27 October 2011 (Thursday) - CPD Archive

It occurred to me that I’ve not done very much with my archive of CPD presentations recently. So this evening I added a couple of talks, and revamped the list so that the presentations now appear alphabetically.

If any of my loyal readers have any suggestions for freely available PowerPoint presentations which are available on-line, please drop me a line giving me the web address (rather than the presentation if possible) and I’ll add them to the list.

25 October 2011 (Tuesday) - Rapid Malaria Testing

Malaria antigen detection tests are a group of commercially available tests that allow the rapid diagnosis of malaria

There are currently over 20 such tests commercially available, but none of the rapid tests are currently as sensitive as a thick blood film, nor as cheap. A major drawback in the use of all current dipstick methods is that the result is essentially qualitative. In many endemic areas of tropical Africa, however, the quantitative assessment of parasitaemia is important, as a large percentage of the population will test positive in any qualitative assay.

Antigen tests look for one of four main markers:

pGluDH

The enzyme pGluDH does not occur in the host red cell and is therefore specific to plasmodium sp.

GluDH activity in P.vivax, P.ovale and P. malariae has never been tested, but given the importance of GluDH as a branch point enzyme, it is assumed that every cell must have a high concentration of GluDH.

Histidine Rich Protein II

The histidine-rich protein II (HRP II) is a histidine - and alanine -rich, water-soluble protein, which is localized in several cell compartments including the parasite cytoplasm.

The antigen is expressed only by P. falciparum trophozoites.

pLDH

P.falciparum lactate dehydrogenase (pLDH) is a 33 kDa oxidoreductase It is the last enzyme of the glycolytic pathway, essential for ATP generation and one of the most abundant enzymes expressed by P.falciparum.

pLDH does not persist in the blood but clears about the same time as the parasites following successful treatment. The lack of antigen persistence after treatment makes the pLDH test useful in predicting treatment failure.

In this respect, pLDH is similar to pGluDH. LDH from P. vivax, P.malariae, and P.ovale exhibit 90-92% identity to pLDH from P.falciparum.

pAldo

Fructose-bisphosphate aldolase catalyzes a key reaction in glycolysis and energy production and is produced by all four species. The P.falciparum aldolase is a 41 kDa protein and has 61-68% sequence similarity to known eukaryotic aldolases.

The presence of antibodies against p41 in the sera of human adults partially immune to malaria suggest that p41 is implicated in protective immune response against the parasite.

However these techniques are not without drawbacks.

Cross-reactions with autoantibodies:

Studies have reported cross reactivity of the various RDTs with autoantibodies such as rheumatoid factor, resulting in false positive tests for malaria. Studies in patients with positive rheumatoid factor have shown that the false positive reactions.

Sensitivity:

RDTs for the diagnosis of P. falciparum malaria generally achieve a sensitivity of >90% at densities above 100 parasites per µL blood and the sensitivity decreases markedly below that level of parasite density.

Many studies have achieved >95% sensitivity at parasitemia of ~500 parasites/µL, but this high parasitemia is seen in only a minority of patients. For the diagnosis of P. vivax malaria, many tests have a lower sensitivity compared to that for P. falciparum malaria; however, the pLDH test has an equal or better sensitivity for P. vivax malaria compared to P. falciparum malaria.

For the diagnosis of P. malariae and P. ovale infections, the sensitivity is lower than that of P. falciparum malaria at all levels of parasitemia on pLDH tests.

The sensitivity of the RDTs at low levels of parasitemia and for non-immune populations remains a problem. Compared to microscopy,many tests were found to be less sensitive in detecting asymptomatic patients, particularly at low parasitemias.

Also, the RDTs have been reported to give false negative results even at higher levels of parasitemia. Therefore, in cases of suspected severe malaria or complex health emergencies, a positive result may be confirmatory but a negative result may not rule out malaria. A negative RDT result should always be confirmed by microscopy.

19 October 2011 (Wednesday) - Lymphoproliferation

A colleague has prepared a case study for staff to use for CPD purposes. I was dead keen to try this out for myself….

Haemoglobin                14.9  g/dl   (11 to 15) White Blood Cells          14.3  10^9/l (4 to 11) Platelets                  124   10^9/l (150 to 400 Red Blood Cells            5.06  10^12/l(3.8 to 4.8) Haematocrit                0.449 ratio  (0.36 to 0.46) Mean Cell Volume           88.7  fl     (80 to 100) Mean Cell Haemoglobin      29.4  pg     (27 to 32) Mean Cell Haemoglobin Con  33.2  g/dl   (32 to 36) Neutrophils                2.6   10^9/l (2 to 7.5) Lymphocytes                11.2  10^9/l (1.5 to 4) Monocytes                  0.4   10^9/l (0.2 to 1) Eosinophils                0.0   10^9/l (0.02 to 0.5) Basophils                  0.0   10^9/l (0 to 0.1)  ESR                        1     mm/h   (1 to 20) Referred to consultant haematologist: Lymphocytosis with mature lymphoid cells with clumped chromatin. smear cells seen.  c/w  Lymphoproliferative disorder, most probably CLL.

Question 1: Discuss what is meant by Lymphoproliferative Disorder?

Lymphoproliferative disorders (LPDs) refer to several conditions in which lymphocytes are produced in excessive quantities. They are sometimes equated with "immunoproliferative disorders", because of the natural function of lymphocytes, but this classification is arguable.

Examples include;

• follicular lymphoma
• chronic lymphocytic leukemia
• acute lymphoblastic leukemia
• hairy cell leukemia
• lymphomas
• multiple myeloma
• Waldenstrom's_macroglobulinemia
• Autoimmune lymphoproliferative syndrome

Question 2: Illustrate the Immunophenotyping Markers to differentiate between the different Lymphoproliferative disorders.

Taken from http://www.cytometry.com.cn/uploadfile/20062615536162.pdf

Disorder/cell type	Marker
B-cell malignancies/Pan-B-cell antigens	CD19, CD20, CD22, CD79b, Igm, Igd, Igk, Igl
Precursor B-cells	CD1d, CD10, CD34, CD45, CD58, TdT, NG2,
Chronic lymphocytic leukaemia	CD5, CD23, CD38, CD52{, ZAP70
Hairy cell leukaemia	CD11c, CD25, CD103
Follicular lymphoma	CD10
Mantle cell lymphoma	CD5
Diffuse large B-cell lymphoma	CD5, CD10
Burkitt’s lymphoma	CD10, TdT
T-cell malignancies/Pan-T-cell antigens	CD2, CD3, CD4, CD5, CD7, CD8, ab TCR, cd TCR
Precursor T cells	CD1a, TdT, Myeloid markers
T-prolymphocytic leukaemia	CD26, CD52
T-large granular lymphocyte leukaemia	CD16, CD56, CD57
Se´zary syndrome/Mycosis fungoides	CD26
Adult T-cell leukaemia/lymphoma	CD25, HLA-DR
Natural killer cell antigens (and natural killer cell malignancies)	CD2, CD7, CD8, CD16, CD56, CD57

Question 3: Discuss the CLL score.

The CLL score is as described in:

http://www.bcshguidelines.com/documents/Lymphoma_disease_app_bcsh_042010.pdf

CLL immunophenotype scoring system (Delgado, et al 2003)
Marker	Score points 1 0
Smlg CD5 CD23 FMC7 CD22 or CD79b	Weak Strong Positive Negative Positive Negative Negative Positive Weak Strong
Score = or > 4 in 92% of cases of CLL
If <= 3 then exclude mantle cell lymphoma by one or both of: (i) Immunostaining of bone marrow trephine sections or lymph node for nuclear cyclin D1 (ii) FISH for t(11:14) using blood, BM or lymph node. Other lymphoproliferative disorders such as marginal zone, follicular and lymphoplasmacytic lymphomas can usually be distinguished by their immunophenotype. In problematic cases, lymph node or other tissue biopsy may be appropriate if the distinction is clinically important.

A score of four or above is obtained in over 90% of cases of CLL. A lower score should be treated with suspicion, but the diagnosis of CLL cannot be ruled out.

However CD5 positivity is present in most (90%+) cases. If this is absent, then alternative diagnoses should be considered.

CLL scoring should not be confused with clinical staging:

	Rai	Binet		Median survival
Lymphocytosis (>15,000/mm3)	0	-		150 months (12.5 years)
Lymphocytosis plus nodal involvement	I	A	<3 node groups	101-108 months (8.5-9 years)
Lymphocytosis plus organomegaly	II	B	>3 node groups	60-71 months (5-6 years)
Anemia (RBCs)	III Hgb <11 g/dL	C	Hgb <10 g/dL	19-24 months (1.5-2 years)
Lymphocytosis plus thrombocytopenia (platelets)	IV PLT <100	PLT <100,000/mm3

Question 4: Discuss the prognosis of patient of being diagnosed with CLL with reference to Lymphocyte doubling time

Lymphocyte Doubling Time is defined as the period of time needed for lymphocytes to double in number the amount found at diagnosis.

http://onlinelibrary.wiley.com/doi/10.1002/1097-0142(19871201)60:11%3C2712::AID-CNCR2820601122%3E3.0.CO;2-1/pdf  quotes that it appears to predict the progression of the disease. Specifically it (and other sources) feel that a doubling in less than a year is of worse prognosis than that of more than a year.

However it doesn’t take long to find doubt cast on the validity of these findings. After all, active therapy will reduce the lymphocyte mass, and consequently prolong the lymphocyte doubling time.