Reticulocitos: Células Sanguíneas Inmaduras y su Conteo Automatizado

Universidad Interamericana de Puerto Rico

Programa de Tecnología Médica

MEDT 4520 Hematología Clínica

• Describir las características físicas de un reticulocito.
• Explicar el significado clínico y la utilidad de los distintos cálculos relacionados a reticulocitos ( retics).
• Calcular contaje de retics (%), contaje corregido de retics (CRC) y el índice de producción de reticulocitos.(RPI)

Objetivos: RETICS

• Young red blood cell; still have small amounts of RNA present in them
• Can be detected using supravital stain
• Tend to stain somewhat bluer than mature RBC's on Wright stain (polychromatophilic)
• Slightly larger than mature RBC
• Effective bone marrow activity , monitoring anemia & response to therapy

PRACTICAL HAEMATOLOGY

tenido

Supravital

con

A B C New methylene Blue D E F G H Fig. 3.4 Photomicrographs of reticulocytes showing stages of maturation. A and B, most immature (group I); C and D, intermediate (group II); E and F, later stage itermediate (group III); G, most mature (group IV); H, haemolytic anaemia, stained supravitally by new methylene blue.Brilliant Checking Blue

• New methylene blue
• 1.0 g of NMB in 100 ml of iso osmotic phosphat buffer pH 6.5 or citrate saline
• A=NaH2po4 2H20(150mmol/L) 23.4 g/l
• B=Na2HPo4 (150mmol/L) 21.3g/l
• 66 ml A + 34 ml B
• 1 part citrate sodium 30g/1 + 4 part NaCl 9 g/l
• Lifespan of stain is one month

El tente no puede tener precipitados staining. 2 or 3 drops of the dye solution with 2-4 volumes of blood

• Keep the mixture at 37º for 15-20 min
• Remixing by 10 complete inversions
• Make at least 2 films on glass slide
• the exact volumes of blood to be added to the dye solution for optimal staining depends upon the red cell count

Method Re0

• Anticoagulant must be EDTA
• 24 hours in 2-6°
• 6-8 hours in RT
• It is recommended retic count is done immediately after sample collection

Toma de muestra. Distribution of cells - cel exparsidas

• Using of miller ocular micrometer disk
• At least 300 RBC are counted = 2700 RBC retic in 20 large squares x 100
• Retic percentage=
• erythrocytes in 20 small squares x 9
• Edge rule : the cells overlapping two of the four borders are not counted.
• The number of cell to be counted to achieve a reasonably precise reticulocyte count

Count & report

The number of cell to be counted to achieve a reasonably precise reticulocyte count

Retic count

Approximate number of cells to be counted in small squares for CV of 10%	Equivalent to total count of
1-2%	1000	9000
3-5%	500	4500
6-10%	200	1800
20-25%	100	900

Important factors that affect the accuracy

• Retic preparation be well spread & well stained
• Visual acuity & patience of the observer
• The quality & resolving power of the microscope

No. Of Reticulocyte

Reticulocyte % X100 1000RBC observed - Retics 1,000 RBC % Retics

RETICULOCYTE COUNT

Absolute Value

• = Retic % x RBC Count -eg 0.01 x 5,000,000 = 50,000
• Normal up to 100,000
• More accurate way to assess body's response to anemia

RETICULOCYTE COUNT

Retic Production Index

• To be useful the reticulocyte count must be adjusted for the patient's hematocrit. Also when the hematocrit is lower reticulocytes are released earlier from the marrow so one can adjust for this phenomenon. Thus:
• Corrected retic. = Patients retic. x (Patients Hct/45)
• RPI = % Retic X Hct/45 X 1/CF Hct Correction factor (CF) % retic x HCT 45 X CF

45	1.0
35	1.5
25	2.0
15	2.5

• Normal RPI = 1 (for non-anemic pt) RPI < 2 : hypoproliferative
• RPI >=2 : hyperproliferative alta

RETICULOCITOS, IPR 2

IPR

RETICULOCITOS ACTO PTE = F.M. L (% RET + ALTO NORMAL HETP F.M.

45%	1
35.1.	25%	1.5
2	2.5
15%.

Retic Production Index

• RPI < 2 hypoproliferative (inadequate response) Causas: - Iron def. anemia - B12/folate def. - Chronic disease - Sideroblastic anemia - Aplastic anemia - Myeloproliferative
• RPI >= 2 : hyperproliferative (adequate response) Causas: - Hemolytic disease - Hemoglobinopathy (including thalassemia) - Treated B12/folate def.

• Retic count = % immature RBC
• Normal 0.5-1.5% (for non-anemic)
• In newborn 2.5-6.5%

Reticulocitos

Días de vida	Reticulocitos (%)	Reticulocitos (109/L)
1 día	3- 7	110 - 450
7 días	< 0,1-3	< 10 -80
4 semanas	<0.1-2	< 10-65
8 semanas	0,1 - 2,9	< 10- 125
12 semanas	0,4 - 1,6	15 - 75
> 12 semanas	0,2 - 2,0	10 - 105

Reticulocyte Manual Count by Supravital Stain: Normal Countwng

Supravital

Differentiating between reticulocytes and other red cell inclusions

• Pappenheimer bodies
• Heinz Bodies
• Howell Jolly body
• Hemoglobin H inclusion

RBC inclusions

Pappenheimer bodies

Wright

• Iron-containing inclusions
• One or more granules towards the periphery of the cell
• May stain a deeper blue than reticulum
• It can be identified by over staining for iron by Perls reaction
• Hyposplenism & Fe overload

Anemia deforme NOes Petic SICKle cell

Heinz Bodies

Supravital

• Denatured Hb
• Larger than Pappenheimer bodies
• Irregular in shape
• Usually attached to the cell membrane and may protrude through it
• They can be stained by a number of vital dyes
• Pale blue

Bajo Quitado

• Splenectomized subjects& G6PD deficient
• Unstable Hb

Wright stain ( no se ven )

con wright

• brilliant green
• Whole blood is mixed with 0.5% neutral red
• Mixture is counterstained with 0.5% brilliant green
• Several thick smears are prepared from the final mixture
• Heinz bodies stain green, while reticulocytes & Howell-Jolly bodies stain a deep red

Specific dye for Heinz bodies

Howell Jolly body

• Nuclear remnant - DNA
• Larger than Pappenheimer bodies
• regular in shape
• Distant from the cell membrane
• Pale blue
• hemolytic anemia
• absent or hypo function spleen

Howell-Jolly bodies This peripheral blood smear shows Howell-Jolly bodies in two red cells (black arrows), nuclear remnants that are normally removed by the spleen. Thus, they are seen in patients who have undergone splenectomy (as in this case) or have functional asplenia (eg, sickle disease disease). Target cells are also seen (blue arrows), another consequence of splenectomy. Courtesy of Carola von Kapff, SH (ASCP).

Hemoglobin H inclusion

• Denatured Hemoglobin H (ß4) in ? thalassemia
• Usually do not form with short incubation periods (2 hours)
• If present they are multiple and spherical, giving a "golf-ball" appearance
• Pale greenish-blue
• in ? thalassemia trait 0.01-1%
• Enrichment

Inclusion bodies

Reticulocyte

• A peripheral blood specimen is stained with a fluorescent dye that binds to RNA
• Dyes: aramin o ,thiazol orange, acridine orange ,thioflavin T
• Large number of cells counted increased precision & accuracy
• Presence of Howell-Jolly bodies, NRBC,giant platelet, malaria parasite false positive
• Immature reticulocyte fraction (IRF)
• IRF indicates BM response to Fe, folic acid, erythropoietin
• Electronic Cell Sizing and Counting/Cytometry/Microscopy

Automated reticulocyte count

Automated Reticulocyte Counting: Methods Available - 1997

Fluoresence Methods

Thiazole Orange (BD) by Flow Cytometry CPO dye (Coulter) by flow cytometry TOA Sysmex R series and SE-Avante by Auramine O . Abbott Cell-Dyn 4000 by CD4K530 ABX Vega by thiazole orange

Light Scatter Methods

Bayer Technicon H3, Advia by oxazine dye Coulter STKS/MAXM and Gen-S with new methylene blue (NMB) . Abbott Cell-Dyn 3500 with NMB

ntages of Automated Reticulocyte An

VSIS

• Amenable to labor efficiencies or robotics -faster analysis per sample allows for batch analysis or random access Improved precision of retic counting superior visual m croscopic counts greater obje
• New parameters of e poiesis ature Reticulocyte Fraction (RF) iculocyte I content

Questions?