INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY

Falsos conteos y falsos resultados en analizadores de

hematologa : una revisin . Parte II: clulas blancas, clulas rojas ,
hemoglobina, indice clulas rojas y reticulocitos
M. ZANDECKI, F. GENEVIEVE, J. GERARD, A. GODON

Haematology Laboratory,University
Hospital of Angers, Angers, France
Correspondence:
M. Zandecki, Haematology Laboratory, University Hospital of Angers,
4, rue Larrey, 49000 Angers,France.
Tel.: +33 241 35 53 53; Fax:
+33 2 41 35 55 99; E-mail:
mazandecki@chu-angers.fr
doi:10.1111/j.1365-2257.2006.00871.x

Received 30 January 2006; accepted

for publication 30 July 2006
Keywords
Haematology analysers, automated
count, cell blood count, spurious
count, white blood cells, haemoglobin, red blood cells, mean cell
volume

RESUMEN
Los analizadores en hematologa proporcionan resultados rpidos y
precisos en la mayora de las situaciones .S i n e m b ar go , falsos
resultados , relacionados ya sea con las plaquetas (parte I de este
informe ) u otros parmetros en el conteo de las clulas sanguneas
(CBC)se pueden observar en varios casos . Glbulos blancos falsamente
bajos se pueden observar debido a la aglutinacin por la presencia de
cido etilendiaminotetractico (EDTA). Crioglobulinas,lipidos, globulos
rojos lo insuficientemente lisados (RBC), eritroblastos y agregacin
plaquetaria s o n s i t u a c i o n e s c o m u n e s q u e i n c r e m e n t a n e l
r e c u e n t o d e WBC. En la mayora de estos casos un marcaje y/o un
anormal WBC diferencial grfico alertara al operador. Muchas
situaciones nos llevaran a una medicin anormal de hemoglobin o
conteo de RBC , incluyendo lipidos, aglutininas, crioglobulinas y
elevados conteo de WBC . El volumen cellular medio (MCV) puede
tambin estar sujeta a una determinacin falsa, a causa de aglutininas, el
exceso de glucosa o sales y consideraciones tecnolgicas. A su vez, la
anormalidad relacionada con un parmetro medido dar lugar a ndices
RBC calculados anormales: celular hemo- globina contenidos (MCHC)
es, sin duda los ndices de glbulos rojos ms importantes a considerar
significante, ya que es tan importante como marcas generados por los
analizadores hematolgicos (HA) para alertar al usuario a un resultado
falso. En muchas circunstancias, varios de los parmetros medidos de
CBC pueden ser alterados, y el descubrimiento de un cambio falso en
un parmetro significa con frecuencia que la validez de otros
parmetros debe ser considerado. Marcas sensibles ahora permitir la
identificacin de varios conteos falsos, pero slo los ms sofisticados HA
tienen marcaje ptimo y ms sencilla , especialmente los que no tienen
un diagrama de dispersin diferencial WBC, no poseen la misma
sensibilidad para la deteccin de resultados anmalos. Los reticulocitos se
integran ahora en el CBC en muchas HA, y varias situaciones pueden dar
lugar a recuentos anormales.

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21

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SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS

INTRODUCTION
Muchos analizadores de hematologa (HA) que
enumera WBC generar un diagrama de
dispersin diferencial de WBC, que es la base
para el diferencial WBC automatizado. Tales
diagramas de dispersin de WBC tambin son
fundamentales para la generacin de
indicadores o alarmas, detectando que el
recuento de glbulos blancos (y el diferencial
WBC) es posiblemente errnea y dar alguna
informacin sobre el posible origen de la
anomala. Como se mencion en la primera
parte de este informe, el diagrama de
dispersin diferencial de WBC tambin es de
impor- tancia crucial identificar anomalas del
conteo de plaquetas (PLT) , PLT siendo los
grumos el ejemplo ms obvio. El conteo de
glbulos rojos (RBC) , hemoglobina (Hb) y
(rojo) el volumen cellular medio (MCV) son
otros parmetros medidos por HA, que
tambin estn sujetas a los valores falsos en
varias situaciones Los indices de clulas rojas
(Wintrobe) aparte de MCV y del hematocrito
(hematocrito; Hct) se calculan por lo general
a partir de estos parmetros medidos, y en la
mayora de circunstancias con estos
parametros erroneos medidos directamente
RBC generarn a su vez indices erroneos
RBC calculados. Estos ndices pueden ser
utilizados como un indicador para los
recuentos anormales. Los reticulocitos se
pueden considerar ahora como una parte de
la CBC, y muchos HA han integrado los
recuentos de reticulocitos en virtud de un
mtodo totalmente automatizado.
CLULAS SANGUNEAS BLANCAS
Para el anlisis de clulas sanguneas, cada
partcula de tamao grande (mayor que el
tamao de un PLT) que no se destruye por
agentes hemolticos ser identificado como
un de WBC en la mayora de HA. Despus de
enumeracin, y de acuerdo con el tipo, la
impedancia con baja y alta frecuencia
electromagntica o corriente directa, de
dispersin de luz lser (en uno o en varios
ngulos), o intensidad de la tincin de
peroxidasa que son usada, ya sea
individualmente o en conjunto, para generar

un cinco -, seis o incluso siete partes del

diferencial (para una revisin, ver Bain &
Bates, 2001). No est en el alcance de este
informe para estudiar cmo se clasifican los
distintos de WBC pero se debe tener en
cuenta que los diagramas de dispersin
generado por el HA para mostrar el diferencial
de WBC debe estar completamente entendido
por los operadores (Bain y Bates, 2001). En
muchos casos, los diagramas de dispersin de
WBC permiten la deteccin de anomalas
relacionadas con recuentos falsos y / o ayuda
para explicarlos. Como regla general, los
instrumentos que no generan diagramas de
dispersin de WBC diferenciales sern ms
propensos a pasar por alto varios puntos
ficticios.
Recuento falsamente bajos WBC
Agregados de polimorfonucleares neutrfilos
Neutrfilos polimorfonucleares (PMN)
agregados pueden ser observados en muestras
de sangre extradas en EDTA. La incidencia es
baja, pero ciertamente subestimado,
correspondiente a 2/65 000 recuentos
completos de sangre en EE.UU. (Epstein y
Kruskal, 1988), 1/9500 en Italia (Bizzaro, 1993),
1/7500 en Francia (Lessive et al., 2000 ).
Muestras anomalas tanto en hombre como
mujeres. Aunque ninguna patologa o ninguna
enfermedad especfica se asocia con la
agrupacin de los PMN, enfermedades
hepaticas en un contexto inflamatoria aguda o
crnica o circunstancias asociadas con la
generacin de aglutininas fras han sido
reportados en muchos casos. El fenmeno
puede o no ser una disminucin transitoria
.Puede ser moderada o clnicamente
significativa, lo que conduce a veces a sospechar
agranulocitosis y para no generar
investigaciones innecesarias (aspiracin de
mdula sea o biopsia) o terapia. No hay
relacin entre el fenmeno descrito aqu, que es
una anomala pura in vitro relacionada con el
muestreo con anticoagulante EDTA , y la
anomala in vivo que ocurre en enfermedades
tales como el sndrome de dificultad respiratoria
del adulto o leucostasis, en el que PMN tienden
a agregarse, como consecuencia de las
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M. ZANDECKI ET AL.

interacciones de membrana con complemento.

El mecanismo que lleva a la aglutinacin no se
aclara totalmente. Siempre es un fenmeno in
vitro, principalmente dependiente de EDTA,
aunque la aglutinacin despus del uso de
citrato de sodio o heparina como
anticoagulantes Tambin se ha informado en
algunos casos. Usando experimentos de
transferencia, ya sea plasma de los pacientes
afectados en contacto con WBC de pacientes
normales o plasma normal en contacto con
PMN de los pacientes afectados, la anomala
ha demostrado estar relacionada con un
componente de plasma, y la incubacin de
plasma con un anti-IgM anticuerpo o con
ditiotreitol suprimido disminuyeron
cuantitativamente la agregacin de PMN. El
uso de citometra de flujo, ha encontrado IgM
en la superficie PMN en un paciente. Algunos
autores han informado de que el tamao de los
agregados fue mayor a baja temperatura y que
desapareci a 37 C. Varios otros autores
informaron que la aglutinacin de glbulos
blancos en sangre anticoagulada con EDTA no
se corrigi por el calentamiento de la muestra
de sangre , al menos en algunos casos, lo que
minimiza la implicacin de un aglutinina fra en
la gnesis de los clusters. Se propuso un alto
nivel de expresin de la integrina (CD18
CD11b-) en la membrana PMN estar
relacionado con la generacin de grumos PMN.
Diagramas de dispersin diferencial de globules
blancos (utilizando el tamao y la complejidad
o el tamao y peroxidasa contenidos) puede
demostrar anomalas, pero hay que tener en
cuenta que los agregados ms grandes se
pasan por alto, y slo el ms pequeo activarn
indicadoresEl tipo de impedancia de HA, la
anomala se sospecha cuando los
acontecimientos estn presentes por encima
de la zona de los PMN (parte superior del
grfico de WBC), o cuando es difcil separar las
clases de WBC (Galifi et al., 1993), y los
indicadores ms frecuentes generado
utilizando tales HA son 'granulocitos inmaduros
"o" clulas de la banda "(Lippi et al., 1994). El
Bayer-Technicon HA, los cmulos ms
pequeos pueden aparecer como una banda
de puntos en la parte superior derecha del
diagrama de dispersin diferencial de WBC:
como estos agregados contienen muchos PMN
que se presentan como partculas ricas en

SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS 23

peroxidasa, y se genera una alarma

correspondiente a elevado contenido
peroxidasa . .En Algunas HA, el anlisis de los
ncleos de WBC se realiza en un canal dedicado
despus de la utilizacin de una solucin
drstica para asegurar la lisis de las membranas
de WBC: nmero de ncleos libres se utiliza
como un control para el recuento de WBC.
Discrepancia entre el recuento obtenido de
WBC de ese canal y que se observa desde el
canal de fluorescencia o peroxidasa .

Agregados de clulas blancas de la sangre se

destruyen despus de la lisis de las membranas
drstica de WBC (Galifi et al., 1993). Pelculas de
sangre pueden mostrar pequeos (hasta cinco
clulas), moderada (hasta 50 clulas) o grandes (>
100 clulas) racimos de PMN (Figura 1). Examen
morfolgico cuidadosa de estos agregados
muestra que unos pocos linfocitos o monocitos
pueden ser a veces atrapadas dentro de los
agregados (Hillyer, Knopf y Berkman, 1990).
Granulocitos inmaduros (mielocitos,
metamielocitos) y clulas de la banda no se
reportan con frecuencia como parte de las clulas
agrupadas, y se propuso que los agregados
pueden desarrollarse alrededor de mielocitos,
mientras PMN solo fall a agruparse (Deol,
Hernndez y Pierre, 1995). Agregados de PMN
estn desprovistos de PLT, en contraste con los
agregados de PLT-PMN (discutidos en la parte I).
Eventualmente, como la agregacin de PMN en
presencia de EDTA conduce a una reduccin en su
nmero, HA puede generar indicadores
correspondientes a los diferenciales falsamente
anormales de WBC, por ejemplo, falsa
agranulocitosis o linfocitosis . Se propusieron
anticoagulantes caseros para superar la
aglutinacin (Schinella, Kodikara y Curci,
1995). Como se mencion anteriormente, el
calentamiento de la muestra a 37 C puede
reducir tanto el tamao y el nmero de
grupos en algunos casos, pero completa
desaparicin est lejos de ser un hallazgo
consistente, y que el mtodo no se puede
proponer para superar la anomala. Pinchazo
en el dedo y la dilucin inmediata de la
muestra de sangre impide la aglutinacin.

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24 M. ZANDECKI ET AL.

SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS

La agregacin de WBC distinta de polimorfos en

presencia de EDTA
Las agrupaciones de linfocitos normales (no
malignas) fueron reportados en un paciente con
infeccin del tracto urinario, en un paciente con
un linfoma de clulas B sin la participacin
torrente sanguneo, en un paciente con ulceras
por presin, y en otro con leucemia
mielomonoctica crnica . Tambin se inform
agregacin de linfocitos que ocurre en pacientes
con leucemia linfoctica crnica, de manera
espontnea cuando el recuento de linfocitos son
extremadamente altos (> 400 109 / l;
O'Flaherty, Kreutzer & Ward, 1978), o no (Bizzaro
y Piazza, 1991). Se observaron agregados de tres
a 50 clulas de linfoma en dos casos de linfoma
esplnico con linfocitos velloso (slvl; Juneja et al.,
1992; Imbing et al., 1995) y en un caso de linfoma
no Hodgkin simulando slvl (Shelton & Frank ,
2000). En estas situaciones, los agregados ms
grandes se pasan por alto por la HA y recuento
de glbulos blancos es falsamente, pero de forma
variable baja. Los agregados ms pequeos
pueden perturbar diferencial WBC y pueden o no
generar un indicador, dependiendo del tipo de
HA utilizado (Shelton & Frank, 2000). En todos
los casos reportados hasta el momento EDTA fue
implicado, aunque tambin se observaron
pequeos grupos de clulas en muestras
extradas heparinizados como controles (Deol,
Hernndez y Pierre, 1996) y citrato de sodio no
cambi sensiblemente la tendencia de
agrupamiento de los linfocitos en algn caso
(Shelton & Frank, 2000). Pinchazo en el dedo y la
dilucin inmediata de sangre parece la mejor
manera de evitar los agregados (Lessive et al.,
2001). El calentamiento a (37 C) se informo para
reducir la formacin de grumos o bien en parte
(Shelton & Frank, 2000) pero no tuvo ningn
efecto significativo sobre el tamao de grumos
(Lessive et al., 2001). A medida que el nmero de
casos reportados es bajo, slo hiptesis sobre el
mecanismo (s) que lleva a la aglutinacin de
linfocitos han sido propuestos, que implican a
varias molculas como la adrenalina (epinefrina),
cido araquidnico, o leucotrieno B4 (Villa et al,
1984;. Shelton & Frank, 2000En un caso se
inform agregados que implican a todas las

clases de leucocitos, en un paciente con

antecedentes de larga data de abuso de alcohol y
la cirrosis alcohlica (Savage, 1989).
Naturaleza y cantidad del anticoagulante
Una disminucin en el recuento de leucocitos no
relacionado con la aglutinacin se inform en situaciones
correspondientes a las muestras que contienen exceso de
K3-EDTA (pero no K2-EDTA) anticoagulante, como
resultado de una muestra insuficiente de sangre despus
de la puncin venosa (Goossens, Van Duppen y
Verwilghen, 1991).

Recuentos falsamente altos de leucocitos

Agregados de plaquetas y grandes plaquetas
Pseudo leucocitosis puede ser secundaria a grupos
PLT lo suficientemente grandes como para simular
tamao de leucocitos. Todo HA moderna que
analizar las subpoblaciones de leucocitos detectar
esta anomala: grumos PLT se localizan como un
rea en forma de cohete de puntos en la esquina
inferior izquierda del diagrama de dispersin
diferencial WBC, y un indicador correspondiente a
la incapacidad para discriminar entre las
categoras WBC, a saber, los linfocitos, se genera
(vase la Figura 2, parte I). Varios autores han
informado de que HA sin WBC diagramas de
dispersin diferenciales son incapaces de detectar
esta anomala . Algunos PLT muy grande puede
encontrarse en trastornos mieloproliferativo y
mielodisplsicos, cuyo tamao y volumen puede
llegar a las de WBC y que pueden ser
enumerados como WBC: indicadores se generan
habitualmente, que mencionan la presencia de
partculas patolgicas que no puede ser
clasificado como WBC (normalmente 'PLT
gigante', o 'agregados' PLT, dependiendo de la
HA).
Glbulos rojos nucleados (NRBC)
Ellos se pueden encontrar en la corriente
sangunea en condiciones fisiolgicas (recin
nacidos) y en circunstancias patolgicas, y en
ocasiones pueden ser mucho ms numerosos
que WBC. Uso de HA, estos NRBC estn en
contacto con agentes de lisis que destruyen su
membrana, dejando libre de ncleos, siendo
este ltimo responsable de las anomalas.
Ncleos NRBC libres son generalmente
partculas <40-50 fl, un tamao que es
superponible a la de grumos PLT o gran PLT.
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M. ZANDECKI ET AL.

Como era de esperar, RBC y PLT grumos

perturban los recuentos WBC idnticos y
ambos artefactos generan igualmente los
indicadores correspondientes (Figura 2). De
acuerdo con el HA, NRBC son incluido en el
recuento de glbulos blancos (los instrumentos
que no generan ningn diagrama de dispersin

SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS 25

diferencial WBC, pero tambin algunos que lo

hacen), o no enumera como WBC, o cuentan por
separado. En varias HA reciente y sofisticado,
NRBC son especficamente identificados y se
puede enumerar utilizando la tecnologa de
fluorescencia

Los glbulos rojos resistentes a la lisis

Glbulos rojos no puede ser destruido por los reactivos de lisis en diversas circunstancias, ya sean
fisiolgicas (neonatos) o patolgicas (hemoglobinas anormales, enfermedad heptica, uremia,
quimioterapia): se consideran como WBC y falsamente aumentan el recuento de glbulos blancos.
Blanco diagramas de dispersin diferenciales de glbulos demuestran puntos de tamao pequeo
ubicado cerca o dentro de la ventana de linfocitos, y la incapacidad para llevar a cabo un diferencial
de WBC conduce a la generacin de un indicador. La imagen es ms o menos superponibles a la
observada con la presencia de NRBC. Este fenmeno parece ser un hallazgo consistente en la
hemoglobina C - que contiene glbulos rojos (ya sea CC, Cbthal, o CS) pero no en pacientes
talasmicos b (ya sea del rasgo o principales) o en la enfermedad de la hemoglobina S (Booth y
Mead, 1983). HA sin WBC diagramas de dispersin diferenciales pueden no detectar la anomala, y
en pacientes sometidos a quimioterapia puede ser de vital importancia, ya que conduce a los
recuentos WBC normales o incluso elevados en los pacientes leucopnicos: la comparacin con los
resultados anteriores (verificacin delta) y examen de frotis de sangre son necesarios en estos
casos. Algunos HA generar un mensaje de resistentes a la lisis RBC y dar acceso a un modo de lisis
prolongada que alarga el contacto de las clulas de la sangre con el reactivo de lisis (Abbott Cell Dyn
4000). En HA usando un canal dedicado para enumerar los ncleos WBC, discrepancia entre el
recuento de WBC total y el recuento de ncleos de WBC es evidente: el canal analizar ncleos WBC
usa agentes de lisis suficientemente fuertes como para destruir todas las membranas (WBC y RBC)
y da el recuento de WBC preciso.
Crioglobulinas
La presencia de crioglobulinas se inform por primera vez como causa WBC cuenta erradas, pero
tambin conduce en muchos casos a los elevados recuentos falsamente PLT, y en ocasiones a los
recuentos de glbulos rojos alterados o Hb mediciones (cf., ver Fohlen-Walter et al., 2002). Las
anomalas que se producen en HA ni son consistente ni en relacin con la cantidad de cryoglobulin
o su naturaleza (cada tipo puede estar involucrado en los recuentos falsos). Sin embargo, las
anomalas generadas deben ser considerados, ya que tienen algunas caractersticas peculiares, y su
reconocimiento puede ser la primera pista que conduce al diagnstico de crioglobulinemia (Zandecki et
al., 1989;. Foh- len-Walter et al, 2002). Crioprecipitados se pueden observar en algunos casos en frotis
de sangre teidos (Figura 3), y en casi todos los casos despus de un examen de la muestra de sangre
fresca utilizando un microscopio de contraste de fase (ms evidente si se realiza a baja temperatura). Se
reportaron diversos aspectos morfolgicos, incluyendo grumos densos amorfos o partculas de escamas,
cristales en forma de aguja, y glbulos rosceas. Como crioglobulinas son inmunoglobulinas que se
precipitan a temperatura inferior a 37 C, los recuentos anormales se observan principalmente en HA
que usan ambos reactivos y realizar anlisis a temperatura ambiente. Sin embargo, HA que utilizan
reactivos precalentados, incluso con reactivos de bajo pH que normalmente evita la precipitacin de
crioglobulinas, no estn libres de anomalas y tambin se observ recuentos falsos. De acuerdo a su
tamao, crioprecipitados slo podrn afectar a los recuentos de PLT, llevando a recuentos falsamente
elevados , hasta ocho veces: histogramas de volmenes PLT muestran gran nmero de partculas de
pequeo tamao, y se genera una alarma general (vase la Figura 8, parte I). Si las partculas
crioglobulinas se agrupan juntos o si su tamao es ms grande, llegando a la de los leucocitos,
recuento de leucocitos se aumenta falsamente: una nube de puntos pequeos se puede
observar en el diagrama de dispersin diferencial de WBC, cerca de los linfocitos o por encima
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26 M. ZANDECKI ET AL.

SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS

del umbral para el PLT, o en el la parte superior de la nube de PMN, dependiendo de la HA

utilizado (Fohlen-Walter et al., 2002). Cuando las muestras son calentaron a 37 C durante al
menos 30 minutos y se volvieron a analizar rpidamente las anomalas desaparecen, pero no es
un hallazgo totalmente consistente y no pocas veces una nueva muestra se deben tomar y se
mantuvieron a 37 C hasta su anlisis para obtener los recuentos WBC precisos (Zandecki et al.,
1989; Infanti et al., 1998; Figura 4). Determinacin de Hb y los recuentos de glbulos rojos a
veces pueden ser alteradas en presencia de crioglobulinas (vase ms adelante). En algunas
situaciones crioglobulinas pueden formar un gel, lo que lleva a la aspiracin inexacta, y una
alarma se puede generar en algunas HA (muestreo insuficiente, o relacionado).

Criofibringeno o relacionados
Las partculas relacionadas con cryofibrinogen o en fibrina se han reportado de elevar el recuento
de leucocitos y en el recuento veces PLT y, segn los informes, PLT y el recuento de leucocitos se
pueden encontrar hasta dos veces y hasta 16 veces el valor exacto, respectivamente (Giuliani, Hyun
y Gabaldn, 1977; Griswold & Champagne, 1985;. Corberand et al, 1991). Un examen cuidadoso de
los frotis de sangre correspondientes muestra fibrillas delgadas, relacionados con hebras de fibrina
tras estudio microscpico de electrones (Figura 5). Estas fibrillas pueden corresponden ya sea a
cryofibrinogen, aunque la investigacin bioqumica puede ser negativa en algunos casos (Griswold &
Champagne, 1985), o una mezcla de crioglobulinas y cryofibrinogen (Giuliani, Hyun y Gabaldn, 1977).
Sin embargo, el anlisis de nuevas muestras de sangre EDTA de los pacientes con frecuencia fallan para
reproducir el fenmeno, se plantearon como hiptesis que tales partculas podran ser el resultado de la
polimerizacin de fibrina despus de una puncin venosa difcil, iniciando la coagulacin in vitro antes
del contacto de la sangre con EDTA (Corberand et al. , 1991). Despus de calentar las muestras a 37 C
conteos anormales o son menos pronunciados o completamente desaparecen.
Lipidos
Como se expuso anteriormente (parte 1), lpidos pueden generar gotas lo suficientemente grandes como para molestar a los recuentos
de PLT, pero tambin el recuento WBC, junto con o independientemente de recuento PLT. Anormales diagramas de dispersin
diferenciales WBC se generan, se presentan diversos cambios, a veces ms o menos superponibles fuera de la presencia de
crioglobulinas (Figura 6). Comentarios sobre los lpidos inquietantes determinacin de Hb se discutirn ms adelante.

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SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS 27

Microorganismos

Recogida de sangre sobrecargados

Las bacterias u hongos pueden ser observados en frotis de

sangre perifrica y, como se indic anteriormente (parte I),
puede dar lugar a recuentos PLT falsamente elevados.
Tambin se informaron como los diferenciales de WBC
perturbadoras si se agruparon, y en estudios in vitro
aadiendo gran nmero de tales microorganismos, se
mostr a aumentar el nmero de WBC (Marshall et al.,
1990) .Hemozoin contenida en PMN y monocitos en los
casos de malaria puede causar despolarizacin de la luz
lser y, posteriormente, la clasificacin errnea de los
tipos WBC en algunas HA, aunque recuento leucocitario
total no cambia claramente.

Como se mencion anteriormente, junto con los

recuentos PLT falsos, llenar demasiado el tubo de
recogida lleva a insuficiente mezcla de muestra y
ajuste del contenido celular: todos los parmetros son
alterados.

Tejido adiposo
Recuento total WBC se inform como artificialmente
elevada, causada por la contaminacin con el tejido
adiposo subcutneo de la muestra de sangre obtenida
por puncin de la vena femoral traumtica: grficos de
dispersin peculiares tambin demostraron un
diferencial de WBC anormal.

Observaciones finales sobre el recuento de

leucocitos falsos
En cuanto a los recuentos de PLT falsos, no siempre es
posible encontrar o identificar el fenmeno que conduce
a recuentos de WBC falsos, incluso si algo es visible en
frotis de sangre perifrica (Savage, 1989). El diagrama
de dispersin WBC de HA es til y debe interpretarse
con cuidado; examen de pelculas de color tambin debe
ser considerado.

HEMOGLOBINA, RBC RECUENTO Y

PARMETROS RBC
HA medir varios parmetros de RBC,
incluyendo conteo de glbulos rojos y MCV
determinado tanto en el mismo canal (s), y la
concentracin de Hb que se mide en otro
canal (s). Hematocrito (Hto), Hb media celular
(MCH), y la concentracin media de Hb
corpuscular (MCHC) se calculan por lo general
a partir de los parmetros medidos. Las
circunstancias que conducen a los parmetros
medidos RBC erradas generaran posteriormente
ndices RBC calculados anormales. As, una
anomala de al menos uno de los ndices de RBC,
al lado de su importancia fisiopatolgica, debe
tambin ser considerado como un indicador
para la deteccin de un error de al menos uno
entre los parmetros medidos de glbulos rojos
(RBC, MCV, Hb). Adems de los ndices
Wintrobe RBC clsicos, otros parmetros
derivados de la tecnologa se han introducido,
que difieren segn el HA, pero su uso tambin
puede ser til para la deteccin de conteos
anormales o mediciones. Un ejemplo es la
evaluacin de la concentracin de Hb de RBC
individuo, tambin llamado hemoglobina celular
concentracin media (CHCM), que
posteriormente se compara con el MCHC
calculado, la generacin de un indicador para
datos discordantes (Abbott, Bayer).

Haemoglobin
Hb concentration is measured using a colorimetric
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28 M. ZANDECKI ET AL.

SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS

(spectrophotometric) method, either by a modification

of the manual cyanmethaemoglobin method or by
addition of various reagents, such as sodium lauryl
sulphate or imidazole. According to the manufacturers, various nonionic detergents are included to insure
rapid RBC lysis and to reduce turbidity because of cell
membranes and plasma lipids.

Lipids and hyperchylomicronemia

Lipemia may cause erroneous PLT (see part I) andWBC
counts (see above), but may also induce inter- ferences by
turbidity (Creer & Ladenson, 1983; Sand- berg, Sonstabo
& Christensen, 1989). An abnormally high MCHC (>36
g/dl) corresponding to an errone- ously high Hb has
been reported for patients with severe constitutional or
acquired hypertryglyceridemia (Gagne et al., 1977; Mayan
et al., 1996), and for patients receiving intravenous
administration of fat emulsions (Nosanchuck, Roark &
Wanser, 1974; Shah, Patel & Rao, 1975; Nicholls,
1975, 1977;
Creer & Ladenson, 1983; Artiss & Zak,
1987; Sandberg, Sonstabo
&
Christensen,
1989;
Cantero, Conejo &

Jimenez, 1996). After the study of various types of

hyperlipoproteinemias, it was observed that erroneous
Hb and MCHC >36 g/dl were observed in patients
with at least 20 g/l of triglycerids, corresponding to
type I and part of type V hyperlipoproteinemias (rich
in chylomicrons) but not to type IV hyperlipoproteinemias (rich in very low density lipoproteins; Gagne
et al., 1977). Samples taken after a meal may at times
demonstrate superimposable spurious Hb measurement. Even the most recent HA are sensitive to
hyperlipemia, although to a variable extent, as for
example Abbott Cell Dyn 4000 that is defined as giving true Hb values for up to 13 and 9 g/l of triglycerid
and cholesterol levels, respectively (M.C. Chrieten,
personal communication). Although excess of lipids
usually spuriously increases Hb, a spurious fall of Hb
was also reported once (Savage, 1989). Similar to the
presence of cold agglutinins, anomaly related to lipids
is suspected when MCHC is >36 g/dl, or when WBC
scattergrams demonstrate high number of particles of
low to moderate size (Figure 6). For laser-beam HA
that measure Hb level within each RBC and generate
the so-called measured MCHC or CHCM, a difference between the CHCM and the calculated MCHC is
observed. Various methods have been proposed to
obviate the abnormality on the relevant sample,
including isovolumetrical replacement of hyperlipemic
plasma with isoosmotic diluent, or ether extraction of
lipids. As previously mentioned, such methods may in
turn lead to erroneous PLT and WBC counts.

High WBC counts

White blood cell may induce excessive turbidity and
disturb Hb measurement if their number is sufficiently
high. There is no clear-cut threshold for WBC counts
associated with spuriously elevated Hb, but one must
be careful with all samples with WBC count over 50
or 100 109/l (Cornbleet, 1983; Sandberg, Sonstabo
& Christensen, 1989), although some HA define
threshold at 250 109/l or even seem to be fully
insensitive to WBC count because of entire WBC lysis
performed before Hb measurement (Sysmex). As for
lipid disturbance, CHCM determined on some HA
may help to find the true Hb value (McVeigh, Faim &
van der Weyden, 1989). However, RBC count may be
also disturbed by high WBC counts (see later), and
one must pay attention to the limits in recalculating

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M. ZANDECKI ET AL.

RBC indices, and a packed cell volume (centrifuged

Hct) should be considered.

SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS 29

2002). However, spurious Hb measurement is far from

being a consistent finding in the presence of cryoglobulins (Fohlen-Walter et al., 2002).

Immunoglobulins
They have been reported to interfere with a number
of clinical laboratory tests (cf., see Roberts, Fontenot
& Lehman, 2000). False elevation of Hb measurement
by automated methods was observed for several
patients with Waldenstro ms macroglobulinemia and
monoclonal IgM, or with multiple myeloma and
monoclonal IgA or IgG (Wallis & Ford, 1987; McMullin, Wilkin & Elder, 1995; Roberts et al., 2000). This
anomaly is related to high levels of Ig that interact
with reagents of the lysis solution. For IgM, this phenomenon has been related to the amount of monomeric component within the circulating paraprotein
(Goodrick et al., 1993). HA employing Hb conversion
to cyanmethaemoglobin seem to be more affected
than others, because of addition of surfactants for
cyanide-free methods. Red blood cell count and MCV
are unaffected in this situation but, as Hb is overestimated, MCHC usually exceeds 36 g/dl. In order to
obtain more accurate results in this instance, it was
proposed to determine plasma Hb after centrifugation
of the sample (which gives turbidity because of the
paraprotein) and to withdraw it from Hb of the whole
blood (Roberts et al., 2000). For Sysmex instruments,
it seems that if the sample is half diluted by the operator before analysis on the HA, the phenomenon does
not occur (Roberts et al., 2000). For laser-beam HA
that determine the measured MCHC (CHCM) a clearcut difference with the calculated MCHC generates an
alarm, and accurate Hb from the sample may be
obtained from measured MCHC.

Cryoglobulins
Spurious Hb values have been reported in some
instances, and several mechanisms are proposed to
explain these abnormal findings. In some cases spuriously high Hb values were related to a mechanism
similar to that described above for immunoglobulins
(Taft et al., 1973; Cornbleet, 1983) or to the disturbance of light transmittance, whereas in other cases a
slight decrease of both Hb measurement and RBC
count was related to a flow anomaly (Taft et al., 1973;
Bremmelgaard & Nygard, 1991; Fohlen-Walter et al.,

Haemolysis
Hb free within plasma is measured together with that
from the RBC, but its amount ranges from 10 to
40 mg/l in normal conditions and does not affect total
Hb measurement. However, in situations related to
major intravascular haemolysis, including chemicals,
mechanical haemolysis associated with heart valves,
and haemolytic anaemias associated with blood transfusion, free plasma Hb may be elevated enough to
affect total Hb measurement. MCHC may be >36 g/dl.
Centrifuged Hct shows a pink or red plasma tinge,
namely if free plasma Hb is >200 mg/l, and is, in some
instances, the only reliable RBC parameter. Some
laser-beam HA directly determine Hb within each
RBC, the Cellular Haemoglobin Concentration Mean
(CHCM), which allows the accurate Hb value to be
calculated. A short time lapse between venepuncture
and analysis is of crucial importance because haemolysis may continue in vitro, leading to a spurious
decrease of RBC count and total Hb with a spurious
increase of free plasma Hb.

Chemical structure of haemoglobin and bilirubin

In physiological situations, Hb is more or less coupled
to oxygen or to carbon-di-oxide and, according to
which molecule is coupled to Hb, the peak of optimal
light absorbance differs slightly. The addition
of
several reagents leave Hb free from coupled molecules
and changes it into one stable molecule (cyanmethaemoglobin is an example), the latter demonstrating a
narrow peak of light absorbance, which allows accurate determination of Hb concentration. However,
high amounts of carbon monoxide coupled to Hb
may not be fully transformed, and in such situations
a spuriously high Hb concentration is reported
(Cornbleet, 1983; Vinatier & Flandrin, 1993). In contrast, sulfhaemoglobin in high amount has
been
reported as lowering Hb measurement (Cornbleet,
1983). Bilirubin: although one must pay attention to
very high amounts of bilirubin within the plasma,
most HA do not presently demonstrate any interference with bilirubin, at least for concentrations up to

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30 M. ZANDECKI ET AL.

SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS

250 mg/l. Above theses values, however, attention is

needed.

MCV, and subsequently lead to abnormal calculated

MCHC (discussed later).

Spurious RBC counts and red cell parameters

Giant platelets

According to the technology used, spurious results

may or may not be observed using some HA, and
some knowledge about the methods analysing blood
parameters is necessary. On impedance type HA an
aliquot of the blood sample is diluted isoosmotically
and the number and height of electric pulses generated by the electrical resistance of RBC that pass
through a small orifice allow the determination of
both the RBC count and the MCV. Using laser-beam
methods, scattering at least at two angles allows the
determination of RBC count and MCV. In order to
improve accuracy, pretreatment of RBC with a specific
reagent that changes them isovolumetrically from discoid to a sphere is performed on some HA. Setting discrimination thresholds is an important consideration:
discriminating the smallest RBC from the largest PLT
is at times a challenge, already discussed (part I). On
the contrary, even in extreme pathological situations,
MCV does not exceed 150160 fl and, as there is no
particle above that size in the blood stream in health
or in disease, HA do not analyse any particle above
200300 fl in volume.

High number of giant PLT may lead to spuriously low

PLT count (see part 1) but, as they are enumerated as
RBC, they may also affect RBC count in a way similar
to that for WBC; however, RBC count is usually only
slightly affected in this instance (Cornbleet, 1983;
Bain & Bates, 2001).

Spuriously elevated RBC counts

High WBC counts
Most HA enumerate RBC and WBC together within
the same channel(s), and the RBC count reported is
the sum of both RBC and WBC counts. In physiological conditions, it is not of any importance, as it corresponds to overestimate RBC count by 0.1% (if we
consider a WBC count of 5 109/l and a RBC count
of 5 1012/l). However,
high
WBC
counts
(>100 109/l) may lead to a significant change in the
RBC count, particularly if the patient is also anaemic
(Bain & Bates, 2001). Moreover, in the latter
instances the MCV reported corresponds to the mean
volume of RBC and of WBC from the sample and
may be spurious, variably according to the nature and
the number of WBC from the relevant sample. So,
high WBC counts may induce several abnormal findings, including Hb (discussed above), RBC count,

Spuriously decreased RBC counts

Cold agglutinins
Cold agglutinins aggregate RBC when the temperature
is lower than 37 C. Unsurprisingly, peculiar anomalies of RBC parameters in the presence of cold agglutinins were reported first on counters acting at room
temperature (Hattersley et al., 1971; Petrucci, Duanne
& Chapman, 1971; Bessman & Banks, 1980). According to the HA, the upper threshold that may consider
particles as RBC into the RBC channel(s) is located
between 200 and 300 fl. So, only particles corresponding either to isolated RBC or to small RBC clumps
(two or three RBC), are analysed, whereas large RBC
clumps are fully neglected by the HA. This leads to
spuriously low RBC counts and to abnormally high
MCV (each small RBC clump is considered as one single particle). Haematocrit (RBC MCV) is erroneous
and spuriously low, contrasting with Hb that is measured after RBC lysis and is unaffected by agglutinins.
As a rule, the MCHC is spurious, usually >36 g/dl. The
peculiar association of low RBC count, high MCV and
MCHC >36 g/dl is almost pathognomonic of the cold
induced artifact on instruments that work at laboratory temperature, and not infrequently helps to diagnose the cold agglutinin in the patient analysed
(Petrucci, Duanne & Chapman, 1971). HA working
with reagents at temperatures near 37 C are not fully
insensitive to cold agglutinins, however, but changes
are less obvious and may remain undiscovered in some
instances (Solanki & Blackburn, 1985). HA that measure directly the amount of Hb within each RBC (measured MCHC, or CHCM) usually show discrepancy
between the measured MCHC and the calculated one.
Whatever the HA used, the common finding is that Hb
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M. ZANDECKI ET AL.

value is unaffected and that anomalies disappear when

the sample is warmed at 37 C and analysed promptly
afterwards. Amplification of the anomalies after the
sample has been cooled at 4 C for 1 or 2 h reinforces
the diagnosis. As viscosity of the sample may be high,
leading to inaccurate aspiration, an alarm may be
generated on some HA (insufficient sampling, or
related). Co-existence of RBC agglutination with
EDTA-dependant thrombocytopenia was reported but
antibodies directed against RBC and those directed
against PLT differed (Bizzaro & Fiorin, 1999).

Warm autoimmune haemolytic anaemia

In some instances warm autoantibodies were also
reported as inducing RBC agglutination, leading to
spurious MCV and RBC count, in a situation superimposable to that observed for cold agglutinins, but not
reversible by warming (Weiss & Bessman, 1984).

Very small RBC and discrimination with PLT

As discussed in part 1, PLT counts may be disturbed
by microcytic cells, namely if RBC volume is <36 fl:
some HA consider particles over this threshold as
RBC, and in severe microcytic anaemias PLT count
may be altered. Underestimation of RBC counts may
be observed in turn, because of the elimination of the
smallest erythrocytes from the RBC count. In such
instances up to 6% lowered RBC counts were reported (Savage et al., 1985).

Cryoglobulins
As previously mentioned, a slight decrease of both Hb
measurement and RBC count was reported in a few
instances, related to a flow anomaly (Taft et al., 1973;
Cornbleet, 1983; Bremmelgaard & Nygard, 1991; Fohlen-Walter et al., 2002).

Considerations related to the quality of the sample.

Clotting, or abnormal mixing secondary to overfilling
of the sample (Pewarchuk et al., 1992), may lead to
an aspirated aliquot unrepresentative of the blood
sample. Cryoglobulins and cold agglutinins may lead
to high viscosity of the sample, leading to inadequate
aspiration.

SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS 31

In vitro haemolysis
As mentioned above, in some situations, namely haemolysis related to chemicals or blood transfusion, RBC
may continue to lyse within the sample, leading to
spuriously low RBC count, together with abnormal
Hb measurement and abnormal red cell indices (see
Hb measurement).

Mean cell volume and haematocrit

Some situations leading to spurious MCV (see Table 1)
are already discussed above [high WBC counts, cold
(warm) agglutinins]. Spurious Hct is a frequent finding, related to abnormal MCV or/and to abnormal
RBC count as it is a parameter that is usually calculated by the HA: situations leading to abnormal Hct,
either increased or decreased, will not be discussed in
details as they may be deducted from MCV changes
(Table 1).

MCV and the technology used for measurement

Using impedance-type HA, disc-shaped RBC become
elongated into a cigar shape as they pass through the
aperture. Elongation is more pronounced for particles
flowing at the periphery of the orifice than at the central part, and also when Hb content is low within the
RBC. Mean (red) cell volume is spuriously lowered
and MCH is spuriously elevated, mainly in hypochromic disorders, and for MCV of 55 fl a spurious
decrease of up to 7 fl may be observed (Mohandas
et al., 1980; Paterakis et al., 1994; Bain & Bates, 2001).
Hydrodynamic focusing, a technical change
that
directs the flow of particles at the centre of the aperture, improves MCV measurement. For laser-beam
HA, the discoid shape of RBC was a major drawback
for peer analysis of RBC until the isovolumetric spherisation was applied, allowing current instruments to
generate accurate MCV (Mohandas et al., 1986). Some
impedance-type HA use both technical improvements
to analyse RBC (Abbott), whereas others measure the
Hct and calculate MCV using the HCT and RBC (Sysmex; ABX, Kyoto, Japan; some Beckman instruments,
Miami, FL, USA). In that latter instance however,
the presence of cold agglutinins, which disturbs RBC
count (see above) leads to erroneous MCV (Sysmex
XE-2100 operators manual revised April 2004).

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M. ZANDECKI ET AL.

SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS 33

Table 1. Situations leading to erroneous results on haematology analysers

Other parameters altered
WBC: spurious decrease
Agglutination of PMN (EDTA - related)
Agglutination of WBC other than PMN
(lymphocytes, lymphoma cells, leukaemic blasts)
Excess amount of K3-EDTA anticoagulant
Shrinkage of RBC: MCV and Hct
Coagulation within the sample
All parameters
WBC: spurious increase
PLT aggregates
PLT
(Very) large PLT
PLT
Nucleated red blood cells
RBC resistant to lysis (newborns, abnormal Hb,
chemotherapy, uraemia, liver disease)
Cryoglobulin, cryofibrinogen, immunoglobulins
PLT
Lipids
PLT, Hb, MCH
Microorganisms (bacterial aggregates)
PLT
Others (adipose tissue, overfilling vacuum tubes)
All parameters
RBC: spurious decrease
Cold agglutinins, warm agglutinins
MCV, MCH
Very small RBC
PLT
Cryoglobulins ( flow, inadequate aspiration)
Hb, WBC, PLT
In vitro haemolysis
Hb, MCH
Coagulation
All parameters
RBC: spurious increase
High WBC counts
MCV, Hct, MCH
Giant PLT
PLT
Haemoglobin: spurious increase
Lipids
WBC, Hb, MCH
High WBC counts
MCH, RBC
Immunoglobulins (and cryglobulins)
WBC, PLT, MCH,
In vitro haemolysis
MCH
Carboxyhaemoglobin (high amount)
Bilirubin (>250300 mg/l)
MCH
Haemoglobin: spurious decrease
Coagulation within the sample
All parameters
Overfilling vaccum tube
All parameters
Veinipuncture near a drip
MCV (glucose drip)
Sulfhaemoglobin
MCV
Cold agglutinins, warm agglutinins:
MCH, RBC, PLT
High WBC counts:
RBC
Hyperglycemia:
MCH
K2 EDTA in excess:
MCH
Hyper- or hyponatremia: or
MCH or
Technology: impedance without hydrodynamic focusing
MCH
(MCV in hypochromic anaemias)
MCH > 36 g/dl (not related to spurious counts in some disorders: spherocytosis, xerocytosis, abnormal Hb: see text)
Cold agglutinins, warm agglutinins
RBC, MCV
Lipids
WBC, Hb
Immunoglobulins
Hb
In vivo and in vitro haemolysis
Hb altered, Hct
Carboxyhaemoglobin (>1020%)
Bilirubin (>300 mg/l, at times less)
Immunosuppressive drugs (see also spuriously high Hb, spuriously low MCV)
MCH <32 g/dl
Hyperglycemia (see also spuriously high MCV, spuriously low Hb)
MCV
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34 M. ZANDECKI ET AL.

SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS

RBC

RBC

50

100
12

RBC =
1. 99 10 /l
MCV = 141.5
fl

200

300 fL
Hb = 6.4 g/dl
MCH = 22.9 g/dl

50
RBC =
MCV =

100
12

3.78 10 /l
97.1
fl

200

300

fL

Hb = 12.5 g/dl
MCH = 34.1 g/dl

Figure 7. Venepuncture performed near a glucose infusion. Blood sample from that patient was diluted (Hb low)
and excess of glucose led to a swelling of RBC: MCV was spuriously high and in turn MCHC spuriously low. Sample drawn correctly by the next morning showed normal values (no transfusion had been performed). RBC histogram on diluted sample (left) and on new sample (right; Beckman Coulter STKS II).

MCV and high WBC counts

As mentioned above, WBC are counted together with
RBC, and MCV reported is the mean of both RBC and
WBC volumes. In situations related to high WBC
counts and low RBC counts (e.g. acute leukaemias),
MCV is altered. Up to a 1520 fl increase may be
observed in some instances. HA which calculate MCV
using the HCT and RBC (Sysmex, ABX, some Beckman instruments) are also sensitive to leukocytosis
over 100 109/l (Sysmex XE-2100 operators manual
revised april 2004).

Severe hyperglycemia
Mean (red) cell volume is reported as spuriously high
in blood samples containing high levels of glucose,
either related to severe hyperglycemia in the relevant
patient or to a sample, which has been drawn near an
intravenous glucose infusion (Morse et al., 1981;
Strauchen et al., 1981; Holt, DeWandler & Arvan,
1982; Evan-Wong & Davidson, 1983; Savage & Hoffman, 1983; Planas, Van Voolen & Kelly, 1985; Van
Duijnhoven & Treskes, 1996). In the latter situation,
the sample is diluted leading at times to a severe spurious macrocytic and hypochromic anaemia (Figure 7).
The calculated Hct is spuriously high and the MCH is
spuriously low: RBC count and Hb are unaffected in
diabetic patients and variably decreased if samples are
diluted because of glucose infusion (Figure 7). This
phenomenon is related to plasma glucose concentration, which balances that of intraerythrocytic, either
in the body or in the sample if an excess of glucose is

present (blood drawn near a glucose drip). Studies

performed on RBC at various concentrations of glucose have shown that within a few seconds after dilution into the HA (diluents from the manufacturers)
water penetrates within RBC rich in glucose, inducing
swelling: return to a normal size is progressively
observed and is complete after up to 5 min. According
to the time spent between dilution and MCV measurement, the anomaly will be more or less conspicuous (Holt, DeWandler & Arvan, 1982). The anomaly
is more obvious if RBC are isovolumetrically sphered,
because stabilization of RBC volume occurs within a
few seconds after the dilution of the sample (Van
Duijnhoven & Treskes, 1996). For the above-mentioned reasons HA are more or less sensitive to this
phenomenon, but glucose concentrations
over
20 mmol/l may begin to increase MCV, and blood
glucose >35 mmol/l may overestimate MCV up to
50 fl (Holt, DeWandler & Arvan, 1982; Planas, Van
Voolen & Kelly, 1985; Van Duijnhoven & Treskes,
1996; Figure 7). Increased MCV leads to an increase
in the calculated Hct and to a spuriously low MCHC.
CHCM measured on some HA is also altered. CBC
demonstrating macrocytic hypochromia may be considered first as related to hyperglycemic sample.

Considerations related to the anticoagulant

The use of either K2- or K3-EDTA salt does not
induce any difference in the CBC, in optimal conditions of sampling. However, when concentration of
anticoagulant is increased, because of insufficient
volume of blood drawn after venepuncture (or in
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M. ZANDECKI ET AL.

neonates), some changes may be observed. Decrease

of centrifuged Hct resulting from shrinking of RBC
was reported in conditions related mainly to excess of
K3 salt (Goossens, Van Duppen & Verwilghen, 1991;
Hinchliffe, Bellamy & Lilleyman, 1992), in contrast
with data obtained on HA: no influence of K3-EDTA
concentration was observed on MCV, while K2-EDTA
at high concentrations resulted in a slight increase
in MCV, and such phenomenon could be observed
using several different HA (Goossens, Van Duppen &
Verwilghen, 1991).

Hyper- and hypo-natremia

Macrocytic and hypochromic RBC changes were
observed on blood samples in conditions related to
hypernatremia, whereas hyponatremia generated a
tendency towards microcytic and hyperchromic RBC
(Cornbleet, 1983). Such situations were also reported
in animals (Boisvert, Tvedten & Scott, 1999). Hyperand hypo-natremia are both situations also reported
as leading to spurious centrifuged Hct (Cornbleet,
1983).

Storage of the sample and MCV

EDTA used as anticoagulant allows the accurate determination of the CBC up to 24 h after the sample has
been drawn. However, after that time, MCV may
increase, namely if the sample is stored at room temperature. This coupled with low haemoglobin could
cause the operator to suspect that a slightly microcytic
anaemia is normocytic and that a normocytic could be
mistaken for a macrocytic anaemia (Cohle, Saleem &
Makkaoui, 1981).

Mean cell haemoglobin content (MCHC)

Measured parameters allow the calculation of mean
cell haemoglobin of individual RBC [MCH
as
expressed in pg Hb (g/l)/RBC (1012/l)] and MCHC
[MCHC in g/dl Hb (g/l) 100/MCV (fl) RBC
(1012/l)]. Some HA measure Hb concentration directly
within RBC, named cellular Hb concentration mean
(CHCM): discordant values (usually difference over
1.5 g/dl) between MCHC and CHCM allow in many
instances the detection of anomalies related to one of
the measured RBC parameters.

SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS 35

MCHC >36 g/dl is infrequent on most impedancetype HA, whereas it may be occasionally observed on
laser-beam HA, in several constitutive disorders in
which RBC are dehydrated, including hereditary
spherocytosis, various haemoglobin disorders (CC, SC,
Cb thal), and some rare RBC disorders (xerocytosis).
Some acquired conditions mimic constitutive ones,
namely acquired
immune
haemolytic
anaemias
caused by warm agglutinins, in which RBC coated
with warm antibodies may transform gradually into
spheres in vivo or in vitro after venepuncture, leading
to more or less dehydrated and spherised cells. Many
situations corresponding to abnormally high Hb values, and/or abnormally low RBC, and/or spuriously
low MCV, also lead to increase MCHC (see the corresponding paragraphs and Table 1). Although the mechanism is unknown, some immunosuppressive drugs
may slightly increase MCHC, usually not above
37.5 g/dl (Cornbleet 1983).
MCHC <32 g/dl. As mentioned above, hyperglycemia leads to artefactual hypochromic
macrocytosis.
Storage of blood samples has been reported to cause a
facticious increase in percentage of hypochromic cells,
and become conspicuous after 24 h of storage
(Murphy, Spaven & Casey 2002). Although changes
are not major, the number of hypochromic cells may
be an important element to consider, as for example
in iron deficiency anaemias or during erythropoietin
therapy (Richardson, Bartlett & Will, 2001).

RETICULOCYTE S
Enumeration of peripheral blood reticulocytes is
essential in the diagnosis and management of anaemic
patients and may be considered now as a part of the
CBC. If manual counting by light microscopy remains
the standard of reticulocyte enumeration, automated
methods developed during the past two decades are
now more accurate, precise, and cost-effective than
manual counting and, in addition, provide a variety of
reticulocyte-related parameters, such as volume, haemoglobin concentration, and maturity, which are
unavailable with light microscopy (Cavill, 1993; Van
Petegem et al., 1993; Davis et al., 1994; Koepke, 1999;
Brugnara, 2000; Siekmeier, Bierlich & Jaross, 2000;
Riley et al., 2001; Bain & Bates, 2001; Pierre, 2002;
Riley et al., 2002). Automated reticulocyte counts can
be performed using general purpose flow cytometers,

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36 M. ZANDECKI ET AL.

SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS

dedicated flow cytometers, or integrated with the

other parameters from the CBC under a fully automatized method. RNA from reticulocytes is stained using
the classical new methylene blue (part of Abbott and
Beckman Coulter instruments), oxazine (Bayer), or
one among various fluorescent dyes, including thiazole orange (Horiba ABX), auramine O (Sysmex),
CD4K530 (Abbott), or coriphosphine O (Beckman
Coulter). According to the HA and to the reagent
used, flow cytometric analysis is performed, using
either the measurement of volume, conductivity, and
light scatter (VCS) or the fluorescence detection (for
review see Riley et al., 2002).
Careful gating of reticulocytes is crucial in determining accurate counts, because dyes stain also RNA
from PLT and WBC, and may also combine with the
DNA of nucleated cells. Red blood cells and reticulocytes being larger than PLTs and smaller than WBC,
both the former are discriminated from both the latter
according to the size. Unsurprisingly, gating may be
difficult in the presence of blood components
of
abnormal size and containing stained RNA/DNA.
Giant PLT, PLT clumps, abnormal WBC, abnormal
number of WBC, and WBC fragments, are situations
reported as a potential source of interference with
automated methods
of
reticulocyte
analysis
(Oyamatsu et al., 1989; Pappas, Owens & Flick, 1992;
Riley et al., 2001). NRBC have been reported as disturbing reticulocyte counts (Pappas, Owens & Flick,
1992; Riley et al., 2001), but, as NRBC themselves are
not counted as reticulocytes in the HA, it has been
hypothesized that abnormal
reticulocyte
count
observed in that instance could be due to increased
number of very young reticulocytes which frequently
appeared with NRBC (Oyamatsu et al., 1989).
After gating of RBCs, reticulocytes are identified
within the RBC population according to the coloured
particles or fluorescent material they contain. Cytoplasmic particles other than RNA that can be stained
by supravital dyes, including HowellJolly bodies,
Pappenheimer bodies, or basophilic stippling, may be
confused with reticulum granules using automated
techniques in HA, as well as they interfere in manual
techniques (Pappas, Owens & Flick, 1992; Ghevaert
et al., 1997; Riley et al., 2001), although such interferences have been found to be more or less conspicuous
according to the machines used (Oyamatsu et al.,
1989; Lofsness, Kohnke & Geier, 1994). Heinz bodies

observed in severe haemolytic anaemias, thalassaemia

major, congenital Heinz-body anaemia, or post-splenectomy disorders, are also situations reported as disturbing automated reticulocyte counts
(Hinchliffe,
1993; Espanol, Pedro & Remacha, 1999; listed in Riley
et al., 2001), as well as sickle cells (Pappas, Owens &
Flick, 1992; Ghevaert et al.,
1997),
spherocytes
(Ghevaert et al., 1997), and haemoglobin H inclusion
bodies (Riley et al., 2001).
Although they are only faintly stained using fluorescent dyes, intraerythrocytic parasites (malaria, babesia) may interfere with automated reticulocyte count,
and value six times higher than the manual count
was reported in a patient demonstrating 70% of RBC
infected with plasmodium falciparum (Laurencet,
Martinez & Beris, 1997).
Intensity of intracellular staining or fluorescence of
reticulocytes is assessed in most HA, the brightest reticulocytes being the youngest, leading to peculiar
indices such as the immature reticulocyte fraction
(IRF; for review see Brugnara, 2000). White blood
cells displaying intense staining were reported as leading to an erroneous estimation of reticulocyte maturation index, the error being directly correlated with
the WBC count (Villamor et al., 1996). Very mature
reticulocytes contain only a few coloured dots and
may be insufficiently detected in neonates using flow
cytometry, possibly due to the low concentration of
the colouring matter used in some HA (Bayer; Wiegand et al., 2004). Although reticulocyte counts are s
after storing blood samples for 72 h at 4 C and 24
48 h at room temperature (Brugnara et al., 1994;
Rudensky, 1997; Lacombe et al., 1999; Bain & Bates,
2001), immature reticulocyte fraction was observed as
stable for only 8 h at 4 C and 6 h at room temperature (Lacombe et al., 1999).
Various other situations are mentioned as generating occasionally spurious reticulocyte counts, including agglutinated cells in the presence of cold
agglutinins (Oyamatsu et al., 1989; Riley et al., 2001),
autofluorescence of RBC (porphyria, drugs) and diagnostic intravenous fluorescent dyes (Riley et al.,
2001), high amount of paraproteins, and haemolysis
(Riley et al., 2001).
Although depending on the software gate corrections, in most of the above described situations the
HA show abnormal flags, prompting the technologist
to perform a manual count. However, exceptions to
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M. ZANDECKI ET AL.

Table 2. Situations leading to possible interference

with automated methods for reticulocyte analysis
Inaccurate gating of RBCs: giant PLTs, PLT clumps,
abnormal WBCs, abnormal number of WBCs, WBC
fragments, nucleated red blood cells
Intraerythrocytic particles: Howell-Jolly bodies,
Pappenheimer bodies, basophilic stippling, Heinz bodies,
sickle cells, spherocytes, Haemoglobin H inclusions,
plasmodium, babesia
Others: cold agglutinin disease, autofluorescence of RBCs
(drugs, porphyria), paraproteins, haemolysis, diagnostic
intravenous fluorescent dyes

flagging have been reported, at least in cold agglutinin

disease (Oyamatsu et al., 1989) and in beta-thalassaemia major (Ghevaert et al., 1997; see Table 2).

CONCLUDING REMARKS
The widespread use of HA has led to major improvement of cellular haematology, because of quick and
accurate results found in most instances, and now
preanalytical and analytical variables should be considered first within the laboratory when spurious
results from the HA are found (see also concluding
remarks from part I). In most situations, HA generate
flags or peculiar scattergrams in response to clinical or
artifactual parameters, independent of the technology
used. Inadequate blood sample may be responsible for
various anomalies, including initiation of coagulation
within the tube because of difficult venepuncture or
to low blood flow, excess of EDTA salt result of insufficient filling of the tube, which generate PLT and/or
WBC aggregates, fibrin precipitates, or others. Overfilling of blood sample and time elapsed from sampling to analysis must also be considered at times. In
several instances the disease itself generates changes
that disturb measurements performed by HA (RBC
agglutination, cryoprecipitates). Some anomalies,
however, are intrinsic to the technology used for analysis, and have led manufacturers to develop technical
changes in analysing blood cells, one example corresponding to the use of hydrodynamic focusing, which
dramatically improved measurement of MCV in
impedance-type HA. Some protocols for measurement
of blood cells have been discarded, such as PLT counts

SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS 37

performed after RBC lysis, leaving intraglobular components free and enumerated together with PLT.
Improvements in analysis of PLT and RBC parameters
has led to an increase in the accuracy of results and
has also led to the generation of several flags in
abnormal situations. The enumeration of PLT in
thrombocytopenias, the identification of NRBC leading
to spurious PLT and WBC counts, and other situations, have led manufacturers to optimise computerized analysis for the detection and analysis of every
blood cell, and moreover to develop further methods
for their specific identification and their specific enumeration on the same sample and on the same HA.
The improvement of WBC analysis and the careful
study of WBC differential scattergrams have led to
another major improvement in blood cell analysis,
allowing WBC differential to be performed automatically but also to exhibit and to give explanation for
several anomalous CBC results. Several peculiar WBC
scattergrams generated were progressively identified,
and related to spurious counts. As discussed all along
both reports, inaccurate identification, analysis, or
enumeration of one or several components from the
CBC leads in many instances to an abnormal WBC
differential scattergram. By the rule the WBC differential is flagged or invalidated, so a blood smear and an
optical count are often needed. Automated abnormal
WBC differentials related to spurious counts must be
included together with the various but insufficiently
reported situations related to the inability of the HA
to perform WBC differential or to identify specifically
one or several cells from the WBC differential. All
these situations should certainly need a specific
report.
So, if some spurious counts were found to be
numerous enough to generate technical improvements to identify them clearly and create other methods for measurement, several other spurious counts
either do not generate specific flags to identify the
anomaly or even do not generate any flag at all.
Moreover, if the most recent and powerful HA are
able to generate several flags related to at least a part
of spurious counts, it is stressed that simpler HA,
namely those without any WBC differential scattergram, will not. So, if acquiring a new HA is a personal
choice, each haematologist must know how his
machine will react when (at least) the situations
reported here occur.

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38 M. ZANDECKI ET AL.

SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS

A CKN OWL EDG EMENT S

CONFLICT OF INTEREST

We are indebted to Mathilde LINARD for kindly

reviewing English manuscript.

No conflict of interest.

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Journal compilation 2007 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 2007, 29, 2141