Nuevas tecnologas: avances en hematimetra

COORDINADORES: J.M. JOU. Barcelona J. VILLARUBIA. Madrid

Resumen del simposio

La funcin principal de los analizadores de hematimetra es proporcionar los resultados de los parmetros necesarios para el diagnstico y seguimiento de los pacientes. Los ms utilizados, desde un punto de vista clnico, son la hemoglobina, el hematcrito, el volumen corpuscular medio (VCM) y los recuentos de leucocitos y plaquetas. As mismo, el recuento diferencial leucocitario (RDL) es solicitado en la gran mayora de los pacientes. El RDL automatizado es realizado por los analizadores y en los que presentan algn tipo de alarmas debe valorarse la realizacin del RDL manual con el consiguiente consumo de material y tiempo. Los laboratorios clnicos de hematologa reciben cada da ms presiones para que sus resultados sean en el menor tiempo posible, con mejor exactitud y precisin y con el menor coste. Las casas comerciales para innovar deben proporcionar parmetros que orienten cada vez mejor la sospecha clnica hacia el diagnstico de una forma rpida, barata y til. La finalidad de todas las nuevas tecnologas deben ser un valor aadido al actual contribuyendo con parmetros nuevos o mejorando los ya existentes. Por lo citado con anterioridad, para la confeccin de este Simposium, se solicit a las cinco empresas de analizadores hematolgicos que son lderes en el mundo, tanto en ventas como en tecnologa, que buscaran un ponente que expusiera lo ms destacado de sus analizadores en la actualidad o los proyectos de futuro inmediato que pudieran contribuir en la mejora del diagnstico de los pacientes. Como se ver en las exposiciones, la finalidad de las innovaciones son: disminuir las revisiones manuales del RDL, la orientacin diagnstica de las trombopenias y trombocitosis, el diagnstico automatizado de las ferropenias y anemias crnicas, el poder orientar sobre el tipo de linfocitosis, el predecir posibles trombosis y el seguimiento de estas alteraciones mediante los analizadores que entregan 120 muestras por hora o incluso ms. El Dr. F. Lacombe, seleccionado por la compaa HORIBA-ABX Diagnostics, presenta una nueva tecnologa para la realizacin del RDL automatizado mediante la utilizacin de la tincin de las clulas de la sangre con naranja de tiazol como sustancia fluorescente combinado con lectura por luz lser a 0 y 90 y que junto con la medida de la resistividad celular permite obtener el RDL de cinco poblaciones, los eritroblastos y las alarmas de granulocitos inmaduros, linfocitos atpicos y blastos. Mediante la misma tecnologa pueden leerse correctamente muestras de mdula sea segn el autor. El Dr. A. Kratz, seleccionado por Bayer Diagnostics, muestra en su exposicin todas las utilidades clnicas demostradas en publicaciones de los parmetros proporcionados por los ADVIA 120/2120. En serie roja, la lectura de la cantidad de hemoglobina de cada hemate junto con su VCM individual, permite realizar el diagnstico diferencial entre anemias ferropnicas y talasemias. El nuevo parmetro CHr (contenido de hemoglobina reticulocitaria) es usado en guas clnicas y en muchos centros para la administracin de hierro junto a eritropoyetina, en el diagnstico diferencial entre anemia ferropnica y anemia crnica y para diagnosticar la ferropenia en nios. El componente plaquetario medio (MPC) de las plaquetas posee muy buena correlacin con la activacin plaquetaria y sirve para monitorizar los pacientes con enfermedades arteriales coronarias, diabticos y atletas. El autor presenta un software en red que contribuye al diagnstico de los distintos tipos de anemia con los resultados del analizador. Por ltimo, el ponente muestra la optimizacin de la revisin manual de los RDL de su laboratorio que le ha permitido disminuir un 41 % de revisiones. El Dr. A. Bentahar, seleccionado por IZASA SA, expone la contribucin que tienen en el diagnstico los 36 parmetros de investigacin de los analizadores Beckman Coulter. Dichos parmetros son las coordenadas tridimensionales de sus lecturas en media y desviacin estndar. As, en las leucemias linfticas crnicas pueden contribuir a la clasificacin entre LLC tpicas y atpicas. En la serie mieloide se han presentado trabajos en los cuales se obtiene que estos parmetros pueden ser ms sensibles y especficos que los tradicionales para el diagnstico de las sepsis y tambin para el diagnstico de los SMD. Los parmetros nuevos de la serie monoltica pueden detectar la malaria y la inmunoparlisis celular de los pacientes de las UCI.
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El Dr. R. Hinzmann, seleccionado por Roche Diagnsticos (Sysmex), presenta la utilidad de la medida de la fraccin plaquetaria inmadura (FPI) cuya lectura se realiza en el canal de reticulocitos y plaquetas pticas de sus analizadores. La medida es con luz lser y fluorescencia con polimetino y oxazina. Con un programa propio, el analizador calcula dicha fraccin aunque no existe un estndar o un mtodo de referencia para dicho parmetro. Segn el autor dicho valor es muy reproducible y segn algunos artculos tiene gran utilidad clnica. En plaquetopenias producidas por falta de produccin de plaquetas o por quimioterapia sus valores seran normales (< 7 %) y en patologas como la prpura trombopnica idioptica, la prpura trombopnica trombocitopnica, los embarazos con hipertensin, y la coagulacin intravascular diseminada sus valores estaran muy aumentados. As mismo, este nuevo parmetro puede predecir la recuperacin de las plaquetas en los trasplantes de mdula sea y tras la quimioterapia antes que el recuento. La Dra. T. Molero, seleccionada por ABBOTT Diagnsticos, presenta la incorporacin de mtodos inmunolgicos a los contadores. Los sistemas de la compaa pueden realizar de forma automatizada, con un software especial, la lectura de CD3/CD4 y CD3/CD8 mediante un mtodo rpido, sencillo y exacto. La autora presenta los resultados de una versin semiautomtica de recuentos inmunolgicos con tubo abierto donde pueden realizarse lecturas de poblaciones linfocitarias como T, B, T/NK y NK con resultados muy correctos en comparacin con la citometra de flujo. Con 100 l de sangre y 5 l de monoclonal los autores han conseguido clasificar las distintas linfocitosis leves o moderadas de su laboratorio en su fase inicial y hacer el cribado sobre lo que precisa un estudio posterior o no. Adems, se presentan diversos estudios con CD61 para deteccin de plaquetas que mostraron un exactitud excelente y tambin con marcadores mieloides, monolticos, eritrocitarios y de clulas inmaduras. La autora sugiere la incorporacin de los marcadores monoclonales de forma automatizada, cuando se precisen, a los analizadores automatizados.

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EXTENSIN DE LAS APTITUDES ANALTICAS DEL CELL-DYN SAPPHIRE MEDIANTE EL USO DE MARCADORES LINFOIDES
Y

T. MOLERO LABARTA, A. LEMES CASTELLANO S. DE LA IGLESIA IIGO

Servicio de Hematologa y Hemoterapia. Hospital Universitario de Gran Canaria Dr. Negrn. Las Palmas de Gran Canaria.

Resumen
El desarrollo de los mtodos inmunolgicos incorporado a los contadores hematolgicos debe suponer un notable avance en el reconocimiento de las clulas sanguneas representadas en el hemograma. Es especialmente relevante para los linfocitos, ya que la linfocitosis moderada es el hallazgo ms frecuente en los hemogramas realizados en un laboratorio hematolgico de rutina. El recuento de linfocitos obtenido en los contadores representa la suma de las principales poblaciones linfocitarias T, B y NK. Una informacin inmunofenotpica sobre estas poblaciones ofrecera una buena aproximacin a la naturaleza de los linfocitos, los distinguira de otras poblaciones celulares y reducira el nmero de frotis para la revisin microscpica, as como las repeticiones de los hemogramas, aumentando de esta manera la eficacia del laboratorio de rutina de hematologa acortando el tiempo de respuesta. Por otro lado evitara la sobrecarga de la unidad de citometra de flujo, quedando reservados estos mtodos a la determinacin de las anomalas celulares especiales y a la resolucin definitiva del inmunofenotipo linfocitario. Hasta la fecha son escasos los autoanalizadores capaces de ofrecer un sistema de deteccin antignica celular y la total automatizacin al respecto es limitada. Sera deseable que la metodologa est en un futuro prximo totalmente automatizada, de manera que la preparacin de las muestras y adquisicin celular sea rpida y sencilla; que un tcnico de laboratorio entrenado pueda procesar las muestras de forma autnoma y que la emisin del resultado final se realice tambin de forma automtica y fiable.

tincin celular (LUC) o atipia linfocitaria (linfocitos variantes)1. Estas clulas pueden ser o no reactivas, o, ni siquiera ser linfocitos, ya que las clulas inmaduras o linfomatosas tienen caractersticas similares de resistencia elctrica y de dispersin de luz. Por otra parte, los datos clnicos incluidos en la peticin del informe aportan a menudo escasa informacin, son poco orientadores y hay adems que tener en cuenta que no todas las desviaciones numricas linfocitarias se asocian con entidades clnicas definidas. En los ltimos aos se han incorporado mtodos inmunolgicos a los autoanalizadores que integran la dispersin de luz con la emisin de fluorescencia (488 nm lser azul) similar a la de los citmetros de flujo convencionales. La combinacin de la tecnologa ptica con la deteccin de fluorescencia mediante el uso de anticuerpos monoclonales (AcMo) marcados con fluorocromos, aplicada a los autoanalizadores hematolgicos, ha supuesto un claro avance en la discriminacin de las distintas clulas hematolgicas. Los contadores hematolgicos Cell-Dyn 4000 y CellDyn Sapphire (Abbott Diagnostic, Santa Clara, CA, USA) disponen de un sistema ptico (dispersin de luz) y de tres canales detectores de fluorescencia FL1, FL2 y FL3. Los datos de adquisicin celular almacenados en modo de lista (List Mode) son capaces de generar mltiples combinaciones de histogramas expresando parmetros de dispersin de luz en ngulo intermedio a 7 Intermediate Angle Scatter (IAS), a 0 Axial Light Loss (ALL), y parmetros de las fluorescencias FL1, FL2 y FL3. La combinacin del doble histograma 0 frente a 7 permite la visualizacin morfolgica de tamao o size frente a complejidad celular o complexity (fig. 1), y, la combinacin de las distintas fluorescencias, muestra la expresin de los distintos

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Introduccin
Los contadores hematolgicos realizan el recuento celular y diferencial leucocitario siguiendo distintas tcnicas nicas o combinadas como la citoqumica, la impedancia, la radiofrecuencia o la dispersin de luz (ptica). Estos mtodos son generalmente insuficientes para ofrecer informacin respecto al origen de los linfocitos. Las alarmas sealan nicamente anomalas cuantitativas, y alguna aproximacin cualitativa como la sospecha de activacin; de tamao con ausencia de
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0 Size 0 0 7 Complexity 255 Figura 1. Doble histograma de dispersin de luz que define tamao (0 size) frente a complejidad celular (7 complexity). Se observa la clara separacin de los eventos correspondientes a los linfocitos (azul), monocitos (morado) y granulocitos (naranja). haematologica/edicin espaola | 2006;91(Supl 1) | 139 |

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104 103

24576 FL1 All

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102 101 100 0 FL1 CD3FITC FL2 CD19PE/HLA-DRPE 101 102 FL2 103 104 Figura 2. A) Distribucin morfolgica. B) Expresin de fluorescencia FITC (FL1) de los linfocitos T CD3+/HLA-DR/CD19 (en rojo) frente a los linfocitos marcados con PE (FL2) CD3/HLA-DR+ (en azul).

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antgenos celulares y su localizacin morfolgica (figs. 2A y 2B). Los resultados se expresan como porcentaje de la poblacin celular en estudio y se transforman en cifras absolutas en referencia al recuento total y diferencial leucocitario suministrados por el mismo autoanalizador. Habitualmente el marcaje con AcMo se realiza con dos fluorocromos FL1 (isotiocianato de fluorescena FITC) y FL2 (ficoeritrina PE). La tercera fluorescencia (FL3) detecta la viabilidad celular (tincin vital con yoduro de propidio), aunque existe una opcin en el Cell-Dyn Sapphire (SRP-CD4) que no incorpora esta tincin dejando as libre el canal FL3, posibilitando el marcaje simultneo con las tres fluorescencias, aunque se perdera la seal de clulas no viables y las caractersticas antignicas podran verse alteradas 2.

Caracterizacin inmunofenotpica de los linfocitos

En los mencionados contadores Cell-Dyn 4000 y Cell-Dyn Sapphire, el anlisis de las subpoblaciones linfocitarias CD3/CD4 y CD3/CD8 est totalmente automatizado precisando la incorporacin de un software o modo de anlisis adecuado. La medicin se realiza en dos tubos, con los correspondientes cdigos de barras para su lectura, y que contienen esferas liofilizadas con los AcMo marcados con FITC y PE, dirigidos contra linfocitos T CD3+, y las subpoblaciones CD4+ colaboradoras y CD8+ supresoras. A continuacin del tubo con sangre total anticoagulado con cido etilendiaminotetraactico (EDTA), se coloca en el rack el tubo CD3FITC/CD4PE seguido del tubo CD3FITC/ CD8PE. El propio sistema aspira el volumen apropiado de la muestra de sangre y lo introduce en los tubos marcados. La lectura y emisin de los resultados es tambin automtica 3. En la evaluacin realizada se concluye que el mtodo es rpido y alto el grado de exactitud. La precisin es comparable con la de otros mtodos, incluso a niveles bajos de recuento3. Es interesante destacar la ventaja que supone la medicin de las poblaciones y subpoblaciones linfocitarias para los recuentos en plataforma nica.
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Adems de las tcnicas de citometra de flujo convencional, estas poblaciones se han definido tambin por otros mtodos como los realizados por capilaridad de un volumen conocido (IMAGN 2000; Biometric Imaging, Mountain View, CA) 4 o en microplaca (TRAxCD4, T cell Diagnostics, Cambridge, MA; and Zymmune, Zynaxis, Malvern, PA) 5, o, de manera similar, en distintos equipos como en el analizador Coulter STKS 6. En los ltimos aos los avances en la determinacin del inmunofenotipo linfocitario en los autoanalizadores Cell-Dyn 4000 y Cell-Dyn Sapphire han ido dirigidos hacia la realizacin de estas tcnicas en tubo abierto. De esta manera se pueden combinar distintos AcMo, detectando la expresin antignica celular, y permitiendo adems una visualizacin de la localizacin morfolgica de las poblaciones, subpoblaciones y de los linfocitos activados (figs. 3A y 3B) 2,7. La tcnica en tubo abierto utiliza una adaptacin del sistema automtico CD3/CD4/CD8 y consiste, brevemente, en sustituir los tubos marcados CD3/CD4 y CD3/CD8 por dos tubos Vacutainer limpios donde se incuba 100 l de sangre total anticoagulada con EDTA con el volumen adecuado de los AcMo a estudio durante 2-10 min a temperatura ambiente. Para la lectura automtica se utilizan los mismos cdigos de barras del sistema CD3/4/8. La adquisicin en el autoanalizador es inmediata y la lisis de los hemates automtica, no precisa lavado y puede ser realizada por un tcnico de laboratorio mnimamente especializado. El tiempo empleado en la adquisicin celular es aproximadamente 10 min por tubo. A continuacin se descargan los datos almacenados en modo de lista en un diskette despus de la transformacin de los datos en formato fcs 2.0 (Cell-Dyn 4000). Estos datos se transfieren a un PC para ser analizados en el programa WinList 4.0 (Becton Dickinson), en el programa WinMDI (software de citometra de flujo) o en el programa FCS Express v3 (De Novo Software, Thornhill, Ontario, Canad)2,7. Los datos del archivo pueden tambin descargarse directamente en un DVD, sin necesidad de transformacin en fcs (Cell-Dyn Sapphire). Utilizando el Cell-Dyn 4000 se analizaron 68 muestras de pacientes procedentes de nuestro laboratorio de rutina con un rango de recuento absoluto de linfocitos amplio: linfopnico (pacientes sometidos a qui00

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A
104 103 Figura 3. A) Imagen de fluorescencias CD3 FITC versus CD19PE/ HLA-DR PE. En rojo los linfocitos T CD3+ DR-CD19; en rosa los linfocitos T activados CD3+ DR+; en azul la poblacin CD3/HLA-DR+. B) Localizacin morfolgica de los linfocitos T activados en el doble histograma morfolgico tamao (ALL) versus complejidad (IAS). FL1 102 101 100 0 All FL1 CD3FITC FL2 CD19PE/HLA-DRPE 101 102 FL2 103 104

B
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20 Nmero absoluto de linfocitos T CD3+ CD4000 18 16 14 12 10 8 6 4 2 0 0

Nmero absoluto de linfocitos B CD+ CD4000

y = 1,0153x - 0,0155 R2 = 0,95

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y = 1,084x - 0,0269 R2 = 0,99

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Nmero absoluto de linfocitos T CD3+ citometra

Nmero absoluto de linfocitos B CD19+ citometra

Figura 4. Correlacin entre el recuento inmunolgico de los linfocitos T en el Cell-Dyn 4000 con el obtenido por citometra de flujo. Test de Passing-Bablock.

Figura 5. Correlacin entre el recuento de linfocitos B en el CD4000 con el obtenido por citometra de flujo. Test de PassingBablock.

mioterapia), normal o alto, y con procesos reactivos o malignos. Para la determinacin de los linfocitos T, B, T/NK y NK se utilizaron las combinaciones de marcadores CD19FITC/HLA-DRPE y CD3FITC/CD56PE. Los resultados mostraron una excelente correlacin entre la determinacin de poblaciones T (fig. 4) y B linfocitarias (fig. 5) con las determinadas por citometra de flujo. La correlacin fue inferior en la determinacin de las poblaciones NK y T-NK 7. Nuestro grupo ha realizado recientemente un estudio de poblaciones y subpoblaciones linfocitarias utilizando el Cell-Dyn Sapphire, en 128 muestras de pacientes infantiles y adultos, procedentes del laboratorio hematolgico de rutina y con linfocitosis leve o moderada (rango 6-20 10 9/l). Los datos preliminares han mostrado la posibilidad de una clasificacin inicial de distintas patologas linfocitarias. La tcnica consisti en la incubacin, en un tubo abierto y durante 5 min, de 100 l de sangre total anticoagulada con EDTA con 5 l de los AcMo CD3FITC/ CD19PE/HLA-DRPE y posterior adquisicin en el autoanalizador. Para el anlisis se utiliz el programa FCS Express v3 aplicando algoritmos para facilitar y agilizar el estudio. Con este sistema se ha logrado dis00

cernir entre linfocitosis T reactiva con expresin de CD3+/HLA-DR+ y no reactiva CD3+/HLA-DR, as como reconocer las posibles expansiones de linfocitos B CD19+/HLA-DR+. En 16 de las muestras se encontr un aumento de linfocitos B superior a 1,5 10 9/l. De ellas, las 8 muestras estudiadas mostraron clonalidad por mtodos citofluoromricos ulteriores. En un segundo tiempo se incluyeron AcMo dirigidos contra clulas NK (CD16FITC/CD56PE) y subpoblaciones T colaboradoras y supresoras (CD4FITC/CD8PE). En 10 muestras se logr objetivar una posible patologa compatible con expansin de clulas NK, de las que dos presentaban un patrn aberrante CD4/CD8. Aplicando rangos de normalidad segn la edad de los pacientes, el anlisis permiti la categorizacin de seis grupos de diagnstico: normal, reactiva, B clonal, linfocitosis NK, linfocitosis de significado incierto y linfocitosis NK persistente 8-10. Estos resultados apuntan hacia la posibilidad de realizar un cribado inicial de las linfocitosis en el autoanalizador, mejorando as la eficiencia del laboratorio de rutina y estableciendo una relacin lgica con el envo de muestras a la unidad de citometra de flujo (datos preliminares no publicados).
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Recuento inmunolgico de plaquetas

Un modo de anlisis similar se ha aplicado al recuento inmunolgico de plaquetas automatizado en el Cell-Dyn 4000 en muestras de sangre total11. La metodologa del recuento plaquetar consiste en la incubacin de sangre total en tubo cerrado que contiene esferas liofilizadas con el AcMo anti-CD61 marcado con el fluorocromo FITC. Este recuento es totalmente automtico y se realiza segn indica el mtodo inmunolgico de referencia (ratio glbulos rojos/plaquetas). Los resultados se expresan en histogramas que combinaron dos ngulos de dispersin de luz a 7 y 90 y con uno de fluorescencia FL1 (FITC). Precisa la incorporacin un modo de anlisis especfico al equipo y ha demostrado una excelente correlacin con los recuentos plaquetarios realizados por el mtodo de referencia (citometra de flujo)12.

focitarias en procesos con linfopenia o linfocitosis, reservando el papel de la citometra de flujo para el diagnstico de la patologa linfocitaria compleja. Estas tcnicas son de especial utilidad en los laboratorios donde el acceso a la citometra de flujo se ve limitada por la distancia fsica o por la sobrecarga de trabajo. El anlisis del inmunofenotipo linfocitario en los mencionados autoanalizadores, exceptuando el modo automtico para las subpoblaciones CD3/CD4/CD8, se realiza parcialmente de forma manual. Por ello es recomendable para facilitar y agilizar el estudio, crear algoritmos en el programa de anlisis. Por ltimo, cabe resaltar el inters en el logro de la total automatizacin de estos procesos en los contadores hematolgicos y la posibilidad de su realizacin de forma sistemtica por un tcnico de laboratorio especializado.

Bibliografa Otras aplicaciones de la caracterizacin inmunofenotpica celular

Adems de poblaciones linfocitarias se han analizado en el Cell-Dyn Sapphire distintos marcadores mieloides, monocticos, plaquetarios, eritrocitarios y de clulas inmaduras, utilizando una amplia batera de AcMo marcados con uno o dos fluorocromos en anlisis en tubo abierto 2. Con respecto a los linfocitos se analizaron las poblaciones y subpoblaciones T, B y NK mediante la combinacin de CD3FITC/CD2PE; CD3FITC/CD4PE; CD3FITC/CD8PE; CD3FITC/ CD19PE; CD3FITC/CD22PE y CD16FITC/CD56PE, mostrando a la vez las poblaciones de acuerdo con las caractersticas de dispersin de la luz segn los distintos tipos celulares. Este trabajo resalta la posibilidad de diferenciar procesos malignos de reactivos, de investigar la naturaleza de las clulas observadas en el frotis y de monitorizar las subpoblaciones durante el seguimiento de una enfermedad o de su tratamiento 2. La investigacin contina en relacin con el estudio de otros marcadores, como refiere el artculo recientemente publicado que determina la expresin de los antgenos CD64 y HLA-DR en poblaciones granulocticas y monocticas 13; con los recuentos de plaquetas citratadas para transfusin14,15 o con la cuantificacin inmunolgica de la hemoglobina fetal16. La combinacin de AcMo podra ser implementada utilizando la tercera fluorescencia FL3 sustituyendo el yoduro de propidio (que determina la viabilidad celular) por otros AcMo.
1. Van der Meer W, Scott CS, Keijzer M. Automated flagging influences the inconsistency and bias of band cell and atypical lymphocyte morphological differentials. Clin Chem Lab Med. 2004;42:371-7. 2. Johannessen B, Roemer B, Flatmoen L, Just T, Aarsand AK, Scott CS. Implementation of monoclonal antibody fluorescence on the Abbott CELL-DYN Sapphire haematology analyser: evaluation of lymphoid, myeloid and platelet markers. Clin Lab Haem. 2006;28:84-96. 3. Marshall P, Hung D, Yuan J, Kim YR. Rapid, automated, closed-tube quantitation of CD4+ and CD8+ T-cell populations on the Cell-Dyn 4000 hematology analyzer. Lab Hematol. 2000;6:137-43. 4. Anonymous. The IMAGN 2000 microvolume fluorimeter, 4T8 assay. Articles available at website www.biometric.com. 5. Nicholson JKA, Vellaca WM, Jubert S, Green TA, Bryan L. Evaluation of alternate CD4 technologies for the enumeration of CD4 lymphocytes. J Immunol Methods. 1994;177:43-54. 6. Machin SJ, Pollard Y, Grant D, Chavda N. CD4+ and CD8+ lymphocyte immunophenotyping using antibody-bound microspheres on Coulter STKS analyzer. Lab Hematol. 1997;3: 60-5. 7. Molero T, Roemer B, Perera lvarez M del Mar, Lemes A, De la Iglesia Iigo S, Palacios G, et al. Analysis and enumeration of T cells, B cells and NK cells using the monoclonal antibody fluorescence capability of a routine haematology analyser (Cell-Dyn CD4000). Clin Lab Haem. 2005;27:224-34. 8. Richards SJ, Scott CS. Classification of large granular lymphocyte (LGL) and NK-associated (NK) disorders. Blood Reviews. 1992;6:220-33. 9. Scott CS, Richards SJ, Sivakumaran M, Short M, Child JA, Hunt KM, et al. Transient and persistent expansions of large granular lymphocytes (LGL) and NK-associated (NKa) cells: The Yorkshire Leukaemia Group study. Br J Haematol. 1993; 83:504-15. 10. Shearer WT, Rosenblatt HM, Gelman RS, Oyomopito R, Plaeger S, Stiehm WR, et al. Lymphocyte subsets in healthy children from birth through 18 years of age: The pediatric AIDS clinical Trails Group P1009 study. J Allergy Clin Immunol. 2003;112:973-80. 11. Gil JE, Davis KA, Cowart WJ, Napacena FU, Kim YR. A rapid and accurate closed tube immunoassay for platelets on an automated hematology analyser. Am J Clin Pathol. 2000;114: 47-56. 12. Kunz D, Kunz WS, Scott CS, Gressner AM. Automated CD61 immunoplatelet analysis of thrombocytopenic samples. Br J Haematol. 2001;112:584-92.
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Conclusin
La incorporacin de la metodologa inmunolgica al laboratorio de rutina hematolgico es ya un hecho real que permite tipificar las distintas poblaciones lin| 142 | haematologica/edicin espaola | 2006;91(Supl 1)

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13. Van der Meer W, Van Dun L, Gunnewiek JK, Roemer B, Scott CS. Simultaneous determination of membrane CD64 and HLA-DR expression by blood neutrophils and monocytes using the monoclonal antibody fluorescence capability of a routine haematology analyser. J Immunol Methods. 2006 (en prensa). 14. Johannessen B, Haugen T, Scott CS. Standardisation of platelet counting accuracy in blood banks by reference to an automated immunoplatelet procedure: comparative evaluation of Cell-Dyn CD4000 impedance and optical platelet counts. Transfus Apher Sci. 2001;25:93-106. 15. Hervig T, Haugen T, Liseth K, Kjeldsen J, Scott CS, Johannessen B. The platelet count accuracy of platelet concentrates obtained by using automated analysis is influenced by instrument bias and activated platelet components. Vox Sang. 2004;87:196-203. 16. Little BH, Robson R, Roemer B, Scott CS. Immunocytometric quantitation of foeto-maternal haemorrhage with the Abbott Cell-Dyn4000 haematology analyser. Clin Lab Haematol. 2005;27:21-31.

LEUKOCYTE DIFFERENTIAL STAINING USING THIAZOLE ORANGE AND FLOW CYTOMETRY ANALYSIS. APPLICATION TO A NEW 5-POPULATION CELL ANALYSER
F. LACOMBE1, M. ROBERT1, S. VRIAC 2, D. LEFEVRE 2 Y F. BELLOC1
Laboratoire dHmatologie, Hpital Haut-Lvque, CHU Bordeaux, Pessac, France. 2Socit Horiba-ABX, Montpellier, France.
1

Nucleic acids of cells can be labeled by Thiazole orange (TO) which is a fluorescent intercalative dye largely used for labeling reticulocytes, reticulated platelets and red cell parasites in flow cytometry. We report here the use of TO for quantifying the intracellular content in nucleic acids of cell lines and peripheral blood leukocytes. Using DNAse and RNAse treatment, we were able to monitor the DNA and RNA intracellular content of K562 cell line by image analysis and flow cytometry. The same study, performed on peripheral whole blood after erythrocyte lysis, showed that leukocyte subpopulations could be discriminated on the basis of their RNA content and Side Scatter (SSC) intensity. Monocytes were the most fluorescent population, lymphocytes the medium fluorescent one and polymorphonuclears (PMN) the less fluorescent. Cell sorting experiments confirmed that CD13, CD3 and CD14 sorted positive cells contained respectively 99 % of dully, 94 % of medium and 97 % of brightly fluorescent cells after TO staining. In other experiments, we proved that the leukocyte discrimination by TO was essentially due to their RNA content and not their DNA content. Using FRET experiments performed with 7-amino-actinomycin D (7-AAD) and TO simultaneous incubation, we measured the green fluorescence of each leukocyte popula00

tion in the presence or not of 7-AAD. We found that the obvious decrease in fluorescence was exactly the same in the three leukocyte subpopulations but the remaining fluorescence was higher in monocytes than in lymphocytes, PMN being the less fluorescent cells. Furthermore, we tested the ability of TO for monitoring the in vitro activation of peripheral blood mononuclear cells (PBMNC). After in vitro activation of PBMNC by phytohemaglutinin (PHA), 44 + /1 % of PHA treated cells had a relative increase of their fluorescence in comparison to the untreated cells. Using this method, we proved that PHA responding lymphocytes could be easily identified from non responding lymphocytes. Since Thiazole orange staining was shown an easy and reliable method to discriminate peripheral blood leukocyte subpopulations, we applied it to leukocyte differential in peripheral blood samples for the determination of normal subpopulations (i.e. white blood cells (WBC), nucleated red blood cells (NRBC)) and abnormal subpopulations (i.e. immature granulocytes (IG), blast cells and atypical lymphocytes). In a first part, we validated the TO staining method by flow cytometry (FCM). The study was performed for two years and the recruitment was as follow: 154 normal blood samples, 100 samples with NRBC, 150 samples with IG in hematological disease, 100 samples with IG in inflammation and/or infectious disease, 175 samples with blast or undifferentiated cells, 135 samples with lymphoproliferative disease. Basophil differential was tested on additional 100 samples. All the samples were analyzed using a manual counting on 200 cells and if discrepancies between manual and automated counts were detected, a new verification was performed on additional 200 cells. All the samples were also analyzed by FCM on an XL flow cytometer (Beckman Coulter) using a combination of TO and CD45-Pe-Cy5 labeling and analyzed using Expo32 analysis software. All the results were compared using the coefficient of correlation of Spearman and the intraclass coefficient of correlation. Excellent results were obtained for normal samples. The correlations between manual and XL counts were very good. From this first study, it was possible to claim that TO is a reliable marker to perform 4-part differential (including basophils). Other parameters were needed for discriminating eosinophils. NRBC were perfectly distinguished on TO/Side Scatter (SS) histograms and correlations between manual and XL counts were good. IG in both hematological disease and inflammatory/infectious disease were well separated from other WBC subpopulations and the TO/SS histogram could be proposed as a new reference method for detecting them. The main remaining problem was the detection and count of blast cells and abnormal lymphocytes. In a second run, the TO staining method was applied to a 5-part differential prototype based on a simultaneous recording of 4 parameters: TO fluorescence intensity, SSC, FSC, and resistivity measurement. The recruitment was as follow: 165 normal blood samples, 95 samples with NRBC, 175 samples with IG in
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hematological or inflammatory and/or infectious disease, 130 samples with blast or undifferentiated cells, 55 samples with lymphoproliferative disease. 50 bone marrow samples were also analyzed. Morphological analysis was performed as previously described and a flow cytometry differential was systematically carried out using a 4 colour staining: CD14-FIT/CD9-PE/ CD45-ECD/CD16-PC5. Data were analysed as previously described. Excellent quantification of peripheral blood normal leukocyte subpopulations, IG and NRBC were obtained. Nevertheless, even if flags could be easily built for detecting blast cells and abnormal lymphocytes, additional markers currently under investigation in our laboratory will be needed for precisely counting these abnormal subpopulations. In bone marrow samples, a good discrimination of cell subpopulation was obtained too. Cell TO staining seems a very promising method for quantification of normal and abnormal leukocyte subpopulations and our evaluation of a prototype based on this method and multiparametric analysis of data confirms that.

Overview
The ADVIA 120/2120 Hematology System is a benchtop analyzer for medium- to large-volume laboratories. A detailed description of this system has recently been published by Harris et al1. Essentially a flow cytometer, the ADVIA 120/2120 uses light scatter, differential white cell lysis, and myeloperoxidase and nucleic acid staining to analyze whole blood samples. In the red cell/platelet channel, red cells are converted to spheres. Using Mie theory, low-angle (2-3) and high-angle (5-15) light scatter analysis allows determination of the size and hemoglobin concentration of individual red cells 2. (Total hemoglobin concentration in a sample is additionally measured in the hemoglobin channel with a cyanide-free method measuring light absorption at 564 nm.) Similarly to the red cell analysis, the size and granularity of individual platelets is determined. White cells are enumerated and counted in two channels, in the peroxidase channel and in the lobularity/nuclear density channel. The peroxidase channel reagents form a dark precipitate with the peroxidase containing granules of white blood cells to distinguish peroxidase positive cells (neutrophils, eosinophils, and monocytes) from peroxidase-negative cells (lymphocytes, basophils, and large unstained cells). In the lobularity/nuclear density channel (also known as the basophil channel), leukocyte nuclei (and basophils) are analyzed according to size, lobularity, and nuclear density. In the separate reticuloctye channel, red cells are stained with oxazine 750, a nucleic acid dye. In addition to the enumeration of the reticulocytes, this method allows the reporting of novel parameters, like the CHr (corpuscular hemoglobin of reticulocytes) at no additional cost. The enumeration of nucleated red cells (NRBC) on the ADVIA 120/2120 system is based on data collected in the two white cell channels already described. No additional hardware or reagents are necessary. A recent international multicenter clinical trial has shown that the method correlates well with microscopy and has very high sensitivity and specificity 3. White cell counts and differentials are corrected automatically if NRBCs are present.

FLOW CYTOMETRY BASED ANALYSIS IN THE ROUTINE HEMATOLOGY LABORATORY

A. KRATZ
Assistant Professor. Harvard Medical School. Director. Clinical Hematology Laboratory. Massachusetts General Hospital. Boston.

Introduction Background
The modern clinical hematology laboratory is facing strong pressures to provide complete blood counts (CBCs), white cell differentials, and reticulocyte enumerations with short turnaround times, high accuracy and reliability, and at the lowest possible cost. These goals can only be achieved with automated cell counters, which allow the rapid analysis of large numbers of patient samples with a minimal amount of labor. This report summarizes the experience of the Massachusetts General Hospital (MGH) in Boston, Massachusetts, in achieving these goals with the Bayer ADVIA 120/2120 system. We are a tertiary care academic teaching hospital with 868 beds, over 42,000 admissions and 1.2 million outpatient visits annually. We have been using the ADVIA 120/2120 system for over 7 years, and are presently using 7 instruments.
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Unique clinical application of the ADVIA 120/2120 System Red cells

The determination of the size and hemoglobin concentration of individual red cells makes it possible to provide users with a red cell cytogram. In this graph, the size of individual red cells is indicated on the y-axis, while their hemoglobin concentration is indicated on the x-axis. Patients with iron deficiency will show an increase in hypochromic and microcytic red cells; thalassemic states are characterized by a more
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pronounced microcytosis and less hypochromasia. The presence of more than one red cell population, for example in transfused patients or patients with correcting iron deficiency, can easily be determined by simple review of the red cell cytogram (fig. 1).

Reticulocytes
The ADVIA 120/2120 System can determine the size as well as hemoglobin- and RNA-content of individual red cells. This allows the reporting of reticulocyte parameters like the CHr (the corpuscular hemoglobin content of reticulocytes) and the comparison of these parameters to their equivalents in all red cells or in mature red cells alone. The CHr is used in many medical centers for the guidance of iron and erythropoietin therapy of patients on hemodialysis 4,5. It can also be used to distinguish between iron deficiency and anemia of chronic disease6. Most recently, Ullrich and colleagues have published in JAMA the results of a prospective study on the use of the CHr for the diagnosis of iron deficiency 7. Their findings show that a CHr of less than 27.5 pg is a more accurate hematological indicator of iron deficiency than hemoglobin measurement. The authors raise the possibility that the CHr, and not hemoglobin determination, should be used as the screening test for iron deficiency in infants.

of platelets from other events (microcytes, cell fragments) and therefore a more accurate platelet count. The determination of platelet granularity allows the reporting of a unique parameter, the mean platelet component (MPC). The MPC, a parameter which is available as a part of every CBC obtained with the ADVIA 120/2120 at no additional cost, is inversely correlated to platelet activation 8. A number of groups have published studies on the use of the MPC for the monitoring of the activational status of platelets, for example in patients with coronary artery disease, in diabetics, and in athletes 9-11.

White cells
Simple review of the myeloperoxidase staining patterns provided by the ADVIA 120/2120 system allows the diagnosis or exclusion of myeloperoxidase deficiency. Combinations of the myeloperoxidase staining patterns and nuclear density of white cells is utilized in the PANDA (Peroxidase Activity Nuclear Density Analysis) Classification, which can be used as a tool in the initial workup of cases of leukemia12,13.

Effects of the use of the ADVIA 120/2120 system on productivity at the Massachusetts General Hospital
At the Massachusetts General Hospital, all blood samples on which a complete blood count and/or a white cell differential is ordered are first analyzed by the

Platelets
The two-dimensional platelet analysis performed by the ADVIA 120/2120 system allows better distinction

C
Figure 1. Red cell cytograms. A) Normal cytogram. B) Patient with iron deficiency: Red cells are hypochromic and microcytic C) Patient with beta thalassemia trait: Red cells are microcytic, but less severely hypochromic than in iron deficiency. D) Patient with iron deficiency who has received a blood transfusion: The patients own red cells are hypochromic and microcytic; the transfused red cells are normocytic and normochromic.
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Table 1. Review rates at the Massachusetts General Hospital

Review rate (%) Before ADVIA 120 After ADVIA 120 1st adjustment of flagging criteria 2nd adjustment of flagging criteria Upgrade to ADVIA 2120 64 47 37 28 23

under way to use a neural network-based software program to analyze the results obtained with the ADVIA 120/2120. This program will allow the preclassification of the cause of a patients anemia. As shown in table 2, the system already has high accuracy for a number of common diseases.

References
1. Harris N, Kunicka J, Kratz A. The ADVIA 2120 Hematology System: Flow cytometry-based analysis of blood and body fluids in the routine hematology laboratory. Lab Hematol. 2005;11:47-61. 2. Mohandas N, Kim YR, Tycko DH, Orlik J, Wyatt J, Groner W. Accurate and independent measurement of volume and hemoglobin concentration of individual red cells by laser light scattering. Blood. 1986;68:506-13. 3. Kratz A, Maloum K, OMalley C, Zini G, Rocco V, Zelmanovic D, et al. Enumeration of Nucleated Red Blood Cells with the ADVIA 2120 Hematology System: An International Multicenter Clinical Trial. Lab Hematol. 2006;12:63-70. 4. Fishbane S, Galgano C, Langley RC Jr, Canfield W, Maesaka JK. Reticulocyte hemoglobin content in the evaluation of iron status of hemodialysis patients. Kidney Int. 1997;52:217-22. 5. Tessitore N, Solero GP, Lippi G, Bassi A, Faccini GB, Bedogna V, et al. The role of iron status markers in predicting response to intravenous iron in haemodialysis patients on maintenance erythropoietin. Nephrol Dial Transplant. 2001;16:1416-23. 6. Thomas C, Thomas L. Biochemical markers and hematologic indices in the diagnosis of functional iron deficiency. Clin Chem. 2002;48:1066-76. 7. Ullrich C, Wu A, Armsby C, Rieber S, Wingerter S, Brugnara C, et al. Screening healthy infants for iron deficiency using reticulocyte hemoglobin content. Jama. 2005;294:924-30. 8. Macey MG, Carty E, Webb L, Chapman ES, Zelmanovic D, Okrongly D, et al. Use of mean platelet component to measure platelet activation on the ADVIA 120 haematology system. Clin Cytometry. 1999;38:250-5. 9. Kennon S, Price CP, Mills PG, Macey M, Cooper J, Clarke H, et al. The central role of platelet activation in determining the severity of acute coronary syndromes. Heart. 2003;89:1253-4. 10. Bae SH, Lee J, Roh KH, Kim J. Platelet activation in patients with diabetic retinopathy. Korean J Ophthalmol. 2003;17:140-4. 11. Kratz A, Wood MJ, Siegel AJ, Hiers JR, Van Cott EM. Effects of marathon running on platelet activation markers: direct evidence for in vivo platelet activation. Am J Clin Pathol. 2005; 125:296-300. 12. Dix CJ, Hassall DG, Bruckdorfer KR. The increased sensitivity of platelets to prostacyclin in marathon runners. Thromb Haemost. 1984;51:385-7. 13. Zini G, dOnofrio G. Neural network in hematopoietic malignancies. Clin Chim Acta. 2003;333:195-201.

Table 2. Accuracy of a neural network used to pre-classify the causes of anemia based on the results of the ADVIA 120/2120 System

# Correct Normal Iron Deficiency Anemia -Thalassemia Heterozygote -Thalassemia Homozygote Hb S Homozygote Hb SC Hereditary Spherocytosis Total 229 69 1580 37 23 12 33 5610

# Incorrect 340 7 340 0 5 2 4 860

Accuracy (%) 87 91 82 1000 82 86 89 87

ADVIA 120/2120 System. Only if the instrument flags the specimen, is a slide prepared and a microscopic review performed. Before the use of the ADVIA 120 at our institution, our review rate (the percentage of samples which required the preparation of a slide) was 64 % (table 1). After we started to use the ADVIA 120, our review rate dropped to 47 %. Subsequent adjustment of the flagging criteria resulted in a further reduction of the review rate to 37 %. A further refinement of the flagging criteria allowed us to achieve a review rate of 28 % on the ADVIA 120. In 2005, we upgraded to the ADVIA 2120. This resulted in an additional reduction of the review rate to 23 %. This reduction in our review rate by 41 % has led to very significant savings in labor cost and allowed us to provide clinicians with a short turnaround time of blood counts and differentials.

Future directions: using neural networks for the analysis of the multitude of parameters provided by the ADVIA 120/2120 System
It is clear that in addition to providing the traditional CBC and white cell differential parameters, modern automated cell counters can provide many novel parameters. The large number of parameters which thus become available as the result of a routine laboratory test like a CBC are best analyzed by sophisticated computer systems. A research project is presently
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NUEVAS APLICACIONES CLNICAS EN LOS ANALIZADORES HEMATOLGICOS DE LA SERIE LH

A. BENTAHAR
IZASA S.A.

Introduccin
Jean Bernard, de la Academia Francesa, describa en su prologo al fascculo de Luis Hernndez Nieto La sangre y sus enfermedades en el Quijote cmo el hombre
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ha tomado conciencia por primera vez de la importancia vital de la sangre: En la pared de una gruta en Altamira, un auriacense de los tiempos paleolticos represent un animal, probablemente un mamut que muere de hemorragia, figurando la sangre derramada una gran mancha roja. Afortunadamente la humanidad ha dado pasos de gigante en el progreso cientfico, pasando de la mera observacin ocular a la utilizacin de tecnologas sofisticadas de anlisis de sangre y de lquidos biolgicos en general.
Figura 4. Contador de partculas Coulter Modelo A.

Breve aproximacin histrica a la descripcin celular

En 1764, el comerciante y cientfico holands Antn Von Leeuwenhoek (fig. 1), utilizando un dispositivo de lentes rudimentarias, realiz la primera descripcin precisa de los glbulos rojos. Y no ha sido hasta el siglo XIX cuando se pudieron observar las partculas ms pequeas de la sangre, gracias al esfuerzo de Alfred Donn (1842) y de Georges Hayem (1875) en su intento de contar las plaquetas. Otro paso significativo se dio, cuando el bacterilogo alemn Paul Ehrlich (fig. 2) pudo, gracias a su tincin de anilinas, diferenciar las subpoblaciones leucocitarias en 1879. El mismo cientfico describi en 1881 la tincin supravital. El siguiente adelanto cualitativo ha sido relacionar las observaciones microscpicas con los hallazgos clnicos. Cientficos como Joseph Arneth (1904) y Schilling (1929) fueron los primeros en establecer una relacin de causa-efecto entre la infeccin-inflamacin y la morfologa de los neutrfilos, principalmente el recuento de los cayados. Ms tarde, Maxwell Wintrobe (fig. 3) en 1934, clasific las anemias basndose en el volumen y la concentracin de hemoglobina de los hemates. Finalmente en 1976 el Grupo FAB (FrenchAmerican-British Cooperative Working Group) propuso una clasificacin de las leucemias basada en la morfologa celular. Hoy en da, como es sabido, la Organizacin Mundial de la Salud considera adems la biologa molecular, la citogentica y el inmunofenotipaje como parmetros para la clasificacin de las hemopatas malignas. y relacionar las alteraciones morfolgicas con posibles patologas, era necesario un recuento celular ms fiable que la apreciacin subjetiva del nmero de clulas al microscopio. As es cmo la armada de Estado Unidos (la US Navy) solicit en 1956, a 2 jvenes ingenieros electrnicos americanos, Joseph y Wallace Coulter, el diseo de un contador automtico de clulas. Ello ha representado el nacimiento del primer contador Coulter Modelo A (fig. 4).

Aportacin de los analizadores de ltima generacin

Hoy en da, adems de cuantificar las subpoblaciones leucocitarias, incluidos los eritroblastos, de generar un perfil de reticulocitos exhaustivo, de generar alarmas morfolgicas de sospecha con una sensibilidad y especificidad adecuadas, los analizadores de Beckman Coulter de ltima generacin informan en cada muestra de 36 parmetros de investigacin (PI): 24 para los leucocitos y 12 para los reticulocitos. Los parmetros de investigacin representan las coordenadas tridimensionales de las subpoblaciones leucocitarias y reticulocitarias. Se entiende por parmetros de investigacin, los datos numricos (ergo objetivos) del volumen medio, conductividad media y dispersin de luz lser media de las subpoblaciones leucocitarias y de los reticulocitos. Al mismo tiempo se informa de la desviacin estndar (DE) de cada uno de estos parmetros (figs. 5 a 7). Se puede establecer un smil con los parmetros eritrocitarios: el volumen corpuscular medio de los hemates correspondera al volumen medio de las subpoblaciones leucocitarias, y el ancho de distribucin (grado de anisocitosis) equivaldra a la DE de las mismas subpoblaciones (reflejo de su grado de homogeneidad) (fig. 8). Una revisin de la literatura cientfica permite evidenciar la utilidad de estos parmetros de investigacin en el cribado de varias patologas. Es, en efecto, lgico inferir que las alteraciones morfolgicas que
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Incorporacin de los contadores celulares al laboratorio de hematologa

Muy pronto, el clnico se dio cuenta de que, adems de describir morfolgicamente las clulas sanguneas
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Parmetros de investigacin Figura 5. Parmetros de investigacin de los leucocitos.

Parmetros de investigacin

Figura 6. Parmetros de investigacin de los reticulocitos.

en el estudio de las leucemias linfticas crnicas (LLC), evaluando el inters del volumen medio de los linfocitos y su conductividad en esta enfermedad. Estos resultados han sido corroboradas por el trabajo de Arroyo et al2, por otro estudio multicntrico3,12-14 y por Silva et al 4. La propuesta, a nuestro juicio, ms innovadora proviene del trabajo de Brtolo et al 5 relativo a la evaluacin de la utilidad de estos parmetros en las LLC tpicas y atpicas. En cuanto a la serie mieloide conviene destacar la investigacin de la utilidad de los PI en el screening de pacientes con sepsis. Chaves et al 6 hallaron una sensibilidad y especificidad de los PI mayores que las de la reaccin en cadena de la polimerasa (PCR), el recuento de leucocitos, el de neutrfilos o el de cayados, en este grupo de pacientes. Las otras evaluaciones son relativas a los sndromes mielodisplsicos (SMD) 7,8 y revelan unas perspectivas interesantes por la alteracin caracterstica de los PI, reflejo de la disgranulopoyesis que acompaa este tipo de sndromes. Gallart et al 9 demuestran, en un estudio prospectivo, la correlacin entre el grado de anisocroma en los SMD y la alteracin de los PI de los reticulocitos. Finalmente en el estudio de los PI de la serie monoctica Fourcade et al 10 y Machin et al [en proceso de publicacin] han determinado un discriminante D = (DE Vol. Lin. DE Vol. Mon.)/100 para la deteccin y screening de pacientes con malaria. La sensibilidad y especificidad de este discriminante han resultado ser similares a las del inmunoensayo. Otra va de investigacin prometedora, ha sido iniciada por Wagner et al11, para evaluar la utilidad de los PI en la deteccin del riesgo de inmunoparlisis en pacientes ingresados en la unidad de cuidados intensivos. Estos autores han encontrado una correlacin significativa entre la disminucin de la expresin HLA-DR de los monocitos de estos pacientes, evidenciada por
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Figura 7. Representacin tridimensional de la distribucin de las subpoblaciones leucocitarias.

acontecen en las distintas patologas deberan traducirse por alteraciones morfomtricas de las clulas en el analizador hematolgico. En lo relativo a la serie linfoide, Mora et al 1 han sido pioneros en investigar la utilidad de estos parmetros
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Figura 8. Analoga entre los parmetros de investigacin y algunos ndices eritrocitarios.

citometra de flujo, y las alteraciones del volumen medio y de la DE (ancho de distribucin) de los monocitos analizados por el LH750.

Conclusin y consideraciones finales

Creemos que los parmetros de investigacin ofrecen interesantes perspectivas de investigacin y de estudios prospectivos. No se pretende aportar otra herramienta de diagnstico, ms bien el uso de los PI como una alarma morfomtrica integrada en el sistema de reglas de validacin de resultados del analizador hematolgico. Ello permitir al usuario llevar a cabo un screening sistemtico de las muestras procesadas, sin ningn coste adicional para el laboratorio y con una sensibilidad y especificidad, probablemente superiores a algunos de los mtodos clsicos en uso actualmente en el laboratorio. Tambin aportan una mayor flexibilidad, en la medida en que abren nuevas perspectivas al usuario imaginativo, para la deteccin y screening de las alteraciones morfolgicas escasamente investigadas. Finalmente sera de gran inters por parte de Beckman Coulter tomar en consideracin estos resultados, con el objeto de desarrollar nuevas alarmas de sospecha que ayuden eficientemente al usuario en su agobiante quehacer diario.

Bibliografa
1. Mora A, Gonzlez FA, Alarcn C, Lpez I, Polo M, Villegas A, et al. Estudio del volumen, conductividad y dispersin (VCS) en los linfocitos de la LLC. Haematologica. 1998;83 Supl 2:172 2. Arroyo JL, Simn R, Garca MA, Bentahar A, San Miguel JF. Utilidad de la pantalla de investigacin del instrumento Coulter Gens, en el estudio etiolgico de linfocitosis y en el diagnstico diferencial de sndromes linfoproliferativos crnicos. Haematologica. 2000;85 Supl 3:49. 3. Lima M, Navarro T, Arroyo JL, Piqueras J, Mill F, San Miguel JF, et al. Multicenter study of the Beckman Coulter Gens investigation screen parameters in the lymphocyte population. Blood. 2000;96(Pt 2):11. 4. Silva M, Fourcade C, Fartoukh C, Lenormand B, Vasse M, et al. Lymphocyte volume and conductivity indices of the haematology analyser Coulter GEN.S in lymphoproliferative disorders and viral diseases. Clin Lab Haem. 2006;28:1-8.
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5. Brtolo R, Ferreira L, Ferreira D, Jorge L, Tavares C, Palmeiro A. Parmetros posicionales del Beckman-Coulter LH750 en las leucemias linfocticas crnicas (LLC) tpica y atpica. Haematologica. 2005;90 Supl 2:84. 6. Chaves F, Tierno B, Xu D. Quantitative determination of neutrophil VCS parameters by the Coulter automated haematology analyzer. Am J Clin Pathol. 2005;124:440-4. 7. Orero M, Miguel A, Marco J, Hueso J, Mora C, Carbonell F, et al. Utilidad de la pantalla de investigacin del Coulter Gens en la valoracin de los signos de disgranulopoyesis en sangre perifrica. Haematologica. 2001;86 Supl 2:108. 8. Caldern M, Dez JL. White blood cell research population data in myelodysplastic sndromes. Abstract 201. ISLH meeting in Amsterdam. 2006. 9. Gallart MA, Gmez-Arbons X, Teixid M, Farr J. Estudio comparativo de los ndices eritrocitarios y reticulocitarios en pacientes con sndrome mielodisplsico y un grupo control mediante tecnologa VCS. Haematologica. 2001;86 Supl 2:108. 10. Fourcade C, Casbas MJC, Belaouni H, Gonzlez JJD, Garca PJJ, Pepio MAE. Automated detection of malaria by means of the haematology analyser Coulter GEN.S. Clin Lab Haem. 2004;26:367-72. 11. Wagner T, Weitzner B, Siegmund R. Detection of immunoparalysed patients using Coulter LH 750 differentials reflexed to the flow cytometric HLA-DR analysis for confirmative analysis. Abstract 55. ISLH meeting in Barcelona. 2004. 12. Calle M, Atalaya J, Andrade A, Carvalho C, Bentahar A, Pereira I, et al. Utilidad de los parmetros posicionales del analizador hematolgico Beckman Coulter LH 750 en el estudio etiolgico de las linfocitosis. Haematologica. 2005;90 Supl 2:84. 13. Piccinini C, et al. Efficacy and diagnostic usefulness of the coulter LH series positional parameters. Abstract 40. ISLH meeting in Barcelona. 2004. 14. Vega R, Redn E, Gallego R, Bentahar A. Estudio paramtrico de linfocitos en el equipo Coulter Gens como discriminante de virasis y otras linfocitosis de origen extrahospitalario. Haematologica. 2003;88 Supl 7:68.

THE CLINICAL UTILITY OF THE QUANTIFICATION OF IMMATURE PLATELETS

R.D. HINZMANN
Head of Medical & Scientific Affairs. Sysmex Europe GmbH. Germany.

Summary
Immature platelets are a marker of the thrombopoietic activity of the bone marrow megakaryocytes.
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They can be measured by flow cytometry, however, standardisation of this test has not been very successful so far. On the Sysmex XE-2100 automated haematology system an immature platelet fraction (IPF) can be measured with high reproducibility between instruments. IPF is a useful tool to assess thrombocytopenia: If thrombocytopenia is caused by destruction or consumption of platelets, like e.g. in immune thrombocytopenia or thrombotic thrombocytopenic purpura, IPF is elevated as a compensatory reaction of the bone marrow. In thrombocytopenia due to bone marrow suppression, e.g. after chemotherapy or stem cell transplantation, IPF is low or normal. IPF can also be used to monitor the above mentioned diseases and can potentially help to save platelet transfusions.

Platelets are derived from megakaryocytes

Platelets are small nucleus-free and disc-shaped cells with an equatorial diameter of 2-3 m. The source of platelets are the megakaryocytes 1. These are derived from pluripotent haematopoietic stem cells and almost all of them are found in the bone marrow where they form a fraction of only less than 1 % of all myeloid cells. A megakaroycyte can form thousands of platelets. Mainly under the influence of thrombopoietin megakaryoctes grow to a size of approximately 100 m while concomitantly multiplying their DNA content and developing an expansive network of cisternae and tubules, called the demarcation membrane system (DMS). These membranes serve as a reservoir for the formation of 100-500 m long proplatelets, long tubular protrusions that originate from the megakaryocyte. From the tips of these proplatelets the platelets are released into the bloodstream. The freshly released platelets contain RNA, which most likely is not produced by the platelets themselves but originates from the megakaryocyte. These young platelets have been termed immature platelets or, synonymously, reticulated platelets or high fluorescent platelets. After 1-2 days immature platelets loose most of their RNA while developing into mature platelets, which have a life-span in the circulation of approximately one week. Platelets contain granules, the content of which may be released following stimulation 2.

RNA and in analogy to the red blood cell reticulocytes were termed reticulated platelets. The RNA inside the reticulated platelets can be measured using flow cytometry and a fluorescent dye binding to RNA, like thiazole orange. In the early 90s it was demonstrated by Kienast and Schmitz 4 that in the clinical condition of thrombocytopenia due to increased consumption or destruction of platelets (with normal or elevated numbers of megakaryocytes in the bone marrow) the percentage of platelets stained with thiazole orange was significantly higher than in cases of thrombocytopenia due to impaired platelet production (with reduced bone marrow megakaryocytes). This observation suggested that the measurement of immature platelets could be very helpful to facilitate the differential diagnosis of thrombocytopenia and to monitor thrombocytopenic conditions, e.g. after cytotoxic chemotherapy 5,6. Additional interest arose in the clinical utility of measuring immature platelets with flow cytometry in thrombotic conditions like stroke 7,8 or in acute coronary syndrome 9.

So far, standardisation of the flow cytometric measurement of immature platelets has not been successful
As a consequence of these potential clinical applications, attempts were made to standardise the flow cytometric measurement of immature platelets. In 2002, the International Society for Haematology (ISLH) Reticulated Platelet Taskforce tried to achieve standardisation by using a well-defined consensus protocol on sets of 50 normal samples in 8 different laboratories 10. The immature platelets were identified by staining with thiazole orange and by using the platelet-specific antibodies anti-CD41 and anti-CD61, both labelled with a fluorescent dye. All raw data were analysed at a central location to avoid differences in the gating procedure. However, despite all these efforts, the means of the investigated patient samples varied strongly between the different laboratories. So, unfortunately, there is still no satisfying flow cytometric procedure that would allow to reproducibly measure immature platelets in different laboratories around the world.

Earlier studies rose interest in measuring immature platelets to investigate thrombocytopenic conditions
In the late 60s Ingram and Coppersmith 3 described that beagle dogs, after an induced acute blood loss, produced platelets that were larger in size than normal platelets and showed a coarse pattern of punctiform dots when stained with methylene blue and looked at under the microscope. The cells contained
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Immature platelets can now be measured as a routine test on the Sysmex XE-2100 haematological analyser
On the Sysmex XE-2100 haematological analyser (Sysmex Corporation, Kobe, Japan) platelets can be measured in the optical (fluorescence) reticulocyte/ platelet channel. The RNA in the cells is stained using a proprietary fluorescent dye containing polymethine and oxazine. The cells pass through a laser beam and the forward scattered light and fluorescence of the cells are measured. In the scattergram
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(fig. 1) the fluorescence is plotted on the abscissa while the forward scattered light is plotted on the ordinate. A computer algorithm identifies the cluster of platelets in the scattergram. Upgrading the XE-2100 with SYSMEXs recently released so-called IPF Master software allows to distinguish the immature from the mature platelets by applying a special gating algorithm. This opens the possibility to easily, quickly and reproducibly measure immature platelets as part of the routine blood count, making this test now available 24 hours a day. The test is called IPF (standing for immature platelet fraction) and results are expressed either in percent of the total platelet concentration (IPF %) or as an absolute concentration (IPF#). In this review IPF is generally expressed in the format of IPF %. Currently, the routine measurement of IPF on a haematological analyser is unique to the Sysmex XE-2100. The availability of a commercial routine test for immature platelets has reignited the interest in the clinical use of immature platelets and, until today, three scientific publications on IPF on the XE-2100 have appeared11-13 and a forth one is in press14. Briggs et al 11 have determined the reference interval for immature platelets by measuring IPF in 50 healthy adults. The reference interval was 1.1-6.1 % (mean 3.4 %). These data were confirmed by Kickler et al 12 who derived a reference interval ranging from 0-6 % (mean 3.1 %) from 80 healthy adults. Zucker et al14 investigated 43 healthy adults and found a reference interval of 1.1-7.1 %. These findings suggest that, contrary to flow cytometric determination of immature platelets, IPF measurement on the XE-2100 is indeed a procedure delivering comparable results on different analysers.

Figure 1. Scattergram of the reticulocyte/platelet channel on the Sysmex XE-2100 haematological analyser. The gate indicating the immature platelet fraction (IPF) is shown only schematically. The patient sample depicted has a high IPF.

The use of immature platelets in immune thrombocytopenic purpura

Immune thrombocytopenic purpura (ITP) 15 is a disease characterised by mucocutaneous bleeding and a low platelet count in which certain B cell clones produce antibodies against various platelet surface proteins. The antibody coated platelets are then eliminated from the circulation by macrophages, resulting in thrombocytopenia. The incidence of ITP is about 100 cases per million per annum, and in half of the cases children are affected, most often following a viral infection. In children, the illness usually resolves within a few weeks or months whereas in adults it is often chronic. The most dangerous complication of ITP is intracranial haemorrhage. In patients with ITP thrombopoiesis is increased as a compensatory reaction to thrombocytopenia. Briggs et al 12 found that immature platelets were highly increased in ITP. If the platelet concentration was below 50,000/ L, 100 % of the 12 patients had an elevated IPF, ranging from 9.2-48.3 % (mean 22.3 %). If the platelet concentration was above 50,000/ L, still 73 % of the 22 patients had an elevated IPF, ranging
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from 2.3-52.1 % (mean 16.8 %). The correlation between platelet concentration and IPF was indirect: The lower the platelet concentration the higher was IPF. Kickler et al13, when looking at all of their ITP patients, found IPF values between 3.6-35.4 % (mean 15.0 %). Despite different national guidelines 16,17, the investigation of the bone marrow to diagnose ITP remains controversial. Although it is not generally required by the guidelines, some authors perform bone marrow examination also for certain patients under 60 year of age 15. In these cases the measurement of IPF could demonstrate a proper thrombopoietic function of the bone marrow and avoid some of these invasive procedures. On the contrary, it has been suggested that all patients with suspected ITP without significantly elevated IPF should undergo bone marrow examination11. Given the fact that immature platelets are larger than mature platelets it has been suggested to use the mean platelet volume (MPV) or other platelet indices to identify ITP 18. However, besides the lower sensitivity and specificity of these parameters as compared to IPF, a major obstacle is that in cases of severe thrombocytopenia haematological analysers often fail to derive the respective platelet parameters from the platelet histogram. Therefore, IPF is clearly superior to MPV and other platelet indices.

The use of IPF in thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is a disease characterised by systemic or intrarenal aggregahaematologica/edicin espaola | 2006;91(Supl 1) | 151 |

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tion of platelets, thrombocytopenia, and mechanical injury to erythrocytes 19. The principal pathological mechanisms underlying the disease have been elucidated recently 19,20: In many cases the activity of the enzyme ADAMTS 13 (a disintegrin and metalloprotease with thrombospondin-1-like domains) is reduced below 5 % of its regular activity. The physiological role of ADAMTS 13 is the cleavage of large von Willebrand factor (vWf) multimeres that are synthesised by the endothelium. In TTP the vWf multimeres accumulate, bind to the platelets and the platelet aggregates are clogging up smaller blood vessels and cause a thrombotic microangiopathy, comprising some of the following symptoms: thrombocytopenia, haemolytic anaemia, neurologic abnormalities, renal failure, and fever. This pathological mechanism for TTP can develop as a drug-induced side-effect of ticlopidine or clopidogrel. TTP, usually without ADAMTS 13 deficiency, is seen after administration of immunosuppressive or chemotherapeutic drugs, total body irradiation, or after organ transplantation. Closely related to TTP with regard to the clinical picture, although with focus on the kidneys, is haemolytic uraemic syndrome (HUS), caused by Shiga toxin in certain E. coli infections (strain O157:H7). Although the incidence of TTP is pretty low with 4 cases per million per annum, a quick diagnosis is required since the disease can be fatal within a very short time-frame. The combination of thrombocytopenia, elevated lactate dehydrogenase concentration and the presence of fragmented erythrocytes (fragmentocytes) points towards the diagnosis of TTP. The measurement of IPF is very helpful if TTP is suspected, since a highly elevated IPF is typical of the disease. All 5 patients with untreated TTP had IPF values above the reference interval:11.2-30.9 % (mean 22.3 %)11. The regular therapy for TTP is daily plasma exchange. To avoid an exacerbation of the microvascular thrombosis platelet transfusions must be strictly avoided, except in cases of life-threatening haemorrhage. IPF can be used to monitor the course of the treatment: As long as IPF is high, the platelet concentration will not rise. The requirement for treatment might persist for months. During this period patients usually suffer episodes of low and normal platelet concentrations which are mirrored by normal and high IPF values, respectively. 6 patients with TTP in remission had normal or only slightly elevated IPF values.

The use of IPF in disseminated intravascular coagulation:

In 25 patients with disseminated intravascular coagulation (DIC), as defined by a clinical setting of sepsis and elevated concentrations of D dimers, IPF was normal or elevated, ranging from 2.3-18.6 % (mean 9.5 %)12. Hence, IPF might not be sensitive enough to detect DIC in all cases, however, high values of IPF within the respective clinical setting will support or suggest the diagnosis of DIC.

The use of IPF in patients receiving cytotoxic chemotherapy, peripheral blood stem cell transplantation or bone marrow transplantation
As a consequence of chemotherapy, the platelet concentration falls. As soon as the bone marrow recovers, IPF will rise. In 30 patients, while their platelet concentrations were falling, IPF was low, ranging from 0.8-3.5 % (mean 3.5 %). 13 of these patients were followed serially. In 8 patients with uncomplicated bone marrow recovery the rise in IPF preceded the rise of the platelet concentration by 1-2 days. At this point IPF ranged from 7.3-18.4 % (mean 12.1 %). For those 5 patients suffering from infections, receiving antibiotic treatment or frequent platelet transfusions, no consistent trend of IPF could be observed13. In another study 79 patients after cytotoxic chemotherapy had IPF values ranging from 1.2-10.9 (mean 4.1)12. In 15 patients receiving autologous and allogeneic peripheral blood stem cell transplantation the rise in IPF occurred after 5-11 days (mean 8 days) and preceded the rise of the platelet concentration by 1-3 days whereas in bone marrow transplantation the bone marrow took considerably longer to recover and IPF rose after 9-27 days (mean 15 days), preceding the rise of the platelet concentration by 2-7 days (mean 4.5 days)13.

IPF could potentially save platelet transfusions

High cost, limited availability of donors, potential formation of anti-platelet antibodies and a small risk of infection are the main issues associated with platelet transfusions. In addition, platelet transfusion temporarily suppresses thrombopoietin production and, thereby, thrombopoiesis 13,21. Although in many countries guidelines suggest platelet transfusions when the platelet concentration falls below 20,000/ l or 10,000/ l, the necessity of this automatism has been challenged 22,23 and it has been argued that many prophylactic platelet transfusions are clinically unnecessary 24. A rise of IPF proves that the bone marrow has recovered again and that a rise in platelets is expected within a short time. Therefore, it is suggested that in patients after peripheral blood stem cell transplanta00

IPF values in late pregnancy

In general, IPF in the third trimester of pregnancy is higher than in non-pregnant women. On average, 6 patients with pregnancy induced hypertension (PIH) demonstrated higher values (7.2-14.6 %, mean 10.8 %) than 6 patients with normal pregnancy (3.29.7 %, mean 6 %) 11. Although the number of cases investigated is too small to allow further conclusions, the overlap between the two groups is noticeable.
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tion when IPF is already elevated and they are neither septic nor bleeding, platelet transfusions should be deferred even at platelet concentrations below 10,000/ L13.

References
1. Patel S, Hartwig J, Italiano J. The biogenesis of platelets from megakaryocyte proplatelets. J Clin Invest. 2005;115:3348-54. 2. McNiol A, Israels S. Platelets and anti-platelet therapy. J Pharmacol Sci. 2003;93:381-96. 3. Ingram M, Coppersmith A. Reticulated platelets following acute blood loss. Br J Haematol. 1969;17:225-9. 4. Kienast J, Schmitz G. Flow cytometric analysis of thiazole orange uptake by platelets: a diagnostic aid in the evaluation of thrombocytopenic disorders. Blood. 1990;75:116-25. 5. Macchi I, Chamlian V, Sadoun A, et al. Comparison of reticulated platelet count and mean platelet volume determination in the evaluation of bone marrow recovery after aplastic chemotherapy. Eur J Haematol. 2002;69:152-7. 6. Wang C, Smith B, Ault K, Rinder H. Reticulated platelets predict platelet count recovery following chemotherapy. Transfusion. 2002;42:368-74. 7. Nakamura T, Uchiyama S, Yamazaki M, Okubo K, Takakuwa Y, Iwata M. Flow cytometric analysis of reticulated platelets in patients with ischemic stroke. Thrombosis Research. 2001; 106:171-7. 8. McCabe D, Harrison P, Sidhu P, Brown M, Machin S. Circulating reticulated platelets in the early and late phases after ischaemic stroke and transient ischaemic attack. Br J Haematol. 2004;126:861-9. 9. Lakkis N, Dokainish H, Abuzahra M, et al. Reticulated platelets in acute coronary syndrome: a marker of platelet activity. J Am Acad Sci. 2004;10:2091-3. 10. Harrison P. Reticulated Platelet Task force report. Lab Haematol. 2003;9:91-2. 11. Briggs C, Kunka S, Hart D, Oguni S, Machin S. Assessment of an immature platelet fraction (IPF) in peripheral thrombocytopenia. Br J Haematol. 2004;126:93-9.

12. Kickler T, Oguni S, Borowitz M. A clinical evaluation of high fluorescent platelet fraction percentage in thrombocytopenia. Am J Clin Pathol. 2006;125:282-7. 13. Briggs C, Hart D, Kunka S, Oguni S, Machin S. Immature platelet fraction measurement: a future guide to platelet transfusion requirement after haematopoietic stem cell transplantation. Transfusion Medicine. 2006;16:101-9. 14. Zucker M, Murphy C, Rachel J, et al. Immature platelet fraction as a predictor of platelet recovery following hematopoietic progenitor cell transplantation. Laboratory Haematology (in press). 15. Cines D, Blanchette V. Immune thrombocytopenic purpura. N Engl J Med. 2002;13:995-1008. 16. British Committee for Standards in Haematology: Guidelines for investigation and management of idiopathic thrombocytopenia purpura in adults, children and pregnancy. Br J Haematol. 2003;120:520-96. 17. George J, Woolf S, Raskob G, et al. Idiopathic thrombocytopenic purpura: a practice guideline developed by explicit methods for the American Society of Hematology. Blood. 1996; 88:3-40. 18. Kaito K, Otsubo H, Usui N, et al. Platelet size deviation width, platelet large cell ratio, and mean platelet volume have sufficient sensitivity and specificity in the diagnosis of immune thrombocytopenia. Br J Haemaol. 2005;128:698-702. 19. Moake J. Thrombotic microangiopathies. N Engl J Med. 2002; 347:589-600. 20. Levy G, Motto D, Ginsburg D. ADAMTS13 turns 3. Blood. 2005;106:11-7. 21. Kuter D, Rosenberg R. The reciprocal relationship of thrombopoietin (c-Mpl ligand) to changes in the platelet mass during busulfan-induced thrombocytopenia in the rabbit. Blood. 1995;85:2720-30. 22. Gmr J, Burger J, Schanz U, Fehr J, Schaffner A. Safety of stringent prophylactic platelet transfusion policy for patients with acute leukaemia. Lancet. 1991;338:1223-6. 23. Wandt H, Frank M, Ehninger G, Gallmeier W. New strategies for prophylactic platelet transfusion in patients with hematologic diseases. Oncologist. 2001;6:446-50. 24. Roy A, Jaffe N, Djerassi I. Prophylactic platelet transfusions in patients with acute leukaemia. Transfusion. 1973;13:283-90.

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