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1

Reticulocyte:

• is an immature RBC
• cell has no nuclei
• RNA is still present (ribosomes and rough ER) in cytoplasm
• RNA is basophilic
• Retic count is a measure of RBC production (regenerative process) in the bone marrow.

2

Theory of • Supravital stain

• Supravital stain is a stain procedure that uses living cells.
• Supervital Stains such as New Methylene Blue Or (Brilliant Cresyl Blue)
 Stain precipitates the cytoplasmic RNA in living cells
 Recticulocytes stain bluish with network of darker granules called a ‘reticulum’
 younger RBC have more RNA

3

Manual Method: -Supravital stain

• Mix equal parts of EDTA blood and stain ( diluted so the angle must be higher and move quicker when making smear)
• Let stand for 15 min
• At least two smear are made of stained cells
• Lay on a flat surface to air dry
• Total 1000 RBCs counted (500 /slide)
• # retics/1000 RBCs is recorded

4

Formula of Reticulocyte count

1. reticulocyte count x100= % retics
total RBC counted

2. absolute reticulocyte count = % retics x RBCs (Necessary because the retic % does not account for the severity of the anemia)

3. Correction of the retic count for anaemia

Corrected reticulocyte count = Patient’s retics % x patient’s Hct / 0.45 (normal Hct for male)

5

Automated Retic Count

• Done by several types of cell counters
• More precise than manual count (large # of cells are counted)
• Uses special fluro-chrome dye that bind to RNA and stains reticulocyte
• Cells pass through a flow cell
• Use laser light and optical sensor detects and count RNA (flow cytometric)
• Reticulocytes cells are detected by the degree of fluorescence
• Measurements are performed that characterize cell size, shape,and morphology.

6


New parameters Retic Count

• mean reticulocyte volume (MRV)- average volume of all retic events
• immature reticulocyte fraction (IRF) – retic /all retic events
• high light scatter reticulocyte (HLR) –retic event/all RBC events

7

Clinical Use for Retic count

1. Investigation of anemia

2. Monitoring the effect of therapy for anemia (eg iron,(iron deficiency) and vitamin B12 (megaloblastic or pernicious anemia) or folic acid

3. Monitoring bone marrow response and the return of normal marrow function following chemotherapy, bone marrow transplant

8

Reference Value

Adults 0.5% – 1.5%
Newborns 2.5-6.5%
Absolute retic count 50x109/L

9

Increased Retic count

• Hemolytic anemia
• Acute blood loss
• Response to replacement therapy

10

Decreased Retic count

• Aplastic anemia
• Marrow suppression by drug, toxin, or viral infection
• Pure red cell aplasia
• Bone marrow replacement (leukemia, lymphoma, carcinoma)

11

Appearance of Reticulocytes on a Blood Film

• Reticulocytes are basophilic
• Reticulocytes stain blue with Wright stain and haemoglobin stains red
• Called polychromatic cells.(bluish)

12

 Polychromasia

corresponds to aggregate retics in a retic stain
 polychromasia can be used as a rough guide to estimate the reticulocyte count

13

Hemoglobin S

abnormal hemoglobin created by a point mutation at the sixth position on the beta globin chain that results in replacement of glutamic acid by valine. It causes sickling of red cells under conditions of reduced oxygen concentration**
very painful
KNOW THE AMINO ACID CHANGE FROM PREVIOUS LECTURE

14

Common RBC issues

sickle cell and thallesemia

15

Sickle Cell Solubility Screen (most common)

• A qualitative test based on the relative insolubility of hemoglobin S compared to other hemoglobin variants.
• Specimen requirement is one lavender top (EDTA) tube.

16

• The hemoglobin solubility test (Sickledex®, Streck Inc)

is a rapid method to detect hemoglobin S in a patient’s blood sample
• HbS mixed with 2.3 M potassium phosphate buffer, reducing reagent (sodium hydrosulfite) and zaponin ( lyse RBC)
• Hb S is insoluble and precipitates in solution (will appear turbid.)
• Deoxygenated Hgb A remains soluble (clear)
• A technologist tries to observe black lines on a card placed behind the sample
• Results are reported as positive or negative.
• Reference value is negative.
• Negative test= black lines are visible because the specimen is clear
• Positive test = the lines are not visible due to turbidity.

17

What does a positive Sickle Cell Solubility Screen mean

A positive test indicates the presence of hemoglobin S (sickle cell anemia or sickle cell trait, in which Hgb S is usually 30-45%) or a non-S sickling hemoglobin such as Hgb C Harlem and C Georgetown

18

Limitations of the SS test
False Positive

• Polycythemia (increase RBC)
• Multiple myeloma ( hypergammaglobulinemia)
• Cryoglobulinemia.
• Very high WBC (leukemias)
• Very Increased platelet counts
• Increased lipids

19

Limitations of the SS test
False-negative

• hemoglobin S is below 15% to 20%,
• increased concentration of fetal hemoglobin (HbF)
• Hemoglobin is less than 80 g/L.
• Recent blood transfusion (repeat test 4 months post transfusion) - testing the new blood instead of your own . testing in 4 months because the transfused blood will expire and your body will have to make more
• Babies up to six months …negative because of the high concentration of HbF.

20

Technical Errors IN ss causing False results :

• too much blood added = false-positive
• too little blood added false= false negative
• holding the test tube too close to the background card
• Using expired and deteriorated reagents = false-negative results

21

Sickle Cell Prep Na metabisulphite Method (not used very often):

• One drop blood mixed with one drop 2 % Na metabisulphite (reducing agent) on a slide
• Seal mixture under coverslip and let sit 30 minutes
• Decreased oxygen will cause HbS to polymerize and cause RBCs to sickle

22

1. Hemoglobin electrophoresis -Confirmatory Tests for Hgb S Indicate disease or trait
Cellulose Acetate Hemoglobin Electrophoresis- LOOK AT PICUTRE ******

 Hemoglobin electrophoresis will indicate if there are any abnormal types of hemoglobin caused by genetic disorders.
 Electrophoresis uses an electrical current to separate normal and abnormal types of hemoglobin in the blood.
 Hemoglobin types have different electrical charges and move at different speeds.
 The amount of each hemoglobin type in the current is measured

If a trait is present you can see Hb A and HbA...if you have disease you have only HbS

23

2. High Performance Liquid Chromatography (HPLC)Confirmatory Tests for Hgb S Indicate disease or trait ******

• Hemoglobins are separated by a cation exchange cartridge
• The hemoglobin fractions separate based on their ionic interaction with the cartridge
• The separated fractions pass through a flow cell, where absorbance is measured at 415 nm and again at 690 nm to reduce background noise.
• Changes in absorbance are monitored over time producing a chromatogram (absorbance vs. time).
• Each hemoglobin has its own characteristic retention time and is measured from the time of sample injection into the HPLC to the maximum point of each peak.
• Identification of unknown hemoglobin is achieved through comparison with known hemoglobin retention times.
• HPLC separats and quantitatifies HbF and HbA2 in addition to screening for variant hemoglobins along with thalassemia.
• HPLC is highly reproducible and gives rapid results.
• Some HPLC instrument programs can identify hemoglobinopathies from both newborns and adult specimens
• To confirm the results of HPLC, genetic testing may be done.

24

3. Genetic DNA Testing Confirmatory Tests for Hgb S Indicate disease or trait

• DNA genetic test can find changes in genes, or it can check the number, order, and structure of chromosomes.
• DNA testing can be done on samples of body tissue, blood, or other body fluids such as urine or saliva.

25

4. Isoelectric focusing (IEF) Confirmatory Tests for Hgb S Indicate disease or trait - look at picture on slide ******

an electrophoresis technique that separates proteins based on their isoelectric point (pI).
• The pI is the pH at which a protein has no net charge and does not move in an electric field.
• The electrophoresis gel effectively create a pH gradient so proteins separate according to their unique pI.
• In the case of hemoglobin, these migrate to a zone in the medium where the pH of the gel matches the hemoglobin’s pI.
• At the pI, the charge of the hemoglobin becomes zero and ceases to migrate.
• The hemoglobin migration order of IEF is similar to alkaline electrophoresis.
• IEF method allows for greater precision and accurate quantification than standard electrophoresis.
• Method is labour-intensive and time consuming.

26

less hemo...how to calc retic

add more blood and less dye

27

more hemo ...how much retic

less blood and more dye