Bone Marrow Aspirate

Written by Mark Hill on .

Bone Marrow aspirates are processed in the Immunophenotyping (cell markers) laboratory.

Please indicate on the request form if immunophenotyping or molecular studies are required by ticking the relevant boxes. Aspirates should be taken into EDTA and transported to the laboratory as soon as possible where slides will be made and processed for morphological examination and reporting by the consultant led team. In addition to routine Romanowsky stains, iron stores will be assessed via the Perl’s Prussian Blue stain. Following reporting, extra investigations may be requested such as immunophenotyping or FISH.

Trephine samples should be directed to the Histopathology Department with an accompanying Histopathology request form.

 

The Bone Marrow Aspirate Request Form (pdf) can be downloaded from here.

Immunophenotyping (Haematology)

Written by Craig Webster on .

The laboratory is equipped with 2 Beckman Coulter Navios flow cytometers with 10 colour capability. These allow the identification of antibody-antigen reactions by identifying light emitted by fluorochromes conjugated to the antibodies used. The pattern of positive and negative reactions provide the Immunophenotype.

We offer an extensive repertoire of investigations which provide evidence for the diagnosis and classification of haematological disorders. We also offer stem cell quantitation for patients undergoing stem cell harvests, PNH diagnosis  and  quantitation of feto-maternal haemorrhage. Further information may be found below.

 

Sample requirement:

Immunophenotyping requires fresh blood or bone marrow drawn into EDTA tubes. Marrow should be first pull. Quality of sample is essential to achieve meaningful results.

Please refer to the specific investigation types below, as the different sample types are not always acceptable. Recommended minimum sample for peripheral blood is 3mls. There is no minimum set for bone marrow. We can also investigate fluid samples such as CSF, but these should reach the lab within 4 hours as the cells degrade quickly in these sample types. Fluids may be taken into EDTA or plain universals.

Please note that the number of leukocytes present may impact on our ability to provide full analysis.

Please telephone if you require advice on 0121 424 0704.

 

Laboratory opening:

The laboratory is open Monday - Friday, 08:30 - 17:30. Latest time of sample receipt for guaranteed same day analysis is 2.00pm. Samples arriving later than this may be refrigerated until the next working day. Analysis may be performed following weekend storage providing cell viability is within acceptable limits.

Samples should be transported to the laboratory as soon as possible after collection following the transport procedure.  For external requests, samples should be transported in suitable containers via courier and labelled for the attention of Cell Markers, Haematology BHH. Samples should not be sent in the post.

 

Contact:

For enquiries telephone 0121 424 0704 to speak with a Senior Biomedical Scientist in the Immunophenotyping Laboratory. For clinical advice please contact the on-call Consultant Haematologist via switchboard 0121 424 2000.

 

Request forms:

Click here to download a request form (pdf) for haematology investigations, or here to download a request form for feto-maternal haemorrhage.

Please avoid using photocopies of old versions of request forms as these may not be UKAS compliant and may not represent the current tests on offer.

Please complete the request form accurately providing as much relevant information as possible. The laboratory will not accept samples/requests with missing or mislabelled PID.

 

Sample acceptance criteria:

There must be a minimum of 3 identifiers which link the sample and the request form and should include the patient’s first name, patient’s surname and either hospital number, date of birth or NHS number..

 

Click for Test Database

 

ADDITIONAL INFORMATION FOR IMMUNOPHENOTYPING

LYMPHOCYTE SCREEN

The lymphocyte screen is designed to screen for lymphoproliferative disorders. If an abnormal population is identified, an extended panel will be performed to allow characterisation of the cells and provide evidence for diagnosis. Screens which do not identify an abnormality will be reviewed with morphology by a member of the consultant team prior to reporting.

 

MDS / ACUTE LEUKAEMIA SCREEN

This panel aims to identify numbers of blasts and monocytoid cells. Blasts are identified as myeloid and abnormal phenotypic expression may indicate dysplasia. However, this is not a diagnostic panel and is designed to monitor blast levels. An additional erythroid panel will be added when indicated from initial results / morphology.

When requesting an MDS panel, please note that peripheral blood is unsuitable.

The panel also provides evidence for acute leukaemia by providing a blast percentage. Following review of results, the sample may be escalated to a full acute leukaemia panel.

All results will be reviewed together with morphology by one of the consultant team prior to reporting.

 

INVESTIGATION OF HAEMATOLOGICAL MALIGNANCIES

 

We have specifically developed panels to give evidence for the diagnosis and classification of haematological malignancies:

B LYMPHOPROLIFERATIVE PANEL for B CLL and B cell Lymphomas

T LYMPHOPROLIFERATIVE PANEL for T cell lymphomas

ACUTE LEUKAEMIA PANEL for all lineages of acute leukaemias

MYELOMA/MGUS PANEL for the identification of neoplastic plasma cells

Relevant panels will be performed as appropriate following initial screening, or as indicated on the request form. The results will indicate approximate percentage of cells and their phenotypic expression. Please note that plasma cells in particular are fragile and easily lost during sample preparation and staining, and thus percentage may be lower than that seen morphologically.

In addition to diagnostic evidence, the results may provide evidence of prognosis.

All results will be reviewed together with morphology by one of the consultant team prior to reporting.

All new diagnosis acute leukaemias will be telephoned through to the requesting consultant –please provide contact details on the request form.

 

MINIMAL RESIDUAL DISEASE (MRD)

 

CLL MRD:

We use a combination of antibodies to identify CLL cells and differentiate them from other B cells. This results in a sensitive measure of CLL MRD and is used to monitor patient’s response to therapy. CLL MRD is detectable in peripheral blood down to 0.01%, and for results below this level, a follow up bone marrow aspirate may reveal residual disease in the marrow.

 

Myeloma MRD:

This utilises the same combination of antisera used in the diagnostic panel, but sensitivity is increased by increasing the number of cells counted.

 

Acute Leukaemia MRD:

There are specific panels for the recognition, characterisation and quantitation of residual disease in acute lymphoblastic leukaemias.

The approach to MRD in acute myeloid leukaemia is to provide a quantitation of myeloid blasts, wherever possible with reference to leukaemia associated antigens.

MRD levels in acute leukaemias by flow are for guidance only and must be viewed alongside other evidence such as cytogenetics.

All results will be reviewed together with morphology by one of the consultant team prior to reporting.

 

PNH SCREEN

Paroxysmal Nocturnal Haemoglobinuria (PNH) is an acquired haematopoietic stem cell disorder resulting in a partial or total deficiency of proteins normally linked to cell membranes by a glycosylphosphatidylinositol (GPI) anchor. Classical features are:

intravascular haemolysis

bone marrow failure

thrombotic tendency

and may also include abdominal pain, dysphagia, erectile dysfunction, renal failure, lethargy. It can often go undiagnosed for months or even years. Flow cytometry can provide a definitive diagnosis.

 

PNH screening can only be performed on peripheral blood (taken into EDTA). Marrow is unsuitable due to the presence of immature cells.

Investigation for PNH involves the analysis of antigens normally linked via the GPI anchor on both red cells and granulocytes. Additionally, granulocyte screening includes FLAER (FLuorescent AERolysin) which binds directly to GPI. Red cell analysis provides classification into PNH types I and II but clone size may not be representative due to transfusion and/or haemolysis. Granulocyte analysis provides an accurate measure of clone size and is used to monitor the disorder over time.

Please indicate recent transfusion history on the request form.

 

Click here for further guidance on PNH testing

 

FETO-MATERNAL HAEMORRHAGE

 

FMH  by flow cytometry identifies and enumerates the bleed of foetal cells into maternal circulation following birth or other traumatic event. Please note that this screen is based on the principle that maternal samples are Rh D negative and baby/cord samples are Rh D positive. In ante-natal patients or patients who have had an IUD it may not be possible to identify a bleed if mother and baby have the same Rh D type. Also as the principle is based on staining using a fluorescent anti-D, patients who have received recent prophylactic anti-D may give reduced values due to antigen blocking by prophylactic anti-D.

To ensure the correct dose of anti-D is administered we report a suggested dosage taking into account the uncertainty of measurement of FMH by flow in our laboratory. The reported bleed (in mls) is the value obtained by flow cytometry. The suggested dosage of anti-D is based on the reported bleed plus the uncertainty of measurement. 

Current guidelines state that FMH quantitation by flow cytometry should be performed on any bleed greater than 2mls as identified by the Kleihauer stain (performed in the Blood Bank). Patients from HEFT will automatically be referred by the blood bank.

 

To download the FMH request form click here

 

For any further advice please telephone the laboratory on 0121 424 0704  

Requesting Guidance for PNH Testing

Written by Mark Hill on .

Paroxysmal Nocturnal Haemoglobinuria (PNH) is an acquired haematopoietic stem cell disorder resulting in a partial or total deficiency of proteins normally linked to cell membranes by a glycosylphosphatidylinositol (GPI) anchor. Classical features are;

  • intravascular haemolysis
  • bone marrow failure
  • thrombotic tendency
  • may also include abdominal pain, dysphagia, erectile dysfunction, renal failure, lethargy

It can often go undiagnosed for months or even years. Flow cytometry can provide a definitive diagnosis.

Click here for further guidance on PNH testing. Contact the laboratory on 0121 424 0704.