Reticulocyte count

 

Artikel ini dapat di unduh melalui link berikut ini : Hitung retikulosit yang kemungkinan bahan ajar dalam bentuk presetasi yang dibuat oleh Dr Nikhil Rabade, Consultant Hematopathologist, SRCC Children’s Hospital Mumbai.

Red cell development

• Bone marrow – 3 days

• Peripheral blood – 1 day

• 90 % of the reticulocytes present in the bloodstream are mature stages

• Erythropoiesis stimulation leads to a “left shift”

Heilmayer stages

Penjelasan gambar dapat dilihat melalui tabel dibawah ini : 

Stage	Morphological description	Frequency
	Orthochromatic normoblast
I	Dense clots of reticulum (A,B)	<0.1%
II	Extended network of loose reticulum (C,D)	~10%
III	Scattered granules with diffusely arranged reticulum (E,F)	~30%
IV	Scattered granules (G)	~60%

Supravital staining 

PRINCIPLE:

• RNA within the ribosomes reacts with basic dyes such as azure B, brilliant cresyl blue or New methylene blue to form a blue precipitate
• This reaction only occurs in ‘vital’ unfixed preparations

On Romanowsky stains: Reticulocytes appear as polychromatophilic (grey/blue) cells

Staining solution

1% Brilliant cresyl blue or 1% New methylene blue

Dissolve 1gm of BCB / NMB in 100ml of 3% trisodium citrate-saline

• Check pH

• Filter with Whatmann’s No.1 filter

• Keep in dry bottle

• Stable for 4 – 6 weeks

Kits reagen hitung retikulosit pewarnaan supravital

Procedure pewarnaan hitung retikulosit

• Mix equal quantities of EDTA anticoagulated blood & retic stain

• Incubate at 37°C for 15-20mins

• Thin smears, dried and observed under oil immersion objectives

• Counter stain to avoid rapid fading

Miller Ocular disc

To be placed in one of the ocular lenses for reticulocyte counting

Counting reticulocytes

• Choose an area with no RBC overlap or distortion

• Cells must contain at least two blue staining particles or one particle linked to a filamentous thread

Average of two results

• % Reticulocytes = Number of reticulocytes(A) × 100

                                 1000 (No of RBC's observed)(B)

• EXAMPLE: 25 reticulocytes in 1,000 total RBC’s

• Reticulocyte count =25 x 100= 2.5%

                                     1000

• Normal range: 0.5-1.5%

Potential errors 

potesial error pembacaan hitung retikulosit

Pappenheimer bodies

Howell-Jolly bodies

Heinz bodies

Hemoglobin H inclusion

Automated Reticulocyte Count

Analyzer	Dye	Technology
ADVIA 2120i	Oxazine 750	Absorbance
CELL DYN 4000	CD4K 530	Fluorescence
SYSMEX	Acridine orange	Fluorescence

Automated counts

• RNA binding dye suchas Acridine orange
• Rapid, highly reproducible results with low CVs
•  Gold standard

Implications

Fairly accurate reflection of erythropoietic activity

• Anemia workup (peripheral destruction vs failure of production)
• Response to therapy (iron, vitamin B-12, folic acid supplementation)
• Bone marrow recovery after bone marrow transplantation or intensive chemotherapy

increased reticulocyte count

• Hemolytic anemia
• Nutritional anemia after treatment
• Acute and chronic blood loss
• Pregnancy

Decreased reticulocyte count

• Untreated nutritional anemia
• Aplastic anemia
• Exposure to radiation or radiation therapy
•  Chronic infection
•  Chemotherapy

Reticulocyte index (RI)

•  Increase in counts may be relative
• The reticulocyte percentage may not be a truereflection of reticulocyte production
•  Correction with reference to normal hematocrit is required

Reticulocyte production index (RPI)

Reticulocyte production index is a factor that is adjusted for both the degree of anemia and the maturation time of reticulocytes.

RPI

• Helps evaluate erythropoiesis efficiency and thus bone marrow productivity

RPI evaluation	RPI value
Normal	1
Anemia with adequate response	> 3
Anemia with inadequate response	< 2

Immature reticulocyte fraction (IRF)

Early marker for evaluating the regeneration of erythropoiesis

• Monitoring treatment response

• IRF and retic count used as monitoring parameters for bone marrow transplant

Take home message

• Reticulocyte count is a simple, easy to perform and inexpensive test
• Along with CBC, reticulocyte count is one of the first tests to be performed while investigating anemia.
• Appropriately stained and evenly smeared slide is essential for proper interpretation.
• Newer promising parameters.