Chapter 7 - SPECIAL HEMATOLOGY PROCEDURES Flashcards
are distorted cells appearing as thin, dense and elongated cells with both ends pointed
Sickle cells
is caused by the precipitation of an atypical hemoglobin (commonly hemoglobin S ), which distort the red blood cells into a sickle or crescent shape.
Sickling
is fully soluble when oxygenated but becomes insoluble when the oxygen level is decreased.
Hemoglobin S
It first polymerizes then forms into crystals which cause the red cell to become rigid
Hemoglobin S
Principle: the red blood cells take on a sickle-like shape when oxygen supply to the red cell is decreased (HbS forms insoluble tactoid crystals when exposed to low oxygen tension)
Sealed Whole Blood Method (Scriver & Waugh)
Degree of sickling depends on the concentration of Hb S in the RBC
Sealed Whole Blood Method (Scriver & Waugh)
Readings are made at an hourly interval for 2 - 3 hrs
Sealed Whole Blood Method (Scriver & Waugh)
A positive result is diagnostic but does NOT distinguish between hb S trait and hb S anemia
Sealed Whole Blood Method (Scriver & Waugh)
Reagent: 2 % Sodium metabisulfite or sodium bisulfate
Sodium Metabisulfite Methods (Daland & Castle)
Principle: A drop of blood is mixed with a drop of 2% sodium metabisulfite (a reducing agent) on a slide, and the mixture is sealed under a coverslip. The hemoglobin inside the RBCs becomes deoxygenated causing polymerization and the resultant sickle cell formation.
Sodium Metabisulfite Methods (Daland & Castle)
Reagent: Sodium hydrosulfite (dithionite)
Solubility test
Specimen: Whole blood with EDTA, heparin or sodium citrate
Solubility test
Principle: Red cells are lysed by saponin allowing Hb to escape. Sodium dithionite binds and removes oxygen from the test environment. HbS polymerizes in the deoxygenated state and forms a precipitate in a high-molarity phosphate buffer solution. The tactoids refract or deflect light and make the solution TURBID.
Solubility test
Observe specimen for turbidity by holding the tube 2.5 cm in front of a newsprint or a card reader with thin black lines.
Solubility test
TURBIDITY indicates PRESENCE of sickling Hb regardless of any genotype
Solubility test
Principle: Hb A & S reagents contain monoclonal antibodies (IgG), which specifically bind to the amino acids at or near the 6th position of the B-chain of hb S and A.
will react with the B-chain of HbS. but not with Hb A
Hb S monoclonal Ab
will react with the B-chain of HbA, but not with Hb S
Hb A monoclonal Ab
are adsorbed to suspended metal sol particles giving the reagent raspberry-like color.
Abs
Confirmatory tests:
Hemoglobin Electrophoresis; High performance liquid chromatography; Capillary
electrophoresis
are young erythrocytes that are in a discrete, penultimate phase of maturation where the nucleus has been removed, however, some of the extranuclear RNA remains in the cytoplasm.
Reticulocytes
The term (?) is derived from the fact that the cell contains a small network of basophilic materials called reticulum which is demonstrable only by supravital stain.
reticulocyte
In the peripheral blood they are called as (?).
polychromatophilic erythrocytes
Reticulocyte count and its associated corrections can be used to assess
bone marrow erythrophoietic activity.
The (?) is stained supravitally. Any non-nucleated RBC that contains 2 or more blue stained particles/granulofilamentous materials, is counted as reticulocyte.
ribosomal RNA
: Wet method and Dr Method (schillings method)
Methods of Staining
Reticulocytes are enumerated among 1000 RBCs in areas where RBCs are close but not overlapping and reticulocytes appear to be well stained.
Light Microscope
Light Microscope Retic % =
The calibrated Miller disc appears in the field with 2 squares: a large square and a small square inside the large square which is 1/9 the size of the larger square.
Miller Disc Method
Reticulocytes are counted in the large square while RBCs are counted in the small square in successive fields on the film until a total of 500 RBCs have been counted
Miller Disc Method
Miller Disc Method Retic % =
Reference Value:
Adults:
Newborn:
Adults: 0.5 - 1.5 %
Newborn: 2.5 - 6.5 %
Inclusions confused with Reticulocytes:
Pappenheimer bodies; Howell-Jolly bodies; Heinz bodies; Artifact
: actual number of Reticulocytes in 1 Liter of whole blood.
Absolute Reticulocyte Count
ARC =
Reference Value:
25.000 - 75,000 /uL
this corrects the Reticulocte count to a normal Hct to allow correction for the degree of patient anemia.
Corrected Retic count / Reticulocyte Index
The percentage of reticuloctes may appear increased because of early release into the circulation or because of a decrease in the number of mature RBCs in the circulation.
Corrected Retic count / Reticulocyte Index
CRC =
Reference value:
1% (depends on the degree of anemia)
: Index calculated to correct for the presence of shift reticulocytes that otherwise may falsely elevate the visual reticulocyte count.
Reticulocyte Production Index/Shift Correction (RPI)
RPI =
Reference value:
1 if hct is 0.45
Clinical Significance of Reticulocytes:
Increased in hemolytic & hemorrhagic anemias
40 - 45
35 - 39
25 - 34
15 - 24
< 15
40 - 45 1.0
35 - 39 1.5
25 - 34 2.0
15 - 24 2.5
< 15 3
measures the ability of the RBCs to take in fluid without lysing.
OFT
It Reflects RBC shape and size (surface area-to-volume ratio).
OFT
This is used to detect spherocytosis, because spherocytes rupture in saline concentrations near the normal level.
OFT
It may also detect target cells, which, owing to their reduced hemoglobin content, are able to withstand osmotic stress and rupture only at very dilute saline concentrations.
OFT
Importance: Diagnosis of (?) and other hemolytic anemias associated with spherocytes.
Hereditary Spherocytosis (HS)
Affected by: shape of the RBC, which in turns depends on the (?).
volume, surface area and functional state of the cell membrane
Specimen: (?); Blood sample should be obtained with minimum trauma and stasis
Heparinized blood or defibrinated blood
Normal Result:
Incomplete hemolysis:
Complete hemolysis:
0.45 %
0.35 %
whole blood is pipetted to each of a series of hypotonic saline solutions of graduated concentration.
Sanford Method
Uses hypotonic saline buffered with PO4 at ph 7.4
Dace Med
Hemolysis is determined spectrophotometrically at 540 nm
Dace Med
: sterile incubation at 37OC for 18 24 hrs to detect mild forms of hereditary spherocytosis.
Incubated OFT
Clinical Significance of OFT:
Increased OFT:
Hereditary spherocytosis; red cells with abnormal membrane; severe G-6-PDH deficiency; Pyruvate kinase deficiency; hemolytic anemias
Clinical Significance of OFT:
Decreased OFT:
Sickle cell anemia, severe Iron deficiency anemia, Thalassemia
Measures the degree of settling of erythrocytes in plasma in an anticoagulated whole-blood during a specified period of time.
ERYTHROCYTE SEDIMENTATION RATE
Importance:
1. An non-specific measure of (?).
2. It is a good index for the presence of a (?)
3. It measures the (?) of the RBCs
4. It measures the (?) of fibrinogen and globulin
inflammation
hidden but active disease
suspension ability
abnormal concentration
Stages of ESR:
- Lag phase/Initial rouleaux occurs during the first 10 minutes.
- Decantation phase/ Period of rapid settling occurs within 40 minutes.
- Final settling/packing occurs during the last 10 minutes.
occurs during the first 10 minutes.
- Lag phase/Initial rouleaux
Period of rapid settling occurs within 40 minutes.
- Decantation phase
occurs during the last 10 minutes.
- Final settling/packing
Erythrocyte factors
RBC size or mass: directly proportional to ESR
Number of RBC: inversely proportional
Presence of Anisocytes & Poikilocytes: slower settling
Plasma factors
Plasma viscosity
Plasma composition
: inversely proportional to ESR
Plasma viscosity
fibrinogen, -1 globulin, -2 globulin
= increase ESR
: decreases ESR
albumin & lecitihin
: should be exactly vertical.
Position of the tube
: should be free from any movement or vibration
Incubation environment
: should be at room temperature
Incubation temperature
of the tube
Length and Diameter
: should not be over-anticoagulated
Anticoagulant
of setting up
Time
Erythrocyte factors
Plasma factors
Mechanical/Technical factors
Factors affecting settling of RBC
Anticoagulant = Ammonium-Potassium Oxalate
Wintrobe & Landsberg Method (Wintrobe tube)
Westergren Method (Westergren tube)
Anticogulant: Original Westergren = 3.8% sodium citrate
Modified Method =
2 ml of EDTA-anticoagulated blood with 0.5 ml NSS or 3.8% sodium citrate
Less than 50 y/o:
0 - 15 mm/hr
0 - 20 mm/hr
More than 50 y/o:
0 - 20 mm/hr
0 - 30 mm/hr
More than 85 y/o:
0 - 30 mm/hr
0 - 42 mm/hr
Children:
0 - 10 mm/hr
Inflammations; acute & chronic infections, tuberculosis, multiple myeloma
Increased ESR
Rheumatic fever, rheumatoid arthritis, myocardial infarction, nephrosis
Increased ESR
SBE, Waldenstroms macroglobulinemia, hepatitis, menstruation, pregnanc
Increased ESR
Polycythemia, spherocytosis, conditions associated with Hb S & Hb C disease
Decreased or Normal ESR
: uses 3.8% sodium citrate and Cutler tube
Graphic-Cutler Method
: uses 3.8% sodium citrate and Linzenmeier tube
- Linzenmeier Method
: uses 5 % sodium citrate
- Micro-Landau Method
: uses 5 % sodium citrate
- Smith Micro Method
: uses heparin
- Roarke-Ernstene Method
: 1.3% sodium oalate
- Bras Method
Automated Erythrocyte Sedimentation Rate
Ves-Matic system
Sedimat 15
ESR STAT PLUS system - based on centrifugation.
Uses (?) (applies controlled centrifugation of blood, producing alternating compaction and dispersion of erythrocytes to measure how closely the erythrocytes approach one another under a specific standardized gravitational force.
zetafuge
Also uses a special capillary tube, (?) in length with an internal diameter of (?
75 mm
2 mm
After centrifugation, (?) is determined at the knee of curve.
Zetacrit%
ZSR (%) =
Reference value: (ZSR) :
Advantages of ZSR:
It requires a (? of blood
It is not affected by (?) and is faster than other methods
Reference range is the same for (?)
smaller amount
anemia
both sexes
Absolute Eosinophil Count:
Specimen:
EDTA or heparinized or capillary BLOOD
Absolute Eosinophil Count:
Equipment:
WBC pipet / Unopette
Fuchs Rosenthal or Speirs Levy Counting chamber
Absolute Eosinophil Count:
Diluting fluid (1:10 dilution)
Phloxine
Propylene glycol
Heparin
Na Carbonate
Alternative stains: Pilots solution; Randolphs stain
(Test for adrenocortical functions)
Thorn test
Thorn test
Perform (?) (fasting simple)
Introduce (?)
After (?)., draw second specimen for absolute eosinophil count
direct absolute eosinophil count
ACTH
4 hrs
uses Cooper and Cruickshank stain (neutral red)
Absolute Basophil Count:
Absolute Basophil Count:
Counting chamber:
Speirs Levy or Fuchs-Rosenthal
Thorn test
Normal = 2nd count should at least be (?) lower than initial count
50%
is a neutrophil or macrophage that has phagocytized (engulfed) the denatured nuclear material of another cell.
LE cell
Its formation is characterized by the production of a number of autoantibodies
LE cell
The most important of these are the (?) which occur in the serum of patients with some autoimmune disorders including systemic lupus erythematosus (SLE).
antinuclear antibodies
Demonstration of LE cells therefore suggests the presence of these antinuclear antibodies also termed as the (?).
LE factor
causes nuclear lysis (lysed nucleus becomes a homogenous amorphous mass) and this material is then phagocytized by a neutrophil.
LE factor
The neutrophil which has phagocytized the lysed nucleus is now called an.
L.E. cell
(seen on a buffy coat smear)
L.E. cell
is NOT considered positive.
Rosette formation
Other Anti-Nuclear Antibody Test:
Fluorescent test
(has replaced the LE test)
Fluorescent test
Uses fluorescein-conjugated antihuman IgG
Fluorescent test
BONE MARROW STUDY:
Collection of specimen:
Adults =
Children =
Adults = posterior iliac crest
Children = tibia
This is taken before aspiration biopsy to avoid any disruption of marrow architecture.
Trephine Biopsy/Core
Imprint biopsy is prepared by touching the specimen on a slide.
Trephine Biopsy/Core
Fixative: Zenker fluid
Trephine Biopsy/Core
Processed within 1hr.
Aspiration
Bone marrow smear preparations:
- Particle smear
- Buffy coat (concentrate) smear
- Histologic section (Cell block)
compensates for hypocellular marrow and allows for examination of large numbers of nucleated cells without interference from fat or RBCs
Buffy coat (concentrate) smear
Fixative: 10% formalin, Zenker glacial acetic acid, or B5 fixative
Histologic section (Cell block)
Bone marrow smear preparation Stains:
Romanowsky; iron stain; H and E
Normal Marrow cells
- Hematopoietic cells
- Lymphoblasts
- Monocytic cells
- Myelocytic cell
- Erythrocytic precurosrs
- Macrophages/ Histiocyte
- Mast Cells / Tissue Basophil
- Osteoblasts
- Osteoclasts
Marrow fat : hematopoietic cell =
1 : 2 (adults)
Myeloid:Erythroid(M:E)ratio =
2:1 - 4:1
include all nucleated hematopoietic cells EXCEPT megakaryocytes & macrophages
Marrow differential
at least 500 cells are counted preferably 1000 cells, 500/slide (2 slides )
Marrow differential:
SMEAR PREPARATION FOR MALARIA EXAMINATION:
Specimen:
EDTA-anticoagulated blood or fresh capillary blood collected before the initiation of treatment
SMEAR PREPARATION FOR MALARIA EXAMINATION: At least (?) should be made.
two thick and two thin peripheral blood films
For morphologic examination and species identification and determination of Percent arasitemia.
Thin blood smear
Thin blood smear Preparation:
same as wedge method
- For screening purposes
Thick blood smear
Thick blood smear Preparation: place (?) of blood close together near one end of the slide. With one
corner of a clean slide, stir the blood for about (?) to mix the three drops over an area approximately (?) in diameter
3 small drops
30 seconds
1 to 2 cm
First dehemoglobinized; air-dried then stained
Thick blood smear
Stains for malarial blood smear:
Wrights; Giemsa