- the rate of settling is slow owing to clogging of the
agglomerates - takes place during the last 10 minutes
Final Phase of Packing
Macromethods for ESR determination
- Wintrobe-Landsberg Method
- Westergren Method
- Graphic and Cutler Method
- Linzenmeir Method
Micromethods for ESR determination
- Micro Landau Method
- Smith Method
- Hellige-Volmer Method or Crista Method
Factors that Influence ESR
A. Intrinsic factors
1. Plasma factors
2. Red cell factors
B. Extrinsic factors
1. mechanical factors
2. technical factors
3. physical factors
Factors that Increased the Rate of Fall
A. Intrinsic factors
1. plasma factors
a. increased fibrinogen concentration
b. increased globulin concentration
c. cholesterol
2. Red cell factors
a. macrocytes
b. anemia
c. hemolysis
Factors that Decrease Rate of Fall
A. Intrinsic factors
1. Plasma Factors
a. increased albumin
b. increased lecithin
c. defibrination
2. Red Cell Factors
a. microcytosis
b. more red cells
c. spherocytosis
d. increased sickle cells and poikilocytes
B. Extrinsic Factors
1. long standing of blood since rbc tends to be spherical
2. excess dry anticoagulant
3. temperature below 20°C
4. short sedimentation tube
5. small bore of sedimentation tube
6. more blood specimen
7. presence of blood clots
8. dirty glass wares
Westergren’s Method normal value
- Men : 0 – 15 mm / hr
- Women : 0 – 20 mm/hr
- Children : 0 – 10 mm /hr
Different Tests That Use Blood
Smears
- Leukocyte differential count
- Morphologic study of normal and
abnormal white cells, red cells and
platelets - Reticulocyte counts
- Platelet count (indirect method)
- (LE) Lupus Erythematosus cell
examination - Bone marrow examination
the simplest and most commonly used method for preparation of smear
Wedge/Push/2-Glass Slide Method
advantages of Wedge/Push/2-Glass Slide Method:
a. slides are not easily broken
b. easy to prepare
c. easy to label and transport
d. allows storages, even without cover slip
e. abnormal cells can easily be found
Characteristics of a Good Smear
- There should be a transition from thick to thin area.
- Smear should occupy ¾ of the length of the slide.
- Most have a smooth even surface, free from waves,
ridges and holes. - White blood cells should not be bunched at the end
or edge of smear. - It should have a feathery edge or tail.
Uses of Thin Smears
- WBC differential count
- Stained red cell examination
- Platelet count (indirect method)
- Reticulocyte count
- Siderocyte count
- Malarial parasite examination
- Thorough study of morphology of blood cells
Requirements to Produce a Proper Blood Films
- Use of a chemically clean glass slides
and cover glass. - Use of not too large nor too small drop
of blood. - Work is done quickly before coagulation
of the blood. - Proper angle and pressure of the
spreader.
Methods of Drying the Blood Films
- Air drying
- Heating in the oven for a low flame
- Chemical drying in ethyl alcohol
Fixatives for Blood Films
- Methanol
- Absolute Ethyl Alcohol
- Absolute Ethyl Alcohol and Ether
- 1% solution of HgCl₂
- 1% Formalin
Factors Affecting Thickness/Thinness of Smear
- Size of the drop of blood used
- Angle of the spreader slide against the stationary slid
- Pressure exerted when pushing the
spreader against the stationary slide - Speed of the spreader slide
large drop
thick smear
small drop
thin smear
heavy pressure
smear
thin smear
light pressure
thick smear
too fast
thick smear
too slow
thin smear
preferred for bone marrow preparation
- even distribution of blood cells
especially
leukocytes is observed
Cover Glass/Ehrlich’s Method
Disadvantages of cover glass method
a. cover glasses are easily broken
b. require chemically clean coverglass
c. difficult to prepare
d. difficult to label, stain and transport
there is even distribution of cells but
yields limited blood smear.
Beacom’s Method/Cover Glass and Slide Method
Automated Method
- spun smear
- Smear Prepared in Miniprep
considered polychrome stain
Wright’s Stain
component of wright’s stain
- methylene blue
- eosin
- sodium phosphate
staining of blood smear
● Wright’s stain
●Giemsa’s stain
● May-Grunwald’s stain
● Leishman’s stain
● Jenner’s stain
● Panoptic stain- combination of Romanowsky stain
and another stain
● Supravital stain
used to stain and inspect living cells which have
been removed from the body
supravital stain
combination of Romanowsky stain and another
stain
panoptic stain
it is the enumeration and determination of
relative proportion or percentage (%) of
each type of leukocyte in the peripheral or
venous blood
Leukocyte Differential
Count
Ways of Scanning Smears for Differential Count
- Strip or Horizontal Method
- Crenellation Method
- Exaggerated Battlement Method
- Two Field Meander Method
- Four Field Meander Method
cells are counted from the upper part
of the smear, the lower part, then
sideways, then to the upper part until
100 cells are differentiated
Crenellation Method
- the presence of an increase in younger
forms of leukocytes. - seen in pyogenic infections
Shift to the left
- the presence of an increase in older forms
of leukocytes - seen in megaloblastic anemia, pernicious
anemia, and in convalescence
Shift to the right
