The reported phenotype is that of mature CD8 + T lymphocytes and combined with considerable cytopenia. Hemophagocytic syndrome and viral infection would be an alternative to thje proposed diagnosis . Are there some morphological features ? (CD8 + T cell lymphomas are rare )

Thank you. The pictures are appended now. Appologies for delay.

The interstitial pattern of bone marrow infiltration by a huge number of CD8+ T-cells, the presence of CD20+ "follicular" structures as well the concomitant agranulocytosis and hypersplenism and modest splenomegaly are very suggestive for your proposed diagnosis. An important issue would be the values of the absolute lymphocytosis, which are probably consistent with LGL, but this should be more clearly documented. A correlation with the peripheral blood smear should be performed.  
 
Are there CD57 and TIA-1 stains?

TIA1 is out of my possibilities. CD57 is in the process. Lymphocytosis is absent in ther documentation- I insist to repeat the peripheral blood evaluation and flow. In BM aspirate 2 population of lymphos were documented: smaller and larger ones without signs of granularity. I request review once more. BM aspirate and PB were evaluated outside my center.

I agree with the others, and favor a CD8+ T cell neoplasm, likely of large granular lymphocytes. The absolute lymphocyte count, given the WBC and the absolute neutrophil count, will not show lymphocytosis; however, this is not unusual in T cell large granular cell lymphomas. Flow cytometry might help show a greatly altered CD4:CD8 ratio, if available. A touch imprint of the core biopsy might have shown the cytologic detail better, and is probably worth adding to your hospital's standard bone marrow procedure.

My experience is limited to T-LGL leukaemia. Essential for this diagnosis is an absolute lymphocyte count of at least 2000/ul, which this patient apparently does not have. In addition, there must be LGL's in the blood film, which are usually easy to identify. The immunophenotype in this case could be consistent with a T-LGL leukaemia, but it is important to have CD16 (+ in 80%) and CD57 (+ in almost 100%).  
The marrow is certainly diffusely infiltrated by a T-lymphoproliferative disorder. This is somewhat unusual in T-LGL leukaemia, in which the infiltration is often subtle and difficult to identify.

Addd data: EBV LMP1(-)(except mega's), CD57+ 10% (interstitial, maybe noneoplastic?). FLOW and TCR rearr. are in progress.

Please find some flow data above.

FLOE: Granulopoesis depression; CD4/CD8~1; Relative lymhocytosis, absolute T and slight CD8+ lymphocytosis;  
Appologies for transcription of FLOW report.

Duration of agranulocytosis for 1 year...

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This is a difficult case, in that both CD4- and CD8-expressing T cells
 
are increased. I'm surprised at that result. A few remaining items may
 
be helpful. One is a careful look at the degree of expression of CD2,
 
CD3, CD4, CD5, CD7, and CD8 in the T cells; decreased/absent expression
 
of one or more of these supports a neoplastic process. If it is
 
possible to do TCR alpha-beta and gamma-delta on the bone marrow (or
 
peripheral blood), it might show an abnormal pattern in the T cells.
 
The TCR gene rearrangement studies should be helpful. Two more clinical
 
questions: does the patient have rheumatoid arthritis or systemic lupus
 
erythematosus (SLE)? Is he on any drugs that could cause drug-induced
 
SLE? I have been fooled by severe drug-induced lupus in the bone marrow.
 
If this should be the case, the neutropenia may be due to a circulating
 
anti-neutrophil antibody. This can be tested, if available to you.
 

Thank you. I will request further data about clin possibility LE and RA. TCR is on progress from BM. On IH we see full house T imunophenotype with maybe exception CD7 (faint -/+).

Thank ypou for theswe nice immunostains . Indeed it is hemophagocytic syndrome and it has a diffuse interstitial infiltration of rather small lymphocytes . Reactive or neoplastic infiltration in this situation can only be diagnosed if TCR clonality and the whole clinical picture and course are considered. T- LGL is an option , but reactive changes are not ruled out .

Add data from clinics: Rheumatoid Factor RF 27,8 (elevated); Antistreptolysin 1020 (normal range 200). Any add symptoms of autoimmune disease are present. Agranulocytosis persists.  
 
Add data about flow (PB): T population CD5+70%; CD7+ 83%; CD2+ 88%; CD16+ 8%.  
So, immunophenotype of T population is "mature" like in BM with CD4/CD8 ratio~1....  
 
To be cont'd with PCR data...

THE LAST POINT: PCR revels TCR monoclonality (beta chain), Thank you for patience in this prolonged evaluation process.

what is your final diagnosis? leukemic mature T-NHL with cytotoxic phenotype = LGL? is the neoplastic population CD8+ or CD8 and CD4(dim)+?

The diagnosis was formulated the same. T proliferation, most probably LGL. By IH immunophenotype was CD8+ CD4-. I hope the patient will be resent to our clinics for further evaluation. I'm still in doubt about possibility to determine final diagnosis from threpine only. Any anti DNA antibodies were found. Usually isolated T proliferations in BM evoke a lot of prpoblems. Sometimes PCR helps, sometimes not. Thank you for continuous discussions and ideas.

The patient has a 2 year history or arthralgia and low ANC numbers. There is a small left cervical l/n (0,5cm) and small increase in spleen size. His ANC 0.06, LYM 1.03, PLT 177 and Hb 136 g/l, few grannular lymphocytes. LDH normal, betta2 microglob 2x normal (creatinine normal). RF 2x normal, polygammaglobulinemia, anti-ds DNA negative.  
His BM by flow is remarkable for 32% of lymphocytes CD2+, CD3+, CD5+, CD7+, CD8+, CD11c+, TCRa/b+, CD20+ (CD3+/CD20+ population), TCRg/d-, CD16-, CD56-, CD57-, CD4-. His TCRb is clonaly rearanged by PCR.  
It looks like the patient has a long standing malignant T lymphoproliferative disorder. The clinical picture (splenomegaly, low ANC, minimal lymphadenopathy, incr. in RF) would favour T-LGL. Unfortunately, immunophenotype does not support the diagnosis. Reactive process could probably also be ruled out (TCRb rearranged, > 6 months, diffuse BM infiltration). However, we will do some viral markers. So far no definitive diagnosis.

Please find some photos from PB and BM aspirate.

The morphology of the blood film and bone marrow aspirate show large lymphocytes with abundant cytoplasm. I cannot see cytoplasmic granules in these pictures, but am not certain whether this is real or not. The morphology is not diagnostic: could be normal large granular lymphocytes, activated lymphocytes or a leukaemia. The infiltrate is of CD8+ T cells (based on the trephine IH). I note that on flow cytometry these CD8 T-cells have normal T cell phenotype. However, on the bone marrow trephine IH there is increased CD57 staining (compared with normal marrow).  
TCRb clonality by PCR is of interest. TCRb is usually of low sensitivity for diagnosing a clonal population of T cells. However, when positive it is of high specificty, although false positive results are common even in very experienced hands. Before diagnosing a clonal T cell disorder, PCR for TCRgamma should be performed. This is the most sensitive for a clonal T cell disorder.  
 
In summary: severe neutropenia with a CD8-positive T cell lymphocytosis (with only low level CD57 expression and CD16 negative) may be reactive or primary. PCR for TCRb is very suggestive of a primary clonal T cell disorder but is not diagnostic. I recommend further PCR for T cell clonality. My laboratory would be happy to assist with this, if required.

Are you sure that the cells we double positive for CD3 and CD20? there are some CD20+ T-cell lymphomas descibed but this is very rare. If this phenotyoe is true it speaks for malignancy. I agree with Wendy - the morphology is not characteristic and can be seen in both neoplastic and ractive conditions. Was TCR gamma also rearranged?

Please, see CD3+/CD20+ results by flow. We will repeat clonality studies on new material. Thank you for your continuous cooperation.

I agree with the others that this is T-LGL leukemia associated with rheumatoid arthritis and splenomegaly, a well-known phenomenon. CD20, when positive on T cells, is expressed on CD8-positive T cells; possibly your other histograms show that. The finding of TCR beta rearrangement and decreased CD7 expression are good indications of clonality. There may be some interesting treatment options for this patient, including G-CSF therapy, splenectomy, and anti-T cell monoclonal antibodies.

TCRβ gene clonality analysis. Positive (clonal) result of the TCRβ chain in the polyclonal background.  
DNA was extracted from the bone marrow aspirate with Qiagen kit according to manufacturer’s protocol. TCRβ gene clonality assessed with IdentiClone™ TCRB Gene Clonality Assay kit (Invivoscribe technologies, USA). TCR gene clonality analysis revealed polyclonal pattern in the  chain (data not shown).  

TCRβ gene clonality analysis. Positive (clonal) result of the TCRβ chain in the polyclonal background.  
DNA was extracted from the bone marrow aspirate with Qiagen kit according to manufacturer’s protocol. TCRβ gene clonality assessed with IdentiClone™ TCRB Gene Clonality Assay kit (Invivoscribe technologies, USA). TCRgamma gene clonality analysis revealed polyclonal pattern in the gamma chain (data not shown).  

Summary of peripheral blood and bone marrow molecular genetic results:  
Blood : TCRG polyclonal ; TCRB clonal (V-J tube A)  
Bone Marrow : TCRG clonal (V-J tube A) and TCRB clonal (V-J tube A; D-J tube C)  
 
These results indicate the presence of a population of clonal T cells within peripheral blood and bone marrow.  
Although additional clonal rearrangements were detected within the bone marrow. This most likely represents sensitivity of the assay rather than clonal evolution.  
For TCRB gene rearrangement, the same V-J fragment was detected within peripheral blood and bone marrow.  
This supports a diagnosis of a primary T-cell disorder.

Thank you for all done. The special question about NOSOLOGY still remains open... What else from WHO T "leukemic" disorders may "fit" into this situation?